CN101978072A

CN101978072A - Rapid test including genetic sequence probe

Info

Publication number: CN101978072A
Application number: CN2009801086971A
Authority: CN
Inventors: 许卫东; 希亚姆·莫哈帕特拉; 阿伦·库马尔
Original assignee: Ultrapid Nanodiagnostics Inc
Current assignee: Ultrapid Nanodiagnostics Inc
Priority date: 2008-01-14
Filing date: 2009-01-14
Publication date: 2011-02-16
Also published as: WO2009128960A2; EP2245186A4; EP2245186A2; WO2009128960A3; ZA201005791B

Abstract

A rapid test kit may have a genetic probe, and an antibody detecting probe or a combination of a genetic probe and an antibody detecting probe disposed within one or more test windows of the test kit. A cellulose filter paper membrane with a flow rate selected in a range of about 0.04 to about 0.4 ml/min/cm2 is used in one example, The test kit provides for rapid screening for DNA, RNA or fragments of DNA or RNA in a bodily fluid or antibodies indicating exposure to such DNA/RNA. The genetic probe may include single stranded DNA or a fragment of single stranded DNA, such as primer, immobilized on the filter paper, and a single stranded DNA, such as the same or a different primer, conjugated with a marker, such as a nanotube or nanoparticle. For example, a gold nanoparticle or a carbon nanotube may be used as a staining agent by conjugating the gold nanoparticle or the carbon nanotube to a genetic probe, such as a DNA primer capable of binding with a complementary DNA or viral RNA or a fratment of one of these. By comparing contrast or intensity of a test spot to a standard, a viral load may be reported. By comparing a test region using the genetic marker and a test region using an antigen to detect antibodies, a sensitive and specific test may be conducted during use of a vaccine to determine the effectiveness of the vaccine, for example.

Description

The rapid detection that contains the genetic sequence probe

Related application

This application requires the U.S. Provisional Application 61/118 of submission on December 1st, 2008, the U.S. Patent application 12/008 that on January 14th, 939 and 2008 submitted to, 861 right of priority, the full content of above-mentioned two applications is introduced into the application by reference, for the unforeseeable advantage of open some example of the present invention and the colour code that is used for quantitative evaluation, U.S. Patent application 12/008,861 photochrome, comprise Fig. 2,3,4A-B, 6, the related description of 11-14 and these accompanying drawings (comprises [0069]-[0070], [0328]-[0331], [0343]-[0344], [0347], [0349]-[0350], [0352]-[0358] and [0361]), incorporate this paper by reference into.

Technical field

This area relates to test kit, and described test kit provides rapid detection and the diagnosis at infectious agent, RNA or DNA in the antibody, RNA, DNA or its segmental certain volume fluid that contain this test kit antagonist of use or DNA, RNA sequential detection q.s.

Background technology

Numerous disease at first is to use screening test to diagnose, and determines by additional survey.Known screening test must have highly sensitive, determines that check then must have high degree of specificity.Known have the rare false negative result of highly sensitive check, and the rare false positive results of the check of high specific.Be difficult to prepare the test kit that has highly sensitive and high degree of specificity simultaneously.Those skilled in the art understand, at the scene, the single agents box that uses of domestic environment or doctor's office can not satisfy sensitivity and specificity simultaneously by detecting in the disease Rapid identification that virus and bacteriocidin diagnose, described antibody for example is AIDS antibody (for example HIV), tuberculosis antibody, malaria antibody and hepatitis antibody.As an alternative, spot inspection only is used for screening, and more specific detection is carried out in controlled laboratory environment.

Be used to screen viral blood and can be saved 7-14 days, in the preservation process, anaphylactoid risk and potassium concn constantly increase, and its oxygen carrier amount constantly reduces.For a long time, exist carrying out the demand of the reagent (agencies) of on-the-spot blood testing the detected person.The use ability of test kit screening body fluid (for example blood, saliva and urine) reliably and fast can't be provided, and need this ability for a long time always.

Modal screening test is enzyme-linked immunosorbent assay (ELISA), is sometimes referred to as enzyme immunoassay (EIA).The most definite check of normal use is a western blotting.If produce antibody in the body, then these checks can be at these antibody of low level detection.For example, Chang Gui HIV detection method in clinical labororatory from sensitive EIA.Can utilize serum, blood plasma, urine or mouth cavity liquid to carry out EIA, and can after 3～4 days, obtain the result.If EIV is negative, think that then the result determines, show to need not further check.

All there is such limitation in any check: a lot of antiviral antibodies were just expressed in infection in back 3 months, infect and detect between cause window phase (window), even use sensitive mensuration.If EIA repeats positive, also need to implement to use the more specific check of western blotting technology to determine.This checkout procedure needs a week from sampling usually to obtaining net result, even the longer time.Finishing a required cost of check and time makes and can't carry out frequent checks, even if also can't carry out in the high risk population.

Western blotting check (WB) utilizes electric field according to each composition in the molecular weight sample separation.This allows to identify the antibody of specific viral antigen, and described antibody demonstrates certifiable " band " on banded test paper.This check shows high degree of specificity, and by utilizing and enzyme link coupled antibody or antigen with easy detection of high turnover number, ELISA combines the susceptibility of simple enzymatic determination and the specificity of antibody.ELISA can provide effective mensuration of antigen or antibody concentration, and this is that the reagent for quickly examining box can't obtain.Therefore, rapid detection is used for the employed blood of check that need finish or the buffered samples of serum within five minutes.

With the amount of ELISA mensuration to the antibody of intact virus, and provide " positive ", " feminine gender " or uncertain detected result difference, western blotting is a kind of more specific detection.It makes visual at the antibody of each viral protein, therefore, is a kind of checking check for the positive check that utilizes ELISA or EIA to carry out.The western blotting check is considered to the golden standard for the checking of the ELISA of the active sample of screening (reactive sample) in a lot of virus infectiones (especially low danger crowd's) the diagnosis and/or rapid detection.Basically all must confirm by any repetition positive findings that the ELISA that is used for a lot of virus infectiones or other rapid screening methods obtain by the more specific mensuration of for example western blotting (WB) check and so on.

This accuracy of using ELISA and the additional strategy of checking further to increase result and diagnosis simultaneously.In principle, any WB test kit that provides the uncertain reaction of high frequency (overwhelming majority is non-specific combination) all is not suitable as the primary screen selection tool of large-scale colony.Its advantage only is as a checking property mensuration under the background of positive or uncertain antiviral antibody ELISA or initial screening check.

In one embodiment, window phase is that virus infection begins and time period between the antiviral antibody occurs detecting.In the sensitive HIV antibody test that current recommendation is used, window phase was about for 3 to 4 weeks.But this time period may be longer.Any blood test based on antibody (for example ELISA, rapid detection and western blotting) of carrying out at window phase may provide false negative result.The expense of these check costs and time mean seldom tests individuality.Although virus can exist and be about the trimestral time most in people's the blood and do not have (detectable) antibody to exist in the blood between whole detection period, but, testing cost rises to 1 year or longer with this window phase, if especially individuality is in the low colony that endangers.In fact, the beginning of most patients disease symptoms first indication (indication) normally.As a rule,, just might infect other people, and significantly reduce the ability that patient is treated in case disease symptoms occurs.This is for AIDS, hepatitis, tuberculosis and be proved to be and utilize for a lot of diseases (some bacterial strain at least) that conventional antiviral therapy or antibiotic therapy more and more be difficult to treat really so.In this window phase and before carrying out subsequent detection, this individuality infects, and may infect other people unintentionally.Need a kind of fast, cheapness and sensitive check and detect infectious diseases, this check allows to detect on-the-spot, blood donation center, even carry out individual conventional sense at home in the clinic (office visit).

The quantity that is used to detect the test kit of infectious agent (for example virus disease and bacteriosis) increases to some extent.Regrettably, a lot of family expenses test kits of selling on the market had not both gone through to be not enough to be used for the disease of test example such as AIDS yet.At the unique approved HIV test kit of the U.S. is to be used for taking a sample and sample being delivered to the laboratory analyze.Not need not sample is delivered to the screening that breadboard reagent for quickly examining box is approved for HIV in the U.S..

Some test kits can be used for serum sample is carried out disease detection.For example, comprise that it is available that effluent is checked the test kit of (lateralflow test).The lateral flow check is also referred to as the immuno-chromatographic test paper strip check, is used for specificity screening or half-quantitative detection to a lot of analytes that comprise antigen and antibody.Can use sample separately, perhaps sample be used with extracting reagent or running buffer, be placed on then on the sample pad of test strip (test strip) end.This test strip also comprises film.Indicator (signal reagent) is soluble, if antigen is arranged in the sample then combine with antigen, and moves to the other end by capillary action by an end of film.This mixture is caught by second antibody then, produces a visual line, shows antigenic existence.Though effluent check is slow, and directly place sample at Examination region and compare, improved the contrast gradient between visible light and the background.Therefore, the effluent check accounts for space of top prominence in the enzyme detection market of body fluid.Known no effluent check can be used blood.

Report as Glynou K (2003), known effluent test paper (Lateral-flow dipstick) test kit can detect DNA, but the shortcoming that the effluent test paper that is used for blood and other body fluid is checked is also unresolved, does not also use the report of effluent test paper inspection RNA.

Circulation check (flow through test) may relate to and contain a plurality of independent box that extracts damping fluid and lavation buffer solution.These checks comprise when analyte (for example antibody or antigen) and utilize reagent to catch described analyte when flowing through film.The common poor contrast of above-mentioned test kit.Working specification may need the user to prepare detected sample, the washing film, add indicator, and the washing film is so that remove any residual from sample from film, to attempt improving background and any screening line or to be used to show contrast gradient between the marker that enzyme or antibody exists.Though known direct circulation test kit is fast, because the complicacy of working specification (requiring provides enough contrast gradients between indicator and background film) is seldom used in the practice.In the field, a common recognition is arranged, promptly the deficiency of contrast gradient makes the sensitivity of circulation test kit be lower than the effluent test kit, thereby the circulation test kit is seldom used.And, although for clean and again for the cleaning reagent box complicated operations and explanation be essential, in fact, it makes compares more inconsistently with the result of effluent test kit, this also brings disadvantageous effect to the circulation test kit.At this, also reported the example that utilizes other several commercial test kits to detect, and compared with the example of the present invention that uses Detection of antigen HIV antibody.The commercial sample that whole blood is tested does not all play a role, and this has limited the operability of these commercial test kits in the spot inspection that does not have laboratory and whizzer.In addition, some embodiment has than commercial test kit better contrast, and this makes them be very easy to read the result.

Chen has described a kind of immunoassay device that is used for detecting at biofluid HIV-1 and HIV-2 antibody in WO 96/21863, provide is hair style immune response and detection that this antibody-like is existed, comprising combined filtering device and the reaction member that utilizes nitrocellulose filter, on described nitrocellulose filter, carry out immune response.By described antibody is combined and observes film (showing the existence of antibody) that red color occurs with albumin A colloid indicator, make thus with the antibody naked eyes of HIV glycoprotein antigen gp41, gp36, gp38 and gpl20 reaction as seen.Chen proposed before the contact nitrocellulose filter effluent and/or makes the blood filtering filtration medium.Carrying out filtering additional step before the contact membrane has increased and has implemented the required time of this check.Chen proposes a kind of allied equipment that needs nitrocellulose filter in another publication WO 95/18624.In this check, Chen only uses a kind of albumen gp41.Western blotting check needs to exist in three kinds of HIV albumen two kinds improving specificity, but increasing detected proteinic quantity might not bring sensitivity and specific raising.In some cases, western blotting may provide a uncertain result to being actually HIV male sample.For example, Abbott Determine ^TMBe early screening reagent, but it does not provide the reagent for quickly examining box that can be used for whole blood at the scene at HIV1 and HIV2.

In No. the 2004/0023210th, U.S. Patent Publication, Mahajan discloses the diagnostic kit that is used at human serum and blood plasma detection antibody of HCV, comprises substrate (base); Be arranged on the nitrocotton immunofiltration film on the absorption pad, described absorption pad places in the described substrate; Top cover, this top cover is connected with substrate movably, and has the centre hole that adapts with the film circumference.Antigen (for example NS3, NS4 and NS5) is fixed on the film, by forming conjugate with albumin A as seen.This reference points out that the aperture of nitrocellulose filter is 0.8～1.5 micron.This aperture and specificity and susceptibility dependency are poor, and described specificity is relevant with contrast gradient (the perhaps ColourIndex number of wherein reporting) with susceptibility.The test kit that only is fit to use with serum or blood plasma is not suitable for use in on-the-spot reagent for quickly examining box.

In No. the 2003/0165970th, U.S. Patent Publication, Hu has proposed to be used for the diagnostic device of the multiple infectious agent of test example simultaneously such as HIV antibody, B-mode and antibody to hepatitis C and syphilis antibody.The disclosed test kit of Hu comprises immune gold filter determinator, damping fluid, and the mixture of colloid gold particle, wherein said device comprises the nitrocellulose filter that utilizes HBsAg monoclonal antibody, HCV antigen, syphilis antigen, HIV antigen and goat anti-mouse IgG antibody trace.This check is not quick test, and needs very complicated rules.

At United States Patent (USP) the 5th, 885, in No. 526, Chu discloses a kind of circulation verifying attachment with reaction film, comprises for example porous material of nitrocellulose.When using nitrocellulose filter, need to be pointed out the small-bore for the area that obtains bigger sessile receptor molecule.As col.5, described in the Ins.53-56.Chu points out that larger aperture causes measuring sensitivity and reduces.The porosity of Chu preferred reaction film is 0.45 micron to 3 microns scope.As col.3, described in the Ins.15-32, Chu points out not utilize pressurization to keep reaction film, because pressurization makes device be unsuitable for the immunoassay that some needs quantitative result, and Chu not disclose any with the example of Mierocrystalline cellulose filter paper to the whole blood use.

The embodiment of Chu also points out not increase flow velocity, and Chu thinks increases the interaction time that flow velocity can reduce the target molecule in the sample and be fixed on the acceptor on the reaction film.So as col.5, Ins.57-60 is disclosed, measures sensitivity and reduce.In addition, the aperture is sensitivity and specific unfavorable factor.

Chu also points out and need form airbag (air pocket) with thick reaction film, to stop lateral flow and directly to flow.As disclosed among the embodiment 2, the work example discloses the nitrocellulose reaction film that the thick 800 microns back side is ply of paper.Chu also discloses has that for example thickness is less than a lot of shortcomings of the existing apparatus of the thin reaction film of 0.1mm, and as col.7, Ins.66-col.8 is described.

Chu discloses film should immobilized antigen and albumin A, and hints that for example the material of nitrocellulose and glass fibre and so on is fit to immobilized antigen and albumin A.Chu required before the check and analysis matter sample, with albumin A and the antigen inoculation different zones at film.Chu also required before being absorbed into serum sample in the film, earlier with albumin A and purpose antigen inoculation to film, and after being absorbed, serum or blood plasma utilize additional step to add albumin A-Radioactive colloidal gold binding substances, this is the preferred version of Chu, this scheme must provide enough contrast gradients, very complicated, not fast fully.In addition, Chu also discloses the edge that albumin A preferably is seeded in device, because the central position of film will comprise interested antigen, for example hepatitis C antigen.Chu is a United States Patent (USP) the 5th, 541 in another patent, discloses use albumin A and antigenic immunoassay apparatus in No. 059.This test kit of Chu is not the reagent for quickly examining box, and the working specification complexity, and the result is unpredictable by untrained working group or individual's operation the time.In No. the 2004/0002063rd, U.S. Patent Publication, disclosed as [0062] section, the preference such as Chu such as the back side are the porousness reaction films of nitrocellulose of paper and so on, and preferred aperture is 0.2 micron to 0.8 micron.By the disclosed film of Chen must be suitable porous-film, for example discloses the example that uses the nitrocellulose of being supported by porous paper.The test of nitrocellulose filter shows that water is very fast by the flow velocity of this film, but nitrocellulose is undesirable in the test of being implemented by the applicant.Though Chen does not get rid of Mierocrystalline cellulose filter paper as film, shown in applicant's result, Mierocrystalline cellulose filter paper has the flow velocity similar to nitrocellulose, can't use.Chen is not provided at and uses the example of Mierocrystalline cellulose filter paper as film in any test kit.In addition, for example at United States Patent (USP) the 5th, 980, in No. 746, Gelman etc. point out, owing to reduce proteinic film adsorptivity at well known cellulosic cpd, so do not use cellulosic cpd.Therefore, the existence of using nitrocellulose filter to detect antibody is known.

In addition, in order to detect blood, Chen discloses a kind of independently more complicated test kit in blood separation district that has, and for example uses the test kit of glass fibre matrix as the blood separation material, for example example of providing of [0089] of No. the 2004/0002063rd, U.S. Patent Publication section.Program that should complexity during spot inspection is infeasible.

At United States Patent (USP) the 6th, 653, in No. 066, Krutzik discloses a kind of effluent check of using the aperture less than 5 microns matrix and nitrocellulose filter, and disapprove uses the wide aperture matrix, obtains the result of difference because of its tendency.

As everyone knows, on Mierocrystalline cellulose filter paper, gather exsiccant whole blood spot, but this is collection and the drying that is used for blood, is not as the reagent for quickly examining box.In fact, for test kit, make Mierocrystalline cellulose filter paper be suitable for preserving the feature and counter-intuitive of blood.Exsiccant blood is washed from filter paper subsequently to be used for detecting.For example, at Ann.Clinical Biochemistry 1991; Among the 28:155-159, people such as Rocks have described and have utilized silver to strengthen gathering blood before the golden label immunoassay assess sample on filter paper in scribbling antigenic microtiter well.At Clinical and Vaccine Immunology, 2006 (1), Patton etc. has described use filter paper and has collected blood sample in the 152-153 page or leaf, utilizing HIV-1p24ELISA to measure is further analyzed, but, do not use Mierocrystalline cellulose filter paper to use Mierocrystalline cellulose filter paper as film or in check.On the contrary, blood is washed from filter paper and is used for separating check.At Journal of Clinical Microbiology, 1989 (6), people such as Fortes has described use cotton filter paper (cotton filter paper) before carrying out next step ELISA check in the 1380-1381 page or leaf.Above-mentioned reference does not all disclose the reagent for quickly examining box of any type.On the contrary, filter paper is used to transmit and storage exsiccant blood, and during detection, described dry blood washes from described filter paper.

One of problem that existence produced of using antibody/antigen detection disease is to have accepted immunization or immunoreactive subject is taken place to have antiviral antibody or bacteriocidin, but does not have disease.For example, the AIDS vaccine comprises the complex mixture of HIV-1 epitope (peptide, protein, DNA expression plasmid and recombinant viral vector) usually, can be in vaccinated volunteer inducing sustained antibody response, this antibody response can be detected by the HIV-1 test kit that FDA ratifies.When utilizing existing serology check and analysis to detect vaccinated volunteer's serum, vaccine-induced antibody can cause false positive or uncertain reactivity.In another example, because baby's immunity system is subjected to the influence of maternal antibody, during this influence is lasting uncertain, may detect antibody positive into HIV so infected mother's of HIV baby.

Utilize advanced PCR and reverse transcription PCR might detect viral genetic, but these technology are not suitable for the reagent for quickly examining box of bedside in-vitro diagnosis (point of care).On the contrary, these technology are expensive and consuming time.The developing into of chip PCR etc. reduce cost and allow bedside in-vitro diagnosis PCR method provide certain may, still, in fact still be difficult to obtain to use the commercial device of these technology.In any case it all is unpractical utilizing the detection method of PCR-based to carry out conventional widely screening.

Nano-substance, for example Single Walled Carbon Nanotube (carbon nanotube, CNT) and the gold (Au) nano particle (gold nanoparticle) known.And the also known functionalized gold nano grain of oligonucleotide that for example how to utilize in the effluent test paper detects DNA.In addition, when the staining technique based on gold nano grain combines with primer extension reaction, successfully be used for single nucleotide polymorphism is carried out gene type.Become known for the nanowires of gold microfluidic procedures platform of the sensitivity detection of blood analyte, but this method needs electrochemical appliance to read.These methods or material all do not have to unite with the reagent for quickly examining box and are used for the bedside in-vitro diagnosis and detect disease.

Summary of the invention

The quick test of detect infecting can detect HIV and infect being less than in 5 minutes, the example of test kit can be used for detecting respectively or simultaneously the antibody in the body fluid or the existence of RNA or dna sequence dna.In one embodiment, an independent test kit detects the antibody of indication disease and the existence of viral RNA sequence simultaneously.In optional embodiment, test kit separately is provided, it carries out antibody screening and utilizes gene probe to detect the sequence of disease specific DNA or RNA.

Therefore, use gene probe test kit can be used for measuring test kit that body fluid sample antibody exists and separate and be used for check.In one embodiment, have only tested sample to be antibody positive, just utilize the test kit test disease that contains gene probe.This check can be used to the level or the concentration of antibody in qualitative and/or the quantitative assay body fluid or RNA or dna sequence dna.

In one embodiment, with contrast scale (contrast scale) relatively with determine the relative level or the concentration of the antibody detected in the body fluid of being checked and/or RNA or dna sequence dna.For example, the quick indication of existence of the RNA in the body fluid of checking or a certain sequence of DNA INFECTION IN DETECTION and/or affirmation can be provided, for example, especially in the acute infection when for example HIV infects.

In certain embodiments, test kit has lower flow velocity and holds back size (relevant with the aperture) with bigger particle, and can finish rapid detection being less than in 3 minutes.For example, the test kit utilization is fixed on antigen or the antigen combination on the film (for example Mierocrystalline cellulose filter paper), and described film is selected to fixing described antigen, and has about 0.04ml/min/cm ²To about 0.4ml/min/cm ²The flow velocity of scope.For example, direct placement, the circulation of the suspension of handling with damping fluid that this test kit can be by PBS buffered blood, serum or blood plasma and so on detect antibody.Other commercialization test kits of testing all can not detect whole blood, use example of the present invention can easily realize whole blood test, and do not influence the result, and have and the similar contrast gradient of identical check of using serum or blood plasma.There is not the known detection that utilizes whole blood to reach and use the identical result of detection of serum or blood plasma in the prior art as yet.In one embodiment, use antigenic particular portion to assign to improve the contrast gradient of positive display area, especially all the more so for whole blood, and, to compare with using antigen integral body, antigenic short-movie section obtains better result.

In one embodiment, diagnostic kit comprises: the Mierocrystalline cellulose filter paper of immobilized antigen, at least a antigen of fixed on described Mierocrystalline cellulose filter paper, detection is at the staining agent of described at least a antigenic antibody, remove the painted decolouring damping fluid of non-specific background, and be arranged on the diagnostic kit bottom a plurality of rapid-curing cutback layers (wicking layer) relative with reaction film.For example, can have as the Mierocrystalline cellulose filter paper of the responding layer of described test kit and be selected from about 6 microns particles and hold back size to about 25 microns scopes.And the assay that variable grain is held back the Mierocrystalline cellulose filter paper of size shows, the paper that have 6,11 and 20～25 particle of (Whatman qualitative/wet intensity level Mierocrystalline cellulose filter paper) is held back size does not show big side-play amount (largedeparture) aspect flow velocity.The reagent for quickly examining box should not have unnecessary low flow velocity, still, has dependency between the ColourIndex number of reporting among flow velocity and the result, and described ColourIndex number is relevant with the sensitivity of the test kit that detects antibody.Therefore, for selecting to have the Mierocrystalline cellulose filter paper of optimum flow rate, there is a preferable range.

In one embodiment, staining agent is and Radioactive colloidal gold bonded albumin A.Use for example decolouring damping fluid raising of phosphate buffered saline (PBS) and so on and the contrast gradient of background.In another embodiment, be used to detect the rapid detection selection Mierocrystalline cellulose filter paper or the equivalent (equivalent) of infection, wherein phosphate buffer soln (PBS) flow velocity is at about 0.04～about 0.4ml/min/cm ²Scope, in order to obtain higher contrast gradient (sensitivity), flow velocity is 0.04～0.2ml/min/cm more preferably ²Aspect determining to measure sensitivity and finishing the experiment required time, the velocity ratio aperture is more important.In one embodiment, can select flow velocity at about 0.1～about 0.2ml/min/cm ²The Mierocrystalline cellulose filter paper of scope, thereby the optimal tradeoff of sensitivity and flow velocity is provided in certain embodiments.

In another embodiment, can select such Mierocrystalline cellulose filter paper, promptly the PBS flow velocity is at about 0.2 ± 0.05ml/min/cm ², to increase flow velocity and reducing sensitivity within reason (being ColourIndex number).When measuring flow velocity, term " about " is used for representing to make Mierocrystalline cellulose filter paper process and testing the difference that produces in the flow velocity process in the ASTM method according to improvement described herein.Those of ordinary skills can measure flow velocity and select Mierocrystalline cellulose filter paper, and those Mierocrystalline cellulose filter paper have the flow velocity roughly the same with scope given herein based on disclosed flow rate test method and flow velocity.

Use Mierocrystalline cellulose to be, can obtain the result of specific infectious agent or a plurality of infectious agents fast, and need not complicated user's scheme (user protocol) as an advantage of the diagnostic kit of responding layer.In fact, in certain embodiments, than serum or blood plasma, the same result that obtains easily of whole blood.

Another advantage is the cost of test kit, and this test kit significantly reduces the screening cost.With the low-cost cheap reagent for quickly examining box of producing and provide, this is especially necessary for the use in remote districts and doctor's office.Can use independent check detect can from blood testing to multiple disease.

Another advantage is to use independent diagnostic kit to detect one or more different body fluid, and for example therefore blood, blood plasma and serum can provide greater flexibility in test process.Spot inspection be can implement, and removable laboratory or whizzer need not.

Another advantage is that the reagent for quickly examining box can provide the result fast and have good sensitivity and good specificity.In one embodiment, comprise gene probe, with RNA, DNA in the detection body fluid or sequence or the fragment of RNA or DNA.For example, can to contain primer right for this probe.A primer of described primer centering can be fixed on the filter paper; and another primer of described primer centering combines with nano particle or nanotube; for example utilize the mercaptanization of another primer of described primer centering to carry out combination, and bonded primer-nano particle or primer-nanotube are comprised in the dyeing damping fluid.An advantage using gene probe for example is that described gene probe can provide RNA and/or the level of dna sequence dna or the qualitative or quantitative analysis of concentration in the volume fluid of surveying (for example being urine, blood, oedema or saliva), and it can be associated with virus load.Another advantage of gene probe is that vaccinated subject or subject with maternal antibody and the subject who catches are made a distinction.

Be used to detect the segmental reagent for quickly examining box of DNA, RNA or DNA or RNA and comprise detection faces and staining agent, described detection faces for example is a film, at the check partial fixing of described detection faces one or more gene probes is arranged.Described gene probe is chosen to and the gene order hybridization that is detected, for example gene order of viral RNA.Described staining agent can comprise different gene probes, for example with check the gene order bonded functionalized nano-particles or the nanotube that are detected.Therefore, when the sample that will contain gene order placed on the detection faces or passes through detection faces, described gene order preferably was fixed on the detection faces zone, and described detection faces for example is a Mierocrystalline cellulose filter paper film.Then, described staining agent is fixed by combining with described gene order, thereby partly and between the background parts produces contrast gradient in the check of film.

In one embodiment, can select to decolour damping fluid with remove with at least a gene probe, described DNA, RNA or described DNA or described RNA fragment and described staining agent between combine irrelevant painted at least a portion of any non-specific background.

For example, described detection faces can be the surface of slide glass or film.For example, described film can be selected Mierocrystalline cellulose filter paper, thereby makes this film when utilizing improvement ASTM standard flow rate measuring method to measure, and the flow velocity of phosphate buffer soln is about 0.04～about 0.4ml/min/cm ²More preferably this film has about 0.04～about 0.2mL/min/cm ²The mensuration flow velocity of scope, to strengthen the contrast gradient between Examination region and the background.For example, the mensuration flow velocity of film can be limited at 0.1mL/min/cm at least ²～be not more than about 0.2mL/min/cm ²Scope, with optimizing check required time and contrast gradient.

In one embodiment; staining agent comprises at least a oligonucleotide functionalized nano-particles or nanotube, and described oligonucleotide functionalized nano-particles or nanotube have the oligonucleotide of at least a gene order area hybridization that can be detected at room temperature and reagent for quickly examining box.

In addition, at least a gene probe can comprise the complementary oligonucleotide of the specific region hybridization that is used for the gene order that detected with test kit.For example, in one embodiment, described complementary oligonucleotide combined with chitosan or chitosan derivatives before being placed on the detection faces, thereby made complementary oligonucleotide be fixed in Mierocrystalline cellulose filter paper film.Provide the example of chitosan and chitosan derivatives in the prior art, for example disclosed chitosan derivatives etc. among the mercaptan chitosan derivatives among the U.S. Patent Publication US 2007/0036867, the U.S. Patent Publication US2008/0087290.

For example; oligonucleotide functionalized nano-particles or nanotube can be the functionalized gold nano grains of mercaptan oligonucleotide, described mercaptan oligonucleotide and gene order distinct portions rather than its target sequence complementation of being fixed in the complementary oligonucleotide target on the film.The mercaptan oligonucleotide can be a primer, and this primer is selected to and is selected from by the viral RNA in HIV virus, hepatitis B virus, hepatitis C virus, SARS virus and its group of forming hybridizes.For example, the example of oligonucleotide can by mercaptanization and with the gold nano grain surface bonding, can also be selected to and the hybridization of the viral RNA of HIV virus.In one embodiment, be fixed in complementary oligonucleotide on the film and can be selected to identical or different area hybridization with the viral RNA of HIV virus, for example LTR sequence.In optional embodiment, staining agent comprises by the functionalized carbon nanotube of the example of oligonucleotide.

No matter whether use nanotube or nano particle, in order to observe positive experimental result, can adjust concentration, thereby under the appropriate illumination condition, provide sufficient contrast gradient.In one embodiment, comprise a plurality of oligonucleotide of a plurality of regional bonded that are used for detected gene order in the staining agent.Each nanotube or nano particle can be functionalized by one or more oligonucleotide.Therefore, a plurality of nanotubes or nano particle can with one or more area hybridizations of individual gene sequence, compare with a zone of only using this single-gene sequence of oligonucleotide target, viewed contrast gradient is amplified.On the other hand, but be fixed in only one or selected several gene region in the described gene order of the one or more oligonucleotide targets zone on the detection faces, to strengthen the specificity of test kit to one or more gene orders of selecting to be used to measure.For example, table 8A-P (for clarity sake, having had a mind to omit name 8I and 8O) discloses the screening of gene order, and described gene order is used to locate distinctive HIV special gene sequence, and this special gene sequence provides the specificity of rapid detection.By the specificity complementary oligonucleotide on the detection faces is combined with a plurality of oligonucleotide that are used to combine each nano particle or nanotube and described gene order; even test with low virus load; this check also can have extraordinary contrast gradient; and in the gene order that is detected, still keep highly sensitive; thereby provide wondrous and beyond thought improvement to any known rapid detection, for example comprised the ability that vaccine is distinguished at the effect and the virus load of antibody.

In addition, can select some complementary oligonucleotide of hybridizing for use with the specific region of gene order.For example, in the rapid detection that is used for the bedside in-vitro diagnosis, preferably select the complementary oligonucleotide of hybridizing under the room temperature.By this way, but any gene order of specific oligonucleotide target for example comprises the viral RNA of HIV virus, hepatitis B virus, hepatitis C virus, SARS virus and combination thereof.In one embodiment, at least a gene probe comprises the oligonucleotide from HIV 89.6 provirus clone's 33-nt, and it can be puted together mutually with chitosan or chitosan derivatives.

In a method, described method comprises the detected level of measuring virus load in the fluid sample volume.This method can comprise the steps: to make above-mentioned at least one gene probe and chitosan or chitosan derivatives to put together the formation conjugate, and the Examination region that described conjugate is fixed in film.In one embodiment, this method comprises utilizes the UV-irradiation film with the check part of reinforcing membrane and the contrast gradient between the background parts, makes UV-light cause and checks part to send fluorescence.For example, can determine the level or the concentration of virus load by described contrast gradient or fluorescence and known level or concentration are compared.For example, in one embodiment, utilize detector and treater to make the mensuration automatization, described treater compares signal and the look-up table (look up table) that described detector receives.For example, the step of report can comprise the contrast gradient of at least a portion of the check of film part or intensity and standard are compared.In a method, staining agent placed on the film so that the gene probe of staining agent in the temperature range that can comprise room temperature with a part of selective binding of gene order.Room temperature is considered to for example from about 15 ℃～about 25 ℃ temperature range.

In one embodiment, gene probe and antibody test are united use.Described antibody test can comprise one or more peptide fragment, and this peptide fragment for example is the gp41 peptide fragment that comprises following sequence:

QLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNAS。

In another embodiment, at least one gene probe is placed the surveyed area of slide glass, tested fluid places on the described surveyed area, and it is nanotube or particulate suspension is functionalized with complementary oligonucleotide, make when described suspension directly places the surveyed area of slide glass, if there is gene order in surveyed area, the then specific region of complementary oligonucleotide and gene order hybridization.For example, can before placing fluid on the slide surface, gene probe be fixed on the slide surface.Can be from surperficial flush away fluid after one period regular time.Then, can in a temperature range (for example room temperature), staining agent be placed surveyed area, and keep one period regular time.Can the flush away staining agent and under the light of for example UV-light, observe slide glass, to measure the contrast gradient between surveyed area and control zone or the background area.Alternatively, detector can be observed the emission light or the absorb light of the surveyed area of slide glass.For example, the functionalized functionalized carbon nanotubes of complementary oligonucleotide can be used for detecting the fluorescence under the UV-light, absorbed light when functionalized gold nano grain can be used for detecting light (for example UV-light) by surveyed area.For example fluorescence or phosphorescence can be absorbed.For example, detector or system can report and relevant numerical value or the output signal of virus load in the sample.

For example, in one embodiment, gene probe comprises the oligonucleotide from HIV 89.6 provirus clone's 33-nt.Gene probe can be fixed on the part of test kit, and the antigen that is used to detect antibody can be fixed on another part of test kit.In one embodiment, these two portions are at same test window, and react with same body fluid sample.In optional embodiment, above-mentioned two portions place different windows.Can use one or more staining agents, it can comprise functionalized nanotube or nano particle.In one embodiment, provide with the functionalized nanotube of complementary oligonucleotide or by the functionalized particle of oligonucleotide.Described gene probe can be can with the complementary oligonucleotide of tested gene order hybridization, for example be the part of the LTR gene order of HIV-1 virus.For example, described staining agent can comprise a plurality of mercaptan oligonucleotide in conjunction with selected gold nano grain, so that a plurality of mercaptan oligonucleotide can be hybridized the different piece of the RNA of HIV-1 virus separately.

Other beyond thought advantages, purposes, device and modification thereof, combination are shown among the embodiment of accompanying drawing and detailed description.

Description of drawings

This patent or application documents comprise at least one color drawings.This patent or the disclosed copy of patent application that can require official to provide to have color drawings, and payment necessary fee.

Accompanying drawing is described the method for some embodiment and the preparation and the use diagnostic kit of quick diagnosis reagent kit.

Figure 1A represents the example of the cross section of diagnostic kit 100.

Figure 1B represents another example of the cross section of diagnostic kit 110.

Fig. 1 C represents the vertical view of example diagnostic kit as shown in Figure 1A and 1B.

Fig. 2 A-2B provides the vertical view of the example of test kit, (A) records antibody and exists for feminine gender, (B) records antibody and exists for the positive.

Fig. 3 A-3B provides the comparison of example (B) with the example (A) of the diagnostic kit that uses glass fibre membrane of the diagnostic kit that uses Mierocrystalline cellulose filter paper, and (A) leads to the failure when testing with whole blood.

Fig. 4 A-4C represents to use the comparison of example and the example of the diagnostic kit that uses nitrocellulose filter of the diagnostic kit of Mierocrystalline cellulose filter paper film.

Fig. 5 represents the relation curve of the flow velocity of ColourIndex number and PBS, and described flow velocity adopts the round Mierocrystalline cellulose filter paper of 7cm that uses in the check, and utilizes the ASTM flow velocity method (modified ASTM flow rateprocedure) of improvement to measure.

Fig. 6 represents to be used for determining the color index chart of ColourIndex number, wherein, is 1 to any identifiable marker assignment that is deeper than background, the any mark assignment darker than 1 is 2, the any mark assignment darker than 2 is 3, is 4 than 3 dark any mark assignment, thereby quantizes the colour intensity of check sample.

Fig. 7 represents the mensuration flow velocity of 6 different Mierocrystalline cellulose filter paper and the relation curve that particle keeps size.

Fig. 8 discloses the ColourIndex number of representing each result according to the order of sample number, comprising the employing test kit, and the check that utilizes blood and blood plasma to carry out, the PBS flow velocity of described test kit is about 0.1mil/min/cm ², also show the color index that contrasts spot.

Fig. 9 is the assay figure that utilizes blood.

Figure 10 diagram relatively use embodiment the reagent for quickly examining box the blood plasma assay and use available commercially available test kit (

G3) assay, described reagent for quickly examining box use flow velocity to be about 0.1ml/min/cm ²Mierocrystalline cellulose filter paper.

Be MedMira Laboratories, the registered trademark of Inc. (Toronto).

Figure 11 uses the assay figure of whole blood.

Figure 12 is to use the assay figure of blood plasma.

Figure 13 is to use the assay figure of blood plasma.

Figure 14 A-14C illustrate have the control test spot and have gene probe simultaneously and and the possible outcome of the test kit of the antibody test spot of antibody, (A) expression adopts two inspection windows that separate that the result is shown; (B) expression adopts an inspection window that the result is shown; (C) all results of above-mentioned two the check spots of diagram (i.e. hypothesis contrast is visual).

Figure 15 illustrates the example of the method for using the reagent for quickly examining box that contains gene probe.

Figure 16 is the functionalized synoptic diagram of gold nano grain.

Figure 17 shows the contrast between following: the ssDNA-gold nano grain that (1) complementary DNA is hybridized, (2) have ssDNA-gold nano grain and (3) independent ssDNA-gold nano grain of ssDNA.

Shown in Figure 18 illustrates and disclosed uv absorption spectra.

Figure 19 is the functionalized synoptic diagram of carbon nanotube.

Figure 20 illustrates uv absorption spectra, wherein, (A) spectrogram of no ssDNA chain or segmental independent carbon nanotube (CNT) is shown; (B) illustrate by the functionalized CNT of strand oligonucleotide; (C) illustrate by the functionalized CNT of strand oligonucleotide, described single stranded oligonucleotide and incomplementarity single stranded oligonucleotide are hybridized; (D) hybridize the back by the functionalized CNT of strand Nucleotide with the complementary oligonucleotide fragment.

Figure 21 A-21C is the atomic force microscope Photomicrograph, (A) is the Photomicrograph of the independent carbon nanotube (CNT) of no any single stranded oligonucleotide; (B) Photomicrograph of the functionalized CNT of single stranded oligonucleotide; (C) the functionalized CNT of single stranded oligonucleotide and with the Photomicrograph of complementary single stranded oligonucleotide fragment hybridization.

Figure 22 A-22B relatively illustrates contrast 784,794 and the incomplementarity composition 786,796 of single stranded oligonucleotide (for example ssDNA or ssDNA fragment) and the complementary combinations thing 788,798 of single stranded oligonucleotide.Fluorescence is sent in zone with complementary single stranded oligonucleotide 788,798, but not complementary oligonucleotide 786,796 do not hybridize, so do not send fluorescence.

Figure 23 utilizes picomole (pmol) scope that the concentration range of carbon nanotube is shown.

Figure 24 contrasted the oligonucleotide that is not fixed in Examination region and with the oligonucleotide that is fixed in Examination region after chitosan or chitosan derivatives combine.

Figure 25 illustrates and is used to measure from the emission light of the Examination region on the slide glass or the detector of transmitted light.

Embodiment

Example of describing and the accompanying drawing that provides are to be used for explanation and should not to be considered to limit scope of the present invention, and described scope is determined by additional claim.Those of ordinary skill will be understood more advantages of the present invention from the description of the example that provided.

Rapid diagnosis analysis provides a kind of quick and cheap being used to detect the screening test of antibody, described antibody is to be caused by the organism that causes disease, and for example virus, bacterium, fungi, mould and other can be by the detected organisms that causes disease of TPPA.Diagnositc analysis is a Rapid identification, means from extracting body fluid to implement the time fast (for example, being less than 10 minutes) to the check of finishing check, and can obtain assay (for example being less than 1 minute) fast behind preparation buffering suspension.The susceptibility and the specific reagent for quickly examining box that have the test kit that is used for example simultaneously are also not known.And, for the present inventor, known test kit all can not begin to provide the result being less than in time of 1 minute from prepare using PBS buffering sample, as obtaining strong positive in the high titre check and shown in the test kit that also obtains excellent results in the low titre check.It is generally acknowledged two time rulers of quick expression (check is prepared to finishing and mixing the time that test kit behind the check sample provides the result) in buffered soln.In addition, the example of test kit provides and utilizes whole blood, serum or the blood plasma rapid diagnosis analysis as experimental material.For all commercially available test kits of being tested, whole blood especially becomes problem.

In one embodiment, a kind of rapid diagnosis analysis method is used the test kit among the embodiment at the scene that need not any medical facilities or laboratory equipment.The ability that can use in remote districts make convenient test and cheapness.

Various antigens may be used to rapid diagnosis analysis.The antibody that rapid diagnosis analysis detected for example can produce when responding to bacterium, fungi, parasite or virus.In the screening group, can separate or use multiple antigen together.Except that catching, rapid diagnosis analysis can also detect the antibody or the antigen of noninfectious disease, and non-infection disease venereal disease for example is cancer, Alzheimer or other noninfectious diseases.

Bacterial antigens

Can utilize reagent for quickly examining box bacterial detection venereal disease former.In one embodiment, antigen is selected from the major outer membrane albumen of Actinobacillus (Actinobacillus) bacterial strain.For example, United States Patent (USP) the 6th, 541, disclosed antigen in No. 011.In another example, bacterial antigens can be from following any bacterium: actinomyces (Actinomyces), for example from the antigen that is rich in ornithine or the United States Patent (USP) the 6th of actinomyces naeslundii (Actinomyces naeslundii), disclosed actinomyces viscosus (Actinomycesviscosus) in 974, No. 700; Gas bacillus (Aerobacter aerogens) or actinomyces Israeli (Actinomyces israelli); Genus bacillus (Bacillus), for example protective antigen, lethal gene or the edema factor of disclosed Bacillus anthracis (Bacillusanthracis), bacillus cereus (Bacillus cereus), Bacillus subtillis (Bacillus subtilis) or Bacillus anthracis among the WO 2004/024067; United States Patent (USP) the 6th, 699, the cell-surface antigens of disclosed bacillus cereus in No. 679; United States Patent (USP) the 5th, 527, the 69Kd protein of disclosed bordetella pertussis (B.pertussis) in No. 529; Bacteroides (Bacteroides); Bordetella (Bordetella); Bordetella pertussis, for example United States Patent (USP) the 6th, 197, the bordetella pertussis of mentioning in No. 548; Molecular weight is respectively parapertussis Bao Te bacterium (B.parapertussis) and the bronchitis Bao Te bacterium (B.bronchiseptica) of 70kDa, 68kDa; Bartonella (Bartonella); Burgdorferi bacterium (Borrelia), for example United States Patent (USP) the 6th, 541, the borrelia obermeyri of mentioning in No. 011 (Borreliarecurrentis) or the outer surface protein A (OspA) of Lyme disease Borrelia burgdoyferi (Borrelia burgdorferi); Brucella (Brucella), for example Bacillus abortus (Brucella abortus) or Bacterium melitense (Brucella melitensis), for example United States Patent (USP) the 6th, outer membrane protein on the Bacterium melitense of mentioning in 541, No. 011 (Omp) 29 or Brucella suis (Brucella suis); Campylobacter (Campylobacter), for example United States Patent (USP) the 5th, 549, the campylobacter pylori of mentioning in No. 051 (Campylobacter pylori); Carbonic acid gas is had a liking for Cellulomonas (Capnocytophaga); Chlamydiaceae (Chlamydia), for example United States Patent (USP) the 6th, 541, chlamydia trachomatis of mentioning in No. 011 (Chlamydiatraqchomatis) or chlamydia psittaci (Chlamydia psittaci), for example 80-90kDa protein and 110kDa protein, the chlamydozoan glycolipid (GLXA) of cell surface display, molecular weight in the Chlamydia pneumoniae species-specific antigen (Chlamydia pneumoniaespecies-specific antigen) of 92-98,51-55,43-46 and 31.5-33kDa scope and scope 12,26 and the genus-specific antigen of 65-70kDa; Clostridium (Clostridium), for example United States Patent (USP) the 5th, Clostridium botulinum of mentioning in 527, No. 529 (Clostridium botulinum) or clostridium perfringens (Clostridium perfingens) or Clostridium tetani (Clostridium tetani) or from the C fragment of Clostridium tetani; Toxoids A, B, C and D from Clostridium perfringens, for example United States Patent (USP) the 6th, 524, toxoid B that mentions in No. 592 or United States Patent (USP) the 6th, 503, that mentions in No. 722 distinguishes the toxin A of clostridium (C.difficile) or from United States Patent (USP) the 6th from difficulty, 849, LT of disclosed Soxhlet clostridium (C.sordellii) and HT toxin or from United States Patent (USP) the 7th in No. 715,037, the alpha toxin of No. 503 clostridium septicum (C.septicum) or from United States Patent (USP) the 6th, 613, disclosed botulinal A-G toxin in No. 329; Corynebacterium (Corynebacterium), for example diphtheria corynebacterium (Corynebacterium diptheria); Coxiella (Coxiella); Dermatophilus (Dermatophilus); Enterococcus spp (Enterococcus); Ehrlichia (Ehrlichia); United States Patent (USP) the 6th, 541, No. 011 disclosed Echinococcus granulosus (Echinococcusgranulosus) antigen 5; Intestinal bacteria (Escherichia coli); Francisella (Francisella); Fusobacterium door (Fusobacteria); Helicobacter pylori (H.pylori), for example United States Patent (USP) the 6th, 541, helicobacter pylori GroES homologue of mentioning in No. 011 (HspA) and the immune-reactive protein of 4 45-65kDa; Hemophilus influenzae (Hemophilus infiuenzae); Haemophilus ducreyi (H.ducreyi); H.hemophilus; Bacterium aegyptiacum (H.aegypticus); Haemophilus parainfluenzae (H.parainfluenzae); Haemobartonella (Haemobartonella); Helicobacterium (Helicobacter); Klebsiella (Klebsiella), for example United States Patent (USP) the 6th, 541, the K antigen of disclosed Klebsiella Pneumoniae (Klebaiellapneumonia) in No. 011; Leptospira icterohemorrhagiae (Leptospira icterohemorrhagiae), for example Leptospira canicola (Leptospira canicola); Leishmania (Leishmania), for example United States Patent (USP) the 6th, 541, the gp63 of disclosed leishmania major (Leishmania major) in No. 011; United States Patent (USP) the 6th, 541, disclosed mycobacterium heat shock protein 65 in No. 011; Leptospira; Listeria (Listeria), for example United States Patent (USP) the 5th, 830, Liszt's rhzomorph O of disclosed Listeria monocytogenes (Listeria monocytogenes) in No. 702; Moraxella (Moraxella), for example United States Patent (USP) the 6th, 541, the CD albumen of the moraxella of mentioning in No. 011; Mycobacterium (Mycobacteria), for example United States Patent (USP) the 6th, 541, the 35kDa protein of disclosed mycobacterium avium in No. 011 (Mycobacterium avium) or Mycobacterium bovis (Mycobacterium bovis) or Mycobacterium leprae (Mycobacterium leprae) or mycobacterium paratuberculosis (Mycobacterium paratuberculosis) or human-like mycobacterium tuberculosis (Mycobacterium tuberculosis hominis) or Mycobacterium leprae, perhaps United States Patent (USP) the 6th, 541, in No. 011 85 of disclosed mycobacterium tuberculosis or 45/47kDa antigen or United States Patent (USP) the 6th, the 18kDa protein of disclosed Mycobacterium leprae in 541, No. 011; Mycoplasma (Mycoplasma).In one embodiment, antigen is from mycoplasma hominis (Mycoplasmahominis).In one embodiment, antigen is from mycoplasma pneumoniae (Mycoplasma pneumoniae).

In one embodiment, bacterial antigens are from neisseria (Neisseria).In one embodiment, antigen is from Diplococcus gonorrhoeae (Neisseria gonorrhea).In one embodiment, antigen is from Neisseria meningitidis (Neisseria meningitidis).In an object lesson, antigen is Por, Rmp or the LOS albumen of Diplococcus gonorrhoeae.In another example, antigen can comprise PorA, PorB, Rmp, Ope, FrpB, TbpB, and is mentioned in No. 273 perhaps as United States Patent (USP) the 6th, 797, can use Nsp; Neorickettsia (Neorickettsia); Nocardia (Nocardia); Pasteurella (Pasteurella), for example bacillus yersini (Pasteurella pestis); Peptococcus (Peptococcus), for example Peptostreptococcus; Streptococcus pneumoniae (Pneumococcus), for example pneumococcus (Diplococcuspneumonia); Proteus (Proteus); Pseudomonas (Pseudomonas); Porphyromonas gingivalis (P.gingivalis), for example United States Patent (USP) the 6th, 541, (41kDa) albumen of the 43-kDa protein of disclosed porphyromonas gingivalis and fimbrin (fimbrilin) in No. 011; Rickettsiae (Rickettsia), for example dermacetor australis (Rickettsia australis) or Bu Nier rickettsia (Rickettsiaburnetii) or Ke Shi rickettsia (Rickettsia conori) or Mohs Rickettsiae (Rickettsiamooseri) or Rickettsia prowazekii (Rickettsia prowazekii) or Rickettsia tsutsugamushi (Rickettsiatsutsugamushi); Luo Sha Lima body belongs to (Rochalimaea); Salmonella (Salmonella), for example United States Patent (USP) the 6th, 541, the O of disclosed Salmonella choleraesuls in No. 011 (Salmonella choleraesus) or Salmonella typhimurium or Salmonella typhi (Salmonella typhosa) or salmonella, SEFl4 fibrial antigen of H and Vi antigen or Salmonella enteritidis (Salmonella enteriditis) and observed flagellum (G) antigen on Salmonella enteritidis and white dysentery Salmonellas (S.pullorum); Shigella (Shigella), for example United States Patent (USP) the 5th, 958, disclosed Shigella arabinotardo or shigella boydii (Shigella boydii) or shigella dysenteriae (Shigella dysenteria) or shigella flexneri (Shigella flexneri) or Shi Micishi Shigellae (Shigella schmitzii) or shigella sonnei (Shigella sonnei) or O-antigen or US Patent No in No. 686,5, disclosed shigella dysenteriae (S.dysenteria) in 204,097; Staphylococcus (Staphylococcus), for example United States Patent (USP) the 6th, disclosed streptococcus aureus in 537, No. 559 (Staphylococcus aureus) or Staphylococcus albus (Staphylococcusalbus) or streptococcus aureus 5 types, 336 types, 4 types, K73 antigen; The hyperimmune response antigen (hyperimmune serum reactive antigen) of the staphylococcus epidermidis (Staphylococcus epidermidis) that proposes as U.S. Patent Publication 2007/0036778; United States Patent (USP) the 6th, 541, ORF-2 antigen or the GipQ of disclosed streptococcus aureus in No. 011; Streptococcus (Streptococcus), for example United States Patent (USP) the 6th, 429, disclosed streptococcus agalactiae in No. 199 (Streptococcus agalactiae) (B group B streptococcus B genus), suis (grass green colour cell (viridans group)), streptococcus faecium (Streptococcus facecalis), streptococcus bovis (Streptococcusbovis), suis (streptococcus anaerobius (anaerobic sps.)) or streptococcus pneumoniae (Streptococcus pneumoniae) or United States Patent (USP) the 6th, the 190kDa proteantigen of the streptococcus mutans of discussing in 541, No. 011 (Streptococcus mutans); The M protein or the C5a peptase of disclosed micrococcus scarlatinae among the WO 2002/050107 (Streptococcus pyogenes); Disclosed in other examples of micrococcus scarlatinae such as the U.S. Patent Publication 2006/0269541, comprise sugar antigen group (groupcarbohydrate antigen), C-material (C-substance), pilin (fimbrial proteins), in conjunction with protein (for example albumen F), the kinase whose cell of marriage chain, SPE A, B and C, α-C albumen, β-C albumen, Rib and Sip albumen or the B group sugar antigen (group B carbohydrateantigens) of fibronectin; The purifying capsular polysaccharide of 7 kinds of serotype (4,9V, 14,19F, 23F, 18C and 6B) streptococcus pneumoniae; Disclosed pneumococcal surface protein A, streptococcus pneumoniae surface adhesion plain A, choline binding protein A, LytB glucosaminidase, LytC N,O-Diacetylmuramidase, PrtA serine protease, PhtA (Histidine triplet A) and Pnu-Imune 23 antigen (pneumococcal vaccineantigen) A among the WO/2004/092209; From US Patent No, 7,128,919 B group B streptococcus B Ema (extracellular matrix attachment proteins polypeptide (extracellular matrix adhesion protein polypeptides)) EmaA, EmaB, EmaC, EmaD and EmaE; United States Patent (USP) the 6th, 541, the Pac antigen of the streptococcus mutans of mentioning in No. 011; United States Patent (USP) the 6th, 541, the MW antigen of the Salmonella typhi of mentioning in No. 011; Treponema pallidum (Treponemapallidum); Yersinia (Yersina), for example United States Patent (USP) the 6th, 541, the V antigen of disclosed yersinia pestis in No. 011 (Yersina pestis) or Fl antigen or pH6 antigen, perhaps United States Patent (USP) the 6th, the disclosed 37kDa secrete polypeptide (secreted polypeptide) of going up coding in the 70kb of pathotype Yersinia virulence plasmid (virulenceplasmid) in 541, No. 011.

Fungal antigen

Fungal pathogens also can be detected by disclosed test kit and method.In one embodiment, antigen is from Candida albicans (Candida albicans).In an object lesson, antigen is the adhesion molecule of fungi, for example from United States Patent (USP) the 6th, 630, and the phosphorylation Mannoproteins (phosphomannoprotein) of the Candida albicans of mentioning in No. 146.In one embodiment, antigen is from the fungal antigen of absidia (Absidia).In one embodiment, antigen is from absidia corymbifera (Absidia corymbifera).In one embodiment, antigen is the fungal antigen from the mould genus of branch top spore (Acremonium).In one embodiment, antigen is the fungal antigen from Alternaria (Alternaria).In one embodiment, antigen is the fungal antigen from Aspergillus (Aspergillus).In one embodiment, antigen is the fungal antigen from frog excrement mould (Basidiobolus).In one embodiment, antigen comes the self-balancing navel and the fungal antigen of spore (Bipolaris).In one embodiment, antigen is the fungal antigen from blastomycete (Blastomyces).In one embodiment, antigen is from blastomycetic fungal antigen.In one embodiment, antigen is the fungal antigen from candidiasis (Candida).An antigenic adhesion molecule that object lesson is a fungi, for example United States Patent (USP) the 6th, 630, the phosphorylation Mannoproteins of mentioning in No. 146 from Candida albicans.In one embodiment, antigen is from oidiomycetic fungal antigen.In one embodiment, antigen is the fungal antigen from Coccidioides (Coccidioides).In one embodiment, antigen is from posadasis spheriforme (Coccidioides immitis).In one embodiment, antigen is the fungal antigen from Conidiobolus (Conidiobolus).In one embodiment, antigen is the fungal antigen from genera cryptococcus (Cryptococcus).In one embodiment, antigen is the fungal antigen from Conidiobolus.In one embodiment, antigen is the fungal antigen from genera cryptococcus.In one embodiment, antigen is from Cryptococcus neoformans (Cryptococcus neoformans).In one embodiment, antigen is the fungal antigen from Curvularia (Curvalaria).In one embodiment, antigen is the fungal antigen from Epidermophyton (Epidermophyton).In one embodiment, antigen is the fungal antigen from Exophiala (Exophiala).In one embodiment, antigen is the fungal antigen from Geotrichum (Geotrichum).In one embodiment, antigen is the fungal antigen from Histoplasma (Histoplasma).In one embodiment, antigen is from Histoplasma capsulatum (Histoplasma capsulatum).In one embodiment, antigen is the fungal antigen from Madura branch Pseudomonas (Madurella).In one embodiment, antigen is the fungal antigen from Malassezia (Malassezia).In one embodiment, antigen is the fungal antigen from microsporum (Microsporum).In one embodiment, antigen is the fungal antigen from Moniliella.In one embodiment, antigen is the fungal antigen from genus mortierella (Mortierella).In one embodiment, antigen is the fungal antigen from Mucor (Mucor).In one embodiment, antigen is the fungal antigen from paecilomyces (Paecilomyces).In one embodiment, antigen is the fungal antigen from Penicillium (Penicillium).In one embodiment, antigen is the fungal antigen from Phialemonium (Phialemonium).In one embodiment, antigen is the fungal antigen from Saksenaea (Phialophora).In one embodiment, antigen is the fungal antigen from Prototheca (Prototheca).In one embodiment, antigen is the fungal antigen from false Escherichia (Pseudallescheria).In one embodiment, antigen is the fungal antigen from Pseudomicrodochium (Pseudomicrodochium).In one embodiment, antigen is the fungal antigen from pythium (Pythium).In one embodiment, antigen is the fungal antigen from Rhinosporidium (Rhinosporidium).In one embodiment, antigen is the fungal antigen from Rhizopus (Rhizopus).In one embodiment, antigen is the fungal antigen that belongs to (Scolecobasisium) from tooth stalk spore.In one embodiment, antigen is the fungal antigen from the mould genus of bunch spore (Sporothrix).In one embodiment, antigen is the fungal antigen that belongs to (Stemphylium) from stemphylium.In one embodiment, antigen is the fungal antigen from trichophyton (Trichophyton).In one embodiment, antigen is the fungal antigen from Trichosporon (Trichosporon).In one embodiment, antigen is the fungal antigen from Xylohypha (Xylohypha).

Parasite antigen

Disclosed test kit and method also can detect the parasitosis substance.In one embodiment, antigen is protozoan parasite, and antigen is from this Eimeria of BABEI (Babesia).In one embodiment, antigen is protozoan parasite, and antigen is from Balantidium (Balantidium).In one embodiment, antigen is protozoan parasite, and antigen is from Balantidium.In one embodiment, antigen is protozoan parasite, and antigen is from Besnoitia (Besnoitia).In one embodiment, antigen is protozoan parasite, and antigen is from Cryptosporidium (Cryptosporidium).In one embodiment, antigen is protozoan parasite, and antigen is from Eimeria (Eimeria).In one embodiment, antigen is protozoan parasite, and antigen is from brain microsporidium (Encephalitozoon), and in one embodiment, antigen is protozoan parasite, and antigen is from entamoeba (Entamoeba).In one embodiment, antigen is protozoan parasite, and antigen is from giardia (Giardia).In one embodiment, antigen is protozoan parasite, and antigen is from Harmon Globidium (Hammondia).In one embodiment, antigen is protozoan parasite, and antigen is from Hepatozoon (Hepatozoon).In one embodiment, antigen is protozoan parasite, and antigen from etc. spore belong to (Isospora).In one embodiment, antigen is protozoan parasite, and antigen is from Leishmania (Leishmania).In one embodiment, antigen is protozoan parasite, and antigen is from microsporidium (Microsporidia).In one embodiment, antigen is protozoan parasite, and antigen belongs to (Neospora) from neospora.In one embodiment, antigen is protozoan parasite, and antigen belongs to from neospora.In one embodiment, antigen is protozoan parasite, and antigen is from Pentatrichomonas (Pentatrichomonas).In one embodiment, antigen is protozoan parasite, and antigen is from plasmodium (Plasmodium).In one embodiment, antigen is protozoan parasite, and antigen is from plasmodium.In an object lesson, antigen can comprise plasmodium falciparum ring spore (P.falciparum circumsporozoite) (PfCSP), the C-terminal (PfLSA1c-term) and output albumen (exportedprotein) 1 (PfExp-1) of sporozoite surface protein (sporozoite surface protein) 2 (PfSSP2), liver state antigen 1.In one embodiment, antigen is from protozoan parasite lung sac worm (Pneumocystis).In one embodiment, antigen is from protozoan parasite sarcocystis (Sarcocystis).In one embodiment, antigen is from protozoan parasite Schistosoma (Schistosoma).In one embodiment, antigen is from protozoan parasite Theileria (Theileria).In one embodiment, antigen is from protozoan parasite toxoplasma (Toxoplasma).In one embodiment, antigen is from protozoan parasite trypanosoma (Trypanosoma).In another example, antigen is from helminthism worm (helminth).In one embodiment, antigen is from Acanthocheilonema (Acanthocheilonema).In one embodiment, antigen belongs to Turbatrix (Aelurostrongylus) from the cat circle.In one embodiment, antigen is from Ancylostoma (Ancylostoma).In one embodiment, antigen is from Angiostrongylus (Angiostrongylus).In one embodiment, antigen is from Ascaris (Ascaris).In one embodiment, antigen is from cloth Shandong Turbatrix (Brugia).In one embodiment, antigen is from Bunostomum (Bunostomum).In one embodiment, antigen is from Hepaticola (Capillaria).In one embodiment, antigen is from summer Bert (Chabertia).In one embodiment, antigen comes cypressCypressus (Cooperia) from ancient times.In one embodiment, antigen comes cypressCypressus from ancient times.In one embodiment, antigen is from ring body nematode (Crenosoma).In one embodiment, antigen is from Dictyocaulus (Dictyocaulus).In one embodiment, antigen belongs to (Dioctophyme) from swollen knot.In one embodiment, antigen is from sour jujube filaria labialis (Dipetalonema).In one embodiment, antigen is from Taenia lata (Diphyllobothrium).In one embodiment, antigen is from Diplydium.In one embodiment, antigen is from Dirofilaria (Dirofilaria).In one embodiment, antigen is from Dracunculus (Dracunculus).In one embodiment, antigen is from pinworm (Enterobius).In one embodiment, antigen is from Filaroides (Filaroides).In one embodiment, antigen is from Haemonchus (Haemonchus).In one embodiment, antigen is from Lagochilascaris (Lagochilascaris).In one embodiment, antigen is from Loa (Loa).In one embodiment, antigen is from Mansonella (Mansonella).In one embodiment, antigen belongs to (Muellerius) from Miu Le.In one embodiment, antigen is from dwarf's shape fluke (Nanophyetus).In one embodiment, antigen is from plate mouth nematode (Necator).In one embodiment, antigen is from Nematodirus (Nematodirus).In one embodiment, antigen belongs to (Oesophagostomum) from esophageal orifice.In one embodiment, antigen is from Onchocerca (Onchocerca).In one embodiment, antigen belongs to (Opisthorchis) from the back testis.In one embodiment, antigen belongs to (Ostertagia) from oersted.In one embodiment, antigen is from Parafilaria (Parafilaria).In one embodiment, antigen is from Paragonimus (Paragonimus).In one embodiment, antigen belongs to (Parascaris) from secondary ascarid.In one embodiment, antigen belongs to (Physaloptera) from the bubble wing.In one embodiment, antigen is from Protostrongylus (Protostrongylus).In one embodiment, antigen is from setaria (Setaria).In one embodiment, antigen is from Spirocerca (Spirocerca).In one embodiment, antigen is from spiral tapeworm (Spirometra).In one embodiment, antigen is from Stephanofilaria (Stephanofilaria).In one embodiment, antigen belongs to (Strongyloides) from the class circle.In one embodiment, antigen is from Strongylus (Strongylus).In one embodiment, antigen is from sucking genus (Thelazia).In one embodiment, antigen belongs to from the bow ascarid.In one embodiment, antigen is from bow roundworm (Toxocara).In one embodiment, antigen belongs to (Trichinella) from Trichinella spiralis.In one embodiment, antigen is from trichostrongylus (Trichostrongylus).In one embodiment, antigen is from Trichocephalus (Trichuris).In one embodiment, antigen is from Ancylostoma (Uncinaria).In one embodiment, antigen is from Wuchereria (Wuchereria).In one embodiment, antigen can comprise the CCA (CCA) in schistosomicide enteron aisle dependency antigen (schistosome gut-associated antigen) CAA (CAA) and Schistosoma mansoni (Schistosoma mansoni), Schistosoma haematobium (Schistosoma haematobium) or the Schistosoma japonicum (Schistosoma japonicum).In one embodiment, antigen can comprise two multiple antigenic peptides (MAP) that different protective antigens is formed of origin autoparasitism worm Schistosoma mansoni.In one embodiment, antigen can comprise third-instar larvae (L3) antigen (Akue et al. (1997), coding annular Taylor worm (Theileria annulata) 30-kDa (Ta) and Tamsl-1 and Tamsl-2 and plasmodium falciparum merozoite surface antigen 1 or 2 of the main merozoite surface antigen of 32-kDa of Leishmania (Leishmania) parasite surface molecular, Loa loa (L.loa).In one embodiment, antigen is plasmodium falciparum (Plasimodium falciparum) antigen Pfs230.In one embodiment, antigen can comprise plasmodium falciparum apical membrane antigen (AMA-I); Plasmodium falciparum albumen Pfs28 and Pfs25; Plasmodium falciparum merozoite surface proteins 4 and 5 is MSP1 in vain; Malaria antigen Pf332; Plasmodium falciparum erythrocyte membrane protein 1; The plasmodium falciparum merozoite surface antigen is PfMSP-1; Plasmodium falciparum antigen SERA, EBA-175, RAP1 and RAP2; Schistosoma japonicum paramyosin (Sj97) or fragment; Parasitic Hsp70.

Virus antigen

Disclosed test kit and method also can detect viral pathogen.In one embodiment, antigen is the virus antigen from adenovirus.In one embodiment, antigen is the virus antigen from alphavirus.In one embodiment, antigen is the virus antigen that belongs to from Calicivirus.In one embodiment, antigen is the virus antigen from the Calicivirus capsid antigen.In one embodiment, antigen is the virus antigen from coronavirus genus.In the object lesson of coronavirus genus, antigen is sars coronavirus.In one embodiment, antigen is from cytomegalovirus.In an object lesson, antigen can comprise cytomegalovirus glycoprotein gB or glycoprotein gH.In one embodiment, antigen is dengue virus.In an object lesson, antigen can comprise that dengue virus envelope protein (Dengue virus envelope) (E) and precursor film antigen (premembrane antigen).In one embodiment, antigen is the virus antigen from canine distemper virus (distempervirus).In one embodiment, antigen is the virus antigen from Ebola virus.In one embodiment, antigen is from Epstein-Barr virus.In an object lesson, antigen is Epstein-Barr virus (EBV) gp340 albumen.In another object lesson, antigen is Epstein-Barr virus (EBV) latent membrane protein LMP2.

In one embodiment, antigen is eb nuclear antigen 1 and 2.In one embodiment, antigen is Measles virus nucleoprotein (N).In one embodiment, antigen be from the virus antigen enterovirus, in one embodiment, antigen is the virus antigen from flavivirus.In one embodiment, antigen is from hepatitis A.In one embodiment, antigen is from hepatitis B.In one embodiment, antigen is the virus antigen from hepatitis B core antibody or surface antigen.In an object lesson, antigen is hepatitis B core antigen and E antigen.In an object lesson, antigen is the hepatitis B surface antigen(HBsAg) that merges with cAg, core-preS2 particle.In one embodiment, antigen is from hepatitis C.In an object lesson, antigen is secretor type or nonsecreting type hepatitis C virus nucleocapsid protein.In another object lesson, antigen can comprise C hepatitis virus antigen: core protein (pC); Independent E1 (pE1) and E2 (ρ E2) or fusion rotein.In one embodiment, antigen is from I type and II type herpes simplex.In one embodiment, antigen is virus antigen or the varicella one varicella zoster virus glycoprotein from hsv.In an object lesson, antigen can comprise ICP0, ICP4, ICP27, ICP47, gB, gD, gE, gG, gH and the gI of hsv.In one embodiment, antigen is the virus antigen from infectious peritonitis virus.In one embodiment, antigen be from virus antigen HIV.In an object lesson, antigen can comprise HIV antigen, for example Gag, Pol, Vif, Nef, p24, gp 120, gp 160, gp41 or gp36.In one embodiment, antigen is the virus antigen from influenza virus.In one embodiment, antigen is from first, second or influenza virus C.In an object lesson, antigen is the virus antigen from influenza A hemagglutinin, neuraminidase or nucleoprotein.In an object lesson, antigen is the N2 neuraminidase of influenza A virus.In one embodiment, antigen is the virus antigen from leukosis virus.In one embodiment, antigen is the virus antigen from Marburg virus.In one embodiment, antigen is from Measles virus.In one embodiment, antigen is from mumps virus.In one embodiment, antigen be from the virus antigen orthomyxovirus.In one embodiment, antigen is the virus antigen from papilloma virus.In an object lesson, antigen can comprise E1, E2, E3, E4, E5, E6 and the E7 albumen of human papillomavirus.In one embodiment, antigen is the virus antigen from parainfluenza virus.In the object lesson from the virus antigen of parainfluenza virus, antigen is hemagglutinin or neuraminidase.In one embodiment, antigen is the virus antigen from paramyxovirus.In one embodiment, antigen is the virus antigen from pestivirus.In one embodiment, antigen is the virus antigen from picornavirus.In the example of picornavirus, antigen can be from Coxsackie virus.In the example of picornavirus, antigen can be from ECHO virus.In the example of picornavirus, antigen can be from poliovirus.In the example of picornavirus, antigen can be from rhinovirus.In the antigenic object lesson of picornavirus, antigen can comprise capsid of poliomyelitis antigen or poxvirus antigen.In one embodiment, antigen is the virus antigen from rabies virus.In an object lesson, antigen comprises rabies virus glucoprotein.In one embodiment, antigen is the virus antigen from reovirus.In one embodiment, antigen is from respiratory syncytial virus.In an object lesson, antigen is respiratory syncytial virus f-protein (PFP-2).In one embodiment, antigen is from rubella virus.In one embodiment, antigen is the virus antigen from rotavirus.In an object lesson, antigen can comprise wheel virus antigen VP4, VP7 or VP7sc.In another object lesson, antigen can comprise by the albumen of the P6 of rotavirus and VP7 genes encoding.In one embodiment, antigen can be from vaccine virus.In one embodiment, antigen is from human T-lymphocyte virus.In an object lesson, antigen can comprise human T-lymphocyte virus I-type gag albumen.

In one embodiment, antigen selection becomes in order to detect noninfectious disease, for example cancer, Alzheimer or other noninfectious diseases.For example, cancer can be a prostate cancer, and selected antigen can be prostate specific antigen (PSA).In the antigenic example from alzheimer's disease, antigen is alzheimer's disease antigen, and promptly United States Patent (USP) the 6th, 864, disclosed A68 or recombinant human tau in No. 062.

Embodiment

Figure 1A-C illustrates testing reagent case assembly cross sectional representation example.Between box top 60 and box bottom 62, for clarity sake, multilayer absorbing material 42 and film 22 are to show in the exploded view in the drawings by concora crush for multilayer absorbing material 42 and film 22.

Shown in Figure 1A, reagent for quickly examining box 100 comprises box top 60 and the box bottom 62 with opening 63.Can tilt as shown in the figure certain angle or can be upright of the wall 61 of opening 63.In addition, what be described is connection section 65, and this connection section for example can provide interlock (snap) or pressing.Mierocrystalline cellulose filter paper 22 can be loaded with one or more antigens.A plurality of absorption layers 42 can be identical or different with filter paper 22.Each absorption layer 42 can specifically identical physics and chemical property, or differs from one another, and described characteristic comprises length, optical density (absorbancy) and thickness.In one embodiment, filter paper 22 and a plurality of absorption layer 42 are of a size of 1 square inch.The length of each layer, width and thickness can be inconsistent.A plurality of absorption layers 42 can be 2 or a plurality of, and this number of plies depends on the thickness of each layer and by the size of box top 60 and box bottom 62 chambers that constitute.Preferred top 60 bottoms 62 compress each layer 42, have reached each other closely physics contact.In one embodiment, layer 42 is a filter paper, and comprises 5～10 layers, and this number of plies depends on the characteristic of filter paper and box.

For example, can pressing or is snapped on the box bottom 62 in box top 60.By device, this opening 63 contains the antigen that is useful on detection antibody via the opening 63 of central authorities for blood plasma, serum, blood, saliva or other body fluid.This antigen can carry before the test kit assembling or after the assembling.In Figure 1B, imbibition pad (wickingpad) 24 replaces one or more absorption layers 42 of test kit 110.

Fig. 1 C illustrates the vertical view of diagnostic kit.Box top 60 comprises the wall 61 of the inclination that limits opening 63.The length of wall 61 can increase by the ring (collar) 67 that extends on box top 60, thereby provides bigger volume in opening 63.

In one embodiment, 1 μ l antigen or antigen mixture are added to the position T (being test position) of circulation device (flow throughdevice), and 1 μ l albumin A (1mg/ml) is added to the different positions C (being control site) of verifying attachment.Then, dry verifying attachment.For example, for the drying of most of test kit, promptly reached thorough drying in air-dry 6-8 hour.For for example check sample of blood, serum or blood plasma, can use the existence of dyeing damping fluid test antibody.In one embodiment, the dyeing damping fluid is the albumin A that is combined with Radioactive colloidal gold.For example, the dyeing damping fluid is can freeze-drying standby, and can use damping fluid (for example IX Dulbecco ' s phosphate buffer soln (DPBS)) to carry out rehydration.

To specify antigenic specific antibody in order detecting, for example can to utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample.In one embodiment, dilution buffer liquid is the ACK lysis buffer Cat#1683 that obtains from Invitrogen.The sample of diluted mistake is placed on the opening 63 neutralization reaction layers 22 of verifying attachment, and described responding layer can comprise the antigen check spot on the Mierocrystalline cellulose filter paper.For blood sample, suggestion is waited until about 3 minutes after blood being joined in the dilution buffer liquid, perhaps becomes uniform transparent red to dilution buffer liquid at least.After being absorbed, dilute sample can add 150 μ l dyeing damping fluid.In one embodiment, the dyeing damping fluid is combined with the albumin A of Radioactive colloidal gold.After being absorbed, the dyeing damping fluid can add 200 μ l decolouring damping fluid.The decolouring damping fluid can be Dulbecco ' s phosphate buffered saline (1X) (DPBS), DPBS for example also is the example of phosphate buffered saline buffer.Can read the result immediately after the decolouring damping fluid flows through system, and need not to postpone again, thereby finish rapid detection.When test position T and control site C show all that when red, assay is positive, there is the antibody of indication specified disease (for example HIV) in expression.Yet if having only control site C to have red point, assay is negative so, and expression exists and antibody by the disease-related of Detection of antigen.If a little or not do not observe control site if observe, it is invalid to check so.If correctly implement check, normally can observe control point.

In an example of treatment process, utilize the lysis buffer dilution with 10 times of blood sample dilutions.In another example of treatment process, use silver to strengthen damping fluid and improve contrast.

For example, as schematically illustrating of Fig. 2, a C spot 102 of first verifying attachment 120 and the 2nd C spot 112 of second verifying attachment 140 be spot in contrast, and this helps the operation of prove device normal.The one T spot 104 of first verifying attachment 120 and second device, 140 the 2nd T spot 114 are the check spots that are used to detect the existence of specific antibody.The check of implementing does not all cause false negative result.

In the example of Fig. 2 A, a T spot 104 does not have punctation, is illustrated in not have any antibody that detects level in the particular test sample.In the example of Fig. 2 B, except contrast spot 112, T spot 114 also all shows punctation, clearly shows the infection of the sample with HIV antibody.

Following example illustrates the various antigens that may be used to the reagent for quickly examining box.The antibody that is present in the sample can combine with specific antigen.These examples also be not intended to limit the antibody type that is verified the test kit test, can detected any antibody can be tested in body fluid (for example blood, serum or blood plasma or other body fluid).

Embodiment 1-actinomycetes

In one embodiment, antigen is actinomycetes.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute 10 μ l serum, blood plasma or whole blood check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

In case dilute sample is absorbed, can add 150 μ l dyeing damping fluid.In one embodiment, described dyeing damping fluid is the albumin A that is combined with Radioactive colloidal gold.In case the dyeing damping fluid is absorbed, can add 200 μ l decolouring damping fluid.The decolouring damping fluid for example can be Dulbecco ' s phosphate buffer soln (1X) (DPBS).In case the decolouring damping fluid flows through system, can read the result immediately.When test position T and control site C all showed redness, test result was positive, and there is the antibody of indication specified disease in expression, promptly to the specific antibody of specific antigen.

Embodiment 2-gas bacillus

In one embodiment, antigen is gas bacillus.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

In case dilute sample is absorbed, can add 150 μ l dyeing damping fluid.In one embodiment, the dyeing damping fluid is the albumin A that is combined with Radioactive colloidal gold.In case the dyeing damping fluid is absorbed, can add 200 μ l decolouring damping fluid.The decolouring damping fluid for example can be Dulbecco ' s phosphate buffer soln (1X) (DPBS).In case the decolouring damping fluid flows through system, can read the result immediately.When test position T and control site C all showed redness, test result was positive, and there is the antibody of indication specified disease in expression, promptly to the specific antibody of specific antigen.

Embodiment 3-genus bacillus

In one embodiment, antigen is genus bacillus.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 4-genera bacillus

In one embodiment, antigen is genera bacillus.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 5-Bartonella bacterium

In one embodiment, antigen is from the Bartonella bacterium.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 6-Borrelia

In one embodiment, antigen is selected from a kind of Borrelia.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 7-Brucella

In one embodiment, antigen is selected from Brucella.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 8-Campylobacter

In one embodiment, antigen is selected from Campylobacter.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

In case dilute sample is absorbed, can add 150 μ l dyeing damping fluid.In one embodiment, the dyeing damping fluid is the albumin A that is combined with Radioactive colloidal gold, in case the dyeing damping fluid is absorbed, can add 200 μ l decolouring damping fluid.The decolouring damping fluid for example can be Dulbecco ' s phosphate buffer soln (1X) (DPBS).In case the decolouring damping fluid flows through system, can read the result immediately.When test position T and control site C all showed redness, test result was positive, and there is the antibody of indication specified disease in expression, promptly to the specific antibody of specific antigen.

Embodiment 9-chlamydozoan

In one embodiment, antigen is selected from a kind of chlamydozoan.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 10-clostridium

In one embodiment, antigen is selected from a kind of clostridium.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 11-coryneform bacteria

In one embodiment, antigen is selected from a kind of coryneform bacteria.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 12-helicobacter pylori

In one embodiment, antigen is selected as helicobacter pylori.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 13-Helicobacterium

In one embodiment, antigen is selected as Helicobacterium.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 14-hemophilus influenzae

In one embodiment, antigen is selected as hemophilus influenzae.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 15-Klebsiella

In one embodiment, antigen is selected from a kind of klebsiella.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 16-Leptospira icterohemorrhagiae

In one embodiment, antigen is selected from a kind of Leptospira icterohemorrhagiae.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 17-leishmania major

In one embodiment, antigen is selected from leishmania major.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

In case dilute sample is absorbed, can add 150 μ l dyeing damping fluid.In one embodiment, the dyeing damping fluid is the albumin A that is combined with Radioactive colloidal gold.In case the dyeing damping fluid is absorbed, can add 200 μ l decolouring damping fluid.For example, the decolouring damping fluid can be Dulbecco ' s phosphate buffer soln (1X) (DPBS).In case the decolouring damping fluid flows through system, can read the result immediately.When test position T and control site C all showed redness, test result was positive, and there is the antibody of indication specified disease in expression, promptly to the specific antibody of specific antigen.

Embodiment 18-Leptospira

In one embodiment, antigen is selected from Leptospira, for detecting one or more antibody at specific antigen, at first utilizes 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 19-listeria

In one embodiment, antigen is selected from a kind of listeria bacteria.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

In case dilute sample is absorbed, can add 150 μ l dyeing damping fluid.In one embodiment, the dyeing damping fluid is the albumin A that is combined with Radioactive colloidal gold.In case the dyeing damping fluid is absorbed, can add 200 μ l decolouring damping fluid.For example, the decolouring damping fluid can be Dulbecco phosphate buffer soln (1X) (DPBS).In case the decolouring damping fluid flows through system, can read the result immediately.When test position T and control site C all showed redness, test result was positive, and there is the antibody of indication specified disease in expression, promptly to the specific antibody of specific antigen.

Embodiment 20-moraxella

In one embodiment, antigen is selected from a kind of catarrhalis.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 21-Mycobacterium

In one embodiment, antigen is selected from a kind of mycobacterium.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 22-Neisseria

In one embodiment, antigen is selected from a kind of Neisseria.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 23-Pasteurella

In one embodiment, antigen is selected from a kind of Pasteurella.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 24-streptococcus pneumoniae

In one embodiment, antigen is selected from a kind of streptococcus pneumoniae for detecting one or more antibody at specific antigen, at first utilizes 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 25-Rickettsiae

In one embodiment, antigen is selected from a kind of Rickettsiae.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 26-Salmonellas

In one embodiment, antigen is selected from a kind of Salmonellas, for detecting one or more antibody at specific antigen, at first utilizes 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 27-Shigella

In one embodiment, antigen is selected from a kind of Shigellae.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 28-Staphylococcus

In one embodiment, antigen is selected from a kind of staphylococcus.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 29-streptococcus

In one embodiment, antigen is selected from a kind of suis.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 30-treponema pallidum

In one embodiment, antigen is selected from treponema pallidum.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 31-Yersinia

In one embodiment, antigen is selected from Yersinia.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 32-Candida albicans

In one embodiment, antigen is selected from Candida albicans.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 33-absidia

In one embodiment, antigen is selected from absidia.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

The mould genus of embodiment 34-branch top spore

In one embodiment, antigen is selected from a mould genus of top spore.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 35-Alternaria

In one embodiment, antigen is selected from Alternaria.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

The mould genus of embodiment 36-frog excrement

In one embodiment, antigen is selected from the mould genus of frog excrement.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 37-blastomycete

In one embodiment, antigen is selected from blastomycete.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 38-Coccidioides

In one embodiment, antigen is selected from a kind of Coccidioides.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 39-genera cryptococcus

In one embodiment, antigen is selected from a kind of genera cryptococcus.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

The Example40-Curvularia

In one embodiment, antigen is selected from a kind of Curvularia.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 41-Epidermophyton

In one embodiment, antigen is selected from a kind of Epidermophyton.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 42-Exophiala

In one embodiment, antigen is selected from a kind of outer Saksenaea vasiformis.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 43-Geotrichum

In one embodiment, antigen is selected from a kind of Geotrichum.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 44-Histoplasma capsulatum

In one embodiment, antigen is selected from a kind of Histoplasma capsulatum.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 45-Madura branch Pseudomonas

In one embodiment, antigen is selected from a kind of Madura branch Pseudomonas.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 47-Malassezia

In one embodiment, antigen is selected from Malassezia.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 48-microsporum

In one embodiment, antigen is selected from microsporum, for detecting one or more antibody at specific antigen, at first utilizes 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 49-candidiasis (Moniliella)

In one embodiment, antigen is selected from candidiasis (Moniliella).For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 50-genus mortierella

In one embodiment, antigen is selected from genus mortierella.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 51-Mucor

In one embodiment, antigen is selected from Mucor.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 52-Phialemonium

In one embodiment, antigen is selected from Phialemonium.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 52-Saksenaea

In one embodiment, antigen is selected from Saksenaea.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 53-Prototheca

In one embodiment, antigen is selected from Prototheca.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

The false Escherichia of embodiment 54-

In one embodiment, antigen is selected from false Escherichia.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 55-Pseudomicrodochium

In one embodiment, antigen is selected from Pseudomicrodochium.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 56-pythium

In one embodiment, antigen is selected from a kind of pythium spp (Phythium).For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 57-Rhinosporidium

In one embodiment, antigen is selected from a kind of Rhinosporidium.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 58-Rhizopus

In one embodiment, antigen is selected from a kind of Rhizopus.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 59-tooth stalk spore belongs to

In one embodiment, antigen is selected from a kind of tooth stalk spore genus.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

The mould genus of embodiment 60-bunch spore

In one embodiment, antigen is selected from a kind of bunch of mould genus of spore.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 61-stemphylium belongs to

In one embodiment, antigen is selected from a kind of stemphylium genus.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 62-trichophyton

In one embodiment, antigen is selected from a kind of trichophyton.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 63-Trichosporon

In one embodiment, antigen is selected from a kind of Trichosporon.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 64-Xylohypha

In one embodiment, antigen is selected from a kind of Xylohypha.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

This Eimeria of embodiment 65-BABEI

In one embodiment, antigen is selected from this Eimeria of a kind of BABEI.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 66-Balantidium

In one embodiment, antigen is selected from a kind of Balantidium, for detecting one or more antibody at specific antigen, at first utilizes 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 67-Balantidium

In one embodiment, antigen is selected from a kind of Balantidium.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 68-Besnoitia

In one embodiment, antigen is selected from a kind of shellfish promise sporozoite.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 69-Cryptosporidium

In one embodiment, antigen is selected from a kind of Cryptosporidium.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 70-Eimeria

In one embodiment, antigen is selected from a kind of of Eimeria.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 71-brain microsporidium

In one embodiment, antigen is selected from a kind of brain microsporidium.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 72-entamoeba

In one embodiment, antigen is selected from a kind of entamoeba.

For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 73-giardia

In one embodiment, antigen is selected from a kind of giardia lamblia.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 74-Harmon Globidium

In one embodiment, antigen is selected from a kind of Harmon coccidia.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 75-Hepatozoon

In one embodiment, antigen is selected from a kind of Hepatozoon.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Spores such as embodiment 76-belong to

In one embodiment, antigen is selected from a kind of spore genus that waits.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 77-Leishmania

In one embodiment, antigen is selected from a kind of Leishmania.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 78-microsporidium

In one embodiment, antigen is selected from a kind of microsporidium, for detecting one or more antibody at specific antigen, at first utilizes 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 79-neospora belongs to

In one embodiment, antigen is selected from a kind of neospora genus.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 80-Pentatrichomonas

In one embodiment, antigen is selected from a kind of Pentatrichomonas, for detecting one or more antibody at specific antigen, at first utilizes 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 81-plasmodium

In one embodiment, antigen is selected from a kind of plasmodium.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 82-lung sac worm

In one embodiment, antigen is selected from a kind of lung sac worm, for detecting one or more antibody at specific antigen, at first utilizes 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 83-sarcocystis

In one embodiment, antigen is selected from a kind of sarcocystis, for detecting one or more antibody at specific antigen, at first utilizes 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 84-Theileria

In one embodiment, antigen is selected from a kind of Taylor worm, for detecting one or more antibody at specific antigen, at first utilizes 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 85-toxoplasma

In one embodiment, antigen is selected from a kind of toxoplasma.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 86-trypanosoma

In one embodiment, antigen is selected from a kind of trypanosoma.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 87-Schistosoma

In one embodiment, antigen is selected from a kind of Schistosoma, for detecting one or more antibody at specific antigen, at first utilizes 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 88-Schistosoma

In one embodiment, antigen is selected from a kind of Schistosoma.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 89-adenovirus

In one embodiment, antigen is selected from a kind of adenovirus.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 90-coronavirus genus

In one embodiment, antigen is selected from a kind of coronavirus.Coronavirus genus antigen for example can be SARS antigen.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 91-cytomegalovirus

In one embodiment, antigen is selected from a kind of cytomegalovirus.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 92-dengue virus

In one embodiment, antigen is selected from a kind of dengue virus.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 93-Ebola virus

In one embodiment, antigen is selected from a kind of Ebola virus.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 94-EB virus

In one embodiment, antigen is selected from a kind of Epstein-Barr virus.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 95-Measles virus

In one embodiment, antigen is from a kind of Measles virus.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 97-bird pox virus

In one embodiment, antigen is from a kind of bird pox virus.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 98-enterovirus

In one embodiment, antigen is selected from a kind of enterovirus.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 99-hepatitis A

In one embodiment, antigen is HA antigen.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 100-hepatitis B

In one embodiment, antigen is Australia antigen(AA).For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 101-hepatitis C

In one embodiment, antigen is hepatitis C antigen.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 102-herpes simplex

In one embodiment, antigen is hsv.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 103-HIV virus

In one embodiment, antigen is HIV1 antigen, for example is used to detect the p24 of HIV-1.P24 antigen also is used to detect HIV-2 antigen.In this embodiment, p24 antigen is made of following sequence:

PIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKARVL

Embodiment 104-HIV virus

In one embodiment, HIV antigen is HIV-1gp 41 Partial Protein, and is made of following sequence:

SELYKYKVVKIEPLGVAPTKAKRRVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVRQGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDR

In case dilute sample is absorbed, can add 150 μ l dyeing damping fluid.In one embodiment, the dyeing damping fluid is the albumin A that is combined with Radioactive colloidal gold.In case the dyeing damping fluid is absorbed, can add 200 μ l decolouring damping fluid.The decolouring damping fluid for example can be Dulbecco ' s phosphate buffer soln (1X) (DPBS).In case the decolouring damping fluid flows through system, can read the result immediately.When test position T and control site C all showed redness, test result was positive, and there is the antibody of indication specified disease in expression, promptly to the specific antibody of specific antigen.Obtained good especially result by the Partial Protein of using this gp41.

Embodiment 105-HIV virus

In one embodiment, being used to detect the antigen that HIV infects is the gp41 peptide fragment, and this fragment is made of following sequence:

QLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNAS

In another example, can before existing, test antigen carry out antigenic preparation.In an example of antigen preparation, the antigen preparation that is used for diagnostic kit comprises at least two kinds of HIV antigens, for example with the gp41 and the p24 of 1: 1 mixed.For example concentration is that the final concentration that the g ρ 41 of 1.6mg/ml and p24 that concentration is 1.47mg/ml can be prepared to gp41 is 0.8mg/ml, and the final concentration of p24 is 0.735mg/ml.

In antigenic another example of preparation, use HIV-1 antigen, HIV-2 antigen or use above-mentioned two kinds of antigens simultaneously.In this example, can prepare antigen mixture.Peptide antigen gp41 and g ρ 36 are dissolved in the distilled water with the concentration of 2mg/ml respectively.The p24 purifying protein is suspended in the 20mM carbonate buffer solution (pH9.6) with the concentration of 2mg/ml.With gp41: gp36: p24=5: 2: 3 (mol ratio) preparation antigen mixture.Then with the preparation the antigen mixture five equilibrium and be kept at-20 ℃.This antigen mixture is fixed on the filter paper that is made of Mierocrystalline cellulose, and described Mierocrystalline cellulose is essentially alpha-cellulose.For example, in one embodiment, this fibre content is 98%.For example, employed fiber filter paper has the particle of 20-25 μ m and holds back size, and ash content per-cent is 0.06%.Can thaw at the antigen mixture that will will freeze before the antigen mixture mounting is to the Mierocrystalline cellulose filter paper.1 microlitre (μ l) antigen (about 2 μ g) mounting is to filter paper, air-dry, it is kept at room temperature before installing in the verifying attachment will being positioned in antigen group on the filter paper.

In antigenic another example of preparation, utilize the peptide antigen gp41 and gp36 (concentration is respectively 2mg/ml) the preparation antigen mixture that are dissolved in the distilled water.The p24 purifying protein is suspended in the 20mM carbonate buffer solution (pH9.6) with the concentration of 2mg/ml.For example, with gp41: gp36: p24=5: 2: 3 ratio prepares antigen mixture.The antigen mixture of preparation can divide five equilibrium and be stored in-20 ℃.Can thaw at the antigen mixture that will will freeze before the antigen mixture mounting is to the fiber filter paper.1 μ l antigen (about 2 μ g) by mounting to Mierocrystalline cellulose filter paper, air-dry, and it is kept at room temperature before installing in the verifying attachment will being positioned in antigen group on the filter paper.

In one embodiment, use two kinds of HIV-1 antigens.In bacterium, express this antigen, and utilize the standard molecular biology method to carry out purifying.As mentioned above, above-mentioned antigen can be HIV-1p24 albumen and HIV-1gp41, and gp41 can be complete albumen, Partial Protein or protein fragments.

For example, in one embodiment, the aminoacid sequence of homologous sequence and gp41 peptide shows 80% above identity.

Embodiment 106-influenza virus

In one embodiment, antigen is influenza antigen, for example influenza A A, B or C antigen.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

In case dilute sample is absorbed, can add 150 μ l dyeing damping fluid.In one embodiment, the dyeing damping fluid is the albumin A that is combined with Radioactive colloidal gold.In case the dyeing damping fluid is absorbed, can add 200 μ l decolouring damping fluid.The decolouring damping fluid for example can be Dulbecco ' s phosphate buffer soln (1X) (DPBS).In case the decolouring damping fluid flows through system, can read the result immediately.When test position T and control site C all showed redness, test result was positive, and there is the antibody of specified disease in expression, promptly to the specific antibody of specific antigen.

Embodiment 107-leukosis virus

In one embodiment, antigen for detecting one or more antibody at specific antigen, at first utilizes 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample from leukosis virus.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 108-Marburg virus

In one embodiment, antigen is from Marburg virus.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 109-mumps virus

In one embodiment, antigen is mumps virus antigen.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 110-papilloma virus

In one embodiment, antigen is papilloma virus.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 111-paramyxovirus

In one embodiment, antigen is a kind of paramyxovirus.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 112-pestivirus

In one embodiment, antigen is a kind of pestivirus.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 113-picornavirus (Picorna)

In one embodiment, antigen is picornavirus antigen.In the antigenic object lesson of picornavirus, antigen can comprise capsid of poliomyelitis antigen or poxvirus antigen.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 114-rabies virus

In one embodiment, antigen is rabies virus antigen.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 115-reovirus

In one embodiment, antigen is reovirus antigen.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 116-respiratory syncytial virus

In one embodiment, antigen is respiratory syncytial viral antigens.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 117-rubella virus (Ruhella)

In one embodiment, antigen is rubella virus antigen.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 118-rotavirus

In one embodiment, antigen wheel virus antigen.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 119-vaccine virus (Vaccinia)

In one embodiment, antigen vaccine virus antigen.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 120-people T-lymphocyte virus

In one embodiment, antigen is people T-lymphocyte virus antigen.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 121-prostate cancer

In one embodiment, antigen is from noninfectious disease, for example cancer.In the object lesson of cancer, cancer is a prostate cancer, and antigen is prostate specific antigen (PSA).For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

Embodiment 122-Alzheimer

In one embodiment, antigen is the A68 antigen from Alzheimer.For detecting one or more antibody, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample at specific antigen.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

The combination of embodiment 123-virus antigen and bacterial antigens

In one embodiment, can detect two or more antigen by test kit.These two kinds of different antigens for example can be virus antigen and bacterial antigens.Bacterial antigens can be mycobacterium tuberculosis (Mycobacterium Tubercolis).Virus antigen for example can be hepatitis antigen or HIV antigen.In order to detect each antigen, at first utilize 150 μ l dilution buffer liquid to dilute serum, blood plasma or the whole blood of 10 μ l as check sample.Then this 150 μ l is diluted good sample and add the central authorities of verifying attachment to.For blood sample, suggestion was waited for about 3 minutes or is transparent red until dilute sample before loading the next step of dilute sample.

In case dilute sample is absorbed, can add 150 μ l dyeing damping fluid.In one embodiment, the dyeing damping fluid is the albumin A that is combined with Radioactive colloidal gold.In case the dyeing damping fluid is absorbed, can add 200 μ l decolouring damping fluid.The decolouring damping fluid for example can be Dulbecco ' s phosphate buffer soln (1X) (DPBS).In case the decolouring damping fluid flows through system, can read the result immediately.When test position T and control site C all showed redness, test result was positive, and expression exists the antibody of specified disease, i.e. antigen-specific antibodies.

The not just combination of virus antigen and bacterial antigens of combination that utilizes test kit to test.In another example, can detect the combination of two or more virus antigens.In another example, can select the combination of virus antigen, parasite antigen, bacterial antigens and fungal antigen.In an example of the composite type of virus antigen, parasite antigen, bacterial antigens and fungal antigen, can detect the combination of fungi infestation and virus infection.Combination described herein is not limited to disclosed specific embodiment.

In one embodiment, will have two or more test point, and show that test result will be positive, there is the antibody of two or more specified diseases in expression.Therefore, two of the combination of Test Virus antigen and bacterial antigens test point show that this person has the antibody of virus disease and bacteriosis.

In the synoptic diagram of Fig. 3 A and 3B, will use the sample result of glass fibre membrane 160 and the result of reagent for quickly examining box to compare, described test kit uses one or more to be used for detecting the HIV antigen of the HIV that is present in plasma sample.The glass fibre membrane 160 of Fig. 3 A has been failed, and reason is that blood plasma can not flow through tunica fibrosa.In contrast, use Fig. 3 B of Mierocrystalline cellulose filter paper 180 other materials similar with physical property that sample is shown, it is positive that the HIV test successfully is shown, and compare with the test kit of prior art and to have better contrast.Contrast spot 172 as seen, and positive test spot 174 is complementary with the ColourIndex number that contrasts spot 172.Table 9 has been described the characteristic of the glass fibre membrane that this check utilized.In Fig. 4 A-C, will use the figure of figure and the sample that uses 220 tests of Mierocrystalline cellulose filter paper of the plasma sample of nitrocellulose filter 200 tests to compare.Shown in the accompanying drawing of Fig. 4 A, the check of nitrocellulose filter failure is compared with the reagent for quickly examining box that utilizes fibrin reaction layer 220 of Fig. 4 C, poor contrast, and finish the long time of test needs.The red residue of colloidal gold solution is retained in the test zone of Fig. 4 A, not by the nitrocellulose reaction film.The nitrocellulose filter 200 that obtains from the Bio-Rad laboratory has 0.45 micron pore size.Utilize fibrin reaction layer 220, shown in Fig. 4 C, be shown clearly in contrast spot 182, positive test spot 184 is clear equally, is complementary with the ColourIndex number that contrasts spot.In Fig. 4 B, device film 205 uses the ester film that is mixed with nitrocellulose with 5 micron pore size.Blood plasma does not pass through, and causes this yet failure of film check.

In Fig. 5, be used to measure the flow velocity of ASTM standard test PBS by filter paper of the improvement of flow velocity, described filter paper is the 7cm circular filter paper that is converted into 1/4th sizes, is suspended from the ring.Utilize several filter paper then and use same antigen and application of sample prepares test kit.Utilize the HIV positive to test, and utilize the ColourIndex number table of Fig. 6 to determine the colour intensity of test spot.Fig. 5 represents the relation curve of ColourIndex number and DPBS flow velocity.Utilization has qualitative filter paper and wet strong filter paper mensuration water and the phosphate buffer soln that various other particles of level keep size.The data of Fig. 5 are shown in table 2.The error bar of Fig. 5 is represented the height data value.The square expression height sample of tiring, circular expression low liter sample.

Flow velocity is relevant with ColourIndex number.Low flow velocity film is sensitiveer than high flow rate film.In this example, six kinds of dissimilar Whatman have been tested ^TMFilter paper, each all has the particle that is distributed between 2.5 microns～30 microns and holds back size.Surprisingly, as shown in Figure 7, a platform area appears in flow velocity between 6～20 microns, and flow velocity is about 0.1～0.2mL/min/cm ²In an example of HIV antibody sensitivity test test kit, this platform area is corresponding to the flow velocity of specimen (no matter being blood, serum or blood plasma) and the best of breed of sensitivity.

Use the ASTM standard test that improves to determine the flow velocity of each film.Filter paper is made the circle of 7cm diameter.This filter paper is placed filtering solution (PBS and water are tested) time enough, so that this filter paper is soaked into fully.Then, the part of this filter paper except the edge lain in the funnel, described edge section is folding upwards.Next, the 5ml filtering solution is added into funnel central authorities, and uses the stopwatch minute.When most of filtering solution passes through filter paper, writing time.

Flow velocity is with mL/min/cm ²Be unit, and calculate: V/Tx60s/1minx1/cm by following manner ², wherein, V=volume (ml), the T=time (second), s=second, min=minute, surface-area was expressed as cm ²

Qualitative filter paper has 2.5,6,11 and 20-25 micron pore size (we show do 20 microns).Wet strong filter paper has the particle of 23 microns and 30 microns and holds back size.Have 2.5 microns～23 microns particle and hold back the water flow velocity of filter paper of scope at about 0.04mL/min/cm ²～about 0.4ml/min/cm ²Scope.The DPBS flow velocity is also at about 0.04mL/min/cm ²～0.4ml/min/cm ²Scope.What statistical significance is mensuration difference between present not clear water and the PBS have.Term " about " is an experimental error of considering that any mensuration known to those of ordinary skills is brought when together using with flow velocity.The flow velocity of ester film that is mixed with nitrocellulose is far above the flow velocity that utilizes Mierocrystalline cellulose filter paper to measure, and is shown among the following table 1B, and unit is mL/min/cm ²

For example, determination data among the table 1A and the contrast gradient between the report data among the table 1B are significant when the water flow velocity of the water flow velocity of the wet strong Mierocrystalline cellulose filter paper of 23 microns of comparisons and the mixed nitrate cellulose ester membrane with 5 micron pore size.The mixed nitrate cellulose ester membrane has the increase of several magnitude.In the past, shown in Fig. 4 B, utilized the sample survey failure of mixed nitrate cellulose ester membrane with 5 micron pore size.In addition, the paperback nitrocellulose with 0.45 micron pore size causes 6ml/min/cm ²Flow velocity faster, reported in [0171] of U.S. Patent Publication the 2004/0002063rd section as Chan etc.

Employed mixed nitrate cellulose ester film filter paper is the Magna that mixes by GE Infrastructure water and treatment technology (GEInfrastructure Water and Process Technology) manufacturing ^TMThe cellulose nitrate ester film filter paper.Used aperture is 5 microns in this example.The water flow velocity of mixed nitrate cellulose ester membrane is with mL/min/cm ²Be unit, and under 520mmHg (10psi), 20 ℃, measure.The air velocity of filtration area is with L/min/cm ²Be unit, and under 520mmHg (10psi), 20 ℃ (68 Fahrenheit temperature), measure.Saturation pressure appears when air at first is forced through the hole of water wet film.

The character of Mierocrystalline cellulose filter paper shown in the table 1C.The table 1C that obtains from the Whatman website illustrates the typical properties of tested Mierocrystalline cellulose filter paper, and for example particle is held back liquid and air flow quantity.Above-mentioned character can be used to select specific filter paper.The rank of when the preparation test kit, reporting among the

utilization table 1C

1,3,4,5,113 and 114.Wet strong qualitative fiber filter paper comprises that a small amount of chemical stability resin is to obtain the wet tenacity of improvement.For these checks, filter paper is cut into the circle of 7cm diameter, is used for hydrometry, and filter paper is made the square of 1 inch x1 inch to be used for test kit.

Table 2 is illustrated in each particle and keeps the every kind of low liter sample of test under size and the flow velocity and the ColourIndex number separately of the high sample of tiring.Fig. 5 illustrates the average specific colour index, and the color index rod illustrates the high low value of color index.Have ColourIndex number and be 1 sample and be considered to the low liter sample, be considered to the height sample of tiring greater than 2 sample and have ColourIndex number.Has about 1.2mL/min/cm ²The Mierocrystalline cellulose filter paper of flow velocity all failed for each low liter sample.At about 0.4ml/min/cm ²Flow velocity the time, the reagent for quickly examining box has successfully detected the height HIV-positive of tiring, still, same test kit has been failed twice in detecting four checks that low liter HIV-positive exists.Therefore, for the fibrin reaction layer, about 0.4ml/min/cm ²Flow velocity be the upper limit that is applied to the HIV screening test.Surprisingly, by adopting the intensity of color index rank quantitative result, an example of reagent for quickly examining box has shown sufficient sensitivity and the specificity that is used to distinguish high low liter HIV-positive.

The data presentation ColourIndex number of Fig. 5 descends along with the flow velocity exponentially ground of Mierocrystalline cellulose filter paper is violent.Therefore, think that more high flow rate may cause failing more.Table 2 has been described the data of high low liter sample.DPBS flow velocity, particle are shown hold back the result of size and check sample and reference substance.

Fig. 6 illustrates the color index that is used for semiquantitative determination sensitivity with the black and white line synoptic diagram, and this color index obtains by measuring ColourIndex number.The background relevant with 0 represents do not have difference between test spot and the background.Any spot darker than background is 1, is shown as light shading in Fig. 6.Be equal to or greater than 1 value and be considered to positive assay.ColourIndex number greater than 2 is corresponding to the high-titer sample, and is shown as dark shading in Fig. 6.Higher contrast gradient between background and the test spot represents that with cross hatch 3 high-contrast is represented with dual crossing hachure 4.Actual color shown in the disclosure of this synoptic diagram and U.S. Patent application 12/008,861 is relevant, and by reference it is incorporated into.

Fig. 6 is the example of color index chart, and from 0 to 4,0 is the specific antibody existence feminine gender of specific antigen, and 4 is the highest semidefinite value.Index value 0 shows that pink dyeing may take place background, but does not still represent the existence of identification point.Index value 1 can be distinguished mutually with background, but dark unlike the color of 1 expression.Index value 2 shows than 1 darker apparent point, and is still dark unlike the colors of 2 expressions.Index value 3 is the very dark points of color, than 2 darker, but dark unlike the reference color that provides in 3.ColourIndex number 4 is darker than the reference color that is labeled as 3.For example, Fig. 9 illustrates from the blood plasma of same donor sample acquisition and the comparison of blood sample.The blood test (a) and (b) have contrast spot 312,332 and test spot 314,334, and this contrast spot and test spot can utilize contrast spot 322,342 and the test spot 324,344 of ColourIndex number and plasma sample (c), (d) to compare.Whole blood and blood plasma can use same test kit and same ColourIndex number chart.

Unless clearly describe, otherwise and other test kits relatively be at the commercial reagent box and use between the example of Mierocrystalline cellulose filter paper and carry out that described Mierocrystalline cellulose filter paper has the flow velocity of about 0.1niL/min/cm.Based on the data shown in the above-mentioned table 2, Fig. 7 provides flow velocity and particle and holds back relation curve between the size.Hole dimension increases the demonstration flow velocity and increases, but as shown in Figure 5, flow velocity increases, and measures sensitivity decline.As preferably, this sensitivity also provides a fast as far as possible check when bringing the result that can distinguish high titre and low titre.

Fig. 8 illustrates the comparison of using blood sample and plasma sample.Measured ColourIndex number.The assay of blood and blood plasma is closely similar, and this is very wondrous and beyond thought.Most of test kit all can not be used to test whole blood.As seeing in the drawings, when utilizing the reaction film of other types to test, when using with blood rather than blood plasma or serum, the reaction film of every other type all is invalid.The use of whole blood allows to test in the place that is not easy to obtain whizzer, and other test kits demonstrate very big advantage relatively.

Table 3 illustrates and illustrates the data of being reported among Fig. 8.Some samples are tested twice, and other samples are once tested.Table 3 relatively uses the sample of blood and uses the data of the sample of blood plasma, and described blood is identical with the blood plasma source, and utilizes the fibrin reaction layer of same type.

Blood plasma, serum and blood sample all have similar visible results.For example, Fig. 9,11,12 and 13 illustrates the comparison of blood plasma and blood sample.Use the example (Fig. 9,11) of whole blood and the example (Figure 12,13) of use blood plasma schematically to be showed, and testedly go out the HIV positive.These images are to utilize the check sample 84 that tabular data are reported in the table 3 to represent.Expression exists the positive test spot of HIV by test spot 314,324,334,344 and 312,322,332,342 expressions of contrast spot.From same donor sample, check sample 260 and 262 have been obtained.One is used blood, and another uses blood plasma.Equally, from same donor sample, obtained check sample 270 and 272, one use blood, and another uses blood plasma.Blood and plasma sample test result all are 3 on the color index scales (scale).

Figure 10 illustrates the histogram of ColourIndex number that expression utilizes each sample of reagent for quickly examining box and commercially available mensuration test kit, and described reagent for quickly examining box has the about 0.1mL/min/cm of DPBS flow velocity ², described commercially available mensuration test kit uses

G3.Use the most of plasma sample and the use of described test kit

The G3 test kit (

With

Be MedMira Laboratories, the plasma sample of Inc. (Toronto, registered trademark Canada)) is compared has better contrast.The box of reagent for quickly examining shown in Figure 11 and

Some of G3 test kit is relatively representative.Having the DPBS flow velocity is about 0.1mL/min/cm ²The reagent for quickly examining box of Mierocrystalline cellulose filter paper tested.Using the method for reagent for quickly examining box to comprise joins 150ml phosphate buffer soln (PBS) in the cryodesiccated dyeing damping fluid.With 150ml PBS solution dilution 10ml blood plasma.Blood plasma, 150ml dyeing damping fluid and the 200ml PBS solution of dilution are placed test kit successively.The check time length is less than 2 minutes, can be described as rapid detection.For the reagent for quickly examining box, except that blood plasma, can also select to use blood and serum.There is not this situation for other commercially available test kits.

The G3 test kit, (Universal Buffer) joins in the test kit with 3 general damping fluids, adds 1 slurry of bleeding then.Afterwards, in test kit, add 3 general damping fluids.On test kit, add gold system lid (gold cap) then immediately, add 12 general damping fluids via this lid.Randomly, can add 3 general damping fluids again.The check time length is less than 3 minutes.Term " general damping fluid " exists

Use in the operation instructions of G3 test kit.Test kit 400 tests out the HIV positive, this with

Coming to the same thing of the test kit 300 of G3.Two kinds of test kit test results all are 1 on the color index scale.Figure 11's (f)

It is negative that G3 illustrates the G3 test kit 320 that detects patient's sample BBI#10.Reagent for quickly examining box 420 test results of sample BBI#10 are positive, and color index is 1.Therefore, uncertain even western blotting shows, reagent for quickly examining box 420 still shows the HIV-positive.As BBI#4 from test kit 440, BBI#25 from sample 460, sample BBI#10 is from the anti-HIV-1PRB204 operating panel (performance panel) available from BBIDiagnostics, and this operating panel is at testing on the different test kits.Utilize the relatively demonstration of the BBI#10 of other competition test kits, use Abbott Determine ^TMThe sample BBI#10 of HIV-1/2 is positive.For example

And Uni-Gold ^TMThe test of other test kits negative (

Be the registered trademark of Orasure Technologies, Uni-Gold ^TMBe the trade mark of Trinity Biotech).The western blotting check of BBI#10 is uncertain.Screening assay PRB 204 shown in the BBI reference table 4 and 5.Though western blotting is a golden standard, a fuzzy western blotting can not judge that the assay of HIV is the positive or feminine gender.

The test kit 440 and use that contain sample BBI#4

The test kit 340 of G3 is identical, and detected result is the HIV positive.Use quick test kit 440 test results of fibrin reaction layer to be 3 on the color index scale, and The test result of test kit 340 is 1 on the color index scale, and Mierocrystalline cellulose test kit 440 shows better contrast, thereby makes test kit 440 easier reading.For example

Also test positive with other test kits of Uni-Gold.Western blotting flat board (panel) data also draw positive findings.Therefore, for positive assay, use the reagent for quickly examining box example of Mierocrystalline cellulose filter paper correctly to identify HIV male assay.

Use sample BBI#25's

G3 test kit 360 test results are negative.On the color index scale that we use, test kit 360 test results are 1 on the color index scale.Test kit 460 tests are negative, are 0 on the color index scale.The 3rd test kit from Abbott, shown positive findings for identical sample BBI#25.

And Uni-Gold ^TMTest all positive.Western blotting verifies as uncertain.But, described test kit, with

The G3 difference except serum or blood plasma, also allows to use blood.Serum and plasma sample and blood relatively need laboratory equipment, and need the more time to prepare sample.In addition, it is fast to be used for the film absorption rate of test kit.Measured by color index, this check has determined that test kit sample 460 is a low liter at ColourIndex number 1 place.Therefore, the reagent for quickly examining box can be used to screening, and utilizes the color index scale can also be as the qualitative test of antibody titer (antibody titer).

Table 4～6 illustrate the more results of all tested BBI samples and the comparison of test kit and western blotting result and competition assays test kit.For example, BBI#1 is corresponding to PRB 204-01, and BBI#2 is corresponding to PRB 204-02 etc.Table 7 and 8 discloses the strip pattern from the western blotting check of BBI flat board.The western blotting strip pattern of each sample of measuring is shown in table 5.

G3 (a) test kit has nitrocellulose filter, therefore, utilizes

The blood test of G3 test kit has been failed, and the reagent for quickly examining box finds that successfully sample is the HIV feminine gender.In Reveal G3 test kit, blood does not flow through, but condenses.The reagent for quickly examining box that successfully detects blood comprises that the DPBS flow velocity is about 0.1mL/min/cm ²Filter paper.In addition, also tested glass fibre membrane.Blood does not flow through glass fibre membrane, but in surface condensation.Used glass fibre membrane is

GF/C.Though Chan points out glass fibre membrane is applied to blood sample in U.S. Patent Publication the 2004/0002063rd, but these checks clearly illustrate: under the situation of the complicated approach that does not use Chen, use glass fibre membrane can't use whole blood to test in test kit. The G3 test kit can not utilize blood sample, falls flat in this.With

The G3 test kit is identical, and the test kit of Mahajan uses nitrocellulose filter among the U.S. Patent Publication No.2004/0023210, also its purposes is limited to serum and blood plasma.Therefore, the reagent for quickly examining box has better contrast when using blood, blood plasma and serum, and this is remarkable and important improvement for the reagent for quickly examining box.

Nitrocellulose is well-known in conjugated protein (binding protein) field, and Here it is, and it is generally used for the reason of western blotting and other mensuration.But these nitrocellulose are measured and are not used the fibrin reaction film, and all are unsuitable for the Rapid identification together used with whole blood.Selection beyond the nitrocellulose also seldom is considered for test kit.In the check that utilizes blood to carry out, nitrocellulose is failed undoubtedly, and at effective flow rates 0.04-0.4ml/min/cm for example ²The effect that the Mierocrystalline cellulose of selecting is brought into play when utilizing blood is good when utilizing blood plasma and serum.The snappiness that has in addition makes check be suitable as spot inspection.Surprisingly, at some embodiment test kit that is used for detecting the HIV-positive, the use of blood does not have reducing sensitivity or specificity.

Table 6 illustrates the feature of glass fibre membrane, and the data of glass fibre membrane are shown.Hold back for particle, suppose: have 2% initial penetration number (initial penetrationvalue) (expression keeps fully in the testing laboratory report of standard) when using the solia particle that is scattered in the water.For flow velocity, suppose: pass through the vacuum filtration of the smooth filter paper of 21/16 inch (5.5cm) down at 100mmHg (1.9psi) through pre-filtered water.Water specific absorption hypothesis has the balance volume (equilibrium volume) of the water that is absorbed by filter paper.

In additional survey, the quick test kit of embodiment compares with using high and low plasma sample of tiring.Herein except as otherwise noted, all test kits shown in the embodiment all use selected PBS flow velocity to be about 0.1mL/min/cm ²Mierocrystalline cellulose filter paper.Embodiment test kit 500,502,504,506,508,510 uses sample #80, #81, #82, #83, #84 and #91 respectively, and with corresponding

Test kit 488,490,492,494,496 and 498 relatively has better visual contrast.The ColourIndex number of sample is shown in table 7.Data presentation: with same detected plasma sample The G3 test kit is compared, and except sample #81, all test kits all have better visual contrast.

Gene probe embodiment

Figure 14 A～C schematically shows except having contrast test spot 494,495, also has the example based on the test kit of the test spot 496 of antibody and gene probe test spot 498.In Figure 14 A, independent test kit has two test windows 1,2, comprises the Examination region of antibody test 1 and gene probe 2 respectively.In Figure 14 B, independent test kit has antibody test zone 496 and gene probe zone 498 in independent inspection window.Test kit among Figure 14 B can have the operation of utilizing single staining procedure, perhaps can have sequencing, and promptly the staining agent of antibody test separates with the application of the staining agent of gene probe.For the check of bedside in-vitro diagnosis, preferably make the numbering of each step and the complicacy of each step reduce to minimum, therefore, preferred combination two kinds of staining agents, antibody and gene probes join an independent window with it in a step, and example is as shown in Figure 14B.For example, the dyeing damping fluid can have and nanotube or particle bonded virus-specific gene probe, and described nanotube or particle for example are colloid gold particle, gold nano grain, silver nano-grain, carbon nanotube etc.When Figure 14 C diagram is correctly shown when hypothesis contrast spot, four kinds of possible results of the test kit of binding antibody and gene probe Examination region 496,498.Positive antibody spot 496 and negative gene probe spot 498 produce first result 504, and this result has shown the existence of antibody, but does not have viral indication.The negative demonstration of this HIV-shows the existence of immunization or maternal antibody.Second negative findings 510 antagonist and RNA may be negative.Two possible positive findingses 506,508 can utilize arbitrary positive demonstration of gene probe Examination region 498 to be proved.Whether the existence of antibody may be an important indication, for example causes the different courses of treatment or clinical monitoring scheme.In one embodiment, the patient may show that the positive of virus existed before the existence of antiviral antibody is shown the positive, and this is owing to for example delay is arranged aspect existing can detecting antibody in the blood.

Probe stationary at RNA or DNA concensus sequence (compatible sequence) can be arrived on trace paper (blotting paper) or the filter paper, with RNA or the DNA that fixedly has concensus sequence, shown in Figure 15 (a), described filter paper for example can be Mierocrystalline cellulose filter paper or nitrocellulose filter paper.The preferred Mierocrystalline cellulose filter paper that uses detects by specific RNA, DNA or its segmental existence in the certain volume body fluid of filter paper, and described Mierocrystalline cellulose filter paper allows to cross effective volume (significant volume) from the filter paper surface current at short notice.For example, can use primer or primer to as gene probe, one of them primer and marker for example gold nano grain link to each other, and it is included in the dyeing damping fluid, another primer is fixed in the spot place of paper or other marker zones of paper, to catch specific RNA or dna sequence dna.Each that primer is right and specific RNA or dna sequence dna complementation, described RNA or dna sequence dna for example are viral RNA or single stranded DNA.

By making the body fluid that contains specific RNA or dna sequence dna by paper, the primer that specific RNA or DNA are fixed on the paper is caught, for example Figure 15 (b) and (c) shown in.Then, by making the dyeing damping fluid by same filter paper, specific RNA or DNA that the primer that is connected with gold nano grain or other particles or nanotube is fixed on the paper fix, and shown in Figure 15 (d), compare with the paper of immobilized primer not, provide visual contrast.Can be by chemical reaction (for example photodevelopment), fluorescence (for example under UV-light) or electrical characteristic (for example electricity is led, resistance or the luck) enhancing contrast ratio.In one embodiment, marker can be nano particle or nanotube, for example when being exposed to UV-light, sends fluorescence or phosphorescence.Can use scavenging solution residual dyeing damping fluid of flush away from the paper, because scavenging solution is only removed the dyeing damping fluid from background, and fixed RNA that tested spot is caught or DNA mixture be by flush away, so strengthened the contrast gradient between spot and the background.Can analyze the contrast gradient between spot and the background then, determine the existence of RNA in the body fluid or dna sequence dna, and in some example, can qualitative or quantitatively determine the level relatively or the concentration of RNA in the body fluid or dna sequence dna.For example, can be with the resulting contrast gradient of test kit and a plurality of known viruse carrying capacity, for example every milliliter 5000,10,000 and 15, the 000 viral concentration that copy) compare.Can utilize a plurality of known viruse carrying capacity that normal intensity or the marker zone of test kit and the normal intensity of the contrast gradient between the background in marker zone are provided.By test kit and standard are compared, can be qualitative or quantitatively determine the relative concentration scope of virus load.

Figure 16 be illustrated schematically in mercaptanization (thiolization) and utilize the oligopolymer of single stranded oligonucleotide for example carry out functionalized before and gold nano grain afterwards.Among Figure 17, the LTR oligonucleotide is as utilizing complementary primer to detecting the example of virus load.For example, primer can be from the LTR zone of HIV-1 isolates (isolate).Because the interaction of ssDNA-Au-RNA and complementary DNA (cDNA) is fixed to the spot on the filter paper with nano particle (being gold nano grain in this example), as shown in figure 15, so under UV-light, can be observed contrast gradient between spot (1) and the background.But the generation of spot (2) is owing to the only interaction between ssDNA-Au and the ssDNA, and the generation of spot (3) only is because ssDNA interacts and there is gene probe.Figure 18 illustrates absorption spectrum, and this absorption spectrum is distinguished sample and independent ssDNA-gold nano grain and the ssDNA-gold nano grain with ssDNA in contrast of the sDNA-gold nano grain-RNA with complementary DNA.For the blood sample that has HIV-1, excellent contrast.Therefore, gene probe can utilize complementary DNA as target, and marker is attached to the marker zone of test kit, thereby can be used in the existence that detects HIV.

Utilize the test case of circulation reagent for quickly examining box to use the specific DNA gene probe of the LTR-that is connected with gold nano grain to detect viral RNA.The DNA oligopolymer that for example can use the gold nano grain of citrate-stable and mercaptan to modify prepares DNA-gold nano grain mixture according to the description of Mirkin etc.In one embodiment, can use 5 '-fluorescein, 3 '-oligonucleotide of mercaptan mark determines surface coverage (surface coverage).For example, as JJ.Storhoff, R.Elghanian, R.C.Mucic, CA.Mirkin, R.L.Letsinger at J.Am.Chem, Soc. (1998) vol.120, disclosed among the pp.1959-1964, can join in the aqueous solution of nano particle by the oligonucleotide that will contain desired amount, prepare Bifunctionalized DNA-gold nano grain conjugate (conjugate).

With 10% bovine serum albumin phosphate buffered saline buffer (BSA) of the functionalized gold nano grain of single stranded oligonucleotide bonded and 0.5% (for example, pH7.4, BD) mix mutually, described functionalized gold nano grain for example can be added drop-wise to and be used to use ultraviolet spectrophotometer to carry out spectroscopic analysis on the glass plate.The result of the ultraviolet spectrophotometer among Figure 18 can make a distinction the flat board (being with or without other single stranded oligonucleotides) of the functionalized gold nano grain puted together to single stranded oligonucleotide to the similar flat board that has by dripping the complementary oligonucleotide that 25 microlitre complementary oligonucleotides add to the mode of the functionalized gold nano grain that is conjugated with single stranded oligonucleotide.

In one embodiment, utilize the functionalized gold nano grain that is combined with single stranded oligonucleotide to prepare the HIV gene probe, and use it for the existence that detects HIV.It is synthetic and be fixed on the spot of Mierocrystalline cellulose filter paper of reagent for quickly examining box of the present invention to have the single stranded DNA primer of functional identical sequence (functionally identical sequence) of HIV-1LTR.For example the body fluid of fresh blood (raw blood), blood plasma, urine, saliva etc. is added in the damping fluid or directly places on the filter paper of reagent for quickly examining box the check of circulating.HIV RNA (for example HIV-1LTR) is hybridized with the dna primer on the filter paper.Then, the dyeing damping fluid that contains the functionalized gold nano grain of puting together with dna probe is added on the filter paper to feasible functionalized gold nano grain and HIV RNA hybridization of puting together with dna probe.Gold nano grain accumulates in HIV RNA place, and background provides and has red contrast gradient relatively.If necessary, can use the decolouring damping fluid further to rinse out the dyeing damping fluid of background, and the gold nano grain mixture with HIV RNA hybridization is not influenced.In one embodiment, a plurality of different dna probes are chosen to a plurality of different zones complementations with HIV RNA, make that a HIV RNA molecule might be in conjunction with several gold nano grains, thereby be verified contrast gradient and the sensitivity that HIV RNA that test kit detects improves the reagent for quickly examining box at every kind.Using the significant advantage of dna probe is that HIV RNA itself is detected, so vaccinated individuality will be negative in the check of use DAN probe, and can directly compare and analyze level or the concentration of HIV in the certain volume body fluid.Only detecting the test kit of HIV antibody can check the vaccinated individual positive that shows, and can only be used to show the individual antibody horizontal that produces, and does not show the level that virus is own.Both can provide detection of antibodies at a spot test antibody and the reagent for quickly examining box that uses dna probe to test another spot, the qualitative or quantitative analysis of HIV virus load in the individual body fluid also can be provided.

Figure 19 schematically shows carbon nanotube and oligonucleotide link coupled example.In this example, carbon nanotube (for example Single Walled Carbon Nanotube or herring-bone (herringbone) carbon nanotube) is scattered in dimethyl formamide under ultrasonic agitation (ultrasonicagitation) effect.For example, for for disperseing 1 milligram of carbon nanotube sample in 2 milliliters of dimethyl formamides, it is sufficient stirring 2 hours.This provides density is 5 milligrams every milliliter black carbon nanotube suspension.Then, the thiol group solution thorough mixing by 2 milliliters of 1mM are had thiol group (positively charged) is in carbon nanotube (electronegative), and the carbon nanotube in the mercaptan suspension, described thiol group solution for example are mercaptan.For collecting for the carbon nanotube of thiol group-functionalized (or mercaptanization) by removing supernatant liquor, with 18,000rpm was sufficient in centrifugal 15 minutes.Can use distilled water to clean the carbon nanotube of mercaptanization, in one embodiment, clean 3 times at least to rinse out the thiol molecule of any not bonding.For example, can add among 1 milliliter of PBS (pH7.0) of 1 milligram of mercaptan carbon nano tube at the single stranded DNA that under 4 ℃ with quality was 10 milligrams through 12 hours.Then, suspension is centrifugal, collect ssDNA-sulphur-carbon mano-tube composite by removing supernatant liquor.Can fully wash the functionalized carbon nanotube of DNA once more to remove all not ssDNA molecules of bonding.As shown in figure 20, independent carbon nanotube (A) has the ultra-violet absorption spectrum that is different from the carbon nanotube (B) that combines single stranded oligonucleotide, than (C) incomplementarity oligonucleotide (DNA) fragment and (D) with the complementary fragment of the single stranded oligonucleotide of the carbon nanotube hybridization that is conjugated with single stranded oligonucleotide, it has different absorption spectrums.Therefore, for example, can use the existence of the qualitative or detection by quantitative complementary DNA of the carbon nanotube of puting together with ssDNA.

In the example of measuring method, the functionalized carbon nanotube of single stranded DNA (ssDNA) can be included in the dyeing damping fluid, described dyeing damping fluid utilizes the damping fluid dilution or does not utilize the damping fluid dilution, after body fluid, be added to reagent for quickly examining box of the present invention, place on the filter paper of test kit.Prepare filter paper by being included in the spot that is fixed with gene probe on the spot, it can fix viral RNA to be detected or DNA.Therefore, for example, the hybridization of the carbon nanotube that viral RNA or complementary DNA and ssDNA are functionalized preferentially combines the functionalized carbon nanotube of ssDNA in the test spot of reagent for quickly examining box, thereby the contrast gradient between test spot and the background is provided.

Spectrum among Figure 20 is to obtain by the slide glass that is prepared as follows, described slide glass in precoating the glass pane surface of bovine serum albumin (BSA) drip the functionalized carbon nanotube of ssDNA and obtain.The functionalized carbon nanotube of described ssDNA is allowed in room temperature (about 25 ℃) dried overnight.Fluorescein isothiocyanate (FITC) mark is added to complementary oligonucleotide, and this complementary oligonucleotide is added dropwise on the functionalized carbon nanotube of ssDNA, is heated to 60 ℃, keeps for 50 seconds.Clean sample then, and examine under a microscope, in 25 seconds, observe hybridization.Use atomic force microscope relatively the carbon nanotube (A) of ssDNA before functionalized, the carbon nanotube (B) after functionalized, subsequently with incomplementarity DNA hybridization after carbon nanotube and subsequently with complementary DNA (cDNA) hybridization after carbon nanotube, this relatively shows, when carbon nanotube is used as gene probe (or marker), take place ssDNA functionalized and with the hybridization of cDNA.This example provides a kind of method of using fluidization sample (fluidized sample) or DNA and RNA fragment detection DNA and the RNA of body fluid or DNA and RNA in the reagent for quickly examining box.

Figure 21 A-21C illustrates following atomic force microscope Photomicrograph image: (A) independent carbon nanotube (CNT), do not have any ssDNA chain or fragment; (B) the functionalized CNT of ssDNA; And (C) ssDNA functionalized and with the CNT of complementary DNA fragment hybridization, thereby the evidence of preparation carbon nanotube as the crossover process of the contrast gradient marker (contrast marking agent) of gene probe test spot is provided.According to an embodiment, any oligonucleotide with complementary oligonucleotide can be according to disclosed method and Single Walled Carbon Nanotube hybridization.

In one embodiment, the reagent for quickly examining box can detect DNA, RNA sample or its fragment, need not DNA in the PCR amplification humoral sample or the quantity of RNA.In another embodiment, can at first utilize polymerase chain reaction technique that small sample quantities is handled, provide the DNA of enough concentration or RNA to detect to utilize the reagent for quickly examining box.Preferably do not use PCR, and the reagent for quickly examining box can relative standard's product determines qualitative comparison or based on the intensity that can measure, contrast gradient or other physical propertiess virus load carried out quantitatively, and described other physical propertiess are based on the concentration of the particle, nanotube of Examination region etc.

Figure 22 A-B provides incomplementarity 786,796 single stranded oligonucleotides and complementary 788,798 single stranded oligonucleotides and contrast 784,794 another embodiment that compare.Complementary oligonucleotide 788,798 hybrid dnas, RNA or DNA or RNA fragment produce fluorescence.Therefore, can detect the complementary single stranded oligonucleotide that is present in the blood sample by hybridization.Figure 23 utilizes the concentration of functionalized carbon nanotubes that the comparison of intensity is shown.The preferred concentration of 750pmol at least, but the concentration that is low to moderate 250pmol can be distinguished.

In one embodiment, gene probe is chosen as the dna primer of viral RNA sequence, and described viral RNA sequence is for example for enumerating a kind of viral RNA sequence in the virus in the summary of the invention.For example a dna primer can combine with nanotube or nano particle by for example mercaptanization, and another dna primer can be fixed on the film, and described film for example is the cellulose filter membrane in the example that uses antigenic reagent for quickly examining box.In one embodiment, can not only detect antigen but also detect the viral RNA sequence, whether produce antibody, and determine virus load level or concentration in the tested fluid sample volume with definite individuality.For example, test kit can be determined the whether infection of uncontrollable HIV of scheme or treatment plan fast, and needs medical treatment and nursing immediately or change treatment plan.

Also tested the hybridization of single stranded oligonucleotide and gold nano grain.Known, the single stranded oligonucleotide that utilizes the gold nano grain of citrate-stable and thiol group to modify in this area prepares the binding substances of gold nano grain and single stranded oligonucleotide.Utilize 5 '-fluorescein, 3 '-single stranded oligonucleotide record (documenting) surface coverage of thiol group mark.Can select to have the multiple single stranded oligonucleotide with detected oligonucleotide sequence complementary oligonucleotide, for example following some, underscore and double underline are represented and complementary single stranded oligonucleotide bonded oligonucleotide sequence.

For example, gene probe is used for the check of bedside in-vitro diagnosis.The reagent for quickly examining box that the result can at room temperature be provided is used in the check of bedside in-vitro diagnosis.For example, the HIV test kit utilizes gene probe to make the HIV specific region hybridization of one or more oligonucleotide and HIV RNA.For example, the LTR zone of HIV has such zone, and guard in multiple HIV strain in this zone, is HIV exclusive (for example comparing with people DNA), and can be in room temperature by oligonucleotide probe hybridization.Identify some peculiar zones (Unique Region) in the example of HIV viral RNA sequence below.Use single underscore to represent distinctive part below.

The peculiar zone of HIV in the sequence (Sequence Context):

GGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTG CTTAAGCC TCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGA TCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACCTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTG CTGAAGCGCGCACGGCAAGAGGCGAGGGG CGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCG TCAGTATTAAGCGGGGGAGAATTAGATCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAA GCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGA AACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGACACAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCAAATG GTACATCAG GCCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGTAGAAGAGAAGGCTTTCAGCCCAGAAGTGA TACCCATGTTTTCAGCATTATCAGAAGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGG GGGACATCAAGCAGCCATGCAAATGTTAAAAGAGACCATC AATGAGGAAGCTGCAGAATGGGATAGAGTG CATCCAGTGCATGCAGGGCCTATTGCACCAGGCCAGATGAGAGAACCAAGGGGAAGTGACATAGCAGGAA CTACTAGTACCCTTCAGGAACAAATAGGATGGATGACAAATAATCCACCTATCCCAGTAGGAGAAATTTATAAAAGATGGATAATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTACCAGCATTCTGGACATAAGACAA GGACCAAAGGAACCCTTTAGAGACTATGTAGACCGGTTCTATAAAACTCTAAGAGCCGAGCAAGCTTCACAGGAGGTAAAAAATTGGATGACAGAAACCTTGTTGGTCCAAAATGCGAACCCAGATTGTAAGACTATTTTAAAAGCATTGGGACCAGCGGCTACACTAGAAGAAATGATGA CAGCATGTCAGGGAGTAGGAGGA CCCGGCCATAAGGCAAGAGTTTTGGCTGAAGCAATGAGCCAAGTAACAAATTCAGCTACCATAATGATGCAGAGAGGCAATTTTAGGAACCAAAGAAAGATTGTTAAGTGTTTCAATTGTGGCAAAGAAGGGCACACAGCCAGAAATTGCAGGGCCCCTAGGAAAAAGGGCTGTTGGAAATGTGGAAAGGAAGGACACCAAATGAAAGATTGTACTGAGAGACAGGCTAATTTTTTAGGGAAGATCTGGCCTTCCTACAAGGGAAGGCCAGGGAATTTTCTTCAGAGCAGACCAGAGCCAACAGCCCCACCAGAAGAGAGCTTCAGGTCTGGGGTAGAGACAACAACTCCCCCTCAGAAGCAGGAGCCGATAGACAAGGAACTGTATCCTTTAACTTCCCTCAGGTCACTCTTTGGCAACGACCCCTCGTCACAATAAAGATAGGGGGGCAACTAAAGGAAGCTCTATTAGATACAGGAGCAGATGATACAGTATTAGAAGAAATGAGTTTGCCAGGAAGATGGAAACCAAAAATGATAGGGGGAATTGGAGGTTTTATCAAAGTAAGACAGTATGATCAGATACTCATAGAAATCTGTGGACATAAAGCTATAGGTACAGTATTAGTAGGACCTACACCTGTCAACATAATTGGAAGAAATCTGTTGACTCAGATTGGTTGCACTTTAAATTTTCCCATTAGCCCTATTGAGACTGTACCAGTAAAATTAAAGCCAGGAATGGATGGCCCAAAAGTTAAACAATGGCCATTGACAGAAGAAAAAATAAAAGCATTAGTAGAAATTTGTACAGAGATGGAAAAGGAAGGGAAAATTTCAAAAATTGGGCCTGAAAATCCATACAATACTCCAGTATTTGCCATAAAGAAAAAAGACAGTACTAAATGGAGAAAATTAGTAGATTTCAGAGAACTTAATAAGAGAACTCAAGACTTCTGGGAAGTTCAATTAGGAATACCACATCCCGCAGGGTTAAAAAAGAAAAAATCAGTAACAGTACTGGATGTGGGTGATGCATATTTTTCAGTTCCCTTAGATGAAGACTTCAGGAAGTATACTGCATTTACCATACCTAGTATAAACAATGAGACACCAGGGATTAGATATCAGTACAATGTGCTTCCACAGGGATGGAAAGGATCACCAGCAATATTCCAAAGTAGCATGACAAAAATCTTAGAGCCTTTTAGAAAACAAAATCCAGACATAGTTATCTATCAATACATGGATGATTTGTATGTAGGATCTGACTTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAGCTGAGACAACATCTGTTGAGGTGGGGACTTACCACACCAGACAAAAAACATCAGAAAGAACCTCCATTCCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACAGTACAGCCTATAGTGCTGCCAGAAAAAGACAGCTGGACTGTCAATGACATACAGAAG TTAGTGGGGAAATTGAATTGGGCAAGTCAGATTTACCCAGGGATTAAAGTAAGGCAATTATGTAAACTCCTTAGAGGAACCAAAGCACTAACAGAAGTAATACCACTAACAGAAGAAGCAGAGCTAGAACTGGCAGAAAACAGAGAGATTCTAAAAGAACCAGTACATGGAGTGTATTATGACCCATCAAAAGACTTAATAGCAGAAATACAGAAGCAGGGGCAAGGCCAATGGACATATCAAATTTATCAAGAGCCATTTAAAAATCTGAAAACAGGAAAATATGCAAGAATGAGGGGTGCCCACACTAATGATGTAAAACAATTAACAGAGGCAGTGCAAAAAATAACCACAGAAAGCATAGTAATATGGGGAAAGACTCCTAAATTTAAACTGCCCATACAAAAGGAAACATGGGAAACATGGTGGACAGAGTATTGGCAAGCCACCTGGATTCCTGAGTGGGAGTTTGTTAATACCCCTCCCTTAGTGAAATTATGGTACCAGTTAGAGAAAGAACCCATAGTAGGAGCAGAAACCTTCTATGTAGATGGGGCAGCTAACAGGGAGACTAAATTAGGAAAAGCAGGATATGTTACTAATAGAGGAAGACAAAAAGTTGTCACCCTAACTGACACAACAAATCAGAAGACTGAGTTACAAGCAATTTATCTAGCTTTGCAGGATTCGGGATTAGAAGTAAACATAGTAACAGACTCACAATATGCATTAGGAATCATTCAAGCACAACCAGATCAAAGTGAATCAGAGTTAGTCAATCAAATAATAGAGCAGTTAATAAAAAAGGAAAAGGTCTATCTGGCATGGGTACCAGCACACAAAGGAATTGGAGGAAATGAACAAGTAGATAAATTAGTCAGTGCTGGAATCAGGAAAGTACTATTTTTAGATGGAATAGATAAGGCCCAAGATGAACATGAGAAATATCACAGTAATTGGAGAGCAATGGCTAGTGATTTTAACCTGCCACCTGTAGTAGCAAAAGAAATAGTAGCCAGCTGTGATAAATGTCAGCTAAAAGGAGAAGCCATGCATGGACAAGTAGACTGTAGTCCAGGAATATGGCAACTAGATTGTACACATTTAGAAGGAAAAGTTATCCTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTTATTCCAGCAGAAACAGGGCAGGAAACAGCATATTTTCTTTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAATACATACTGACAATGGCAGCAATTTCACCGGTGCTACGGTTAGGGCCGCCTGTTGGTGGGCGGGAATCAAGCAGGAATTTGGAATTCCCTACAATCCCCAAAGTCAAGGAGTAGTAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAAATCCACTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAGGTGAAGGGGCAGTAGTAATACAAGATAATAGTGACATAAAAGTAGTGCCAAGAAGAAAAGCAAAGATCATTAGGGATTATGGAAAACAGATGGCAGGTGATGATTGTGTGGCAAGTAGACAGGATGAGGATTAGAACATGGAAAAGTTTAGTAAAACACCATATGTATGTTTCAGGGAAAGCTAGGGGATGGTTTTATAGACATCACTATGAAAGCCCTCATCCAAGAATAAGTTCAGAAGTACACATCCCACTAGGGGATGCTAGATTGGTAATAACAACATATTGGGGTCTGCATACAGGAGAAAGAGACTGGCATTTGGGTCAGGGAGTCTCCATAGAATGGAGGAAAAAGAGATATAGCACACAAGTAGACCCTGAACTAGCAGACCAACTAATTCATCTGTATTACTTTGACTGTTTTTCAGACTCTGCTATAAGAAAGGCCTTATTAGGACACATAGTTAGCCCTAGGTGTGAATATCAAGCAGGACATAACAAGGTAGGATCTCTACAATACTTGGCACTAGCAGCATTAATAACACCAAAAAAGATAAAGCCACCTTTGCCTAGTGTTACGAAACTGACAGAGGATAGATGGAACAAGCCCCAGAAGACCAAGGGCCACAGAGGGAGCCACACAATGAATGGACACTAGAGCTTTTAGAGGAGCTTAAGAATGAAGCTGTTAGACATTTTCCTAGGATTTGGCTCCATGGCTTAGGGCAACATATCTATGAAACTTATGGGGATACTTGGGCAGGAGTGGAAGCCATAATAAGAATTCTGCAACAACTGCTGTTTATCCATTTTCAGAATTGGGTGTCGACATAGCAGAATAGGCGTTACTCGACAGAGGAGAGCAAGAAATGGAGCCAGTAGATCCTAGACTAGAGCCCTGGAAGCATCCAGGAAGTCAGCCTAAAACTGCTTGTACCAATTGCTATTGTAAAAAGTGTTGCTTTCATTGCCAAGTTTGTTTCATAACAAAAGCCTTAGGCATCTCCTATGGCAGGAAGAAGCGGAGACAGCGACGAAGAGCTCATCAGAACAGTCAGACTCATCAAGCTTCTCTATCAAAGCAGTAAGTAGTACATGTAATGCAACCTATACCAATAGTAGCAATAGTAGCATTAGTAGTAGCAATAATAATAGCAATAGTTGTGTGGTCCATAGTAATCATAGAATATAGGAAAATATTAAGACAAAGAAAAATAGACAGGTTAATTGATAGACTAATAGAAAGAGCAGAAGACAGTGGCAATGAGAGTGAAGGAGAAATATCAGCACTTGTGGAGATGGGGGTGGAGATGGGGCACCATGCTCCTTGGGATGTTGATGATCTGTAGTGCTACAGAAAAATTGTGGGTCACAGTCTATTATGGGGTACCTGTGTGGAAGGAAGCAACCACCACTCTATTTTGTGCATCAGATGCTAAAGCATATGATACAGAGGTACATAATGTTTGGGCCACACATGCCTGTGTACCCACAGACCCCAACCCACAAGAAGTAGTATTGGTAAATGTGACAGAAAATTTTAACATGTGGAAAAATGACATGGTAGAACAGATGCATGAGGATATAATCAGTTTATGGGATCAAAGCCTAAAGCCATGTGTAAAATTAACCCCACTCTGTGTTAGTTTAAAGTGCACTGATTTGAAGAATGATACTAATACCAATAGTAGTAGCGGGAGAATGATAATGGAGAAAGGAGAGATAAAAAACTGCTCTTTCAATATCAGCACAAGCATAAGAGGTAAGGTGCAGAAAGAATATGCATTTTTTTATAAACTTGATATAATACCAATAGATAATGATACTACCAGCTATAAGTTGACAAGTTGTAACACCTCAGTCATTACACAGGCCTGTCCAAAGGTATCCTTTGAGCCAATTCCCATACATTATTGTGCCCCGGCTGGTTTTGCGATTCTAAAATGTAATAATAAGACGTTCAATGGAACAGGACCATGTACAAATGTCAGCACAGTACAATGTACACATGGAATTAGGCCAGTAGTATCAACTCAACTGCTGTTAAATGGCAGTCTAGCAGAAGAAGAGGTAGTAATTAGATCTGTCAATTTCACGGACAATGCTAAAACCATAATAGTACAGCTGAACACATCTGTAGAAATTAATTGTACAAGACCCAACAACAATACAAGAAAAAGAATCCGTATCCAGAGAGGACCAGGGAGAGCATTTGTTACAATAGGAAAAATAGGAAATATGAGACAAGCACATTGTAACATTAGTAGAGCAAAATGGAATAACACTTTAAAACAGATAGCTAGCAAATTAAGAGAACAATTTGGAAATAATAAAACAATAATCTTTAAGCAATCCTCAGGAGGGGACCCAGAAATTGTAACGCACAGTTTTAATTGTGGAGGGGAATTTTTCTACTGTAATTCAACACAACTGTTTAATAGTACTTGGTTTAATAGTACTTGGAGTACTGAAGGGTCAAATAACACTGAAGGAAGTGACACAATCACCCTCCCATGCAGAATAAAACAAATTATAAACATGTGGCAGAAAGTAGGAAAAGCAATGTATGCCCCTCCCATCAGTGGACAAATTAGATGTTCATCAAATATTACAGGGCTGCTATTAACAAGAGATGGTGGTAATAGCAACAATGAGTCCGAGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCCTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCCTTGGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCTCAAATATTGGTGGAATCTCCTACAGTATTGGAGTCAGGAACTAAAGAATAGTGCTGTTAGCTTGCTCAATGCCACAGCCATAGCAGTAGCTGAGGGGACAGATAGGGTTATAGAAGTAGTACAAGGAGCTTGTAGAGCTATTCGCCACATACCTAGAAGAATAAGACAGGGCTTGGAAAGGATTTTGCTATAAGATGGGTGGCAAGTGGTCAAAAAGTAGTGTGATTGGATGGCCTACTGTAAGGGAAAGAATGAGACGAGCTGAGCCAGCAGCAGATAGGGTGGGAGCAGCATCTCGAGACCTGGAAAAACATG GAGCAAT CACAAGTAGCAATACAGCAGCTACCAATGCTGCTTGTGCCTGGCTAGAAGCACAAGAGGAGGAGGAGGTGGGTTTTCCAGTCACACCTCAGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAGAACTACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGATAGAAGAGGCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGAGAGAA GTGTTAGAGTG GAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGC TGACATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTG CCTGTACTGGGTCTCTCTGGTTAG ACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTC

The most suitable on-the-spot test of test kit that can at room temperature use and The check of bedside in-vitro diagnosis (Point ofcare testing).Usually, the hybridization of gene probe and viral RNA is not at room temperature carried out.For example, following HIV RNA part demonstrates some with double underline and is accredited as the part that can at room temperature hybridize.

The peculiar zone of HIV that the check of bedside in-vitro diagnosis is identified:

GGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTG CTTAAGCC TCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGT

CCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACCTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTG CTGAAGCGCGCACGGCAAGAGGCGAGGGG CGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCG TCAGTATTAAGCGGGGGAGAATTAGATCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAA GCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGA AACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGACACAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCAAATG GTACATCAG G

AATGCAT

AAGGCTTTCAGCCCAGAAGTGA TACCCATGTTTTCAGCATTATCAGAAGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGG GGGACATCAAGCAGCCATGCAAATGTTAAAAGAGACCATC AATGAGGAAGCTGCAGAATGGGATAGAGTG CATCCAGTGCATGCAGGGCCTATTGCACCAGGCCAGATGAGAGAACCAAGGGGAAGTGA

CCCTTCAGGAACAAATAGGATGGATGACAAATAATCCACCTATCCCAGTAGGAGAAATTTATAAAAGATGGATAATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTACCAGCATTCTGGACATAAGACAAGGACCAAAGGA ACCCTTTAGAGACTATGTAGACCGGT

GAGCAAGCTTCACAGGAGGTAAAAAATTGGATGACAGAAACCTTGTTGGTCCAAAATGCGAACCCAGATTGTAAGACTATTTTAAAAGCATTGGGACCAGCGGCTACACTAGAAGAAATGATGA CAGCATGTCAGGGAGTAGGAGGA CCCGGCCATAAGGCAAGAGTTTTGGCTGAAGCAATGAGCCAAGTAACAAATTCAGCTACCATAATGATGCAGAGAGGCAATTTTAGGAACCAAAGAAAGATTGTTAAGTGTTTCAATTGTGGCAAAGAAGGGCACACAGCCAGAAATTGCAGGGCCCCTAGGAAAAAGGGCTGTTGGAAATGTGGAAAGGAAGGACACCAAATGAAAGATTGTACTGAGAGACAGGCTAATTTTTTAGGGAAGATCTGGCCTTCCTACAAGGGAAGGCCAGGGAATTTTCTTCAGAGCAGACCAGAGCCAACAGCCCCACCAGAAGAGAGCTTCAGGTCTGGGGTAGAGACAACAACTCCCCCTCAGAAGCAGGAGCCGATAGACAAGGAACTGTATCCTTTAACTTCCCTCAGGTCACTCTTTGGCAACGACCCCTCGTCACAATAAAGATAGGGGGGCAACTAAAGGAAGCTCTATTAGATACAGGAGCAGATGATACAGTATTAGAAGAAATGAGTTTGCCAGGAAGATGGAAACCAAAAATGATAGGGGGAATTGGAGGTTTTATCAAAGTAAGACAGTATGATCAGATACTCATAGAAATCTGTGGACATAAAGCTATAGGTACAGTATTAGTAGGACCTACACCTGTCAACATAATTGGAAGAAATCTGTTGACTCAGATTGGTTGCACTTTAAATTTTCCCATTAGCCCTATTGAGACTGTACCAGTAAAATTAAAGCCAGGAATGGATGGCCCAAAAGTTAAACAATGGCCATTGACAGAAGAAAAAATAAAAGCATTAGTAGAAATTTGTACAGAGATGGAAAAGGAAGGGAAAATTTCAAAAATTGGGCCTGAAAATCCATACAATACTCCAGTATTTGCCATAAAGAAAAAAGACAGTACTAAATGGAGAAAATTAGTAGATTTCAGAGAACTTAATAAGAGAACTCAAGACTTCTGGGAAGTTCAATTAGGAATACCACATCCCGCAGGGTTAAAAAAGAAAAAATCAGTAACAGTACTGGATGTGGGTGATGCATATTTTTCAGTTCCCTTAGATGAAGACTTCAGGAAGTATACTGCATTTACCATACCTAGTATAAACAATGAGACACCAGGGATTAGATATCAGTACAATGTGCTTCCACAGGGATGGAAAGGATCACCAGCAATATTCCAAAGTAGCATGACAAAAATCTTAGAGCCTTTTAGAAAACAAAATCCAGACATAGTTATCTATCAATACATGGATGATTTGTATGTAGGATCTGACTTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAGCTGAGACAACATCTGTTGAGGTGGGGACTTACCACACCAGACAAAAAACATCAGAAAGAACCTCCATTCCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACAGTACAGCCTATAGTGCTGCCAGAAAAAGACAGCTGGACTGTCAATGACATACAGAAG TTAGTGGGGAAATTGAATTGGGCAAGTCAGATTTACCCAGGGATTAAAGTAAGGCAATTATGTAAACTCCTTAGAGGAACCAAAGCACTAACAGAAGTAATACCACTAACAGAAGAAGCAGAGCTAGAACTGGCAGAAAACAGAGAGATTCTAAAAGAACCAGTACATGGAGTGTATTATGACCCATCAAAAGACTTAATAGCAGAAATACAGAAGCAGGGGCAAGGCCAATGGACATATCAAATTTATCAAGAGCCATTTAAAAATCTGAAAACAGGAAAATATGCAAGAATGAGGGGTGCCCACACTAATGATGTAAAACAATTAACAGAGGCAGTGCAAAAAATAACCACAGAAAGCATAGTAATATGGGGAAAGACTCCTAAATTTAAACTGCCCATACAAAAGGAAACATGGGAAACATGGTGGACAGAGTATTGGCAAGCCACCTGGATTCCTGAGTGGGAGTTTGTTAATACCCCTCCCTTAGTGAAATTATGGTACCAGTTAGAGAAAGAACCCATAGTAGGAGCAGAAACCTTCTATGTAGATGGGGCAGCTAACAGGGAGACTAAATTAGGAAAAGCAGGATATGTTACTAATAGAGGAAGACAAAAAGTTGTCACCCTAACTGACACAACAAATCAGAAGACTGAGTTACAAGCAATTTATCTAGCTTTGCAGGATTCGGGATTAGAAGTAAACATAGTAACAGACTCACAATATGCATTAGGAATCATTCAAGCACAACCAGATCAAAGTGAATCAGAGTTAGTCAATCAAATAATAGAGCAGTTAATAAAAAAGGAAAAGGTCTATCTGGCATGGGTACCAGCACACAAAGGAATTGGAGGAAATGAACAAGTAGATAAATTAGTCAGTGCTGGAATCAGGAAAGTACTATTTTTAGATGGAATAGATAAGGCCCAAGATGAACATGAGAAATATCACAGTAATTGGAGAGCAATGGCTAGTGATTTTAACCTGCCACCTGTAGTAGCAAAAGAAATAGTAGCCAGCTGTGATAAATGTCAGCTAAAAGGAGAAGCCATGCATGGACAAGTAGACTGTAGTCCAGGAATATGGCAACTAGATTGTACACATTTAGAAGGAAAAGTTATCCTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTTATTCCAGCAGAAACAGGGCAGGAAACAGCATATTTTCTTTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAATACATACTGACAATGGCAGCAATTTCACCGGTGCTACGGTTAGGGCCGCCTGTTGGTGGGCGGGAATCAAGCAGGAATTTGGAATTCCCTACAATCCCCAAAGTCAAGGAGTAGTAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAAATCCACTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAGGTGAAGGGGCAGTAGTAATACAAGATAATAGTGACATAAAAGTAGTGCCAAGAAGAAAAGCAAAGATCATTAGGGATTATGGAAAACAGATGGCAGGTGATGATTGTGTGGCAAGTAGACAGGATGAGGATTAGAACATGGAAAAGTTTAGTAAAACACCATATGTATGTTTCAGGGAAAGCTAGGGGATGGTTTTATAGACATCACTATGAAAGCCCTCATCCAAGAATAAGTTCAGAAGTACACATCCCACTAGGGGATGCTAGATTGGTAATAACAACATATTGGGGTCTGCATACAGGAGAAAGAGACTGGCATTTGGGTCAGGGAGTCTCCATAGAATGGAGGAAAAAGAGATATAGCACACAAGTAGACCCTGAACTAGCAGACCAACTAATTCATCTGTATTACTTTGACTGTTTTTCAGACTCTGCTATAAGAAAGGCCTTATTAGGACACATAGTTAGCCCTAGGTGTGAATATCAAGCAGGACATAACAAGGTAGGATCTCTACAATACTTGGCACTAGCAGCATTAATAACACCAAAAAAGATAAAGCCACCTTTGCCTAGTGTTACGAAACTGACAGAGGATAGATGGAACAAGCCCCAGAAGACCAAGGGCCACAGAGGGAGCCACACAATGAATGGACACTAGAGCTTTTAGAGGAGCTTAAGAATGAAGCTGTTAGACATTTTCCTAGGATTTGGCTCCATGGCTTAGGGCAACATATCTATGAAACTTATGGGGATACTTGGGCAGGAGTGGAAGCCATAATAAGAATTCTGCAACAACTGCTGTTTATCCATTTTCAGAATTGGGTGTCGACATAGCAGAATAGGCGTTACTCGACAGAGGAGAGCAAGAAATGGAGCCAGTAGATCCTAGACTAGAGCCCTGGAAGCATCCAGGAAGTCAGCCTAAAACTGCTTGTACCAATTGCTATTGTAAAAAGTGTTGCTTTCATTGCCAAGTTTGTTTCATAACAAAAGCCTTAGGCATCTCCTATGGCAGGAAGAAGCGGAGACAGCGACGAAGAGCTCATCAGAACAGTCAGACTCATCAAGCTTCTCTATCAAAGCAGTAAGTAGTACATGTAATGCAACCTATACCAATAGTAGCAATAGTAGCATTAGTAGTAGCAATAATAATAGCAATAGTTGTGTGGTCCATAGTAATCATAGAATATAGGAAAATATTAAGACAAAGAAAAATAGACAGGTTAATTGATAGACTAATAGAAAGAGCAGAAGACAGTGGCAATGAGAGTGAAGGAGAAATATCAGCACTTGTGGAGATGGGGGTGGAGATGGGGCACCATGCTCCTTGGGATGTTGATGATCTGTAGTGCTACAGAAAAATTGTGGGTCACAGTCTATTATGGGGTACCTGTGTGGAAGGAAGCAACCACCACTCTATTTTGTGCATCAGATGCTAAAGCATATGATACAGAGGTACATAATGTTTGGGCCACACATGCCTGTGTACCCACAGACCCCAACCCACAAGAAGTAGTATTGGTAAATGTGACAGAAAATTTTAACATGTGGAAAAATGACATGGTAGAACAGATGCATGAGGATATAATCAGTTTATGGGATCAAAGCCTAAAGCCATGTGTAAAATTAACCCCACTCTGTGTTAGTTTAAAGTGCACTGATTTGAAGAATGATACTAATACCAATAGTAGTAGCGGGAGAATGATAATGGAGAAAGGAGAGATAAAAAACTGCTCTTTCAATATCAGCACAAGCATAAGAGGTAAGGTGCAGAAAGAATATGCATTTTTTTATAAACTTGATATAATACCAATAGATAATGATACTACCAGCTATAAGTTGACAAGTTGTAACACCTCAGTCATTACACAGGCCTGTCCAAAGGTATCCTTTGAGCCAATTCCCATACATTATTGTGCCCCGGCTGGTTTTGCGATTCTAAAATGTAATAATAAGACGTTCAATGGAACAGGACCATGTACAAATGTCAGCACAGTACAATGTACACATGGAATTAGGCCAGTAGTATCAACTCAACTGCTGTTAAATGGCAGTCTAGCAGAAGAAGAGGTAGTAATTAGATCTGTCAATTTCACGGACAATGCTAAAACCATAATAGTACAGCTGAACACATCTGTAGAAATTAATTGTACAAGACCCAACAACAATACAAGAAAAAGAATCCGTATCCAGAGAGGACCAGGGAGAGCATTTGTTACAATAGGAAAAATAGGAAATATGAGACAAGCACATTGTAACATTAGTAGAGCAAAATGGAATAACACTTTAAAACAGATAGCTAGCAAATTAAGAGAACAATTTGGAAATAATAAAACAATAATCTTTAAGCAATCCTCAGGAGGGGACCCAGAAATTGTAACGCACAGTTTTAATTGTGGAGGGGAATTTTTCTACTGTAATTCAACACAACTGTTTAATAGTACTTGGTTTAATAGTACTTGGAGTACTGAAGGGTCAAATAACACTGAAGGAAGTGACACAATCACCCTCCCATGCAGAATAAAACAAATTATAAACATGTGGCAGAAAGTAGGAAAAGCAATGTATGCCCCTCCCATCAGTGGACAAATTAGATGTTCATCAAATATTACAGGGCTGCTATTAACAAGAGATGGTGGTAATAGCAACAATGAGTCCGAGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCCTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCCTTGGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCTCAAATATTGGTGGAATCTCCTACAGTATTGGAGTCAGGAACTAAAGAATAGTGCTGTTAGCTTGCTCAATGCCACAGCCATAGCAGTAGCTGAGGGGACAGATAGGGTTATAGAAGTAGTACAAGGAGCTTGTAGAGCTATTCGCCACATACCTAGAAGAATAAGACAGGGCTTGGAAAGGATTTTGCTATAAGATGGGTGGCAAGTGGTCAAAAAGTAGTGTGATTGGATGGCCTACTGTAAGGGAAAGAATGAGACGAGCTGAGCCAGCAGCAGATAGGGTGGGAGCAGCATCTCGAGACCTGGAAAAACATG GAGCAAT CACAAGTAGCAATACAGCAGCTACCAATGCTGCTTGTGCCTGGCTAGAAGCACAAGAGGAGGAGGAGGTGGGTTTTCCAGTCACACCTCAGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAGAACTACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGATAGAAGAGGCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGAGAGAA GTGTTAGAGTG GAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGC TGACATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTG CCTGTACTGGGTCTCTCTGGTTAG ACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTC

Following embodiment provides the specific oligonucleotides sequence that is applicable to bedside in-vitro diagnosis test kit.

Sequence 1-44 (before the GAG zone)

GGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGC

Sequence 63-179 (before the GAG zone)

CTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGC

Sequence 252-364 (before the GAG zone and the sequence in the GAG zone)

CTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGG

Sequence 448-539 (sequence in the GAG zone)

GCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTT

Sequence 762-929 (sequence in the GAG zone)

GTACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGTAGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGTTTTCAGCATTATCAGAAGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATCAAGCAGCCATG

Sequence 951-1113 (sequence in the GAG zone)

AATGAGGAAGCTGCAGAATGGGATAGAGTGCATCCAGTGCATGCAGGGCCTATTGCACCAGGCCAGATGAGAGAACCAAGGGGAAGTGACATAGCAGGAACTACTAGTACCCTTCAGGAACAAATAGGATGGATGACAAATAATCCACCTATCCCAGTAGGAG

Sequence 1197-1255 (sequence in the GAG-POL zone)

GGACCAAAGGAACCCTTTAGAGACTATGTAGACCGGTTCTATAAAACTCTAAGAGCCGA

Sequence 1378-1422 (sequence in the GAG-POL zone)

CAGCATGTCAGGGAGTAGGAGGACCCGGCCATAAGGCAAGAGTTT

Sequence 2873-2930 (sequence in the GAG-POL zone)

TTAGTGGGGAAATTGAATTGGGCAAGTCAGATTTACCCAGGGATTAAAGTAAGGCAAT

Sequence 8464-8515 (sequence in the NEF zone)

GAGCAATCACAAGTAGCAATACAGCAGCTACCAATGCTGCTTGTGCCTGGCT

Sequence 8880-9001 (sequence after in the NEF zone and the NEF zone)

GTGTTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTGACATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTT

Sequence 9077-9129 (sequence after the NEF zone)

CCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGC

With single underscore and double underline mark, described unique portion is used to use the hybridization and the detection of the HIV viral RNA of gene probe in other examples of some of the unique portion of HIV viral RNA sequence below:

GGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTG CTTAAGCC CAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGT

AATGCAT

CCCTTCAGGAACAAATAGGATGGATGACAAATAATCCACCTATCCCAGTAGGAGAAATTTATAAAAGATGGATAATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTACCAGCATTCTGGACATAAGACAA GGACCAAAGGA

ACCGGT

GAGCAAGCTTCACAGGAGGTAAAAAATTGGATGACAGAAACCTTGTTGGTCCAAAATGCGAACCCAGATTGTAAGACTATTTTAAAAGCATTGGGACCAGCGGCTACACTAGAAGAAATGATGA CAGCATGTCAGGGAGTAGGAGGA CCCGGCCATAAGGCAAGAGTTTTGGCTGAAGCAATGAGCCAAGTAACAAATTCAGCTACCATAATGATGCAGAGAGGCAATTTTAGGAACCAAAGAAAGATTGTTAAGTGTTTCAATTGTGGCAAAGAAGGGCACACAGCCAGAAATTGCAGGGCCCCTAGGAAAAAGGGCTGTTGGAAATGTGGAAAGGAAGGACACCAAATGAAAGATTGTACTGAGAGACAGGCTAATTTTTTAGGGAAGATCTGGCCTTCCTACAAGGGAAGGCCAGGGAATTTTC TTCAGAGCAGACCAGAGCCAACAGCCCCACCAGAAGAGAGCTTCAGGTCTGGGGTAGAGACAACAACTCCCCCTCAGAAGCAGGAGCCGATAGACAAGGAACTGTATCCTTTAACTTCCCTCAGGTCACTCTTTGGCAACGACCCCTCGTCACAATAAAGATAGGGGGGCAACTAAAGGAAGCTCTATTAGATACAGGAGCAGATGATACAGTATTAGAAGAAATGAGTTTGCCAGGAAGATGGAAACCAAAAATGATAGGGGGAATTGGAGGTTTTATCAAAGTAAGACAGTATGATCAGATACTCATAGAAATCTGTGGACATAAAGCTATAGGTACAGTATTAGTAGGACCTACACCTGTCAACATAATTGGAAGAAATCTGTTGACTCAGATTGGTTGCACTTTAAATTTTCCCATTAGCCCTATTGAGACTGTACCAGTAAAATTAAAGCCAGGAATGGATGGCCCAAAAGTTAAACAATGGCCATTGACAGAAGAAAAAATAAAAGCATTAGTAGAAATTTGTACAGAGATGGAAAAGGAAGGGAAAATTTGAAAAATTGGGCCTGAAAATCCATACAATACTCCAGTATTTGCCATAAAGAAAAAAGACAGTACTAAATGGAGAAAATTAGTAGATTTCAGAGAACTTAATAAGAGAACTCAAGACTTCTGGGAAGTTCAATTAGGAATACCACATCCCGCAGGGTTAAAAAAGAAAAAATCAGTAACAGTACTGGATGTGGGTGATGCATATTTTTCAGTTCCCTTAGATGAAGACTTCAGGAAGTATACTGCATTTACCATACCTAGTATAAACAATGAGACACCAGGGATTAGATATCAGTACAATGTGCTTCCACAGGGATGGAAAGGATCACCAGCAATATTCCAAAGTAGCATGACAAAAATCTTAGAGCCTTTTAGAAAACAAAATCCAGACATAGTTATCTATCAATACATGGATGATTTGTATGTAGGATCTGACTTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAGCTGAGACAACATCTGTTGAGGTGGGGACTTACCACACCAGACAAAAAACATCAGAAAGAACCTCCATTCCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACAGTACAGCCTATAGTGCTGCCAGAAAAAGACAGCTGGACTGTCAATGACATACAGAAG TTAGTGGGGAAATTGAATTGGGCAAGTCAGATTTACCCAGGGATTAAAGTAAGGCAATTATGTAAACTCCTTAGAGGAACCAAAGCACTAACAGAAGTAATACCACTAACAGAAGAAGCAGAGCTAGAACTGGCAGAAAACAGAGAGATTCTAAAAGAACCAGTACATGGAGTGTATTATGACCCATCAAAAGACTTAATAGCAGAAATACAGAAGCAGGGGCAAGGCCAATGGACATATCAAATTTATCAAGAGCCATTTAAAAATCTGAAAACAGGAAAATATGCAAGAATGAGGGGTGCCCACACTAATGATGTAAAACAATTAACAGAGGCAGTGCAAAAAATAACCACAGAAAGCATAGTAATATGGGGAAAGACTCCTAAATTTAAACTGCCCATACAAAAGGAAACATGGGAAACATGGTGGACAGAGTATTGGCAAGCCACCTGGATTCCTGAGTGGGAGTTTGTTAATACCCCTCCCTTAGTGAAATTATGGTACCAGTTAGAGAAAGAACCCATAGTAGGAGCAGAAACCTTCTATGTAGATGGGGCAGCTAACAGGGAGACTAAATTAGGAAAAGCAGGATATGTTACTAATAGAGGAAGACAAAAAGTTGTCACCCTAACTGACACAACAAATCAGAAGACTGAGTTACAAGCAATTTATCTAGCTTTGCAGGATTCGGGATTAGAAGTAAACATAGTAACAGACTCACAATATGCATTAGGAATCATTCAAGCACAACCAGATCAAAGTGAATCAGAGTTAGTCAATCAAATAATAGAGCAGTTAATAAAAAAGGAAAAGGTCTATCTGGCATGGGTACCAGCACACAAAGGAATTGGAGGAAATGAACAAGTAGATAAATTAGTCAGTGCTGGAATCAGGAAAGTACTATTTTTAGATGGAATAGATAAGGCCCAAGATGAACATGAGAAATATCACAGTAATTGGAGAGCAATGGCTAGTGATTTTAACCTGCCACCTGTAGTAGCAAAAGAAATAGTAGCCAGCTGTGATAAATGTCAGCTAAAAGGAGAAGCCATGCATGGACAAGTAGACTGTAGTCCAGGAATATGGCAACTAGATTGTACACATTTAGAAGGAAAAGTTATCCTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTTATTCCAGCAGAAACAGGGCAGGAAACAGCATATTTTCTTTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAATACATACTGACAATGGCAGCAATTTCACCGGTGCTACGGTTAGGGCCGCCTGTTGGTGGGCGGGAATCAAGCAGGAATTTGGAATTCCCTACAATCCCCAAAGTCAAGGAGTAGTAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAAATCCACTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAGGTGAAGGGGCAGTAGTAATACAAGATAATAGTGACATAAAAGTAGTGCCAAGAAGAAAAGCAAAGATCATTAGGGATTATGGAAAACAGATGGCAGGTGATGATTGTGTGGCAAGTAGACAGGATGAGGATTAGAACATGGAAAAGTTTAGTAAAACACCATATGTATGTTTCAGGGAAAGCTAGGGGATGGTTTTATAGACATCACTATGAAAGCCCTCATCCAAGAATAAGTTCAGAAGTACACATCCCACTAGGGGATGCTAGATTGGTAATAACAACATATTGGGGTCTGCATACAGGAGAAAGAGACTGGCATTTGGGTCAGGGAGTCTCCATAGAATGGAGGAAAAAGAGATATAGCACACAAGTAGACCCTGAACTAGCAGACCAACTAATTCATCTGTATTACTTTGACTGTTTTTCAGACTCTGCTATAAGAAAGGCCTTATTAGGACACATAGTTAGCCCTAGGTGTGAATATCAAGCAGGACATAACAAGGTAGGATCTCTACAATACTTGGCACTAGCAGCATTAATAACACCAAAAAAGATAAAGCCACCTTTGCCTAGTGTTACGAAACTGACAGAGGATAGATGGAACAAGCCCCAGAAGACCAAGGGCCACAGAGGGAGCCACACAATGAATGGACACTAGAGCTTTTAGAGGAGCTTAAGAATGAAGCTGTTAGACATTTTCCTAGGATTTGGCTCCATGGCTTAGGGCAACATATCTATGAAACTTATGGGGATACTTGGGCAGGAGTGGAAGCCATAATAAGAATTCTGCAACAACTGCTGTTTATCCATTTTCAGAATTGGGTGTCGACATAGCAGAATAGGCGTTACTCGACAGAGGAGAGCAAGAAATGGAGCCAGTAGATCCTAGACTAGAGCCCTGGAAGCATCCAGGAAGTCAGCCTAAAACTGCTTGTACCAATTGCTATTGTAAAAAGTGTTGCTTTCATTGCCAAGTTTGTTTCATAACAAAAGCCTTAGGCATCTCCTATGGCAGGAAGAAGCGGAGACAGCGACGAAGAGCTCATCAGAACAGTCAGACTCATCAAGCTTCTCTATCAAAGCAGTAAGTAGTACATGTAATGCAACCTATACCAATAGTAGCAATAGTAGCATTAGTAGTAGCAATAATAATAGCAATAGTTGTGTGGTCCATAGTAATCATAGAATATAGGAAAATATTAAGACAAAGAAAAATAGACAGGTTAATTGATAGACTAATAGAAAGAGCAGAAGACAGTGGCAATGAGAGTGAAGGAGAAATATCAGCACTTGTGGAGATGGGGGTGGAGATGGGGCACCATGCTCCTTGGGATGTTGATGATCTGTAGTGCTACAGAAAAATTGTGGGTCACAGTCTATTATGGGGTACCTGTGTGGAAGGAAGCAACCACCACTCTATTTTGTGCATCAGATGCTAAAGCATATGATACAGAGGTACATAATGTTTGGGCCACACATGCCTGTGTACCCACAGACCCCAACCCACAAGAAGTAGTATTGGTAAATGTGACAGAAAATTTTAACATGTGGAAAAATGACATGGTAGAACAGATGCATGAGGATATAATCAGTTTATGGGATCAAAGCCTAAAGCCATGTGTAAAATTAACCCCACTCTGTGTTAGTTTAAAGTGCACTGATTTGAAGAATGATACTAATACCAATAGTAGTAGCGGGAGAATGATAATGGAGAAAGGAGAGATAAAAAACTGCTCTTTCAATATCAGCACAAGCATAAGAGGTAAGGTGCAGAAAGAATATGCATTTTTTTATAAACTTGATATAATACCAATAGATAATGATACTACCAGCTATAAGTTGACAAGTTGTAACACCTCAGTCATTACACAGGCCTGTCCAAAGGTATCCTTTGAGCCAATTCCCATACATTATTGTGCCCCGGCTGGTTTTGCGATTCTAAAATGTAATAATAAGACGTTCAATGGAACAGGACCATGTACAAATGTCAGCACAGTACAATGTACACATGGAATTAGGCCAGTAGTATCAACTCAACTGCTGTTAAATGGCAGTCTAGCAGAAGAAGAGGTAGTAATTAGATCTGTCAATTTCACGGACAATGCTAAAACCATAATAGTACAGCTGAACACATCTGTAGAAATTAATTGTACAAGACCCAACAACAATACAAGAAAAAGAATCCGTATCCAGAGAGGACCAGGGAGAGCATTTGTTACAATAGGAAAAATAGGAAATATGAGACAAGCACATTGTAACATTAGTAGAGCAAAATGGAATAACACTTTAAAACAGATAGCTAGCAAATTAAGAGAACAATTTGGAAATAATAAAACAATAATCTTTAAGCAATCCTCAGGAGGGGACCCAGAAATTGTAACGCACAGTTTTAATTGTGGAGGGGAATTTTTCTACTGTAATTCAACACAACTGTTTAATAGTACTTGGTTTAATAGTACTTGGAGTACTGAAGGGTCAAATAACACTGAAGGAAGTGACACAATCACCCTCCCATGCAGAATAAAACAAATTATAAACATGTGGCAGAAAGTAGGAAAAGCAATGTATGCCCCTCCCATCAGTGGACAAATTAGATGTTCATCAAATATTACAGGGCTGCTATTAACAAGAGATGGTGGTAATAGCAACAATGAGTCCGAGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCCTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCCTTGGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCTCAAATATTGGTGGAATCTCCTACAGTATTGGAGTCAGGAACTAAAGAATAGTGCTGTTAGCTTGCTCAATGCCACAGCCATAGCAGTAGCTGAGGGGACAGATAGGGTTATAGAAGTAGTACAAGGAGCTTGTAGAGCTATTCGCCACATACCTAGAAGAATAAGACAGGGCTTGGAAAGGATTTTGCTATAAGATGGGTGGCAAGTGGTCAAAAAGTAGTGTGATTGGATGGCCTACTGTAAGGGAAAGAATGAGACGAGCTGAGCCAGCAGCAGATAGGGTGGGAGCAGCATCTCGAGACCTGGAAAAACATG GAGCAAT CACAAGTAGCAATACAGCAGCTACCAATGCTGCTTGTGCCTGGCTAGAAGCACAAGAGGAGGAGGAGGTGGGTTTTCCAGTCACACCTCAGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAGAACTACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGATAGAAGAGGCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGAGAGAA GTGTTAGAGTG GAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGC TGACATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTG CCTGTACTGGGTCTCTCTGGTTAG ACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTC

In order quantitatively to manifest gold nano grain, on slide glass, prepare sample, be used for absorption spectroanalysis.Add the aqeous suspension of nano particle to or contain in the solution of nanoparticle precursor by the mixture that will contain the desired amount single stranded oligonucleotide, in conjunction with selected single stranded oligonucleotide and gold nano grain.The gold nano grain that oligonucleotide is functionalized mixes with 0.5%BSA, and is placed on the sheet glass, carries out absorption spectroanalysis.Alternatively, the functionalized gold nano grain of oligonucleotide can place on the filter paper, with detected complementary oligonucleotide sequence hybridization.As shown in figure 18, utilize pipettor complementary and incomplementarity single stranded oligonucleotide to be moved on to the following zone of sheet glass, placed the functionalized gold nano grain of oligonucleotide before this zone, then utilize the ultraviolet spectrophotometer inspection with the absorption spectrum of measuring the gold nano grain by slide glass and hybridization before, kept for 60 seconds in room temperature.In Figure 18, (ssDNA-gold+cDNA) is different from incomplementarity DNA (ssDNA-gold+ssDNA) or the functionalized gold nano grain (ssDNA-Au is only arranged) of independent oligonucleotide easily with the hybridization of complementary DNA.This method of testing hybridization on slide glass is the mode of the particular complementary oligonucleotide sequence of functionalized nano particle (for example gold nano grain) hybridization of a kind of quick and effective screening and oligonucleotide.

In one embodiment, use the test kit for preparing viral RNA from the 33-mer oligonucleotide of HIV-1LTR sequence.This LTR sequence is conservative relatively in several HIV-1 strains that comprise clade C HIV 1084i, and because vaccine constructs is not an attenuated live vaccine, so this LTR sequence is not included in any vaccine constructs.Therefore, there is the HIV-1 strain of living in the positive expression of the test of this LTR sequence, and does not show that for antibody or the inoculation that is used for vaccine test is positive.Other gene orders, for example env or gag/pol, can select to be used to detect the viral RNA of HIV-1, but, these sequences are included in the vaccine constructs, because the existence of vaccine constructs may cause the positive of gene probe to show, when detecting the vaccine Clinical Laboratory of the virus load in the humoral sample, the body fluid of the viral RNA that described body fluid for example detects for blood, serum, blood plasma or other test kits with enough concentration.Be used for having of vaccine constructs oligonucleotide and possibly can't distinguish HIV-individuality that infects and the individuality that uses the candidate vaccine inoculation, described candidate vaccine comprises the oligonucleotide that is used to detect in vaccine constructs.Therefore, for the check of detection, preferably avoid being used for the oligonucleotide of such vaccine constructs from the existence of the viral RNA of live virus.

At the preparation method's of the test kit that is used for detecting HIV a embodiment, synthetic and 33-merHIV-1LTR RNA sequence complementary single stranded oligonucleotide, and hybridize test.At first, for example by this oligonucleotide of mercaptanization, it can be used for functionalized carbon nanotubes or gold nano grain.Then, utilize absorption spectroanalysis, test the hybridization of the particular sequence of this oligonucleotide and HIV LTR RNA.Strong absorption spectrum is represented the hybridization of complementary oligonucleotide, and does not have absorption spectrum or very weak absorption spectrum shows the hybridization failure.One of failure cause is that this oligonucleotide is the incomplementarity oligonucleotide.For example, Figure 22 A and 22B illustrate the zone 784,794 between adding and the Single Walled Carbon Nanotube bonded oligonucleotide.Then, complementary oligonucleotide can be fixed on the matrix (matrix) of cellulose filter membrane, described filter membrane for example is the film that is used for antibody test kit shown in Figure 14 A-14B.When the sample that for example by placement body fluid (dilution or undiluted) HIV-1 is infected joins checkbox (test cassette), complementary oligonucleotide will be hybridized with the RNA (HIV-1LTR) from infected sample, thereby be fixed on the matrix of film.

Carbon nanotube or gold nano grain can be by functionalized with second single stranded oligonucleotide of another regional complementarity of HIV RNA.If the film surface of adding functionalized carbon nanotube or gold nano grain to test kit, then itself and HIV RNA hybridization, thus carbon nanotube or gold nano grain are combined with Examination region on the film.When test spot is accumulated enough a large amount of nanotubes or nano particle, it is clear that the contrast gradient between background and the test spot will become.Figure 23 illustrates the screening test of the concentration of the functionalized single walled carbon nanotubes that is identified for the reagent for quickly examining box.For example, be less than 5～10 minutes and promptly can be observed test spot in such inspection period.Can strengthen contrast gradient by utilizing multiple different functionalized nanotube of complementary oligonucleotide probe or nano particle, described complementary oligonucleotide probe target is to the different zones (for example different zones of LTR sequence) of HIV RNA sequence.Because HIV RNA is limited to undiscovered particular sequence in the vaccine with combining of film, unless there is virus in the tested body fluid, otherwise contrast gradient will not appear.But HIV RNA chain or sequence can be in conjunction with multiple functionalized nanotube or nano particles, if utilize functionalized such nanotube of multiple complementary oligonucleotide or nano particle, then contrast gradient improves.By this way, need not for example to pass through the concentration of round pcr cloning RNA, get final product amplifying signal (being contrast gradient), but have only the sample of infected by HIV can provide positive findings.Therefore, the positive findings in the viral RNA check will show that real HIV-1 infects, and from will the viral RNA check, being negative by the sample of the individuality collection of any candidate vaccine construct of inoculation.

In one embodiment, the complementary oligonucleotide that is used for functionalized nanotube or nano particle 5 '-terminal by mercaptanization, and mix with gold nano grain.Complementary oligonucleotide can with for example LTR zone HIV RNA complementation of the zone of HIV virus.In one embodiment, utilize functionalized dissimilar nano particle of different markers or nano particle to be added on the particular complementary oligonucleotide.Then, the existence of the concentration of the nano particle of particular type or the marker relevant with specific nano particle will show the existence of one type complementary oligonucleotide.Because a kind of complementary oligonucleotide can be selected and the HIV of particular type or the HIV hybridization of particular strain, thus test kit can test example as the HIV RNA that is present in the particular type in the humoral sample and the HIV RNA of particular strain.In one embodiment, selecting for a lot of different HIV strains and/or HIV-1 and HIV-2 is common complementary oligonucleotide.Can be on film (for example Mierocrystalline cellulose filter paper) 1 or more than the fixing several different complementary oligonucleotides in 1 zone, and can select multiple complementary oligonucleotide each fix one or more HIV dissimilar or strain.

In one embodiment, the gold nano grain that dyestuff used in the HIV antibody test kit is added in use to utilizes Glynow, K etc. are at Oligonucleotide-functionalized gold nanoparticles asprobes in a dry-reagent strp biosensor for DNA analysis by hybridization, Anal.Chem., 75 (16), (2003) p.4155-60 middle scheme of describing prepares functionalized gene probe.Test kit comprises single window, as shown in Figure 14B, and has the test spot of the existence that is used to detect antibody and HIV RNA.For example, can synthetic oligonucleotide and carry out mercaptanization.Join in the suspension (about 1.5pmol) of 10 μ l gold nano grains through 24 hours dna probes at 4 ℃ then 0.9nmol mercaptanization.Adding sodium-chlor to concentration in this mixture is 90nmol/L, and allows to keep other 24 hours at 4 ℃.Can be with centrifugal 45 minutes of the 2800g gold nano grain that oligonucleotide is functionalized, and it can be suspended in 600 μ l, 30% sucrose that contains 45nmol/L NaCl.In one embodiment, with the freeze-drying of functionalized gold nano grain probe and prolonged preservation in room temperature.

In a method that detects viral RNA, dilute the blood that 50 μ l HIV-infect with 150 μ l hybridization buffers.This sample can be joined in the checkbox of assembling in advance, this checkbox has the LTR-complementary oligonucleotide that is fixed on the test spot.After blood sample is absorbed, in test hole, add the functionalized gold nano grain of 200 μ l as gene probe.This gene probe makes the complementary region hybridization of gold nano grain and viral RNA.Add 200 μ l cleaning buffer solutions then, salts solution (saline solution) for example so that background color dies down, thereby strengthens contrast gradient between background and the test spot.All the viral RNAs check can both be finished being less than in 5～10 minutes.The color of test kit can at room temperature be stablized maintenance 48 hours.

The optimization of ULTraPID-RNA (Au) platform

In one embodiment, will be fixed on the film test spot of HIV test kit from HIV 89.6 provirus clone's 33-nt oligonucleotide.Because this fixed dna primer and the HIV LTR regional complementarity that comprises clade C HIV1084i, so can and be fixed on the test spot with HIV LTR RNA hybridization.Except this oligonucleotide, can select to use and another dna sequence dna of LTR complementary.In one embodiment, optimize the length of oligonucleotide, effectively to catch HIVRNA with complementary LTR zone.

In one embodiment, directly with fluorescigenic oligonucleotide (siGLO, from Thermo ScientificDharmacon, CO) be added on the film of ULTraPID box, this combination be not enough to stop the 0.3M NaCl that is generally comprised within the gold nano grain dyeing damping fluid with fluorescent marker from film surface flush away.Therefore, complementary oligonucleotide directly puts on the film surface and is not suitable for immobilized oligonucleotide.In another example, the fluorescence oligonucleotide is at first puted together with chitosan nano particle, and this conjugate is put on the film surface.Figure 24 is illustrated in that conjugate is stably fixed on the film in this example.Figure 24 has the green fluorescence 607 (only changing white into for purposes of illustration in this image) of fluorescigenic oligonucleotide, and uniform black (no fluorescence) background of the sample for preparing 606 does not form contrast with at first puting together the fluorescence oligonucleotide with chitosan nano particle for this.In background 606 images, fluorescigenic oligonucleotide is rinsed out from Mierocrystalline cellulose filter paper, and the oligonucleotide that is conjugated with chitosan and chitosan derivatives is fixed on fluorescigenic oligonucleotide the part of Mierocrystalline cellulose filter paper.The contriver thinks that when chitosan and oligonucleotide were puted together, chitosan formed the cationic polymers of puting together with membrane matrix.Therefore, chitosan helps complementary oligonucleotide is fixed to the film that is used to test detected viral RNA or other RNA or DNA.By with suitably the puting together and RNA or DNA target sequence fixing of film, one or more functionalized complementary oligonucleotides of nanotube or nano particle can be hybridised on the target RNA or DNA that is detected, and then between gene probe test spot (G) and Mierocrystalline cellulose filter paper background, provide contrast gradient.Therefore, gene probe can be used to detect the existence of specific RNA or dna sequence dna, this sequence be fixed on filter paper on and the complementary oligonucleotide that is combined with nanotube and/or nano particle combine and the situation that can be used to distinguish viral DNA and only have viral DNA antibody.

In other embodiments, use bovine serum albumin (BSA) or Streptavidin or put together coupling with chitosan nano particle and strengthen 33-nt oligonucleotide combining on Mierocrystalline cellulose filter paper film (film that for example is used for the example of antibody test kit) separately.In another embodiment, use the part of Streptavidin coated film, and biotinylated 33-nt oligonucleotide is applied to the part that scribbles Streptavidin of film.

Figure 25 draws the synoptic diagram of detector 2500, and this detector can be measured the light that surveyed area 2510 sends or see through the light of the surveyed area 2510 of slide glass 2511.Light source 2501 is provided in base 2502, provides charge coupled device or other photodetectors or spectrum analyzer 2505, be used to catch and analyze light with cover 2515 or point instrumentation.For such detector, can provide spectral filter (Filter) and camera lens (optic) according to the general knowledge of this area.

The alternative combination of the embodiment that is provided and modification all will be conspicuous on this disclosed basis.The all possible combination of the embodiment that can not provide a description and the object lesson of modification, still, such combination and modification can require the right that finally obtains.

Table 1A illustrates the hydrometry that specific particle keeps size.

Table 1BThe flow speed data of the mixed nitrate cellulose ester membrane with various pore sizes is shown.

Table 1C

The typical properties of Mierocrystalline cellulose filter paper

Table 2

Table 3

Table 4: the test of commercial kit and embodiment test kit relatively

Table 5: the test of commercial kit and embodiment test kit is (band pattern) relatively

I.D.#PRB204	RL37-is with pattern
		01	There is not band
02	18，24，31，41，55，51，65，120，160
		03	There is not band
04	18，24，31，41，55，51，65，120，160

05	24，31，41，51，55，65，120，160
		06	18，24，31，41，55，51，65，120，160
07	18，24，31，41，55，51，65，120，160
		08	f18，24，31，41，51，55，120，160
09	There is not band
		10	24，51，55，f160
11	18，24，31，41，55，51，65，120，160
		12	18，24，31，41，55，51，65，120，160
13	24，f51，f55，f160
		14	18，24，31，41，55，51，65，120，160
15	18，24，31，41，55，51，65，120，160
		16	24，f41，51，55，160
17	24，51，55，f120，160
		18	f24，f51，f55，160
19	24，f31，51，55，f120，160
		20	18，24，31，41，51，55，65，120，160
21	24，51，55，160
		22	24，31，41，51，55，65，120，160
23	There is not band
		24	24，f51，f55，f160
25	f51，f55

Table 6

The Whatman rank	GF/C
		Particle keeps	1.2μm
Flow velocity	10.5sec./100mL
		Thickness	0.26mm
Substance	53g/m ²
		Top temperature	500℃(932°F)
Water absorbs	250mL/m ²
		Sterilization	Autoclavable

Table 7

Claims

1. be used to detect the segmental reagent for quickly examining box of DNA, RNA or DNA or RNA, described test kit comprises:

Film;

At least a gene probe, described gene probe is fixed on the check part of described film, make when containing the described film of the direct filtration of described DNA, RNA or described DNA or the segmental fluid of RNA described at least a gene probe fixing described DNA, RNA or described DNA or RNA fragment; And

Staining agent, if but it is chosen to feasible described DNA, RNA or described DNA or the RNA fragment that has detection level, described staining agent preferentially is fixed on the check part of described film, makes it possible to observe the check part of described film and the contrast gradient between the background parts.

2. reagent for quickly examining box according to claim 1, it also comprises:

The decolouring damping fluid, it is chosen to be used for to remove the irrelevant non-specific background dyeing that combines between at least a portion and described at least a gene probe, described DNA, RNA or described DNA or RNA fragment and the described staining agent, but, can be observed described contrast gradient if make described DNA, RNA or described DNA or the RNA fragment that has detection level.

3. reagent for quickly examining box according to claim 1, wherein, the phosphate buffer soln flow velocity that described film is chosen to make the ASTM standard flow rate measurement of described film utilization improvement to record is about 0.04～about 0.4ml/min/cm ²

4. reagent for quickly examining box according to claim 3, wherein, described film is chosen to make the determined flow velocity of described film at about 0.04mL/min/cm ²～about 0.2mL/min/cm ²Scope in.

5. reagent for quickly examining box according to claim 4, wherein, institute's velocity measurement of described film is at 0.1mL/min/cm at least ²～0.2mL/min/cm ²Scope in.

6. reagent for quickly examining box according to claim 1; wherein; described staining agent comprises nano particle or the nanotube that oligonucleotide is functionalized, and described nano particle or nanotube have described DNA, the RNA that can detect with described reagent for quickly examining box or the oligonucleotide of described DNA or the hybridization of RNA fragment.

7. reagent for quickly examining box according to claim 6, wherein, described at least a gene probe comprises the complementary oligonucleotide that is used for described DNA, RNA or described DNA or RNA segmental specific region hybridization.

8. reagent for quickly examining box according to claim 7, wherein, described complementary oligonucleotide and chitosan or chitosan derivatives are puted together, and make described complementary oligonucleotide be fixed on the described film.

9. reagent for quickly examining box according to claim 8; wherein; nano particle that described oligonucleotide is functionalized or nanotube comprise the gold nano grain that the oligonucleotide of mercaptanization is functionalized; the segmental different piece complementation of the oligonucleotide of described mercaptanization and described DNA, described RNA or described DNA or RNA, described different piece do not comprise that described DNA, described RNA or described DNA or RNA fragment are fixed on the part of the complementary oligonucleotide hybridization on the described film.

10. reagent for quickly examining box according to claim 9, wherein, the oligonucleotide of described mercaptanization is chosen to be used for the primer of hybrid virus RNA, and described viral RNA is selected from by HIV virus, hepatitis B virus, hepatitis C virus, SARS virus and the group that constitutes thereof.

11. reagent for quickly examining box according to claim 10, wherein, described primer is chosen to be used for hybridizing the viral RNA of described HIV virus.

12. reagent for quickly examining box according to claim 11 wherein, is fixed on the zone that described complementary oligonucleotide on the described film is chosen to be used for hybridize the viral RNA of described HIV, described zone is in the LTR of the viral RNA of described HIV virus sequence.

13. reagent for quickly examining box according to claim 6, wherein, described staining agent comprises the carbon nanotube that oligonucleotide is functionalized, and the segmental part of described oligonucleotide and described DNA, described RNA or described DNA or RNA is complementary.

14. reagent for quickly examining box according to claim 13, wherein, the viral RNA that described specific complementary DNA or viral RNA are selected from the viral RNA group, described viral RNA group is by the viral RNA of HIV virus, hepatitis B virus, hepatitis C virus, SARS virus and constitute.

15. reagent for quickly examining box according to claim 1, wherein, described at least a gene probe comprises the 33-nt oligonucleotide from HIV 89.6 provirus clone.

16. reagent for quickly examining box according to claim 1, wherein, described at least a gene probe is puted together with chitosan or chitosan derivatives.

17. utilize the method for the described reagent for quickly examining box of claim 1, but comprise the virus load that detects detection level in the certain volume sample fluid.

18. method according to claim 17 also comprises: described at least a gene probe and chitosan or chitosan derivatives are puted together the formation conjugate; Fix this conjugate at the Examination region of described film.

19. method according to claim 17 also comprises: utilize the described film of UV-irradiation with the contrast gradient between the background parts that strengthens described check part and described film.

20. method according to claim 17 also comprises virus load level or concentration described in the described fluid sample volume of report.

21. method according to claim 20, wherein, described reporting step comprises the contrast gradient of at least a portion of the check of described film part or intensity and standard substance is compared.

22. method according to claim 17, wherein, described detection step comprises described staining agent placed on the described film, makes gene probe in the described staining agent optionally in conjunction with the described DNA of described at least a gene probe institute's fixed, RNA or described DNA or RNA fragment on the check part of described film.

23. be used for detecting the test kit of subject's DNA, RNA or DNA or the RNA fragment antibody relevant with the existence of at least a and described DNA or RNA, described test kit comprises:

The mensuration flow velocity that Mierocrystalline cellulose filter paper with detection faces and opposing face, described Mierocrystalline cellulose filter paper are selected from the phosphate buffer soln of the ASTM standard flow rate mensuration of utilizing improvement is about 0.04～about 0.4ml/min/cm ²Mierocrystalline cellulose filter paper;

At least a gene probe, it is fixed on the first check part of described Mierocrystalline cellulose filter paper, make that described at least a gene probe is fixed described DNA, RNA or described DNA or RNA fragment when containing described DNA, RNA or described DNA or the segmental fluid of RNA and directly filter described Mierocrystalline cellulose filter paper;

At least a antibody test probe, it is fixed on the second check part of described Mierocrystalline cellulose filter paper, makes when the fluid that contains described antibody directly during the described Mierocrystalline cellulose filter paper of filtration the described antibody on the described filter paper of described at least a antibody test probe stationary; And

At least a staining agent, it is chosen to comprise and nanotube or particle bonded oligonucleotide, but if make the described DNA that has detection level, RNA or described DNA or RNA fragment, described at least a staining agent preferentially is fixed on the described first check part of described Mierocrystalline cellulose filter paper, thereby can observe first check part of described Mierocrystalline cellulose filter paper and the contrast gradient between the background parts, but if there is the described antibody of detection level, described at least a staining agent preferentially is fixed on the described second check part of described Mierocrystalline cellulose filter paper, thereby can observe the described second check part of described Mierocrystalline cellulose filter paper and the contrast gradient between the described background parts.

24. test kit according to claim 23, wherein, described at least a gene probe is chosen to be used for distinguishing described DNA, RNA or described DNA or the RNA fragment and the described antibody that is produced by vaccination of existence.

25. a test kit that is used to detect antibody, described test kit comprises:

At least a antibody test probe, it is fixed on the check part of described Mierocrystalline cellulose filter paper, makes when the fluid that contains described antibody directly during the described Mierocrystalline cellulose filter paper of filtration the described antibody on the described filter paper of described at least a antibody test probe stationary; And

At least a staining agent, if but described staining agent is chosen to the feasible described antibody that has detection level, described at least a staining agent preferentially is fixed on the described check part of described Mierocrystalline cellulose filter paper, thereby can observe the described check part of described Mierocrystalline cellulose filter paper and the contrast gradient between the described background parts.

26. test kit according to claim 25, wherein, described at least a antibody test probe or described at least a staining agent comprise the gp41 peptide fragment, and described fragment comprises following sequence:

QLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNAS。

27. a disease test comprises:

At least a gene probe, it places on the surveyed area of slide glass, make that described DNA, RNA or described DNA or described RNA fragment were hybridized with described at least a gene probe when the segmental fluid contain DNA, RNA or described DNA or described RNA directly placed the described surveyed area of described slide glass;

Nanotube that complementary oligonucleotide is functionalized or particulate suspension make that described complementary oligonucleotide is hybridized the fragment of described DNA, RNA or described DNA or RNA when described suspension directly places the described surveyed area of described slide glass; And

Detector is used to detect from the luminous of the described surveyed area of described slide glass or the light that absorbed by the described surveyed area of described slide glass.

28. check according to claim 27, wherein, described suspension comprises by the functionalized carbon nanotube of complementary oligonucleotide.

29. check according to claim 28, wherein, described detector comprises ultraviolet source; Described detector detects the fluorescence that sent by described surveyed area or the level of phosphorescence, makes that the segmental virus load of described DNA, RNA in the tested sample or described DNA or RNA can be by quantitative assay.

30. check according to claim 29, wherein, described detectors measure is from the fluorescence of surveyed area.

31. check according to claim 30, wherein, described detector provides the result relevant with virus load.

32. check according to claim 27, wherein, described suspension contains gold nano grain.

33. check according to claim 32, wherein, described detector is measured the absorption through the light of described surveyed area.

34. check according to claim 27, wherein, described gene probe comprises the 33-nt oligonucleotide from HIV 89.6 provirus clone.

35. be used to detect the test kit of antibody, described test kit comprises:

At least a antibody test probe, it is fixed on the check part of described Mierocrystalline cellulose filter paper, makes when the fluid that contains described antibody directly during the described Mierocrystalline cellulose filter paper of filtration the described antibody on the described filter paper of described at least one antibody test probe stationary; And

36. test kit according to claim 35, wherein, described at least a antibody test probe or described at least a staining agent comprise the gp41 peptide fragment.

37. test kit according to claim 36, wherein, described gp41 peptide fragment comprises following fragment:

QLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNAS。

38. according to the described test kit of claim 37, wherein, described at least a dyeing damping fluid comprises the albumin A that is combined with Radioactive colloidal gold.

39. test kit according to claim 35, it also comprises:

Gene probe, it is fixed on another part of described Mierocrystalline cellulose filter paper; And,

Described at least a staining agent comprises nanotube or the functionalized particle of oligonucleotide that complementary oligonucleotide is functionalized, make when containing DNA, when RNA or described DNA or the segmental fluid of described RNA directly filter described Mierocrystalline cellulose filter paper, described at least a gene probe is fixed described DNA, RNA or described DNA or RNA fragment, described staining agent and described DNA, RNA or described DNA or the hybridization of RNA fragment, if described gene probe and described staining agent comprise and described DNA, the oligonucleotide of RNA or described DNA or RNA fragment complementation then provides the contrast gradient between the background parts of the described another part of described Mierocrystalline cellulose filter paper and described Mierocrystalline cellulose filter paper.

40. according to the described test kit of claim 39, wherein, described gene probe is the complementary oligonucleotide with described DNA, RNA or described DNA or the hybridization of RNA fragment.

41. according to the described test kit of claim 40, wherein, the hybridization of the part of the described LTR sequence of described complementary oligonucleotide and described HIV-1 virus.

42. according to the described test kit of claim 41, wherein, described at least a staining agent comprises the multiple oligonucleotide that is combined with the mercaptanization of gold nano grain, and the oligonucleotide of described mercaptanization is chosen to make a plurality of parts hybridization of described RNA of the oligonucleotide of described multiple mercaptanization and described HIV-1 virus.

43. a method comprises:

Vaccination; And

Utilize claim 1,23,25,35 described test kits, the described check of claim 27 or their combination,

Wherein, the step of described utilization comprises evaluation vaccine safety or vaccine potency.