CN101426931A - Nucleic acid detection system - Google Patents

Abstract

The present application discloses a system for detecting target nucleic acid comprising: a container comprising a nucleic acid amplification mix comprising a primer labeled with different haptens at its 5' and 3' ends, and optionally dNTP labeled with a hapten to form a nucleic acid complex; and a lateral flow test device comprising a reservoir area comprising reagent conditions suitable for binding of a specific binding partner with the nucleic acid complex; a dye area comprising a specific binding partner to the nucleic acid complex, wherein the specific binding partner is linked or conjugated to a reporter dye or another hapten; and a test area comprising a different specific binding partner specific to a different aspect of the nucleic acid complex.

Description

Nucleic acid detection system

The cross reference of related application

The application requires in No. the 60/560th, 197, the U.S. Provisional Application of submission on April 7th, 2004 and the 60/567th, No. 845 the right of priority of submitting on May 3rd, 2004, and its full content is hereby expressly incorporated by reference.

About the statement of governmental interests in the present invention

This research work obtains the support of United States Army medical research and material headquarters, and contract number is DAMD17-03-C-0098.

Technical field

The present invention relates to detect the field of nucleic acid molecule by the hypersensitivity that utilizes effluent (lateral flow) nucleic acid detection system (sensitivity).

Background technology

The progress of preventive medicine goes far towards to protect modern soldier to avoid disease, the mosquito matchmaker singapore hemorrhagic fever (mosquito-bornedengue fever) when being deployed to tropical area or subtropical area as soldier.Yet although taked preventive measures at the scene, part soldier still infects singapore hemorrhagic fever or more serious dengue hemorrhagic fever (dengue hemorrhagic fever)/dengue shock syndrome (dengue shock syndrome) (DHF/DSS).It is crucial that the quick in-situ horizontal (field-level) of disease detects for timely treatment and the further a large amount of breedings of prevention.

Flaviviridae (family Flaviviridae) comprises almost 70 kinds of viruses, comprising those viruses that cause yellow jack, singapore hemorrhagic fever, west Nile fever and Japanese encephalitis.Because the growth of whole world tourism, a large amount of breedings of these viruses occur beyond having begun more continually in the torrid areas.At ConUS, a large amount of breedings of St. Louis encephalitis virus and start from New York recently and expand to a large amount of breedings of west Nile virus of the whole East Coast of the U.S..In addition, be to have two kinds of mosquitos that carry disease germs in the U.S., Aedes aegypti (Aedes Aegypti) and aedes albopictus (Aedes albopictus), and under some environment, every kind of mosquito can be infected dengue fever virus.According to CDC, such infection is detected in 1980,1986 and nineteen ninety-five at Texas's downstate, and relevant with the dengue epidemic in Mexico the north.

Singapore hemorrhagic fever and DHF/DSS have formed virus disease (Gubler and Clark, the Infect Dis.1995Apr-Jun of human most important arthropod-borne infection; 1 (2): 55-7).Four kinds of different dengue fever virus types (DEN-1, DEN-2, DEN-3 and DEN-4) are arranged, and every type can both cause human diseases.Conservative 3 ＇-non-coding sequence of four kinds of dengue virus serotype types successfully is used to develop RT-PCR (2002 to 2003, subsidized by MIDRP STO A/L) based on TaqMan to identify (identification) dengue fever virus from different areas, the world quantitatively.

Polymerase chain reaction (PCR) provide Causal Agent Identification accurately, but need that the complex reaction component of strict specimen preparation, limited staging life, precise dose are regulated, numerous and diverse hardware, complicated testing process and through the staff of training, all these are at the scene or all be difficult to assurance under " in real time " condition as under the situation at bio-terror attack.

This is suitable for laboratory diagnosis, but the utilization under " (real-time) in real time " field condition is then limited, and under " in real time " field condition, the character of rapid evaluation biological pollution and existence are the keys that its influence is reduced to minimum level.Chromatography side direction detection (chromatographic lateral assay) principle as protein detection method can be developed as the DNA detection system.

In most of detection systems, key link is to obtain unique, detectable signal, and this signal is by means of based on the response to particular target sequence (from the biopathogen body that exists) of the method for non-polymeric enzyme chain reaction nucleic acid.

Summary of the invention

The present invention relates to signal amplifying system, as the particulate and/or the specific conductivity of the europium encapsulate that detects together with the chromatography effluent.This system is a kind of improved nucleic acid detection system, and it keeps all advantages that traditional immunochromatography detects: 1) step; 2) can on-the-spotly use; 3) utilize stable reagent; 4) not special storage requirement; 5) result fast.Amplifying signal is detected by less portable reader, obtains quantitatively or result qualitatively.

The present invention relates to increase in proportion the production of the fluorescence nucleic acid test kit that is used for the detection of serotype specificity target nucleic acid.The invention further relates to cryodesiccated test kit and form, it is responsive, specific and stable.Can detect the cultivation pathogenic agent of various concentration, as comprise the virus of dengue fever virus or dengue fever virus cDNA.

In a kind of embodiment, system of the present invention is used for the ready-made available freeze-dried reagent pad (analoids, reagent pad) of gene amplification.Response matrix can comprise damping fluid, dNTP, RNA enzyme inhibitors, ThermoScript II, mark Auele Specific Primer such as dengue virus serotype type specificity primer (DEN-1 ,-2 ,-3 ,-4), downstream primer, vitamin H dUTP, Taq polysaccharase, internal contrast mRNA and control mRNA Auele Specific Primer.Add vitamin H dUTP with the labeling nucleic acid amplified production, so that utilize the effluent detection technique to detect and amplify indicator signal.In the time of in being incorporated into double-stranded PCR product, fluorescent primer is used for increasing their fluorescence intensity (Nazarenko et al., Nucleic Acid Res.2002 May 1; 30 (9): e37) and with the PCR product be used for the detection of sidestream immune chromatography to identify singapore hemorrhagic fever serotype.

In vote of the present invention, used time resolved fluorescence technology (time-resolved fluorescence technology), preferred such system, the temporal resolution confocal scanning device that it embeds (embedding) particulate and be used for signal detection based on the europium that is combined with antihapten antibody.This be characterized as to detect other susceptibility is provided.This extremely sensitive chromatography effluent signal amplifying system is than fluorescence detecting system needs threshold cycle number (threshold cycle number) (C still less ^t).PCR product detection system of the present invention be fast (＜30min), a step form and stable (down storing more than 1 year) at＜30 ℃.Easily operation of this detection, cheap, convenient, use thermally-stabilised reagent and store requirement not especially.These features make its become under real-time conditions can by untrained personnel use fast and be easy to, available RT-PCR test kit immediately.

Detection system of the present invention can also be used to detecting any organism, includes but not limited to significant virus in pathogenic agent such as the military affairs, comprises Flavivirus, as japanese encephalitis virus, yellow fever virus, west Nile virus, smallpox etc.Other pathogenic agent comprises bacterium, as anthrax bacillus (Bacillus anthracis).

The invention still further relates to by utilizing and detect based on europium and/or based on the target nucleic acid based on the non-genomic amplification of the target nucleic acid detection method of specific conductivity.

Detection system based on nucleic acid of the present invention has been amplified the signal of the dna sequence dna (from the biopathogen body) from lower concentration specific gene group, and need not the complex reaction component of strict specimen preparation, limited staging life, numerous and diverse hardware, complicated testing process or the staff through training.This method also is unique, specific, simple, and this amplification system is operated in the mode of scene adoptable (field-deployable) module easily.

In specific embodiments of the invention, the present invention relates to be modified into based on the detection system of real-time ThermoScript II polymerase chain reaction (RT-PCR) technology of singapore hemorrhagic fever 3 ＇-non-coding region that available immediately (the ready-made use, dengue fever virus ready-to-use) detects and diagnositc system.Show the on-the-spot adoptable and user-friendly diagnostic device that utilizes the sidestream immune chromatography to detect, it has and does not have real-time fluorescence thermo cycler (thermal cycler).The present invention can be applied to PCR in real time and/or traditional PCR doubly.

The invention further relates to detailed analysis result together with the serotype specificity information that the RT reaction is arranged.Polynary PCR reaction can be carried out (Fig. 4) in single test tube.The reactive component of the RT that is useful on reaction and PCR reaction all be frozen dry in having the matrix that proper formulation designs.Add the Oligonucleolide primers of vitamin H dUTP or mark and fluorescent mark beacon primer (beacon primer) and be used for mark PCR product and utilize real-time fluorescence thermo cycler or effluent detection kit to detect.In single PCR reaction, can identify four kinds of serotypes of dengue fever virus.This reaction kit can at room temperature be stored more than 1 year.

Should be appreciated that and can use any suitable detection system, comprise system based on fluorescence, chemoluminescence, enzyme or any other dyestuff, if utilize lateral flow systems of the present invention can detect the combining of target nucleic acid in ground detection probes and the sample.Such fuel system can comprise having based on fluorescein-labeled molecular beacon type system, or it can comprise other mark as the avidin that has Radioactive colloidal gold and cooperate or the biotin labeling of streptavidin part or any other detectable chemical substance that cooperates such as lanthanon (as europium).The certification mark of other type and method belong in the scope of the present invention.

The invention further relates to from carrying out (carrying out device self-performing) certainly.This quick nucleic acid detection system comprises with all reagent of accurate amount and component and produces test result to add the back at sample.This system can also have the Nucleotide target sequence evaluation that built-in heating cushion (heating pad) is formed based on probe oligonucleotides with accurate coupling.

The target DNA of biopathogen body can react with the oligonucleotide of europium particle mark, the peptide nucleic acid(PNA) (PNA) of europium particle mark in the sample, its have light addressing direct electron reader (Light addressable Direct Electrical Readout, LADER) or specific conductivity.Compare the susceptibility (Fig. 5) that this europium mark oligomer of working together with the effluent detection system and specific conductivity detection method can increase by 10 to 1,000 times with the Radioactive colloidal gold particulate.These two kinds of signal amplifying systems can increase susceptibility be higher than 103 targets/100 μ l (=10aM).In addition, detect (equilibrium plate assay) with balancing plate and compare, these detection systems can make susceptibility increase (Hamplet al.Anal Biochem 2001 more than 10 times with combining of chromatography effluent detection; 176-187).

In one aspect, the present invention relates to be used to detect the lateral flow devices of target nucleic acid, this device comprises: storage area (reservoir area), dye area (dye area) and test site (detection zone, test area), wherein the storage area comprises available nucleic acid amplification mixture immediately, and this nucleic acid amplification mixture comprises with hapten-marked primer and/or with hapten-marked dNTP; Dye area comprises the haptenic specific binding ligand of the first kind (specific binding partner) that connects report dyestuff (reporter dye); And the test site comprises the haptenic specific binding ligand of second type.This device can comprise that (storage tank reservoir), detects the target nucleic acid of various ways to the different haptenic holder of second type with more than one therein.The haptens of the first kind can be vitamin H (biotin), and its specific binding ligand can be streptavidin (streptavidin), and the report dyestuff can be europium or gold.In addition, the haptens of second type can be vitamin H, fluorophore (fluorophore) or oligopeptides, if and detected the target nucleic acid of single form, then its specific binding ligand could be streptavidin or be specific antibody to fluorophore or oligopeptides.In addition, the haptens of second type can be polytype fluorophore or oligopeptides, and its specific binding ligand can be to be specific antibody to every type fluorophore or oligopeptides.The source of target nucleic acid can be a pathogenic agent, and it can be viral as dengue fever virus, and this dengue fever virus particulate serotype can utilize the haptens of the second different types to be detected.Equally, primer can be the molecular beacon type.

The invention still further relates to and be used for the method that there is target nucleic acid in test sample, this method comprises the storage area of sample contact according to above-described device, if wherein in sample, there is target nucleic acid, then target nucleic acid is increased and carry out mark with at least two types haptens, be transported to dye area then, first haptens is bonded to its specific binding ligand (it is connected in the report dyestuff) therein, further carried by wicking action then and passed through the test site, and being bonded to second type haptens-specific binding ligand on the test site, its detection shows have target nucleic acid in sample.Holder can comprise more than one the second different type haptens, wherein detects the target nucleic acid of various ways.First kind haptens can be a vitamin H, and its specific binding ligand can be a streptavidin, and the report dyestuff can be europium or gold.The second type haptens can be vitamin H, fluorophore or oligopeptides, and if detect the target nucleic acid of single form, then its specific binding ligand can be streptavidin or be specific antibody to fluorophore or oligopeptides.The second type haptens can be polytype fluorophore or polytype oligopeptides, and their specific binding ligand can be to be specific antibody to every type fluorophore or oligopeptides.The source of target nucleic acid can be a pathogenic agent.

The invention further relates to and be used for the method that there is target nucleic acid in test sample, this method comprises the storage area that sample is contacted lateral flow devices, if wherein in sample, there is target nucleic acid, then target nucleic acid is increased and carry out mark with the haptens of at least a type, be transported to dye area then, haptens is bonded to its specific binding ligand (it is connected in the report dyestuff) therein, further carried by capillary action then and passed through the test site, and the target-specific oligonucleotide that in the test site, is bonded to, its detection shows have target nucleic acid in sample, wherein detects by light addressing direct electron reader or by detecting the report dyestuff and finishes.Hybridization in the test site (hybridization) can be controlled by the built-in heating cushion in the lateral flow devices.Haptens can be a vitamin H, and its specific binding ligand is streptavidin, and the report dyestuff can be europium or gold.

The invention still further relates to and be used for the method that there is target nucleic acid in test sample, this method comprises the storage area that sample is contacted lateral flow devices, if wherein in sample, there is target nucleic acid, then carries target nucleic acid by the test site and be bonded to target-specific oligonucleotide with the europium mark by capillary action.In addition, can amplifying target nucleic acid before being transported to the test site.If, then can not need to increase, wherein use specific conductivity and europium to detect as same responsive detection.

Description of drawings

Can understand the present invention more fully according to the following detailed description and accompanying drawing, wherein accompanying drawing only is illustrative rather than limitation of the present invention, and wherein:

Figure 1A shows modified serotype specificity upstream primer and Figure 1B shows the hairpin structure that utilizes these primers to make.

Fig. 2 shows the structure of molecular beacon dengue virus serotype type specificity upstream primer.

Fig. 3 shows the downstream primer that is used for PCR antisense primer and RT reaction primer.

Fig. 4 is the synoptic diagram of multiplex PCR amplification.

Fig. 5 A-5B shows detection system of the present invention.Fig. 5 (A) shows test (test) device of the system before the test.This device has at least two major portions: at the sample well and the window of reading as a result in the middle part of device of bottom of device.Fig. 5 (B) shows the test-results after the detection.Article two, line represent positive (male, positive) and line is represented to bear (negative, negative).Control line is as inner built-in (built-in) contrast.If correctly detect, then it should occur.

Fig. 6 shows the multiple PCR products identification and detection.

Fig. 7 shows the picture of PCR product identification apparatus.

Fig. 8 shows the structure of lateral flow devices.

Fig. 9 shows the structure of the amido modified factor of 5 ＇ that connects 2 type forward primers.

Figure 10 shows the structure (wiht strip-lattice type (strip format)) of the immunochromatography PCR product identification and detection of utilizing mark PCR product.

Figure 11 shows the structure (device form (device format)) of the immunochromatography PCR product identification and detection of utilizing mark PCR product.

Figure 12 shows the structure of 5 ＇ biotinylation forward primers.

Figure 13 shows the structure of the reverse primer of 5 ＇ rhodamine redss (rhodamine red) mark.

Figure 14 shows the structure of the forward primer of 5 ＇ synthetic oligopeptide marks.

Figure 15 shows the determination step that is used for bar test (strip test).

Figure 16 shows the determination step that is used for the device format detection.

Figure 17 shows and is used for the PCR in real time that dengue virus serotype type 1 (serotype 1) detects and the comparative result of fast PCR product analysis test kit.Two kinds of tests all are the MgCl that uses 1.5mM ₂Carry out.A road (lane): 4,340,000 copy/test, B road: 868,000 copy/tests, C road: 173,600 copy/tests, D road: 34,720 copy/tests, E road: 6,944 copy/tests, F road: 1,388 copy/tests, G road: 277 copy/tests, H road: 55 copy/tests, I road: do not have template.

Figure 18 shows and is used for the PCR in real time that dengue virus serotype type 2 detects and the comparative result of fast PCR product analysis test kit.A road: 3,720,000 copy/test, B road: 1,240,000 copy/tests, C road: 413,000 copy/tests, D road: 138,000 copy/tests, E road: 45,900 copy/tests, F road: 15,300 copy/tests, G road: 5,100 copy/tests, H road: 1,700 copy/test, I road: 567 copy/tests, J road: 189 copy/tests, K road: 63 copy/tests, L road: 21 copy/tests, M road: do not have template.

Figure 19 shows and is used for the PCR in real time that dengue virus serotype type 3 detects and the comparative result of fast PCR product analysis test kit.Two kinds of tests all are the MgCl that uses 1.5mM ₂Carry out.A road: 5,090,000 copy/test, B road: 1,018,000 copy/test, C road: 203,600 copy/tests, D road: 40,720 copy/tests, E road: 8,144 copy/tests, F road: 1,628 copy/test, G road: 325 copy/tests, H road: 65 copy/tests, I road: do not have template.

Figure 20 shows the principle of light addressing direct electron reader (LADER).

The enlarged view that Figure 21 illustrates the synoptic diagram of reading device and is used for the contact head of CBT chip (with the EDDA form).

Figure 22 shows before and after hybridizing with coupling target (itself is modified with iron cerium (FcAc) mark), is modified the also electrochemical behavior of the electrode of covalently bound Os mark with the long capture probe (capture probe) of 20 Nucleotide.

Figure 23 shows the structure that carries out quick nucleic acid detection system certainly that utilizes specific conductivity to detect.

Figure 24 shows the synoptic diagram that utilizes time resolved fluorescence emission and/or conductivity variations to detect the detection principle of detection of biological pathogen specific dna sequence dna.

Figure 25 A-25C shows the structure that is used for heating system.

Figure 26 A and B show the structure that is used for heating system.

Figure 27 A and Figure 27 B show the structure that is used for heating system.

Figure 28 shows the synoptic diagram of the testing apparatus with the position (space) that is used for the instrument junctor.

Figure 29 shows the gene spot inspection, and (gene real-time test, gene point of caretesting GPOCT) install, and it is a kind of integrated DNA sampling thief.

Figure 30 shows a kind of detailed embodiment of GPOCT device.

Figure 31 shows a kind of detailed embodiment of GPOCT device.

Embodiment

In this application, " one " is used to refer to single and a plurality of objects.

As employed in this article, " target nucleic acid of various ways " is meant the target nucleic acid of multiple isotype or serotype, and as dengue fever virus, it can have four kinds of serotypes.

As employed in this article, when being used for probe unit, term " nucleic acid " or " oligonucleotide " are meant any Nucleotide string.Thereby, comprise DNA, RNA or PNA.

As employed in this article, " nucleic acid amplification " is meant that polymerase chain reaction (PCR), ligase chain reaction (LCR), Q β replicative enzyme, chain substitute detection (strand displacementassay, SDA), based on the amplification (NASBA) of nucleotide sequence or cascade rolling cyclic amplification (cascade rolling cycle amplification, CRCA).

As employed in this article, " available immediately (the ready touse) " in the nucleic acid amplification scope be meant with carry out target nucleic acid needed all reagent of amplification and enzyme after the sample that comprises target nucleic acid contacts.

In one aspect, the present invention relates to strengthen the signal that is used for detection of nucleic acids.This method can comprise utilizes various marks or haptens to detect this signal sensitively.

In one aspect of the invention, available nucleic acid amplification premixture can be included in the container immediately.Sample can be added premixture, carry out nucleic acid amplification then.Premixture can comprise the reagent that is used for nucleic acid amplification, comprising at 5 ＇ and/or the 3 ＇ end primer of mark in addition differentially.Use for PCR in real time, 5 ＇ of single primer and 3 ＇ end can be with molecular beacon mode marks in addition differentially, wherein fluorophore at one end and quencher at the other end.Each of these marks can be thought haptens, because can obtain and can produce antibody with respect to these marks.In addition, the main body of amplified production can be alternatively with hapten-marked dNTP (as vitamin H) mark in addition.Premixture can be dried or lyophilize, and by isothermal duplication PCR and/or PCR in real time, premixture goes for amplification.

Detect for effluent, amplification of nucleic acid haptenization or mark or non-amplifying genom DNA (comprising the target nucleic acid that can flow to dye area (usually with cutting)) be at storage area contact side flow detection device, wherein the storage area comprise be suitable for hybridization conditions or antibody/antigen in conjunction with other specificity of conditioned disjunction in conjunction with reagent to condition.

Nucleic acid can be transported to dye area then, and this district comprises the specific binding ligand of target nucleic acid mixture of nucleic acid and the haptens that is connected to nucleic acid.Therefore, specific binding ligand can be antibody or the high affinity ligands such as the streptavidin of one of target-specific oligonucleotide, haptens, is incorporated into vitamin H its high-affinity.Specificity can connect or be matched with dyestuff in conjunction with the streptavidin part, and the dyestuff of preferred heights sensitivity is as europium or golden to form the mixture of target nucleic acid specific binding ligand-dyestuff.Replacedly, specific binding ligand can be with the further haptenization in addition of a kind of different mark.In addition, for the oligonucleotide specific binding ligand, mark can be europium, fluorophore, FITC, vitamin H, oligopeptides etc., as long as this mark can be detected by any way.

When mixture moves to the test site, can fix the test site with another specific binding ligand of one of other haptens, wherein above-mentioned other haptens be at the target nucleic acid mixture as being in the specific oligonucleotide to target nucleic acid, so the combination of the different specific binding ligands on mixture and the test site shows and has target nucleic acid.In the dye area use and at test site fixed oligonucleotide can be peptide nucleic acid(PNA) or RNA.Under the situation that is fixed on the DNA oligonucleotide on the test site, the hybridization of target nucleic acid mixture and the oligonucleotide on the test site produces conductivity signal, and it can be detected by extremely sensitive LADER detection method.In addition, when the oligonucleotide of europium mark is hybridized oligonucleotide on being fixed on the test site or PNA, can combine the extremely sensitive mensuration of the susceptibility of LADER detection method and europium detection method.For example, if the as one man combined europium so of europium mark part will produce strong signal.If the europium mark zone is not incorporated into fixed nucleic acid, and only be that the non-europium mark part of nucleic acid is combined, can use the LADER specific conductivity so as a kind of interchangeable mensuration.

In another aspect of the present invention, the test site can be fixed with target-specific oligonucleotide and/or at least a other the specific binding ligand that is used for one of haptens, with further difference and definite target nucleic acid.Therefore, in a kind of embodiment, the specific binding ligand of one of haptens on the target nucleic acid (as being specific to 5 ＇ marks or 3 ＇ marks) can be fixed on the test site, and the oligonucleotide probe that is used for target nucleic acid also can be fixed, so that can target nucleic acid be detected having under the bigger specific situation.

Below describe exemplarily explanation and utilize the nucleic acid amplification method of PCR to detect dengue fever virus, yet, should be appreciated that the nucleic acid amplification method (as PCR, LCR, NASBA etc.) that utilizes any chemistry to drive can detect any pathogenic agent.

ThermoScript II polymerase chain reaction (RT-PCR)

ThermoScript II polymerase chain reaction (RT-PCR) provides accurate viral pathogens to identify, but as conventional use at present, specimen preparation that need be strict, the complex reaction component of limited staging life, precise dose regulation and control, numerous and diverse hardware, complicated testing process and staff through training.This is applicable to laboratory diagnosis, but the application under " in real time " field condition is limited.

System of the present invention can optimize and increase in proportion the production that is used for RT-PCR test kit general and serotype specificity microorganism detection (comprising the dengue fever virus detection).Developed special-purpose lyophilize scheme to produce exsiccant and available singapore hemorrhagic fever detection kit immediately, it comprises the component that the RT of be useful in single test tube reacts multi-PRC reaction.Test kit can be used for general thermo cycler (thermal cycler) or PCR in real time thermo cycler such as fluorescent PCR thermo cycler.

The extraction of viral RNA

(Qiagen Inc.Valencia, CA91355), viral RNA is the viral suspension that extracts from the serum of the serum of virus infection such as dengue infection according to QiAamp Viral RNA Handbook.

Molecular beacon serotype specificity primer design is synthetic

Molecular beacon is " hair clip " oligonucleotide that forms stem-ring structure and have the report not for public consumption signal, as fluorescence reporter molecule and quencher molecule.When they formed hairpin structure, fluorescence reporter molecule and quencher molecule can be close together, and fluorescence is suppressed.When they were bonded to complementary target sequence, their experience were connected comformational switch (Bonnetet al., the Proc Natl Acad Sci USA.1999 May 25 of their fluorescence; 96 (11): 6171-6.).The hair clip molecular beacon can be used as fluorescence PCR primer or fluorescent probe.When Oligonucleolide primers is modified when having the hairpin structure of fluorophore with formation, this primer can provide the low initial fluorescence of this primer, it can increase to 8 times of (Nazarenko et al., Nucleic Acids Res.2002 May 1 after forming the PCR product; 30 (9): e37).Hairpin oligonucleotide is can be the same with the straight chain primer effective and can provide other specificity to PCR by preventing primer-dimer and primer mispairing (mispriming).

Based on this previous research, can modify to form hairpin structure (Fig. 1) upstream primer.In order to make the hair clip primer, 5 ＇ tails are stretched, and it is complementary to the 3 ＇ end of primer.5 ＇ tails form flush end (blunt-end) hairpin structure being lower than under its temperature of unwinding.To report fluorescein (6-FAM ^TM(520nm), HEX ^TM(556nm), Texas (603nm),

(640nm) etc.) mark is to the 3rd or the 4th base (T) from 3 ＇ end.With quencher fluorescein (Iowa black ^TM, the quencher scope is 520nm to 700nm) mark to stretch 5 ＇ end (complementary sequence of 3 ＇ end).All report dyestuffs and quenchers can be available from Integrated DNA Technology, and Inc. (Coralville, IA) and MolecularProbes, Inc. (Eugene, OR).The secondary structure and the fluorescent mark position of estimating are shown among Fig. 2.

In another aspect of the present invention, fluorescein also plays the antigenic work of haptens in order to react with antihapten antibody, identifies serotype specificity PCR product thereby utilize effluent quick to go on foot the characterization test test kit.

Antisense primer and RT primer design are synthetic

The general antisense primer DV-L1 (nucleotide residue 368-388) that has good homology between 3 ＇-non-coding sequence of singapore hemorrhagic fever 1-3 is designated as and is used for these viral RT primers.Though singapore hemorrhagic fever 4 genomes and DV-L1 sequence are enjoyed significant homology, as shown in Figure 3, in the DV-L1 primer, there are 5 Nucleotide mispairing.Therefore, the antisense primer DV-L2 that separates is used for the RT reaction that singapore hemorrhagic fever 4 viruses detect single-mindedly.These two kinds of antisense primers (DV-L1 and DV-L2) are as general RT primer sets (primer set), to transcribe the RNA (Houng et al., 2001 J.Virol.Meth.95:24-35) that is used for all four kinds of dengue virus serotype types.

Multiple RT-PCR reaction in single test tube

RT reaction and multi-PRC reaction can carry out (Fig. 4) in single test tube.All reactive components that are used for RT reaction and PCR reaction are frozen dry in the matrix with proper formulation design.Add the Oligonucleolide primers of vitamin H dUTP and mark and fluorescently-labeled beacon primer with mark PCR product and utilize the real-time fluorescence thermo cycler or the effluent detection kit detects.In single PCR reaction, can identify four kinds of serotypes of dengue fever virus.This reaction kit can at room temperature store more than 1 year.

PCR result identifies

Commercially available real-time fluorescence PCR system can be used for this test kit of available immediately.Depend on fluorescence detecting system, the available test kit can be adjusted by user's customizations immediately.In addition, this test kit can use under the imperfect laboratory condition of real-time thermo cycler not having.

It is a kind of replaceable system that identifies amplification PCR product that the sidestream immune chromatography detects.This method is unique, specific, and is easy to operate in the adoptable at the scene system.This system can be used in fact all biopathogen body and viruses, and at the scene or under " in real time " condition (as being deployed under the situation of singapore hemorrhagic fever epidemic regions) Americans in uniform reliable result can be provided.

Non-PCR effluent detection of nucleic acids

Fast, certainly the structure that carries out nucleic acid detection system is shown among Figure 23, and it comprises all reagent and components with accurate amount and produces test-results to add the back at sample.This system also has built-in heating cushion is formed (composition) based on probe oligonucleotides with accurate coupling Nucleotide target sequence evaluation.Detect principles illustrated (Figure 24) in the diagram.At first by storing pad (tablet storage, reservoir pad), it comprises sample: the stain remover of all components and generation are by the suitable mobile porous filter material of device in the damping fluid of optimization sample pH value, the suspended sample.Sample flow to dye area by capillary action thereafter, and the oligonucleotide of the target DNA of the biopathogen body in the sample and europium particle mark or peptide nucleic acid(PNA) (PNA) react therein.So the reaction mixture that forms moves then by wicking film (wicking action film, wicking membrane), catches the oligonucleotide that is embedded in conductive test district and check plot therein.Initial europium label probe reaction makes can open target nucleotide sequences.

Utilize the effluent of time resolved fluorescence to detect

Hypersensitization time resolved fluorescence (TRF) dyestuff is used for effluent and detects form.This can guarantee the useful aspect that the susceptibility of improving keeps effluent to detect simultaneously.Nano level TRF dyestuff comprises by 30 of beta-diketon trapping, 000-2,000,000 europium molecule, it has a kind of (Harma, Technology Review126/2002, the TEKES of the known lanthanide chelate of the sub-productive rate of maximum amount, National Technology Agency, Helsinki 2002).This encapsulate (is sealed, encapsulation) basically to the not influence of the fluorescence efficiency of dyestuff.For the europium particle of typical 100nm size, fluorescence quantum yield is equivalent to about 3,000 fluorescein molecules.By comparing, phycobiliprotein (phycobiliprotein) B-PE (perhaps being the material of known maximum fluorescence) has the fluorescence quantum yield that is equivalent to about 30 fluorescein molecules.Because the particle of 100nm is about 10 times and big thousands of times aspect volume/mass of phycobiliprotein B-PE diameter,, but only be 10% (Seradyn, Color-Rich based on volume/mass so Seradyn europium particulate fluorescence is big 100 times based on mol ratio B-PE ^TMDyed and Fluoro-Max ^TMFlorescent Microparticles Technical Bulletins1999).Utilize the effluent detection of this TRF dye granule can increase the level of detection sensitivity, this means threshold cycle number (C up to pg/ml ^t) can be lowered and reach 10 circulations.This feature can provide other specificity.

This system needs simply and the one step process that carries out certainly, and this method does not need complicated and self-reacting device costliness.Figure 5 illustrates less plastic sheet (comprise of the present invention from carry out detector bar with and test result) example.

By means of detection system provided herein, can and not have in single sampling to detect multiple assay under the situation of further process steps.The immunochromatography detection system is preferred.If various antibody-dye conjugatess are embedded in dyestuff pad district (dye pad area) and be that specific antibody is separately fixed in the disengaging zone on the film to every kind of report dyestuff, then each detection line (test wire, test line) will provide the distinctiveness information (Fig. 6) about every specific specificity singapore hemorrhagic fever serotype.

On-the-spot adoptable detection system

It is contemplated that on-the-spot adoptable detection system.The bar shaped reader can be used for the existence of test sample.For example, can use and be used for the instrument that Measuring Time is differentiated the lanthanon emission, the mechanical chopper (mechanical chopper) that it is equipped with nitrogen laser, diffraction grating spectrometer, charge-coupled device (CCD) and is used for time gating (time gating).

Scope of the present invention is not limited to embodiment described herein.In fact, except that described herein, according to above description and accompanying drawing, various changes of the present invention to those skilled in the art will be conspicuous.These changes belong in the scope of appended claims.The embodiment that below provides only is used for illustrating the present invention, rather than restriction the present invention.

Embodiment

Below be step-by-step procedure, the rabbit globin mRNA that its explanation is used for the dengue fever virus cDNA of positive control and is used for internal contrast.The whole design of amplification PCR product identification systems has been described.

Embodiment 1

The molecular beacon primer design

The molecular beacon primer that utilized Beacon Designer 2 (Bio-Rad) primer and probe design software design.

The preparation of double-tagging molecular beacon primer

With various report fluorescein (Oregon

(517nm), 6-FAM ^TM(520nm), HEX ^TM(556nm), TAMRA ^TM(573nm), Texas

(603nm),

(640nm) etc.) mark is to the 3rd or the 4th base (T) from 3 ＇ end.With quencher fluorescein (Iowa black ^TM, quencher scope 520nm to 700nm) and mark is to the 5 ＇ end that stretches (complementary sequence of 3 ＇ end).All report dyestuffs and quencher can be available from Integrated DNA Technology, and Inc. (Coralville, IA) with Molecular Probes, Inc. (Eugene, OR).

The internal soundness contradistinction system

The available test kit comprises as the 1ng rabbit globulin mRNA of internal-response contrast and each of two kinds of sphaeroprotein specific PCR primers immediately.Detect by the PCR product of effluent identification kit in the check plot rabbit globulin mRNA.This check plot intensity can be as the reference of semi-quantitative results.

Available RT-PCR reaction kit immediately

Various substrate mediums such as Mierocrystalline cellulose, glass fibre, polymkeric substance or regenerated fiber etc. have been estimated.The formulation components of below listing is in addition dry in lyophilizer, under constant vacuum.Reformulate and RT-PCR reacts and selects matrix with respect to optimum reagent.

The tabulation of formulation components:

The RT-PCR damping fluid,

The MgCl of proper concn ₂

dNTP

Vitamin H dUTP (vitamin H-16-dUTP or vitamin H-21-dUTP)

The RNA enzyme inhibitors

ThermoScript II

Mark dengue virus serotype type specificity primer (1,2,3,4 type)

The mark downstream primer

Rabbit globulin mRNA

Two kinds of sphaeroprotein specific PCR primers

The Taq polysaccharase

Stablizer (not having the RNA enzyme/DNA enzyme of BSA and carbohydrate etc.).

The main purpose of this step is to find the condition of storage of best lyophilize and key ingredient (main ingredient) (being combined with the release and the reformulation of all components under the RT-PCR reaction).Tested various chemical substances (as but be not limited to damping fluid, stain remover, carbohydrate and polymkeric substance and biomaterial such as protein and other biological polymer) various combinations to find top condition.Use iCycler IQ ^TM(BioRAD) and ABI 7700 real-time fluorescence gene thermo cyclers tested available RT-PCR test kit immediately.Effluent for success detects MgCl ₂Concentration be an important factor.MgCl ₂The preferable range of concentration is not to be higher than about 1.5mM, because be higher than a certain amount of MgCl ₂Just can see primer dimer and false positively charged (false positive) result.The adding of vitamin H dUTP increases susceptibility, this or because bigger capture ability or because bigger indicator binding ability (table 1).By control mark and unmarked ratio and use too much catching to overcome substrate with dose indicating and suppress.

Table 1 show be used for the amplification PCR result that dengue virus serotype type 2 detects with labeled primer and have labeled primer and amplification PCR result that vitamin H dUTP adds between comparative data.

Comparative data between the table 1 dengue fever virus amplification PCR result

Copy number/test	Labeled primer	Labeled primer and vitamin H dUTP add
			1000/ test	++	+++
200/ test	+	++
			50/ test	+/-	+

Embodiment 2

Effluent fast PCR product identification kit

The structure of test

Test kit of the present invention is designed to from carrying out device.Comprise all reagent and components at storage pad place such as the effluent fast PCR product qualification test test kit of describing among Fig. 7 and produce test-results to add the back at sample with accurate amount.Detection principle and structure have been described in Fig. 8.At first by the storage piece, it comprises sample: optimize all components in the damping fluid, suspended sample of sample pH value stain remover, separate the column material (as agarose beads etc.) of unreacted matrix (as labeled primer and biotinylation dUTP) and produce suitable mobile porous holder by device.Sample flow to dye area in capillary action subsequently, and the streptavidin of the serotype specificity PCR product of the dengue fever virus DNA in the sample and europium particle mark reacts therein.The reaction mixture migration that so forms then is by the embedding wicking film that anti-fluorescence dye antibody test district and check plot are arranged.

This test can be carried out at the scene by the personnel that were subjected to the minimum level training, but and in that one or two DNA extraction samples are added to disposal type (accessible, disposal) test card (test card) can obtain result fast in later 10 minutes.Mark streptavidin and target DNA mixture are caught by test site and check plot.Each serotype specificity PCR product can be distinguished in the test site, and when europium was used as the signal detection mark, this district can detect by the reader that is equipped with the time resolved fluorescence scanning device.As the internal soundness contradistinction system, any amplification PCR product can be detected in the check plot that separates.This check plot reaction guarantees that crucial test component correctly works.

Catch the preparation of anti-dyestuff antibody immobilization

The antibody of fluorescence dye strengthens for signal and secondary detection provides unique chance with the PCR product of fluorochrome label.Various anti-fluorophore antibody can be available from MolecularProbes, and Inc. (Eugene, OR).0.5 and 2mg/ml between different concns under utilize every kind of antibody of printing press (BioDot Corp.) printing, its thickness is 0.5 to 1mm.Can check the quality of visible waveband (visible band) and the binding characteristic of antibody (Hassan, J., et al, J.Clin.Lab.Immunol., 24:104,1987) with the Ponceau method.

The antihapten antibody yoke closes the preparation of particulate

The europium particle

The europium particle of various particle diameters (50nm-300nm) buy from Seradyn (Indianapolis, Indiana) and Molecular Probes (Eugene, Oregon).Activate europium chelated nano particulate carboxyl 30 minutes with N-(3-dimethylaminopropyl)-the N '-ethyl carbodiimide of 10mmol/L and the N-hydroxysulphosuccinimide of 100mmol/L.With 50mM MES damping fluid (pH is 6.1) washing activatory particle once.The antibody or the streptavidin that add 5mg/L.Be incubated after 2 hours, with the particle of 50mM MES damping fluid (pH is 6.1) washing antibody or streptavidin coating three times.

Gold grain

The gold grain of various particle diameters (20nm-200nm) is bought from British BiocellInternational (UK).Close in order to obtain best yoke, with the pH regulator to 6.5 of colloidal gold solution.The antibody or the streptavidin that add 5mg/L.Be incubated after 15 minutes, add the bovine serum albumin(BSA) of 2g/L.With twice of the particle of 10mM phosphate buffered saline buffer (pH is 7.4) washing antibody or streptavidin coating.

Yoke closes in the optimization of the particulate of streptavidin

Detected the influence (suppressing the storage that conjugates is formed (composition) and conjugates) of streptavidin concentration under various conditions.Also determined covalent cross-linking.The feasibility of conjugation productive rate and increase in proportion is important determinative in the system of selecting and optimizing.

Yoke closes in the optimization of the particulate of antihapten antibody

Detected the influence (suppressing the storage that conjugates is formed (composition) and conjugates) of antihapten antibody concentration under various conditions.Also determined covalent cross-linking.The feasibility of conjugation productive rate and increase in proportion is important determinative in the system of selecting and optimizing.

Test kit is formed (kit formulation)

Dye area

The main purpose of this step is to find the condition of storage of optimum dye-streptavidin or haptens-antibody conjugates (being combined with the streptavidin or the antibody of release mark under wet testing conditions).Tested various chemical substances (as but be not limited to damping fluid, stain remover, carbohydrate and polymkeric substance and biomaterial such as protein and other biological polymer) various combinations to find top condition.

The storage area

Because sample is applied to the storage area, so utilize the various combinations of chemical substance and biomaterial sample to be adjusted to the top condition that is used to detect.

The wicking film

Because this component can promote the capillary migration of nucleic acid samples, so it should flow effectively nucleic acid, make and can carry out immune response between the tagged target nucleic acid of catching streptavidin and long strong wool capillary action is provided being incorporated into.Find the optimum concn of stain remover also very important with suitable stablizer.

Effluent detects

The structure of this system is similar to the Ag-Ab immunochromatography and detects, but streptavidin closed in colloid gold particle by yoke, and can interact by streptavidin-vitamin H and produce signal.Come mark serotype 2 Auele Specific Primers with fluorescence haptens such as rhodamine reds.Anti-rhodamine reds antibody is fixed on the nitrocellulose membrane.Sample at first flow to dye area by capillary action then by the storage piece, and the streptavidin that closes of serotype specificity PCR product of (there) dengue fever virus and Radioactive colloidal gold yoke reacts therein.The reaction mixture migration that so forms then is the anti-fluorescence dye antibody that embeds test site and check plot by the wicking film therein.

2 successes of the test of effluent dengue virus serotype type are used for detecting and identify the amplification of dengue virus serotype type 2 specific PCRs.The detection limit of dengue virus serotype type 2 tests is＜the 100pg/ test that it is corresponding to＜1 femto mole/test.In theory, this susceptibility is enough to detect the amplification PCR product of single digit (single digit) copy from dengue fever virus cDNA.

2 tests of the dengue virus serotype type reported can be carried out at the scene by the personnel that were subjected to the minimum level training, but and can obtain result fast in the test card that the PCR product is added to disposal type in later 10 minutes.Simultaneously, this system be do not need complicated and expensive laboratory condition simply and the one step process that carries out certainly.

Embodiment 3

(FITC) the yoke preparation of closing oligonucleotide of fluorescein isothiocyanate (isothiocyanate)

Fluorescein isothiocyanate (FITC) is bought from Sigma.For yoke closes, modify the 5 ＇ ends (Fig. 9) of dengue fever virus 2 type forward primers with the amido modified connexon of 12 carbochains (C12) (modifier linker).Utilize ordinary method, FITC is connected to modified oligomer.

Effluent detects the preparation of thing

The streptavidin yoke is closed in colloid gold particle, can produce signal by antigen-antibody reaction then.The biotin molecule yoke is combined in the 5 ＇ end of reverse primer (DV.L1).FITC molecule yoke is combined in the 5 ＇ end of forward primer (DV2.U).Have the forward of those modifications and the amplification PCR product of reverse primer and comprise vitamin H and FITC molecule.Combination vitamin H dUTP during pcr amplification.When this sample was applied to test bar, the biotin label of amplification PCR product reacted with the Radioactive colloidal gold that closes with the streptavidin yoke.PCR product-Jin conjugates mixture passes through nitrocellulose membrane by means of chromatography capillary force (chromatographiccapillary power) migration then.Anti--FITC antibody capture that this mixture is fixed.Depend on the gold or the europium particulate amount of catching, produce visible signal (Figure 10).Device form formation is shown among Figure 11.

The preparation of the biotin labeling forward primer of 1 type, 2 types and 3 types

The biotin molecule yoke is combined in the 5 ＇ end of dengue fever virus forward primer (DV1.U, DV2.U and DV3.U).Modify every kind of forward primer (Figure 12) at 5 ＇ end with 6 carbochains (C6) connexon.

The preparation of rhodamine reds mark reverse primer (DV.L1)

Be combined in the rhodamine reds molecular modification reverse primer (Figure 13) of the 5 ＇ end of reverse primer (DV.L1) with yoke.

The preparation of oligopeptides mark reverse primer (DV2.U)

Be combined in the synthetic oligopeptide molecular modification forward primer (Figure 14) of the 5 ＇ end of primer (DV2.U) with yoke.

Be used for the determination step (Figure 15) that wiht strip-lattice type detects

1) sample applies

Amplification PCR product, gold or europium conjugates and the damping fluid of various template concentrations are mixed, be added on the nitrocellulose membrane contiguously then.

2) detect damping fluid

The PBS that comprises stain remover according to the ordinary method preparation.

3) measure

Read test-results later at 10 minutes

The measuring method (Figure 16) that is used for the device format detection

1) sample applies

The amplification PCR product (as the PCR product of 2 μ l) of various template concentrations is added sample well.

2) detect damping fluid

Utilize ordinary method preparation to comprise the PBS of stain remover, then specific two developing solutions of signal detection mark that are used for are contacted with device on the nitrocotton.

3) measure

Read test-results later at 10 minutes

Figure 17, Figure 18 and Figure 19 provide the PCR in real time that is used for singapore hemorrhagic fever serotype 1,2 and 3 and the comparative result of fast PCR product analysis test kit.

Embodiment 4

Utilize the effluent of specific conductivity and europium labeled oligonucleotide to detect

The principle that light addressing direct electron is read (LADER) is based on the specific conductivity difference of the oligonucleotide of strand (" insulating ") and double-stranded (" conduction ") form.This specific conductivity difference can be considered to switch.Just can make up molecular circuit by capture probe being connected in electrode, connecting electron donor/electron acceptor(EA) mixture (DA) and adding the dissolved active ingredient.Electron donor/electron acceptor(EA) mixture serves as other, photoinduction switch.If probe is hybridized, incident light can induce electric charge to transfer to A and electronics can be from being sent to electrode here from D.The electric charge of the DA mixture by active ingredient fill again (chargerefilling) but closed circuit, and circulation produces the highly read output signal of amplification continuously.The LADER principles illustrated is in EP 1144685 B1; it has described protection photosynthetic reaction center and similar as the system that is used for the electrochemistry mixture of LADER technology, the operation of DE19921940 C2 (content of its disclosure is incorporated into this paper as a reference with the way of reference) specific conductivity of then touching upon technology.

The donor/acceptor mixture is applicable to pigment/protein complex (known its be reactive center in light compositing) well, can in the 100ps after photoinduction electronics be delivered to quinone acceptor (chinon-acceptor) from the porphyrin donor.This pigment/protein complex can easily be divided into quinone acceptor and remaining (apo-) protein.Modify the quinone acceptor in the following manner: on the one hand it can be covalently attached to oligonucleotide capture probe and can reformulate to probe at (apo-) protein on the other hand and be connected in (apo-) protein later in conjunction with the quinone acceptor.

This mode is described in the reaction scheme of Figure 20.The ubiquinone (UQ) that will exhaust the light compositing reactive center is recombinated and is cross-linked to the UQ that is incorporated into double-stranded DNA.Donor is represented by the P of porphyrin system and ubiquinone partly forms acceptor A.Irradiation in the first step (1) produces excited state P*-UQ, and it proceeds to temporary transient stable charge separation state P in further step (2) ⁺UQ ^-Be attracted to electrode (4) at this state electronics from DNA (3), thereby with P ⁺-UQ ^-Be oxidized to P ⁺-UQ also produces measurable anode photoelectric current.That gets back to original state cycles through the reductive agent cytochrome c ²⁺(X) come closure, and reductive agent itself is oxidized to P-UQ by reduction (5) system.

By in the reference of each independent spot and the automatic comparison between the measurement signal, can read and conciliate the textual research and explain number and carry out qualitative and quantitative evaluation.The amplification diagrammatic sketch that Figure 21 illustrates the sketch of reading device and is used for the contact head of CBT chip (with the EDDA form).Reader is partly developed and can be to being carried out necessary all liquid treatment step by simple " pressing the end (press bottom) " mode.The reference measure data of barcode chip (before the hybridization) are stored in the internal buffer, and compare with actual measurement data after the hybridization.The difference of each electrode is displayed on the outer computer screen and can be further handled.

Utilize the electrochemical impedance spectrometry, further surveyed double-stranded DNA and the specific conductivity of single stranded DNA and relevant parameters for tunnel (tunnelling parameter) β with double-stranded electron transfer rate constant by the dna single chain that is identified for variation length.Figure 22 shows and is carrying out the front and back of hybridization with coupling target (itself is modified with iron cerium (FcAc) mark), and the chemical property of the electrode of Os mark is modified and be covalently attached to the capture probe of growing with 20 Nucleotide.As expected, under the strand situation, have electrochemical response, it is attributable to the mark at the Os-of capture probe far-end.Occur other peak after hybridization, it is because iron cerium mark (FcAc-peak).This not only proves and can electrochemically scan the mark at the Os-of capture probe far-end, and the efficient of proof hybridization near 100% (represent capture probe and target mark two peaks height much at one).

Utilize the chromatography of conductivity detector to detect

Comprise that from the structure that carries out quick nucleic acid detection system (Figure 23) all reagent and component with accurate amount produce test-results after adding sample.This system also has the Nucleotide target sequence evaluation that built-in heating cushion is formed based on probe oligonucleotides with accurate coupling.Detect principles illustrated in the synoptic diagram of Figure 24.At first by storing pad, it comprises sample: optimize the damping fluid of sample pH value, and the stain remover of all components in the suspended sample, and generation is by the suitable mobile porous filter material of device.Sample flow to dye area by capillary action thereafter, and the oligonucleotide of the target DNA of biopathogen body and europium particle mark reacts in the sample therein.The reaction mixture migration that so forms then is by the wicking film, and embedding therein have a capture oligo, is applicable to conductive test and check plot.Initial europium label probe reaction makes can open target nucleotide sequences.

The test that proposes can be carried out at the scene by the personnel that were subjected to the minimum level training, but and can obtain result fast in later 30 minutes one or two DNA extraction samples being added to the disposal type test card.Labeled oligonucleotide probe and target DNA mixture are caught by the test site and the check plot of specific conductivity mark.Test site display target sequence, and this district can detect by the reader that is equipped with time resolved fluorescence scanning device and/or conductivity readings device.Check plot separately shows the anti-oligonucleotide sequence of europium labeled oligonucleotide.This check plot reaction guarantees that crucial test component correctly works.

It is a kind of amplification system that the chromatography effluent detects.When the target DNA in the liquid sample by capillary force when the film, transparently constantly focus on electrically charged test site on the film by electroaffinity.Even under the low-down situation of the concentration of molecule, the actual molecules concentration in the test site is also much higher than the molecular conecentration in the sample.

Built-in heating cushion (built in heating pad)

Effluent detects thing and can be installed on the heating PCB/PCP (printed wiring paper), and it goes up the high-resistance metal (for example, nickel/Chrome metal powder can be used for heating purposes) of printing at scrub board (base board).This system can have relative precise dose monitor and low-cost control.Sample arrangement is shown among Figure 25 A-25C, Figure 26 A-26B and Figure 27 A-27B.

The synoptic diagram of testing apparatus

Test kit (test kit) is made by the accessible plastic housing with two windows (plastics casing, plastic case), and one of them window is used for observing check plot and test site, and another window provides the hole that receives sample, as shown in figure 28.

In device inside is the test bar with two pads.First pad comprises damping fluid, stain remover and the chemical substance of guaranteeing consistent test-results, and simultaneously its target oligonucleotide yoke of also being included in the buffering system of specific preparation closes the europium particle.Second pad is used to remove the excess liq by reaction film.Two types oligonucleotide are separately fixed at the test site and the check plot of nitrocellulose membrane as fine rule (thinline).To be fixed on the test site for the specific oligonucleotide of target biopathogen body, will be fixed on the check plot to the specific another kind of oligonucleotide of europium label probe simultaneously.Also comprise the instrument junctor, can be connected in device so that be used for the instrument that Measuring Time differentiates fluorescence or specific conductivity (under the situation of using the specific conductivity mark).

Embodiment 5

Utilize the effluent of the specific conductivity of europium mark peptide nucleic acid(PNA) (PNA) to detect

All methods described in embodiment 4 (with the probe of nucleic acid as the detection target DNA) and step also are applicable to uses peptide nucleic acid(PNA) as probe, comprises wherein described various heating cushions of use and testing apparatus.

Embodiment 6

The detection of HLA-DQ α sequence multiform state property

HLA-DQ α 2 genetic locis (genetic locus) can be used as genetic marker (genetic marker) and are used for the individual forensic analysis of identifying.Various analytical technologies have been used for detecting the heritable variation in the pcr amplified dna.

Can be at primer and various haptens of probe place mark or fluorescence haptens.Therefore, the oligopeptides of various sequences and size can be used as haptens; (epi-position, other small molecules epitope) can be used as haptens, and can use various fluorescence haptens such as those to be listed in haptens in the table 2 to can be used as epitope.

Table 2

Antihapten antibody is as above-mentioned haptenic counterpart (counter part).Many antihapten antibodies can be commercial available from such company such as Molecular Probes, Inc.In addition, can be fixed on antihapten antibody on the solid substrate or yoke closes in various particles." various particle " is meant that Radioactive colloidal gold, latex particle, magnetic or paramagnetic particle, europium embed latex particle etc.

Embodiment 7

The gene spot inspection

In another kind of embodiment of the present invention, the present invention relates to gene spot inspection device (GPOCT), its objective is the gene alaysis system that a step or easy steps combination are provided.Absorption pad (absorbent pad) is connected in matrix (being connected in reacting pad (reactionpad)), and antibody of wherein catching or antigen are fixed on the matrix and detect as line that is used for gene spot inspection device or some effluent.Be reagent area on reacting pad, wherein apply response sample (amplification sample) then response sample move to down a kind of medium.

Embodiment 1-sample extraction system.

With reference to Figure 29, a kind of limiting examples of extraction system can comprise:

Be used for removing the stocking system of solution and be used for the buffer components of concentration of DNA or RNA sample, and can comprise multilayer absorption agent through special processing, and including but not limited to the extraction solution of stain remover and ealkaline buffer.

Sort buffer liquid can destroy cell walls or cytolemma and gene samples such as DNA or RNA are exposed in the solution.It is contemplated that, can add proteolytic enzyme to remove histone proteinoid and cell walls.Replacedly, can use solution-type tunicle (solutioncapsule) or multilayer film separately.

In addition, can use various holders, activated charcoal filter, ion-exchange filter.In addition, strainer can be that the molecule scope of blocking is 60,000 to 300,000D or higher semi-permeable membranes and can be the non-sticky coating.

This filter system is removed all potential inhibitor, small molecules, ion and protein.Therefore, sample can be concentrated 10 to 1000 times from initial extraction solution.

Embodiment 2-is used for crude samples or extracts the integrated system (integrated system) of DNA sample.

As shown in figure 29, integrated system comprises the sample input field, and it is connected in the multi-bed filter system, and wherein filter unit absorbs most of ions and small molecules and protein.This part is equivalent to functional filtration and thickener.Filter system further is connected in the very thin pipe that Rapid Thermal is transmitted that is used for, as kapillary.For easy and quick controlled temperature, the built-in metal that is printed on the tube-surface can play heater coil.Can be to be used for placing the zone that some is embedded in solid substrate and is frozen the exsiccant component in tubule.Tubule is connected in reacting pad, and it further is connected in nitrocellulose membrane, and it further is connected in absorption pad.

Embodiment 3-amplification and test section general introduction.

Apply purification of samples, and in a kind of interchangeable embodiment, apply sample and pass through roller system (roller system), the flow rate of its control sample.Sample is by thermal sensor and thermal control circuit then, and by the amplification chamber, it can be made up of following: the pad that 1) comprises all required reagent; 2) cryodesiccated preformulation reagent pad; And 3) PCR reaction.This chamber (pad) comprises Taq polysaccharase, dNTP, primer (mark), other labeled monomer or the like.

When sample from the amplification chamber when coming out, it contacts with the reacting pad of effluent detection.When carrying out the effluent detection, sample flow is crossed results window, and wherein the result can show with the detectable signal of spot or line or any kind of, flows on the absorption pad then.

Hereinafter describe the parts of device of the present invention in detail.

1. thermal sensor and thermal control circuit unit.

Various conductive ink materials (conductive ink) can print on the surface of flexible matrix (as polyester, polycarbonate or polystyrene).Replacedly, can be with various conductive ink materials, as carbon, carbon/silver-colored mixture, silver, silver/silver chloride, gold, platinum, ultraviolet curing dielectric medium, the dielectric conductive ink material of thermofixation, being printed on the matrix and producing suitable heat is 30 ℃ to 100 ℃ thermal cycling to carry out range of temperature.Therefore, just can realize the control of temperature sensor and temperature by the electric current and the resistance that detect and control them.

Can by two or three different temperature are set and under this temperature recirculation realize thermal cycling 20-40 time.Thermal cycle conditions needs optimised, and this depends on their primer sequence.

2. (amplification pad) filled up in amplification

The amplification pad comprises all reagent that are used for isothermal duplication such as PCR or RFPCR.All reagent can be carried out the premix merging prepares and lyophilize best.All reagent are meant the basal component that is used for amplified reaction.Such reagent can be including but not limited to following component: ThermoScript II, archaeal dna polymerase, Taq polysaccharase, PNA primer, labeled primer, dNTP, mark dUTP, damping fluid and stablizer such as protein, BSA (bovine serum albumin(BSA)) and EDTA.The amplification pad can further be reformulated by means of the sample that applies specific volume.Can fix primer to strengthen amplified reaction and to stop substrate to suppress with granulated glass sphere or latex beads." substrate inhibition " is meant that unreacted primer can suppress the reaction between anti-tag antibody (anti-tag antibody) and the polymeric primer." primer " is meant oligonucleotide or PNA (peptide nucleic acid(PNA)) and oligonucleotide hybridization thing." granulated glass sphere " or " latex beads " is meant microparticle.Can obtain the microparticle that the diameter scope is 0.01-10.0 μ m.Preferably, being used for the diameter of this embodiment of the present invention can be in the scope of 0.1-10.0 μ m.

United States Patent (USP) the 6th, 153 has disclosed a kind of detection system No. 425, yet, should ' 425 ' patent not have to disclose or the particulate of prompting in the amplification pad can be used to prevent the substrate inhibition.

Gene chip

At gene spot inspection device on the other hand, the present invention relates to protein chip and gene chip.About gene chip, the embodiment of when describing gene spot inspection device, having described.In a kind of preferred embodiment, this device can comprise the nucleic acid extraction that is connected in DNA or RNA purifying chamber, and it further is connected in amplification or hybridization chamber, and amplification or hybridization chamber are connected in results window to observe the signal (Figure 30) that produces.Gene spot inspection device can make up test bar described above.

In a specific embodiment of the present invention, sample is joined in the extraction buffer container.The DNA sample that extracts further can be added to Gene POCT, can in 30 minutes, read result (Figure 31) then by naked eyes or signal reader.Gene POCT method is fast with extremely sensitive.

All reference of quoting herein all are hereby expressly incorporated by reference.

*****

Those skilled in the art will recognize that or only utilize normal experiment just can determine many equivalent transformations of the specifically described specific embodiment of the present invention of this paper.Such equivalent transformation is also included within the scope of appended claims.

Sequence table

＜110〉Access Bio Inc. (ACCESS BIO, INC.)

＜120〉nucleic acid detection system

<130>13010-05CN

<150>US?60/560,197

<151>2004-04-07

<150>US?60/567,845

<151>2004-05-03

<160>12

<170>PatentIn?version?3.3

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<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be used for the downstream primer that PCR antisense primer and RT react primer

<400>9

<210>10

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be used for the downstream primer that PCR antisense primer and RT react primer

<400>10

<210>11

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉at 5 ' the synthetic oligopeptide molecule (DV2.U) that closes of end yoke of primer

<400>11

<210>12

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉at 5 ' the synthetic oligopeptide molecule (DV2.U) that closes of end yoke of primer

<400>12

Claims (20)

1. system that is used to detect target nucleic acid comprises:

Container comprises nucleic acid amplification mixture, its be included in its 5 ' with 3 ' end with different hapten-marked primers and optionally with hapten-marked dNTP with the formation nucleic acid complexes; And

The lateral flow assay device, described device comprises the storage area, it comprises and is suitable for specific binding ligand and described nucleic acid complexes carries out bonded reagent condition; Dye area, it comprises the specific binding ligand of described nucleic acid complexes, wherein said specific binding ligand is connected or yoke is bonded to report dyestuff or another kind of haptens; And the test site, it comprises that the different piece to described nucleic acid complexes is specific different specific binding ligand.

2. system according to claim 1, wherein, described nucleic acid amplification mixture is dried or lyophilize, and is suitable for isothermal duplication, PCR and/or PCR in real time.

3. system according to claim 1, wherein, described premixture comprises polytype haptens, wherein the described target nucleic acid of various ways is detected.

4. system according to claim 1, wherein, the described specificity bonded nucleic acid complexes that is used for is included in haptens or described nucleic acid on the described nucleic acid.

5. system according to claim 4, wherein, described haptens is vitamin H, fluorophore or oligopeptides.

6. system according to claim 5, wherein, described specific binding ligand be streptavidin, hapten specificity antibody or with described target nucleic acid complementary oligonucleotide.

7. system according to claim 6, wherein, described report dyestuff is europium, gold or fluorophore.

8. system according to claim 3, wherein, described polytype haptens is polytype fluorophore or oligopeptides.

9. system according to claim 1, wherein, be fixed on described test site be with at the different specific binding ligand of the specific binding ligand of described dye area.

10. system according to claim 9, wherein, the described ligands specific that is fixed on described test site be haptenic antibody and/or with described target nucleic acid complementary oligonucleotide or PNA.

11. system according to claim 10, wherein, the described specific binding ligand that is fixed on the described test site is DNA.

12. system according to claim 11 comprises being used for the junctor that specific conductivity detects reading.

13. system according to claim 1, wherein, the source of described target nucleic acid is a pathogenic agent.

14. system according to claim 13, wherein, described pathogenic agent is the virus that belongs to flaviviridae.

15. system according to claim 14, wherein, described virus is dengue fever virus.

16. system according to claim 15, wherein, described dengue fever virus particulate serotype is by using multiple different hapten-marked primer to be detected in order to distinguish them.

17. system according to claim 1, wherein, described primer at one end carries out mark and carries out mark at the other end with quencher with fluorophore, and described optional mark dNTP is with vitamin H mark in addition.

18. one kind is used for the method that there is target nucleic acid in test sample, comprises: make described sample contact comprise the container of premixture, wherein said premixture comprises the primer and the selectable marker dNTP according to claim 1 of target nucleic acid specific marker; Make thus obtained described amplification of nucleic acid contact the described storage area of described lateral flow devices; And detect combining of described nucleic acid complexes and described specific binding ligand on described test site.

19. one kind is used for the method that there is target nucleic acid in test sample, comprise the described storage area that makes described sample contact lateral flow devices according to claim 1, wherein said dye area comprises the target nucleic acid specific oligonucleotide with the report dye marker, and the oligonucleotide of described mark is incorporated into the described target nucleic acid that forms nucleic acid complexes, and wherein said nucleic acid complexes flow to described test site, wherein said test site immobilized oligonucleotide, the oligonucleotide of wherein said mark produce with combining of fixed oligonucleotide and are used for the positive signal that described target nucleic acid exists

20. method according to claim 19, wherein, described signal is read by report dyestuff or specific conductivity.

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