CN100475976C - Reagent kit for inspecting hepatitis and AIDS virus nucleic acid by synchronous amplification - Google Patents

Abstract

A method for inspecting hepatitis and AIDS nucleic acid by synchronized amplification and its reagent knit are disclosed. The process is carried out by taking magnetic ball as automatic medium, specific synchronized capturing HBV, CHV and HIV nucleic acid, accelerating biotin primer construction and purification by RNA external label and internal label, real-time synchronized inspecting and T-PCR amplifying based on Tagman probe. The reagent knit consists of dis-inhibitor, cracking liquid, magnetic ball suspension, washing liquor, internal check, RT-PCR reactive liquor, enzyme mixture, fluorescent mixture, positive check and negative check. It's accurate and automatic, has single-tube operation, closed inspection AND synchronized extraction, and it has better sensitivity and specific performance and can be used for large-scale blood screening and large-capacity clinical inspection.

Description

A kind of synchronous amplification detects the test kit of hepatitis and AIDS virus nucleic acid

Technical field

The invention belongs to diagnostic reagent diagnostic nucleic acid technical field, be specifically related to a kind of highly sensitive, high specificity, be suitable for the simultaneous extraction of conventional blood nucleic acid screening and detect the method and the test kit of hepatitis (comprising hepatitis B and hepatitis C) and AIDS virus nucleic acid synchronously in real time.

Background technology

According to The World Health Organization's statistics, surpass half country and the blood that the blood donor dedicates to is not thoroughly checked, cause diseases such as acquired immune deficiency syndrome (AIDS), hepatitis, malaria and syphilis to be spread unchecked in a lot of places.Among the annual 560 sides people living with AIDS who increases in the whole world, there is 5% to 10% people to infect via blood transfusion or blood products.Virus carrier in African children and women, this ratio is up to 20%-25%.Since the eighties in 20th century, France, Japan, Romania, the U.S., Germany and China etc. all once caused serious consequence because of the blood screening shortcoming.Blood-borne diseases are in case local a generation, tend to cause explosive the propagation, so transfusion safety has extremely special meaning to the people's safety and society stable.Clinical blood detects and also faces the detecting pressure that increases severely day by day.Estimate that according to WHO China's HIV (human immunodeficiency virus) infection number to 2010 year will reach 1,000 ten thousand, and grow continuously and fast.China has the people more than 60% once to be subjected to the infection of hepatitis B virus, finally has the people about 1/10 to become hepatitis b surface antigen positive.In addition, the third the highest liver of chronicity degree in all hepatitis, number of the infected also reaches about 4,000 ten thousand.

Compare with the extreme importance of blood safety, present detection means also lags behind needs of society far away.The hepatitis B virus (HBV) that transfusion safety is constituted a serious threat, hepatitis C virus (HCV), main enzyme immunity (EIA) method that adopts of acquired immune deficiency syndrome (AIDS) (HIV) virus detect at present.Though passed through the improvement of several generations, obtained significant progress at aspects such as sensitivity, specificity, precision and stability, as a kind of detection method of indirect hysteresis, what it detected is human body antibody rather than the virus itself that virus is caused.So it can not eliminate the omission to " window phase " sample, so-called " window phase " promptly infected early stage, also do not produce antibody or antibody below detectability in the body, and the serology of this moment detects and is generally feminine gender.And the window phase of HBV, HCV, HIV virus infection generation antibody is long especially, wherein the window phase of hepatitis C virus (HCV) is about 66-82 days, and hepatitis B virus (HBV) and human immunodeficiency syndrome's virus (being acquired immune deficiency syndrome (AIDS)) window phase (HIV) is respectively about 59 days and 22 days.In addition, because the generation of the incidents such as sudden change of virus has also caused partial immunity method to detect the possibility of omission.So there is the inherent defective in amynologic diagnostic method, can not stop omission.The hysteresis of the method for inspection brings serious threat for transfusion safety and Clinical Laboratory.

In recent years, the development of nucleic acid technology and the ripe gradually existence that directly detects virus for people provide the strongest instrument.(Nucleic acid AmplificationTechnology NAT) is exactly the general designation of this class technology to nucleic acid amplification technologies.Detection of nucleic acids be virogene itself, be to the detection of virus on the most basic aspect.Detection of nucleic acids has shortened the window phase that virus detects greatly, and has high sensitivity, specificity, precision and stability.Fact proved that NAT detects can be with HBV, and average " window phase " that HCV and HIV infect shorten 25 days respectively, 59 days and 11 days, respectively shortening " window phase " 45%, 89% and 50%.Just because of this, more and more countries is recognized the importance that blood nucleic acid detects, and carries out in clinical blood transfusion and blood product as mandatory index in countries such as West Europe, and simultaneously, the conventional examination project of including the blood donor in detects with NAT in the U.S., Japan, European Union.

External existing a plurality of individual event detection of nucleic acids launch products.Countries such as the U.S., Australia, Canada have product to come out in succession.Roche Holding Ag has also developed the diagnostic nucleic acid test kit.But these test kits or be opening PCR-ELISA detecting pattern, or complex operation easily take place to detect to pollute, and all are that single index single detects, and these defectives make it to be difficult to adapt to the needs of the market status.Analyze the present situation of these diagnostic nucleic acid reagent, exist following wretched insufficiency: 1) reagent is open, and contamination of heavy is big; Level of automation is low, and the technical bottleneck that automatization and totally-enclosed property are the detection of nucleic acids most criticals.2) reagent is that individual event detects, the none blood screening reagent can simultaneous extraction, detect multiple viral nucleic acid simultaneously in real time (as HBV, HCV, HIV), Luo Shi blood screening reagent as the FDA approval is the individual event operation, and the amplification program of use has nothing in common with each other, and testing process also is not quite similar; The employed TMA technology of GEN-PROBE company of another family of FDA approval can increase simultaneously HCV and HIV, but the HBV that can not increase simultaneously; Detect in addition or be different pipe operations, each surveys each; Be with pipe operation, in case but measurement result can not be differentiated HCV or HIV when positive.Up to now, U.S. FAD only ratified above-mentioned two products and can be used for blood nucleic acid screening.3) complex operation, poor repeatability, flux is low, is unsuitable for Clinical Laboratory and large-scale blood screening.4) poor compatibility provides the producer of nucleic acid extracting reagent often not provide augmentation detection reagent or the two well not to mate mostly, needs the user to adjust even optimizes; The reagent that coupling can be provided simultaneously that has, but specific nucleic acid automatic extracting instrument not necessarily be applicable to; Even above-mentioned condition is met, the user also will select specified instrument such as COBAS AMPLICOR etc. for use according to the requirement of augmentation detection reagent.5) reagent often is not instant, need to dissolve dry powder before for example using, in some bottles Bottle ﹠ Can jar, add organic reagents such as the ethanol of specified amount or Virahol, before using, the reagent that has also has complicated preparation work, the careless slightly utter failure that just causes test.The operate miss that reagent is prepared this step is also introduced in different personnel's operation; Even and some reagent is in case the Kaifeng back or the back cryopreservation validity period of redissolving also have only several days, is unsuitable for multiple batches of under the less situation of specimen amount especially and uses repeatedly.Aforesaid all situations have seriously hindered the extensive use of blood nucleic acid screening in mechanisms such as blood station and inspection and quarantine, also be unfavorable for the widespread use of detection of nucleic acids, become one of bottleneck of the blood screening and clinical nucleic acid method of inspection development in big-and-middle-sized hospital inspection section office.

Therefore; this area presses for exploitation and is suitable for the automatization, high-throughput, the synchronous nucleic acid extraction of multinomial purpose of conventional blood nucleic acid screening and augmentation detection technology platform and can be applicable to the stable performance of large-scale production, the test kit of workable, instant reagent in real time, and has relevant instrument, consumptive material to match.

Summary of the invention

The object of the present invention is to provide the test kit of a kind of energy simultaneous extraction HBV, HCV, the nucleic acid of HIV virus, synchronously real-time augmentation detection HIV, HBV, HCV viral nucleic acid, to overcome traditional Protein Detection inherent defective and deficiency, remedy insurmountable critical defect on many deficiencies of existing similar products at home and abroad and even the methodology simultaneously.

The present invention proposes method and the test kit that whether HIV (acquired immune deficiency syndrome (AIDS)), HBV (hepatitis B), HCV (hepatitis C) viral nucleic acid exist in a kind of energy simultaneous extraction and synchronous The real time measure serum or the plasma specimen.Comprise following four relatively independent contents that are mutually related again according to the method described in the present invention, and provide optional combination for complete realization test kit of the present invention.1. the invention discloses a kind of based on hybridization principle select for use magnetic bead as the automatization medium, use the magnetic bead separometer to realize method and the modification method and the reagent of the multiple viral nucleic acid of specificity synchronization acquistion, it is characterized in that having selected for use with follow-up augmentation detection in the oligonucleotide of a corresponding to biotin modification of primer sequence as the capture probe of nucleic acid extracting reagent, its advantage is not only to have avoided the PCR of omission but also non-interfere with subsequent to detect.2. the present invention also discloses the quick add-on PCR structure and the purification process of a kind of novel RNA external standard (being positive control) and RNA internal reference (mark in competitive), do not see any bibliographical information or exemplary application as yet, be characterized in fast, efficiently, advantage is not have the pollution of template DNA or its pollution to hang down to detecting in the RNA goods, especially is suitable as the validity of the omnidistance monitoring of Quality Control nucleic acid extraction, RT reaction, pcr amplification detection.3. the invention allows for the basis of design environmental type test kit--the genetically engineered of-positive control and internal reference makes up method or PCR structure method, makes positive control and internal reference in the test kit all not have infectivity.4. at last also the most key is to the invention discloses a kind of TaqMan pcr amplification method that is suitable for the two multinomial joint-detection of enzyme of single stage method single tube of blood screening, this system contains the anti-pollution composition of UNG-dUTP " built-in ", the project that is detected is HBV DNA, HCV RNA, three viral nucleic acids of HIVRNA, existing DNA, RNA is arranged again, and have in the system and detect the internal reference component whether false negative takes place, the advantage that this invention is the most outstanding is the outstanding sensitivity of this detection reagent and the fraction of coverage of specificity and each hypotype, and different subtype amplification efficiency Approximate Equivalent makes it can satisfy the extremely harsh requirement of blood nucleic acid screening; Its outstanding feature is to use cheap two enzymes (promptly containing reversed transcriptive enzyme and hot resistant DNA polymerase) system, is different from and single enzyme systems such as the rTth of Roche company or ZO5 enzyme.Conspicuous, to abandon outside the price factor of single enzyme system raw material costliness, the optimization difficulty of single enzyme system and the optimization difficulty of dual-enzyme system cannot be mentioned in the same breath.In the past, use relatively inexpensive dual-enzyme system use to sensitivity, specificity, antipollution, multinomial order have thought by majority in the conventional blood screening of requirement infeasible.

1, the invention provides a kind of angling and get principle based on hybridization, HBV is extracted in specificity synchronization acquistion, HCV, the method of HIV nucleic acid, it is characterized in that having selected for use with follow-up augmentation detection in the oligonucleotide of a corresponding to biotin modification of primer sequence as the capture probe of nucleic acid extracting reagent, its advantage is not only to have avoided omission but also do not disturb PCR to detect: the fragment that every in theory PCR can increase all can not extracted this step owing to the defective of sequence capture probe design is missed at sample, not only because the conservative property of the primer sequence that is used to increase and specificity live through strict screening and clinical practice check, the more important thing is, the positive sample that every PCR can increase necessarily can be caught by this probe; On the other hand, final step based on hybrid capture method nucleic acid purification is the high temperature wash-out, be difficult to avoid the magnetic bead of biotinylated probes and streptavidin parcel to separate and enter elutriant, might there be the probe of μ M level concentration to enter elutriant, if the sequence of probe is not the primer part, just might disturb pcr amplification as redundant oligonucleotide.Otherwise, only be that the primer ratio is not an equal proportion in the PCR reaction at most, the PCR under the PCR fundamental sum equal proportion under this particular case is as broad as long.In addition, this invention also provides a kind of universal capture method of accommodation, promptly design probe in the middle of a section, its 3 ' termination of literalness common oligonucleotide (consistent with a primer sequence in the follow-up fluorescent PCR system) has a series of A such as Oligo (d A) _18-25,, use the Oligo (d T) of biotin modification as the trunk probe of middle intermolecular hybrid ₂₅As general hybrid capture probe; 5 ' end nucleotide sequence of middle probe is as " the identification arm " of the viral nucleic acid in the specific recognition sample, and hybridize and catch viral nucleic acid, and Oligo (d A) of its 3 ' end _18-25As biotinylated Oligo (d T) ₂₅Recognition site, by it is caught, the complex body that terminal crossing forms is that the magnetic bead of streptavidin parcel is adsorbed, through washings A, B, the cleaning successively of C is removed non-specific hybridization thing and PCR/RT-PCR inhibition such as carbohydrate, metaprotein, fat, impurity such as high salt or heavy metal, last magnetic bead enters elutriant through heating, and nucleic acid-templated eluting from hybridization complex enters elutriant. and still adsorbing biotinylation Oligo (d T) ₂₅Useless magnetic bead is removed automatically through instrument, and elutriant promptly can be used as the template that fluorescent PCR detects.The advantage of this method is biotin labeled Oligo (d T) ₂₅Be consistent, joint efficiency only depends on the hybridization efficiency of the middle probe of design.This invention also provides the method for another kind of accommodation, synthetic one comprises above-mentioned HBV, HCV, the oligonucleotide of the linear array of HIV specific probe sequence, as one the three same probe of allying the communists, the centre can between be separated with connecting arm such as 3-8 T or A, the modification of vitamin H still is positioned at 5 ' end of common probe, can save the amount of some streptavidin parcel magnetic beads like this, the corresponding amount of magnetic bead that improves is to excessive, help the further raising of detection sensitivity, be particularly suitable for the saturated test of magnetic bead usage quantity (extracting) as multinomial purpose, because magnet absorption magnetic bead has a saturation ratio, just can't adsorb totally more than a certain amount of.This invention is not got rid of the combination or the composite combined of aforesaid method, and as synthetic oligonucleotide that comprises the linear array of above-mentioned HBV, HCV, HIV specific probe sequence, its 3 ' termination has a series of A such as Oligo (d A) _18-25,, use the Oligo (d T) of biotin modification as the trunk probe of middle intermolecular hybrid ₂₅As general hybrid capture probe etc.

Use the remarkable advantage of above-mentioned hybridization principle specific extraction purifying to be to improve the specificity that detects, avoided the false positive of PCR.Though PCR is as the high detection method of a specific specificity, but the false positive report that still has a certain proportion of uncontamination factor to cause, got rid of primer sequence and designed after irrational factor, be considered to the template of extracting in to contain other a large amount of heteronuclear acid relevant.And thereby the specific extraction method is only extracted the nucleic acid of being studied and has been avoided interfering factors.In addition, this method is based on the hybridization principle, is used for extracting being rich in the PCR inhibitory substance such as hyperlipidemia, height haemolysis, jaundice even heparinization blood specimen are all noiseless; And traditional pellosil extracting method is because the similar linear molecule that is rich in negative charge that is to nucleic acid of heparin, all is easy to be adsorbed onto on the pellosil at last wash-out and goes into elutriant and suppress PCR/RT-PCR as template.In the conventional blood sieve since the daily test mark these are many, workload is big, even the PCR false positive low to ten thousand/, the sample probability that is rich in inhibition is ten thousand/, will run into one less than several days, so both lost time and be used for repetition measurement, waste blood again, also might cause false-negative generation because the PCR inhibition exists.The disclosed specificity simultaneous extraction of this invention method provides the dual specificity guarantee of hybridization extraction with TaqMan probe method PCR in conjunction with following synchronous amplification detection reagent for blood screening, and effectively removed the PCR inhibition that may contain in the extremely rare sample, avoid false negative to take place.

2, the present invention also provides the PCR that adds end biotin modification primer fast of a kind of novel RNA external standard (being positive control) and RNA internal reference (mark in competitive) to make up and purification process, does not see any bibliographical information or exemplary application as yet.Be characterized in fast, efficiently not having the pollution of template DNA or its pollution low in the RNA goods to detecting.Be used to make up the primer of RNA internal reference, it 5 ' adds end and is modified with vitamin H, and wherein a primer 5 ' adds end and is the additional transcripting promoter recognition site sequence that the T7 RNA polymerase is arranged.Can prepare the RNA internal reference fast, after PCR/RT-PCR obtains to contain the DNA of target sequence, the magnetic bead of streptavidin parcel is directly added, in conjunction with having biotinylated PCR product, discard liquid, after PCR damping fluid washing 2 times, damping fluid is transcribed in adding and the T7 RNA polymerase is diluted the RNA that transcribe out of gained and original dna profiling mixture with the PCR damping fluid 37 ℃ of reactions after 50-60 minute, after the thermally denature in quenching on ice, the biotin labeling that has on the dna profiling is by the magnetic bead of mortise in the streptavidin parcel, and the RNA that transcribes out does not have biotin labeling and can not be incorporated into magnetic bead, and because sex change separates itself and dna profiling fully, centrifuging and taking supernatant or separate magnetic bead with magnet, remaining purified RNA goods can be further concentrated and purified with ethanol sedimentation.Present method is used to make up RNA external standard and RNA internal reference, as long as a positive sample extract, PCR/RT-PCR or twice PCR/RT-PCR add two hours purifying work, can make purified RNA goods as Quality Control.Traditional method is that pcr amplification obtains fragment, and access carrier such as T carrier behind the purifying are chosen positive colony extraction plasmid and with the recombinant plasmid linearization for enzyme restriction, in-vitro transcription is carried out in repurity, uses dnase digestion again, repurity RNA.The breadboard many equipment of whole process need genetically engineered needs carrier, competent cell, restriction enzyme, needs culturing bacterium, whole flow process length consuming time.It is simple and direct much quick that method disclosed by the invention is by contrast wanted, and present method biggest advantage is that also the RNA goods that provide are pure in traditional method far away.Because short segmental dna profiling digestion is always halfway, through repeatedly digestion and purifying even use post method purifying are still of no avail, no RT contrast PCR test still detects a considerable number of DNA.And the RNA contrast that contains DNA is difficult to the outer contrast or the internal reference that detect as RNA viruses truly, because of it can not actual response RT efficient; If set up quantitatively contrast based on this, then there is quantitative inaccurate risk, because RNA viruses detects is its RNA, and quantitatively contain DNA/RNA in the contrast, certainly will cause to detect asynchronously to be tending towards distortion, and be difficult to quantitative Quality Control as virus load.

3, the invention allows for the basis of design environmental type test kit--the genetically engineered of-positive control and internal reference makes up method or PCR makes up method:-----according to hardware condition designs such as the requirement of customers, plant and instrument used in the preparation of positive control and the preparation of internal reference, the independent use of positive control and internal reference or mix.The present invention has significant advantage aspect biological safety.

The present invention is according to the nucleic acid type of detect virus, and the FFI nucleic acid fragment of external preparation is as the positive control (RNA external standard) and the internal reference (mark in the RNA) of test kit; Positive control preparation is that (as HBV is that the plasmid that contains HBV C district is the plasmid that contains HCV 5 ' NCR district through HCV for the nucleic acid fragment that will contain the amplified target sequence, HIV is the plasmid that contains HIV GAG district) through linearization for enzyme restriction DNA, become RNA through in-vitro transcription again, and obtain the RNA goods with purifying behind the dnase digestion.Certainly can use method of the present invention to make up fast, here the use of only narrating general traditional method is to emphasize the basis as green test kit: can not introduce communicable factor in test kit, although its operator must be considered as operating of infectivity sample according to rules.Above-mentioned HBV, HCV, HIV positive control both can use separately, can mix use again, also can use engineered method that three above-mentioned slice groups are installed to linearizing again in the carrier, use its DNA or RNA after the in-vitro transcription as required, or the two mixes use.

Internal reference adopts competitive interior mark, just equally have identical PBR with positive control, but the nucleotide sequence of primer transcribed spacer or arrange different, make its can not with detection probes (positive control, can be described as the external standard probe) combination, but can combine with interior mark probe.This internal reference can make up by the rite-directed mutagenesis of positive control template, also can use the method for block PCR to make up.Equally, internal reference is built into corresponding D NA or RNA according to the nucleic acid type that detects virus, can use separately, also can mix use, also can three internal reference slice groups be installed in the fragment by the method for engineered method or add-on PCR.In a word, select only scheme as required, both be particular about the effective monitoring of internal reference to the whole process of whole nucleic acid extraction process, RT process, pcr amplification testing process, the introducing of being particular about it does not again influence the sensitivity of detection; Also to be particular about the introducing amount of each test internal reference simultaneously: too high then can not the downtrod situation of actual response sample, do not see as slight inhibition; Cross the low then mortality height of internal reference, poor repeatability; The general high 2-20 of the detection sensitivity internal reference amount doubly of selecting can change specific to practice as monitoring.So specific to application scheme, the internal reference of appropriate amount joined participate in the nucleic acid extraction step in the lysate, this is a kind of embodiment of standard, but not repelling the user who has directly adds internal reference when configuration PCR reaction solution, and in the sample leaching process, do not introduce internal reference, this practice also can realize whether containing in the sample extract effective monitoring of PCR/RT-PCR inhibition, therefore also should be subjected to certainly.The other user detects wavelength because the fluorescent PCR instrument is a single passage, and whether mark probe in detecting false negative takes place in can't using, though also can use test kit provided by the present invention, should be careful to result's judgement.

4, the present invention also proposes a kind of RT-PCR TRAP based on the TaqMan probe of synchronous multinomial detection in real time, be characterized in using the single stage method RT-PCR dual-enzyme system reagent of single tube, the project of multinomial detection had both contained DNA, contained RNA again, and selected antipollution system is classical UNG-dUTP system.Employed single stage method RT-PCR reagent is dual-enzyme system, promptly contains reversed transcriptive enzyme and hot resistant DNA polymerase, and reversed transcriptive enzyme is thermo-labile, and the RT temperature can not be higher than 60 ℃, and preferred version is 45-55 ℃, especially 50 ℃.And the UNG (as the at present commercially available overwhelming majority's UNG) of conventional genetically engineered preparation under this temperature has the DNA that contains U (being called U-DNA) that the degraded reverse transcription comes out, therefore can not use this UNG, to be heat labile UNG have a liking for cold marine bacteria BMTU3346 (heat-labile UNG/heat-labile UNG:EC3.2.2.3Roche-derives from and has a liking for cold bacterium BMTU3346, USB--and come from Gadus morhau, Epicenter--and come from Gadus morhau etc., and these companies all have product to sell) as coming to preferred version.It is medium-term and long-term stable and in case only two minutes its transformation period when temperature is 40 ℃ in the PCR damping fluid to be characterized in being stored in certain substrate, need not do specific program setting during application, because (when mixing) this enzyme just works in the preparation work of RT-PCR reagent and RNA template, rapidly with last time RT-PCR pollute as residual U-DNA degraded, best reaction be 20--30 ℃ 5-20 minute.When beginning to carry out RT-PCR, the rapid inactivation of this UNG and do not influence RT or PCR.The most outstanding advantage of synchronous multinomial detection reagent of the present invention is the outstanding sensitivity of this detection reagent and the fraction of coverage and the different subtype amplification efficiency Approximate Equivalent of its specificity and each hypotype.Still not having at present a kind of the report uses two enzyme single tube RT-PCR systems to introduce the antipollution scheme and can be successfully used to the multinomial synchronous detection (promptly having DNA that RNA is arranged again) that sensitivity is had requirement, have only one piece of Japanese Roche Holding Ag to be used for the Preliminary Applications report of blood sieve about many inspection ZO5 enzyme list enzyme RT-PCR TaqMan systems of automatization by retrieval, and its use is single enzyme system, its shortcoming is conspicuous, this system costs an arm and a leg and for the said firm's patent owns, is difficult to apply; Dual-enzyme system is owing to use two kinds of cheap enzymes, because two kinds of enzymes are mutually independent system separately, the peak optimization reaction condition has nothing in common with each other, and highly sensitive two enzyme single tube RT-PCR systems in the industry cycle are acknowledged as the problem with certain difficulty always, simultaneously one of focus of the feasible relevant scholar's research in recent years of its relatively inexpensive cost.Multinomially be considered to have a challenging difficult problem equally with inspection.

The quality that the present invention is outstanding depends on selected HBV primer to a great extent, HBV TaqMan probe; The HCV primer, HCV TaqMan probe; The HIV primer, HIV TaqMan probe sequence.Therefore, the primer that the present invention announced, probe sequence undoubtedly should be subjected within the rights protection applying for requiring.

In sum, the invention reside in and propose a kind of test kit of measuring HIV (acquired immune deficiency syndrome (AIDS)), HBV (hepatitis B), HCV (hepatitis C) viral nucleic acid, the magnetic bead that comprises simultaneous extraction HBV-HCV-HIV nucleic acid extracts reagent and medical disposable material, wherein contain the agent of disinthibiting, lysate, magnetic bead suspension, washings A, washings B, washings C, elutriant etc., with HBV-HCV-HIV nucleic acid amplification reagent, wherein contain RT-PCR reaction solution, probe, enzyme mixture, positive control, negative control and internal reference etc.The coupling instrument can use KHB DP-1000, the KHB DP-2000 etc. of Shanghai section China test apparatus system manufacturing, but be not limited to these, may also have suitable with it instrument of the suitable with it instrument of MagNA pure LC instrument, the suitable with it instrument of TECAN, the ThermoLabsystem company of ROCHE, instrument, ABI company that Beckman-Coulter is suitable with it etc.

The above-mentioned lysate of mentioning is the guanidine thiocyanate solution of virion in the humoral specimens such as cracking serum, blood plasma, be specially the 4-6M guanidine thiocyanate, 20-50mM Tris, PH6.0-8.0,1-20%NP-40,1-20%TritonX-100,0.1-10%SDS, 0.01-1 μ M biotinylated probe (capture probe), 1-10% polymkeric substance etc.Wherein capture probe is: 5 ' BIOTIN-acc acc aaa tgc ccc tat-3 ' (to HBV) (SEQ.ID.NO.28), 5 ' BIOTIN-agtacc aca agg cct ttcg-3 ' (to HCV) (SEQ.ID.NO.29), 5 ' BIOTIN-cta tgt cac ttcccc ttg gtt ctc tc-3 ' (to HIV) (SEQ.ID.NO.30), can entrust Shanghai to give birth to the precious biosynthesizing of worker or Dalian, purity is PAGE or HPLC; Described magnetic bead suspension is the magnetic bead of streptavidin parcel, be specially the magnetic bead of the super paramagnetic material that contains TWEEN20, the surface is surrounded by even one deck streptavidin, diameter is 0.1-10 μ M, contain compositions such as BSA, NaN3, as the magnetic bead of E.Merck company, Roche company, Invitrogen company, Toyabo company, Agowa company, Chemogen company, Promega company, the production of Seradyn company; Described washings A, B, C are the buffered soln that contains lithium chloride, be specially washings A:LiCI, Tris PH7.0, the dodecyl sodium sulfonate lithium, dispersion agents such as sanitas and Tween 20, pigment is as pinkish red or other dyestuff etc., wherein the concentration of LiCI can be from 0.15M-3M, Tris concentration can be 10-100mM, PH can be 6.0-8.0, the concentration of dodecyl sodium sulfonate lithium can be from 1%, and sanitas can be sodium azide or PROCLIN300, PROCLIN150, Thiomersalate, microbiotic etc., and the concentration of Tween 20 can be from 0.001-0.1%; Washings B: composition and washings A are similar but do not contain the dodecyl sodium sulfonate lithium, and pigment is different or do not contain pigment; Washings C: the concentration of the similar but LiCI of composition and washings B reduces (as being 0.1-1M) and does not contain pigment etc.; The described agent of disinthibiting is the alcoholic solution that contains proteolytic enzyme, the agent of disinthibiting: contain proteolytic enzyme and glycerine etc.; Described elutriant is low salt buffer or water, and the Tris damping fluid of the purified water preparation that elutriant: DEPC handled is added with Tween 20 dispersion agents such as grade and EDTA, NaN3, DTT etc.

Among the present invention, sample extracts (containing in the lysate) used middle probe and is

5’-acc?acc?aaa?tgc?ccc?tat?ttt?ttt?agt?acc?aca?agg?cct?ttc?g?aaa

aaa?cta?tgt?cac?ttc?ccc?ttg?gtt?ctc?tc?aaa?aaa?aaa?aaa?aaa?aaa

Aaa aaa-3 ' (SEQ.ID.NO.15) and 5 ' Biotin-Oligo (d T) ₂₅(SEQ.ID.NO.16).

Described primer: hepatitis B virus detects primer at the fragment gene fragment in the encoding gene in hepatitis B virus core district (C district), BF:5 '-acc acc aaa tgc ccc tat-3 ' (SEQ.ID.NO.1), BR:5 '-ttc tgc gacgcg gcg a-3 ' is (SEQ.ID.NO.2).Hepatitis C virus detects the 5 ' non-translational region of primer at the hepatitis c virus gene sequence, two sections primer sequences are: CF:5 '-cgg gag agc cat agt gg-3 ' (SEQ.ID.NO.4), CR:5 '-agtacc aca agg cct ttc g-3 ' is (SEQ.ID.NO.5).Virus of AIDS detects the gene order of primer at HIV gag district, two sections primer sequences are: IF:5 '-gac atc aag cag cca tgc aaa t--3 ' (SEQ.ID.NO.7), IR:5 '-cta tgt cac ttc ccc ttg gtt ctc tc-3 ' is (SEQ.ID.NO.8).

Described probe is the Taqman probe, the detection probes that comprises interior mark (being internal reference) probe and three kinds of viruses, respectively by different fluorophor marks or identical fluorophor mark, but the fluorophor of mark probe is different from the fluorophor of detection probes at least, and preferred TaqMan probe is the TaqMan probe of TaqMan-MGB probe or non-fluorescent quenching group, as BHQ-1 or BHQ-2 etc.Because conventional TAMRA quenching group is the fluorescent quenching group, self has fluorescence, and this fluorescence is understood mutual superposition in disturbing in multiple reaction, and itself is the passage that a fluorophor has occupied instrument, makes multiple detection go a re-detection less; The probe sequence that adopts among the present invention is: the HBV probe is: 5 ' FAM (Ned)-cgacga ggc agg acc cct aga aga a-NFQ3 ' (SEQ.ID.NO.3); The HCV probe is: 5 ' FAM (Vic)-ctg cggaac cgg tga gta cac-NFQ-3 ' (SEQ.ID.NO.6); The HIV probe is: 5 ' FAM (Fam)-cca tca atg aggaag ctg cag aat ggg ata-NFQ3 ' (SEQ.ID.NO.9); Interior mark probe is that ICP:5 ' Vic (Rox)-aac cttgga acc ttg gaa ctg gag aga gaa-NFQ3 ' (SEQ.ID.NO.14).

Described RT-PCR reaction solution is for containing d ATP, d CTP, d GTP, d UTP, damping fluid, KCI, sanitas and stablizer, above-mentioned primer etc.; Described enzyme mixture is a multienzyme component system, specifically contain reversed transcriptive enzyme, archaeal dna polymerase, RNasin, heat-labile UNG, RT-PCR promotor (as heat shock protein(HSP), gp32 albumen etc.) and enzyme stabilizers (as various carbohydrate molecules and tensio-active agent, DTT, proteinase inhibitor) etc.; Be specially: contain 1.8Mm d NTPs (containing d UTP) among the RT-PCR reaction solution buffer, 3.6mM DTT, 3.5mM MgCl ₂, each 0.4 each primer of μ M (BF, BR, CF, CR, IF, IR) etc.; Enzyme mixture: 0.6U/ μ l AMV reversed transcriptive enzyme, 0.6U/ μ l warm start Taq archaeal dna polymerase, 1U/ μ l RNasin; 0.1U/ μ l UNG (heat-labile) etc.; Probe: 10 μ M probe HBV Probe, HCV Probe, mark probe, stablizer and damping fluid in the HIV Probe, 5 μ M.

Described positive control is: contain section H BV DNA, HCV RNA, the segmental mixture of HIV RNA, be a fragment gene DNA or a RNA in the amplifying target genes outside, wherein the HBV positive control does not only contain HBV RNA by section of DNA, the HCV positive fragment only has one section RNA not contain HCV DNA, the HIV positive fragment only has one section RNA not contain HIV DNA, and three's mixture suitably is stored in the nucleic acid stability liquid positive control as test kit after the dilution; Described internal reference be competitive in mark RNA, include the identical PBR of test item, but different with the probe land of test item, can not be in conjunction with detection probes, but can be in conjunction with interior mark probe.For example internal reference can be the in-vitro transcription RNA that contains one section HIV PBR, but probe area can not be discerned the HIV probe through sudden change (PCR sudden change method or other genetic engineering means), and the interior mark probe in can the identification agent box, this internal reference is RNA, does not contain DNA; The mark probe is the oligonucleotide that has no to concern with test item of one section synthetic in described, its TaqMan of 5 ' modifies the fluorophor of the detection probes of the different arbitrary positive controls of fluorophor, be convenient to distinguish detected result and internal reference result, and the result of internal reference is necessary for the positive when requiring detection result of specimen negative with a sample.Described negative control is the blood plasma of donors with normal.

Test kit of the present invention sample disposal adopted is the magnetic bead extraction method.This technology is used streptavidin magnesphere (based on the nucleic acid hybridization principle), because DNA all has identical cross performance with RNA, so it is generally acknowledged this method DNA and RNA there is identical or proximate yield, by repeatedly removing impurity after the washing, at last nucleic acid is eluted the template that detects as nucleic acid amplification in less salt or water.Can use KHB DP-1000 or 2000 instruments, also not get rid of the use on other self-reacting device simultaneously.After this technology is applied to this diagnostic kit, toxic reagents such as phenol, chloroform, primary isoamyl alcohol have been avoided using in the traditional method on the one hand, introduce mechanized operation on the other hand, high throughput automated operation or semi-automatic operation have reduced the influence of manual operation factor to the result.The training of those skilled in the art by the short period of time can very fast its operating process of grasp and understanded its technical key point; can also be according to content provided by the invention; in addition the modification of a little is flexible forms new techniqueflow and test kit, but in these rights protection scopes that all fall into the present invention and claimed.

In the test kit of the present invention augmentation detection employed be the single stage method RT-PCR TaqMan dual-enzyme system of single tube, this technology adopts reversed transcriptive enzyme such as AMV or MMLV or Superscript I/Superscript II/SuperscriptIII or Ominiscript, hot resistant DNA polymerase adopts Taq enzyme or gold medal Taq enzyme or so-called warm start Taq enzyme (as having its monoclonal antibody or chemically modified or the aglucon oligonucleotide Taq enzyme of inactivation in advance), also can select other heat-resisting polymerase such as Tfl enzyme etc. for use.The PCR buffer that selects for use is TRIS-HCI or TRIS-H ₂SO ₄Or TRIS-HOAc or Good ' s buffer B icine, Tricine, contain Repone K, also can contain ammonium sulfate, methyl-sulphoxide, methane amide, glycerine, TMAC, urea, gp32 albumen, trimethyl-glycine, bovine serum albumin, nonionogenic tenside, TWEEN-20, TRITON X-100, NP-40, Brij-35, polyoxyethylene glycol, PVP, DTT, Aptamer, protamine, HSA, SSB, Alpha Casine, topoisomerase 1 and 2, sucrose, seminose, sorbyl alcohol, trehalose, pectinose, ficoll, glycogen, gelatin, salmon sperm DNA, calf thymus DNA, rRNA, tRNA, PolyA RNA, OLIGO (dT), additives such as compound agent MP voluntarily.

The present invention design be used for synchronous multinomial detection in real time, based on the RT-PCR TRAP of TaqMan probe, being characterized in designing primer and probe sequence, to be characterized as virus of AIDS, hepatitis B virus, hepatitis C virus institute peculiar.Wherein, two sections primer sequences of hepatitis B virus are: BF:5 '-acc acc aaa tgc ccc tat-3 ' (SEQ.ID.NO.1), BR:5 '-ttctgc gac gcg gcg a-3 ' (SEQ.ID.NO.2), probe adopts the Taqman probe type, promptly at probe base two ends difference mark fluorescent reporter group and quenching group, the HBV probe sequence is: 5 ' FAM (Ned)-cga cga ggc agg acccct aga aga a-NFQ3 ' (SEQ.ID.NO.3).

Two sections primer sequences of hepatitis C virus are: CF:5 '-cgg gag agc cat agt gg-3 ' (SEQ.ID.NO.4), CR:5 '-agt acc aca agg cct ttc g-3 ' (SEQ.ID.NO.5), probe adopts the Taqman probe type, promptly at probe base two ends difference mark fluorescent reporter group and quenching group, the HCV probe sequence is: 5 ' FAM (Vic)-ctg cgg aac cgg tga gta cac-NFQ-3 ' (SEQ.ID.NO.6).

Two sections primer sequences of virus of AIDS are: IF:5 '-gac atc aag cag cca tgc aaat--3 ' (SEQ.ID.NO.7), IR:5 '-cta tgt cac ttc ccc ttg gtt ctc tc-3 ' is (SEQ.ID.NO.8).Probe adopts the Taqman probe, promptly at probe base two ends difference mark fluorescent reporter group and quenching group.The Taqman probe sequence is: 5 ' FAM (Fam)-cca tca atg agg aag ctg cag aat ggg ata-NFQ3 ' (SEQ.ID.NO.9).Interior mark probe is that ICP:5 ' Vic (Rox)-aac ctt gga acc ttg gaa ctg gag agagaa-NFQ3 ' (SEQ.ID.NO.14).

When pcr amplification, the fluorescent PCR instrument detects the fluorescent signal that each reaction tubes produces, strength of signal surpasses the positive that is reported as of detector threshold values, and template content has negative logarithmic relation in the pcr amplification cycle index (being called CT) of this moment and the sample, is the record automatically of instrument institute.The real-time fluorescence PCR instrument can be realized the fluorescent signal variation diagram of the whole process of sample template amplification, can very judge the positive and negative of sample visually and can distinguish its power.

Because detection of nucleic acids complicated operation, whole flow process are subject to multiple factor affecting and may make the amplification failure, test kit behaviour may be drawn thus with personnel detect the negative wrong conclusion of sample.For avoiding the generation of this kind situation, must set up contrast in each detector tube is internal reference, even like this because extremely rare mistake such as a certain specimen tube forgotten to have added template, internal reference can be monitored out, perhaps a certain hole of PCR instrument temperature efficiency has problem, and internal reference also can monitor out.

Use the internal reference quality control system in the optimum composition of test kit of the present invention, promptly in lysate, add the internal reference RNA of particular sequence, detect each step through cracking, purifying, reverse transcription, amplification, TaqMan jointly with sample.This internal reference sequence contain one with the irrelevant nucleic acid fragment of sample to be measured (as the rearrangement of the sequence fragment immediately of artificial fully design or test item probe sequence or one section plasmid such as PUC18 sequence etc.), two ends contain primer sequence used in the test kit, so can be shared with a pair of primer amplification with sample to be measured.Used internal reference probe is the Taqman probe of specificity at the irrelevant sequence of this section.If it is all negative that test-results is sample, internal reference, then show test failure, test-results is invalid; Other situation of test-results then can take place and also test-results effective.Do not lack quality control standard one although do not contain the test kit of internal reference, do not influenced normal use.

Test kit of the present invention is as a kind of reagent of qualitative detection, but do not get rid of the reagent that can be used as detection by quantitative.Because the method applied in the present invention is TaqMan PCR, only need to introduce the test kit that outer standard substance can become quantitative analysis, though the meaning of blood nucleic acid screening is qualitative analysis, lay particular emphasis on the sensitivity and the specificity of reagent; Clinical detection and curative effect of medication monitoring then lay particular emphasis on quantitatively, the fastidious quantitative accuracy of carrying capacity test.

Test kit of the present invention need cooperate nucleic acid magnetic bead extraction apparatus and fluorescent PCR instrument to use.

The present invention has the advantage of following several respects: (1) closure detects, and the level of automation height has reduced manual operation, has avoided possible pollution and artificial error; (2) simultaneous extraction of multiple nucleic acid virus detects synchronously in real time; (3) highly sensitive, specificity is good, the precision height, and good stability is convenient to operation; (4) be applicable to extensive high-throughout blood screening, carry out nucleic acid screening, also check applicable to the clinical nucleic acid of high capacity highly effective rate as Blood Center and blood products manufacturing enterprise.(5) whole flow process time weak point, the Energy Efficiency Ratio height.Only test several hrs for 96 and just finish and report the result that confidence level is high fast.

Description of drawings:

Fig. 1 is a Streptavidin magnetic capture nucleic acid schematic diagram.

Wherein (A) is for combining the magnetic bead structure iron of biotinylated probes;

(B) caught by the Streptavidin magnetic bead for hybridization complex, hybridization complex-magnetic bead is by the synoptic diagram of magnet institute enrichment;

(C) be the schematic diagram of whole hybrid capture magnetic bead.

Fig. 2 is the synoptic diagram that makes up of DNA/RNA reference substance fast

Fig. 3 is the synoptic diagram that makes up of internal reference DNA/RNA quality control product fast

Embodiment

Embodiment 1 biotin modification primer add-on PCR method prepares internal reference and positive control (basis of environmental type test kit) fast,

1, HIV internal reference and positive control is prepared as follows

1.1 the quick structure of positive control: entrust Shanghai to give birth to the synthetic following primer of worker, sequence is:

5’BIOTIN-AAT?TCT?AAT?ACG?ACT?CAC?TAT?AGG?GAG?gac?atc?aag?cag?cca?tgc?aaa?t-3’

(5 ' end italicized item is a T7 RNA pol promoter region, and 3 ' end is the upstream primer in HIV GAG district)

(SEQ.ID.NO.10)

5 ' BIOTIN-cta tgt cac ttc ccc ttg gtt ctc tc-3 ' (downstream primer in HIV GAG district)

(SEQ.ID.NO.11)

Get the sample portion of the HIV nucleic acid strong positive clinically plasmid portion of HIV GAG gene (or contain), extract viral nucleic acid, strictly operate by its working instructions requirement with QIAamp ViralRNA Mini Kit (QIAGEN company).The single stage method RT-PCR reagent that extract product is set up through the present invention, with above-mentioned two primer amplifications, operate by following loop parameter:

50 ℃ 30 minutes--94 ℃ 4 minutes---94 ℃ 20 seconds,

55 ℃ of 20 seconds, circulation 30-40 time, extend at last 72 ℃ of 5 minutes ends.

72 ℃ 30 seconds,

Can get the band that partial reaction product electrophoresis detection is viewed as a 160bp size.Add the long-pending 1XPCR Buffer of decaploid in the amplified production; the magnetic bead that adds the streptavidin parcel of 100-500 μ g; room temperature is 10 minutes behind the mixing; low-speed centrifugal removes supernatant; add 1XPCR Buffer 400 μ l washing magnetic bead 2 times; collect magnetic bead; use transcript reagent such as NTPs in the RiboMAXTM Large ScaleRAN Production Systems-T7 test kit (Promega company); Rnasin; DTT; transcribe damping fluid; T7 RNA Pol is to specifications behind the application of sample; transcribed 1 hour for 37 ℃; dilute with 1XPCRBuffer equally after the taking-up; but do not wash and need denaturing treatment; can adopt 90 ℃ of thermally denatures or NaOH sex change also can select the guanidine thiocyanate sex change for use; the last centrifugal template DNA that will be combined on the magnetic bead is removed; get its supernatant in a new clean centrifuge tube; add the dehydrated alcohol precipitated rna, will transcribe matrix such as NTPs etc. and remove totally, be dissolved in the RNA protective material.Certainly also can use the DNA enzyme further to digest.Resulting RNA is HIV RNA positive control mother liquor, and its gradient dilution can be participated in nucleic acid extraction, RT reaction, PCR TaqMan augmentation detection whole process in certain liquid, as the positive control of the green of the Biosafety of test kit.

1.2 the structure of internal reference: based on the positive control dna of above-mentioned structure (also can a sample of HIV nucleic acid strong positive clinically), entrust Shanghai life worker to synthesize following primer, sequence be:

5 '-AAC CTT GGA ACC TTG GAA CTG GAG AGA GAA gagtgcatccagtgcatgcag-3 ' (reset for HIV detection probes sequence, with detection probes identical GC% arranged, similar Tm value by its 5 ' end of SEQ.ID.NO.12; 3 ' end is for adjoining the nucleotide sequence of detection probes 3 ' end position on the HIV positive control)

(its 5 ' end of SEQ.ID.NO.13 is complementary with the 5 ' end of SEQ.ID.NO.12 for 5 '-TTC TCT CTC CAG TTC CAA GGT TCC AAG GTT tct ctt taa tta aca ttt gc-3 '; The nucleic acid array complementation that adjoins detection probes 5 ' end position on 3 ' end and the HIV positive control)

With the positive control dna is pcr template, is that a pair of primer and SEQ.ID.NO.11, SEQ.ID.NO.12 go amplification for another to primer with SEQ.ID.NO.10, SEQ.ID.NO.13 respectively, and the loop parameter of use is:

94 ℃ 2 minutes---94 ℃ 10 seconds,

45 ℃ of 15 seconds, circulation 20 times, extend at last 72 ℃ of 5 minutes ends.

72 ℃ 10 seconds,

Will be behind above-mentioned two kinds of amplified production purifying mixed and gradient dilution, respectively as the template of next round PCR.This take turns PCR use SEQ.ID.NO.10 and SEQ.ID.NO.11 as primer to amplification, program is the same, cycle number is increased to 30-40.This takes turns amplified production and is internal reference DNA, can get the band that the portion of product electrophoresis detection is viewed as a 160bp (HIVDNA134BP that contains, other contains TTRNA promoter sequence 27BP) size.Same its magnetic beads for purifying, transcribe, preserve all, be prepared into the pure product of HIV internal reference RNA at last with above-mentioned positive control (RNA).HIV sequence that can reference is as follows, schematic diagram such as accompanying drawing 3 that can reference.

gaca?tcaagcagcc?atgcaaatgt?taattaaagagac?catcaatgag?gaagctgcag?aatgggatag

agtgcatcca?gtgcatgcag?ggcctattgc?accaggccag?atgagagaac?caaggggaag?tgacatag

(SEQ.ID.NO.25)

2, HCV internal reference and positive control is prepared as follows

2.1 the quick structure of positive control: entrust Shanghai to give birth to the synthetic following primer of worker, sequence is:

5’BIOTIN-AAT?TCT?AAT?ACG?ACT?CAC?TAT?AGG?GAG?cgg?gag?agc?cat?agt?gg-3’

(5 ' end italicized item is a T7 RNA pol promoter region, and 3 ' end is the upstream primer in HCV NCR district)

(SEQ.ID.NO.17)

5 ' BIOTIN-agt acc aca agg cct ttc g-3 ' (downstream primer in HCV NCR district)

(SEQ.ID.NO.18)

Get the sample portion of HCV nucleic acid strong positive clinically (or contain HCV 5 ' NCR plasmid portion), extract viral nucleic acid, strictly operate by its working instructions requirement with QIAamp ViralRNA Mini Kit (QIAGEN company).Extract product, is operated by following loop parameter with above-mentioned two primer amplifications through this single stage method RT-PCR reagent:

50 ℃ 30 minutes--94 ℃ 4 minutes---94 ℃ 20 seconds,

50 ℃ of 20 seconds, circulation 30-40 time, extend at last 72 ℃ of 5 minutes ends.

72 ℃ 30 seconds,

Can get partial reaction product electrophoresis detection and be viewed as a 187bp bright band.Prepare the positive control of the green of Biosafety with 1.1 operations.

2.2 the structure of internal reference: based on the positive control dna of above-mentioned structure, entrust Shanghai to give birth to the synthetic following primer of worker, sequence is:

5 '-AAC CTT GGA ACC TGG ACC GGG CGGAATTGCCAGGACGACCGGG-3 ' (reset for HCV detection probes sequence, with detection probes identical GC% arranged, similar Tm value by its 5 ' end of SEQ.ID.NO.19; 3 ' end is for adjoining the nucleotide sequence of detection probes 3 ' end position on the HCV positive control)

(its 5 ' end of SEQ.ID.NO.20 is complementary with the 5 ' end of SEQ.ID.NO.19 for 5 '-CCC GGT CCA GGT TCC AAG GTT accactatggctctcc-3 '; The nucleic acid array complementation that adjoins detection probes 5 ' end position on 3 ' end and the HCV positive control) be pcr template with the positive control dna, be that a pair of primer and SEQ.ID.NO.18, SEQ.ID.NO.19 go amplification for another to primer with SEQ.ID.NO.17, SEQ.ID.NO.20 respectively, the loop parameter of use is:

94 ℃ 2 minutes---94 ℃ 10 seconds,

45 ℃ of 15 seconds, circulation 20 times, extend at last 72 ℃ of 5 minutes ends.

72 ℃ 10 seconds,

Will be behind above-mentioned two kinds of amplified production purifying mixed and gradient dilution, respectively as the template of next round PCR.This take turns PCR use SEQ.ID.NO.17 and SEQ.ID.NO.18 as primer to amplification, program is the same, cycle number is increased to 30-40.This takes turns amplified production and is internal reference DNA, can get the band that the portion of product electrophoresis detection is viewed as a 187bp size.Same its magnetic beads for purifying, transcribe, preserve all, be prepared into the pure product of HCV internal reference RNA at last with above-mentioned positive control (RNA).HCV sequence that can reference is as follows, schematic diagram such as accompanying drawing 3 that can reference.

C?GGGAGAGCCA?TAGTGGTCTG?CGGAACCGGT?GAGTACACCG?GAATTGCCAG

GACGACCGGG?TCCTTTCTTG?GATCAACCCG?CTCAATGCCT?GGAGATTTGG?GCGTGCCCCC

GCGAGACTGC?TAGCCGAGTA?GTGTTGGGTC?GCGAAAGGCC?TTGTGGTACT

(SEQ.ID.NO.26)

3, HBV internal reference and positive control is prepared as follows

3.1 the quick structure of positive control: entrust Shanghai to give birth to the synthetic following primer of worker, sequence is:

5’AGG?GAG?acc?acc?aaa?tgc?ccc?tat-3’(SEQ.ID.NO.21)

(5 ' end italicized item is irrelevant sequence, prevents dna degradation since 5 ' the PBR degraded that causes positive control, and 3 ' end is the upstream primer of HBV)

5’AGG?GAG?ttc?tgc?gac?gcg?gcg?a-3’(SEQ.ID.NO.22)

(5 ' end italicized item is irrelevant sequence, and 3 ' end is the downstream primer of HBV)

Get the sample portion of HBV nucleic acid strong positive clinically (or contain HBV DNA plasmid portion), extract viral nucleic acid, strictly operate by its working instructions requirement with QIAamp ViralMini Kit (QIAGEN company).The PCR reagent that extract product is set up through this testing laboratory, with above-mentioned two primer amplifications, operate by following loop parameter:

94 ℃ 2 minutes---94 ℃ 20 seconds,

50 ℃ of 20 seconds, circulation 30-40 time, extend at last 72 ℃ of 5 minutes ends.

72 ℃ 30 seconds,

Can get partial reaction product electrophoresis detection and be viewed as a 131bp bright band.Use glue to reclaim purification kit and electrophoretic band is reclaimed purifying be HBV positive control mother liquor, preserve the liquid gradient dilution with DNA/RNA and prepare that the green of Biosafety is stable, the positive control of suitable concn.

3.2 the structure of internal reference: based on the positive control dna of above-mentioned structure, entrust Shanghai to give birth to the synthetic following primer of worker, sequence is:

5 '-TACGACGACGACGACGACGACGAGA gaactccc tcgcctcgc-3 ' (reset for HBV detection probes sequence, with detection probes identical GC% arranged, similar Tm value by its 5 ' end of SEQ.ID.NO.23;

3 ' end is for adjoining the nucleotide sequence of detection probes 3 ' end position on the HBV positive control)

(its 5 ' end of SEQ.ID.NO.24 is complementary with the 5 ' end of SEQ.ID.NO.23 for 5 '-TCTCGTCGTCGTCGTCGTCGTCGTA tctaacaacagtagtttc-3 '; The nucleic acid array complementation that adjoins detection probes 5 ' end position on 3 ' end and the HBV positive control)

With the positive control dna is pcr template, is that a pair of primer and SEQ.ID.NO.23, SEQ.ID.NO.22 go amplification for another to primer with SEQ.ID.NO.21, SEQ.ID.NO.24 respectively, and the loop parameter of use is:

94 ℃ 2 minutes---94 ℃ 10 seconds,

45 ℃ of 15 seconds, circulation 20 times, extend at last 72 ℃ of 5 minutes ends.

72 ℃ 10 seconds,

Will be behind above-mentioned two kinds of amplified production purifying mixed and gradient dilution, respectively as the template of next round PCR.This take turns PCR use SEQ.ID.NO.21 and SEQ.ID.NO.22 as primer to amplification, program is the same, cycle number is increased to 30-40.This takes turns amplified production and is internal reference DNA, can get the band that the portion of product electrophoresis detection is viewed as a 131bp size.Use glue to reclaim purification kit and electrophoretic band is reclaimed purifying be HBV internal reference DNA mother liquor, preserve the liquid gradient dilution with the DNA/RNA of our company and prepare that the green of Biosafety is stable, the HBV internal reference DNA of suitable concn.HBV sequence that can reference is as follows, schematic diagram such as accompanying drawing 3 that can reference.

tatagaccac?caaatgcccc?tatcttatca?acacttccgg?aaactactgt

tgttagacga?cgaggcagga?cccctagaag?aagaactccc?tcgcctcgca?gacgaaggtc

tcaatcgccg?cgtcgcagaa?gatctcaatc

(SEQ.ID.NO.27)

Embodiment 2 sample disposal unit: the paramagnetic particle method of nucleic acid extraction and reagent preparation

Sample disposal unit reagent composition based on the hybridization principle comprises:

Lysate: 4.8M guanidine thiocyanate, 50mM Tris, PH7.0, NP-40, Triton X-100, SDS, biotinylated probe, polymkeric substance etc.Wherein capture probe is:

5’BIOTIN-acc?acc?aaa?tgc?ccc?tat-3’(HBV)

5’BIOTIN-agt?acc?aca?agg?cct?ttc?g-3’(HCV)

5’BIOTIN-cta?tgt?cac?ttc?ccc?ttg?gtt?ctc?tc-3’(HIV)

The magnetic bead suspension: the magnetic bead of 1mg/ml streptavidin parcel, contain dispersion agents such as sanitas and Tween 20

Washings A:LiCI, Tris PH7.0, dodecyl sodium sulfonate lithium, dispersion agents such as sanitas and Tween 20, pigment etc.

Washings B:LiCI, Tris PH7.0, dispersion agents such as sanitas and Tween 20, pigment etc.

Washings C:LiCI, Tris PH7.0, dispersion agents such as sanitas and Tween 20, pigment etc.

The agent of disinthibiting: contain proteolytic enzyme and glycerine etc.

The Tris damping fluid of the purified water preparation that elutriant: DEPC handled is added with Tween 20 dispersion agents such as grade and EDTA, NaN3, DTT etc.

Internal reference: the solution that contains 40000 copy/ml internal reference RNA approximately, during use its ratio with 1: 20 is joined in the lysate, the concentration of the internal reference in lysate is about 2000 copy/ml, and each test adds the lysate 200 copy internal references participations just of 0.1ml and extracts.Amplification detects owing to the template amount of only getting 1/5 participates in RT-PCRTaqMan, so the amount of participating in of internal reference RNA is lower than 40 copies in each detector tube.

Operating process:

Get six special use 96 orifice plates (test kit provides), be numbered 1. 2. 3. 4. 5. 6..

1. add 20 μ ls disinthibite agent with the volley of rifle fire or common pipettor at plate in, add sample serum to be measured or blood plasma 100 μ l, inhale repeatedly and make a call to about 1 time, whenever add a sample and change a suction nozzle, attention should be used the suction nozzle of being with filter core; Add lysate 100 μ l at last.

2. add 100 μ l magnetic bead suspensions with the volley of rifle fire or common pipettor at plate in, note using preceding one or two minute of magnetic bead suspension with its turn upside down for several times up to magnetic bead be uniformly dispersed do not form any macroscopic precipitation till.

3. add 200 μ l washings As with the volley of rifle fire or common pipettor at plate in; The 4. middle 200 μ l washings B that add of plate; The 5. middle 200 μ l washings C that add of plate; The 6. middle 80 μ l elutriants that add of plate.

Above-mentioned six blocks of plates are moved into the 1-6 position in KHB DP-2000 instrumentation cabin respectively by number order, and move into the deep hole Tip Comb of personality board at the 4th plate on 4..

Start " Start " key on the KHB DP-2000 instrument, make its steering routine.

Operation approximately needs 70 minutes, finishes the back and takes out plate 1.-6., and wherein the liquid of plate in 6. is nucleic acid-templated, and 1.-5. plate is useless plate, unified discarding through sterilizing after.

Embodiment 3 nucleic acid amplification detecting units: the pattern and the reagent preparation of the many corpse or other object for laboratory examination and chemical testing of two enzyme one-step method RT-PCR TaqMan probes of single tube system

The nucleic acid amplification detecting unit adopts fluorescence TaqMan PCR, requires to cooperate the fluorescent PCR instrument to use.Its component comprises as follows:

RT-PCR reaction solution: contain 1.8Mm d NTPs (containing d UTP) among the buffer, 3.6mM DTT, 3.5mM MgCl ₂, primer BF, BR, CF, CR, each 0.4 μ M of IF, IR.

Enzyme mixture: 0.6U/ μ l AMV reversed transcriptive enzyme, 0.6U/ μ l warm start Taq archaeal dna polymerase, 1U/ μ l RNasin; 0.1U/ μ l UNG (heat-labile) etc.

Probe: 10 μ M probe HBV Probe, HCV Probe, mark probe, stablizer and damping fluid in the HIV Probe, 5 μ M.

Reagent proportioning and preparation: according to every test RT-PCR reaction solution: enzyme mixture: the ratio of probe=8: 6: 1 is mixed with 15 μ l/ test, adds the nucleic acid-templated 15 μ l amplification that promptly is available on the machine and detects in real time.

With ABI7500 is example, and the amplification parameter of instrument is provided with as follows:

Loop parameter is set: (can be provided with reference to the function software of each quasi-instrument)

The application of one of embodiment 4 detecting patterns

According to the actual experiment hardware condition, the employed fluorescent PCR instrument of the user of this example is twin-channel MJ Opticon2 fluorescent PCR instrument, preferred detection probes is all used the FAM fluorescent mark, interior mark probe uses the HEX mark, and internal reference preferably joins in the preparation of RT-PCR reagent and do not add in lysate.And only contain the primer of each test item in the RT-PCR/PCR reaction solution, probe then each project is the detection probes of this project and the mixture of interior mark probe, internal reference select for use comprise three test item PBRs, middle probe land is the common internal reference RNA of interior mark probe binding sequence.

Sample to be measured is numbered P1, P2, and P3..PN, other has 2 reference substances (negative control and positive control) to participate in parallel laboratory test.Contain very HBV DNA or the HCV RNA or the HIV RNA of low copy number in the sample to be measured.Wherein weak positive sample P1 is that HBV DNA 1000 copy/ML, P2 are that HCV RNA 1000 copy/ML, P3 are that HIV RNA 1000 copy/ML, P4 are that P1 comes with ten times of dilutions of negative blood plasma, P5 is P2 with ten times of dilutions of negative blood plasma, and P6 is P3 with ten times of dilutions of negative blood plasma; P7 is P1 with 20 times of dilutions of negative blood plasma, and P8 is P2 with 20 times of dilutions of negative blood plasma, and P9 is P3 with 20 times of dilutions of negative blood plasma; P10 is P1 with 40 times of dilutions of negative blood plasma, and P11 is P2 with 40 times of dilutions of negative blood plasma, and P12 is P3 with 40 times of dilutions of negative blood plasma.

Numbering	Sample
		P1	Sample 1 to be measured
P2	Sample 2 to be measured
		P3	Sample	3 to be measured
...	...
		PN	Treat side sample N
NC	Negative control
		PC	Positive control
X	Diluting plasma

It is all positive that the result detects the internal reference of sample, and the HBV sample is all positive when carrying capacity is 1000 copy/ML, 100 copy/ML, 50 copy/ML, 25 copy/ML, and detected results such as negative control, diluting plasma are negative.The HCV sample is all positive when carrying capacity is 1000 copy/ML, 100 copy/ML, 50 copy/ML, and detected results such as 25 copy/ML, negative control, diluting plasma are negative.The HIV sample is all positive when carrying capacity is 1000 copy/ML, 100 copy/ML, 50 copy/ML, 25 copy/ML, and detected results such as negative control, diluting plasma are negative.

Two application of embodiment 5 detecting patterns

This detecting pattern is best embodiment the of the present invention: select the fluorescent PCR instrument such as the ABI7500 of four looks and even the five colors for use, the inspection probe is used FAM, VIC, NED fluorescent mark respectively, and interior mark uses the ROX mark.Internal reference adds in lysate.The primer that contains each test item in the T-PCR reaction solution, probe contain the detection probes and the interior mark probe of each project.Internal reference select for use comprise the HIV PBR, middle probe land is the internal reference RNA of interior mark probe binding sequence.Sample is with embodiment 3, and experimental result is also identical with embodiment 3, but Quality Control is more strict, operates more conveniently, and detecting pattern and sample extract coupling more, and it is higher to detect flux.

Three application of embodiment 6 detecting patterns

This detecting pattern is the compromise proposal of embodiment 3 and 4: instrument can be used the two-color fluorescence PCR instrument, and the flux of detection and rigorous Quality Control are identical with embodiment 4, but the positive amplification that in a single day comes out, it is positive to distinguish which test item.Its preferred main points are that detection probes is all used the FAM fluorescent mark, and interior mark uses HEX or marks such as VIC or NED or JOE or TET.Internal reference adds in lysate.The primer that contains each test item in the RT-PCR reaction solution, probe contain the detection probes and the interior mark probe of each project.Internal reference select for use comprise the HIV PBR, middle probe land is the internal reference RNA of interior mark probe binding sequence.

Sample is with embodiment 3, and experimental result is also identical with embodiment 3, the Quality Control strictness, easy to operate, detecting pattern and sample extract phase coupling detect the flux height, but the HBV positive or the HCV positive or the HIV positive or two kinds of while positives and even three kinds of common positives all can not be known.Yet this what it counts in blood screening shortcoming because the purpose of blood sieve is to filter out underproof blood, is superseded blood no matter which kind of viral nucleic acid is positive or associating is positive.

Four application of embodiment 7 detecting patterns

Synthesize common centre (catch) probe of following nucleic acid as HBV, HCV, HIV, internal reference RNA:

5’-acc?acc?aaa?tgc?ccc?tat?ttt?ttt?agt?acc?aca?agg?cct?ttc?g?aaa

aaa?cta?tgt?cac?ttc?ccc?ttg?gtt?ctc?tc?aaa?aaa?aaa?aaa?aaa?aaa

Aaa aaa-3 ' is purity (99bp): PAGE or HPLC (SEQ.ID.NO.15)

Biotinylated probe: 5 ' Biotin-Oligo (d T) ₂₅(SEQ.ID.NO.16).

Above-mentioned two kinds of probes are cofabrication in lysate, and the fixed ratio is arranged, and the optimization of the amount of common middle (catching) probe has conclusive effect to extracting sensitivity.

Reagent is all with embodiments of the invention 2.1

Testing process is with embodiment 2 (sample extraction), and detecting pattern is pressed embodiment 4, and result and embodiment 4 detected results are identical.

According to disclosure of the present invention, those skilled in the art need not too much experiment and can implement the test kit of the present invention's blood screening project required for protection, and produce a desired effect.Embodiment disclosed by the invention only is that present invention is described, but is not construed as limiting the invention.Those skilled in the art are with conspicuous similar surrogate or transformation; or substitute preparation described here at the relevant preparation of structure function chemically or on the biology with some; or related content of the present invention changed; but do not exceed spirit of the present invention, scope and thought, all fall into the scope of protection of present invention.

Sequence table

SEQ.ID.NO.1：

5’-acc?acc?aaa?tgc?ccc?tat-3’，

SEQ.ID.NO.2：

5’-ttc?tgc?gac?gcg?gcg?a-3’，

SEQ.ID.NO.3：

5′FAM-cga?cga?ggc?agg?acc?cct?aga?aga?a-NFQ3′，

SEQ.ID.NO.4：

5’-cgg?gag?agc?cat?agt?gg-3’，

SEQ.ID.NO.5：

5’-agt?acc?aca?agg?cct?ttc?g-3’，

SEQ.ID.NO.6：

5′FAM-ctg?cgg?aac?cgg?tga?gta?cac-NFQ3′，

SEQ.ID.NO.7：

5′-gac?atc?aag?cag?cca?tgc?aaa?t-3’，

SEQ.ID.NO.8：

5′-cta?tgt?cac?ttc?ccc?ttg?gtt?ctc?tc-3’，

SEQ.ID.NO.9：

5′Vic(Rox)-aac?ctt?gga?acc?ttg?gaa?ctg?gag?aga?gaa-NFQ3’，

SEQ.ID.NO.10：

5’BIOTIN-AAT?TCT?AAT?ACG?ACT?CAC?TAT?AGG?GAG?gac?atc?aag?cag?cca?tgc?aaa?t-3’，

SEQ.ID.NO.11：

5’BIOTIN-cta?tgt?cac?ttc?ccc?ttg?gtt?ctc?tc-3’，

SEQ.ID.NO.12：

5’-AAC?CTT?GGA?ACC?TTG?GAA?CTG?GAG?AGA?GAA?gagtgcatccagtgcatgcag-3’，

SEQ.ID.NO.13：

5’-TTC?TCT?CTC?CAG?TTC?CAA?GGT?TCC?AAG?GTT?tct?ctt?taa?tta?aca?ttt?gc-3’，

SEQ.ID.NO.14：

5′Vic(Rox)-aac?ctt?gga?acc?ttg?gaa?ctg?gag?aga?gaa-NFQ3’，

SEQ.ID.NO.15：

5’-acc?acc?aaa?tgc?ccc?tat?ttt?ttt?agt?acc?aca?agg?cct?ttc?g?aaa

aaa?cta?tgt?cac?ttc?ccc?ttg?gtt?ctc?tc?aaa?aaa?aaa?aaa?aaa?aaa

aaa?aaa-3′，

SEQ.ID.NO.16：

5’Biotin-Oligo(d?T) ₂₅，

SEQ.ID.NO.17：

5’BIOTIN-AAT?TCT?AAT?ACG?ACT?CAC?TAT?AGG?GAG?cgg?gag?agc?cat?agt?gg-3’

SEQ.ID.NO.18：

5’BIOTIN-agt?acc?aca?agg?cct?ttc?g-3’

SEQ.ID.NO.19：

5’-AAC?CTT?GGA?ACC?TGG?ACC?GGG?CGGAATTGCCAGGACGACCGGG-3’，

SEQ.ID.NO.20：

5’-CCC?GGT?CCA?GGT?TCC?AAG?GTT?accactatggctctcc-3’，

SEQ.ID.NO.21：

5’AGG?GAG?acc?acc?aaa?tgc?ccc?tat-3’，

SEQ.ID.NO.22：

5’AGG?GAG?ttc?tgc?gac?gcg?gcg?a-3’，

SEQ.ID.NO.23：

5’-TACGACGACGACGACGACGACGAGA?gaactccc?tcgcctcgc-3’，

SEQ.ID.NO.24：

5’-TCTCGTCGTCGTCGTCGTCGTCGTA?tctaacaacagtagtttc-3’，

SEQ.ID.NO.25：

gaca?tcaagcagcc?atgcaaatgt?taattaaagagac?catcaatgag?gaagctgcag?aatgggatag

agtgcatcca?gtgcatgcag?ggcctattgc?accaggccag?atgagagaac?caaggggaag?tgacatag

SEQ.ID.NO.26：

C?GGGAGAGCCA?TAGTGGTCTG?CGGAACCGGT?GAGTACACCG?GAATTGCCAG

GACGACCGGG?TCCTTTCTTG?GATCAACCCG?CTCAATGCCT?GGAGATTTGG?GCGTGCCCCC

GCGAGACTGC?TAGCCGAGTA?GTGTTGGGTC?GCGAAAGGCC?TTGTGGTACT

SEQ.ID.NO.27：

5′FAM-cca?tca?atg?agg?aag?ctg?cag?aat?ggg?ata-N?FQ3’，

SEQ.ID.NO.28：

5’BIOTIN-acc?acc?aaa?tgc?ccc?tat-3’，

SEQ.ID.NO.29：

5’BIOTIN-agt?acc?aca?agg?cct?ttcg-3’，

SEQ.ID.NO.30：

5’BIOTIN-cta?tgt?cac?ttc?ccc?ttg?gtt?ctc?tc-3’。

Claims (8)

1, a kind of test kit that detects acquired immune deficiency syndrome (AIDS), hepatitis B, hepatitis C nucleic acid, it is characterized in that comprising disinthibite agent, lysate, magnetic bead suspension, washings, elutriant, internal reference, RT-PCR reaction solution, enzyme mixture, fluorescent probe, positive control, negative control, wherein, the described agent of disinthibiting is the alcoholic solution that contains proteolytic enzyme; Described lysate is the guanidine thiocyanate solution of virion in cracking serum, the plasma specimen, and contains by the oligonucleotide of the biotin modification capture probe as nucleic acid extraction; Described washings is the solution of sodium chloride-containing; Described elutriant is low salt buffer or water; Described magnetic bead suspension contains streptavidin parcel magnetic bead; Described RT-PCR reaction solution contains HBV, HCV, HIV primer and dNTP and buffer salt solution; Described fluorescent probe is the Taqman probe; Described enzyme mixture is a multienzyme component system; Described positive control is section of DNA or the RNA that contains HBV, HCV, the HIV amplifying target genes outside; Described negative control is the blood plasma of donors with normal; Described internal reference be competitive in mark RNA, include the identical PBR of test item, but different with the probe land of test item, can not but can be in conjunction with interior mark probe in conjunction with detection probes; Wherein:

The HBV primer is in the PT-PCR reaction solution:

5’-acc?acc?aaa?tgc?ccc?tat-3’，

5’-ttc?tgc?gac?gcg?gcg?a-3’，

The Taqman probe of HBV is:

5′FAM-cga?cga?ggc?agg?acc?cct?aga?aga?a-NFQ3′，

The HCV primer is:

5’-cgg?gag?agc?cat?agt?gg-3’，

5’-agt?acc?aca?agg?cct?ttc?g-3’，

The Taqman probe of HCV is:

5′FAM-ctg?cgg?aac?cgg?tga?gta?cac-NFQ3′，

The HIV primer is:

5′-gac?atc?aag?cag?cca?tgc?aaa?t-3’，

5′-cta?tgt?cac?ttc?ccc?ttg?gtt?ctc?tc-3’，

The Taqman probe of HIV is:

5′FAM-cca?tca?atg?agg?aag?ctg?cag?aat?ggg?ata-NFQ3’，

Interior mark probe is SEQ.ID.NO.14.

2, test kit according to claim 1 is characterized in that using the TaqMan of reversed transcriptive enzyme, the two enzyme single tube single stage method RT-PCR of hot resistant DNA polymerase multinomial with corpse or other object for laboratory examination and chemical testing system, and test item comprises DNA and RNA viruses; Contain the anti-pollution component of UNG-dUTP; Also contain the whole process monitoring Quality Control of internal reference as each test.

3, test kit according to claim 1, it is characterized in that using biotin modification add that the end primer makes up fast, purifying internal reference RNA, positive control RNA pure rna.

4, test kit according to claim 1 is characterized in that containing in the said lysate sample and extracts used capture probe:

For HBV be: 5 ' BIOTIN-acc acc aaa tgc ccc tat-3 ',

For HCV be: 5 ' BIOTIN-agt acc aca agg cct ttc g-3 ',

For HIV be: 5 ' BIOTIN-cta tgt cac ttc ccc ttg gtt ctc tc-3 '.

5, test kit according to claim 1 is characterized in that containing in the said lysate sample and extracts used middle probe:

5’-acc?acc?aaa?tgc?ccc?tat?ttt?ttt?agt?acc?aca?agg?cct?ttc?g?aaa

aaa?cta?tgt?cac?ttc?ccc?ttg?gtt?ctc?tc?aaa?aaa?aaa?aaa?aaa?aaa

Aaa aaa-3 ' and 5 ' Biotin-Oligo (d T) ₂₅

6, test kit according to claim 1 is characterized in that consisting of of lysate: 4-6M guanidine thiocyanate, 20-50mMTris, PH6.0-8.0,1-20%NP-40,1-20%Triton X-100,0.1 a 10%SDS, 0.01-1 μ M capture probe, 1 one 10% polymkeric substance; Wherein capture probe to HBV is: 5 ' BIOTIN-acc acc aaa tgc ccc tat-3 ', to HCV be: 5 ' BIOTIN-agt acc aca agg cct ttcg-3 ', to HIV be: 5 ' BIOTIN-cta tgt cac ttcccc ttg gtt ctc tc-3 ', purity is PAGE or HPLC; Described magnetic bead suspension is the magnetic bead that contains the super paramagnetic material of TWEEN20, the surface is surrounded by even one deck streptavidin, diameter is 0.1-10 μ M, contain BSA, NaN3 composition, described elutriant is the Tris damping fluid of the purified water preparation handled of DEPC, is added with Tween 20 dispersion agents and EDTA, NaN3 and DTT.

7, test kit according to claim 1 is characterized in that described RT-PCR reaction solution is to contain 1.8Mmd NTPs in the damping fluid, 3.6mM DTT, 3.5mM MgCl ₂, primer BF, BR, CF, CR, each 0.4 μ M of IF, IR.

8, test kit according to claim 1 is characterized in that described enzyme mixture contains reversed transcriptive enzyme, archaeal dna polymerase, RNasin, heat-labile UNG, RT-PCR promotor and enzyme stabilizers.

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