The kit of HCV gene typing and methodTechnical field
The present invention relates to a kind of disease pathogen technique of gene detection, relate in particular to a kind of HCV gene typingKit and method, for detection of the type 1a of the HCV of domestic generation, 1b, 2a, 2b, 3a, it is auxiliary that 6a infectsHelp diagnosis.
Background technology
HCV (hepatitisCvirus, HCV), hepatitis C is worldwide distribution, and crowd is generally easySense is serious harm human health, Epidemic Scope infectious disease widely. Its pathogen is HCV (hepatitisCVirus, HCV). At present, its infection rate is 0.1%-10%, average out to 3%. The whole world has and exceedes 1.7 hundred million people's HCV infection, whereinHave every year to exceed 100,000 routine HCV patients and develop into liver cancer, and then occur hemorrhage of digestive tract and ascites. According to WHO2002Report, in calendar year 2001, chronic liver disease causes that 1,400 ten thousand examples are dead, comprise 79.6 ten thousand examples by cirrhosis cause, 61.6 ten thousand examples are by formerProperty liver cancer causes.
HCV is positivity single stranded RNA flavivirus, comprises a 9400 left and right nucleotides. HCV genome has independent openingPut reading frame, gene structure is made up of 5 '-NCR-C-E1-E2-NS1-NS2-NS3-NS4-NS5-N-CR-3 ', and coding one has3010 amino acid whose polyprotein bodies, are divided into virus replication and virion and form necessary structure and non-structure after translationAlbumen. HCV has variability highly, and (its aberration rate is up to 1.4-1.9 × 10-3/ nucleotides/year) and itself there is negative selectionThe feature of effect, high sudden change is because replicase is without calibration function; Easily mistake property RDRP (error-proneRDRP, EP-RDRP)Existence and the reason such as selection effect just. Show as and itself there is negative selection effect: various bases in viral genomeBecause of structure and function difference, some region height is conservative. The not whole or portion of homophyletic of HCV separating according to different regions, the whole worldDivide genomic phylogenetic analysis, HCV is divided into 6 kinds of main genotype (representing with 1-6), and the various hypotype of dividing is again (with a, b, cDeng expression). HCV hypotype has exceeded 100, and wherein modal is 1a, 1b, and 2a, 2b, 3a, 6a, between various nucleotide sequenceDiffer 31-34%, it is about 30% that amino acid sequence differs, and between hypotype sequence, differ about 20-23%. There are several HCV diseasesStrain is mainly separated from Southeast Asia, is named as HCV7, and 8,9,10,11 types, except HCV10a type (is put at presentHCV3 type), due to the similitude on its phyletic evolution, all the other various HCV6 types that are put into.
Research shows, between the different types of HCV, the susceptibility of interferon therapy is differed, and particularly 1 type is compared with other typeSusceptibility is particularly poor. The infectious hepatitis of different type HCV, the moderate that it is acute, chronic and severe, posthepatitic cirrhosis and formerProperty liver cancer incidence has difference, and the clinical state of an illness of HCV genotype and hepatitis C patients and the order of severity thereof are closely related. In view ofDifferent HCV genotype the infecteds' clinical manifestation, the hepatopathy order of severity and chronicity course advancement are all variant, antiviral therapyEffect also different. Therefore detect complexity, formulation individuation antiviral therapy side that HCV genotype contributes to judgement treatmentCase.
At present HCV methods of genotyping is mainly contained: restricted length polymorphism analysis method (RFLP), Serotype-dependentPrimer PCR method, Serotype-dependent probe nucleic acid hybridization analysis method, the methods such as direct sequencing, but these methods are deposited mostlyThe shortcomings such as at complex operation, high to equipment requirement, detection time is long, and flux is low, also exists sensitivity low, and specificity is not high.
Summary of the invention
The object of the invention is to overcome defect and the deficiency of prior art, manufacture third type with high flux, high accuracyThe genetic chip of virogene of hepatitis somatotype and kit and method, can disposablely accurately divide HCV in multiple samplesType detects, and saves detection time.
Technical solution of the present invention, for a kind of kit of HCV gene typing is provided, mainly comprises PCR reactionLiquid and DNA hybond membrane bar, described DNA hybond membrane bar comprises nylon membrane, on described nylon membrane, is fixed with probe, described probe is canWith the nucleotides of the nucleic acid hybridization of the various subtype virus of HCV, described probe sequence is as follows respectively:
For HCV1a type designing probe Y1:5 '-AATTGCCAGGACGACCGG-3 ';
For HCV1b type designing probe Y2:5 '-AATTGCCAGGATGACCGGG-3 ';
For HCV1b type designing probe Y3:5 '-CCCGGAATTGCCAGGAC-3 ';
For HCV2a type designing probe Y4:5 '-CAATGCCTGGAGATTTGGGCG-3 ';
For HCV2a type designing probe Y5:5 '-CAATGCCTGGAGATTTGGGC-3 ';
For HCV2b type designing probe Y6:5 '-CTCAATGCCTGGAGATTTGG-3 ';
For HCV2b type designing probe Y7:5 '-GCTCAATGCCTGGAGATTTGG-3 ';
For HCV3a type designing probe Y8:5 '-CAATGCCTGGAGATTTGGG-3 ';
For HCV3a type designing probe Y9:5 '-CGAGGTAGACGTCAGCC-3 ';
For HCV3a type designing probe Y10:5 '-ACCTCGAGGTAGACGTCAGCC-3 ';
For HCV6a type designing probe Y11:5 '-AACCTCGAGGTAGACGTCAGCC-3 ';
For HCV6a type designing probe Y12:5 '-GCAACCTCGAGGTAGACGT-3 ';
For HCV6a type designing probe Y13:5 '-TAGACGTCAGCCTATCCCCAA-3 ';
For HCV1-6 type design general probe (being positive probe HP) Y14:5 '-TAGACGCCAGCCTATTCCCA-3 ';
Negative probe HN:5 '-GGTCCTGTTTAACTGGCG-3 ';
Described each probe 5 ' end carry out amino labeled with nylon membrane coupling.
Preferably, in the kit of above-mentioned HCV gene typing, on described nylon membrane, be also provided with colour developing controlManufacturing probe CC, described colour developing control probe CC sequence is as follows: 5 '-GCGCAGGGCCACAATAATGG-3 ', described cc probe5 ' end carries out amino labeled, and 3 ' end carries out biotin labeling.
Preferably, in the kit of above-mentioned HCV gene typing, described PCR reactant liquor comprises amplification the third type liverThe primer of the nucleic acid of scorching viral various subtype virus, described primer sequence is as follows:
Reverse transcriptase primer RT:5 '-CGTCTAGCCATGGCGTTAGT-3 ';
Upstream primer F:5 '-GAAAGCGTCTAGCCATGGCGTTAGT-3 ';
Downstream primer R:5 '-CGCAAGCACCCTATCAGGCAGTA-3 '.
5 ' the end of described upstream primer F carries out biotin labeling.
Preferably, in the kit of above-mentioned HCV gene typing, described PCR reactant liquor also comprises internal reference systemSystem, described internal reference system is arabidopsis internal reference system, comprising:
Internal reference primers F 1:5 '-TCATCGTCGCTGGAGCTGGTT-3 ';
Internal reference primer R1:5 '-CGGCGGTTTGTCAAGCTGAT-3 ';
Internal reference probe PC:5 '-TGCCGATATAGGTCACAGCATGGGC-3 ';
5 ' end of described internal reference primers F 1 carries out biotin labeling.
Preferably, in the kit of above-mentioned HCV gene typing, described concentration and probe concentration is 5-20 μ M.
Another technical scheme of the present invention is for providing a kind of method of HCV gene typing, and the method comprisesFollowing concrete steps:
A, design serotype specific primer and probe, described probe is as shown in claim 1 probe Y1-Y14, and described primer comprises contraryTranscribe primer RT:5 '-CGTCTAGCCATGGCGTTAGT-3 ';
Upstream primer F:5 '-GAAAGCGTCTAGCCATGGCGTTAGT-3 ';
Downstream primer R:5 '-CGCAAGCACCCTATCAGGCAGTA-3 ';
5 ' end of probe described in b, use active amino markers step a, and it is fixed on nylon membrane successively, detection madeFilm bar, carries out biotin labeling to the 5 ' end of described downstream primer R;
C, utilize primer described in step a to carry out HCVDNA amplification, by amplified production with detect the hybridization of film bar, with peroxideCompound enzyme and tetramethyl benzidine colour developing;
D, employing scanner, scanning hybridization signal, judges Genotyping result.
Preferably, the method for above-mentioned HCV gene typing, is also provided with colour developing and controls on described nylon membraneProbe CC, described colour developing control probe CC sequence is as follows: 5 '-GCGCAGGGCCACAATAATGG-3 ', described colour developing control5 ' the end of probe CC carries out amino labeled, and 3 ' end carries out biotin labeling.
Preferably, the method for above-mentioned HCV gene typing, the primer in described step a also comprises:
Internal reference primers F 1:5 '-TCATCGTCGCTGGAGCTGGTT-3 ';
Internal reference primer R1:5 '-CGGCGGTTTGTCAAGCTGAT-3 ';
Probe in described step a also comprises:
Internal reference probe PC:5 '-TGCCGATATAGGTCACAGCATGGGC-3 ';
5 ' end of described internal reference primers F 1 carries out biotin labeling.
The genetic chip of HCV gene typing of the present invention and kit and method have quick, sensitive, accurateReally, high-throughout feature, one time hybridization reaction can detect multiple target sequence, draws materials conveniently, without special installation, instrument drops intoLow, have fast and convenient, susceptibility is high and the feature of high specificity.
Brief description of the drawings
Fig. 1: the arrangement schematic diagram of HCV probe of the present invention on nylon membrane;
Fig. 2: 1 type HCV Virus Sample testing result scintigram;
Fig. 3: 2 type HCV Virus Sample testing result scintigrams;
Fig. 4: 3 type HCV Virus Sample testing result scintigrams;
Fig. 5: 6 type HCV Virus Sample testing result scintigrams.
Detailed description of the invention
By describing technology contents of the present invention, structural feature in detail, being realized object and effect, below in conjunction with embodimentAnd coordinate accompanying drawing to be explained in detail.
The object of the invention is to prepare a kind of HCV (HCV) gene typing DNA hybond membrane bar, a large amount of to meetBlood sample carries out the quick and precisely needs of somatotype of HCV.
For this reason, the present invention by the following technical solutions: adopt reversal point hybridization technique (ReverseDotBlot, RDB),With nylon membrane, as solid phase carrier, the specific oligonucleotide probe designing for HCV different genotype, by each HCV specificityProbe is fixed on film bar in the mode of round dot, is prepared into DNA hybond membrane bar, and with biotin labeling primer, sample to be checked increasesObject nucleic acid fragment, obtain with biotin labeled amplified production; Assorted with the film bar of fixing various oligonucleotide probes againHand over, by integrated enzyme reaction and colour developing, after scanning, with professional spot-analysis software, image is identified, removed background, carries outObjectively signal value analysis, thus judge in amplified production whether have the gene order for various oligonucleotide probes, at knotIn fruit judgement, reduce the subjective factors of artificial identification, be more suitable for Clinical detection application.
The present invention is directed to other gene order of HCV different shaped, design a set of specific oligonucleotide probe (as shown in table 1For sequence oligonucleotide probe and the type that detects), adopt automatic point sample instrument, probe is printed on to the spy of a nylon membraneDetermine region, judge the type of HCV according to the difference in colour developing site.
On the basis of above-mentioned DNA hybond membrane bar, be equipped with again other necessary component, as PCR primer (is as shown in table 2lyThe primer sequence that the present invention is used), form complete product. In the time that reality detects, get the HCV sample of nucleic acid solution of extraction,Adopt biotin labeling primer and asymmetric PCR method, amplify the HCV genome biotin labeling target sheet that may existSection. Get one of DNA hybond membrane bar, add PCR product 45 μ L to hybridize, hybridization temperature is set as 45 DEG C, 60 points of hybridization timeClock, through hybridization-washing-colour developing. Take out film bar and dry, put into scanner and scan, use analysis software to scan imageSignal carries out view data conversion process, and analyzes generation sample HCV type result.
According to another preferred embodiment of the present invention, introducing interior reference for the PCR system of sample amplificationThing, and internal reference template, and time prepared by film bar, introduced the probe that can hybridize with internal reference template, can be to PCR processCarry out effective mass control.
According to another preferred embodiment of the present invention, in the time preparing film bar, introduce colour developing and controlled probe (CC.),At one end of probe mark-NH2, with nylon membrane coupling, the other end is introduced biotin labeling, can directly develop the color. CCIntroducing can carry out effective mass control to process color.
Another preferred embodiment according to the present invention, the sheet base carrier of DNA hybond membrane bar can be slide, nylon membraneOr other can adhesion probe carrier.
Table 1
Table 2
Embodiment
1,5 ' UTR district design RT and PCR primer in HCV genome, adopts asymmetric RT-PCR system, amplification somatotype instituteThe target fragment needing; As shown in table 1, within the scope of target fragment, 3 of design 1 type probes, 24 of type probes, 3 type probes 3Bar, 1 of 6 three of type probes and general probe.
2, refer to Fig. 1 by nylon membrane by the format print grid designing, adopt automatically some film instrument, 14 somatotypes are visitedPin and 2 contrast probe points system, to the specific region of film bar, is made DNA hybond membrane bar.
Point sample probe solution: by probe dilution to 5 × SSC, 0.05%SDS solution, final concentration is 10 μ M;
Point sample matrix: by probe points on film bar, each probe point sample 0.5pM, every row 8 points, totally 2 row (referring to Fig. 1).
3, use product of the present invention to detect clinical sample
Get various other clinical sample 6 examples and positive and negative and contrast each portion, extract RNA, prepare RT-PCR according to table 3 anti-Answer system, each primer is as shown in table 3, carries out pcr amplification, obtains DNA hybridization template.
Table 3
| Reagent | 1 person-portion (μ L) |
| Pure water | 21.5 |
| 10×PCR buffer | 5 |
| 25mM MgCl2 | 6 |
| dNTP(10mM) | 1 |
| 10μM F | 2 |
| 10μM R | 1 |
| 10μM F1 | 2 |
| 10μM R1 | 1 |
| Reverse transcriptase/Taq enzyme | 0.5 |
| RNA template | 10 |
| Total amount | 50 |
Pcr amplification program is as follows:
Adopt HCV Genotyping hybond membrane bar of the present invention, above sample is detected.
Get a hybond membrane bar, marker samples numbering, puts into hybrid pipe, get respectively the each 45 μ L of 8 pipe PCR product be added to rightIn the pipe that should number, set 45 DEG C of hybridization temperatures, hybridization time 60 minutes. After hybridization, wash, coupling, colour developing and air-dry.Concrete operations are as follows:
1) hybridization:
Get 15mL centrifuge tube, put into 1 of label film bar, add 5-8ml preheating A liquid (2*SSC, 0.5%SDS), corresponding40ulPCR product, covers tightly pipe lid, puts into vibration tank, 50 DEG C of jogs (100～150 revs/min), 40 minutes. Discard liquid.
2) coupling: (at every turn can simultaneously process 5 film bars)
Get 50mL centrifuge tube, preheating B liquid (0.5*SSC, 0.5%SDS) 20ml, C liquid (0.01%TMB) 1ml, mixes, and putsEnter hybond membrane bar, 50 DEG C of jogs (40～50 revs/min) 10 minutes, discard liquid;
Add preheating B liquid (0.5*SSC, 0.5%SDS) 40ml, 50 DEG C of jogs 3 minutes, discard liquid; Add D liquid (0.5M lemonAcid sodium, pH5.5) 40ml, 50 DEG C of jogs 3 minutes, discard liquid.
3) colour developing: (can process 5-10 at every turn simultaneously and open film bar)
Get 50mL centrifuge tube, add D liquid (0.5M natrium citricum, pH5.5) 20ml, E liquid 1ml (0.025%POD), F liquid(0.3H2O2) 1ml, mix and be nitrite ion, put into film bar and leave standstill 8 minutes in room temperature lucifuge, discard liquid;
Add D liquid (0.5M natrium citricum, pH5.5) 40ml, jog 3 minutes, discards liquid. Hybridization completes.
Adopt scanner, scanning hybridization signal, obtains results of hybridization scintigram (seeing Fig. 2-Fig. 5), and image is converted toData.
Adopt analysis software, above data analysis is converted into the gene type of each sample, meanwhile, by 8 partsSample DNA order-checking, contrasts as testing result. Result shows, 8 these testing results of increment conform to completely with sample sequencing result(in table 4).
Embodiment has illustrated the advantage of product of the present invention aspect detection flux, once can make the sample of arbitrary number; InspectionSurvey the reliability (testing result conforms to completely with sample sequencing result) of accuracy aspect.
Table 4
| Sample number | Sequencing result | This result of the test | Whether conform to |
| 01 | 1 type | 1 type | Be |
| 02 | 2 types | 2 types | Be |
| 03 | 1 type | 1 type | Be |
| 04 | 3 types | 3 types | Be |
| 05 | 3 types | 3 types | Be |
| 06 | 6 types | 6 types | Be |
| 07 | 1 type | 1 type | Be |
| 08 | 6 types | 6 types | Be |
The foregoing is only embodiments of the invention, not thereby limit the scope of the claims of the present invention, everyly utilize thisThe equivalent structure that bright description and accompanying drawing content are done or the conversion of equivalent flow process, or be directly or indirectly used in other relevant skillsArt field, is all in like manner included in scope of patent protection of the present invention.