CN103966363B - A kind of kit of HCV gene typing - Google Patents
A kind of kit of HCV gene typing Download PDFInfo
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- CN103966363B CN103966363B CN201410208083.3A CN201410208083A CN103966363B CN 103966363 B CN103966363 B CN 103966363B CN 201410208083 A CN201410208083 A CN 201410208083A CN 103966363 B CN103966363 B CN 103966363B
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Abstract
The present invention relates to a kind of disease pathogen technique of gene detection, relate in particular to the kit that a kind of HCV gene typing detects, can be used for the auxiliary diagnosis of 1a, 1b, 2a, 2b, 3a, 3b and the infection of 6a type of the HCV that detects domestic generation. The present invention adopts nylon membrane carrier, for HCV different genotype design oligonucleotides probe, is prepared into genetic chip, then is equipped with PCR primer and other components, can quick and precisely carry out somatotype to HCV in blood sample. The kit of HCV gene typing of the present invention has quick, sensitive, accurate, high-throughout feature, one time hybridization reaction can detect multiple target sequence, draws materials conveniently, without special installation, instrument drop into low, have fast and convenient, susceptibility is high and the feature of high specificity.
Description
Technical field
The present invention relates to a kind of disease pathogen technique of gene detection, relate in particular to a kind of HCV gene typingThe kit detecting, infects for detection of 1a, 1b, 2a, 2b, 3a, 3b and the 6a type of the HCV of domestic generationAuxiliary diagnosis.
Background technology
Hepatitis C is worldwide distribution, and the general susceptible of crowd is that serious harm human health, Epidemic Scope pass widelyCatch an illness, 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, wherein has every year to exceed 100,000 routine HCV patients and develop into liver cancer, and thenThere is hemorrhage of digestive tract and ascites. According to WHO2002 annual report, in calendar year 2001, chronic liver disease causes that 1,400 ten thousand examples are dead, comprises79.6 ten thousand examples by cirrhosis cause, 61.6 ten thousand examples cause by primary carcinoma of liver. HCV is positivity single strand RNA virus, comprises 9400 left sidesRight nucleotides number. HCV genome has an independent ORFs, and gene structure is by 5 '-NCR-C-E1-E2-NS1-NS2-NS3-NS4-NS5-N-CR-3 ' composition, coding one has 3010 amino acid whose polyprotein bodies, is divided into disease after translationPoison copies with virion and forms necessary structure and non-structural protein. HCV has the variability of height and itself has negative choosingSelect the feature of effect, high sudden change is because replicase is without calibration function; Easily mistake property RDRP (error-proneRDRP, EP-The reasons such as existence RDRP) and just selection effect. Show as and itself there is negative selection effect: each in viral genomePlant gene structure and function difference, some region height is conservative. Not homophyletic whole of the HCV separating according to different regions, the whole worldOr the phylogenetic analysis of portion gene group, HCV is divided into 6 kinds of main genotype (representing with 1-6), various divide again hypotype (withThe expressions such as a, b, c). HCV hypotype has exceeded 100, and wherein modal is 1a, 1b, 2a, 2b, 3a, 3b, 6a, various nucleic acidBetween sequence, differ 31-34%, it is about 30% that amino acid sequence differs, and between hypotype sequence, differ about 20-23%. Have severalPlant HCV Strain and be mainly separated from Southeast Asia, be named as HCV7,8,9,10,11 types, except HCV10a type (quilt at presentList HCV3 type in), 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
Technical problem to be solved by this invention is to provide that a kind of time of detection is short, flux is high, highly sensitive, specificityThe kit of high HCV Genotyping.
For addressing the above problem, the present invention discloses a kind of kit of HCV gene typing, a kind of the third type liverThe kit of scorching gene typing, comprises PCR reactant liquor and DNA hybond membrane bar, and described DNA hybond membrane bar comprises nylon membrane,On described nylon membrane, be fixed with probe, described probe be respectively with the core of the nucleic acid hybridization of the various subtype virus of HCVThuja acid, described probe sequence is as follows:
For HCV1a type designing probe Y1:5 '-GCAGGGGCCCTAGATTGGGT-3 ';
For HCV1b type designing probe Y2:5 '-CGTGGAAGGCGACAACCTA-3 ';
For HCV1b type designing probe Y3:5 '-AACCTCGTGGAAGGCGACAACC-3 ';
For HCV2a type designing probe Y4:5 '-ATTGCCGGGAAGACTGGGTC-3 ';
For HCV2a type designing probe Y5:5 '-ACTTCGGAGCGGTCCCA-3 ';
For HCV2b type designing probe Y6:5 '-CAGCCCATCCCGAAAGATCGG-3 ';
For HCV2b type designing probe Y7:5 '-GAATTACCGGAAAGACTGGGT-3 ';
For HCV3a type designing probe Y8:5 '-AATCGCTGGGGTGACCGG-3 ';
For HCV3a type designing probe Y9:5 '-TCTGAACGGTCACAGCCTC-3 ';
For HCV3a type designing probe Y10:5 '-CCCGCGAGATCACTAGCC-3 ';
For HCV3b type designing probe Y11:5 '-CGCTCAATGCCCGGAAATT-3 ';
For HCV6a type designing probe Y12:5 '-GACCGGGTCCTTTCCATTGG-3 ';
For HCV6a type designing probe Y13:5 '-GAGCGATCCCAGCCCAGAG-3 ';
For HCV6a type designing probe Y14:5 '-GAACGGTCCCAGCCCAGAG-3 ';
For HCV1-6 type design general probe Y15:5 '-TTGGGTGTGCGCGCGAC-3 ';
Negative probe HN:5 '-GGTCCTGTTTAACTGGCG-3 ';
Described probe 5 ' end carry out amino labeled with nylon membrane coupling.
Beneficial effect of the present invention is: the design of HCV detector probe of the present invention is under Big Clinical Samples checking, sensitiveDegree is (True Positive Rate) 99.00%, and specificity (true negative rate) is 100%, and the degree of accuracy is 99.63%, and false positive rate is 0, vacationNegative rate is 1.00%, have highly sensitive, specificity is high, accuracy is high, zero false positive rate, low false negative rate, diagnosis are consistentThe advantage that property is fabulous.
Brief description of the drawings
Fig. 1: the arrangement schematic diagram of HCV probe of the present invention on nylon membrane;
Fig. 2: HCV1a type Virus Sample testing result scintigram;
Fig. 3: HCV2a type Virus Sample testing result scintigram;
Fig. 4: HCV3a type Virus Sample testing result scintigram;
Fig. 5: HCV6a type Virus Sample testing result scintigram.
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 design of most critical of the present invention is: design has the probe sequence of high specific, from primer amplification and probe spyIn the root of opposite sex identification, ensure high sensitivity, specificity and the accuracy that HCV detects.
The present invention discloses a kind of kit of HCV gene typing, comprises PCR reactant liquor and DNA hybond membraneBar, described DNA hybond membrane bar comprises nylon membrane, on described nylon membrane, is fixed with probe, described probe for respectively with hepatitis CThe nucleotides of the nucleic acid hybridization of the various subtype virus of virus, described probe sequence is as follows:
For HCV1a type designing probe Y1:5 '-GCAGGGGCCCTAGATTGGGT-3 ';
For HCV1b type designing probe Y2:5 '-CGTGGAAGGCGACAACCTA-3 ';
For HCV1b type designing probe Y3:5 '-AACCTCGTGGAAGGCGACAACC-3 ';
For HCV2a type designing probe Y4:5 '-ATTGCCGGGAAGACTGGGTC-3 ';
For HCV2a type designing probe Y5:5 '-ACTTCGGAGCGGTCCCA-3 ';
For HCV2b type designing probe Y6:5 '-CAGCCCATCCCGAAAGATCGG-3 ';
For HCV2b type designing probe Y7:5 '-GAATTACCGGAAAGACTGGGT-3 ';
For HCV3a type designing probe Y8:5 '-AATCGCTGGGGTGACCGG-3 ';
For HCV3a type designing probe Y9:5 '-TCTGAACGGTCACAGCCTC-3 ';
For HCV3a type designing probe Y10:5 '-CCCGCGAGATCACTAGCC-3 ';
For HCV3b type designing probe Y11:5 '-CGCTCAATGCCCGGAAATT-3 ';
For HCV6a type designing probe Y12:5 '-GACCGGGTCCTTTCCATTGG-3 ';
For HCV6a type designing probe Y13:5 '-GAGCGATCCCAGCCCAGAG-3 ';
For HCV6a type designing probe Y14:5 '-GAACGGTCCCAGCCCAGAG-3 ';
For HCV1-6 type design general probe Y15:5 '-TTGGGTGTGCGCGCGAC-3 ';
Negative probe HN:5 '-GGTCCTGTTTAACTGGCG-3 ';
Described probe 5 ' end carry out amino labeled with nylon membrane coupling.
Described general probe Y15 is positive probe HP.
From foregoing description, beneficial effect of the present invention is: the present invention adopts reversal point hybridization technique (ReverseDotBlot, RDB), use nylon membrane as solid phase carrier, for the specific oligonucleotide probe of HCV different genotype design,Each HCV specific probe is fixed on film bar in the mode of round dot, is prepared into DNA hybond membrane bar, with biotin labeling primer,The increase object nucleic acid fragment of sample to be checked, obtains with biotin labeled amplified production; Again with fixing various oligonucleotidesThe film bar hybridization of probe, by integrated enzyme reaction and colour developing, identifies, goes image with professional spot-analysis software after scanningExcept background, carry out objective signal value analysis, thereby judge in amplified production, whether there is the base for various oligonucleotide probesBecause of sequence, in result 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
Wherein, in SEQIDNO:21, Y is degenerate primer, Y=T/C, and the design of degenerate primer can increase the spy of detectionThe opposite sex, detects effect thereby improve HCV.
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 8 examples and positive and negative and contrast each portion, add interior mark template, extract RNA, according to table 3Preparation RT-PCR reaction system, each primer is as shown in table 2, carries out pcr amplification, obtains DNA hybridization template.
Table 3
| Reagent | 1 person-portion (μ L) |
| Pure water | 20.5 |
| 10×PCR buffer | 5 5 --> |
| 25mM MgCl2 | 6 |
| dNTP(10mM) | 1 |
| 10μM F | 2 |
| 10μM R | 1 |
| 10 μ M F1 and RT | 2 |
| 10μM R1 | 1 |
| 10μM F2 | 2 |
| 10μM R2 | 1 |
| Reverse transcriptase or Taq enzyme | 0.5 |
| RNA template | 10 |
| Internal reference template | 1 |
| 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, ((compound method can be referring to molecule for 2*SSC to add 5-8ml preheating A liquidClone), 0.5%SDS (commercially available AR)), corresponding 40ulPCR product, cover tightly pipe lid, put into vibration tank, 50 DEG CJog (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, (0.01%TMB (the pure examination of commercially available analysis of C liquidAgent)) 1ml, mix, put into 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, (0.025%POD (can be purchased from for E liquid 1mlROCHE company), F liquid (0.3H2O2) 1ml, mixes and is nitrite ion, puts into film bar and leaves standstill 8 minutes in room temperature lucifuge, discards liquidBody;
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.
Refer to Fig. 2, Fig. 3, Fig. 4 and Fig. 5, CC in Fig. 2, PC probe site hybridization signal are positive, illustrate this time anti-The PCR environment of answering is normal and chromogenic reaction is normal, the hybridization signal of H1 and H15 probe detected simultaneously, and this sample sense is describedDye HCV1a type virus; In like manner, Fig. 3 detects the hybridization signal of H4, H5 and H15 probe simultaneously, illustrates that this sample is for infectingHCV2a type Virus Sample; Fig. 4 detects the hybridization signal of H8, H8, H10 and H15 probe simultaneously, illustrates that this sample is for infectingHCV3a type Virus Sample; Fig. 5 detects the hybridization signal of H12, H12, H14 and H15 probe simultaneously, illustrates that this sample is for infectingHCV6a type Virus Sample.
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 | 1a | 1a | Be |
| 02 | 2a | 2a | Be |
| 03 | 1b | 1b | Be |
| 04 | 3a | 3a | Be |
| 05 | 3b | 3b | Be |
| 06 | 6a | 6a | Be |
| 07 | 1b | 1b | Be |
| 08 | 6a | 6a | Be |
4. use product of the present invention to carry out the checking of large sample
According to the definite experimental implementation of embodiment 3 and method, product of the present invention is carried out to clinical verification. This clinical verificationAdopt the experimental design of blind method, contrast, collect and quantitatively detect positive sample and a part from the HCV of three front three hospitalsNegative sample, adopts kit of the present invention to carry out somatotype detection, uses PCR sequencing PCR, as goldstandard, this product is detected to knot simultaneouslyFruit is verified. Detecting altogether effective sample number is 1092 examples, wherein case group sample 402 examples, control group sample 690 examples, sampleType is serum. Result: the testing result of 4 routine sample product of the present invention and goldstandard method is not inconsistent. Statistical analysis, the present inventionThe sensitivity of product is 99.00%, and specificity is 100%, and the degree of accuracy is 99.63%. Kappa check consistency analysis result isUnder the inspection level of α=0.05, Kappa value=0.992, P=0.000, the uniformity of product of the present invention and contrast method has statisticsLearn meaning, diagnosis uniformity is fabulous. Clinical research result shows that this kit is reliable, accurately, safety, easy, stable, haveHigh clinical value.
Table 5 is that each type distributes and coincidence rate statistical form, and table 6 is Authentic Assessment data to arrange tables, concrete statisticsAs follows.
Table 5
Table 6
Sensitivity (True Positive Rate)=A/ (A+C) × 100%=398/ (398+4)=99.00%
Specificity (true negative rate)=D/ (B+D) × 100%=690/ (0+690)=100%
The degree of accuracy=(A+D)/(A+B+C+D) × 100%=(398+690)/(398+0+4+690)=99.63%
False positive rate=B/ (B+D) × 100%=0%
False negative rate=C/ (A+C) × 100%=4/402=1.00%
Application Kappa evaluation method treats examination kit and goldstandard result is diagnosed consistency analysis, inspection waterStandard is α=0.05. The reference of Kappa value is evaluated to principle as follows: 0.75 < κ≤1 o'clock, diagnosis uniformity is fabulous; 0.4 < κ≤0.75 o'clock, diagnosis high conformity; 0≤κ≤0.4 o'clock, diagnosis uniformity is poor.
SymmetricMeasures
aNotassumingthenullhypothesis.
bUsingtheasymptoticstandarderrorassumingthenullhypothesis.
Above-mentioned consistency analysis table, for using SPSS13.0 software gained, those skilled in the art know that each of this formThe implication of project, therefore no longer carry out translator of Chinese.
Through SPSS13.0 software Kappa inspection, Kappa value=0.992, P=0.000. Under the inspection level of α=0.05, thisThe uniformity of kit and goldstandard method has statistical significance, and diagnosis uniformity is fabulous.
In sum, the design of HCV detector probe of the present invention is under Big Clinical Samples checking, and sensitivity is (true positivesRate) 99.00%, specificity (true negative rate) is 100%, and the degree of accuracy is 99.63%, and false positive rate is 0, and false negative rate is1.00%, diagnosis uniformity is fabulous; HCV gene typing detecting reagent kit provided by the invention has detectionTime is short, flux is high, highly sensitive, specificity is high, the advantage of diagnosis high conformity, and one time hybridization reaction can detect multiple targetSequence, draws materials conveniently, and without special installation, instrument drops into low, have fast and convenient, susceptibility is high and high specificity have a beneficial effectReally.
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.
Claims (5)
1. a kit for HCV gene typing, comprises PCR reactant liquor and DNA hybond membrane bar, it is characterized in that,Described DNA hybond membrane bar comprises nylon membrane, on described nylon membrane, is fixed with probe, described probe for respectively with HCVThe nucleotides of the nucleic acid hybridization of various subtype virus, described probe sequence is as follows:
For HCV1a type designing probe Y1:5 '-GCAGGGGCCCTAGATTGGGT-3 ';
For HCV1b type designing probe Y2:5 '-CGTGGAAGGCGACAACCTA-3 ';
For HCV1b type designing probe Y3:5 '-AACCTCGTGGAAGGCGACAACC-3 ';
For HCV2a type designing probe Y4:5 '-ATTGCCGGGAAGACTGGGTC-3 ';
For HCV2a type designing probe Y5:5 '-ACTTCGGAGCGGTCCCA-3 ';
For HCV2b type designing probe Y6:5 '-CAGCCCATCCCGAAAGATCGG-3 ';
For HCV2b type designing probe Y7:5 '-GAATTACCGGAAAGACTGGGT-3 ';
For HCV3a type designing probe Y8:5 '-AATCGCTGGGGTGACCGG-3 ';
For HCV3a type designing probe Y9:5 '-TCTGAACGGTCACAGCCTC-3 ';
For HCV3a type designing probe Y10:5 '-CCCGCGAGATCACTAGCC-3 ';
For HCV3b type designing probe Y11:5 '-CGCTCAATGCCCGGAAATT-3 ';
For HCV6a type designing probe Y12:5 '-GACCGGGTCCTTTCCATTGG-3 ';
For HCV6a type designing probe Y13:5 '-GAGCGATCCCAGCCCAGAG-3 ';
For HCV6a type designing probe Y14:5 '-GAACGGTCCCAGCCCAGAG-3 ';
For HCV1-6 type design general probe Y15:5 '-TTGGGTGTGCGCGCGAC-3 ';
Negative probe HN:5 '-GGTCCTGTTTAACTGGCG-3 ';
Described probe 5 ' end carry out amino labeled with nylon membrane coupling;
Described PCR reactant liquor comprises the primer of the nucleic acid of amplification HCV various subtype virus, described primer sequence asShown in lower:
Reverse transcriptase primer RT:5 '-GAAAGCGTCTAGCCATGGCGTTAGT-3 ';
Upstream primer F1:5 '-GAAAGCGTCTAGCCATGGCGTTAGT-3 ';
Downstream primer R1:5 '-CGCAAGCACCCTATCAGGCAGTA-3 ';
Upstream primer F2:5 '-TGGTCAGATCGTYGGYGGAGT-3 ';
Downstream primer R2:5 '-ACGAGCGGAATGTACCCCATGAG-3 ';
5 ' the end of described downstream primer R1, R2 carries out biotin labeling;
Described PCR reactant liquor also comprises internal reference system, and described internal reference system is arabidopsis internal reference system, and described internal reference isTurnkey is drawn together:
Internal reference primers F: 5 '-TCATCGTCGCTGGAGCTGGTT-3 ';
Internal reference primer R:5 '-CGGCGGTTTGTCAAGCTGAT-3 ';
Internal reference probe PC:5 '-TGCCGATATAGGTCACAGCATGGGC-3 ';
5 ' end of described internal reference primers F carries out biotin labeling.
2. the kit of HCV gene typing as claimed in claim 1, is characterized in that, described upstream primer F2Middle Y is degenerate primer, described Y=T/C.
3. the kit of HCV gene typing as claimed in claim 1, is characterized in that, on described nylon membrane, goes backBe provided with colour developing and control probe CC, described colour developing control probe CC sequence is as follows: 5 '-GCGCAGGGCCACAATAATGG-3 ', the 5 ' end of described colour developing control probe CC carries out amino labeled, and 3 ' end carries out biotin labeling.
4. the kit of HCV gene typing as claimed in claim 1, is characterized in that, described concentration and probe concentration is5-20μM。
5. the kit of HCV gene typing as claimed in claim 1, is characterized in that, described PCR reactant liquorAlso comprise 10 times of PCR buffer solutions, 25mMMgCl2,10mMdNTP and enzymes, described enzyme is reverse transcriptase or Taq enzyme.
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| CN101660002A (en) * | 2008-08-27 | 2010-03-03 | 中山大学达安基因股份有限公司 | HCV genotyping DNA microarray chip |
| CN102766701A (en) * | 2012-07-04 | 2012-11-07 | 福州泰普生物科学有限公司 | Kit and method for genotyping of hepatitis C virus |
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| CN101660002A (en) * | 2008-08-27 | 2010-03-03 | 中山大学达安基因股份有限公司 | HCV genotyping DNA microarray chip |
| CN102766701A (en) * | 2012-07-04 | 2012-11-07 | 福州泰普生物科学有限公司 | Kit and method for genotyping of hepatitis C virus |
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