CN103966363A - Hepatitis C virus (HCV) genotyping kit - Google Patents
Hepatitis C virus (HCV) genotyping kit Download PDFInfo
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Abstract
The invention relates to a disease pathogen gene detecting technology and particularly relates to a hepatitis C virus (HCV) genotyping kit which can be used for auxiliary diagnosis for infection of types 1a, 1b, 2a, 2b, 3a, 3b and 6a of HCV happening in China. According to the invention, a nylon membrane carrier is adopted, an oligonucleotide probe is designed according to the different genotypes of the HCV, a gene chip is prepared, and then, a PCR (Polymerase Chain Reaction) primer and other components are matched, so that the HCV in a blood sample can be rapidly and accurately typed. The HCV genotyping kit provided by the invention has the characteristics of rapidness, sensitivity, accuracy, high flux, simplicity, convenience, high sensitivity and strong specificity, and is capable of detecting various target sequences in hybridization reaction once, convenient to take materials, free of special equipment and low in instrument investment.
Description
Technical field
The present invention relates to a kind of disease pathogen technique of gene detection, relate in particular to the test kit that a kind of HCV gene typing detects, the auxiliary diagnosis infecting for detection of 1a, 1b, 2a, 2b, 3a, 3b and the 6a type of the hepatitis C virus of domestic generation.
Background technology
Hepatitis C is worldwide distribution, and the general susceptible of crowd is serious harm human health, Epidemic Scope transmissible disease widely, and its pathogenic agent is hepatitis C virus (hepatitis C virus, 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 then occurs digestive tract hemorrhage and ascites.According to WHO2002 annual report, in calendar year 2001, chronic hepatopathy causes that 1,400 ten thousand examples are dead, comprise 79.6 ten thousand examples by liver cirrhosis cause, 61.6 ten thousand examples cause by primary hepatocarcinoma.HCV is positivity single strand RNA virus, comprises 9400 left and right Nucleotide numbers.HCV genome has an independent open reading frame, gene structure is made up of 5 '-NCR-C-E1-E2-NS1-NS2-NS3-NS4-NS5-N-CR-3 ', coding one has 3010 amino acid whose polyprotein bodies, is divided into virus replication and virion and forms necessary structure and Nonstructural Protein after translation.HCV has the variability of height and itself has the feature of negative selective action, and high sudden change is because replicative enzyme is without correct functioning; The easily reason such as the existence of mistake property RDRP (error-prone RDRP, EP-RDRP) and positive selective action.Show as and itself there is negative selective action: range gene structure and function difference in viral genome, some region height is conservative.The not all or part of genomic phylogenetic analysis of homophyletic of HCV separating according to different areas, the whole world, HCV is divided into 6 kinds of main genotype (representing with 1-6), the various hypotype (representing with a, b, c etc.) of dividing again.HCV hypotype has exceeded 100, and wherein modal is 1a, 1b, and 2a, 2b, 3a, 3b, 6a, differs 31-34% between various nucleotide sequence, and it is about 30% that aminoacid sequence differs, and between hypotype sequence, differ about 20-23%.There are several HCV virus strain to be mainly separated from South East Asia, are named as HCV7,8,9,10,11 types, except HCV10a type (being put at present HCV3 type), due to the similarity 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 particularly poor compared with other type susceptibility.The infectious hepatitis of different type HCV, the moderate that it is acute, chronic and severe, posthepatitic cirrhosis and primary hepatocarcinoma incidence have difference, and the clinical state of an illness of HCV genotype and hepatitis C patients and severity thereof are closely related.All variant in view of different HCV genotype the infecteds' clinical manifestation, hepatopathy severity and chronicity course advancement, the effect of antiviral therapy is also different.Therefore detect complexity, formulation individuation antiviral therapy scheme that HCV genotype contributes to judgement treatment.
At present HCV methods of genotyping is mainly contained: restricted length polymorphism analysis method (RFLP), PCR with genotype-specific primers method, Serotype-dependent probe nucleic acid hybridization analysis method, the methods such as direct sequencing, but mostly there is complex operation in these methods, high to equipment requirements, detection time is long, the shortcomings such as 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 the test kit of the HCV gene type that a kind of time of detection is short, flux is high, highly sensitive, specificity is high.
For addressing the above problem, the present invention discloses a kind of test kit of HCV gene typing, a kind of test kit of HCV gene typing, comprise PCR reaction solution and DNA Hybond membrane bar, described DNA Hybond membrane bar comprises nylon membrane, on described nylon membrane, be fixed with probe, described probe be respectively with the Nucleotide of the nucleic acid hybridization of the various subtype virus of hepatitis C 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.
Beneficial effect of the present invention is: the design of HCV detection probes of the present invention is under Big Clinical Samples checking, sensitivity is (True Positive Rate) 99.00%, specific degree (true negative rate) is 100%, accuracy is 99.63%, false positive rate is 0, false negative rate is 1.00%, have advantages of highly sensitive, specificity is high, accuracy is high, zero false positive rate, low false negative rate, diagnosis consistence are 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 C-type virus C pattern detection result scintigram;
Fig. 3: HCV2a C-type virus C pattern detection result scintigram;
Fig. 4: HCV3a C-type virus C pattern detection result scintigram;
Fig. 5: HCV6a C-type virus C pattern detection result scintigram.
Embodiment
By describing technology contents of the present invention, structural attitude in detail, being realized object and effect, below in conjunction with embodiment and 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, ensures from the root of primer amplification and probe specificity identification high sensitivity, specificity and the accuracy that HCV detects.
The present invention discloses a kind of test kit of HCV gene typing, comprise PCR reaction solution and DNA Hybond membrane bar, described DNA Hybond membrane bar comprises nylon membrane, on described nylon membrane, be fixed with probe, described probe be respectively with the Nucleotide of the nucleic acid hybridization of the various subtype virus of hepatitis C 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 (Reverse Dot Blot, RDB),,, 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 as solid phase carrier with nylon membrane, be prepared into DNA Hybond membrane bar, with biotin labeling primer, the object nucleic acid fragment of the sample to be checked that increases, obtains with biotin labeled amplified production; Hybridize with the film bar of fixing various oligonucleotide probes again, by integrated enzyme reaction and colour developing, after scanning, with professional spot analysis software, image is identified, removed background, carries out objective signal value analysis, thereby judge and in amplified production, whether have the gene order for various oligonucleotide probes, 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 1 is sequence oligonucleotide probe and the type that detects), adopt automatic point sample instrument, probe is printed on to the specific region of a nylon membrane, judges 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 (as shown in table 2 is the present invention's primer sequence 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 fragment that may exist.Get one of DNA Hybond membrane bar, add PCR product 45 μ L to hybridize, hybridization temperature is set as 45 DEG C, and hybridization time 60 minutes, through hybridization-washing-colour developing.Take out film bar and dry, put into scanner and scan, use analysis software to carry out view data conversion process to scan image signal, and analyze generation sample HCV type result.
According to another preferred embodiment of the present invention, introducing internal reference primer for the PCR system of sample amplification, and internal reference template, and time prepared by film bar, introduced the probe that can hybridize with internal reference template, can carry out virtual mass control to PCR process.
According to another preferred embodiment of the present invention, in the time preparing film bar, having introduced colour developing control probe (CC), at one end of probe mark-NH2, with nylon membrane coupling, the other end introduce biotin labeling, can directly develop the color.The introducing of CC can be carried out virtual mass control to process color.
Another preferred embodiment according to the present invention, the sheet base carrier of DNA Hybond membrane bar can be the carrier that slide, nylon membrane or other can adhesion probes.
Table 1
Table 2
Wherein, in SEQ ID NO:21, Y is degenerated primer, Y=T/C, and the design of degenerated primer can increase the specificity of detection, detects effect thereby improve HCV.
Embodiment
1,5 ' UTR district design RT and PCR primer in HCV genome, adopts asymmetric RT-PCR system, the needed target fragment of amplification somatotype; As shown in table 1, within the scope of target fragment, 3 of design 1 type probes, 24 of type probes, 33 of type probes, 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 typing probes and 2 contrast probe points systems, to the specific region of film bar, are made to 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, prepare RT-PCR reaction system according to table 3, 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 |
| 25mM MgCl 2 | 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 |
| Reversed transcriptive enzyme or Taq enzyme | 0.5 |
| RNA template | 10 |
| Internal reference template | 1 |
| Total amount | 50 |
Pcr amplification program is as follows:
Adopt HCV gene type Hybond membrane bar of the present invention, above sample is detected.
Get a Hybond membrane bar, marker samples numbering, puts into hybrid pipe, gets respectively the each 45 μ L of 8 pipe PCR product and is added in the pipe of reference numeral, sets 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 (compound method can referring to molecular cloning), 0.5%SDS (commercially available analytical reagent)), corresponding 40ul PCR product, cover tightly pipe lid, put 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 (commercially available analytical reagent)) 1ml, mixes, and puts 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 Trisodium Citrate, 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 Trisodium Citrate, pH5.5) 20ml, E liquid 1ml (0.025%POD (can purchased from ROCHE company), F liquid (0.3H2O2) 1ml, mixes and is nitrite ion, put into film bar and leave standstill 8 minutes in room temperature lucifuge, discard liquid;
Add D liquid (0.5M Trisodium Citrate, 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 converts image to data.
Refer to Fig. 2, Fig. 3, Fig. 4 and Fig. 5, CC in Fig. 2, PC probe site hybridization signal are positive, illustrate that the PCR environment of this secondary response is normal and color reaction is normal, the hybridization signal of H1 and H15 probe detected simultaneously, this sample HCV infection 1a C-type virus C is described; In like manner, Fig. 3 detects the hybridization signal of H4, H5 and H15 probe simultaneously, illustrates that this sample is HCV infection 2a C-type virus C sample; Fig. 4 detects the hybridization signal of H8, H8, H10 and H15 probe simultaneously, illustrates that this sample is HCV infection 3a C-type virus C sample; Fig. 5 detects the hybridization signal of H12, H12, H14 and H15 probe simultaneously, illustrates that this sample is HCV infection 6a C-type virus C sample.
Adopt analysis software, above data analysis is converted into the gene type of each sample,, by 8 these DNA sequencings of increment, contrast as detected result meanwhile.Result shows, 8 these detected results of increment and sample sequencing result conform to completely (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; The reliability (detected result conforms to completely with sample sequencing result) of detection accuracy aspect.
Table 4
| Sample number | Sequencing result | This test-results | 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 verification adopts the test design of blind method, contrast, collect sample and a part of negative sample from the HCV detection by quantitative positive of three front three hospitals, adopt test kit of the present invention to carry out somatotype detection, use sequencing as gold standard, this product detected result to be verified simultaneously.Detecting altogether effective sample number is 1092 examples, wherein case group sample 402 examples, and control group sample 690 examples, sample type is serum.Result: the detected result of 4 routine sample product of the present invention and gold standard method is not inconsistent.Statistical analysis, the sensitivity of product of the present invention is 99.00%, and specificity is 100%, and accuracy is 99.63%.Kappa check consistency analytical results is under the inspection level of α=0.05, Kappa value=0.992, and P=0.000, the consistence of product of the present invention and contrast method has statistical significance, and diagnosis consistence is fabulous.Clinical study result shows that this test kit is reliable, accurately, safety, easy, stable, there is higher clinical value.
Table 5 is that each type distributes and coincidence rate cartogram, and table 6 is Authentic Assessment data compilation tables, and concrete statistics is as follows.
Table 5
Table 6
Sensitivity (True Positive Rate)=A/ (A+C) × 100%=398/ (398+4)=99.00%
Specific degree (true negative rate)=D/ (B+D) × 100%=690/ (0+690)=100%
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 test kit and gold standard result is diagnosed consistency analysis, and inspection level is α=0.05.The reference of Kappa value is evaluated to principle as follows: 0.75 < κ≤1 o'clock, diagnosis consistence is fabulous; 0.4 < κ≤0.75 o'clock, diagnosis high conformity; 0≤κ≤0.4 o'clock, diagnosis consistence is poor.
Symmetric Measures
a Not assuming the null hypothesis.
b Using the asymptotic standard error assuming the null hypothesis.
Above-mentioned consistency analysis table, for using SPSS13.0 software gained, those skilled in the art know that the implication of each project of this form, 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, this test kit has statistical significance with the consistence of gold standard method, and diagnosis consistence is fabulous.
In sum, the design of HCV detection probes of the present invention is under Big Clinical Samples checking, sensitivity is (True Positive Rate) 99.00%, specific degree (true negative rate) is 100%, accuracy is 99.63%, false positive rate is 0, and false negative rate is 1.00%, and diagnosis consistence is fabulous; HCV gene typing detecting reagent kit provided by the invention has advantages of that the time of detection is short, flux is high, highly sensitive, specificity is high, diagnosis high conformity, one time hybridization can detect multiple target sequence, draw materials conveniently, without specific installation, instrument drop into low, have fast and convenient, susceptibility is high and the beneficial effect of high specificity.
The foregoing is only embodiments of the invention; not thereby limit the scope of the claims of the present invention; every equivalent structure or conversion of equivalent flow process that utilizes specification sheets of the present invention and accompanying drawing content to do; or be directly or indirectly used in other relevant technical fields, be all in like manner included in scope of patent protection of the present invention.
Claims (7)
1. the test kit of a HCV gene typing, comprise PCR reaction solution and DNA Hybond membrane bar, it is characterized in that, described DNA Hybond membrane bar comprises nylon membrane, on described nylon membrane, be fixed with probe, described probe be respectively with the Nucleotide of the nucleic acid hybridization of the various subtype virus of hepatitis C 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.
2. the test kit of HCV gene typing as claimed in claim 1, is characterized in that, described PCR reaction solution comprises the primer of the nucleic acid of the various subtype virus of amplification hepatitis C virus, and described primer sequence is as follows:
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.
3. the test kit of HCV gene typing as claimed in claim 2, is characterized in that, in described upstream primer F2, Y is degenerated primer, described Y=T/C.
4. the test kit of HCV gene typing as claimed in claim 1, it is characterized in that, on described nylon membrane, be also provided with colour developing and control probe CC, described colour developing control probe CC sequence is as follows: 5 '-GCGCAGGGCCACAATAATGG-3 ', 5 ' the end of described colour developing control probe CC carries out amino labeled, and 3 ' end carries out biotin labeling.
5. the test kit of HCV gene typing as claimed in claim 1, is characterized in that, described PCR reaction solution also comprises internal reference system, and described internal reference system is Arabidopis thaliana internal reference system, and described internal reference system comprises:
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.
6. the test kit of HCV gene typing as claimed in claim 1, is characterized in that, described concentration and probe concentration is 5-20 μ M.
7. the test kit of HCV gene typing as claimed in claim 1, is characterized in that, described PCR reaction solution also comprises 10 times of PCR damping fluids, 25mM MgCl2,10mM dNTP and enzyme, and described enzyme is reversed transcriptive enzyme or Taq enzyme.
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Cited By (3)
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| CN106282404A (en) * | 2015-05-28 | 2017-01-04 | 达雅高生物科技有限公司 | Quick and the Sensitive Detection of hepatitis C virus and genotype identification |
| CN107083449A (en) * | 2017-05-08 | 2017-08-22 | 英科新创（苏州）生物科技有限公司 | A kind of HCV gene typing immue quantitative detection reagent box |
| CN109593887A (en) * | 2018-12-29 | 2019-04-09 | 中山大学达安基因股份有限公司 | A kind of kit of hepatitis C virus nucleic acid quantitative detection |
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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 |
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Cited By (3)
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| CN106282404A (en) * | 2015-05-28 | 2017-01-04 | 达雅高生物科技有限公司 | Quick and the Sensitive Detection of hepatitis C virus and genotype identification |
| CN107083449A (en) * | 2017-05-08 | 2017-08-22 | 英科新创（苏州）生物科技有限公司 | A kind of HCV gene typing immue quantitative detection reagent box |
| CN109593887A (en) * | 2018-12-29 | 2019-04-09 | 中山大学达安基因股份有限公司 | A kind of kit of hepatitis C virus nucleic acid quantitative detection |
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