CN102766701A - Kit and method for genotyping of hepatitis C virus - Google Patents
Kit and method for genotyping of hepatitis C virus Download PDFInfo
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- CN102766701A CN102766701A CN2012102292359A CN201210229235A CN102766701A CN 102766701 A CN102766701 A CN 102766701A CN 2012102292359 A CN2012102292359 A CN 2012102292359A CN 201210229235 A CN201210229235 A CN 201210229235A CN 102766701 A CN102766701 A CN 102766701A
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Abstract
The invention relates to a disease pathogen gene detection technology and particularly relates to a kit and method for genotyping of hepatitis C virus, which can be used for auxiliary diagnosis for detecting type 1a, 1b, 2a, 2b, 3a and 6a infection of the hepatitis C virus occurring in China. According to the kit disclosed by the invention, a nylon membrane carrier is adopted, oligonucleotide probes are designed against different genotypes of the HCV (hepatitis C virus), a gene chip is prepared, and a PCR (polymerase chain reaction) primer and other components are further matched, so that the hepatitis C virus in blood samples can be fast and accurately typed. The gene chip, the kit as well as the method for genotyping of the hepatitis C virus, disclosed by the invention, have the characteristics of high speed, sensitivity, accuracy and high flux, one-time hybridization reaction can detect a variety of target sequences, materials can be taken conveniently, no special equipment is required, the instrument investment is low, and the characteristics of high speed, simplicity, convenience, high sensitivity and strong specificity can be further realized.
Description
Technical field
The present invention relates to a kind of disease pathogen technique of gene detection, relate in particular to a kind of test kit and method of HCV gene typing, be used to detect the type 1a of the hepatitis C virus of domestic generation, 1b, 2a, 2b, 3a, the auxiliary diagnosis that 6a infects.
Background technology
Hepatitis C virus (hepatitis C virus, HCV), hepatitis C is worldwide distribution, and the general susceptible of crowd is serious harm human health, popular far-ranging transmissible disease.Its pathogenic agent be hepatitis C virus (hepatitis C virus, HCV).At present, its infection rate is 0.1%-10%, average out to 3%.There are 1.7 hundred million people's HCV infection of surpassing in the whole world, and wherein has every year the 100000 routine HCV patients of surpassing to develop into liver cancer, and then digestive tract hemorrhage and ascites occur.According to the 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 a positivity single stranded RNA flavivirus, comprises about 9400 Nucleotide.The HCV genome has an independent ORFs; 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 after the translation and forms necessary structure and Nonstructural Protein.HCV has the variability (its aberration rate is up to 1.4-1.9 * 10-3/ Nucleotide/year) of height and itself has the characteristic of negative selective action, and high sudden change is because replicative enzyme does not have correct functioning; Be prone to mistake property RDRP (error-prone RDRP, reasons such as existence EP-RDRP) and positive selective action.Show as and itself have negative selective action: range gene structure and function are different in the viral genome, and some region height is conservative.According to the isolating HCV in different areas, the whole world all or part of genomic phylogenetic analysis of homophyletic not, HCV is divided into 6 kinds of main genotype (representing with 1-6), the various hypotype (with expressions such as a, b, c) of dividing again.The HCV hypotype has surpassed 100, and wherein modal is 1a, 1b, and 2a, 2b, 3a, 6a differs 31-34% between the various nucleotide sequence, and it is about 30% that aminoacid sequence differs, and differ about 20-23% between the hypotype sequence.There are several kinds of HCV virus strain mainly to be separated, are named as HCV7 from South East Asia, 8,9,10,11 types, except HCV10a type (being put into the HCV3 type at present), since the similarity on its phyletic evolution, all the other various HCV6 types that are put into.
Research shows that the susceptibility to interferon therapy between the different types of HCV differs, and particularly 1 type is particularly poor than other type susceptibility.The infectious hepatitis of different type HCV, moderate that it is acute, chronic and severe, posthepatitic cirrhosis and primary hepatocarcinoma incidence all 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 of disease progress, the effect of antiviral therapy is also different.Therefore detect complexity, formulation individuation antiviral therapy scheme that the HCV genotype helps to judge treatment.
At present the HCV methods of genotyping is mainly contained: restricted length polymorphism analysis method (RFLP), genotype Auele Specific Primer PCR method, genotype specific probe nucleic acid hybridization analysis method; Methods such as direct sequencing, but there is complex operation mostly in these methods, high to equipment requirements; Detection time is long; Flux is low, also exists sensitivity low, the not high shortcoming of specificity.
Summary of the invention
The objective of the invention is to overcome the defective and the deficiency of prior art; Manufacturing has gene chip and the test kit and the method for the HCV gene typing of high-throughput, high accuracy; Can disposablely carry out accurate somatotype and detect, save detection time HCV in a plurality of samples.
Technical scheme of the present invention is the test kit that a kind of HCV gene typing is provided; Mainly comprise PCR reaction solution and DNA Hybond membrane bar; Said DNA Hybond membrane bar comprises nylon membrane; Be fixed with probe on the said nylon membrane, said probe is can be respectively and the Nucleotide of the nucleic acid hybridization of the various subtype virus of hepatitis C virus, and said probe sequence is as follows:
To HBV1a type designing probe Y1:5 '-AATTGCCAGGACGACCGG-3 ';
To HBV1b type designing probe Y2:5 '-AATTGCCAGGATGACCGGG-3 ';
To HBV1b type designing probe Y3:5 '-CCCGGAATTGCCAGGAC-3 ';
To HBV2a type designing probe Y4:5 '-CAATGCCTGGAGATTTGGGCG-3 ';
To HBV2a type designing probe Y5:5 '-CAATGCCTGGAGATTTGGGC-3 ';
To HBV2b type designing probe Y6:5 '-CTCAATGCCTGGAGATTTGG-3 ';
To HBV2b type designing probe Y7:5 '-GCTCAATGCCTGGAGATTTGG-3 ';
To HBV3a type designing probe Y8:5 '-CAATGCCTGGAGATTTGGG-3 ';
To HBV3a type designing probe Y9:5 '-CGAGGTAGACGTCAGCC-3 ';
To HBV3a type designing probe Y10:5 '-ACCTCGAGGTAGACGTCAGCC-3 ';
To HBV6a type designing probe Y11:5 '-AACCTCGAGGTAGACGTCAGCC-3 ';
To HBV6a type designing probe Y12:5 '-GCAACCTCGAGGTAGACGT-3 ';
To HBV6a type designing probe Y13:5 '-TAGACGTCAGCCTATCCCCAA-3 ';
To HBV1-6 type design general probe (being positive probe HP) Y14:5 '-TAGACGCCAGCCTATTCCCA-3 ';
Negative probe HN:5 '-GGTCCTGTTTAACTGGCG-3 ';
Said each probe 5 ' end carry out amino labeled with the nylon membrane coupling.
Preferably; In the test kit of above-mentioned HCV gene typing; Also be provided with colour developing control probe CC on the said nylon membrane; Said colour developing control probe CC sequence is as follows: 5 '-GCGCAGGGCCACAATAATGG-3 ', and said cc probe 5 ' end carries out amino labeled, and 3 ' end carries out biotin labeling.
Preferably, in the test kit of above-mentioned HCV gene typing, said PCR reaction solution comprises the primer of the nucleic acid of the various subtype virus of amplification hepatitis C virus, and said 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 said upstream primer F carries out biotin labeling.
Preferably, in the test kit of above-mentioned HCV gene typing, said PCR reaction solution also comprises the confidential reference items system, and said internal reference system is an Arabidopis thaliana internal reference system, comprising:
Confidential reference items primers F 1:5 '-TCATCGTCGCTGGAGCTGGTT-3 ';
Confidential reference items primer R1:5 '-CGGCGGTTTGTCAAGCTGAT-3 ';
Confidential reference items probe PC:5 '-TGCCGATATAGGTCACAGCATGGGC-3 ';
5 ' end of said confidential reference items primers F 1 carries out biotin labeling.
Preferably, in the test kit of above-mentioned HCV gene typing, said concentration and probe concentration is 5-20 μ M.
Another technical scheme of the present invention is the method that a kind of HCV gene typing is provided, and this method comprises following concrete steps:
A, design serotype specific primer and probe, said probe is shown in claim 1 probe Y1-Y14, and said primer comprises reverse transcriptase primer RT:5 '-CGTCTAGCCATGGCGTTAGT-3 ';
Upstream primer F:5 '-GAAAGCGTCTAGCCATGGCGTTAGT-3 ';
Downstream primer R:5 '-CGCAAGCACCCTATCAGGCAGTA-3 ';
B, with 5 ' end of the said probe of active amino markers step a, and it is fixed on the nylon membrane successively, process and detect the film bar, the 5 ' end of said downstream primer R is carried out biotin labeling;
C, utilize that primer carries out the HBV DNA cloning described in the step a, with amplified production with detect the hybridization of film bar, develop the color with px and TMB;
D, employing scanner, the scanning hybridization signal is judged the gene type result.
Preferably; The method of above-mentioned HCV gene typing; Also be provided with colour developing control probe CC on the said nylon membrane, said colour developing control probe CC sequence is as follows: 5 '-GCGCAGGGCCACAATAATGG-3 '; 5 ' the end of said colour developing control probe CC carries out amino labeled, and 3 ' end carries out biotin labeling.
Preferably, the method for above-mentioned HCV gene typing, the primer among the said step a also comprises:
Confidential reference items primers F 1:5 '-TCATCGTCGCTGGAGCTGGTT-3 ';
Confidential reference items primer R1:5 '-CGGCGGTTTGTCAAGCTGAT-3 ';
Probe among the said step a also comprises:
Confidential reference items probe PC:5 '-TGCCGATATAGGTCACAGCATGGGC-3 ';
5 ' end of said confidential reference items primers F 1 carries out biotin labeling.
The gene chip of HCV gene typing of the present invention and test kit and method have fast, sensitive, accurate, high-throughout characteristics; One time hybridization can detect multiple target sequence; Draw materials conveniently; Need not specific installation, it is low that instrument drops into, have fast and convenient, susceptibility is high and the characteristics of high specificity.
Description of drawings
Fig. 1: the arrangement synoptic diagram of HCV probe of the present invention on nylon membrane;
Fig. 2: 1 type HCV Virus Sample detected result scintigram;
Fig. 3: 2 type HCV Virus Sample detected result scintigrams;
Fig. 4: 3 type HCV Virus Sample detected result scintigrams;
Fig. 5: 6 type HCV Virus Sample detected result scintigrams.
Embodiment
By specifying technology contents of the present invention, structural attitude, realized purpose and effect, give explanation below in conjunction with embodiment and conjunction with figs. are detailed.
The objective of the invention is to prepare a kind of hepatitis C virus (HCV) gene typing DNA Hybond membrane bar, carry out the HCV needs of somatotype quick and precisely to satisfy a large amount of blood samples.
For this reason, the present invention adopts following technical scheme: and employing reversal point hybridization technique (Reverse Dot Blot, RDB); As solid phase carrier, the specific oligonucleotide probe to the design of HCV different genotype is fixed on the mode of each specific probe of HCV with round dot on the film bar with nylon membrane; Be prepared into DNA Hybond membrane bar; With the biotin labeling primer, the purpose nucleic acid fragment of the sample to be checked that increases obtains to have biotin labeled amplified production; Hybridize with the film bar of fixing various oligonucleotide probes again; Through integrated enzyme reaction and colour developing; The scanning back is discerned, is removed background, carries out objective signal value analysis image with the spot analysis software of specialty; Thereby judge the gene order that whether has in the amplified production to various oligonucleotide probes, in the result judges, reduced the subjectivity factor of artificial identification, more be applicable to the clinical detection application.
The present invention is directed to other gene order of HCV different shaped; Design one cover specific oligonucleotide probe (as shown in table 1 be sequence oligonucleotide probe and the type that detected); Adopt automatic point sample instrument; Probe is printed on 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 other necessary component again,, promptly form complete product like PCR primer (as shown in table 2 is the used primer sequence of the present invention).When actual detected, 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 possibly exist.Get one of DNA Hybond membrane bar, add PCR product 45 μ L and hybridize, hybridization temperature is set at 45 ℃, and hybridization time 60 minutes is through hybridization-washing-colour developing.Take out the film bar and dry, put into scanner and scan, operational analysis software carries out the view data conversion process to scan image signal, and analyzes generation sample HCV type result.
According to another preferred embodiment of the present invention; Introduced the confidential reference items primer being used for sample amplification PCR system; And the confidential reference items template, and when the film bar prepares, introduced the probe that can hybridize with the confidential reference items template, can carry out virtual mass control to the PCR process.
According to another embodiment preferred of the present invention, when preparation film bar, having introduced colour developing control probe (CC.), promptly at an end mark-NH2 of probe, with the nylon membrane coupling, the other end is introduced biotin labeling, can directly develop the color.The introducing of CC can be carried out virtual mass control to process color.
Another embodiment preferred according to the present invention, the sheet base carrier of DNA Hybond membrane bar can be the carriers that slide, nylon membrane or other can adhesion probes.
Table 1
Table 2
Embodiment
1,5 ' UTR district design RT and PCR primer in the HCV genome adopt asymmetric RT-PCR system, the needed target fragment of amplification somatotype; As shown in table 1, in the target fragment scope, design 3 of 1 type probes, 4 of 2 type probes, 3 of 3 type probes, 1 of three of 6 type probes and general probe.
2, see also Fig. 1 with nylon membrane by the format print grid that designs, adopt some film appearance automatically, 14 typing probes and 2 contrast probe points systems to the specific regions of film bar, are processed DNA Hybond membrane bar.
The point sample probe solution: in probe dilution to 5 * SSC, 0.05%SDS solution, final concentration is 10 μ M;
The point sample matrix: with probe points on the film bar, each probe point sample 0.5pM, every row 8 points, totally 2 the row (referring to Fig. 1).
3, use product of the present invention to detect clinical sample
Get various other clinical sample 6 examples and each portion of positive and negative contrast, extract RNA, according to table 3 preparing RT-PCR reaction 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?MgCl 2 | 6 |
| dNTP（10mM） | 1 |
| 10μMF | 2 |
| 10μMR | 1 |
| 10μMF1 | 2 |
| 10μMR1 | 1 |
| Reversed transcriptive enzyme/Taq enzyme | 0.5 |
| The RNA template | 10 |
| Total amount | 50 |
The pcr amplification program is following:
Adopt HCV gene type Hybond membrane bar of the present invention, above sample is detected.
Get a Hybond membrane bar, the marker samples numbering is put into hybrid pipe, gets 8 each 45 μ L of pipe PCR product respectively and is added in the pipe of reference numeral, sets 45 ℃ of hybridization temperatures, hybridization time 60 minutes.Wash after the hybridization, coupling, colour developing and air-dry.Concrete operations are following:
1) hybridization:
Get the 15mL centrifuge tube, put into 1 of label film bar, add the 40ul PCR product of 5-8ml preheating A liquid (2*SSC, 0.5%SDS), correspondence, the tight pipe lid of lid is put into the vibration tank, 50 ℃ of jogs (100～150 rev/mins), 40 minutes.Discard liquid.
2) coupling: (at every turn can handle 5 film bars simultaneously)
Get the 50mL centrifuge tube, and preheating B liquid (0.5*SSC, 0.5%SDS) 20ml, C liquid (0.01%TMB) 1ml, mixing is put into the Hybond membrane bar, and 50 ℃ of jogs (40～50 rev/mins) 10 minutes discard liquid;
(50 ℃ of jogs 3 minutes discard liquid for 0.5*SSC, 0.5%SDS) 40ml to add preheating B liquid; (50 ℃ of jogs 3 minutes discard liquid for 0.5M Trisodium Citrate, pH5.5) 40ml to add D liquid.
3) colour developing: (can handle 5-10 at every turn simultaneously and open the film bar)
Get the 50mL centrifuge tube, add D liquid (0.5M Trisodium Citrate, pH5.5) 20ml, E liquid 1ml (0.025%POD), F liquid (0.3H
2O
2) 1ml, mixing is colour developing liquid, puts into the film bar and leaves standstill 8 minutes in the room temperature lucifuge, discards liquid;
(jog 3 minutes discards liquid for 0.5M Trisodium Citrate, pH5.5) 40ml to add D liquid.Hybridization is accomplished.
Adopt scanner, the scanning hybridization signal obtains the results of hybridization scintigram and (sees Fig. 2-Fig. 5), and image transitions is become data.
Adopt analysis software, above data analysis is converted into the gene type of each sample, simultaneously,, contrast as detected result with 8 these dna sequencings of increment.The result shows, 8 these detected results of increment and sample sequencing result conform to fully (seeing table 4).
Embodiment has explained the advantage of product of the present invention aspect the detection flux, once can make the sample of arbitrary number; The safety of detection accuracy aspect (detected result conforms to the sample sequencing result fully).
Table 4
| Sample number | Sequencing result | This test-results | 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 above is merely embodiments of the invention; Be not so limit claim of the present invention; Every equivalent structure or equivalent flow process conversion that utilizes specification sheets of the present invention and accompanying drawing content to be done; Or directly or indirectly be used in other relevant technical fields, all in like manner be included in the scope of patent protection of the present invention.
Claims (8)
1. the test kit of HCV gene typing; Mainly comprise PCR reaction solution and DNA Hybond membrane bar; It is characterized in that said DNA Hybond membrane bar comprises nylon membrane, is fixed with probe on the said nylon membrane; Said probe is can be respectively and the Nucleotide of the nucleic acid hybridization of the various subtype virus of hepatitis C virus, and said probe sequence is as follows:
To HBV1a type designing probe Y1:5 '-AATTGCCAGGACGACCGG-3 ';
To HBV1b type designing probe Y2:5 '-AATTGCCAGGATGACCGGG-3 ';
To HBV1b type designing probe Y3:5 '-CCCGGAATTGCCAGGAC-3 ';
To HBV2a type designing probe Y4:5 '-CAATGCCTGGAGATTTGGGCG-3 ';
To HBV2a type designing probe Y5:5 '-CAATGCCTGGAGATTTGGGC-3 ';
To HBV2b type designing probe Y6:5 '-CTCAATGCCTGGAGATTTGG-3 ';
To HBV2b type designing probe Y7:5 '-GCTCAATGCCTGGAGATTTGG-3 ';
To HBV3a type designing probe Y8:5 '-CAATGCCTGGAGATTTGGG-3 ';
To HBV3a type designing probe Y9:5 '-CGAGGTAGACGTCAGCC-3 ';
To HBV3a type designing probe Y10:5 '-ACCTCGAGGTAGACGTCAGCC-3 ';
To HBV6a type designing probe Y11:5 '-AACCTCGAGGTAGACGTCAGCC-3 ';
To HBV6a type designing probe Y12:5 '-GCAACCTCGAGGTAGACGT-3 ';
To HBV6a type designing probe Y13:5 '-TAGACGTCAGCCTATCCCCAA-3 ';
To HBV1-6 type design general probe (being positive probe HP) Y14:5 '-TAGACGCCAGCCTATTCCCA-3 ';
Negative probe HN:5 '-GGTCCTGTTTAACTGGCG-3 ';
Said each probe 5 ' end carry out amino labeled with the nylon membrane coupling.
2. the test kit of HCV gene typing as claimed in claim 1; It is characterized in that; Also be provided with colour developing control probe CC on the said nylon membrane; Said colour developing control probe CC sequence is as follows: 5 '-GCGCAGGGCCACAATAATGG-3 ', and the 5 ' end of said colour developing control probe CC carries out amino labeled, and 3 ' end carries out biotin labeling.
3. the test kit of HCV gene typing as claimed in claim 1 is characterized in that, said PCR reaction solution comprises the primer of the nucleic acid of the various subtype virus of amplification hepatitis C virus, and said 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 said downstream primer R carries out biotin labeling.
4. the test kit of HCV gene typing as claimed in claim 1 is characterized in that, said PCR reaction solution also comprises the confidential reference items system, and said internal reference system is an Arabidopis thaliana internal reference system, comprising:
Confidential reference items primers F 1:5 '-TCATCGTCGCTGGAGCTGGTT-3 ';
Confidential reference items primer R1:5 '-CGGCGGTTTGTCAAGCTGAT-3 ';
Confidential reference items probe PC:5 '-TGCCGATATAGGTCACAGCATGGGC-3 ';
5 ' end of said confidential reference items primers F 1 carries out biotin labeling.
5. the test kit of HCV gene typing as claimed in claim 1 is characterized in that, said concentration and probe concentration is 5-20 μ M.
6. the method for HCV gene typing is characterized in that, this method comprises following concrete steps:
A, design serotype specific primer and probe, said probe is shown in claim 1 probe Y1-Y14, and said primer comprises reverse transcriptase primer RT:5 '-CGTCTAGCCATGGCGTTAGT-3 ';
Upstream primer F:5 '-GAAAGCGTCTAGCCATGGCGTTAGT-3 ';
Downstream primer R:5 '-CGCAAGCACCCTATCAGGCAGTA-3 ';
B, with 5 ' end of the said probe of active amino markers step a, and it is fixed on the nylon membrane successively, process and detect the film bar, the 5 ' end of said downstream primer R is carried out biotin labeling;
C, utilize that primer carries out the HBV DNA cloning described in the step a, with amplified production with detect the hybridization of film bar, develop the color with px and TMB;
D, employing scanner, the scanning hybridization signal is judged the gene type result.
7. the method for HCV gene typing as claimed in claim 6; It is characterized in that; Also be provided with colour developing control probe CC on the said nylon membrane; Said colour developing control probe CC sequence is as follows: 5 '-GCGCAGGGCCACAATAATGG-3 ', and said CC probe 5 ' end carries out amino labeled, and 3 ' end carries out biotin labeling.
8. the method for HCV gene typing as claimed in claim 6 is characterized in that, the primer among the said step a also comprises:
Confidential reference items primers F 1:5 '-TCATCGTCGCTGGAGCTGGTT-3 ';
Confidential reference items primer R1:5 '-CGGCGGTTTGTCAAGCTGAT-3 ';
Probe among the said step a also comprises:
Confidential reference items probe PC:5 '-TGCCGATATAGGTCACAGCATGGGC-3 ';
5 ' end of said confidential reference items primers F 1 carries out biotin labeling.
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| CN103710464A (en) * | 2013-12-30 | 2014-04-09 | 湖南圣湘生物科技有限公司 | HCV (Hepatitis c virus) genotype detection kit |
| CN103966363A (en) * | 2014-05-16 | 2014-08-06 | 福州泰普生物科学有限公司 | Hepatitis C virus (HCV) genotyping kit |
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