CN105112562A - Reagent kit and method for typing HBV (hepatitis B virus) genes and analyzing drug-resistant sites - Google Patents
Reagent kit and method for typing HBV (hepatitis B virus) genes and analyzing drug-resistant sites Download PDFInfo
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Abstract
The invention provides a reagent kit and a method for typing (hepatitis B virus) genes and analyzing drug-resistant sites. The reagent kit comprises 10xPCR (polymerase chain reaction) Permix, HBV target DNA (deoxyribonucleic acid) amplification primers, positive control standard substances and deionized water. The reagent kit and the method have the advantages that the method is used for carrying out sequencing analysis on sequences of genomes of hepatitis B viruses HBV on the basis of DNA first generation sequencing techniques of sanger processes, the base sequence sequencing and reading accuracy can reach 99.9%, accordingly, the viruses can be typed according to sequenced genome sequences of the hepatitis B viruses HBV, and the relevant drug-resistant sites can be analyzed according to the sequenced genome sequences of the hepatitis B viruses HBV; results obtained by the aid of the method are extremely high in accuracy, the method is high in sensitivity and can be advantageously applied to providing accurate guidance for long-term chronic hepatitis hepatitis B treatment in precision medicine, and high-throughput operation can be carried out.
Description
Technical field
The invention belongs to biotechnology service industry, relate to a kind of DNA generation sequencing technologies that utilizes to the test kit of hepatitis b virus hbv gene type and resistance Locus Analysis in Shoots.
Background technology
Hepatitis B virus (HepatitisBVirus, HBV) belongs to Hepadnaviridae, and genome is about 3.2KB, is partially double stranded cyclic DNA, and current fixed hepatitis b virus hbv genotype has A, B, C, D, E, F, G, H eight kinds.Hepatitis b virus hbv infects and causes host's liver inflammatory lesion to be main, and can cause the disease that multiple organ damages, hepatitis B, is commonly called as hepatitis B.According to WHO Report, the whole world about has 2,000,000,000 people once to infect hepatitis b virus hbv, wherein 3.5 hundred million is chronic HBV HBV infection person, about have every year 1000000 people die from hepatitis b virus hbv infect caused by liver failure, liver cirrhosis and primary hepatocarcinoma.The common recognition of the domestic and international treatment for chronic HBV HBV infection patient is at present antiviral long-term treatment, and hepatitis b virus hbv is the high virus of a kind of mutation rate, natural mutation rate 10
10-11point mutation/sky, under long-term drug therapy particularly nucleotide analog medicament selection pressure, hepatitis b virus hbv medicament-resistant mutation occurrence frequency is high, and clinical drug-resistant sudden change becomes one of principal element affecting treating chronic hepatitis B effect.The nucleotide analog medicine that China SFDA ratifies listing has 4 kinds: lamivudine (Lamivudine, LAM), Telbivudine (Telbivudine, LdT), adefovir ester (Adefovir, and Entecavir (Entecavir, ETV) ADV); According to the different somatotype of hepatitis B patient, and resistance site type, determine to use which kind of medicine, therefore detection and genotyping and the covariation of early discovery hepatitis b virus hbv resistance have great importance.The hepatitis b virus hbv somatotype of current routine detects and mainly uses serotype, other antigenic epitopes of different shaped is different, utilize these monoclonal antibodies with type specific epitopes to distinguish different types by enzyme linked immunological, also have restriction fragment length polymorphism (RFLP), type specificity probe in detecting, gene microarray analysis, real-time quantitative PCR method etc. in addition; And the method for drug resistance analysis mainly applying gene chip is carried out.
The method of application serotype is formed comparatively early, is applicable to the phenotypic analysis of great amount of samples, but due to the hepatitis B virus core antigen limited amount in serum in hepatitis B patient body, make the efficiency of enzyme linked immunoassay extremely low, and step is numerous and diverse, relatively consuming time longer; Drug resistance analysis is higher for the accuracy requirement of gene order simultaneously, so traditional gene chip detects relative to genome sequence order-checking, easily pollute, occur false positive, accuracy is poor, consuming time longer, and the method for order-checking is more intuitive and reliable.
Summary of the invention
The object of the present invention is to provide a kind of test kit to HBV gene somatotype and resistance Locus Analysis in Shoots and its method, the result accuracy utilizing this test kit to obtain is high, highly sensitive, can operate by high-throughput, be conducive to being applied in accurate medical treatment, give to instruct accurately to chronic viral hepatitis B long-term treatment.
For realizing object of the present invention, the present invention by the following technical solutions:
To a test kit for HBV gene somatotype and resistance Locus Analysis in Shoots, comprise 10xPCRPermix, hepatitis b virus hbv target DNA amplimer, positive control standard substance and deionized water, described hepatitis b virus hbv target DNA amplimer is:
HBV-forwardprimer(HBV-FP)：GGTGGACTTCTCTCAATTTTCTAGG
HBV-reverseprimer(HBV-RP)：GTGCAGAGGTGAAGCGAAGTG
Described 10xPCRPermix comprises: the Taq enzyme of dNTPs, 1U of Mgcl2,2mM of 10xTaqbuffer, 25mM and deionized water.
As preferably, described test kit also comprises hepatitis b virus hbv DNA extraction reagent, positive control standard substance and negative control.
As preferably, described hepatitis b virus hbv DNA extraction reagent comprises the Proteinase K of 20mg/ml, the RNaseA of 50mg/ml, erythrocyte cracked liquid, dehydrated alcohol, adsorption column, washingbuffer and deionized water.
As preferably, described positive control standard substance are other hepatitis b virus hbv DNA sample of known type, and described negative control is deionized water.
A method for hepatitis b virus hbv gene type and resistance Locus Analysis in Shoots, comprises the following steps:
1) the genomic extraction of hepatitis b virus hbv: with the serum containing deactivation hepatitis b virus hbv for sample, get 200ul serum sample, add 20ul Proteinase K Solution in 1.5ml centrifuge tube, add 5ulRNaseA solution in addition, mix, add 200ul erythrocyte cracked liquid, mixing fully, low speed brief centrifugation is placed in 56 DEG C of water-baths, temperature bath 15min, and put upside down mixing once every 3min, add 200ul dehydrated alcohol, concussion 10s, mixed solution all being transferred to cover has in the adsorption column of 2ml collection tube, under room temperature, the centrifugal 1min of 10000g, outwell waste liquid in collection tube, adsorption column is put in a new 2ml collection tube, use washingbuffer500ul, the centrifugal 1min of the centrifugal 13000g of room temperature, repeat wash-out twice, the centrifugal 2min of 13000g under room temperature, remove the waste liquid and .washingbuffer that remain in above adsorption column, then adsorption column is placed in the 1.5ml centrifuge tube of a clean sterilizing, deionized water is used to be eluted by the DNA on adsorption column pellosil,
2) genome sequence amplification: according to known hepatitis b virus hbv genomic sequence analysis, obtain P district and S region sequence, P district and S district through sequence analysis, design and synthesis pair of primers amplification object region sequence:
Primer sequence:
HBV-forwardprimer(HBV-FP)：GGTGGACTTCTCTCAATTTTCTAGG
HBV-reverseprimer(HBV-RP)：GTGCAGAGGTGAAGCGAAGTG
PCR reaction solution wherein comprises: the Taq enzyme of dNTPs, 1U of HBV-FP and HBV-RP of 10xTaqbuffer, 10pmol/ul, Mgcl2,2mM of 25mM, hepatitis b virus hbv DNA and deionized water;
3) hepatitis b virus hbv somatotype: the DNA obtained checks order to increasing, the base sequence of order-checking gained P district (comprising S district) carries out phenotypic analysis by the online typing data storehouse of NCBI:
Somatotype website: http://www.ncbi.nlm.nih.gov/projects/genotyping/formpage.cgi;
4) hepatitis b virus hbv resistance Locus Analysis in Shoots: if based on the drug main nucleoside medicine for the treatment of hepatitis B at present, its treatment principle suppresses hepatitis b virus hbv polymerase active by competing to reach with the natural substrate dNTP of hepatitis b virus hbv polymerase, thus reach the effect suppressing hepatitis b virus hbv to copy; Measure the genetic analysis to following site, determine Drug resistance status:
A, lamivudine (LAM): rtM204V/I, rtL180M, rtV173L,
B, adefovir ester (ADV): rtA181T/V, rtN236T, rtI233V,
C, Entecavir (ETV): rtT184G, rtS202G/I, rtI169T, rtM250V,
D, Telbivudine (LdT): rtM204I/V, rtL180M.
The beneficial effect that the present invention compared with prior art has is: method of the present invention is based on sanger method DNA generation sequencing technologies, sequence analysis is carried out to the genome of hepatitis b virus hbv, the rate of accuracy reached 99.9% of base sequence, according to the genome sequence of the hepatitis b virus hbv obtained that checks order, somatotype and relevant resistance Locus Analysis in Shoots thereof are carried out to virus, reliable results, highly sensitive.
Accompanying drawing explanation
Fig. 1 is HBV target dna fragment amplification agarose gel electrophoresis;
Fig. 2 is HBV target DNA sequence sequencing result splicing situation;
Fig. 3 is the online genotyping result of HBVNCBI.
Fig. 4 is that HBV resistance site mutation analyzes situation.
Embodiment
For the understanding the present invention making those skilled in the art more clear and intuitive, below the present invention is further illustrated.
Embodiment 1
To a test kit for HBV gene somatotype and resistance Locus Analysis in Shoots, comprise 10xPCRPermix, hepatitis b virus hbv target DNA amplimer, positive control standard substance and deionized water, described hepatitis b virus hbv target DNA amplimer is:
HBV-forwardprimer(HBV-FP)：GGTGGACTTCTCTCAATTTTCTAGG
HBV-reverseprimer(HBV-RP)：GTGCAGAGGTGAAGCGAAGTG
Described PCR reaction solution comprises: the Taq enzyme of dNTPs, 1U of HBV-FP and HBV-RP of 10xTaqbuffer, 10pmol/ul, Mgcl2,2mM of 25mM, hepatitis b virus hbv DNA and deionized water; Described test kit also comprises hepatitis b virus hbv DNA extraction reagent, positive control standard substance and negative control; Described positive control standard substance are other hepatitis b virus hbv DNA sample of known type, and described negative control is deionized water; Described hepatitis b virus hbv DNA extraction reagent comprises the Proteinase K of 20mg/ml, the RNaseA of 50mg/ml, erythrocyte cracked liquid, dehydrated alcohol, adsorption column, washingbuffer and deionized water.
Embodiment 2
The test kit of embodiment 1, to a method for hepatitis b virus hbv gene type and resistance Locus Analysis in Shoots, comprises the following steps:
1) the genomic extraction of hepatitis b virus hbv: with the serum containing deactivation hepatitis B virus for sample, get 200ul serum sample, add 20ul Proteinase K Solution in 1.5ml centrifuge tube, add 5ulRNaseA (50mg/ml) solution in addition, mix, add 200ul erythrocyte cracked liquid, mixing fully, brief centrifugation is placed in 56 DEG C of water-baths, temperature bath 15min, and put upside down mixing once every 3min, add 200ul dehydrated alcohol, concussion 10s, mixed solution all being transferred to cover has in the adsorption column of 2ml collection tube, under room temperature, the centrifugal 1min of 10000g, outwell waste liquid in collection tube, adsorption column is put in a new 2ml collection tube, use washingbuffer500ul, the centrifugal 1min of the centrifugal 13000g of room temperature, repeat wash-out twice, the centrifugal 2min of 13000g under room temperature, remove the waste liquid and .washingbuffer that remain in above adsorption column, then adsorption column is placed in the 1.5ml centrifuge tube of a clean sterilizing, deionized water is used to be eluted by the DNA on adsorption column pellosil,
2) genome sequence amplification: according to known hepatitis b virus hbv genomic sequence analysis, obtain P district and S region sequence, P district and S district through sequence analysis, design and synthesis pair of primers amplification object region sequence:
Primer sequence:
HBV-forwardprimer(HBV-FP)：GGTGGACTTCTCTCAATTTTCTAGG
HBV-reverseprimer(HBV-RP)：GTGCAGAGGTGAAGCGAAGTG
PCR reaction solution wherein comprises: the Taq enzyme of dNTPs, 1U of HBV-FP and HBV-RP of 10xTaqbuffer, 10pmol/ul, Mgcl2,2mM of 25mM, hepatitis b virus hbv DNA and deionized water, amplification as shown in Figure 1, wherein, M:markerDL2000; 1: positive control standard substance; 2: sample 1; 3: sample 2; 4: negative control.
3) hepatitis b virus hbv somatotype: the DNA obtained checks order to increasing, the base sequence of order-checking gained P district (comprising S district) carries out phenotypic analysis by the online typing data storehouse of NCBI:
Somatotype website: http://www.ncbi.nlm.nih.gov/projects/genotyping/formpage.cgi;
Sequencing result splicing as shown in Figure 2.
As shown in Figure 3, sample 1 is Type B to positive control standard substance genotyping result, and sample 2 is C type;
4) hepatitis b virus hbv resistance Locus Analysis in Shoots: if based on the drug main nucleoside medicine for the treatment of hepatitis B at present, its treatment principle suppresses hepatitis b virus hbv polymerase active by competing to reach with the natural substrate dNTP of hepatitis b virus hbv polymerase, thus reach the effect suppressing hepatitis b virus hbv to copy; Measure the genetic analysis to following site, determine Drug resistance status:
A, lamivudine (LAM): rtM204V/I, rtL180M, rtV173L,
B, adefovir ester (ADV): rtA181T/V, rtN236T, rtI233V,
C, Entecavir (ETV): rtT184G, rtS202G/I, rtI169T, rtM250V,
D, Telbivudine (LdT): rtM204I/V, rtL180M.
For standard substance, the comparison of resistance site as shown in Figure 4.
Obtaining from the order-checking peak map analysis of accompanying drawing 4, does not all undergo mutation in the resistance site of four kinds of medicines, so medication in early stage can put aside drug resistance problems.
With this: sample 1 is rtM204V sudden change, is LAM resistance; Sample 2 does not have resistance mutation to occur.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (5)
1., to a test kit for HBV gene somatotype and resistance Locus Analysis in Shoots, this test kit comprises 10xPCRPermix, hepatitis b virus hbv target DNA amplimer and deionized water, and described hepatitis b virus hbv target DNA amplimer is:
HBV-forwardprimer(HBV-FP)：GGTGGACTTCTCTCAATTTTCTAGG
HBV-reverseprimer(HBV-RP)：GTGCAGAGGTGAAGCGAAGTG
Described 10xPCRPermix comprises: the Taq enzyme of dNTPs, 1U of Mgcl2,2mM of 10xTaqbuffer, 25mM and deionized water.
2. the test kit to hepatitis b virus hbv gene type and resistance Locus Analysis in Shoots according to claim 1, is characterized in that: described test kit also comprises hepatitis b virus hbv DNA extraction reagent, positive control standard substance and negative control.
3. the test kit to hepatitis b virus hbv gene type and resistance Locus Analysis in Shoots according to claim 2, is characterized in that: described hepatitis b virus hbv DNA extraction reagent comprises the Proteinase K of 20mg/ml, the RNaseA of 50mg/ml, erythrocyte cracked liquid, dehydrated alcohol, adsorption column, washingbuffer and deionized water.
4. the test kit to hepatitis b virus hbv gene type and resistance Locus Analysis in Shoots according to claim 2, it is characterized in that: described positive control standard substance are other hepatitis b virus hbv DNA sample of known type, and described negative control is deionized water.
5., to a method for hepatitis b virus hbv gene type and resistance Locus Analysis in Shoots, it is characterized in that, comprise the following steps:
1) the genomic extraction of hepatitis b virus hbv: with the serum containing deactivation hepatitis b virus hbv for sample, get 200ul serum sample, add 20ul Proteinase K Solution in 1.5ml centrifuge tube, add 5ulRNaseA solution in addition, mix, add 200ul erythrocyte cracked liquid, mixing fully, low speed brief centrifugation is placed in 56 DEG C of water-baths, temperature bath 15min, and put upside down mixing once every 3min, add 200ul dehydrated alcohol, concussion 10s, mixed solution all being transferred to cover has in the adsorption column of 2ml collection tube, under room temperature, the centrifugal 1min of 10000g, outwell waste liquid in collection tube, adsorption column is put in a new 2ml collection tube, use washingbuffer500ul, the centrifugal 1min of the centrifugal 13000g of room temperature, repeat wash-out twice, the centrifugal 2min of 13000g under room temperature, remove the waste liquid and .washingbuffer that remain in above adsorption column, then adsorption column is placed in the 1.5ml centrifuge tube of a clean sterilizing, deionized water is used to be eluted by the DNA on adsorption column pellosil,
2) genome sequence amplification: according to known hepatitis b virus hbv genomic sequence analysis, obtain P district and S region sequence, P district and S district through sequence analysis, design and synthesis pair of primers amplification object region sequence:
Primer sequence:
HBV-forwardprimer(HBV-FP)：GGTGGACTTCTCTCAATTTTCTAGG
HBV-reverseprimer(HBV-RP)：GTGCAGAGGTGAAGCGAAGTG
PCR reaction solution wherein comprises: the Taq enzyme of dNTPs, 1U of HBV-FP and HBV-RP of 10xTaqbuffer, 10pmol/ul, Mgcl2,2mM of 25mM, hepatitis b virus hbv DNA and deionized water;
3) hepatitis b virus hbv somatotype: the DNA fragmentation obtained carries out sequencing analysis to increasing, the base sequence of order-checking gained P district (comprising S district) carries out phenotypic analysis by the online typing data storehouse of NCBI:
Somatotype website: http://www.ncbi.nlm.nih.gov/projects/genotyping/formpage.cgi;
4) hepatitis b virus hbv resistance Locus Analysis in Shoots: if based on the drug main nucleoside medicine for the treatment of hepatitis B at present, its treatment principle suppresses hepatitis b virus hbv polymerase active by competing to reach with the natural substrate dNTP of hepatitis b virus hbv polymerase, thus reach the effect suppressing hepatitis b virus hbv to copy; Measure the genetic analysis to following site, determine Drug resistance status:
A, lamivudine (LAM): rtM204V/I, rtL180M, rtV173L,
B, adefovir ester (ADV): rtA181T/V, rtN236T, rtI233V,
C, Entecavir (ETV): rtT184G, rtS202G/I, rtI169T, rtM250V,
D, Telbivudine (LdT): rtM204I/V, rtL180M.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107475461A (en) * | 2017-10-11 | 2017-12-15 | 广州立菲达安诊断产品技术有限公司 | A kind of primer, kit and method for detecting hepatitis B virus drug resistance gene |
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| CN102286645A (en) * | 2011-08-30 | 2011-12-21 | 解码（上海）生物医药科技有限公司 | Detection probe, detection kit and detection method for hepatitis B virus resistant gene mutation |
| CN102286635A (en) * | 2011-07-15 | 2011-12-21 | 广东凯普生物科技股份有限公司 | Hepatitis B virus nucleoside analog drug resistant mutation detection kit |
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Patent Citations (3)
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| CN101654714A (en) * | 2009-08-10 | 2010-02-24 | 上海中优医药高科技有限公司 | Detecting method for identifying hepatitis B virus drug resistance |
| CN102286635A (en) * | 2011-07-15 | 2011-12-21 | 广东凯普生物科技股份有限公司 | Hepatitis B virus nucleoside analog drug resistant mutation detection kit |
| CN102286645A (en) * | 2011-08-30 | 2011-12-21 | 解码（上海）生物医药科技有限公司 | Detection probe, detection kit and detection method for hepatitis B virus resistant gene mutation |
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| 覃霞等: ""焦磷酸测序技术检测乙型肝炎病毒多个基因型耐药突变方法的建立及初步评价"", 《CHINESE HEPATOLOGY》 * |
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| CN107475461A (en) * | 2017-10-11 | 2017-12-15 | 广州立菲达安诊断产品技术有限公司 | A kind of primer, kit and method for detecting hepatitis B virus drug resistance gene |
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Application publication date: 20151202 |