CN101565756B - Detection kit for nucleoside analogue drug-resistant correlated mutation of hepatitis B viruses - Google Patents
Detection kit for nucleoside analogue drug-resistant correlated mutation of hepatitis B viruses Download PDFInfo
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Abstract
The invention relates to a detection kit for the nucleoside analogue drug-resistant correlated mutation type hepatitis B viruses, in particular to a detection kit for quickly and accurately differentiating wild type and/or nucleoside analogue drug-resistant correlated mutation type hepatitis B viruses in a clinical blood sample by using a DNA reverse dot blot technology.
Description
Technical field
The present invention relates to a kind of hepatitis B virus detection kit for nucleoside analogue drug-resistant correlated mutation type, particularly relate to and use the DNA reverse dot blot hybridization technique, distinguish rapidly and accurately wild-type in the clinical blood sample with (or) detection kit of the relevant mutant hepatitis B virus of nucleoside analogue drug-resistant.
Background technology
Hepatitis B virus (HBV) is the main pathogens that causes viral hepatitis.Be a kind of serious communicable disease that endangers by the caused hepatitis B of HBV.At present, whole world chronic HBV infection person has 3.5 hundred million at least, and Chinese occurred frequently popular district as hepatitis B, the crowd (about 1.2 hundred million) who accounts for total population about 9% is the chronic HBV infection person.Lasting chronic HBV infection can show as various dissimilar diseases clinically, comprises asymptomatic carrier state, urgency or chronic hepatitis, liver cirrhosis etc., and serious may develop into primary hepatocarcinoma (HCC).The whole world has 1,200,000 people to die from the disease that the HBV infection causes every year at present.。
Studies have shown that, continue high carrying capacity HBV and be chronic viral hepatitis B state of an illness progress and the main diseases that develops into liver cirrhosis and hepatocellular carcinoma (HCC) because of, so the treatment of chronic viral hepatitis B should get down to long term inhibition or remove virus.The medicine that is used for anti-HBV treatment at present has Interferon, rabbit and nucleoside analog two big classes, and nucleoside analog then is the fastest antiviral of developed recently.So far, the nucleoside analog medicine that drugs approved by FDA can be used for treating hepatitis B mainly contains lamivudine, Adefovir, Entecavir, Telbivudine and according to bent Xi Taping, Clevudine is still under test, and a clinical line medicine is based on lamivudine, Adefovir and Entecavir.
According to the study, medicament-resistant mutation mainly concentrates on the viral dna polymerase sequence in most of hepatitis B virus, and wherein the medicament-resistant mutation that produces under the selective pressure of different pharmaceutical is not quite similar, and is summarized as follows:
Lamivudine (LAM) medicament-resistant mutation mainly concentrates on tyrosine-methionine(Met)-aspartic acid-aspartic acid (YMDD) motif of viral dna polymerase, the Nucleotide generation base that shows as the coding methionine(Met) substitutes (ATG → ATT/GTG), thereby make its amino acids coding take place corresponding the variation (M → I/V, rtM204I/V).Adefovir (ADV) resistance concentrates on sites such as HBV polymerase D district N236T and B district A181V, and the variation of rtV173L site also produces certain synergy to the Adefovir resistance; Simultaneously, it is reported and find that a routine rtA181S variation is relevant with the Adefovir resistance that it is relatively poor to lamivudine and Adefovir combination therapy susceptibility that rtA181S+N236T unites variation.Prove according to present data, on YMDD sudden change basis, add one of following resistant mutational site: T184G, S202I, M250V, Entecavir (ETV) resistance can take place.Therefore, the medicament-resistant mutation of ETV only occurs among the patient of LAM resistance, and prompting ETV controls the patient of LAM resistance, and the ETV medicament-resistant mutation might take place.Telbivudine (LdT) YMDD sudden change resistance also can occur in therapeutic process, treated for 52 weeks after, the sudden change resistant rate occurring is 2/44 (4.5%), main mutant phenotype is M204I and L180M+M204V, with LAM crossing drug resistant is arranged.It is reported that according to bent Xi Taping (FTC) YMDD sudden change resistant rate reaches 12.6%, with the LAM crossing drug resistant after 48 weeks for the treatment of.
The variant sites that present hepatitis-B virus drug-tolerant gene mutation detection kit both domestic and external detects is generally rtL180M, rtA181V, rtM204V and rtN236T etc. and lamivudine and Adefovir resistance related locus, though also can cover part drug-resistance of hepatitis patient group, but there is certain loss, and can not detects the Entecavir resistant mutational site.The present invention intend detecting rtV173L, rtL180M, rtA181V, rtA181S, rtT184G, rtS202I, rtM204V/I, rtI233V, rtN236T and rtM250V etc. almost contain at present known all can cause nucleoside analog drug tolerance nucleotide diversity site, and can detect wild-type and the polyinfection of medicament-resistant mutation type more accurately, the inferior position type accounts for 10% and can detect in population mixture; Design a kind of system at short notice, comprehensively detect and analyze the hepatitis B virus detection kit for nucleoside analogue drug-resistant correlated mutation type.
The detection method of medicament-resistant mutation mainly contains following several at present: mass spectroscopy, fluorescence polarization, PCR-peptide nucleic acid(PNA), restriction fragment length polymorphism method RFLP, linear probe analytical method, quantitative fluorescent PCR melting curve analytical method and biochip technology.Yet, the most cost costliness of these methods, complex operation is unfavorable for that vast basic hospital carries out.Therefore, the inventor is on the basis of the achievement in research in early stage (CN200410052531.1), in conjunction with automanual interpreting system as a result, set up a kind of test kit that can detect and analyze the multiple nucleoside analog drug resistance of hepatitis B virus mutator gene type at short notice.
Summary of the invention
The purpose of this invention is to provide a kind of hepatitis B virus detection kit for nucleoside analogue drug-resistant correlated mutation type, particularly relate to and use the DNA reverse dot blot hybridization technique, distinguish rapidly and accurately wild-type in the clinical blood sample with (or) detection kit of the relevant mutant hepatitis B virus of nucleoside analogue drug-resistant.
The detection step of test kit is as follows: (1) provides sample to be checked and therefrom extracts DNA; (2) use synthetic specific oligonucleotide primers, utilize polymerase chain reaction amplification to comprise one section target DNA sequence of purpose motif; (3) make target DNA sequence and the specific oligonucleotide probe hybridization that is labeled; (4) the Hybond membrane bar is after colour developing, scanning result and with software analysis and judge existence and the relative quantity thereof of resistance of hepatitis B virus medicine sudden change.
In order to reach above-mentioned detection step, the component of hepatitis B virus detection kit for nucleoside analogue drug-resistant correlated mutation type provided by the invention comprises: (1) extracts the nucleic acid extraction agent of sample nucleic acid; (2) pcr reagent, the yin, yang quality control product; (3) be used for nucleic acid hybridization reagent, the Hybond membrane bar of nucleic acid hybridization reaction, wherein pcr reagent is made up of the deoxyribonucleoside triphosphate (dNTPs) of forward primer, biotin labeled reverse primer, deoxyuridine triphosphoric acid (dUTP), heat-resisting Taq enzyme and uridylic-DNA-glycosylase (UDG enzyme), it is characterized in that the sequence of forward primer and biotin labeled reverse primer is respectively:
(1)5’-TAGACTCGTGGTGGACTTCTCTCARTTT-3’(SEQ ID NO：1)
(2)5’-TGGATGTRTCTGCGGCGTTT-3’(SEQ ID NO：2)
(3) vitamin H-5 '-GCCTTRTAAGTTGGCGAG-3 ' (SEQ ID NO:3)
(4)5’-CGACGTGCAGAGGTGAAGCGAAGTGCA-3’(SEQ ID NO：4)。
According to a preferred embodiment of the invention, described primer is all possible base length and position grouping in 15～28bp base scope in SEQ ID NO:1～4.
According to a preferred embodiment of the invention, the sequence that is used for the oligonucleotide probe of 5 of nucleic acid hybridization ' end amino labeled in the Hybond membrane bar is respectively:
(5)NH
2-5’-TWCCTATGGGAGTGGGCCTCAGYCCG-3’(SEQ ID NO：5)
(6)NH
2-5’-GYCCGTTTCTCYTGGCTCAGTTTACT-3’(SEQ ID NO：6)
(7)NH
2-5’-CGTTTCTCHTGGCTCAGTTTACTWGY-3’(SEQ ID NO：7)
(8)NH
2-5’-TGGCTCAGTTTACTWGYGCMATTTGT-3’(SEQ ID NO：8)
(9)NH
2-5’-GYYTGGCTTTYAGTTATATKGATGAY-3’(SEQ ID NO：9)
(10)NH
2-5’-GCTTTYAGYTATATGGATGATSTGGT-3’(SEQ ID NO：10)
(11)NH
2-5’-TRTCTBTGGGYATMCATTTRAAYMCY-3’(SEQ ID NO：11)
(12)NH
2-5’-TWCCTATGGGATTGGGCCTCAGYCCG-3’(SEQ ID NO：12)
(13)NH
2-5’-GYCCGTTTCTCYTGGCTCAGTTTACT-3’(SEQ ID NO：13)
(14)NH
2-5’-CGTTTCTCHTGACTCAGTTTACTWGY-3’(SEQ ID NO：14)
(15)NH
2-5’-TGGCTCAGTTTGGTWGYGCMATTTGT-3’(SEQ ID NO：15)
(16)NH
2-5’-GYYTGGCTTTYATTTATATKGATGAY-3’(SEQ ID NO：16)
(17)NH
2-5’-GCTTTYAGYTATATHGATGATSTGGT-3’(SEQ ID NO：17)
(18)NH
2-5’-TRTCTBTGGGYGTGCATTTRAAYMCY-3’(SEQ ID NO：18)
(19)NH
2-5’-TCTBTGGGYAAYCATTTRAAYCCTMA-3’(SEQ ID NO：19)
(20)NH
2-5’-TCTBTGGGYACMCATTTRAAYCCTMA-3’(SEQ ID NO：20)
(21)NH
2-5’-CGTTTCTCHTGATTCAGTTTACTWGY-3’(SEQ ID NO：21)
(22)NH
2-5’-CCCTTAAYTTYATGGGHTAYRTNATY-3’(SEQ ID NO：22)
(23)NH
2-5’-CCCTTAAYTTYGTGGGHTAYRTNATY-3’(SEQ ID NO：23)
(24)NH
2-5’-GCTTTYAGYTATGTGGATGATSTGGT-3’(SEQ ID NO：24)
(25)NH
2-5’-ATTGTTCAGTGGTTCGTAGGGCTTTC-3’(SEQ ID NO：25)
And the colour developing control oligonucleotide probe that is used for the monitoring crossover process of 5 ' end biotin labeling and 3 ' end amino labeled is:
(26) vitamin H-5 '-GCCGTAACCGTCACAATCCGT-3 '-NH
2(SEQ ID NO:26).
According to a preferred embodiment of the invention, described probe is all possible base length and position grouping in 15～26bp base scope in SEQ ID NO:5～26.
According to a preferred embodiment of the invention, wherein the sample to be tested of test kit refers to serum, blood plasma, the combination of any known varient of hepatitis B virus or wild-type and anomaly in ascites and the hepatic tissue, or the combination of multiple anomaly.
According to a preferred embodiment of the invention, wherein said extraction sample nucleic acid reagent is to form with the sodium-chlor of 3%～8% polyoxyethylene glycol and 1mol/L formulated concentrated solution and conventional DNA extraction liquid.
According to a preferred embodiment of the invention, wherein said nucleic acid hybridization reagent by hybridization solution I (2 * SSC-0.1%SDS) and II (0.5 * SSC-0.1%SDS), solution I (the streptavidin solution of 250u/ml coupling horseradish peroxidase), II (sodium citrate solution of 0.1mol/L), III (the tetramethyl biphenyl amine aqueous solution of 2mg/ml) and IV (3% superoxol) form.
According to a preferred embodiment of the invention, the probe of wherein being fixed on the said Hybond membrane bar site that can detect hepatitis B virogene group pol gene wild-type comprises: rt173V, rt180L, rt181A, rt181A, rt184T, rt202S, rt204M, rt233L, rt236N and rt250M; The mutant site comprises: rt173L, rt180M, rt181V, rt181S, rt184G, rt202I, rt204V/I, rt233V, rt236T and rt250V.
According to a preferred embodiment of the invention, wherein said Hybond membrane bar is nylon membrane or nitrocellulose filter, and hybridization is to carry out hybridization in constant temperature water bath apparatus, and proofing unit comprises water bath with thermostatic control shaking table or automatic, semi-automatic hybridization instrument.
Owing to increased a lot of recent newfound nucleoside analogue drug-resistant relevant mutational sites among the present invention, and these sites are distributed on the detection film bar simultaneously, therefore test kit of the present invention can not only have more hepatitis B virus polyinfection or the multiple ability of uniting sudden change of detecting for detection of single hepatitis b virus infected.On the other hand, because test kit of the present invention uses shaking bath to detect, it is simple to operate, flux is big, cheap, and semi-automatic hybrid device is convenient and swift, efficient is high, use manpower and material resources sparingly, but both all have a good application prospect and generalization.Particularly design rigorous nucleic acid probe owing to having used, make the hepatitis B virus detection of nucleoside analogue drug-resistant have better sensitivity and specificity.
In order to realize the present invention, the detailed step division is as follows:
1. primer design is with synthetic
For special, increase goal gene in the nucleic acid samples to be checked efficiently, we transfer A～H type hepatitis B virus complete genome DNA sequence from Gene Bank, the site that under nucleotide analog medicament selection pressure, is easy to make a variation at the hepatitis B virus pol gene, general principle according to design of primers, adopt primer premier5.0 and oligo6.0 software to design respectively and synthesized used forward and the reverse primer of nest-type PRC, wherein 5 ' of reverse primer end uses biotin labeling.
(1)5’-TAGACTCGTGGTGGACTTCTCTCARTTT-3’(SEQ ID NO：1)
(2)5’-TGGATGTRTCTGCGGCGTTT-3’(SEQ ID NO：2)
(3) vitamin H-5 '-GCCTTRTAAGTTGGCGAG-3 ' (SEQ ID NO:3)
(4)5’-CGACGTGCAGAGGTGAAGCGAAGTGCA-3’(SEQ ID NO：4)。
2. probe synthesizes and mark
The site that under nucleotide analog medicament selection pressure, is easy to make a variation at the hepatitis B virus pol gene, 21 probes and a colour developing probe (probe 22 that is used for the control color reaction have been designed and synthesized, SEQ ID:24), with its in contrast so as to control color reaction.In order to guarantee that each probe all can be under same temperature successfully hybridize with specific target DNA, the present invention has designed many degeneracy probes and the Tm value of probe and GC content is strict controlled in the specified range.Synthetic probe is used for the hybridization of back behind the separation of 20% polyacrylamide gel electrophoresis and purifying.The oligonucleotide probe that uses in the hybridization is respectively:
(5)NH
2-5’-TWCCTATGGGAGTGGGCCTCAGYCCG-3’(SEQ ID NO：5)
(6)NH
2-5’-GYCCGTTTCTCYTGGCTCAGTTTACT-3’(SEQ ID NO：6)
(7)NH
2-5’-CGTTTCTCHTGGCTCAGTTTACTWGY-3’(SEQ ID NO：7)
(8)NH
2-5’-TGGCTCAGTTTACTWGYGCMATTTGT-3’(SEQ ID NO：8)
(9)NH
2-5’-GYYTGGCTTTYAGTTATATKGATGAY-3’(SEQ ID NO：9)
(10)NH
2-5’-GCTTTYAGYTATATGGATGATSTGGT-3’(SEQ ID NO：10)
(11)NH
2-5’-TRTCTBTGGGYATMCATTTRAAYMCY-3’(SEQ ID NO：11)
(12)NH
2-5’-TWCCTATGGGATTGGGCCTCAGYCCG-3’(SEQ ID NO：12)
(13)NH
2-5’-GYCCGTTTCTCYTGGCTCAGTTTACT-3’(SEQ ID NO：13)
(14)NH
2-5’-CGTTTCTCHTGACTCAGTTTACTWGY-3’(SEQ ID NO：14)
(15)NH
2-5’-TGGCTCAGTTTGGTWGYGCMATTTGT-3’(SEQ ID NO：15)
(16)NH
2-5’-GYYTGGCTTTYATTTATATKGATGAY-3’(SEQ ID NO：16)
(17)NH
2-5’-GCTTTYAGYTATATHGATGATSTGGT-3’(SEQ ID NO：17)
(18)NH
2-5’-TRTCTBTGGGYGTGCATTTRAAYMCY-3’(SEQ ID NO：18)
(19)NH
2-5’-TCTBTGGGYAAYCATTTRAAYCCTMA-3’(SEQ ID NO：19)
(20)NH
2-5’-TCTBTGGGYACMCATTTRAAYCCTMA-3’(SEQ ID NO：20)
(21)NH
2-5’-CGTTTCTCHTGATTCAGTTTACTWGY-3’(SEQ ID NO：21)
(22)NH
2-5’-CCCTTAAYTTYATGGGHTAYRTNATY-3’(SEQ ID NO：22)
(23)NH
2-5’-CCCTTAAYTTYGTGGGHTAYRTNATY-3’(SEQ ID NO：23)
(24)NH
2-5’-GCTTTYAGYTATGTGGATGATSTGGT-3’(SEQ ID NO：24)
(25)NH
2-5’-ATTGTTCAGTGGTTCGTAGGGCTTTC-3’(SEQ ID NO：25)
And the colour developing control oligonucleotide probe that is used for the monitoring crossover process of 5 ' end biotin labeling and 3 ' end amino labeled is:
(26) vitamin H-5 '-GCCGTAACCGTCACAATCCGT-3 '-NH
2(SEQ ID NO:26).
3. the preparation of hybrid vector
Film as hybrid vector can be the film of being made by materials such as nitrocellulose, nylon or cellulose acetates, and size is about 3.2cm * 2.5cm, and breaks into the form of 3 row * 8 row with printer.EDC (N-ethyl-N '-[dimethyl aminopropyl]-carbodiimide) solution with 10% before the film bar uses fully soaks activation treatment.New synthetic probe (100pmol/ μ l) after dilution, by every hole 1.0 μ l on the appropriate location of film bar with the set form point sample.Behind the point sample with NaOH solution-treated film bar and fully washing, be stored in then-20 ℃ standby.
4. the extraction of nucleic acid
The about 1ml of aseptic collection hepatitis B patient's to be checked venous blood, leave standstill for some time respectively at room temperature and 4 ℃ after, centrifugation is also collected serum specimen.Therefrom get an amount of serum sample then, to wherein adding equivalent DNA extraction liquid, abundant mixing was also placed 10 minutes in boiling water bath, and centrifugal and collection supernatant liquor is used for further PCR reaction.
5. the optimization of nucleic acid amplification system
In order further to improve sensitivity, specificity and the repeatability of test kit, we have carried out repeatedly design and preferred to the polymerase chain reaction the primer, and the system of polymerase chain reaction has been carried out groping making it to reach best specificity and the highest amplification efficiency with optimizing.Found that this method can make the sample DNA of low copy number effectively be increased, it detects lower limit and reaches 1 * 10
3～5 * 10
3IU/ml.Simultaneously, pollute in order to reduce as much as possible, we use the dUTP-UNG enzyme to handle nucleic acid to be checked.Particularly, we have adjusted the primer that uses in the pcr amplification and its concentration, and adjust and optimized Mg
2+With other concentration of reactants such as templates, also be conducive to prevent and reduce pollution.
6. the optimization of nucleic acid hybridization system
The operating process of nucleic acid hybridization comprises: at first will put into the good 15mlEP pipe of numbering as the film bar of preceding preparation, the mixture that adds PCR product and hybridization solution I is then accordingly hybridized at a certain temperature.By to the groping of hybridization system, hybridization solution, enabling hybridization reaction reaches optimization and best hybridization efficiency.After hybridization is finished, with demonstration the existing of hybridization spot, and come the transgenation of judgement sample according to the difference of colour developing spot position probe through washing and development step.Experimental results show that the detection reagent among the present invention can detect hepatitis B virus lamivudine resistance relevant mutational site (rt173, rt180 and rt204), Adefovir resistance relevant mutational site (rt181A, rt233 and rt236N) and Entecavir resistance relevant mutational site (rt184, rt202 and rt250M).Simultaneously, the present invention also can detect whether there is hepatitis B virus in the biological specimen.In addition, in the invention implementation process, by groping result that different hybridization temperatures and concentration and probe concentration make detection more accurately and reliably, and make it to have better repeatability.
In sum, test kit of the present invention mainly for detection of with differentiate wild-type and anti-nucleoside analog mutant hepatitis B virus, so as to judging that hepatitis B virus is to the tolerance situation of medicine.Whole testing process comprises that the HBV DNA, the pcr amplification that extract in the serum sample comprise the target DNA segment of HBVDNA polysaccharase rt173～rt250 regional sequence, and the key steps such as hybridization of pcr amplification product.In order to finish these steps, test kit of the present invention can be divided into PCR reaction kit and two parts of reverse dot blot hybridization test kit again.Wherein, the former comprises nucleic acid extraction agent, pcr reagent, yin, yang quality control product (wild-type and anti-nucleoside analog mutant HBV DNA plasmid).The latter comprises nucleic acid hybridization reagent and Hybond membrane.Hybridizing reagent is mainly conventional 20 * SSC, sodium laurylsulfonate (SDS), streptavidin-horseradish peroxidase binding substances (POD), tetramethyl benzidine (TMB), test kit form comprise hybridization solution I (2 * SSC-0.1%SDS), hybridization solution II (0.5 * SSC-0.1%SDS), solution I (250u/mlPOD), solution II (0.1mol/L Trisodium Citrate), solution III (2mg/mlTMB), solution IV (3%H
2O
2), and the film bar that is used as the hybridization carrier.
Description of drawings
Fig. 1 shows the Pareto diagram of employed probe on the film bar.The rt173V probe has the sequence shown in the SEQ ID NO:5, the rt180L probe has the sequence shown in the SEQ ID NO:6, the rt181A probe has the sequence shown in the SEQ ID NO:7, the rt184T probe has the sequence shown in the SEQ ID NO:8, the rt202S probe has the sequence shown in the SEQ ID NO:9, the rt204M probe has the sequence shown in the SEQ ID NO:10, the rt233I probe has the sequence shown in the SEQ ID NO:11, the rt173L probe has the sequence shown in the SEQ ID NO:12, the rt180M probe has the sequence shown in the SEQ ID NO:13, the rt181T probe has the sequence shown in the SEQ ID NO:14, the rt184G probe has the sequence shown in the SEQ ID NO:15, the rt202I probe has the sequence shown in the SEQ ID NO:16, the rt204I probe has the sequence shown in the SEQ ID NO:17, the rt233V probe has the sequence shown in the SEQ ID NO:18, the rt236N probe has the sequence shown in the SEQ ID NO:19, the rt236T probe has the sequence shown in the SEQ ID NO:20, the rt181S probe has the sequence shown in the SEQ ID NO:21, the rt250M probe has the sequence shown in the SEQ ID NO:22, the rt250V probe has the sequence shown in the SEQ ID NO:23, the rt204V probe has the sequence shown in the SEQ ID NO:24, positive control probe (P.C) has the sequence shown in the SEQ ID NO:25, and colour developing contrast probe (C.C) has the sequence shown in the SEQ ID NO:26.
Fig. 2 display target DNA cloning fragment is through 2% agarose gel electrophoresis figure.Wherein the 1-6 swimming lane is the specific target dna fragmentation that amplifies from the hepatitis B virus clinical sample; The 7th negative contrast.
Fig. 3 shows six kinds of different HBV genotype serum sample reverse dot blot hybridization detected results.Wherein: A: wild-type; The B:rt204I sudden change; The C:rt204V sudden change; D:rt181T and rt236T sudden change; The E:rt236T sudden change; F:rt180M, rt202I and rt204V sudden change.
Fig. 4 is determining of hepatitis B virus wild-type and the polyinfection simulated experiment of medicament-resistant mutation type and sensitivity thereof.
Embodiment
The following example is intended to illustrate rather than limit the present invention.
Embodiment 1: the extraction of serum DNA
The about 1ml of aseptic collection hepatitis B patient's to be checked venous blood, leave standstill 2 hours and 1 hour respectively at room temperature and 4 ℃ after, centrifugal (8,000rpm, 5 minutes) are separated and are also collected serum specimen 200 μ l.Therefrom get serum sample 40 μ l then, to wherein adding equivalent DNA extraction liquid, abundant mixing was also placed 10 minutes in boiling water bath.In order to guarantee that virion contained in the serum can further be continued this mixture to leave standstill 10 hours by abundant cracking under 4 ℃.Centrifugal (10,000rpm, 5 minutes) and collect supernatant liquor 2 μ l and use it for further PCR reaction then.
Embodiment 2: comprise the nest-type PRC amplification of the target DNA segment of HBV archaeal dna polymerase rt173～rt250 regional sequence.
Get single part of some pipes of PCR reaction solution of single tube, every pipe directly adds 2 μ l templates (or yin and yang attribute standard substance), and fully instantaneous (3 seconds) are centrifugal behind the mixing.Then each reaction tubes is put into the PCR instrument, 50 ℃ of pre-treatment 3 minutes, by following condition amplification: 93 ℃ 5 minutes, then by 93 ℃ 30 seconds, 68 ℃ 1 minute, totally 25 circulations, then by 93 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 45 seconds, totally 30 circulations, last 72 ℃ were extended 7 minutes.Amplified production detects (referring to accompanying drawing 2) through 2% agarose gel electrophoresis.Employed pcr amplification system comprises: 1 * qualitative PCR damping fluid, 0.2mM dNTPs, 6U Taq enzyme, 0.1pmol primer 1, primer 4 (SEQ ID NO:1,4) and 0.3pmol primer 2, primer 3 (SEQ ID NO:2,3).
Embodiment 3: adopt the water-bath hybrid method that three kinds of genotypic samples are carried out reverse dot blot hybridization and detect
Before the hybridization, shaking bath (or water-bath) temperature is transferred to hybridization temperature, get hybridization solution I (2 * SSC-0.1%SDS) with hybridization solution II be preheated to 40～50 ℃ standby.Get several 15ml centrifuge tubes according to the number of sample to be checked, each pipe adds 8-10ml hybridization solution I respectively and preheats to 40～50 ℃.Get 1000 μ l hybridization solution I+2 μ l solution I (1000: 2) mixing solutionss then as standby in conjunction with 4 ℃ of preservations of liquid, and get solution II: solution III: the mixture of solution IV (1900: 200: 1) (1.9ml solution II+0.2ml solution III+1 μ l solution IV) keeps in Dark Place standby as colour developing liquid.
At first the hybridization solution I of preheating is added in the pipe until not having the film bar, press film bar reference numeral and add the PCR product, put into after covering tightly on the shaking bath and hybridize 40～50min with medium rotating speed; Then take out the film bar with tweezers and put into the 50ml sluicing pipe, wash 3 times with the hybridization solution II of preheating, each 3～5 minutes, 40～50 ℃ were carried out wash-out in shaking bath; Add then and use down biochemistry to wave instrument in conjunction with liquid at normal temperature (20～30 ℃) to carry out binding operation; In conjunction with after, wave on the instrument wash-out 3 times with normal temperature (20～30 ℃) hybridization solution I biochemistry, each 1～3 minute; The dried residual liquid of control added solution II and waves on the instrument pre-colour developing 2min at biochemistry after wash-out was finished; Add colour developing liquid colour developing 5～8 minutes at last, outwelling after colour developing is finished develops the color liquid and add a certain amount of solution II stops 5min, washes twice with pure water 30ml again and has namely finished the hybridization whole process.Results of hybridization is preserved with jpeg format after adopting scanner scanning.
Embodiment 4: the result of test kit of the present invention judges
Result's decision principle is that the colour developing contrast probe (SEQ ID NO:26) that is used for the monitoring crossover process should all develop the color in all detections.Single colour developing occurs as rt204M site (SEQ ID NO:10), rt204I site (SEQ ID NO:17), rt204V site (SEQID NO:24), represent respectively that then the YMDD wild-type is arranged in the sample, the existence of YIDD mutant, the single mutant HBV DNA of YVDD; If rt204M site (SEQ ID NO:10) and rt204I site (SEQ ID NO:17) probe develop the color simultaneously, then show to have YMDD wild-type and the polyinfection of YIDD mutant in the sample; Develop the color simultaneously as rt204M site (SEQ ID NO:10) and rt204V site (SEQ ID NO:24) probe, then show to have YMDD wild-type and the polyinfection of YVDD mutant in the sample; Develop the color simultaneously as rt204I site (SEQ ID NO:17) and rt204V site (SEQ IDNO:24) probe, then show the polyinfection that has YVDD and two kinds of mutant HBV of YIDD in the sample; Develop the color simultaneously as rt204M site (SEQ ID NO:10), rt204I site (SEQ ID NO:17) and rt204V site (SEQ ID NO:24) three kinds of probes, namely show the polyinfection that has YMDD wild-type, YIDD and YVDD mutant HBV in the sample simultaneously.
The formulation that the invention of this test kit can be clinical nucleoside analog therapeutic regimen provides molecular biology experiment data science, individuation, for hepatitis B patient's treatment provides reliable Molecular Virology evidence.If occur certain medicament-resistant mutation C-type virus C among the patients serum, then to reduce consumption even the drug withdrawal of this kind nucleoside analog immediately, use other drug treatment instead, the manpower and materials that avoid waste, the state of an illness that also avoids delay and treatment and produce unnecessary consequence.The situation of interpretation part as a result of the present invention is as follows:
1, positive control probe, colour developing contrast probe (SEQ ID NO:25,26) and probe rt173V, rt180L, rt181A, rt184T, rt202S, rt204M, rt233I, rt236N, rt250M (SEQ ID NO:5,6,7,8,9,10,11,19,22) colour developing are judged to be the infection of HBV wild-type;
2, positive control probe, colour developing contrast probe (SEQ ID NO:25,26) and probe rt173V, rt180L, rt181A, rt184T, rt202S, rt204I, rt233I, rt236N, rt250M (SEQ ID NO:5,6,7,8,9,11,17,19,22) colour developing are judged to be the rt204I sudden change, to lamivudine resistance;
3, positive control probe, colour developing contrast probe (SEQ ID NO:25,26) and probe rt173V, rt180L, rt181A, rt184T, rt202S, rt204V, rt233I, rt236N, rt250M (SEQ ID NO:5,6,7,8,9,17,19,22,24) colour developing are judged to be the rt204V sudden change, to lamivudine resistance;
4, positive control probe, colour developing contrast probe (SEQ ID NO:25,26) and probe rt173V, rt180L, rt181T, rt184T, rt202S, rt204M, rt233I, rt236T, rt250M (SEQ ID NO:5,6,8,9,10,11,20,22) colour developing are judged to be rt181T and rt236T sudden change, to the Adefovir resistance;
5, positive control probe, colour developing contrast probe (SEQ ID NO:25,26) and probe rt173V, rt180L, rt181A, rt184T, rt202S, rt204M, rt233I, rt236T, rt250M (SEQ ID NO:5,6,7,8,9,10,11,20,22) colour developing are judged to be the rt236T sudden change, to the Adefovir resistance;
6, positive control probe, colour developing contrast probe (SEQ ID NO:25,26) and probe rt173V, rt180M, rt181A, rt184T, rt202I, rt204I, rt233I, rt236N, rt250M (SEQ ID NO:5,7,8,11,13,16,17,19,22) colour developing are judged to be rt180M, rt202I and rt204V sudden change, to lamivudine or Entecavir resistance; (referring to table 1 and accompanying drawing 3)
The different types of table 1 HBV infect and corresponding resistance situation
| Sample number | Probe combinations | The |
| 1 | Rt173V, rt180L, rt181A, rt184T, rt202S, rt204M, rt233I, rt236N, rt250M, positive control probe, colour developing contrast probe | YMDD infects (wild-type) |
| 2 | Rt173V, rt180L, rt181A, rt184T, rt202S, rt204I, rt233I, rt236N, rt250M, positive control probe, colour developing contrast probe | YIDD infects (LAM resistance) |
| 3 | Rt173V, rt180L, rt181A, rt184T, rt202S, rt204V, rt233I, rt236N, rt250M, positive control probe, colour developing contrast probe | YVDD infects (LAM resistance) |
| 4 | Rt173V, rt180L, rt181T, rt184T, rt202S, rt204M, rt233I, rt236T, rt250M, positive control probe, colour developing contrast probe | The relevant sudden change of ADV resistance (rt181T+rt236T) |
| 5 | Rt173V, rt180L, rt181A, rt184T, rt202S, rt204M, rt233I, rt236T, rt250M, positive control probe, colour developing contrast probe | The relevant sudden change of ADV resistance (rt236T) |
| 6 | Rt173V, rt180M, rt181A, rt184T, rt202I, rt204I, rt233I, rt236N, rt250M, positive control probe, colour developing contrast probe | The relevant sudden change of ETV resistance (rt180M+rt202I+rt204I) |
Embodiment 5: the determining of hepatitis B virus wild-type and the polyinfection simulated experiment of medicament-resistant mutation type and sensitivity thereof.
The resistance of hepatitis B virus is to produce also progressively development and change under the continuous action of medicine and human immunity selective pressure gradually, generally have from less to more, become the progressively conversion process of advantage from inferior position, therefore, detection to the hepatitis B virogene group just need have quite good detecting and resolving power to viral wild-type and the polyinfection of medicament-resistant mutation type with monitoring, the generation of the viral resistance of reflection dynamically, development and the state of an illness are returned and are turned over journey, for clinical further understanding and the viral resistance of research produce, development and control conditions of patients development in time produce positive influence, simultaneously clinical accurate and effective medication are had great importance.
Choose YMDD, YIDD and YVDD type serum sample (has been diluted to 10
4Copy/ml) each pipe, YMDD and YIDD are the A group, YMDD and YVDD are the B group, respectively according to 1: 9,2: 8,3: 7,4: 6,5: 5,6: 4,7: 3,8: 2,9: 1 mixed, extracting DNA, PCR and RDB then.Experiment showed, that wild-type of the present invention and mutant polyinfection detect lower limit and is 10% (seeing accompanying drawing 4).
Embodiment 6: the hybridization probe sequence determine that the sequence of middle probe of the present invention is about 15～26 bases, its length is relevant with intensity of hybridization signal, its Tm value is relevant with hybridization temperature.In the sequence scope of (SEQ ID NO:5～26), the combination of selected different lengths probe sequence all can produce hybridization signal and differentiation distinguishing ability preferably in listed 21 probes.As in the invention implementation process, when the sequence of three probes (SEQ ID NO:10,17,24) was following sequence, hybridization can produce good signal and separating capacity when 55 spend.
NH
2-5’-GCTTTYAGYTATATGGATGATSTGGT-3’(SEQ ID NO：10)
NH
2-5’-GCTTTYAGYTATATHGATGATSTGGT-3’(SEQ ID NO：17)
NH
2-5’-GCTTTYAGYTATGTGGATGATSTGGT-3’(SEQ ID NO：24)
Remove 5 bases at 5 ' end, remove 5 bases or remove two bases at 5 ' end at 3 ' end, 3 ' end removes 3 bases; The probe of these three kinds of sequences all can produce good signal and distinguishing ability wild and sudden change when 42 degree hybridization.
Sequence table
Claims (3)
1. hepatitis B virus detection kit for nucleoside analogue drug-resistant correlated mutation type, this test kit comprises the nucleic acid extraction agent that extracts sample nucleic acid, pcr reagent, cloudy, the sun quality control product, the nucleic acid hybridization reagent that is used for nucleic acid hybridization reaction, the Hybond membrane bar, wherein pcr reagent is by forward primer, biotin labeled reverse primer, the deoxyribonucleoside triphosphate that contains the deoxyuridine triphosphoric acid, heat-resisting Taq enzyme and uridylic-DNA-glycosylase is formed, and it is characterized in that the sequence of forward primer and biotin labeled reverse primer is respectively:
1)5’-TAGACTCGTGGTGGACTTCTCTCARTTT-3’
2)5’-TGGATGTRTCTGCGGCGTTT-3’
3) vitamin H-5 '-GCCTTRTAAGTTGGCGAG-3 '
4)5’-CGACGTGCAGAGGTGAAGCGAAGTGCA-3’，
The sequence that is used for the oligonucleotide probe of 5 of nucleic acid hybridization ' end amino labeled in the Hybond membrane bar is respectively:
5)NH
2-5’-TWCCTATGGGAGTGGGCCTCAGYCCG-3’
6)NH
2-5’-GYCCGTTTCTCYTGGCTCAGTTTACT-3’
7)NH
2-5’-CGTTTCTCHTGGCTCAGTTTACTWGY-3’
8)NH
2-5’-TGGCTCAGTTTACTWGYGCMATTTGT-3’
9)NH
2-5’-GYYTGGCTTTYAGTTATATKGATGAY-3’
10)NH
2-5’-GCTTTYAGYTATATGGATGATSTGGT-3’
11)NH
2-5’-TRTCTBTGGGYATMCATTTRAAYMCY-3’
12)NH
2-5’-TWCCTATGGGATTGGGCCTCAGYCCG-3’
13)NH
2-5’-GYCCGTTTCTCYTGGCTCAGTTTACT-3’
14)NH
2-5’-CGTTTCTCHTGACTCAGTTTACTWGY-3’
15)NH
2-5’-TGGCTCAGTTTGGTWGYGCMATTTGT-3’
16)NH
2-5’-GYYTGGCTTTYATTTATATKGATGAY-3’
17)NH
2-5’-GCTTTYAGYTATATHGATGATSTGGT-3’
18)NH
2-5’-TRTCTBTGGGYGTGCATTTRAAYMCY-3’
19)NH
2-5’-TCTBTGGGYAAYCATTTRAAYCCTMA-3’
20)NH
2-5’-TCTBTGGGYACMCATTTRAAYCCTMA-3’
21)NH
2-5’-CGTTTCTCHTGATTCAGTTTACTWGY-3’
22)NH
2-5’-CCCTTAAYTTYATGGGHTAYRTNATY-3’
23)NH
2-5’-CCCTTAAYTTYGTGGGHTAYRTNATY-3’
24)NH
2-5’-GCTTTYAGYTATGTGGATGATSTGGT-3’
25)NH
2-5’-ATTGTTCAGTGGTTCGTAGGGCTTTC-3’
And the sequence of the colour developing control oligonucleotide probe that is used for the monitoring crossover process of 5 ' end biotin labeling and 3 ' end amino labeled is: vitamin H-5 '-GCCGTAACCGTCACAATCCGT-3 '-NH
2
2. according to the test kit of claim 1, be further characterized in that nucleic acid hybridization reagent is made up of hybridization solution I, hybridization solution II, solution I, solution II, solution III and solution IV, wherein hybridization solution I is 2 * SSC-0.1%SDS, and hybridization solution II is that 0.5 * SSC-0.1%SDS, solution I are the streptavidin solution of 250u/ml coupling horseradish peroxidase, sodium citrate solution that solution II is 0.1mol/L, the solution III is 2mg/ml tetramethyl biphenyl amine aqueous solution, solution IV are 3% superoxol.
3. according to the test kit of claim 1, be further characterized in that Hybond membrane is nylon membrane or nitrocellulose filter.
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| 丁静娟 等.乙型肝炎病毒耐拉米夫定多聚酶基因变异检测方法研究.《中华实验和临床病毒学杂志》.2004,第18卷(第1期),24-27. * |
| 黄燕萍 等.逆向斑点杂交检测乙型肝炎病毒基因型及YMDD变异.《中华检验医学杂志》.2006,第29卷(第2期),140-142. * |
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Application publication date: 20091028 Assignee: Zhongshan Bio-Tech Co., Ltd. Assignor: Daan Gene Co., Ltd., Zhongshan Univ. Contract record no.: 2013440000537 Denomination of invention: Detection kit for nucleoside analogue drug-resistant correlated mutation type hepatitis B viruses Granted publication date: 20130710 License type: Exclusive License Record date: 20131213 |
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