CN1588066A - Fluorescent PCR detecting method for hepatitis B virus gene parting and reagent kit - Google Patents
Fluorescent PCR detecting method for hepatitis B virus gene parting and reagent kit Download PDFInfo
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Abstract
The invention relates to a fluorescent PCR detecting method for a HBV type seperating and the reagent box. The characteristics of the method lies in finding out the HBV DNA complete sequence which has been seperatedd from the GenBank to make a sequence; according to the result of the sequence matching, finding out the gathering zone of the type specific base of the BHA gene type, designing a probe and a pair of primers according the gathering zone. Extract HBV DNA from the blood serum sample to make PCR amplification to realize HBV gene type seperating detection in the fluorescent PCR when the probe and primer exist. The invention provides a reagent box and the type positive standard used in it. The accuracy of type parting reaches to 98% and can reach to 100% by adjusting the reaction condition., simpler and quicker, the test procedure and the time consuming is less, the cost is lower than the complete sequence analysis and more quick and accurate than the regular relevant PCR and RFLP method, besides, the closed check adopted in the invention can avoid the contamination efficiently.
Description
Technical field
The present invention relates to a kind of hepatitis type B virus (Hepatitis B virus, HBV) fluorescent polyase chain reaction of Genotyping (PCR) detection method and kit thereof.
Background technology
Hepatitis type B virus mainly causes liver damage by host immune mechanism, and body cell is discerned viral antigen and attacked infected liver cell and causes inflammation, and this process is comprised host and viral a plurality of factor affecting.The viral gene heterogeneity affects the expression of antigen, may also therefrom play the part of important role.The frequency difference of some variation appears in different strains, and different strains are removed different and other the factor of resistibility to immunity of organism and may have been caused different genotype to have spectrum of disease after the different infection.From present research, HBV C genotype is relevant with heavier hepatic disease, and the Type B then pathology with lighter is relevant; The A type is relevant with chronic hepatitis, and the D type is relevant with acute self-limiting hepatitis; The relation of other genotype and spectrum of disease is still waiting special discovery.Kao finds that the C type is relevant with hepatocellular carcinoma with severe such as cirrhosis, and Type B is present in the young non-liver cirrhosis patient more.Mostly C type patient is that HBeAg (+) and serum dna content are higher than Type B, and C type patient postpones than Type B in the immune clearance stage seroconversion of HBV, and the C type has higher C gene promoter mutation rate than Type B simultaneously.In antiviral therapy, Type B to drawing miaow furan pyridine sensitivity, is the same but amphitypy produces the probability of medicine resistance than C type.The genotype that Mayerat etc. have compared among 35 routine oxyhepatitises and the 30 routine chronic hepatitis patients distributes, and finds that the A type accounts for 80% (28/35) in the chronic hepatitis group, and the D type accounts for 11% (4/35); Oxyhepatitis group D type accounts for 80% (24/30), the A type accounts for 10% (3/30), the prompting virogene type has certain difference in virus-host interacts, this may be because the antigenicity that genotype A is produced will be weaker than genotype D, induce ability that body produces immune clearance also a little less than, cause infecting delay.HBV chronic infection person's genotype and studies show that of CP variation that Lindh etc. are B and C to 43 genotype of East Asia Region, the T1762 variation more is prone to the genotype at C, more is prone to heavier liver inflammation after infecting the C type.
HBV is carried out Genotyping help epidemiology, aetiology and clinical diagnosis and treatment that HBV infects are carried out more intensive research, have the clinical disease course of the HBV infection that studies show that different subtype now and the reaction for the treatment of is all existed certain difference.Virus variation is one of biological heredity Fundamentals of evolving, and hepatitis B virus variation is abiogenous for adapting to living environment in chronic infection process, also can betide drug application or vaccine inoculation after.HBV utilizes the RNA intermediate in duplicating, varial polymerases and reverse transcriptase activity are effectively and rapidly, and varial polymerases lacks the check and correction enzymatic activity, takes place to be difficult to revise after the nucleotide substitution variation.The variation of having found the HBV sequence all can take place in each zone of genome.Different patient's bodies and different medicines show different genotype with different variation strains long-term the interaction.Though do not have definite data to show the variation ratio of HBV, virus variation is a high frequency and eternal, has now developed into A～H eight types by initial A～D four types, virus more make a variation focus and genotype will occur with the continuation struggle of body.Although HBV somatotype, variation and clinical sign have certain degree of association, still do not illustrate clear at present fully.The epidemic disease credit subcharacter generaI investigation of large-scale system will more help the Dialectic Relationship that people are familiar with HBV and body and medicine, and this just depends on simple and rapid HBV classifying method.
The HBV carrier of China is numerous at present, accounts for 10%.In the today that does not have specific drug, the still difficult standard scheme that a widespread usage is arranged of treatment hepatitis B causes different patients' result of treatment to differ, and long-term host carries with the drug abuse meeting and improves the risk that variant occurs.Hepatitis B is the main result of virus and immune response, needs to work out for a long time therapeutic scheme targetedly.HBV is carried out the detection of Genotyping and variation focus, important guidance is all arranged at aspects such as selecting suitable medicine or formulation therapeutic scheme.At present clinical detection market presses for relevant proven technique scheme especially at the method for viruses molecule feature and mechanism, further instruct clinical treatment and the discovery and the structure specific medicament of hepatitis B, though a lot of laboratories are arranged, on the domestic and international market blank out all in the detection method of research HBV somatotype and variation.
Both at home and abroad the method that HBV is carried out Genotyping can be divided into complete genome sequence comparison and fragment gene sequence alignment two big classes.It is based on the dna homolog in the biosystem that complete genome sequence is compared, it mainly is to measure by the HBV complete genome sequence to carry out Genotyping, in addition, also can use the full gene of HBV is carried out restriction fragment length polymorphism analytical technology (RFLP).The fragment gene sequence alignment is to utilize the sequence type of the representative segment among the HBV to reflect the type of complete genome sequence, and the major technique type has fragment gene sequencing, PCR-RFLP, type special primer (SSP) TRAP and type specific probe (SSO) detection method etc.Though the sequencing result accurately and reliably,, only use at present as laboratory study because its technical sophistication, experiment flow is long, requirement for experiment condition is high, length consuming time and expense costliness are difficult to do routine clinical use; The RFLP technology is simple relatively, but restriction enzyme site is subject to genetic mutation influence, and meets mixed infection or enzyme is cut not exclusively, complicated band can occur, influences somatotype result judgement; Application SSP method is carried out pcr amplification needs a plurality of amplification pipes, uses the method for conventional PCR afterproduct electrophoresis also to pollute easily, and its sensitivity is lower than specificity probe; Use the SSO method and can detect somatotype, therefore have characteristics such as the simple relatively and expense of operation is lower, have practical value most by PCR product to single tube.Biochip technology is a new developing technology in recent years, it is by using for reference robot calculator machine chip array principle, utilizing making nucleic acid molecular hybridization and chemiluminescence, make testing result have highly sensitive, required sample trace and characteristics such as easy and simple to handle, is a kind of advanced person's SSO method.In theory, the method for utilizing biochip technology to set up the hepatitis B virus gene typing diagnosis is fully possible, but expense is higher, also in the experimental study stage, has not yet to see the research report that genetic chip carries out the HBV Genotyping.
The real-time fluorescence PCR reaction is outside a pair of primer of conventional PCR, adds two ends and has fluorescently-labeled oligonucleotide probe.Under the intact state of probe, the exciting light of 5 ' end report fluorophor is suppressed by the cancellation fluorophor of 3 ' end.In the PCR course of reaction, extension along with chain, the Taq enzyme moves to the binding site of fluorescence labeling probe along dna profiling, because its 5 ' → 3 ' exonuclease activity, fluorescence probe is cut off, discharge the fluorescence signal of report fluorophor, whenever synthetic nascent strand, just have the signal of a reporter group to discharge, d/d number and the PCR product that swashs from the report fluorophor is man-to-man relation.By regularly each circulation of dynamic monitoring of fluorescent PCR instrument, can obtain the actual amplification curve of sample, find the logarithmic phase of pcr amplification, can make qualitative and quantitative detection to sample.Only yet there are no the report that fluorescent PCR is used for the HBV Genotyping up till now.The variation of HBV is absolute, and will constantly produce and accumulate various variations, and all methods except the genom sequence analysis can not guarantee that all 100% somatotype is accurate at present.
Summary of the invention
The object of the present invention is to provide a kind of detection method of more effective easy again hepatitis B virus gene typing; Another object of the present invention is to be provided for the kit of this method.
The present invention is on the basis of the existing HBV dna sequence dna of somatotype of macromethod, find out the regularity of distribution of the genotypic type specificity base of HBV, taked the fluorescence PCR detecting method of Auele Specific Primer and specific probe combination, somatotype sensitivity, accuracy had both been improved, continue the fast and convenient performance of fluorescence PCR detecting method again, realized purpose of the present invention.
The technical characterictic of the fluorescence PCR detecting method of a kind of hepatitis B virus gene typing of the present invention is, in the presence of the probe that obtains of design and corresponding primer as follows, in the fluorescent PCR instrument, the hepatitis B virus DNA that extracts from blood serum sample is carried out pcr amplification, realize that hepatitis B virus gene typing detects;
Described probe and primer design method are as follows:
1. sequence alignment: from GenBank, find out the HBV DNA complete sequence of having made Genotyping and carry out sequence alignment;
2. find out the aggregation zone of the genotypic type specificity base of HBV according to the sequence alignment result;
3. determine the HBV dna sequence dna segment that type is special according to the aggregation zone of type specificity base;
4. the HBV dna sequence dna segment designing probe special according to type;
5. according to the probe design primer.
The present invention finds out HBV DNA complete sequence as much as possible from GenBank, in respect of more than 530, that has wherein done the Genotyping description has 143, and wherein the A type is 23,34 of Type Bs, 52 on C type, 13 on D type, 3 on E type, 14 on F type, 2 on G type, 2 on H type; Format Series Lines is organized into the FASTA form; Then 143 sequences making Genotyping are submitted to EBI (European Bioinformatics Institute, Europe bioinformatics research institute) contigency of contributing a foreword is joined (Multiple Sequence alignment claims the multisequencing comparison again) to find a shade of difference of HBV DNA on variation is accumulated and evolved.
Our analytical sequence connection join the result on the complete sequence scope, find out the type specificity base (so-called type specificity base be meant various on same position separately with respect to its alloytype and different base, that is to say for example some fixing bases of the most appearance of A type on a certain position, and its alloytype can not be this base), further find out the aggregation zone of various HBV DNA type specificity base again, the principle that the present invention follows is to have at least the type specificity base more than 6 just to think type specificity base aggregation zone in the 100bp.The distribution majority of type specificity base is random distribution, but long-term virus evolution development and distribute with type specificity base that host's mutual relationship makes virus be certain rule in some zone, and this is the most basic foundation of this method somatotype.For example A type and D type, this amphitypy has feature clearly, and the A type has more about 6 bases than its alloytype near the 2355th base, and the D type lacks about 33 bases near the 2845th base.It is the type specificity base that this insertion and disappearance are considered as by the present invention too.The type specificity base that anatomizes the BC amphitypy again is: there is type specificity base aggregation zone in Type B between the 1611st～1677 base, and there is type specificity base aggregation zone in the C type between the 1314th～1390 base.
(probe Tm value is between 68～70 ℃ according to the rule designing probe of fluorescent PCR probe design, GC content can not have 6 identical bases of continuous appearance between 30%～80%, 5 ' end can not be bases G, avoid secondary structure), make probe comprise the type specificity base as far as possible.On the basis of probe, (primer Tm value is lower about 10 ℃ than probe for the rule design primer that designs according to fluorescence PCR primer, with probe that primer in the same way should be close with probe, preferably be separated by and be no more than 2 bases, avoid secondary structure), making primer 3 ' hold last base is the type specificity base as far as possible.More than describe and be primarily aimed at the B/C amphitypy, because the type specificity base difference is too big, need only just passable on several bases of insertion/disappearance probe design for the A/D amphitypy.The primer probe of design becomes with reaction result over against answering, and that is to say in a certain type reaction has amplification just to be decided to be a certain type.
Probe of the present invention and primer design method are applicable to each genotype of hepatitis type B virus, but at the common B in Southeast Asia especially Chinese, C, D and A type, (FAM is a luminophore only to provide best probe of these several types and primer design below, BHQ1 is a quenching group, be the title of primer or probe before the colon, the back is concrete sequence; Various primer probe is respectively the arrangement mode of upstream primer, probe, downstream primer).
The hepatitis B virus C genotype, the special HBV dna sequencing fragment of its type is:
GAAACTTATCGGCACCGACAACTCTGTTGTCCTCTCTCGGAAATACACCTCCTTTCCATGGCTGCTAGGCTGTG
The sequence of described primer and probe is:
BVc1314pri：GAAACTTATCGGCACCGACAAC
BVc1340pro：FAM-CTGTTGTCCTCTCTCGGAAATACACCTCCT-BHQ1
BVc1390rpri：CACAGCCTAGCAGCCATGG
Hepatitis type B virus B genotype, the special HBV dna sequencing fragment of its type is:
AGACCACCGTGAACGCCCACCGGAACCTGCCCAAGGTCTTGCATAAGAGGACTCTTGGACTTTCAG
The sequence of described primer and probe is:
BVb1611pri：AGACCACCGTGAACGCCC
BVb1650rpro：FAM-AGACCTTGGGCAGGTTCCGGTG-BHQ1
BVb1677rpri：CTGAAAGTCCAAGAGTCCTCTTATGC
Hepatitis type B virus D genotype, the special HBV dna sequencing fragment of its type is:
GTGGGTCACCATATTCTTGGGAACAAGAGCTACAGCATGGGGCAGAATCTTTCCACCAGCAATCCTCTG
The sequence of described primer and probe is:
BVd2811pri：GTGGGTCACCATATTCTTGGGA
BVd2835pro：FAM-CAAGAGCTACAGCATGGGGCAGAATC-BHQ1
BVd2879rpri：CAGAGGATTGCTGGTGGAAA
Hepatitis type B virus A genotype, the special HBV dna sequencing fragment of its type is:
ATCACTTCCGGAAACTACTGTTGTTAGACGACGG(A)GACCGAGGCAGGTCCCCTAGAAGAAGAACTCCTCGCCTC
The sequence of described primer and probe is:
BVa2322pri：ATCAGTTCCGGAAACTACTGTTGTTA
BVa2349pro：FAM-ACGACGGGACCGAGGCAGGTC-BHQ1
BVa2390rpri：GAGGCGAGGGAGTTCTTCTTCTA
Fluorescent PCR reaction of the present invention is the same with general fluorescent PCR reaction, contains the PCR reaction buffer, Mg in the required fluorescent PCR reactant liquor
2+Solution, dNTP, Taq archaeal dna polymerase, and probe and pairing a pair of upstream and downstream primer; All in the required concentration range of common PCR reaction, described PCR reaction buffer is that 1 * PCR reaction buffer comprises 10mmol/L Tris-HCl to the concentration of above-mentioned substance, and pH 8.9,50mmol/L KCl, 5% glycerine; Mg
2+Solution concentration is 2.5mmol/L; Described dNTP concentration is 0.2mmol/L; The Taq archaeal dna polymerase is 1U; Concentration and probe concentration is 0.12 μ mol/L; The upstream and downstream primer concentration respectively is 0.6 μ mol/L; For preferably also containing UNG (UNG) 0.1U in the pre-anti-pollution PCR reactant liquor.
The pcr amplification condition that the present invention determines is:
55～65 ℃ of 30s of 94 ℃ of 1min → 94 ℃ 5s → annealing temperatures wherein carry out 30～50 circulations between 94 ℃ of 5s → 55～65 ℃ 30s; Described annealing temperature is preferably 60 ℃, and described circulation is preferably 40 times.According to our company's experience in the past, utilize the fluorescent PCR instrument to detect HBV, the length of amplification can adopt two-step approach in 200bp.Because the PCR most critical is annealing temperature, so attempt different annealing temperatures with the primer probe of having selected according to above system, Tm value according to design primer and probe, in 55～65 ℃ of scopes of annealing temperature, reaction result to 55 ℃, 58 ℃, 60 ℃, 61 ℃ and 62 ℃ five temperature is made comparisons, find that making annealing temperature with 60 ℃ is fit to 98% HBV DNA type specificity amplification, minority can clear and definite somatotype after 60 ℃ of HBV DNA that do not have an amplification are being adjusted to 55 ℃, and minority Type B C type all has the HBV DNA of amplification can clear and definite somatotype after being adjusted to 61 ℃.
Hepatitis B virus DNA required for the present invention can extract from blood serum sample with usual way, preferably adopts method provided by the invention, sees embodiment for details.
The fluorescent dye of label probe of the present invention is that common fluorescent PCR detects employing, preferably FAM (Carboxyfluorescein, Fluoresceincarboxylic acid, absorbing wavelength 492nm, emission wavelength 518nm, green); Quencher is that (its effective light absorbing wavelength is at 500-580nm for Black Hole Quencher 1, a kind of light absorption material that cries the black hole for BHQ1.)。
The judgement of measurement result of the present invention: the expected result of primer and probe design judges and is over against answering, and promptly which type has amplified fluorescence just can differentiate to be this kind of.Because A type and D type and its alloytype have apparent in view sequence difference, thus in result's judgement, see that earlier the A/D type does not have not amplification, the A/D type amplification is arranged and the B/C type all what do not increase just should be the A/D type; Which has amplification to see the B/C type again under the prerequisite that the A/D type does not have to increase, and is which type just.Sometimes can meet BC and all not have amplification, this phenomenon is by reducing still somatotype again of reaction annealing temperature (about 2～5 ℃); Sometimes also have BC that the phenomenon of amplification is all arranged, this phenomenon is by the still somatotype again of reaction annealing temperature (about 1～2 ℃) that raises.But this is at the common ABCD type of Chinese, for all do not have amplification can not be disappointed under assert because may be type such as EFGH; For the also careful of amplification all arranged, because may there be the chimera of mixed type virus infections or veriform HBV.So recommend to carry out complete sequence analysis.
A kind of kit that is used for the fluorescence PCR detecting method of hepatitis B virus gene typing of the present invention contains the PCR reactant liquor, comprises the PCR reaction buffer, Mg
2+Solution, dNTP, the Taq archaeal dna polymerase, and by arbitrary the probe and the pairing a pair of upstream and downstream primer of said method of the present invention design.All in the required concentration range of common PCR reaction, described PCR reaction buffer is 1 * PCR reaction buffer: comprise 10mmol/L Tris-HCl, pH 8.9,50mmol/LKCl, 5% glycerine for the concentration of above-mentioned substance; Described Mg
2+Solution concentration is 2.5mmol/L; Described dNTP concentration is 0.2mmol/L; Described Taq archaeal dna polymerase is 1U; Described concentration and probe concentration is 0.12 μ mol/L; Described upstream and downstream primer concentration respectively is 0.6 μ mol/L; Preferably also contain UNG (UNG) 0.1U in the PCR reactant liquor.Kit of the present invention preferably also contains DNA extraction reagent, comprises A liquid: 6mol/L guanidinium isothiocyanate, 30mmol/L sodium citrate, 0.58% sodium dodecylsulphonate, 1mmol/L disodium ethylene diamine tetraacetate, 2% glycogen; B liquid: isopropyl alcohol; C liquid: 75% ethanol water.
Effect of the present invention and advantage:
Through surpassing the checking of 100 parts of samples, the accuracy of the inventive method somatotype reaches 98%, can reach 100% accuracy by adjusting reaction conditions; Than complete sequence analysis method simple and fast, experiment flow is few, weak point consuming time, and expense is low again, and more relevant than the PCR of routine and RFLP again method is accurate, and the enclosed detection of the inventive method can effectively be avoided polluting.
Embodiment
Following is that example further specifies the present invention with the common HBV gene A of Chinese, B, C and the experiment of D type only, should not be used as limitation of the present invention.
Embodiment 1: the fluorescent PCR assay method and the kit thereof of hepatitis B virus gene typing of the present invention.
1. kit is formed:
(1) DNA extraction reagent: comprise A liquid: 6mol/L guanidinium isothiocyanate, 30mmol/L sodium citrate, 0.58% sodium dodecylsulphonate, 1mmol/L disodium ethylene diamine tetraacetate, 2% glycogen; B liquid: isopropyl alcohol; C liquid: 75% ethanol water.
(2) PCR reaction reagent:
A type PCR reactant liquor (everyone part consumption):
The addition final concentration
10 * PCR damping fluid (100mmol/L Tris-HCl, 2.5 μ L, 1 * PCR damping fluid
PH 8.9,500mmol/L KCl, 50% glycerine)
25mmol/L?MgCl
2 2.5μL 2.5mmol/L
5mmol/L?dNTP 1μL 0.2mmol/L
100pmol/ μ L A type primer 1 0.15 μ L 0.6 μ mol/L
100pmol/ μ L A type primer 2 0.15 μ L 0.6 μ mol/L
100pmol/ μ L A type probe 0.03 μ L 0.12 μ mol/L
5U/ μ L Taq enzyme 1U
1U/μL?UNG 0.1U
Be settled to 23 μ L with purified water.
The sequence of described A type primer and probe is:
Primer 1:ATCACTTCCGGAAACTACTGTTGTTA
Primer 2: GAGGCGAGGGAGTTCTTCTTCTA
Probe: ACGACGGGACCGAGGCAGGTC
Type B PCR reactant liquor (everyone part consumption):
Removing primer and probe is Type B, and outside the sequence of its sequence and A type was different, other reagent and concentration thereof were identical with A type PCR reactant liquor.
The sequence of described Type B primer and probe is:
Primer 1:AGACCACCGTGAACGCCC
Primer 2: CTGAAAGTCCAAGAGTCCTCTTATGC
Probe: AGACCTTGGGCAGGTTCCGGTG
C type PCR reactant liquor (everyone part consumption):
Removing primer and probe is the C type, and outside the sequence of its sequence and A type was different, other reagent and concentration thereof were identical with A type PCR reactant liquor.
The sequence of described C type primer and probe is:
Primer 1:GAAACTTATCGGCACCGACAAC
Primer 2: CACAGCCTAGCAGCCATGG
Probe: CTGTTGTCCTCTCTCGGAAATACACCTCCT
D type PCR reactant liquor (everyone part consumption)
Removing primer and probe is the D type, and outside the sequence of its sequence and A type was different, other reagent and concentration thereof were identical with A type PCR reactant liquor.
The sequence of described D type primer and probe is:
Primer 1:GTGGGTCACCATATTCTTGGGA
Primer 2: CAGAGGATTGCTGGTGGAAA
Probe: CAAGAGCTACAGCATGGGGCAGAATC
(3) type positive criteria product:
Hepatitis B virogene A type standard items (10
5～10
7Copies/mL), hepatitis B virogene Type B standard items (10
5～10
7Copies/mL), hepatitis B virogene C type standard items (10
5～10
7Copies/mL), hepatitis B virogene D type standard items (10
5～10
7Copies/mL).
2. the fluorescent PCR of hepatitis B virus gene typing is measured
(1) clinical sample sampling: extract person under inspection's venous blood 1mL with asepsis injector, inject aseptic 1.5mL centrifuge tube, left standstill 2 hours, change 4 ℃ of placements in room temperature.If there is not tangible serum supernatant, then 5000r/min is centrifugal 5 minutes, gets supernatant and changes (red blood cell is not brought in attention into) in another aseptic centrifuge tube over to, is serum specimen.
(2) DNA extraction of serum specimen is handled: get 120 μ L DNA extraction A liquid in the 0.5mL centrifuge tube, every pipe adds serum to be checked, the negative control sera 60 μ L that examined and determine to the HBV DNA positive respectively, carries out mark, mixing, 98 ℃ of heating cracking in 10 minutes; Add 180 μ L DNA extraction B liquid, abundant mixing, centrifugal 8 minutes of 13000r/min removes supernatant; Add 150 μ L DNA extraction C liquid, put upside down mixing for several times, 13000 went out r/min centrifugal 3 minutes, inhaled and abandoned supernatant (200 μ L application of sample rifle rifle points are extended the pipe end once to be blotted), and the room temperature of uncapping is placed and dried in 5 minutes; Add 15 μ L purified water dilutions suspendible sediment, the of short duration centrifugal liquid that makes falls within the pipe bottom, standby (extract used A, B and C liquid is seen kit).
Get various reactant liquor respectively in each PCR reaction tube by mentioned reagent box addition, add the dna profiling of 2 μ L extraction and various positive reference substance more respectively, whole reaction system is 25 μ L, does blank simultaneously; The lid upper tube cap places full-automatic fluorescent PCR ABI7000 detector with reaction tube, carries out the PCR reaction with reference to parameters such as instrumentation explanation setting blank, various positive control, samples to be checked.
The fluorescent PCR reaction conditions is set: 94 ℃ 1 minute; 94 ℃ 5 seconds, 60 ℃ 30 seconds, 40 circulations.Fluorescein is set at FAM.
3. the result judges:
Every data that composite analyser provides are set rational threshold value (Threshold) and baseline (Baseline), make instrument provide correct result.Blank and negative control are negative; Various positive control is all positive.Sample to be tested has amplification to be the A type in A type reactant liquor, has amplification to be the D type in D type reactant liquor; The amplification that has in the Type B reactant liquor of A/D type feminine gender is Type B, has amplification to be the C type in C type reactant liquor.
Embodiment 2: clinical trial
This experiment has detected 103 parts of samples altogether, A type 1 example wherein, D type 1 example, Type B 46 examples, C type 53 examples.There is 1 routine sample all models reactant liquor all negative, suitably reduces temperature of reaction (55 ℃) and carry out the PCR reaction again, be found to be the C type.There are 1 routine B, the reaction of C type that amplification is all arranged, improve temperature of reaction (61 ℃) and carry out the PCR reaction again, find it also is the C type.Also occurred the situation that many types of HBV mixed infection or mosaic type HBV infect in clinical, therefore if the situation of amplification is all arranged in a plurality of type reactant liquor, and the adjustment of the reaction conditions obvious somatotype of failing should carry out the HBV complete sequence analysis.
Claims (11)
1. the fluorescence PCR detecting method of a hepatitis B virus gene typing, it is characterized in that, in the presence of the probe that obtains of design and corresponding primer as follows, in the fluorescent PCR instrument, the hepatitis B virus DNA that extracts from blood serum sample is carried out pcr amplification, realize that hepatitis B virus gene typing detects;
Described probe and primer design method are as follows:
(1) sequence alignment: from GenBank, find out the hepatitis B virus DNA complete sequence of having made Genotyping and carry out sequence alignment;
(2) find out the aggregation zone of the type specificity base of genotype of hepatitis B virus according to the sequence alignment result;
(3) determine the hepatitis B virus DNA sequence fragment that type is special according to the aggregation zone of type specificity base;
(4) the hepatitis B virus DNA sequence fragment designing probe special according to type;
(5) according to the probe design primer.
2. the fluorescence PCR detecting method of a kind of hepatitis B virus gene typing according to claim 1, it is characterized in that described genotype of hepatitis B virus is the C genotype, the special hepatitis B virus DNA sequence fragment of described type is: GAAACTTATCGGCACCGACAACTCTGTTGTCCTCTCTCGGAAATACACCTCCTTTC CATGGCTGCTAGGCTGTG
The sequence of described primer and probe is:
Primer 1:GAAACTTATCGGCACCGACAAC
Primer 2: CACAGCCTAGCAGCCATGG
Probe: CTGTTGTCCTCTCTCGGAAATACACCTCCT
3. the fluorescence PCR detecting method of a kind of hepatitis B virus gene typing according to claim 1, it is characterized in that described genotype of hepatitis B virus is the B genotype, the special hepatitis B virus DNA sequence fragment of described type is: GGGAGCATTCGGGCCAGGGTTCACCCCTCCCCATGGGGGACTGTTGGGGTGGAGCC CTC
The sequence of described primer and probe is:
Primer 1:AGACCACCGTGAACGCCC
Primer 2: CTGAAAGTCCAAGAGTCCTCTTATGC
Probe: AGACCTTGGGCAGGTTCCGGTG
4. the fluorescence PCR detecting method of a kind of hepatitis B virus gene typing according to claim 1, it is characterized in that described genotype of hepatitis B virus is the D genotype, the special hepatitis B virus DNA sequence fragment of described type is: GTGGGTCACCATATTCTTGGGAACAAGAGCTACAGCATGGGGCAGAATCTTTCCAC CAGCAATCCTCTG
The sequence of described primer and probe is:
Primer 1:GTGGGTCACCATATTCTTGGGA
Primer 2: CAGAGGATTGCTGGTGGAAA
Probe: CAAGAGCTACAGCATGGGGCAGAATC
5. the fluorescence PCR detecting method of a kind of hepatitis B virus gene typing according to claim 1, it is characterized in that described genotype of hepatitis B virus is the A genotype, the special hepatitis B virus DNA sequence fragment of described type is: ATCACTTCCGGAAACTACTGTTGTTAGACGACGG (A) GACCGAGGCAGGTCCCCTAGAAGAAGAACTCCTCGCCTC
The sequence of described primer and probe is:
Primer 1:ATCACTTCCGGAAACTACTGTTGTTA
Primer 2: GAGGCGAGGGAGTTCTTCTTCTA
Probe: ACGACGGGACCGAGGCAGGTC
6. according to the fluorescence PCR detecting method of any hepatitis B virus gene typing described in the claim 1～5, it is characterized in that described pcr amplification condition is:
55～65 ℃ of 30s of 94 ℃ of 1min → 94 ℃ 5s → annealing temperatures wherein carry out 30～50 circulations between 94 ℃ of 5s → 55～65 ℃ 30s.
7. the fluorescence PCR detecting method of a kind of hepatitis B virus gene typing according to claim 6 is characterized in that described annealing temperature is 60 ℃, and described circulation is 40 times.
8. the fluorescence PCR detecting method of a kind of hepatitis B virus gene typing according to claim 1, it is characterized in that described hepatitis B virus DNA extracting method is as follows: get 120 μ L DNA extraction A liquid in the 0.5mL centrifuge tube, every pipe adds serum to be checked, negative control sera 60 μ L respectively, carry out mark, mixing, 98 ℃ of heating cracking in 10 minutes; Add 180 μ L DNA extraction B liquid, abundant mixing, centrifugal 8 minutes of 13000r/min removes supernatant; Add 150 μ L DNA extraction C liquid, put upside down mixing for several times, centrifugal 3 minutes of 13000rpm/min inhales and abandons supernatant, and the room temperature of uncapping is placed and dried in 5 minutes; Add 15 μ L purified water dilution suspendible sediment, the of short duration centrifugal liquid that makes falls within the pipe bottom; Described A liquid comprises: 6mol/L guanidinium isothiocyanate, 30mmol/L sodium citrate, 0.58% sodium dodecylsulphonate, 1mmol/L disodium ethylene diamine tetraacetate, 2% glycogen; Described B liquid is isopropyl alcohol; Described C liquid is 75% ethanol water.
9. a kit that is used for the fluorescence PCR detecting method of hepatitis B virus gene typing is characterized in that this kit contains the PCR reactant liquor, comprising: PCR reaction buffer, Mg
2+Solution, dNTP, Taq archaeal dna polymerase, and arbitrary group of probe described in the claim 1～5 and pairing a pair of upstream and downstream primer.
10. a kind of kit that is used for the fluorescence PCR detecting method of hepatitis B virus gene typing according to claim 9, it is characterized in that described PCR reaction buffer is 1 * PCR reaction buffer: comprise 10mmol/L Tris-HCl, pH8.9,50mmol/L KCl, 5% glycerine; Described Mg
2+Solution concentration is 2.5mmol/L; Described dNTP concentration is 0.2mmol/L; Described Taq archaeal dna polymerase is 1U; Described concentration and probe concentration is 0.12 μ mol/L; Described upstream and downstream primer concentration respectively is 0.6 μ mol/L; Also contain UNG 0.1U in the described PCR reactant liquor; Described kit also contains DNA extraction reagent, comprises A liquid: 6mol/L guanidinium isothiocyanate, 30mmol/L sodium citrate, 0.58% sodium dodecylsulphonate, 1mmol/L disodium ethylene diamine tetraacetate, 2% glycogen; B liquid: isopropyl alcohol; C liquid: 75% ethanol water.
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| CN100374564C (en) * | 2005-10-27 | 2008-03-12 | 云南大学 | Defining for specificity DNA sequence of hepatitis B viruse and application thereof |
| CN100436598C (en) * | 2006-02-14 | 2008-11-26 | 中国科学院武汉病毒研究所 | Biological articles for detecting hepatitis B virus |
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| JP2002355098A (en) * | 2000-08-14 | 2002-12-10 | Genome Science Laboratories Co Ltd | Method for classifying genotype of hepatitis b virus, and primer and probe for the same |
| US6583279B1 (en) * | 2001-01-26 | 2003-06-24 | Becton, Dickinson And Company | Sequences and methods for detection of hepatitis B virus |
| CN1126819C (en) * | 2001-12-26 | 2003-11-05 | 南开大学 | Homogeneous molecular lampmark fluorescent probe PCR hepatitis B virus detecting method and reagent kit |
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