CN102140537A - Polymerase chain reaction detection method of hepatitis B virus genotyping - Google Patents
Polymerase chain reaction detection method of hepatitis B virus genotyping Download PDFInfo
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Abstract
The invention provides a polymerase chain reaction (PCR) detection method of hepatitis B virus (HBV) genotyping. The method comprises the following steps: a primer is designed according to the entire genome sequence of the HBV genotype; in the existence of the primer, DNAs are extracted from a serum sample to perform PCR amplification in a fluorescence PCR meter, the PCR product is analyzed according to the melting curve, the genotype is judged according to the Tm value of the PCR product and the HBV genotyping can be realized. The PCR melting curve method used in the invention is convenient to operate, the detection result of the method has higher coincidence rate with the detection result obtained by adopting a commercial genotyping kit; and the method can be used in the clinical detection of the HBV genotype. On the basis of the PCR technology, the PCR melting curve method which is to judge the HBV genotype according to the Tm value, is provided in the PCR detection method, thus the HBV genotype can be identified conveniently, rapidly and accurately.
Description
Technical field
The present invention relates to a kind of PCR melting curve detection method of HBV gene type.
Background technology
Hepatitis B virus (Hepatitis B virus, HBV) mainly cause liver damage by host immune mechanism, body immune system is discerned virus antigen and is attacked infected liver cell and causes inflammation, and this process is comprised host and viral a plurality of factor affecting.The virogene heterogeneity affects antigenic expression, is also playing the part of important role in this process.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 all may have been caused different genotype to have spectrum of disease after the different infection.
Usually HBV is divided into eight kinds of genotype of A～H, the somatotype foundation is complete genome sequence heterogeneity 〉=8%, or S gene order heterogeneity 〉=4%.The HBV genotype is certain geographical areal distribution.Northern China is based on the C type, and south is based on Type B, and the D type is more common in the minority area, as Tibet, Xinjiang, and the pathogenic difference of different genotype.Genotype that there are some researches prove HBV not only influences the hepatitis B patient disease progression, and with antiviral curative effect and clinical prognosis certain correlationship is arranged.Therefore, the HBV gene type is studied further clear and definite pathogenesis of hepatitis B, searching treatment countermeasure, realization epidemiology is controlled and resist viral therapeutic response and state of an illness prognosis evaluation is all significant.The method that some HBV gene types have been arranged now, wherein the information that provides of sequencing is reliable comprehensively, but this method is loaded down with trivial details, time-consuming, experiment condition and requirement height, is not suitable for extensively carrying out.And the result of additive method is all reliable not as sequencing, and at present still not by stdn and commercialization, as the PCR-RFLP complex operation, and the restriction enzyme site influence that is subject to genovariation influences the result and judges; PCR microplate making nucleic acid molecular hybridization-ELISA method operative technique is easy, is applicable to common laboratory mensuration, carries out gene type but can't infect the HBV of serum HBsAg feminine gender; Type specificity primer PCR operation is simple relatively, the result is accurate, but still need carry out two-wheeled PCR, and there is the possibility of product pollution in the electrophoretic step of product.
Summary of the invention
The object of the present invention is to provide a kind of PCR detection method of HBV gene type, this method can be identified the HBV genotype easy, quickly and accurately.
Technical scheme of the present invention is as follows:
Whole genome sequence design primer according to genotype of hepatitis B virus; In the presence of above-mentioned primer, in the fluorescent PCR instrument, the DNA that extracts from serum sample is carried out pcr amplification, utilize melting curve to analyze the PCR product, judge genotype according to the Tm value of PCR product, realize the gene type of hepatitis B virus.
Wherein said genotype of hepatitis B virus is genotype of hepatitis B virus B, and primer sequence is as follows:
Upstream primer BF:AGTTAATCATTACTTCCAGACGC
Downstream primer BR:CTGTAGATCTTGTTCCCAAGAAT
Described genotype of hepatitis B virus is genotype of hepatitis B virus C, and primer sequence is as follows:
Upstream primer CF:TGTTCCGACTACTGCCTCAC
Downstream primer CR:GGACAAATTGGAGGACAAGAG.
Above-mentioned primer design step is: according to document (Wu Guanghua, Ding Huiguo, Ceng Changqing. common core is calculated the foundation of database hepatitis B virus whole genome sequence overview and Chinese HBV canonical sequence. the natural science progress, 2008,18:121-129.) the Chinese HBV B, the whole genome sequence of C type of report, according to the primer design principle, utilize Primer Premier 5.0 softwares to design B, C type specificity primer voluntarily, to the Pubmed database, carry out blast afterwards, confirm that the purpose amplified fragments of primer is the gene order of HBV; Tm value according to amplified fragments is judged genotype.
The condition of described pcr amplification is: adopt the reaction system of 50 μ L, and 2 times of spissated SYBR Premix Ex Taq 25 μ L, primer is to each 1 μ L of 10 μ M/L, 50 times of spissated ROX Reference Dye 1 μ L, HBV dna profiling 4 μ L, dH
2O 16 μ L; React and detect the PCR reaction conditions in ABI7000 fluorescent PCR instrument: 95 ℃ of 30s, by 95 ℃ of 5s, 60 ℃ of 31s carry out 40 circulations then, and fluoroscopic examination is at 60 ℃.Fluorescence channel detects selects SYBR.
Mainly based on the characteristics of Type B and C type, the present invention has only designed the primer of B, C type at China HBV genotype, has set up that the PCR melting curve detects HBV B, C and B/C mixes genotypic method.Present method adopts the melting curve of SYBR Green I to detect the HBV genotype, SYBR Green I is a kind of asymmetric nitrile fluorescein, can be embedded in the ditch in the dna double spirane structure non-specificly, generally do not combine with single stranded DNA, the fluorescence intensity of bonding state increases thousands of times than the fluorescence of unbound state.The HBV double chain DNA molecule is identified because of combining with SYBR Green I, when two dna molecular renaturation in conjunction with the time fluorescence the strongest, reduce and occur one with temperature rising fluorescence intensity than the constant gradient, fluorescence intensity sharply descends when temperature reaches the Tm value.Can obtain melting curve figure through software analysis, the temperature at its crest place is represented the Tm value of double chain DNA molecule.The size of Tm value depends on G/C content in the length of dna molecular and the sequence thereof.The dna molecular fragment length difference of two pairs of type specificity primer amplifications of the present invention design, Type B 130bp wherein, C type 273bp has different Tm values, so can judge genotype according to the Tm value of PCR product.PCR melting curve method is added with two pairs of primers simultaneously in a PCR reaction system, method is simple, consuming time less, can detect 96 samples simultaneously at every turn, 2 hours consuming time, because whole amplification and testing process are the stopped pipe operation, the absorption amplified production of need not uncapping carried out electrophoresis, avoid the pollution of pcr amplification product, reduced false positive results.The primer of a certain type only with mutually isostructural template in conjunction with pcr amplification reaction just takes place, thereby guaranteed the specificity that detects, simultaneously, amplified fragments of the present invention is lacked, and adopts higher annealing temperature, also can guarantee the specificity that increases.Reaction system can be differentiated the HBV genotype easy by the Tm value, apace after the quantitative real time PCR Instrument amplification.
Remarkable advantage of the present invention:
The PCR melting curve method that the present invention sets up is easy and simple to handle, and comparing with the detected result of commercial gene type test kit has higher coincidence rate, can be used for the genotypic detection of HBV clinically.
The present invention is utilizing on the basis of round pcr, has set up a kind of according to the genotypic PCR melting curve of Tm value judgement HBV method, the easy HBV genotype of identifying quickly and accurately of energy.
Description of drawings
Fig. 1 is the typical plot that PCR melting curve method detects HBV genotype B, C, wherein X-coordinate be temperature (℃), ordinate zou is the second derivative of fluorescent value.
Fig. 2 is the typical plot that PCR melting curve method detects HBV genotype B/C mixed type, wherein X-coordinate be temperature (℃), ordinate zou is the second derivative of fluorescent value.
Embodiment
Be specific embodiments of the invention below, further describe the present invention, but the present invention be not limited only to this.
Embodiment 1
One, object
The First Affiliated Hospital, Fujian Medical University in 2009 is in hospital and reaches 186 routine HBV infected patients of outpatient service, male 124 examples, and 35.21 years old mean age, women 69 examples, 35.71 years old mean age, its serum fluorescence quantitative PCR detection is the HBV DNA positive (10
3～10
8Copy/ml), be stored in-80 ℃.All patients have all got rid of the disease that first, the third and hepatitis e virus infection and other cause hepatic disease, and diagnosis meets the Case definition that Chinese Medical Association's transmissible disease in 2000 and parasitology branch (Xi'an) revise.
Two, method
1. main laboratory apparatus and reagent: ABI7000 quantitative real time PCR Instrument (U.S.), virus genom DNA extracts test kit (available from Shanghai Jierui Biology Engineering Co., Ltd), SYBR Premix Ex Taq (available from the precious biotechnology in Dalian company limited), hepatitis B virus gene typing fluorescence PCR detection reagent kit (available from Fuxing Medical Science-Technology Development Co., Ltd., Shanghai), other chemical reagent is homemade chemical pure.
2. design of primers: according to document (Wu Guanghua, Ding Huiguo, Ceng Changqing. common core is calculated the foundation of database hepatitis B virus whole genome sequence overview and Chinese HBV canonical sequence. the natural science progress, 2008,18:121-129.) B that provides, the whole genome sequence of C type, design four primers altogether, company limited is synthetic by the precious biotechnology in Dalian, and particular case sees Table 1.
Table 1 PCR melting curve of the present invention method the primer
3. serum HBV DNA extraction: get the object serum specimen, the virus genom DNA of producing according to Shanghai Jierui Biology Engineering Co., Ltd extracts the step of test kit and extracts hepatitis B virus DNA in the serum, as the template of PCR reaction, be kept at-20 ℃ standby.
4. HBV DNA increases: promptly adopt the reaction system of 50 μ L, and 2 times of spissated SYBR Premix Ex Taq 25 μ L, primer is to each 1 μ L of 10 μ M/L, 50 times of spissated ROX Reference Dye 1 μ L, HBV dna profiling 4 μ L, dH
2O 16 μ L; React and detect the PCR reaction conditions in ABI7000 fluorescent PCR instrument: 95 ℃ of 30s, by 95 ℃ of 5s, 60 ℃ of 31s carry out 40 circulations then, and fluoroscopic examination is at 60 ℃.Fluorescence channel detects selects SYBR.
5. positive and negative contrast: with H
2O is that template is a negative control as various blank.Hepatitis B virogene B, C, the B/C mixed type sample of getting sequence verification be as positive reference substance, and detects serum specimen and detect simultaneously.
6. genotypic judgement: with ABI7000 software the result is analyzed, can obtain melting curve figure through software analysis, the temperature at its crest place is represented the Tm value (melting temperature (Tm)) of double chain DNA molecule.Tm value size depends on G/C content in the length of dna molecular and the sequence thereof, and the different corresponding Tm values of dna molecular sequence are difference also.The Type B Auele Specific Primer of the present invention's design is 130bp to amplified fragments, and C type specificity primer is 273bp to amplified fragments, and the B/C mixed type has two kinds of amplified productions to occur, and two Tm values promptly can appear in 130bp and 273bp; The dna molecular sequence difference of amplified production, corresponding Tm value are also different, and as shown in Figure 1, 2, wherein Type B is 79.9 ± 0.5 ℃; 84.6 ± 0.4 ℃ on C type, and two Tm value places that occur among the melting curve figure of B/C mixed type all have peak value to occur.
7. the comparison of PCR melting curve method of the present invention and commercialization fluorescence PCR detection reagent kit (being the hepatitis B virus gene typing fluorescence PCR detection reagent kit) (hereinafter to be referred as the A test kit) measurement result: PCR melting curve method detects the HBV genotype of 186 routine samples, picked at random 40 routine samples therefrom, Type B 14 examples wherein, C type 21 examples, B/C mixed type 5 examples, carry out the HBV gene type with the A test kit, two kinds of resulting detected results of method are analyzed comparison, relatively both coincidence rates.
8. statistical analysis: adopt SPSS 11.5 statistical softwares to carry out statistical study.3 kinds of genotype (Type B, C type, B/C mixed type) coincidence rate that 2 kinds of HBV genotype detection methods draw relatively adopts the Kappa consistency check.P＜0.05 has statistical significance for difference.
Three, result
1, HBV DNA positive patient PCR melting curve method detects the genotype result
The present invention judges genotype, 79.9 ± 0.5 ℃ of Type Bs according to the Tm value; 84.6 ± 0.4 ℃ on C type; Use PCR melting curve method and detect genotype (Fig. 1), among the 186 routine patients, Type B accounts for 71.51%(133/186); The C type accounts for 19.89%(37/186), the B/C mixed type accounts for 8.60%(16/186).
2, the comparison of PCR melting curve method of the present invention and A test kit detected result
14 parts are detected with PCR melting curve method of the present invention is in the sample of Type B, and the A test kit detects 14 parts of Type Bs, and promptly the detected result of two methods is in full accord; It all is the C type that 21 parts of C type samples detect with the A test kit; In the sample of 16 parts of B/C mixed types, the detected result of A test kit is 1 part of Type B, 2 parts of C types, 13 parts of B/C mixed types.Two kinds of methods compare, and Type B coincidence rate as a result is 100%, and C type coincidence rate as a result is 100%, and the B/C mixed type is coincidence rate 81.25% as a result, and overall coincidence rate is 94.12%(48/51, Kappa=0.909, P＜0.05), see Table 2.
Table 2 PCR melting curve method and A test kit detect HBV genotype result's comparison
＜110〉The First Affiliated Hospital, Fujian Medical University
＜120〉a kind of PCR detection method of HBV gene type
<160>?4
<210>?1
<211>?23
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉upstream primer BF
<400>?1
agttaatcat?tacttccaga?cgc?23
<210>?2
<211>?23
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉downstream primer BR
<400>?2
ctgtagatct?tgttcccaag?aat?23
<210>?3
<211>?20
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉upstream primer CF
<400>?3
tgttccgact?actgcctcac?20
<210>?4
<211>?21
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉downstream primer CR
<400>?4
ggacaaattg?gaggacaaga?g?21
Claims (4)
1. the PCR detection method of a HBV gene type, it is characterized in that: the step of described method is: according to the whole genome sequence design primer of genotype of hepatitis B virus; In the presence of above-mentioned primer, in the fluorescent PCR instrument, the DNA that extracts from serum sample is carried out pcr amplification, utilize melting curve to analyze the PCR product, judge genotype according to the Tm value of PCR product, realize the gene type of hepatitis B virus.
2. the PCR detection method of HBV gene type according to claim 1 is characterized in that: described genotype of hepatitis B virus is genotype of hepatitis B virus B, and primer sequence is as follows:
Upstream primer BF:AGTTAATCATTACTTCCAGACGC
Downstream primer BR:CTGTAGATCTTGTTCCCAAGAAT.
3. the PCR detection method of HBV gene type according to claim 1 is characterized in that: described genotype of hepatitis B virus is genotype of hepatitis B virus C, and primer sequence is as follows:
Upstream primer CF:TGTTCCGACTACTGCCTCAC
Downstream primer CR:GGACAAATTGGAGGACAAGAG.
4. the PCR detection method of HBV gene type according to claim 1, it is characterized in that: the condition of described pcr amplification is: the reaction system that adopts 50 μ L, 2 times of spissated SYBR Premix Ex Taq 25 μ L, primer is to each 1 μ L of 10 μ M/L, 50 times of spissated ROX Reference Dye 1 μ L, HBV dna profiling 4 μ L, dH
2O 16 μ L; React and detect the PCR reaction conditions in ABI7000 fluorescent PCR instrument: 95 ℃ of 30s, by 95 ℃ of 5s, 60 ℃ of 31s carry out 40 circulations then, and fluoroscopic examination is at 60 ℃.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586473A (en) * | 2012-01-12 | 2012-07-18 | 泰普生物科学（中国）有限公司 | Hepatitis B virus genotyping PCR (polymerase chain reaction) test kit |
| CN103667439A (en) * | 2013-08-02 | 2014-03-26 | 福建医科大学附属第一医院 | Real-time quantitative polymerase chain reaction (PCR) method using double probe and capable of distinguishing B-type and non-B-type hepatitis B virus (HBV) |
| CN104789700A (en) * | 2015-04-03 | 2015-07-22 | 中国农业科学院上海兽医研究所 | DHAV (duck hepatitis A virus) typing detection method based on fluorescent quantitative PCR (polymerase chain reaction) melting curve method |
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2011
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Non-Patent Citations (2)
| Title |
|---|
| 《Journal of Molecular Diagnostics》 20100531 Fatima Marin et al. Simultaneous Genotyping of GSTT1 and GSTM1 Null Polymorphisms by Melting Curve Analysis in Presence of SYBR Green I 300-304 1-4 第12卷, 第3期 * |
| 《Journal of Virological Methods》 20040607 Sunchai Payungporn et al. Simultaneous quantitation and genotyping of hepatitis B virus by real-time PCR and melting curve analysis 131-140 1-4 第120卷, * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586473A (en) * | 2012-01-12 | 2012-07-18 | 泰普生物科学（中国）有限公司 | Hepatitis B virus genotyping PCR (polymerase chain reaction) test kit |
| CN102586473B (en) * | 2012-01-12 | 2013-09-18 | 泰普生物科学（中国）有限公司 | Hepatitis B virus genotyping PCR (polymerase chain reaction) test kit |
| CN103667439A (en) * | 2013-08-02 | 2014-03-26 | 福建医科大学附属第一医院 | Real-time quantitative polymerase chain reaction (PCR) method using double probe and capable of distinguishing B-type and non-B-type hepatitis B virus (HBV) |
| CN103667439B (en) * | 2013-08-02 | 2015-05-13 | 福建医科大学附属第一医院 | Real-time quantitative polymerase chain reaction (PCR) method using double probe and capable of distinguishing B-type and non-B-type hepatitis B virus (HBV) |
| CN104789700A (en) * | 2015-04-03 | 2015-07-22 | 中国农业科学院上海兽医研究所 | DHAV (duck hepatitis A virus) typing detection method based on fluorescent quantitative PCR (polymerase chain reaction) melting curve method |
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