[1]张 孟a,李 倩b,周华君b,等.巢氏PCR扩增低拷贝HBV全基因组方法的建立及应用[J].现代检验医学杂志,2018,33(03):44-49.[doi:10.3969/j.issn.1671-7414.2018.03.013]
　ZHANG Menga,LI Qianb,ZHOU Hua-junb,et al.Establishment and Application of Nested PCR Amplification Method for the Whole Genome of HBV with the Low Copy[J].Journal of Modern Laboratory Medicine,2018,33(03):44-49.[doi:10.3969/j.issn.1671-7414.2018.03.013]
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巢氏PCR扩增低拷贝HBV全基因组方法的建立及应用()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第33卷
期数:
2018年03期
页码:
44-49
栏目:
论著
出版日期:
2018-07-17

文章信息/Info

Title:
Establishment and Application of Nested PCR Amplification Method for the Whole Genome of HBV with the Low Copy
文章编号:
1671-7414(2018)03-044-06
作者:
张 孟a; 李 倩b; 周华君b; 孙长贵b; 成 军b; 戴玉柱b
中国人民解放军第一一七医院a.财经中心; b.检验科,杭州 310013
Author(s):
ZHANG Menga; LI Qianb; ZHOU Hua-junb; SUN Chang-guib; CHENG Junb; DAI Yu-zhub
a.Financial Services Center; b.Department of ClinicalLaboratory, the 117th Hospital of PLA,Hangzhou 310013,China
关键词:
乙型肝炎病毒;  低拷贝;  全基因;  巢式聚合酶链式反应
分类号:
R512.62; Q503
DOI:
10.3969/j.issn.1671-7414.2018.03.013
文献标志码:
A
摘要:
目的 建立一种用于低拷贝乙型肝炎病毒(HBV)不同基因型全基因组扩增的巢式聚合酶链式反应(nested PCR)方法及应用评价。方法 通过设计和改良一组对HBV各基因型(A,B,C,D,E,F,G)DNA均具有较高扩增效率的通用巢式简并引物,采用两步法分六段(Ⅰa,Ⅰb,Ⅱa,Ⅱb,Ⅲ,Ⅳ)扩增低拷贝HBV基因组,比较不同反应条件对于PCR扩增产物的影响(退火温度,引物浓度),建立一种适合于低拷贝HBV全基因组扩增的方法; 同时采用该方法对57份低浓度HBsAg乙肝感染者(HBsAg<10IU/ml)的全基因组进行扩增、测序、拼接并进行评价。结果 该方法的灵敏度可达25IU/ml,重复性好,对57份低浓度HBsAg乙肝感染者进行扩增,其中全基因扩增49例(86.0%),各片段(Ⅰa,Ⅰb,Ⅱa,Ⅱb,Ⅲ,Ⅳ)扩增分别为:53例(93.0%),52例(91.2%),54例(94.7%),54例(94.7%),55例(96.5%)和49例(86.0%),说明该方法适用于慢性低浓度HBsAg乙肝感染者HBV DNA全基因扩增。结论 该文为慢性低浓度HBsAg乙肝感染者的全基因系列分析提供了高灵敏度、高特异度的方法,可在低浓度HBsAg感染者流行病学调查中发挥重要作用,同时为巢氏PCR扩增反应体系的优化提供了参考。
Abstract:
Abstract:Objective To establish and evaluate a nested polymerase chain reaction(nested PCR)method for the whole genome amplification of hepatitis B virus(HBV)with low copy in the different genotypes.Methods By designing and improving a set of universal nested degenerate primers with high amplification efficiency for all HBV genotypes(A,B,C,D,E,F,G),and the low copy HBV genome was divided into six segments(Ⅰa,Ⅰb,Ⅱa,Ⅱb,Ⅲ,Ⅳ)which were amplified by the two step method.The effects ofdifferent reaction conditions of the PCR amplification products(annealing temperature,primer concentration)were compared.A method suitable for the whole genome amplification of low copy HBV was established,and amplified,sequenced,spliced and evaluated of the whole genome of 57 hepatitis B infected persons with low-level HBsAg(HBsAg<10 IU/ml)was completed by this method.Results The sensitivity of the method was 25IU/ml and reproducible,and 57 cases of hepatitis B infection with low-level HBsAg were amplified,in whichtotal gene amplification was 49(86%)cases.Each segment(Ⅰa,Ⅰb,Ⅱa,Ⅱb,Ⅲ,Ⅳ)was 53 cases(93%),52 cases(91.2%),54 cases(94.7%),54 cases(94.7%),55 cases(96.5%)and 49 cases(86.0%),respectively.This method was suitable for the amplification of HBV DNA of chronic hepatitis B infection with low-level HBsAg.Conclusion This study provides a highly sensitive and highly specific method for the analysis of the whole gene sequence of chronic hepatitis B infection with low-level HBsAg.It can play an important role in the epidemiological investigation of HBV infection with low-level HBsAg,and provides a reference for the optimization of the nesting PCR amplification reaction system.

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备注/Memo

备注/Memo:
基金项目:浙江省自然科学基金项目(LY15H200001)。 作者简介:张 孟(1965-),女,本科,副主任技师,主要从事临床免疫学检验诊断研究,E-mail:zhangm117@sina.cn。 通讯作者:戴玉柱(1985-),男,硕士,副主任技师,主要从事临床免疫学检验诊断研究,E-mail:dyz5895@qq.com。
更新日期/Last Update: 2018-04-16