巢氏PCR扩增低拷贝HBV全基因组方法的建立及应用  

Establishment and Application of Nested PCR Amplification Method for the Whole Genome of HBV with the Low Copy

作　　者：张孟[1] 李倩 周华君 孙长贵[2] 成军[2] 戴玉柱[2] ZHANG Meng;LI Qian;ZHOU Hua-jun;SUN Chang-gui;CHENG Jun;DAI Yu-zhu(Financial Services Center;Department of Clinical Laboratory, the 117th Hospital of PLA , Hangzhou 310013, China)

机构地区：[1]中国人民解放军第一一七医院财经中心,杭州310013 [2]中国人民解放军第一一七医院检验科,杭州310013

出　　处：《现代检验医学杂志》2018年第3期44-49,共6页Journal of Modern Laboratory Medicine

基　　金：浙江省自然科学基金项目(LY15H200001)

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。Objective To establish and evaluate a nested polymerase chain reaction （nested PCR） method for the whole ge- nome amplification of hepatitis B virus （HBV） with low copy in the different genotypes. Methods By designing and impro ring 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 of different 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 estab- lished,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 re- producible,and 57 cases of hepatitis B infection with low-level HBsAg were amplified,in which total 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 refer- ence for the optimization of the nesting PCR amplification reaction system.

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[医药卫生—内科学] Q503[医药卫生—临床医学]