血清1型鸭甲型肝炎病毒实时定量PCR检测方法的建立与初步应用

Establishment and application of real-time quantitative PCR to detect Duck hepatitis A virus serotype 1

关键词:  血清1型鸭甲型肝炎病毒  实时定量PCR  TaqMan探针  

Key words:  Duck hepatitis A virus serotype 1  Real-time quantitative PCR  TaqMan probe  

摘要:

为建立了一种可检测血清1型鸭甲型肝炎病毒（DHAV-1）的实时定量PCR方法，根据GenBank中DHAV-1 5，非编码区的保守区，设计合成1对引物和1条TaqMan探针，以构建的重组质粒作为标准品，绘制标准曲线，并对所建立方法进行了特异性、敏感性和可重复性试验以及临床病料检测初步应用。结果，该方法与血清3型鸭甲型肝炎病毒、鸭瘟病毒、新城疫病毒、禽流感病毒、呼肠孤病毒、传染性支气管炎病毒等无交叉反应性；最低可以检测到10copies/μL；组内和组间变异系数均小于3%；临床病料的检测结果与测序检测结果一致。结果表明，所建立的实时定量检测方法具有特异、敏感、稳定等优点，可用于DHAV-1的快速检测与定量分析。

Abstract:

In order to develop a real-time quantitative PCR to detect duck hepatitis A virus serotype 1(DHAV-1)，a pair of primers and one TaqMan probe were designed and synthesized, according to the 5' untranslated sequences of DHAV-1published on GenBank, The recombinant plasmid was built as a standard control for the method, the specificity, sensitivity, and repeatability of the method were determined, and also preliminarily applicated in the detection of clinical samples. The results showed that the detection assay was specific for DHAV-1, and there was no cross reaction between duck hepatitis A virus serotype 3(DHAV-3) and the other viruses including duck plague virus, Newcastle disease virus, avian influenza virus, duck reovirus, avian infectious bronchitis virus, etc. The method also showed high sensitivity, as low as 10 copies virus could be detected for each reaction, and good reproducibility, there was a coefficient of variations less than 3% for both inter-assay and intra-assay. The detection results of clinical samples were consistent with the sequencing. These results indicated that the developed TaqMan fluorescence quantitative PCR assay had the advantages of good specificity, sensitivity and repeatability; it was useful for the rapid diagnosis and quantification analysis of DHAV-1.