鸭圆环病毒实时定量PCR检测方法的建立及流行毒株全基因组遗传变异分析

Establishment of real-time quantitative PCR for detection of duck circovirus and analysis of genome-wide genetic variation of the epidemic strains

为了解近年来鸭圆环病毒(duck circovirus,DuCV)的流行现状,本研究在DuCV的Rep基因保守区内设计一对特异性荧光定量引物,建立SYBR Green I实时荧光定量PCR检测方法。结果显示:该方法最低可检测到拷贝数为3.46×10^(1)copies/μL的目的基因,比常规PCR灵敏;选取Ct值最低的阳性样品进行全基因组扩增及进化分析,成功扩增出1988 bp的病毒全基因,序列分析结果显示该病毒基因型为DuCV-1,亚型为1 b。本研究建立的实时荧光定量PCR检测方法为DuCV的流行病学调查提供了有效的监测手段,同时通过分析不同基因型的遗传进化特点与Cap蛋白氨基酸位点突变特征,为DuCV感染的防控研究提供参考。

In order to understand the epidemic status of duck circovirus(DuCV)in recent years,an efficient and sensitive detection method was established.Herein,primers targeting the conserved regions of DuCV were designed and the detection method of real-time quantitative PCR was established;and,we detected DuCV with this method.We designed a pair of primers in the conserved region of the Rep gene to establish an SYBR Green I real-time quantitative PCR detection method.The minimum detectable copy number of the target gene was 3.46×10^(1)copies/μL,which was more sensitive than the conventional PCR.Positive samples with the lowest Ct value were selected for complete genome amplification and evolutionary analysis.A complete viral gene with a length of 1988 bp was successfully amplified.The analysis of its sequences showed that the genotype of the virus was DuCV-1 and the subtype was 1 b.To sum up,the real-time quantitative PCR detection method established in this study provided an effective detection tool for the clinical diagnosis and epidemiological investigation of DuCV.By analyzing the genetic evolution characteristics of different genotypes and the amino acid site mutation characteristics of the Cap protein,this method provided reference for the further research on DuCV.