针对猪瘟病毒E2蛋白的实时荧光定量PCR方法的建立及应用

DEVELOPMENT AND APPLICATION OF REAL-TIME FLURESCENCE QUANTITATIVE PCR FOR DETECTION OF CLASSICAL SWINE FEVER VIRUS E2 PROTEIN

根据猪瘟病毒(Classical swine fever virus,CSFV)E2基因的保守序列,设计合成一套特异性引物和TaqMan探针,通过对反应体系和反应条件进行优化后,检测其特异性和灵敏性。结果显示,本研究建立的实时荧光定量PCR在101～108范围内线性相关系数为0.997,能够检测到相当于10 copies/μL的病毒核酸,比常规PCR检测方法敏感性强;采用建立的实时荧光定量PCR方法检验猪繁殖与呼吸综合征病毒、猪伪狂犬病毒和猪圆环病毒2型呈现阴性结果,特异性强;变异系数为0.09%～0.7%,小于1%,具有良好的重复性。本研究建立的猪瘟病毒实时荧光定量PCR方法为临床上猪瘟病毒的诊断和定量研究提供了重要的检测工具。

A real-time fluorescence quantitative PCR(qPCR)assay was innovated and developed for Classical swine fever virus(CSFV)for convenient,effective and rapid detection of clinical cases.A primer pairs and a probe based on the CSFV E2 gene were designed and its sensitivity and specificity were detected.The assay showed good specificity and linear relationship at a range of 101~108 with the detection limit at 10 copies/μL of viral genome.The real-time qPCR developed here was highly specific to CSFV as it did not react with Porcine reproductive and respiratory syndrome virus(PRRSV),Porcine circovirus type 2(PCV2)and Pseudorabies virus(PRV).The distributions with a coefficient of variation to be 0.09%~0.7%were less than 1%,indicating that the assay had good reproducibility.In this study,the real-time RT-qPCR assay provided a very important tool for CSFV investigation and quantitative research.