鸡传染性喉气管炎病毒SYBR Green Ⅰ实时荧光定量PCR检测方法的建立与应用

Establishment and Application of SYBR GreenⅠReal-time Fluorescent Quantitative PCR Method for Chicken Infectious Laryngotracheitis Virus

鸡传染性喉气管炎病毒(Infectious laryngotracheitis virus,ILTV)以往多感染蛋鸡,近年来在蛋种鸡、商品肉鸡中连续多发,表明该病毒感染谱有扩大风险,因此有必要加强对ILTV的监测。为建立高效、灵敏的ILTV检测方法,本研究以ILTV的TK基因为靶基因设计引物,扩增并构建pMD18-T-TK质粒标准品,建立SYBR GreenⅠ实时荧光定量PCR检测方法和标准曲线,对其特异性、敏感性和重复性进行验证,并对临床疑似病例进行检测。结果发现,该检测方法特异性良好,与其他常见症状相似病原无交叉反应;最低检测浓度为1.55拷贝/μL,灵敏度是普通PCR方法的10倍;重复性好,批内、批间变异系数在0.79%~1.83%之间。表明本研究建立的ILTV实时荧光定量PCR方法特异性强、灵敏度高,可为ILTV的临床诊断和流行病学研究等提供有效的检测方法。

The avian infectious laryngotracheitis virus(ILTV)had been prevalent infected layer in the past,but it has been continuously isolated in breeders and broilers in recent years,indicating the risk of expanding the infection spectrum of the virus..Therefore,it is necessary to strengthen the monitoring of ILTV.To establish an efficient and sensitive ILTV detection method,a pair of specific primers was designed based on the TK gene of ILTV,and then the gene was amplificated and pMD18-T-TK plasmid was constructed.The fluorescence quantitative SYBR GreenⅠPCR standard method was established based on the pMD18-T-TK plasmid as the standard production.The specificity,sensitivity,and repeatability of the fluorescence quantitative PCR method were tested and the diseased tissue samples was detected using the established method.The results showed that the method had good specificity,no cross reactivity with other pathogens exhibiting similar symptoms.The minimum detection concentration was 1.55 copies/μL 10-times higher than conventional PCR.The method has good repeatability with the coefficient of variation ranges from 0.79 to 1.77%for intra-and inter batch.This indicates that the real-time fluorescence quantitative PCR method for ILTV established in this study has strong specificity and high sensitivity,providing an effective detection method for clinical diagnosis and epidemiological research of ILTV.