犬瘟热病毒一步法荧光定量PCR检测方法的建立

Development of real-time PCR assay for detection and quantification of the canine distemper virus

为建立犬瘟热病毒(CDV)TaqMan实时荧光定量PCR检测方法,根据CDV核衣壳蛋白(NP)基因序列,设计合成2对通用引物和1条通用探针,通过体外转录构建绝对定量标准品RNA。对荧光定量PCR方法的各项条件进行优化,建立CDV荧光定量PCR检测方法,该方法检测灵敏度可达4.02拷贝/μL,比普通RT-PCR方法的灵敏度高出100倍,具有良好的重复性。对犬细小病毒(CPV)、犬腺病毒(CAV-1)、犬冠状病毒(CCV)、犬副流感病毒(CPIV)核酸检测为阴性,具有很好的特异性。研究结果表明,建立的CDV荧光定量PCR方法具有敏感性高、特异性强、重复性好的特点,为犬瘟热的早期诊断提供了重要的技术支撑。

To develop the TaqMan real-time quantitative PCR assay for detection of canine distemper virus（CDV）,two pairs of universal primes and a universal TaqMan probe were designed on conservative sequence of canine distemper virus nucleocapsid protein gene.A standard RNA used in absolute quantification assay was constructed through vitro transcript.A TaqMan real-time PCR was established by optimizing the variety of conditions and the minimum detection limit was 4.02copies/μL,100 times higher than that of the conventional RT-PCR.This method has good repetitiveness.Canine parvovirus（CPV）,canine adenovirus（CAV-1）,canine coronavirus（CCV）and canine parainfluenza virus（CPIV）were not amplified by this method,demonstrating its excellent specificity.The result indicated the real-time PCR assay for detecting and quantifying the CDV has high detection specificity and sensitivity,and good reproducibility as well,which provides technical support for the diagnosis of dog infected with CDV.