检测猪繁殖与呼吸综合征病毒SYBR GreenⅠ实时荧光定量PCR方法的建立及初步应用

Establishment and preliminary application of a SYBR GreenⅠ real-time PCR method for detection of PRRSV

利用反转录PCR(RT-PCR)技术扩增出猪繁殖与呼吸综合征病毒(PRRSV)GP5基因部分片段,并克隆到pGEM-TEasy载体上,得到重组质粒作为荧光定量PCR检测的标准模板,以10倍梯度稀释模板,进行SYBR GreenⅠ荧光定量PCR扩增并制作标准曲线,建立PRRSV的荧光定量PCR检测方法。该方法检测灵敏度可达10拷贝/μL,与猪圆环病毒、猪乙型脑炎病毒、猪伪狂犬病毒、猪瘟病毒、猪流感病毒和猪细小病毒不发生交叉反应,具有良好的特异性和重复性;对16份临床疑似病料进行了检测,发现15份均为荧光定量PCR阳性,而常规RT-PCR只能检测出12份阳性。结果表明,建立的实时荧光定量PCR具有特异、敏感、快速、定量、重复性好等优点,可用于临床PRRSV感染的早期诊断以及分子流行病学调查。

Partial region of the PRRSV GP5 gene was amplified by RT-PCR and cloned into pGEM-T Easy vector.Serial dilutions of the recombinant plasmid were used as standard templates for RT-PCR to quantify the virus genomic copy number.A SYBR GreenⅠreal-time PCR was developed to detect PRRSV.Sensitivity analysis showed that the developed SYBR Green I real-time PCR could detect 10 copy/μL.The specificity assay exhibited that the other porcine pathogens,such as PPV,PCV,JEV,PRV,HCV and SIV could not be detected by this method.Then the established method was used to detect the clinical samples.The results showed that 15 positive samples out of 16 suspicious positive samples could be observed by real-time PCR and 12 positive samples could be detected by normal RT-PCR.All the results suggested that the real-time PCR we established in current study showed the characteristics of sensitivity and specificity,and could be used in clinical diagnosis and epidemiological investigation.