猪嵴病毒实时荧光定量RT-PCR方法的建立

Establishment of a real-time RT-PCR method based on SYBR GreenⅠfor diagnosis of porcine kobuvirus

目的本研究根据GenBank中登录猪嵴病毒(porcine kobuvirus,PKV)株的3D蛋白序列基因特征,设计特异性引物,建立基于SYBR GreenⅠ检测PKV的实时荧光定量RT-PCR方法。结果该方法检测PKV的3D基因在6.42×102～6.42×108拷贝/μL范围内有很好的线性关系,其扩增相关系数为0.999,扩增效率为100%,扩增产物的融解曲线分析只出现1个单特异峰,融解温度为(84.94±0.24)℃,对传染性胃肠炎病毒、猪轮状病毒、猪圆环病毒、猪繁殖与呼吸综合症病毒、猪伪狂犬病毒、猪瘟病毒均检测不到荧光信号,特异性强。所建立实时荧光定量RT-PCR方法组内变异系数为0.26%～1.14%,组间变异系数0.63%～1.79%,重复性好。结论本方法的建立为PKV的早期诊断及定量分析PKV感染水平和确定感染靶器官提供新的检测方法。

A pair of specific primers targeted to the 3D gene of porcine kobuvirus was designed and a real-time reverse-transcription polymerase chain reaction(RRT-PCR) based on SYBR GreenⅠfluorescent was developed for quantization of the porcine kobuvirus infection.The standard curve generated a wide dynamic range of 6.42×102-6.42×108 DNA copies/μL with a linear correlation(R2) of 0.999 and efficiency of 100% between the Ct value and the logarithm of the plasmid copy number.The melting curve analysis using SYBR GreenⅠ dye showed one specific peaked with a melting temperature(Tm) of(84.94±0.24)℃ with no primer-dimer peak represent.No amplificon was detected from unrelated DNA samples by this method,such as porcine transmissible gastroenteritis virus,porcine rotavirus,porcine circovirus,porcine reproductive and respiratory syndrome,pseudorabies virus,and classical swine fever virus.Excellent reproducibility was obtained for detecting constructed positive plasmid DNA with intra-assay of 0.26%-1.14%% and inter-assay of 0.63%-1.79%.A real-time RT-PCR method established in this study may be used for earlier diagnosis and quantitative analysis of the infection status and the target organs infected with porcine kobuvirus.