荧光RT-LAMP法检测猪流行性腹泻病毒方法的建立

Establishment of Fluorescent Reverse Transcription Loop Mediated Isothermal Amplification for Detecting Porcine Epidemic Diarrhea Virus

猪流行性腹泻病是一种急性传染病,在我国各省均有发生,幼龄猪的发病率和死亡率较高。本研究拟建立一种能够快速检测猪流行性腹泻病毒的荧光RT-LAMP方法,为早期诊断和治疗提供技术支持。根据GenBank公布的猪流行性腹泻病毒核蛋白基因设计一套特异性LAMP引物,使用荧光PCR仪对待检样品进行检测,根据扩增结果判断所检测样品是否含有猪流行性腹泻病毒。除已确诊为猪流行性腹泻病的病猪样品外,其余参考样品在本方法中均未出现特异性扩增曲线,检测下限为102拷贝的质粒模板,病猪小肠样品的阳性检出率为100%。本研究建立的方法特异性强、灵敏度高、检测速度快,可广泛应用于猪流行性腹泻的快速筛查及实验室检测。

Porcine epidemic diarrhea is an acute infectious disease which can cause high mortality rate in neonatal pigs that occurs sporadically in all provinces of China.The aim would be establish a rapid detection method for PEDV with fluorescent RT-LAMP technology.A group of specific primers were designed based on the nucleocapsid protein gene of the nucleic acid sequences of PEDV provided by GenBank.The quarantine samples with the primers and reaction systems would be tested by the fluorescent PCR device which could display the testing results,and the testers could making a judgment on PEDV of these samples according to the amplification curves of the testing results.None amplification curve appeared in the testing results except all PEDV samples and positive controls.It can also be seen that the inferior limit of the method could be 100 copies of plasmid RNA template.The established method is appropriate for the screening of PEDV in farm and the detection in laboratory because of its stability,sensitivity and veracity.