猪流行性腹泻焦磷酸测序检测方法的建立

Establishment of Pyrosequencing Assay Detection Method of Porcine Epidemic Diarrhea Virus

本研究旨在建立一种基于RT-PCR的焦磷酸测序方法,以便对猪流行性腹泻(PED)进行高效准确地检测。利用PSQ Assay Design SW软件分析了猪流行性腹泻病毒(PEDV)N蛋白基因的保守区域,设计了一对扩增引物及一条测序引物,建立了PEDV N蛋白基因的焦磷酸测序检测方法。实验结果表明,该方法可以直接通过PSQ的序列结果直观地判定PEDV,大大提高了PEDV的检出率和准确性。该方法特异性强,不与其他猪源病毒发生交叉反应,最低核酸检测限为0.05 pg/μL。本研究所建立的焦磷酸测序方法灵敏度高、稳定性好,能从基因序列水平上精确鉴定PEDV,避免了假阳性结果的出现,对于PED的快速确诊具有重要意义。

The pyrosequencing（PSQ）assay based on reverse transcription-polymerase chain reaction（RT-PCR）was developed for rapid detection of porcine epidemic diarrhea virus（PEDV） efficiently and accurately.The pair of amplification primers and a sequencing primer were designed for the conserved region of the N protein gene of PEDV by the analysis of the software PSQ Assay Design SW to establish the pyrosequencing assay for the detection of PEDV.The results showed that this assay could be used for the identification of PEDV by the sequences of the PSQ directly and the rates of detection and the accuracy could be improved greatly.The results of specification assay showed that this assay had good specificity,no cross reaction with other porcine viruses.The minimum detection limit of the nuclei acid was 0.05 pg/μL.The pyrosequencing assay with high sensitivity and stability could accurately identify PEDV at the level of gene to avoid the occurrence of false positive results.It was of great significance for the rapid diagnosis of porcine epidemic diarrhea.