Abstract: In this study，aLNA ? based fluorescent quantitation PCR method was developed for the rapid，accurate and trace detection of porcine epidemic diarrhea virus（PEDV）by targeting the M gene. Special primers and LNA ? TaqMan probe were designed according to the M gene，and the reaction conditions have been optimized. The LNA?based fluorescent quantitation PCR method was compared with the conventional TaqMan probe method. Results showed that the detection limit of the method was 3.2 copies/μL，improved by 10 times compared to conventional TaqMan probe method. The method was specific of PEDV and no cross reaction to TGEV，PoRV，and PDCoV. The method had a good repeatability with variation coefficient of intra? and inter?group less than 1%. The method was used to detect 103 clinical samples，the results showed that 47 samples were positive for PEDV，with a positive rate of 45.63%，and the positive coincidence rate with the conventional TaqMan probe PCR assay was 90.38%. This LNA? based fluorescent quantitation PCR method provides a new technical for accurate detection and epidemiological investigation of PEDV.

Key words: PEDV, Mgene, LNA?TaqMan real?time PCR

摘要： 为建立一种快速、灵敏的猪流行性腹泻病毒（porcine epidemic diarrhea virus, PEDV）早期痕量检测方法，该研究以 PEDV 的 M 基因为靶基因，设计了特异性引物和锁核酸(LockedNucleic Acid，LNA)?TaqMan 探针，经过条件优化，建立了基于 LNA?TaqMan 探针的 PEDV 荧光定量 PCR 方法，并与常规 TaqMan 探针法进行了比对。结果显示，所建立的 PEDV 的 LNA?Taq?Man 探针荧光定量 PCR 方法最低检测限为 3.2 拷贝/微升，较常规 TaqMan 探针法提高了 10 倍；与猪传染性胃肠炎病毒、猪轮状病毒和猪德尔塔冠状病毒等多种病原不存在交叉反应，特异性良好；批内和批间重复性结果的变异系数均小于 1%，重复性较好。应用该方法对 103 份临床样品进行检测，结果显示，共检出 PEDV 阳性样品 47 份，阳性率 45.63%，与常规 TaqMan 探针法检测结果的阳性符合率为 90.38%。本研究建立的基于 LNA?TaqMan 探针的荧光定量 PCR 方法为 PEDV 的精准检测和流行病学调查提供了一种新的技术选择。

关键词: 猪流行性腹泻病毒, M 基因, LNA?TaqMan 探针荧光定量 PCR