猪流行性腹泻病毒RT-LAMP快速检测方法的建立及应用

Establishment and application of RT-LAMP rapid detection assay for porcine epidemic diarrhea virus

为建立一种适用于现场快速检测猪流行性腹泻病毒(PEDV)的环介导等温扩增(RT-LAMP)检测方法,本研究以PEDV N基因的保守序列为靶基因设计引物,优化并建立了基于RT-LAMP的PEDV快速检测方法。结果显示,RT-LAMP方法的最佳反应条件为:反应温度为61℃,反应时间为50 min,Mg2+和dNTPs浓度分别为8.0 mmol/L和4.0 mmol/L,外引物和内引物最佳浓度比为1.4。特异性试验结果显示,该方法只能检测出PEDV,对其他病原检测均为阴性,具有良好的特异性;敏感性试验结果显示,该方法能够检测出的最低RNA浓度为6.52×10^(-5)mg/L,而常规RT-PCR能检测到最低浓度为6.52×10-3mg/L,表明RT-LAMP敏感性是RT-PCR的100倍;重复性试验结果显示,批内和批间重复性试验结果无明显差异,重复性较好。利用RT-LAMP和RT-PCR方法分别对3份已知阳性样品,5份已知阴性样品以及52份未知临床样品进行检测。阳性样品经RT-LAMP和RT-PCR方法均检测到PEDV,阴性样品均未检测到PEDV。52份临床样品中,RT-LAMP对PEDV检测阳性率为23.08%(12/52);RT-PCR检测阳性率为17.31%(9/52),二者的符合率为94.23%。以上结果表明,建立的RT-LAMP方法具有成本低、检测快、灵敏度高、特异性强等优点。且结果易于判定、便于现场检测,在病原检测方面具有良好的应用前景,为该病的流行病学调查及综合防制提供重要的实验依据。

Porcine epidemic diarrhea(PED) can cause severe dehydration in pigs, and the fatality rate of suckling piglets can reach 100%. In order to establish a loop-mediated isothermal amplification(RT-LAMP) detection method suitable for rapid detection of porcine epidemic diarrhea virus(PEDV) in the field, a rapid PEDV detection method based on RT-LAMP was optimized and established by using the conserved sequence of PEDV N gene as the target gene to design primers in this study. The results showed that the optimal reaction conditions of RT-LAMP were as follows: the reaction temperature was 61℃, the reaction time was 50 min, the concentration of Mg2 +and dNTPs were 8.0 mmol/L and 4.0 mmol/L, respectively, and the optimal concentration ratio of external primer and internal primer was 1:4. The specificity test results showed that the established method could only detect PEDV, but could not detect other pathogens, which had good specificity. The sensitivity test results showed that the lowest RNA concentration detected was 6.52×10^(-5) mg/L, however, the lowest RNA concentration detected by reverse transcription PCR(RTPCR) was 6.52×10-3 mg/L, indicating that the sensitivity of RT-LAMP was 100 times more sensitive than RT-PCR. The results of repeatability test showed that this method showed no significant difference in both in-batch and inter-batch repeatability, which showed good repeatability. Both RT-LAMP and RT-PCR were used to detect 3 positive samples, 5 negative samples and 52 clinical samples, respectively. PEDV could be detected in two positive samples by RT-LAMP and RT-PCR, while no PEDV was detected in negative samples. RT-LAMP detection results showed that 12 of the 52(23.08%) unknown clinical samples were positive, the positive rate of RT-PCR for detecting unknown virus clinical samples was 9/52(17.31%), and the coincidence rate was 94.23%.The above results indicate that the established RT-LAMP assay has advantages of low cost, rapid amplification, high sensitivity and specificity, easy to determine, convenient to detect in the field. It has potential applications for clinical diagnosis, which provides an important experimental basis for the epidemiological investigation and comprehensive control of PEDV.