PDCoV、SADS-CoV与SVA三重RT-PCR检测方法的建立与应用

Establishment and application of a triplex RT-PCR method for detection of PDCoV, SADS-CoV, and SVA

摘要:

【背景】猪丁型冠状病毒(porcine deltacoronavirus, PDCoV)、新型猪急性腹泻综合征冠状病毒(swine acute diarrhea syndrome coronavirus, SADS-CoV)与塞内卡病毒A型(seneca virus A, SVA)均为猪的新发病原，严重危害养猪业的发展。猪感染这3种新发病原难以根据临床症状诊断，因此亟须建立多重反转录-聚合酶链式反应(reverse transcription-polymerase chain reaction, RT-PCR)检测方法对疑似患病猪进行快速诊断，以降低经济损失。【目的】建立能同时检测PDCoV、SADS-CoV和SVA单一或混合感染的三重RT-PCR检测方法。【方法】参考GenBank中登录的PDCoV和SADS-CoV N基因、SVA L/P1基因保守区域序列设计3对特异性引物，以温度梯度PCR法确定最适退火温度(T_m)；采用方阵法优化其引物浓度；构建重组质粒PMD-PDCoV、PMD-SADS-CoV和PMD-SVA作为标准品确定最小检测量；以猪传染性胃肠炎病毒、猪流行性腹泻病毒、猪繁殖和呼吸综合征病毒等6种常见感染猪的病毒核酸样本为模板，确定三重RT-PCR法的特异性；以批间和批内试验验证其重复性；经检测临床样本并与已报道的检测方法进行比较，评估其临床应用效果。【结果】建立的三重RT-PCR检测方法最佳退火温度为58.3 ℃，引物最佳浓度分别为0.50、0.25和0.25 μmol/L；其敏感性高，PMD-PDCoV、PMD-SADS-CoV与PMD-SVA最低检测限分别为1、1和10 copies/μL；其特异性强，仅对PDCoV、SADS-CoV和SVA有特异性条带，对其他病毒均无扩增条带；该方法重复性好，批间和批内试验检测结果均一致。最后经临床样本检测PDCoV、SADS-CoV和SVA的阳性率分别为4.4%、0%和0.73%，与已报道的检测方法一致。【结论】建立了一种能够同时快速检测PDCoV、SADS-CoV和SVA的三重RT-PCR方法，为临床上检测上述3种病毒提供了技术支撑。

Abstract:

[Background] Porcine deltacoronavirus (PDCoV), swine acute diarrhea syndrome coronavirus (SADS-CoV), and senecavirus A (SVA) are emerging pathogens which seriously endanger the development of the pig industry. The clinical symptoms of pigs infected with the three pathogens are difficult to be distinguished. Therefore, it is urgent to establish a multiplex reverse transcription-polymerase chain reaction (RT-PCR) method for the rapid diagnosis of suspected pigs and reduce economic losses. [Objective] To establish a triplex RT-PCR method for simultaneous detection of single or mixed infection of PDCoV, SADS-CoV, and SVA. [Methods] Three pairs of specific primers were designed according to the conserved regions of the N genes of PDCoV and SADS-CoV and the L/P1 genes of SVA registered in GenBank, and the optimal annealing temperature (T_m) was determined by the temperature gradient PCR method. The primer concentration was optimized by the array method. The recombinant plasmids PMD-PDCoV, PMD-SADS-CoV, and PMD-SVA were constructed as standards to determine the limits of detection (LODs). The specificity of the triplex RT-PCR method was determined with the nucleic acid samples of six common pig viruses including porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and porcine reproductive and respiratory syndrome virus. The repeatability of the established method was verified by inter-batch and intra-batch tests. Finally, we employed the triplex PCR method to detect the clinical samples and compared the results with those obtained with the reported detection methods, thus evaluating the clinical application performance of this method. [Results] The optimal T_m was 58.3 ℃, and the optimal primer concentrations were 0.50, 0.25, and 0.25 μmol/L, respectively. The established method had high sensitivity, with the LODs of 1, 1, and 10 copies/μL for PMD-PDCoV, PMD-SADS-CoV, and PMD-SVA, respectively. It had strong specificity, with specific bands only for PDCoV, SADS-CoV, and SVA and no bands for other viruses. Moreover, the method had good repeatability as the test results were consistent between and within batches. Finally, the positive rates of PDCoV, SADS-CoV, and SVA in the clinical samples detected by the established method were 4.4%, 0%, and 0.73%, respectively, which were consistent with the results obtained with the reported detection methods. [Conclusion] The triplex RT-PCR method established in this study is accurate and reliable for the simultaneous detection of PDCoV, SADS-CoV, and SVA in clinical samples.