Development of a novel SYBR green I-based quantitative RT-PCR assay for Senecavirus A detection in clinical samples of pigs
Development of a novel SYBR green I-based quantitative RT-PCR assay for Senecavirus A detection in clinical samples of pigs
复制标题
开发一种新型基于 SYBR green I 的定量 RT-PCR 检测方法，用于检测猪临床样本中的塞内卡病毒 A
DOI：
10.1016/j.mcp.2020.101643
复制
发表时间：
2020-10-01
影响因子：
3.3
通讯作者：
Sun, Shiqi
中科院分区：
文献类型：
--
作者：
Mu, Suyu;Abdullah, Sahibzada Waheed;Sun, Shiqi
猪水泡病是由A型Senecavirus A（SVA）引起的一种新发疾病。仅根据临床症状，将SVA感染与其他类似疾病（如口蹄疫、猪水泡病和水泡性口炎）区分开来是非常具有挑战性的。因此，建立SVA感染的检测方法对临床诊断SVA感染至关重要。本研究根据SVA高度保守的L/VP 4基因序列设计了一对特异性引物。建立的SYBR绿色I定量逆转录聚合酶链反应（qRT-PCR）方法用于检测临床样本中的SVA核酸。通过qRT-PCR检测SVA核酸的最低限为6.4 x 10（1）copies/mu L，这显著高于凝胶电泳的6.4 x 10（3）copes/mu L。该方法特异性强，与其它7种猪病毒无交叉反应。采用SYBR绿色I荧光定量RT-PCR方法检测实验动物和现场样品中SVA的阳性率分别为67.60%（96/142）和80%（24/30）。结果表明，基于SYBR绿色I的qRT-PCR是一种快速、特异的方法，可用于SVA引起的相关水泡性疾病的临床诊断和流行病学调查。
Porcine vesicular disease caused by Senecavirus A (SVA) is a newly emerging disease in many countries. Based on clinical signs only, it is very challenging to distinguish SVA infection from other similar diseases, such as foot and mouth disease, swine vesicular disease, and vesicular stomatitis. Therefore, it is crucial to establish a detection assay for the clinical diagnosis of SVA infection. In this study, a pair of specific primers were designed based on the highly conserved L/VP4 gene sequence of SVA. The established SYBR green I-based quantitative reverse transcription polymerase chain reaction (qRT-PCR) method was used to detect SVA nucleic acids in clinical samples. The limit of detection SVA nucleic acids by qRT-PCR was 6.4 x 10(1) copies/mu L, which was significantly more sensitive than that by gel electrophoresis of 6.4 x 10(3) copes/mu L. This assay was specific and had no crossreaction with other seven swine viruses. Using SYBR green I-based qRT-PCR, the SVA positive rates in experimental animal samples and field samples were 67.60% (96/142) and 80% (24/30) respectively. The results demonstrate that SYBR green I-based qRT-PCR is a rapid and specific method for the clinical diagnosis and epidemiological investigation of related vesicular diseases caused by SVA.