Wang Hongmei, Hou Peili, Zhao Guimin, Yu Li, Gao Yu-Wei, He Hongbin
Ruminant Diseases Research Center, Key Laboratory of Animal Resistant Biology of Shandong, College of Life Sciences, Shandong Normal University, Jinan, 250014, China.
Division of Livestock Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Harbin, 150001, China.
BMC Vet Res. 2018 Nov 20;14(1):359. doi: 10.1186/s12917-018-1644-4.
Foot-and-mouth disease (FMD) caused by foot-and-mouth disease virus (FMDV) is one of the most highly infectious diseases in livestock, and leads to huge economic losses. Early diagnosis and rapid differentiation of FMDV serotype is therefore integral to the prevention and control of FMD. In this study, a series of serotype-specific reverse transcription recombinase polymerase amplification assays combined with lateral flow dipstick (RPA-LFD) were establish to differentiate FMDV serotypes A, O or Asia 1, respectively.
The serotype-specific primers and probes of RPA-LFD were designed to target conserved regions of the FMDV VP1 gene sequence, and three primer and probe sets of serotype-specific RPA-LFD were selected for amplification of FMDV serotypes A, O or Asia 1, respectively. Following incubation at 38 °C for 20 min, the RPA amplification products could be visualized by LFD. Analytical sensitivity of the RPA assay was then determined with ten-fold serial dilutions of RNA of VP1 gene and the recombinant vector respectively containing VP1 gene from FMDV serotypes A, O or Asia1, the detection limits of these assays were 3 copies of plasmid DNA or 50 copies of viral RNA per reaction. Moreover, the specificity of the assay was assessed, and there was no cross reactions with other viruses leading to bovine vesicular lesions. Furthermore, 126 clinical samples were respectively detected with RPA-LFD and real-time PCR (rPCR), there was 98.41% concordance between the two assays, and two samples were positive by RPA-LFD but negative in rPCR, these were confirmed as FMDV-positive through viral isolation in BHK-21 cells. It showed that RPA-LFD assay was more sensitive than the rPCR method in this study.
The development of serotype-specific RPA-LFD assay provides a rapid, sensitive, and specific method for differentiation of FMDV serotype A, O or Asia1, respectively. It is possible that the serotype-specific RPA-LFD assay may be used as a integral protocol for field detection of FMDV.
由口蹄疫病毒（FMDV）引起的口蹄疫（FMD）是家畜中传染性最强的疾病之一，会导致巨大的经济损失。因此，口蹄疫病毒血清型的早期诊断和快速鉴别对于口蹄疫的预防和控制至关重要。在本研究中，建立了一系列血清型特异性逆转录重组酶聚合酶扩增试验结合侧向流动试纸条（RPA-LFD），分别用于鉴别口蹄疫病毒A、O或亚洲1型。
RPA-LFD的血清型特异性引物和探针设计靶向口蹄疫病毒VP1基因序列的保守区域，分别选择三组血清型特异性RPA-LFD引物和探针用于扩增口蹄疫病毒A、O或亚洲1型。在38℃孵育20分钟后，RPA扩增产物可通过LFD可视化。然后分别用VP1基因RNA和分别含有口蹄疫病毒A、O或亚洲1型VP1基因的重组载体进行十倍系列稀释来确定RPA试验的分析灵敏度，这些试验的检测限为每个反应3个质粒DNA拷贝或50个病毒RNA拷贝。此外，评估了该试验的特异性，与其他导致牛水疱性病变的病毒无交叉反应。此外，分别用RPA-LFD和实时PCR（rPCR）检测126份临床样本，两种检测方法的一致性为98.41%，两份样本RPA-LFD检测为阳性但rPCR检测为阴性，通过在BHK-21细胞中进行病毒分离证实这些样本为口蹄疫病毒阳性。结果表明，在本研究中RPA-LFD试验比rPCR方法更灵敏。
血清型特异性RPA-LFD试验的开发为分别鉴别口蹄疫病毒A、O或亚洲1型提供了一种快速、灵敏和特异的方法。血清型特异性RPA-LFD试验有可能用作口蹄疫病毒现场检测的完整方案。
