Wang Jianchang, Liu Libing, Wang Jinfeng, Pang Xiaoyu, Yuan Wanzhe
Hebei Academy of Science and Technology for Inspection and Quarantine, Shijiazhuang 050051, China; Center of Inspection and Quarantine, Hebei Entry-Exit Inspection and Quarantine Bureau, Shijiazhuang 050051, China.
Hebei Academy of Science and Technology for Inspection and Quarantine, Shijiazhuang 050051, China.
Anal Biochem. 2018 Feb 15;543:122-127. doi: 10.1016/j.ab.2017.12.012. Epub 2017 Dec 13.
The objective of this study was to develop a dual real-time recombinase polymerase amplification (RPA) assay using exo probes for the detection and differentiation of pseudorabies virus (PRV). Specific RPA primers and probes were designed for gB and gE genes of PRV within the conserved region of viral genome. The reaction process can be completed in 20 min at 39 °C. The dual real-time RPA assay performed in the single tube was capable of specific detecting and differentiating of the wild-type PRV and gE-deleted vaccine strains, without cross-reactions with other non-targeted pig viruses. The analytical sensitivity of the assay was 10 copies for gB and gE genes. The dual real-time RPA demonstrated a 100% diagnostic agreement with the real-time PCR on 4 PRV strains and 37 clinical samples. Through the linear regression analysis, the R value of the real-time RPA and the real-time PCR for gB and gE was 0.983 and 0.992, respectively. The dual real-time RPA assay provides an alternative useful tool for rapid, simple, and reliable detection and differentiation of PRV, especially in remote and rural areas.
本研究的目的是开发一种使用外切酶探针的双重实时重组酶聚合酶扩增（RPA）检测方法，用于伪狂犬病病毒（PRV）的检测和鉴别。针对PRV病毒基因组保守区域的gB和gE基因设计了特异性RPA引物和探针。反应过程可在39℃下20分钟内完成。在单管中进行的双重实时RPA检测能够特异性地检测和鉴别野生型PRV和gE缺失疫苗株，与其他非靶向猪病毒无交叉反应。该检测方法对gB和gE基因的分析灵敏度为10个拷贝。双重实时RPA与实时PCR对4株PRV毒株和37份临床样本的诊断一致性为100%。通过线性回归分析，实时RPA与实时PCR对gB和gE的R值分别为0.983和0.992。双重实时RPA检测为PRV的快速、简单、可靠检测和鉴别提供了一种有用的替代工具，尤其适用于偏远农村地区。
