College of Veterinary Medicine, Hebei Agricultural University, No.2596 Lekai South Street, Baoding, Hebei, 071001, People's Republic of China.
Technology Center of Shijiazhuang Customs District, No.318 Heping Xi Lu, Shijiazhuang, 050051, People's Republic of China.
BMC Vet Res. 2022 Sep 8;18(1):339. doi: 10.1186/s12917-022-03437-8.
Bovine rotavirus A (BRVA) is considered to be the most common pathogen of severe diarrhea in cattle worldwide, which could lead to the death of newborn calves and cause the significant economic losses to the cattle industry. As a novel isothermal nucleic acid amplification technique, recombinase polymerase amplification (RPA) has been applied widely for the rapid detection of different important pathogens in human and animals.
An RT-RPA assay based on the real time fluorescence monitoring (real-time RT-RPA) and an RT-RPA assay combined with a lateral flow strip (LFS RT-RPA) were successfully developed by targeting the VP6 gene of BRVA. The RT-RPA assays allowed the exponential amplification of the target fragment in 20 min. After incubation of the LFS RT-RPA on a metal bath at 40 °C, the results were displayed on the lateral flow strip within 5 min, while real-time RT-RPA allowed the real-time observation of the results in Genie III at 42 °C. Both of the two assays showed high specificity for BRVA without any cross-reaction with the other tested pathogens causing diarrhea in cattle. With the standard RNA of BRVA serving as a template, the limit of detection for real-time RT-RPA and LFS RT-RPA were 1.4 × 10 copies per reaction and 1.4 × 10 copies per reaction, respectively. In the 134 fecal samples collected from cattle with diarrhea, the BRVA positive rate were 45.52% (61/134) and 46.27% (62/134) in real-time RT-RPA and LFS RT-RPA, respectively. Compared to a previously published real-time PCR, the real-time RT-RPA and LFS RT-RPA showed a diagnostic specificity of 100%, diagnostic sensitivity of 98.39% and 100%, and a kappa coefficient of 0.985 and 1.0, respectively.
In this study, BRVA was successfully detected in cattle fecal samples by the developed real-time RT-RPA and LFS RT-RPA assays. The developed RT-RPA assays had great potential for the rapid detection of BRVA in under-equipped diagnostic laboratory and the point-of-need diagnosis at quarantine stations and farms, which is of great importance to control BRVA-associated diarrhea in cattle herds.
牛轮状病毒 A（BRVA）被认为是全球导致牛严重腹泻的最常见病原体，可导致新生牛犊死亡，并给牛养殖业造成重大经济损失。重组酶聚合酶扩增（RPA）作为一种新型等温核酸扩增技术，已广泛应用于人类和动物中不同重要病原体的快速检测。
本研究基于 BRVA 的 VP6 基因，成功建立了实时荧光监测的 RT-RPA 检测方法（实时 RT-RPA）和结合横向流条带（LFS RT-RPA）的 RT-RPA 检测方法。RT-RPA 检测方法可在 20 分钟内实现靶片段的指数扩增。将 LFS RT-RPA 在 40°C 的金属浴中孵育后，可在 5 分钟内在横向流条带中显示结果，而实时 RT-RPA 则可在 42°C 的 Genie III 中实时观察结果。两种检测方法均对 BRVA 具有高度特异性，与引起牛腹泻的其他检测病原体无交叉反应。以 BRVA 的标准 RNA 为模板，实时 RT-RPA 和 LFS RT-RPA 的检测限分别为 1.4×10 拷贝/反应和 1.4×10 拷贝/反应。在从腹泻牛采集的 134 份粪便样本中，实时 RT-RPA 和 LFS RT-RPA 的 BRVA 阳性率分别为 45.52%（61/134）和 46.27%（62/134）。与先前发表的实时 PCR 相比，实时 RT-RPA 和 LFS RT-RPA 的诊断特异性均为 100%，诊断敏感性分别为 98.39%和 100%，kappa 系数分别为 0.985 和 1.0。
本研究成功建立了用于检测牛粪便中 BRVA 的实时 RT-RPA 和 LFS RT-RPA 检测方法。所建立的 RT-RPA 检测方法具有在设备不足的诊断实验室和检疫站和农场的现场进行快速检测 BRVA 的潜力，这对控制牛群中 BRVA 相关腹泻具有重要意义。
