Li Ruiwen, Wang Jinfeng, Sun Xiaoxia, Liu Libing, Wang Jianchang, Yuan Wanzhe
College of Veterinary Medicine, Hebei Agricultural University, Baoding, China.
Food Microbiology and Animal Quarantine Laboratory, Technology Center of Shijiazhuang Customs District, Shijiazhuang, China.
Front Cell Infect Microbiol. 2021 Feb 25;11:639083. doi: 10.3389/fcimb.2021.639083. eCollection 2021.
This study aimed to detetct () in bovine milk quickly and directly by developing and validating isothermal recombinase polymerase amplification (RPA) assays. Targeting the gene of , an RPA assay based on the fluorescence monitoring (real-time RPA) and an RPA assay combined with a lateral flow strip (LFS RPA) were conducted. It took 20 min for the real-time RPA to finish in a Genie III at 39°C, and 15 min were required to perform the LFS RPA in an incubator block at 39°C, followed by the visualization of the products on the lateral flow strip within 5 min. Both of the two assays showed high specificity for without any cross-reaction with the other tested pathogens. With the standard recombinant plasmid pMbovis-uvrC serving as a template, both RPA assays had a limit of detcion of 1.0 × 10 copies per reaction, equivalent to that of a real-time PCR assay. In the 65 milk samples collected from cattle with mastitis, the genomic DNA was detected in 24 samples by both the real-time RPA and the LFS RPA assays. The developed RPA assays could detect in bovine milk in an efficient, convenient, and credible manner as attractive and promising tools, and the assays would be helpful in the rapid response to infection causing bovine mastitis.
本研究旨在通过开发和验证等温重组酶聚合酶扩增（RPA）检测方法，快速、直接地检测牛乳中的（）。针对（）基因，进行了基于荧光监测的RPA检测（实时RPA）和结合侧向流动条的RPA检测（LFS RPA）。实时RPA在Genie III中于39°C下完成检测需要20分钟，而LFS RPA在39°C的恒温块中进行检测需要15分钟，随后在5分钟内在侧向流动条上观察产物。这两种检测方法对（）均显示出高特异性，与其他测试病原体均无交叉反应。以标准重组质粒pMbovis-uvrC作为模板，两种RPA检测的检测限均为每个反应1.0×10个拷贝，与实时PCR检测相当。在从患乳腺炎的奶牛采集的65份乳样中，实时RPA和LFS RPA检测均在24份样品中检测到了（）基因组DNA。所开发的RPA检测方法可作为高效、便捷且可靠的工具，以有吸引力且有前景的方式检测牛乳中的（），这些检测方法将有助于对引起奶牛乳腺炎的（）感染做出快速反应。
