Silva Marta Filipa, Kienesberger Sabine, Pereira Gonçalo, Mateus Luísa, Lopes-da-Costa Luís, Silva Elisabete
Faculdade de Medicina Veterinária, Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Universidade de Lisboa, Lisbon, Portugal.
Associate Laboratory for Animal and Veterinary Science (AL4AnimalS), Lisbon, Portugal.
Front Microbiol. 2022 Sep 9;13:969825. doi: 10.3389/fmicb.2022.969825. eCollection 2022.
Bovine Genital Campylobacteriosis (BGC) is a worldwide spread venereal disease of cattle caused by subsp. (). Although several real-time PCR assays were developed for identification, most target mobile genetic elements, which may lead to false-positive diagnosis. In this study, a real-time PCR assay coupled with High-Resolution Melting analysis (HRM) was developed for the identification of subspecies and application in BGC diagnosis. Two HRM assays targeting different single nucleotide polymorphisms were validated using 51 strains, including 36 and 15 subsp. (). The specificity was assessed in 50 preputial samples previously tested as negative for and in 24 strains from other species. The analytical sensitivity was determined with ten-fold dilutions of genome copies and in preputial samples spiked with cells. Both HRM assays accurately identified the 51 strains, showing 100% concordance with the previous identification. subspecies identification by HRM showed concordant results with the glycine test in 98.0% of the isolates. No amplification was obtained in negative preputial samples as well as in strains from other species. The assays were able to detect 10 genome copies of , while for preputial washing samples the limit of detection was 10 CFU/mL. These novel HRM assays represent a highly specific and sensitive tool for the identification of subspecies and show potential for direct use in bull preputial samples for BGC diagnosis.
牛生殖道弯曲菌病（BGC）是一种由亚种（）引起的在全球范围内传播的牛性病。尽管已经开发了几种用于鉴定的实时PCR检测方法，但大多数针对的是移动遗传元件，这可能导致假阳性诊断。在本研究中，开发了一种结合高分辨率熔解分析（HRM）的实时PCR检测方法，用于亚种的鉴定及其在BGC诊断中的应用。使用51株弯曲杆菌菌株对两种针对不同单核苷酸多态性的HRM检测方法进行了验证，其中包括36株亚种和15株亚种（）。在50份先前检测为弯曲杆菌阴性的包皮样本和24株来自其他弯曲杆菌物种的菌株中评估了特异性。通过对弯曲杆菌基因组拷贝进行十倍稀释以及在接种了弯曲杆菌细胞的包皮样本中测定分析灵敏度。两种HRM检测方法均准确鉴定了51株弯曲杆菌菌株，与先前的鉴定结果显示出100%的一致性。通过HRM进行的亚种鉴定在98.0%的分离株中与甘氨酸试验结果一致。在弯曲杆菌阴性的包皮样本以及来自其他弯曲杆菌物种的菌株中均未获得扩增。这些检测方法能够检测到10个弯曲杆菌基因组拷贝，而对于包皮冲洗样本，检测限为10 CFU/mL。这些新型HRM检测方法是用于鉴定弯曲杆菌亚种的高度特异性和灵敏的工具，并显示出直接用于公牛包皮样本进行BGC诊断的潜力。
