Departments of Veterinary Microbiology and Preventative Medicine (McDaniel) and Veterinary Diagnostic and Production Animal Medicine (Derscheid), College of Veterinary Medicine, Iowa State University, Ames, IA, 50011, USA.
BMC Vet Res. 2021 Apr 17;17(1):170. doi: 10.1186/s12917-021-02870-5.
Mycoplasma bovis is an important pathogen of cattle worldwide. Many different clinical manifestations of infection can occur, including respiratory disease, arthritis, and mastitis, causing heavy losses to beef and dairy industries. Because Mycoplasma species are slow-growing and fastidious, traditional identification methods are not cost- or time-effective, and improved methods are sought to streamline laboratory processes. High-resolution melting PCR (HRM-PCR) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) are 2 relatively recent tools that are rapid and inexpensive to use; we tested 9 isolates of M. bovis using both assays. The HRM-PCR assay used universal mycoplasma primers for the 16S-23S intergenic spacer region (IGSR).
The resulting melting profiles of the field isolates were indistinguishable from the reference strain, indicating accurate identification. For the MALDI-TOF MS, each M. bovis isolate was accurately identified. Mycoplasma arginini and Mycoplasma alkalescens isolates did not identify as M. bovis when tested by either assay.
Our work shows that either assay could be used to identify unknown M. bovis isolates. For future work, the MALDI-TOF MS library should be expanded to include more mycoplasmas, and the HRM-PCR assay should be tested on additional mycoplasmas to ensure that the melting profiles are sufficiently distinctive.
牛支原体是一种全球性的重要牛病原体。感染可能导致多种不同的临床表现，包括呼吸道疾病、关节炎和乳腺炎，给牛肉和奶制品行业造成重大损失。由于支原体生长缓慢且挑剔，传统的鉴定方法既不经济也不省时，因此需要寻求改进的方法来简化实验室流程。高分辨率熔解 PCR（HRM-PCR）和基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）是两种相对较新的快速、廉价的工具；我们使用这两种方法检测了 9 株牛支原体。HRM-PCR 检测使用针对 16S-23S 基因间隔区（IGSR）的通用支原体引物。
田间分离株的熔解曲线与参考株无法区分，表明鉴定准确。对于 MALDI-TOF MS，每个牛支原体分离株都被准确识别。在用任一方法检测时，支原体精氨酸和支原体碱性分离株均未鉴定为牛支原体。
我们的工作表明，这两种方法都可用于鉴定未知的牛支原体分离株。未来的工作应扩大 MALDI-TOF MS 文库，以包括更多的支原体，并测试 HRM-PCR 检测方法，以确保熔解曲线具有足够的特征性。
