Loganbill Jessie K, Wagner April M, Besselsen David G
Department of University Animal Care, The University of Arizona, Tucson, Arizona, USA.
Comp Med. 2005 Oct;55(5):419-24.
Mycoplasma pulmonis induces persistent infections in laboratory mice and rats and can contaminate biological materials. We developed a fluorogenic nuclease polymerase chain reaction (fnPCR) assay to detect M. pulmonis specifically. Primer and probe sequences for the assay were targeted to 16S rRNA sequences specific to M. pulmonis. The assay consistently detected the equivalent of fewer than 10 copies of template DNA. When evaluated against a panel of 24 species of bacteria, the M. pulmonis assay detected only M. pulmonis isolates. Evaluation of 10-fold serial dilutions of cultured M. pulmonis showed that the M. pulmonis fnPCR assay and culture on Dutch agar had comparable sensitivity in detecting viable M. pulmonis organisms, whereas the mouse antibody production test displayed positive serologic results at dilutions higher than those in which viable organisms could be detected. Finally, the M. pulmonis fnPCR assay was able to detect M. pulmonis DNA in nasopharyngeal wash fluid and trachea, lung, and uterus tissue collected from mice naturally infected with M. pulmonis but did not detect the organism in similar samples collected from uninfected, negative control mice. The M. pulmonis fnPCR assay provides a high-throughput, PCR-based method to detect M. pulmonis in infected rodents and contaminated biological materials.
肺支原体可在实验小鼠和大鼠中引发持续性感染，并能污染生物材料。我们开发了一种荧光核酸酶聚合酶链反应（fnPCR）检测方法，用于特异性检测肺支原体。该检测方法的引物和探针序列靶向肺支原体特异的16S rRNA序列。该检测方法始终能检测到少于10个拷贝的模板DNA。在针对一组24种细菌进行评估时，肺支原体检测方法仅能检测到肺支原体分离株。对培养的肺支原体进行10倍系列稀释评估表明，肺支原体fnPCR检测方法与在荷兰琼脂上培养在检测活的肺支原体生物体方面具有相当的灵敏度，而小鼠抗体产生试验在高于能检测到活生物体的稀释度时显示出阳性血清学结果。最后，肺支原体fnPCR检测方法能够在从自然感染肺支原体的小鼠收集的鼻咽洗液、气管、肺和子宫组织中检测到肺支原体DNA，但在从未感染的阴性对照小鼠收集的类似样本中未检测到该生物体。肺支原体fnPCR检测方法提供了一种基于PCR的高通量方法，用于检测感染啮齿动物和受污染生物材料中的肺支原体。
