实验小鼠四种常见病原微生物多重PCR检测体系的建立

Establishment of Multiple PCR Detection System for Four Common Pathogenic Microorganisms in Laboratory Mouse

摘要: 目的：建立实验小鼠四种常见病原微生物的多重PCR快速检测方法。方法：对四种病原微生物引物浓度、特异性以及DNA模板的敏感性进行测试，优化多重PCR反应条件；对活体实验动物口腔及粪便样本进行优化处理，简化DNA模板的提取方法。结果：四种病原微生物的多重PCR反应在引物浓度0.15 μmol/L和退火温度56℃的反应条件下扩增，灵敏度高(绿脓杆菌的敏感性为100 pg，金黄色葡萄球菌的敏感性为1 pg，肺炎克雷伯杆菌的敏感性为10 pg，嗜肺巴斯德杆菌的敏感性为1 pg)且特异性好，与实验动物常见的致病菌无交叉反应。活体实验动物口腔及粪便样本经培养煮沸1 min后即能快速获取检测模板。结论：该方法灵敏特异、简便快速，可为活体实验动物大规模筛查和检测四种病原微生物提供借鉴。

Abstract: Objective: This study aimed to establish a rapid multiplex PCR method for the detection of four common pathogenic microorganisms in laboratory mouse. Methods: The primer concentration, specificity and the sensitivity of the DNA template for the four pathogenic microorganisms were tested to optimize the multiple PCR reaction conditions; optimized processing of oral and fecal samples from live experimental animals was performed to simplify the extraction method of the DNA template. Result: The multiple PCR reaction of the four pathogenic microorganisms amplified efficiently at the reaction conditions of primer concentration of 0.15 μmol/Land annealing temperature of 56˚C, demonstrating high sensitivity (sensitivity of Pseudomonas aeruginosa is 100 pg, sensitivity of Staphylococcus aureus is 1 pg, the sensitivity of Klebsiella pneumoniae is 10 pg, and the sensitivity of Pasteurella pneumophila is 1 pg) and good specificity, there is no cross-reaction with the common pathogenic bacteria in experimental animals. Rapid template extraction was achieved from oral and fecal samples of live experimental animals after boiling for 1 min. Conclusion: The method was sensitive, specific, simple and fast, providing a reference for large-scale screening and detection of the four pathogenic microorganisms in live experimental animals.