国家科技支撑计划:实验动物质量检测体系的完善与检测关键技术研究(2013BAK11B00), 实验动物质量检测关键技术研究(2013BAK11B01)。
目的 建立念珠状链杆菌的实时荧光定量PCR检测方法, 实现该菌在多种实验动物中的快速检测。方法 根据NCBI公布的念珠状链杆菌序列, 设计特异性引物和探针, 通过对24株标准参考菌株的扩增, 验证其特异性、敏感性和重复性。运用所建立的方法对小鼠、大鼠、豚鼠、地鼠、兔、长爪沙鼠和树鼩的共823份呼吸道样本进行检测。结果 用所建Taqman MGB荧光定量PCR方法在小鼠、大鼠、豚鼠、地鼠和兔中未检出念珠状链杆菌;检出普通级长爪沙鼠和树鼩中念珠状链杆菌的阳性率分别为1.5%(1/65)和61.7%(37/60)。结论 本研究所建立的念珠状链杆菌Taqman MGB荧光定量PCR检测方法, 能够对实验动物中念珠状链杆菌进行快速准确的检测, 敏感性优于国标方法, 为实验动物质量检测体系的完善奠定了基础。
Objective To establish a real-time quantitative PCR (qPCR) method for detection of Streptobacillus moniliformis, which can be used to rapidly detect this pathogen in laboratory animals. Method According to the S. moniliformis sequences published in NCBI, we designed specific primers and MGB probe. The specificity, sensitivity and stability of this method were evaluated using 24 standard reference strains. Total of 823 respiratory specimens of animals including mice, rats, guinea pigs, hamsters, rabbits, Mongolian gerbils and tree shrews, were detected by this established Taqman MGB qPCR method. Results We had successfully established the S. moniliformis Taqman MGB qPCR method. S. moniliformis was not detected in the samples of mice, rats, guinea pigs, hamsters and rabbits. The positive rate of S. moniliformis was 1.5% (1/65) and 61.7% (37/60) in conventional Mongolian Gerbils and tree shrews, respectively. Conclusions Our developed qPCR method can be used to effectively detect S. moniliformis in laboratory animals. Moreover, its accuracy and sensitivity are better than the national standard method. This study laid the foundations for optimizing the quality inspection system of laboratory animals.
邢进,冯育芳,岳秉飞,贺争鸣,戴方伟,萨晓婴,代解杰.念珠状链杆菌Taqman MGB荧光定量PCR检测方法的建立与初步应用[J].中国比较医学杂志,2015,25(8):62~67.
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