实验树鼩空肠弯曲菌、沙门氏菌和志贺菌多重荧光定量PCR方法的建立

Establishment of a multiplex real-time PCR method for quantitative detection of Campylobacter jejuni,Salmonella and Shigella in tree shrews

目的为实验树鼩微生物检测提供一种快速、简便、灵敏,并且特异性强的空肠弯曲菌、沙门氏菌和志贺菌多重荧光定量PCR检测方法。方法根据空肠弯曲菌的Hip O区域、沙门菌的in V区域和志贺菌的ipa H区域设计特异性引物和探针,并进行单病原定量PCR检测验证;后分析多重PCR的灵敏度和特异性,并使用该多重PCR方法检测实验树鼩样品。结果本研究的PCR要素可用于空肠弯曲菌、沙门氏菌及志贺菌等单种菌的实时荧光定量PCR扩增。多重定量PCR扩增出了空肠弯曲菌、沙门氏菌和志贺氏菌标准扩增曲线;该方法的灵敏度为1×103ng/μL;特异性检测未从其他参考菌株中检出阳性。结论该多重定量PCR方法在实验树鼩微生物检测中具有很好的应用和推广前景。

Objective To establish a rapid,simple,sensitive,and specific multiplex real-time PCR method for quantitative detection of Campylobacter jejuni,Salmonella and Shigella in tree shrews. Methods Specific primers and probes were designed,according to the Hip O gene of Campylobacter jejuni,in V gene of Salmonella and ipa H gene of Shigella. The primers were confirmed by single pathogen quantitative PCR,and the sensitivity and specificity of the multiplex PCR were analyzed. Finall,the samples of experimental tree shrews were detected by this multiplex PCR method. Results The PCR element of Taq Man-MGB real-time PCR assay was able to quantitatively amplify the Campylobacter jejuni,Salmonella or Shigella. Appropriate standard amplification curves of Campylobacter jejuni,Salmonella and Shigella in the multiplex quantitative PCR were obtained. The sensitivity of this method was 1 × 10^3ng/μL. There was no false positive detection from other bacterial strains. Conclusions This multiplex quantitative real-time PCR method has good application and development prospects in the detection of microorganisms in tree shrews.