国家科技支撑计划（2015BAI07B02-05）。
目的 建立金黄色葡萄球菌、绿脓杆菌和肺炎克雷伯杆菌多重PCR检测方法，为实验动物细菌检测提供快速、灵敏、简便的方法。方法 根据金黄色葡萄球菌nuc基因、绿脓杆菌LasI基因、肺炎克雷伯杆菌PhoE基因和细菌通用16S rRNA基因设计出特异性引物；经过多重PCR引物浓度和退火温度的优化，特异性和敏感性的检测，建立多重PCR体系。应用该PCR体系对人工感染标本、实验动物粪便样本进行验证检测，与传统检测方法对比。结果 多重PCR分别扩增出金黄色葡萄球菌（153 bp）、绿脓杆菌（600 bp）、肺炎克雷伯杆菌（368 bp）和通用（520 bp）的目的条带。多重敏感性为10 pg，特异性检测未从其他细菌中检测出目的条带。应用建立的多重PCR体系检测出不同组合的人工感染标本，从76只粪便检测出绿脓杆菌阳性1例，与传统检测方法结果一致。结论 本文建立的多重PCR方法具有特异、灵敏、简便、快速等优点，为实验动物微生物的快速检测提供了可靠的方法。
Objective Aiming at detecting Staphylococcus aureus、Pseudomonas aeruginosa and Klebsiella pneumoniae in laboratory animals,the paper provides a rapid,sensitive and simple test method.Methods According to Staphylococcus aureus nuc gene,Pseudomonas aeruginosa LasI gene,Klebsiella pneumonia PhoE geneand general 16S rRNA gene,designed specific primers; Through the optimization of multiplex PCR primer concentrations and annealing temperature, the specificity and sensitivity of detection, establishing multiplex PCR system.Application of the PCR system test specimens of artificial infections and experiment animal feces is compared with traditional test method.Results Multiplex PCR amplification of Staphylococcus aureus (153 bp), Pseudomonas aeruginosa (600 bp) with Klebsiella pneumoniae (368 bp) and general (520 bp).The multiplex sensitivity for the purpose of 10pg, specificity of detection was not detected from other pathogens.Application of establishing multiplex PCR system to detect the artificial positive samples, and detect 1 Pseudomonas aeruginosa positive case in 76 fecals. Conclusions This paper established the multiplex PCR method which has the advantages of specific,sensitive,simple and rapid, and provides a reliable way for rapid test in laboratory animals microbiology.
祝岩波,郑龙,尤红煜,王璇,梁晓亮,刘健敏,张东明,连伟光,栗彦宁,王俊霞.金黄色葡萄球菌、绿脓杆菌和肺炎克雷伯杆菌多重PCR方法的建立与初步应用[J].中国比较医学杂志,2017,27(5):94~98.
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