多重荧光定量PCR快速检测6种常见下呼吸道感染病原菌

Rapid detection of six common pathogens of lower respiratory tract infection by multiplex fluorescent quantitative PCR

目的:探讨多重实时荧光定量PCR(MRT-PCR)检测6种常见呼吸道病原菌的检测方法。方法:采用BeaconDesigner7.9设计引物和探针建立MRT-PCR法,通过构建质粒标准品分析该方法的最低检出限。对176例临床标本中大肠埃希菌、金黄色葡萄球菌、铜绿假单胞菌、肺炎克雷伯菌、阴沟肠杆菌和鲍曼不动杆菌进行回顾性检测,并用一代基因测序验证。结果:MRT-PCR法检测病原菌的最低检出限达103 copies/mL,大肠埃希菌19例,金黄色葡萄球菌30例,铜绿假单胞菌37例,肺炎克雷伯菌53例,阴沟肠杆菌15例,鲍曼不动杆菌22例,与传统鉴定方法报告病原菌一致,特异性达100%。通过一代基因测序结果完全符合。结论:使用多荧光通道PCR仪,采用多重PCR技术检测痰液标本中6种常见病原菌的基因检测方法,敏感性和特异性高,提高了检测效率。

Objective:To explore a multiplex real-time fluorescent quantitative PCR(MRT-PCR)method for the detection of six common respiratory pathogens.Methods:The primers and probes were designed with beacon designer 7.9 establish MRT-PCR.The lowest detectable limit and specificity of MRT-PCR were analyzed by plasmid standard constructing.Escherichia coli,Staphylococcus aureus,Pseudomonas aeruginosa,Klebsiella pneumoniae,Enterobacter cloacae and Acinetobacter baumannii in 176 clinical specimens were retrospectively detected by the established method,and the results were verified by gene sequencing of first generation.Results:The lowest detectable limit of MRT-PCR for six pathogens was 103 copies/mL.There were 19 cases of Escherichia coli,30 cases of Staphylococcus aureus,37 cases of Pseudomonas aeruginosa,53 cases of Klebsiella pneumoniae,15 cases of Enterobacter cloacae,22 cases of Acinetobacter baumannii,which were consistent with the traditional identification methods,and the specificity was 100%.The results of gene sequencing of the first generation were completely consistent.Conclusion:The gene detection of six common pathogens in sputum samples by multiplex PCR technology with multi fluorescent channel PCR instrument has high sensitivity and specificity,which improves the detection efficiency.