基于多重PCR和第二代高通量测序技术快速检测下呼吸道感染病原微生物方法的建立和应用

Establishment and application of rapid detection of pathogenic bacteria in lower respiratory tract infection based on multiplex PCR and next generation sequencing technology

目的针对引起下呼吸道感染的20种常见病原微生物,建立一种基于多重PCR和第二代高通量测序技术的快速、高通量检测方法AC-seq。方法经生物信息学分析,设计20种下呼吸道感染常见病原微生物特异性引物并通过PCR评估引物的敏感性和特异性。建立可进行第二代高通量测序技术的多重PCR扩增体系。采集34例临床肺泡灌洗液标本,对检测方法进行初步测试,并与临床培养结果进行对比评估检测效果。结果所有引物均具有良好的敏感性和特异性。在临床标本检测中AC-seq共检出13种病原微生物,培养法检出8种;AC-seq检测阳性率为79.41%,培养法为32.35%,差异有统计学意义(P<0.05)。AC-seq检测灵敏度为100%,与培养法的一致率为50%。检测时间比培养法缩短至少1 d。结论基于多重PCR和第二代高通量测序技术,成功建立了一种检测下呼吸道感染20种常见病原微生物的快速、高通量的方法。

Objective To establish a rapid and high-throughput detection method AC-seq based on multiplex PCR and next generation sequencing technology for 20 common pathogenic bacteria that caused lower respiratory tract infections.Methods According to bioinformatics analysis,20 kinds of common pathogens of lower respiratory tract infection specific primers were designed and the sensitivity and specificity of primers by PCR reaction were evaluated.A multiplex PCR amplification system that could perform next generation sequencing technology was established.Thirty four clinical alveolar lavage fluid samples were collected,and preliminary tests on the detection methods were conducted,and the clinical culture results were compared to evaluate the detection effect.Results All primers had good sensitivity and specificity.In the clinical specimen detection,AC-seq detected 13 kinds of pathogenic bacteria,culture method detected 8 kinds;AC-seq detection positive rate was 79.41%,culture method was 32.35%,the difference was statistically significant(P<0.05).AC-seq detection sensitivity was 100%,and the agreement rate with culture method was 50%.The detection time was shorter than the culture method by at least 1 day.Conclusion Based on multiplex PCR and next generation sequencing technology,a rapid and high-throughput detection method for 20 common pathogenic bacteria in lower respiratory tract infections has been successfully established.