基于多重PCR靶向测序技术建立临床病原菌检测方法  

Establishment of clinical pathogen detection method based on multiplex PCR⁃targeted sequencing technology

作　　者：黄晓园 郑凯文 张俊杰 徐鸿绪[2] 王菊芳[1] HUANG Xiaoyuan;ZHENG Kaiwen;ZHANG Junjie;XU Hongxu;WANG Jufang(School of Bioscience and Bioengineering,South China University of Technology,Guangzhou,Guangdong,China,510006;Department of Laboratory Medicine,the First Affiliated Hospital,Sun Yat-sen University,Guangzhou,Guangdong,China,510080)

机构地区：[1]华南理工大学生物科学与工程学院,广东广州510006 [2]中山大学附属第一医院医学检验科,广东广州510080

出　　处：《分子诊断与治疗杂志》2023年第9期1473-1477,共5页Journal of Molecular Diagnostics and Therapy

基　　金：广东省重点研发计划(2020B1111160004)。

摘　　要：目的针对不同临床样本(肺泡灌洗液、痰液、血液)中常见的22种病原菌,建立一种基于多重PCR靶向二代测序(mPCR⁃NGS)的高通量检测方法。方法选取2018年10月至2019年2月中山大学附属第一医院和广州军区广州总医院共84例临床样本和5种病原菌混合的模拟感染样本,对检测方法进行测试,并根据测序序列区分病原菌种类。通过生物信息学方法筛选和设计三种临床样本中常见22种病原菌的特异性引物,并通过多重PCR和二代测序调整引物扩增效率,建立多重PCR靶向二代测序高通量检测体系。结果PCR验证结果表明,针对22种病原菌所设计的44对引物均具有良好特异性;引物终浓度经调整,扩增均一值在0.33~2.37之间。本研究建立的mPCR⁃NGS方法对鲍曼不动杆菌、白色念珠菌、肺炎链球菌、大肠埃希氏菌和金黄色葡萄球菌5种菌混合的模拟样本进行验证,其检测下限是3 copies。在11例血液样本中,有6例样本属于单一感染,与临床结果完全一致;5例样本属于混合感染,其中样本1、3、7、10的病原菌感染种类与临床结果不完全一致。在84例临床样本检测中,mPCR⁃NGS共检测出16种病原菌,其中检出最多的病原菌是鲍曼不动杆菌(28/84),次之是缓症链球菌(13/84),第三位是嗜麦芽窄食单胞菌(12/84);mPCR⁃NGS检测阳性率为69.05%,高于临床培养结果的阳性率(53.57%);两种检测方法结果完全一致的样本共39例,总一致率为46.43%。结论多重PCR靶向二代测序检测技术是一种快速、准确、高效的检测方法,可用于肺泡灌洗液、痰液、血液中22种临床常见病原菌的快速、高通量检测。Objective To Establish a high⁃throughput detection method based on multiplex PCR⁃targeted next⁃generation sequencing(mPCR⁃NGS)for 22 common pathogenic bacteria in different clinical samples(alveolar lavage fluid,sputum,blood).Methods From October 2018 to February 2019,a total of 84 clinical samples and simulated infection samples mixed with 5 pathogenic bacteria were selected from the First Affiliated Hospital of Sun Yat⁃sen University and Guangzhou General Hospital of Guangzhou Military Region to test the detection method,and the types of pathogens were distinguished according to the sequencing results.The specific primers for 22 common pathogens in three clinical samples were screened and designed by bioinformatics methods,and the amplification efficiency of the primers was adjusted by multiplex PCR and next⁃generation sequencing to establish a high⁃throughput detection system for multiplex PCR⁃targeted next⁃generation sequencing.Results The results of PCR verification showed that the 44 pairs of primers designed for 22 pathogenic bacteria had good specificity.The final primers concentration was adjusted,and the average amplification value was between 0.33 and 2.37.The mPCR⁃NGS method established in this study was validated against the simulated samples mixed with 5 species of Acinetobacter baumannii,Candida albicans,Streptococcus pneumoniae,Escherichia coli and Staphylococcus aureus,and the detection limit was 3 copies.Among the 11 blood samples,6 samples belonged to single infection,which were completely consistent with the clinical results.5 samples belonged to mixed infections,and the types of pathogenic bacteria in samples 1,3,7,and 10 were not completely consistent with the clinical results.In the detection of 84 clinical samples,mPCR⁃NGS detected a total of 16 pathogenic bacteria,among which Acinetobacter baumannii(28/84)was the most detected pathogen,followed by Streptococcus mitis(13/84).Three were Stenotrophomonas maltophilia(12/84);the positive rate of mPCR⁃NGS was 69.05%,whic

关 键 词：二代测序 多重PCR 临床病原菌 高通量检测 

分 类 号：R446.5[医药卫生—诊断学]