摘要
目的建立一种宏基因组二代测序(mNGS)DNA流程检测BALF标本的性能确认方法。方法以Hela细胞代表宿主细胞,向无菌生理盐水中加入不同浓度Hela细胞、7种病原体(肺炎链球菌、流感嗜血杆菌、肺炎克雷伯菌、白色念珠菌、新型隐球菌、烟曲霉和腺病毒)和干扰物质制备模拟BALF标本,同时收集临床BALF标本,以传统检测方法为参比方法,评价mNGS检测结果,确定mNGS的最低检出限、精密度、抗干扰能力、稳定性和准确度。结果模拟标本中,肺炎链球菌、流感嗜血杆菌、肺炎克雷伯菌、白色念珠菌、新型隐球菌、烟曲霉和腺病毒7种病原体的最低检出限分别为150、262、102、67、96、83 CFU/ml和439拷贝/ml。模拟阳性标本中所有病原体重复检测结果符合率均为100%。抗干扰试验结果显示,人源核酸浓度越高,mNGS检出病原体序列数越少。以大肠埃希菌和宋内志贺菌评价mNGS对近源物种分辨能力,结果显示,当宋内志贺菌浓度为4000 CFU/ml时,该系统能稳定分辨大肠埃希菌和宋内志贺菌。稳定性试验结果显示,标本进行1、2、3次冻融或在4℃、-20℃、-80℃放置36 h后病原体序列数无明显变化。与传统检测方法相比,17份临床标本的初步准确度为82.4%(14/17)。2021年10月25日至2022年9月14日同济医院同时进行mNGS和传统方法检测的临床BALF标本的持续评估结果显示,mNGS相对细菌培养、真菌培养、分枝杆菌培养、结核分枝杆菌培养和常规PCR技术的准确度分别为67.5%(472/699)、81.5%(570/699)、92.3%(335/363)、96.4%(350/363)和86.8%(132/152)。与常规PCR技术比较,mNGS检测耶氏肺孢子菌、腺病毒、肺炎支原体的准确度分别为89.4%(84/94)、93.3%(56/60)和87.1%(61/70)。结论通过制备模拟BALF标本以及以传统检测方法为参比方法,可以初步评价mNGS检测BALF标本的性能特征。
Objective To establish a performance validation method for mNGS applied in BALF samples.Method Hela cells were used as a representative of host cells,and simulated BALF samples were prepared by adding different concentrations of Hela cells,seven species of isolated pathogens(including Streptococcus pneumonia,Hemophilus influenza,Klebsiella pneumonia,Candida albicans,Cryptococcus neoformans,Aspergillus fumigatus,and Adenovirus),and interfering substances to sterile normal saline.Clinical BALF samples were collected simultaneously,and the results of mNGS were evaluated using traditional detection methods as a reference.The limit of detection(LOD),precision,anti-interference ability,stability,and accuracy of mNGS were determined.Results In the simulated samples,the LOD of Streptococcus pneumoniae,Haemophilus influenzae,Klebsiella pneumoniae,Candida albicans,Cryptococcus neoformans,Aspergillus fumigatus,and Adenovirus were 150,262,102,67,96,83 CFU/ml,and 439 copies/ml,respectively.The repeatability of the detection results for all pathogens of simulated positive BALF samples was 100%.The anti-interference test showed that the higher the concentration of human DNA,the fewer pathogen sequences detected by mNGS.Escherichia coli and Shigella sonnei were used to evaluate the ability of mNGS to distinguish closely related species.The results showed that the system could stably distinguish Escherichia coli and Shigella sonnei when the concentration of Shigella sonnei was 4,000 CFU/ml.The stability test results showed that there was no significant change in the number of pathogen sequences detected whether after 1 to 3 freeze-thaw cycles or storage at 4℃,-20℃,or-80℃for 36 h.Compared with traditional detection methods,the accuracy of 17 clinical samples was 82.4%(14/17).Continuous evaluation of clinical BALF samples simultaneously tested by mNGS and traditional methods at Tongji Hospital from October 25,2021,to September 14,2022,showed that the accuracy of mNGS compared to bacterial culture,fungal culture,mycobacterial
作者
宋慧娟
鲁艳军
田磊
陈中举
汪玥
刘为勇
毛莉妍
孙自镛
彭静
Song Huijuan;Lu Yanjun;Tian Lei;Chen Zhongju;Wang Yue;Liu Weiyong;Mao Liyan;Sun Ziyong;Peng Jing(Department of Laboratory Medicine,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430000,China)
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2023年第10期1067-1073,共7页
Chinese Journal of Laboratory Medicine
基金
国家科技基础资源调查专项(2019FY01206)。
关键词
支气管肺泡灌洗液
宏基因组二代测序
性能确认
准确度
结核分枝杆菌
Bronchoalveolar lavage fluid
Metagenomic next-generation sequencing
Performance validation
Accuracy
Mycobacterium tuberculosis
