细菌及真菌血流感染宏基因组学检测标本预处理方法探索  

Exploration of pretreatment methods for metagenomics detection specimens of bacterial and fungal bloodstream infection

作　　者：朱盈 杨启文[1] ZHU Ying;YANG Qiwen(Department of Clinical Laboratory,Peking Union Medical College Hospital,Chinese Academy of Medical Sciences,Beijing 100730,China;Peking Union Medical College,Chinese Academy of Medical Sciences,Beijing 100730,China)

机构地区：[1]中国医学科学院北京协和医院检验科,北京100730 [2]中国医学科学院北京协和医学院,北京100730

出　　处：《国际检验医学杂志》2021年第23期2834-2838,共5页International Journal of Laboratory Medicine

基　　金：国家自然科学基金资助项目(82072318);国家重点研发计划(2018YFC1200105);北京市临床重点专科医学检验科卓越项目(ZK201000)。

摘　　要：目的本研究主要针对血流感染患者标本存在大量人类核酸,对细菌及真菌宏基因组学测序检测带来的干扰进行探讨,设计了3种去宿主(人源)核酸方法:皂素去宿主核酸法(Saponin法)、SDS去宿主核酸法(SDS法)和水洗法,从中选出最优方法。方法向无菌血液标本中分别注入金黄色葡萄球菌、肺炎克雷伯菌、白色念珠菌模拟临床血流感染患者血液标本,对标本进行去宿主核酸处理、核酸提取、实时荧光定量聚合酶链反应,通过比较对照组及实验组标本的ΔCt值评价各种方法的去人源核酸效果及病原菌核酸丢失量。结果Saponin法和SDS法人源核酸去除效果较好:GAPDH基因ΔCt分别为10.13和11.13;β-actin基因ΔCt分别为12.76和14.30;水洗法中人源基因核酸发生了富集,GAPDH基因ΔCt=-4.23,β-actin基因ΔCt=-4.05。同时Saponin法和水洗法中肺炎克雷伯菌和白色念珠菌的核酸均发生富集:Saponin法ΔCt分别为4.24、1.07,水洗法ΔCt分别为2.41、0.88,金黄色葡萄球菌的核酸发生丢失;SDS法中肺炎克雷伯菌、白色念珠菌和金黄色葡萄球菌均发生了丢失:ΔCt分别为-1.09、-1.75、-4.56。结论综合考虑去宿主核酸及病原菌核酸丢失量2个方面,使用Saponin法处理含菌血液标本效果最理想。Objective This study mainly explores the interference caused by a large number of human nucleic acids in the samples from bloodstream infection patients on bacterial and fungal metagenomics sequencing detection.Three methods for de-host(human source)nucleic acids are designed:saponin de-host nucleic acid method(Saponin method),SDS de-host nucleic acid method(SDS method)and wash method,select the best method.Methods Injected Staphylococcus aureus,Klebsiella pneumoniae and Candida albicans into sterile blood samples to simulate the blood samples of patients with clinical blood stream infection,the samples were treated with de-host nucleic acid,nucleic acid extraction and real-time fluorescence quantitative polymerase chain reaction.By comparing theΔCt value of the samples of the control group and the experimental group,the effect of various methods of depleting human-derived nucleic acid and the amount of nucleic acid loss of pathogenic bacteria were evaluated.Results Saponin method and SDS method human-derived nucleic acid removal effect was better:GAPDH geneΔCt were 10.13 and 11.13,respectively;β-actin geneΔCt were 12.76 and 14.30,respectively.In the wash method,human-derived gene nucleic acid was enriched,GAPDH geneΔCt=-4.23;β-actin geneΔCt=-4.05.At the same time,the nucleic acids of Klebsiella pneumoniae and Candida albicans in the Saponin method and the wash method were enriched:ΔCt of Saponin method was 4.24,1.07,respectively,andΔCt of wash method was 2.41,0.88,respectively,and the nucleic acid of Staphylococcus aureus lost.In the SDS method,Klebsiella pneumoniae,Candida albicans and Staphylococcus aureus were all lost,ΔCt were-1.09,-1.75,-4.56,respectively.Conclusion Considering both the de-host nucleic acid and the loss of pathogenic nucleic acid,Saponin method is the best method to treat blood samples containing bacteria.

关 键 词：血流感染 去宿主核酸 病原菌核酸 标本前处理 宏基因组学 

分 类 号：R446.5[医药卫生—诊断学]