一种病原宏基因高通量测序方法的开发与性能确认  

Development and performance verification of a high throughput sequencing method for pathogenic microbial metagenomics

作　　者：柴海云 黄月眉 谭海燕 王威 CHAI Haiyun;HUANG Yuemei;TAN Haiyan;WANG Wei(Guangdong Seehow Medical Laboratory Co.,Ltd.,Foshan 528200,Guangzhou,China)

机构地区：[1]广东芯海医学检测实验室有限公司,广东佛山528200

出　　处：《临床检验杂志》2025年第1期1-6,共6页Chinese Journal of Clinical Laboratory Science

基　　金：佛山市自筹经费类科技计划项目(2020001005263)。

摘　　要：目的建立一种病原宏基因高通量测序方法,并验证其检测性能。方法筛选不同核酸投入量的文库构建条件,分别测试5 ng、50 ng、100 ng 3种核酸投入量,20 min、25 min、30 min 3种片段化时间,7个、5个、3个3种扩增循环数,通过分析文库浓度和片段大小,确定最佳建库条件。选择真菌、革兰阳/阴性细菌的代表菌:白念珠菌、金黄色葡萄球菌、表皮葡萄球菌、大肠埃希菌和格特隐球菌,按照1∶1的投入量,制备3种不同浓度混合菌悬液,分别添加10^(6)个/mL的T细胞悬液,制备参考品。通过参考品确认自建方法(方法1)的性能,并以方法2(DNA样本文库构建试剂盒联合探针锚定聚合测序法)为对照,确认两种方法一致性。结果确定了新建病原宏基因测序鉴定方法的最佳建库条件:核酸投入量50 ng,核酸打断时间20 min,PCR扩增循环数5个。方法1阳性符合率(准确性)、阴性符合率(特异性)可达100%,检出限为500 CFU/mL,线性样本中各种微生物检出每百万序列数(reads per million,RPM)的相关系数(r)>0.9,细菌检出变异系数(CV)在10%以内,真菌CV约为20%。以方法2对照,二者检测结果一致性为100%。结论建立一种病原微生物宏基因测序鉴定方法,该方法结果准确、稳定可靠,适用于肺泡灌洗液类样本的病原微生物检测。Objective To develop a high throughput sequencing method for pathogenic microbial metagenomics and verify its detection performance.Methods First,the library construction conditions with different input levels of nucleic acid were studied.Three different input levels of nucleic acid such as 5 ng,50 ng,and 100 ng,three fragmentation times such as 20 min,25 min,and 30 min,and three amplification cycles such as 7,5,and 3 cycles were tested.The optimal library construction conditions were determined by the library concentration and fragment size.The representative bacteria covering fungi and gram-positive/negative bacteria,including Candida albicans,Staphylococcus aureus,Staphylococcus epidermidis,Escherichia coli and Cryptococcus Gattii,were selected.Three different concentrations of mixed bacterial suspensions were prepared at a 1∶1 ratio.Then,10^(6)/mL of T-cell suspensions were added to prepare reference samples.The performance of the self-established method(Method 1)was verified by the reference samples,and its consistency with Method 2(DNA sample library construction kit combined with probe anchored polymerase chain reaction sequencing method)was compared.Results The optimal library construction conditions of the sequencing method for pathogenic microbial metagenomics were determined as follows:a nucleic acid input of 50 ng,a nucleic acid fragmentation time of 20 min,and 5 amplification cycles for PCR.The positive coincidence rate(accuracy)and negative coincidence rate(specificity)of Method 1 were 100%,and its detection limit was 500 CFU/mL.The correlation coefficients(r)of the reads per million(RPM)in linear samples of various microorganisms were greater than 0.9.The CV of bacterial detection were less than 10%,while those of fungal detection were around 20%.The consistency between Method 1 and Method 2 was 100%.Conclusion A sequencing method for pathogenic microbial metagenomics is established successfully,which can acquire accurate,stable,and reliable results and is suitable for the pathogen detection of a

关 键 词：病原微生物 高通量测序 性能验证 

分 类 号：R446.5[医药卫生—诊断学]