二代测序技术在人工关节感染关节液病原菌检测中的应用

摘要

Objective To investigate the role of next generation sequencing technology in the detection of pathogenic bacteria in synovial fluid of prosthetic joint infection.Methods Nine samples of synovial fluid specimens of prosthetic joint infection patients with positive microbial culture from October,1 2016 to April 1,2017 were collected.Each specimen (200 μl) was used for next generation sequencing.Total DNA was extracted from synovial fluid samples.The collected DNA samples were amplified by PCR in the V4 region of 16S rDNA gene.The amplified products were sequenced using the Illumina Miseq platform,2× 250 bp double-end sequencing strategy.The sequencing results were compared with the SILVA database to analyze the types of bacteria and relative abundance in the DNA samples.A total of 200 μl sterile double-distilled deionized water was used as control.Results Nine cases of microbial culture positive prosthetic joint infection synovial fluid DNA samples were sequenced by 16S rDNA amplicon sequencing and yielded 3 132 415 high-quality reads and 3 752 operational taxonomic units (OTU).At the level of bacteria,a total of 9 different bacterial gates were detected on 9 DNA samples.At the level of bacteria,34 different bacteria were detected by 16S rDNA amplicon sequencing.Each DNA sample was detected by 16S rDNA amplicon sequencing and the bacterial genus was identical to that of laboratory culture.16S rDNA amplicon sequencing detected more species of bacteria [6(3,9.5)] than bacterial cultures [(1.0(1.0,1.0)].There was statistically significant difference in the number of bacteria detected in the same specimen between the 16S rDNA amplicon sequencing and the laboratory culture (Z=2.533,P=0.011).Among them,the dominant bacterial population (highest abundance) detected by 16S rDNA amplicon sequencing in four DNA samples was consistent with the results of laboratory culture.Conclusion In the prosthetic joint infection,the 16S rDNA amplicon sequencing technology can accurately detect pathogens that are consistent with the laboratory culture,and can detect other bacteria outside the laboratory culture.This technology can provide the basis for clinical diagnosis and antibiotic selection.%目的 探讨二代测序技术在人工关节感染关节液病原菌检测中的作用.方法 收集2016年10月1日至2017年4月1日微生物培养阳性的人工关节感染患者的关节液标本9例,每例标本取200μl用于二代测序,提取关节液标本总DNA,对收集的DNA标本行16S rDNA基因V4区PCR扩增,应用Illumina Miseq平台,2×250 bp双端测序策略对扩增产物进行测序,测序结果与SILVA数据库进行比对,分析DNA标本中细菌的种类及相对丰度,取200μl无菌双蒸去离子水作空白对照.结果 9例微生物培养阳性人工关节感染关节液DNA标本通过16S rDNA扩增子测序共产生3 132415条高质量的序列和3 752个可操作分类单元(operational taxonomic units,OTU).在细菌门水平,9个DNA标本共检出9种不同的细菌门;在细菌属水平,16SrDNA扩增子测序共检出34种不同的细菌;每个DNA标本通过16SrDNA扩增子测序都检出与实验室培养一致的细菌属;16S rDNA扩增子测序检出的细菌属种类[平均每个标本6.0(3.0,9.5)种]显著多于细菌培养检出的细菌属种类[平均每个标本1.0(1.0,1.0)种],16S rDNA扩增子测序和实验室培养在同一份标本细菌检出数目的差异有统计学意义(Z=2.533,P=0.011).其中,4例DNA标本采用16S rDNA扩增子测序检出的优势菌群(丰度最高)与实验室培养结果一致.结论 在人工关节感染中,16S rDNA扩增子测序技术可准确检出与实验室培养一致的病原菌,还可检出培养之外的其他细菌,为临床诊断及抗生素选择提供依据.