基于细菌16S rRNA基因扩增临床不常见病原菌的价值

目的探讨16S rRNA基因扩增与测序在临床不常见病原菌鉴定中的价值,指导临床相关感染的诊治。方法选择临床微生物实验室常规方法难以准确鉴定、无法鉴定或有特殊表型的细菌12株,采用聚合酶链反应（PCR）扩增其16S rRNA基因,测序后进行BLAST比对鉴定菌种,并分析相关感染的临床特点。结果12株菌经PCR扩增均得到阳性条带（约1 500 bp）,均鉴定到种（相似度≥99%）,分别为产单核细胞李斯特菌、马耳他布鲁杆菌各2株,死亡梭杆菌、空间罗氏菌、鼻疽奴卡菌、解糖葡萄球菌、放射性根瘤菌、二路普雷沃菌、解甘露醇罗尔斯顿菌及阴道阿托波菌各1株。16S rRNA基因扩增灵敏度高,大肠埃希菌ATCC 25922的最低检测限为1.5×101 CFU/mL。12例患者临床资料显示上述菌可引起临床多部位、多类型感染,选用针对性抗菌药物治疗后11例好转,1例死亡。结论16S rRNA基因测序方法可快速、准确鉴定少见菌、厌氧菌及难培养细菌,为临床不同类型感染的病原学诊断和治疗提供实验室依据。

Value of 16S rRNA gene amplification for identification of clinical rare pathogens

ObjectiveTo evaluate the value of amplification and sequencing of 16S rRNA gene in the identification of clinical rare pathogenic bacteria, and guide the diagnosis and treatment for related clinical infection.Methods12 bacterial isolates that were difficult, or unable to be identified with conventional laboratory methods, or with special phenotypes were collected. The 16S rRNA gene was amplified by polymerase chain reaction (PCR), then sequenced for identifying bacterial species through BLAST comparison, clinical characteristics of related infection were analyzed.Results12 clinical isolates were all positive for PCR amplification (about 1 500 bp), species were all identified (similarity ≥99%), the identified strains were Listeria monocytogenes(n=2), Brucella melitensis(n=2), Fusobacterium mortiferum(n=1), Rothia aeria(n=1), Nocardia farcinica(n=1), Staphylococcus saccharolyticus(n=1), Rhizobium radiobacter(n=1), Prevotella bivia(n=1), Ralstonia mannitolilytica(n=1), and Atopobium vaginae(n=1). The sensitivity of 16S rRNA gene amplification was high, and the minimum detection limit of Escherichia coli ATCC 25922 was 1.5×101 CFU/mL. Clinical data of 12 patients revealed that these strains can cause multi sites and multi types of infection,after patients received targeted antimicrobial therapy, 11 improved, and 1 died.ConclusionSequencing for 16S rRNA gene can rapidly and accurately identify rare, anaerobic, and difficult cultured bacteria, provide laboratory evidence for etiological diagnosis and treatment of different types of infection.