基于细菌16SrRNA基因的PCR扩增与测序分析在临床不常见菌鉴定中的应用

Application of broad-spectrum PCR amplification and direct sequencing for identification of the infrequent bacterial cultures from clinical sources, targeting the bacterial 16S rRNA gene with universal primes

目的探讨细菌16SrRNA基因的广谱PCR扩增与测序分析在临床分离的少见病原菌的鉴定及分类中的价值。方法收集2010年12月至2011年9月来自7家不同医院和机构，常规方法难以鉴定或具有特殊表型的少见菌48株，以及仪器法连续监测报警阳性、但二次传代无细菌生长的液体增菌培养瓶7个，采用细菌16S核糖体核糖核酸（rRNA）基因MicroSeq500试剂盒、通用引物27f-1492r、27f-1525r进行广谱PCR扩增及目的片段的基因测序。运用美国国立生物信息中心“基于局部比对算法的搜索工具（Blast）”，结合“具有法定分类学地位的原核生物名称目录（http：／／W^Ufir．bacterio．cict．fr／）”提供的分类学信息，比较待鉴定细菌与近缘种模式菌株基因序列的相似程度；参考美国临床与实验室标准化协会（CLSI）MM18-A的解释标准，以确定待鉴定细菌的种属及分类学位置。结果采用广谱PCR测定，7份假阳性血培养瓶中，2份扩增到细菌的16SrRNA基因，经序列分析鉴定均为肺炎链球菌。48株不同来源、不同种属的少见菌，均扩增到16SrRNA基因目的片段并成功测序，参考CLSIMM18-A的解释标准，35株（72．9％）直接鉴定到“种”，11株（22．9％）鉴定到“属”，另2株鉴定为可能的新属新种。进一步结合细菌的生化反应特征及其他管家基因序列分析结果，则可鉴定到“种”的细菌可增加到42株（87．5％），其中包括链杆菌、嗜二氧化碳噬纤维菌、玫瑰单胞菌、奴卡菌、弯曲菌、分枝杆菌等具有明确临床意义的少见菌，以及多株近十年内命名的新细菌，如小不动杆菌、富西亚分枝杆菌、黏液玫瑰单胞菌、Halomonasjohnsoniae等。此外，还发现了1个新亚型的胎儿弯曲菌。结论16SrRNA基因测序鉴定能明确提供细菌的遗传学信息，且无种属选择性，对临床少见菌、苛养菌、以及固体培养基不生长的纯培养物等具有独特的优势，是病原微生物鉴定的发展责向.

Objectives To identify the infrequent strains in clinical isolates by broad-spectrum PCR amplification and direct sequencing targeting the bacterial 16S rRNA gene. Methods Total 48 clinical isolates and 7 false-positive blood culture samples were collected from 7 different hospitals or institutions from December 2010 to September 2011. The bacterial 16S rRNA gene were amplified and sequenced by universal prime sets of 27f-1492r and 27f-1525r, and MicroSeq 500 16S rRNA gene kit. The homology analysis was used by the Basic Local Alignment Search Tool, and comparing to gene sequence of the type strain, provided by the List of Prokaryotic names with Standing in Nomenclatm＇e. The criteria for the bacterial identification was interpreted according to the Clinical and Laboratory Standards Institute （CLS1） MM18-A. Results All of the 48 cultured strains were succeeded amplifying and sequencing the targeted 16S rRNA genes. According to the criteria of CLSI MM18-A, total 35 strains were specified to the species level, 1 l strains were specified to the genus level, and the other 2 strains were specified to possible novel genus and species. Combining the analysis the sequence of other housekeeping gene with the results of biochemical results, total 42 strains can be specified to the species level, including some clinical important pathogens, such as Streptobacillus, Capnocytophaga, Nocardia, Mycobacterium, Roseomonas and Campylobacter. Two false-positive blood culture samples were managed to amplify 16S rRNA genes and finally identified as Streptococcus pneumoniae. We also identified one novel subspecies of Campylobacter fetus, and some new valid-published species, such as Acinetobacter parvus, Mycobacterium phocaicum, Roseomonas mucosa and ttalomonas johnsoniae. Conclusions The 16S rRNA gene sequence based identification has unique advantages over the phenotypic methods. It is universal to almost of all the bacteria, and can provide the genetic classified information. It is very suitable for the clinical infrequent and special bacterial cultures, such as the slow-growing, fastidious, or un-cuhured bacteria. （ Chin J Lab Med ,2012,35：612-619）