16S rRNA基因序列鉴定标本中病原细菌的方法学研究

16S rRNA gene sequencing for pathogen identification from clinical specimens

目的研究16S rRNA基因序列鉴定标本中病原微生物的关键技术，评价序列分析技术作为病原微生物检测方法的可行性和实用件。方法对117份不同来源的临床标本分别采用形态学、细菌培养、16S rRNA基因扩增与测序分析，比较不同方法对病原菌检出的灵敏度与特异性。分子技术通过改良的微量核酸提取方法，扩增16S rRNA基因两个区域（BSF8-BSR534和BAK11 w-BAK2）。结果细菌培养和PCR检测的阳性率分别是49％（57／117）和72％（84／117），63％（53／84）的PCR产物通过直接测序鉴定到种；60例（52％）培养阴性标本中有7例（12％）经序列分析再次鉴定出病原菌；形态学检查阳性率为64％（75／117），与PCR结果接近，两者结合可提供推测性报告。第1对引物（扩增区：BSF8-BSR534）较适合革兰阳性细菌分类，第2对引物（扩增区：BAK11w-BAK2）对临床常见病原菌均可获得理想的序列。结论改进核酸提取技术、筛选恰当的引物、优化基因扩增和测序流程，通过16S rRNA基因序列直接鉴定标本中病原微生物有着广阔的应用前景。

Objective To investigate the key point technique with 16S rRNA gene sequence for identifying the pathogens in clinical specimens, and to evaluate the feasibility and practicability as a routine tool for determining pathogens in clinical laboratory. Methods 117 clinical specimens from different sources underwent morphological examination, bacterium culture, and 16S rRNA gene PCR and sequencing. Two parts of 16S rRNA gene （BSF8-BSR534 and BAKllw-BAK2） were amplified by PCR, the products of PCR were purified by gel isolation, sequencing was conducted for the fragments, and then the sequence was put in GenBank BLAST to compare with data-base in NCBI. If the similarity of different original sequences was more than 99% , the bacterium could be considered as the homogeneous species. The sensitivity and specificity for pathogens identification were compared among different methods. Results The positive rates of bacterium culture and PCR test were 49% （57/117） and 72% （84/117） respectively. 7 bacteria could be classified as the pathogens from the 60 cases negative by culture methods. The positive rate of direct smearing was 64% （75/117） ; similar to that of the PCR consequence, and the combination of both methods could provide inferential reports. The 16S rRNA gene was perfect relativity with bacterial evolution and phylogenesis, and it could be classified species by the sequencing. The first pair of primer of the 16S rRNA gene （amplification area： BSF8-BSR534） was suitable for the Gram positive bacterium, and second pair of primer （amplification area： BAK11w-BAK2） was suitable for the common pathogens in the clinical specimens. Conclusion By improving the extract DNA method, selecting suitable primer, developing the gene amplification and sequencing procedure, direct 16S rRNA gene sequencing can identify the pathogens from clinical specimens.