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 BAK11w-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.