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

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 Nomenclature.The criteria for the bacterial identification was interpreted according to the Clinical and Laboratory Standards Institute （CLSI） 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, 11 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 Halomonas 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-cultured bacteria.（Chin J Lab Med,2012,35:612-619） Key words: RNA，ribosomal，16S; Polymerase chain reaction; Sequence analysis; Bacteriological techniques