Development of a PCR assay for the identification of staphylococci by cpn60 and tuf genes
Gergal Nagabushana Rao, Rakesh Rao |
Molekuliniai metodai tapo labai svarbūs identifikuojant bakterijas. Per pastariuosius metus PGR ir DNR sekvenavimas tapo pagrindiniai įrankiai nustatant tiksliai bakterijų rūšį. Bakterijų ir jų taksonomijos tyrimams dažnai naudojoma 16S ribosominė RNR geno sekoskaitos analizė. Kaip filogenitikos žymuo bakterijų indentifikacijai naudojamas 16S rRNR genas, dėl savo labai konservatyvių ir ypač variabilių regionų. Stafilokokų identifikavimui dažniausia pasirenkami: SodA, tuf, cpn60, gap, rpoB genai. Tyrime pritaikytas PGR metodas. PGR reakcijoms buvo pasirinkti specifinis Stafilokoko CoNS genas cpn60 ir universalus tuf genas. Atrinkti buvo išbandyti su klinikiniais pavyzdžiais. Sekvenavimo rezultatai buvo palyginti su 16S rRNR rezultatais. Sekvenavimo analizė parodė, kad cpn ir tuf genai yra tinkami naudojant diskriminavimo metodą tam tikroms CoNS rūšims. Taip pat šis metodas tinka nustatyti ir klinikiniams izoliatams.
Molecular techniques have gained importance over the conventional biochemical tests for the identification of bacteria. Over the years PCR and DNA sequencing, have played a pivotal role in the accurate detection of bacterial species. Sequence analysis of the 16S ribosomal RNA gene has been widely used to identify bacterial species and taxonomic studies. Hence 16S rRNA gene are common used as a phylogenetic marker (highly conserved and hypervariable regions) which is helpful for the identification of bacteria. As the region of 16Sr RNA has low discriminatory power. With advance in research the possibility to work on various loci has been exploited (SodA, tuf, cpn60, gap, rpoB) which have being useful for the staphylococcus identification. In this study, we development a PCR based assay for the identification of staphylococci especially CoNS by cpn60 (Staphylococcus species specific) and tuf genes (universal). Both the primers were optimized with reference strains and were cycle sequenced. It was also tested on clinical samples (isolates and direct clinical samples).Sequencing results were compared with results of 16S rRNA. Sequencing analysis of the cpn and tuf proved to be more discriminative for certain CoNS species. Further, this method exhibited better distinction in the identification of clinical isolates.