16s PCR is quite enough.

16s PCR is quite enough but this is a very sensitive test and can easily get false positive results. Therefore, you should have a positive, reliable control and a negative control for each test run

Detection of 16S rRNA gene can indicate that this organism is Staph.aureus after using of Basic Local Alignment Search Tool Programme that will makes cluster analysis.Also with using specific primers,you can make different runs to differentiate between VRSA,VISA and VRSA.A lot of PCR runs also can be carried out to elucidate virulence genes which could be used as strain markers.

Goodluck

And all Staph. aureus are not pathogenic too. Therefore, other tests besides 16s RNA are required for true epidemiology of pathogenic staph as suggested by Gamal Enan.

@Gamal Enan, i was also thinking the way you answered. You are right. Thank you very much for your valuable input.

Goodluck Amjad

Whether or not a Staph aureus is pathogenic depends on the clinical context and on the presence of virulence factors. 16S rDNA PCR, however, may help you in identifying the bug but not in rating its actual virulence/pathogenicity.

A Staph aureus isolated from the skin surface or mucous membranes may reflect carrier status (i.e. not being virulent under that specific circumstances). The same Staph aureus may be rated virulent (“pathogenic”) if isolated from an infected wound or from mastitis. The same Staph. aureus may be able to express enterotoxins in dairy products, thus rendering it virulent from a food making perspective.

If you want to go for characterization of virulence of Staph aureus isolates, you need PCRs to detect the genes for enterotoxins, PVL, TSST, and so on. Multiplex PCRs have been published and may easily be found on MedLine.

16S rDNA PCR with subsequent sequencing will only allow you to identify the species (Staph aureus vs. other staphylococci). However, be aware that you need a good sequence of at least the first 500 bp to discriminate reliably between members of the genus Staphylococcus.

If preventative measures and/or epidemiology are of interest, 16S and even multiplex of virulence factors may not be helpful. Rapid identification is crucial (16S or MALDI, mecA/mecC PCR for MRSA) but epidemiology requires more elaborate techniques (pulsed-field gele electrophoresis, MLST or whole genome sequencing).

Oliver Nolte, Your response is very informative. I am intending to optimize multiplex PCR for prevalent strains of staph aureus involved in mastitis (as you mentioned in last paragraph). For more betterment in epidemiological study of s aureus, i will look into PFGE or MLST etc,.

Full length or V3-V5 regions of 16S PCR sequencing should be enough to identify S. aureus. Avoid only region V3 sequencing as used in PCR-SSCP, because it doesn't discriminate S. delphini/intermedious/pseudointermedious group. However V3 region may accurately identify S. aureus. I agree with Oliver Nolte in defining the pathogenic potential.