Direct identification of mycobacteria from culture media using a multiplex real-time PCR assay: report on its application in a clinical laboratory in a region of high tuberculosis endemicity.

There are few commercial assays that easily and correctly identify the mycobacteria from culture in a clinical laboratory with a high workload. Thus, we developed and evaluated a scheme for the identification of mycobacteria using a multiplex real-time PCR assay and report on its application in our laboratory. The scheme consisted of 3 stepwise PCRs.

Multiplex real-time PCR assay for detection of methicillin-resistant Staphylococcus aureus (MRSA) strains suitable in regions of high MRSA endemicity.

A multiplex real-time PCR assay that simultaneously detects the mecA, staphylococcal cassette chromosome (SCCmec)-open reading frame X (orfX) junction, and staphylococcal 16S rRNA genes was developed and evaluated using 444 staphylococcal strains. We demonstrated that this assay resulted in fewer false-positive results than a single-locus real-time PCR assay that amplified the SCCmec-orfX junction. This assay would be useful in a clinical laboratory in a region of high endemicity for methicillin-resistant Staphylococcus aureus (MRSA) infections.

Multiplex real-time PCR assay and melting curve analysis for identifying Mycobacterium tuberculosis complex and nontuberculous mycobacteria.

A multiplex real-time PCR assay and melting curve analysis for identifying 23 mycobacterial species was developed and evaluated using 77 reference strains and 369 clinical isolates. Concordant results were obtained for all 189 (100%) isolates of the Mycobacterium tuberculosis complex and 169 (93.9%) isolates of nontuberculous mycobacteria.

[Detection of vancomycin-resistant enterococci using multiplex real-time PCR assay and melting curve analysis].

Background: We developed and evaluated the utility of a multiplex real-time PCR assay that uses melting curve analysis and allows simultaneous identification of vancomycin-resistant genotypes and clinically relevant enterococci.

Methods: The specificity of the assay was tested using 4 reference strains of vancomycin-resistant enterococci (VRE) and 2 reference strains of vancomycin-susceptible enterococci. Ninety-three clinical isolates of enterococci with different glycopeptide-resistant phenotypes were genotyped and identified using a multiplex real-time PCR assay and melting curve analysis.