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Development and validation of multiplex polymerase chain reaction assay for concomitant detection of genus Staphylococcus and clinically relevant methicillin resistance determinants

Nimita Venugopal, Feroze Ganaie, Susweta Mitra, Rituparna Tewari, Tushar K. Dey, Rakshith Ojha, Rajeswari Shome, Bibek R. Shome.

Cited by 1 Articles

The increasing emergence of methicillin-resistant staphylococci (i.e., methicillin-resistant Staphylococcus aureus [MRSA] and methicillin-resistant coagulase-negative staphylococci [MRCoNS]) has become a threat globally for both human and animal populace. Phenotypic detection of MRSA and MRCoNS is a less sensitive and time-consuming approach which affects the treatment outcome. Thus, a rapid and accurate method is needed for an early diagnosis of MRSA/MRCoNS infections. The present study aimed at standardization and validation of a multiplex polymerase chain reaction (mPCR) assay to detect genus Staphylococcus (16s rRNA gene) and methicillin-resistance determinants (mecA and mecC genes) simultaneously. The assay characteristics were evaluated against 53 well characterized strains comprising of 40 Staphylococcus and 13 non-Staphylococcus strains. Among Staphylococcus strains, 32 were mecA positive and one strain was mecC positive. The lower limit of detection of the mPCR assay was 1ng/mL (Genome copies: 16S rRNA = 1.1 × 109; mecA = 3.17 × 109; mecC = 1.6 × 109), with analytical sensitivity and specificity of 100%. The mPCR assay developed in the study is useful for rapid and accurate diagnosis of MRSA/MRCoNS infections. The assay can be an important diagnostic as well as surveillance tool to investigate the emergence and dissemination of methicillin-resistant staphylococci which is of both clinical and public health significance.

Key words: Multiplex polymerase chain reaction, Staphylococcus, methicillin resistance, mecA/mecC genes

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