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

Abstract

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.