A Multiplex PCR Assay for the Rapid and Sensitive Detection of Methicillin‐Resistant Staphylococcus aureus and Simultaneous Discrimination of Staphylococcus aureus from Coagulase‐Negative Staphylococci

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is a global health concern, which had been detected in food and food production animals. Conventional testing for detection of MRSA takes 3 to 5 d to yield complete information of the organism and its antibiotic sensitivity pattern. So, a rapid method is needed to diagnose and treat the MRSA infections. The present study focused on the development of a multiplex PCR assay for the rapid and sensitive detection of MRSA. The assay simultaneously detected 4 genes, namely, 16S rRNA of the Staphylococcus genus, femA of S. aureus, mecA that encodes methicillin resistance, and one internal control. It was rapid and yielded results within 4 h. The analytical sensitivity and specificity of the multiplex PCR assay was evaluated by comparing it with the conventional method. The analytical sensitivity of the multiplex PCR assay at the DNA level was 10 ng DNA. The analytical specificity was evaluated with 10 reference staphylococci strains and was 100%. The diagnostic evaluation of MRSA was carried out using 360 foodborne staphylococci isolates, and showed 99.1% of specificity, 96.4% of sensitivity, 97.5% of positive predictive value, and 97.3% of negative predictive value compared to the conventional method. The inclusion of an internal control in the multiplex PCR assay is important to exclude false-negative cases. This test can be used as an effective diagnostic and surveillance tool to investigate the spread and emergence of MRSA.

Microbiologic Spectrum and Antibiotic Susceptibility Pattern among Patients with Urinary and Respiratory Tract Infection

“…The isolated colony was confirmed by the pattern of biochemical reactions using the standard method [10] and also by PCR for the type of microorganism [11]. Antibiotic susceptibility was evaluated by Kirby-Bauer disc diffusion method on Mueller-Hinton agar according to the guidelines of Clinical and Laboratory Standards Institute (CLSI) [12] and antibiogram was obtained.…”

Species Diversity and Pheno‐ and Genotypic Antibiotic Resistance Patterns of Staphylococci Isolated from Retail Ground Meats

“…They are ubiquitous in soil, water, air, dust, and foods such as meat and milk (Marino and others ). Frequently, members of the genes are identified genetically based on their 16S or 23S rRNA gene regions, and Staphylococcus aureus is differentiated from other staphylococci by examining the nuc gene that encodes extracellular thermostable nuclease as a marker for S. aureus (Stuhlmei and Stuhlmei ; Xu and others ). Both coagulase‐positive and coagulase‐negative staphylococci (CoNS) are important public health problems worldwide (Miko and others ).…”

“…Both coagulase‐positive and coagulase‐negative staphylococci (CoNS) are important public health problems worldwide (Miko and others ). Toxins synthesized by staphylococci are involved in food poisoning, postsurgical infections, septicemia, and toxic shock syndrome (Marino and others ; Xu and others ; Miko and others ). One of these toxins, Panton‐Valentine Leukocidin (PVL), possesses cytotoxic properties and causes leukocyte fragmentation and tissue necrosis (Argudin and others ).…”

Incidence of Methicillin-Resistant Staphylococci in Fresh Seafood

“…DNA of Staphylococcus aureus ATCC BAA-976) was used as the positive control in all PCR reactions. The thermocycling conditions for the amplifications of 16S rDNA, femA, nuc and mecA genes were same as previously described [23]- [25]. For the amplification of lmrS gene using the primers designed in this study, the thermocycling conditions consisted of 1 min denaturation at 94˚C, 1 min annealing at 55˚C and 1 min extension at 72˚C.…”