Multiplex Real-Time PCR for Rapid Staphylococcal Cassette Chromosome mec Typing

Abstract

Rapid identification and typing of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is important for understanding the molecular epidemiology and evolution of MRSA and offers many advantages for controlling transmission in both health care and community settings. We developed a rapid molecular beacon real-time PCR (MB-PCR) assay for staphylococcal cassette chromosome mec (SCCmec) typing. The design of this system is based on the established definition of SCCmec types, namely, the combination of the mec class complex with the ccr allotype. The assay consists of two multiplex panels, the combination of which results in two targets (mec class, ccr) for each SCCmec type. MB-PCR panel I targets mecA, ccrB2, mecI, and the ⌬mecR1-IS1272 junction (mec class B); it can definitively identify SCCmec types II and IV. MB-PCR panel II detects ccrC, ccrB1, ccrB3, ccrB4, and the ⌬mecR1-IS431 junction (mec class C2) and is therefore capable of identifying SCCmec types I, III, V, and VI in combination with panel I. The method can also detect the recently described novel SCCmec type VIII (ccrAB4 with mec class A). Our assay demonstrated 100% concordance when applied to 162 MRSA strains previously characterized by traditional SCCmec typing schemes. Four geographically and temporally diverse S. aureus collections were also successfully classified by our assay, along with 1,683 clinical isolates comprising both hospital-and community-associated MRSA and methicillin-susceptible S. aureus strains. As many as 96 isolates can be classified easily within 3 to 4 h, including DNA isolation, PCR cycling, and analysis. The assay is rapid, robust, sensitive, and cost-effective, allowing for high-throughput SCCmec typing of MRSA isolates.

Prevalence of Panton Valentine Leukocidin (pvl) Gene in Methicillin Resistant Staphylococcus aureus Isolated from Market Samples of Chicken Meat

“…Methicillin resistance in Staphylococcus aureus (MRSA) in humans as well as in foods of animal origin and its prevalence has been increasing worldwide. This resistance is due to the acquisition of genes encoding a unique penicillin-binding protein (PBP2'or PBP2a) (Chen et al, 2009). Studies in different countries have strongly suggested that consumption of under cooked MRSA contaminated meat could be responsible for the prevalence of MRSA in the community (Ogata et al, 2102).…”

Naturally occurring IgG antibody levels to theStaphylococcus aureusprotein IsdB in humans

“…Methicillin resistance was determined using standard laboratory techniques and SCCmec typing performed as previously described. 28 Serology assay. A direct binding assay was developed for the detection of total IgG antibodies to IsdB, using Luminex ® technology.…”

Real-Time Nucleic Acid Sequence-Based Amplification Assay for Rapid Detection and Quantification of agr Functionality in Clinical Staphylococcus aureus Isolates

“…Four fungal strains (Candida albicans) were also included. All bacterial and fungal strains were selected from our previous studies (1,17). Statistical analysis.…”

“…In a previous study, agr functionality among 269 single-patient MRSA isolates was determined by screening for synergistic and direct hemolysis (15). All 269 isolates were previously genotyped by a combination of methods, including spa typing, SCCmec typing, and pulsed-field gel electrophoresis (PFGE) (1,10,16). Although the majority of strains were deemed positive for ␦-hemolysin production (synergistic hemolysis), 40 strains were identified as nonhemolytic, while 13 exhibited a mixture of both hemolytic and nonhemolytic colonies (15).…”