LAUSR

CI, confidence interval; CV, coefficient of variation; RT-PCR, reverse transcription polymerase chain reaction; SD, standard deviation. a Number of batches of 30 tests analysed for mecA gene using standard suspensions of methicillin resistant Staphylococcus aureus (A and B). Sir, Testing isolated bacterial colonies from plate cultures by quantitative polymerase chain reaction (qPCR) assays is commonly performed in our laboratory for rapid identification, sub-typing and detection of antibiotic resistance genes. Assays incorporating either TaqMan probes or SYBR Green for product detection are used for this purpose and currently cost our organisation AU$2.86 and AU$3.22 for reagents (including primers/probes) per each test, respectively. In addition, bacterial DNA is extracted for both methods (InstaGene Matrix; BioRad, USA) costing AU$1.25 per isolate and taking about 30 min for completion. We describe here a novel qPCR method for detecting mecA in plate cultures of Staphylococcus species. It is based on an application of SYBR Green intercalating dye that is significantly cheaper and quicker to perform than conventional qPCR assays. Validation was based on tests performed on clinical isolates of staphylococci previously tested for susceptibility to methicillin using the CDS method, clones of epidemic methicillin resistant Staphylococcus aureus (MRSA) (provided by Dr Geoffrey Coombs, Australian Group on Antimicrobial Resistance), and isolates representative of other genera. The application of SYBR Green in qPCR assays circumvents the use of hybridisation probes for product detection. This dye intercalates with dsDNA and expresses enhancement of fluorescence during binding. However, its use in qPCR assays is thwarted by potential to bind non-specifically to other DNA moieties. In this novel assay, the limitation was addressed by using a heavy suspension of the test bacterium to increase the DNA template. This allows for earlier detection of target (i.e., a reduced crossing-point) distinguishable from non-specific products which are usually amplified after prolonged cycling. For this purpose, individual colonies from a fresh culture were sampled sufficiently to cover the surface area of a 1.0 mL sterile loop (Copan, Italy) and suspended in 2.5 mL of sterile water (Braun, Germany). Measurements of 129 suspensions showed that this equates to an OD640nm of 0.495e0.562 (CI95%) or approximately 10 e 10 CFU/mL as determined by plate viable counts. A volume of 3.5 mL of this suspension was introduced directly (i.e., without preliminary extraction) to 1.5 mL of reaction mix comprising 1.0 mL of LightCycler FastStart DNAMaster Plus SYBR Green 1 (Roche Diagnostics, Switzerland); 0.75 mM each forward and reverse primers targeting themecA gene designated mecA2F and mecA2R, respectively; and 0.25 U uracil N-glycosylase (Roche Diagnostics). Assay was performed in a LightCycler 2.0 (Roche) as programmed: 40 C for 10 min to degrade uracil incorporated amplicons; 95 C for 10 min to extract bacterial DNA and activate polymerase; 40 amplification cycles at 95 C for 10 s, 50 C for 10 s, and 72 C for 20 s with single acquisition in Channel F1 (530 nm); melt curve analysis commencing at 95 C, reducing