A simple and expedient PCR format for rapid molecular screening of methicillin-resistant Staphylococcus aureus in Amies gel swabs.

Screening patients for carriage of methicillin-resistant Staphylococcus aureus (MRSA) is commonly undertaken in hospital laboratories using phenotypic methods. This work is labour-intensive, costly and may take several days to complete. We report on the validation of a novel rapid screening approach for direct testing of Amies gel swabs for MRSA. The method is based on two quantitative real time-PCR (qRT-PCR) assays for the detection of the nuc and mecA genes of MRSA. Based on SYBR Green technology, the assays use significantly less reagents than conventional qRT-PCR methods and are applied to testing templates derived directly from aqueous suspensions of swabs. Notwithstanding the occurrence of false-positives due to non-specific fluorescence generated by the SYBR Green dye, the novel assays showed a high negative predictive value enabling earlier reporting of negative findings and selection of swabs for confirmatory phenotypic testing for MRSA. In a blinded trial of 461 swabs, of which 34 (7.4%) were previously shown to be culture-positive for MRSA, the novel assays selected 121 (26.2%) swabs (inclusive of the known MRSA-positive swabs) for phenotypic testing. This enabled early reporting of negative findings for 340 (73.8%) of the 461 swabs tested. Application of this method has implications for screening strategies for large laboratories whilst achieving cost benefits.

Development of an internal amplification control system for a real-time PCR assay for detection of Neisseria meningitidis in CSF and EDTA blood.

The aim of this study was to assemble and assess a non-competitive internal amplification control (IAC) system targeting the Escherichia coli alanine racemase (alr) gene to include in a real-time polymerase chain reaction (PCR) assay for Neisseria meningitidis. Primers and hybridisation probes specific for the IAC were designed and assessed for specificity. Amplification efficiency and limit of detection for the assembled assay was extrapolated using standard curves constructed with serial dilutions of N. meningitidis in saline, pooled cerebrospinal fluid (CSF) and EDTA blood. The 95% confidence limits (CI) were calculated for IAC crossing-points recorded for assays for N. meningitidis ctrA in saline (negative blank), and N. meningitides-negative samples of CSF and EDTA blood. These limits served as a reference range against which the IAC crossing-points recorded for prospective assays are compared to detect sample inhibition. This system was used in testing consecutive EDTA blood samples from two cases of meningococcal disease. The IAC system is specific for Escherichia coli and Shigella species. The amplification efficiency of the assembled assay for N. meningitidis and ability to detect low target DNA levels was not compromised with the inclusion of the IAC system. The IAC crossing-points varied in clinical samples of CSF and EDTA blood. The elucidated reference range for EDTA blood was used to detect sample inhibition in one of the two clinical cases investigated.The IAC system monitors the performance of all processes in the assembled assay for N. meningitidis. Measuring IAC crossing-points serves as an indicator of sample stability and inhibitory properties when testing single or multiple samples from the same patient. Specificity for E. coli and Shigella species enables inclusion in assays of different targets within the same laboratory. Reporting PCR assay results in the context of the IAC crossing-points and reference ranges validates against sample inhibition and suitability for detection of low levels of target DNA in random and multiple samples.