BD GeneOhm MRSA assay

1. BD GeneOhmMRSA assay Julia Robbins August 11, 2009

2. Objectives • Clinical Significance of MRSA in Healthcare Setting • Principle of assay • Assay Procedure • Assay Perfomance

3. BD GeneOhm™ MRSA assay is a qualitative in vitro diagnostic test for the direct detection of nasal colonization by methicillin-resistant Staphylococcus aureus (MRSA) to aid in the prevention and control of MRSA infections in healthcare settings. This rapid (within two hours) test allows for faster detection of MRSA colonization compared to culture which requires 24 to 72 hours enabling swift implementation of appropriate intervention.

4. The test performed on the SmartCycler® instrument with a nasal swab specimen from patients at risk for colonization, utilizes polymerase chain reaction (PCR) for the amplification of MRSA DNA and fluorogenic target-specific hybridization probes for the detection of the amplified DNA.

5. Following glass bead lysis, amplification of the target, a gene sequence near the insertion site of Staphylococcal Cassette Chromosome mec (SCCmec) that is unique to the methicillin-resistant strain of Staph aureus DNA, will occur.

6. Amplification of the internal control (IC), a DNA fragment of 335-bp including a 277-bp sequence not found in MRSA, will also take place unless there are PCR inhibitory substances present.

7. The amplified DNA targets are detected with molecular beacons, hairpin-forming single-stranded oligonucleotides labeled at one end with a quencher and at the other end with a fluorescent reporter dye (fluorophore).

8. For the detection of MRSA amplicons, the molecular beacon contains the fluorophore FAM at the 5’ end and the non-fluorescent quencher moiety DABCYL at the opposite end of the oligonucleotide.

9. For the detection of MRSA amplicons, the molecular beacon contains the fluorophore FAM at the 5’ end and the non-fluorescent quencher moiety DABCYL at the opposite end of the oligonucleotide.

10. For the detection of the IC (internal control) amplicons, the molecular beacon contains the fluorophore TET at the 5’ end and the quencher DABCYL at the 3’ end.

11. Each beacon-target hybrid fluoresces at a wavelength characteristic of the fluorophore used in the particular molecular beacon. The amount of fluorescence at any given cycle, or following cycling, depends on the amount of specific amplicons present at that time.

12. The SmartCycler simultaneously monitors the fluorescence emitted by each beacon, interprets all data and at the end of the cycling program provides a final result.

13. Specimen Preparation/Concentration • Place swab in sample buffer tube • Break swab • Vortex at high speed • Transfer cell suspension to a lysis tube • Centrifuge at a minimum of 14,000 x g at room temperature • Discard supernatant • Add sample buffer to the lysis tube

14. Lysis –DNA Extraction • Vortex, centrifuge briefly, then heat to inactivate potential inhibitors • Place lysis tube on a cooling block

15. Reconstitution of Molecular Reagents • Reconstitute 1 master mix for up to 6 specimens and 2 controls • Add diluent to lyophilized master mix and transfer master mix to the reservoir of each reaction tube • Transfer lysate solution to the specimen reaction tube • Add control DNA to positive control tube • Add sample buffer to negative control tube • Centrifuge all tubes briefly • Place tubes on specially adapted cooling block until ready to load

16. Real-Time PCR Analysis • Insert each reaction tube into the instrument • Start run and obtain results in approximately 1 hour • No interpretation required • Internal control monitors inhibition

17. Example of Test Results

18. Assay Performance • Sensitivity: 93% • Specificity: 96% • Negative Predictive Value: 98% • Positive Predictive Value: 85%