Julia RobbinsAugust 11, 2009

Objectives

Clinical Significance of MRSA in Healthcare Setting

Principle of assay

Assay Procedure

Assay Perfomance

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

Specimen Preparation/Concentration

Place swab in sample buffer tube Break swabVortex at high speedTransfer cell suspension to a lysis tubeCentrifuge at a minimum of 14,000 x g at

room temperature Discard supernatantAdd sample buffer to the lysis tube

Lysis –DNA Extraction

Vortex, centrifuge briefly, then heat to inactivate potential inhibitors

Place lysis tube on a cooling block

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 tubeAdd sample buffer to negative control tubeCentrifuge all tubes brieflyPlace tubes on specially adapted cooling block until

ready to load

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

Example of Test Results

Assay PerformanceSensitivity: 93%

Specificity: 96%

Negative Predictive Value: 98%

Positive Predictive Value: 85%