Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2014

A novel 3base™ multiplex PCR assay for the rapid detection and stratification of MRSA  (#360)

John R Melki 1 , Shoo Peng Siah 1 , Kiran Kaur 1 , Jiny Nair 1 , Christopher French 1 , William R Rawlinson 2 , Douglas S Millar 1
  1. Genetic Signatures, Sydney, NSW, Australia
  2. Director of Virology, Prince of Wales Hospital, Randwick, NSW, Australia

Objective: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important nosocomial pathogens worldwide. Patients carrying MRSA are at a greater risk of developing a more serious infection, compared with methicillin-susceptible S. aureus (MSSA) in the intensive care unit. In addition, the recent discovery of a mecA homologue (mecALGA251) suggests that MRSA strains harboring only the mecALGA251 homologue may be wrongly identified as MSSA using current molecular methods. Furthermore, nuc deficient MRSA strains have also been identified again possibly leading to false negative results in molecular assays only targeting the nuc gene. We aimed to produce a rapid real time PCR assay that could detect the presence of MRSA from clinical samples in less than 3 hours that addresses the current limitations of molecular methods.

Methods: 100µl of liquid sample (Ames transport medium or saline) was transferred to 300ul of lysis buffer, vortexed briefly then heated at 95oC for 15 minutes. Purification was completed using the Kingfisher automated nucleic acid extraction platform. Alternatively the assay can be implemented on a dedicated automated sample processing/PCR set up robot (GS1) that reduces hands on time to 2-3 minutes.

Results: Sensitivity experiments were assessed using synthetic oligonucleotides containing the PCR target region for the individual MREJ genotypes, mecA, mecALGA25, nuc and femA . The sensitivity of all targets was determined to be in the range of 5-20 copies in the PCR reaction. No cross reactivity was observed with a wide range of non-target bacteria including MSSA, MRCoNS and MSCoNS. Results from testing over 400 swab samples and reference strains were in agreement with previous assays. 

Conclusion: The assay was able to sensitively and specifically identify MRSA from primary patient material in under 3 hours from patients. In addition, the test can be carried out in virtually any clinical laboratory that is equipped with a sample extraction platform and real-time instrument without a further capital outlay.