Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2017

A Real-Time Multiplex-PCR for the detection of Extended-Spectrum β-Lactamases (ESBL) and Carbapenemase-Producing Organisms (CPO) using 3baseTM technology (#68)

Dilshan M Abeysekera 1 , Damien J Stark 2 , Doug S Millar 1 , John R Melki 1
  1. Genetic Signatures, Newtown, NSW, Australia
  2. Microbiology Department, St. Vincent's Hospital, Sydney, NSW, Australia

Background

Beta-lactam and carbapenem antibiotics are the most commonly used worldwide in the treatment of bacterial infections. The recent emergence of ESBL and CPO is a significant global concern in healthcare settings, as standard treatments may be rendered ineffective. Thus accurate and rapid detection of these resistant organisms will have a significant impact on patient management. We have developed a real-time multiplex-PCR assay to detect most significant and commonly encountered bacterial resistance genes TEM, CTX-M, SME, GES, IMP, NDM, OXA 23, OXA48, OXA51, MCR-1, DHA, SHV, VIM, IMI, CMY, KPC and their subtypes.

Methods/Materials

To test the assay sensitivity synthetic DNA constructs were designed. In addition, validation organisms and panels were obtained from Vircell, Zeptometrix and QCMD to test the performance of the assay on known reference material. Over 130 clinical isolates were obtained from St. Vincent’s Hospital (Sydney, Australia) and University Hospital Galway (Galway City, Ireland). DNA extraction and PCR setup were performed on a GS1 and a KingFisher automated extraction platform. PCR was performed on a CFX384 TouchTM real-time PCR instrument.

Results

Multiplexing sensitivity of each component was found to be less than 25 copies of the target. Mixed infections could be easily detected using the different channels of the PCR instrument. Specificity of the assay was assessed by a cross-reactivity panel. Results from the validation panels yielded 100% concordance with the expected resistance patterns.

Conclusions

The 3baseTM technique expands the detection capacity of multiplex-PCR for some target genes (CTX-M) to detect various subtypes within the target by affecting the DNA sequence homology. Also, novel variants or new resistant markers can readily be incorporated into existing assays easily given the properties of the 3baseTM converted DNA sequences, thus improving the throughput of such assays. The optimized assay provides a sensitive and specific alternative for the detection of ESBL and CPO and can be carried out in less than 3 hours with minimal hands-on time for laboratory technicians.