Robust, rapid tests are essential to a successful screening strategy and PCR detection of pathogens can be used as an effective infection control tool. Existing commercial PCR tests only detect one pathogen per sample and may not detect resistant organisms other than VRE and MRSA. As horizontal transfer of vancomycin and methicillin resistance genes plays a role in their dissemination, detection of resistance markers rather than specific organisms is more comprehensive.
Objective: To develop a rapid multiplex screening assay capable of detecting both vancomycin and methicillin resistant organisms in the same specimen.
Methods: Using unique, well-conserved regions of the vancomycin and methicillin resistance genes, IntelligentMDx’s bioinformatics process was used to design primer/probe sets for a multiplexed real-time PCR assay. Primer/probe sets were analyzed to determine the likelihood that they would interfere with each other when combined in a single assay format. The specificity of primers and probes for their intended targets was also determined.
A process/internal control was also designed to monitor sample extraction and reagent integrity. The dynamic range, amplification efficiency, LOD, and analytical specificity against single and multiple resistance targets were determined.
Results: In silico analysis showed no interference or crossreactivity between the multiplexed primer/probe sets based on the predicted affinity of the various primers to each other vs. their intended genetic targets and the affinity of primers and probes to non-target DNA sequences.
The assay for each target was linear across seven logs, with amplification efficiencies greater than 95% and a run-time of less than 2 hours. In addition, the multiplexed PCR assay was able to detect targets at a LOD of 10 copies input per reaction. Primer/probe sets were specific for their intended targets and did not cross-react with the other targets or normal flora organisms.
Conclusions: (1) Single assay format for the detection of multiple antibiotic resistance markers combines speed with broad detection coverage; (2) The LOD and amplification efficiency indicate that the assay is both sensitive and robust; (3) Together, these suggest that the assay will identify patients who are colonized or co-colonized with vancomycin and/or methicillin resistant organisms with high specificity and sensitivity.