Cost-Effective Frozen Master Mix Modification of a Commercial Methicillin-Resistant Staphylococcus aureus PCR Assay

Abstract

The expense inherent to molecular diagnostics may be an overriding concern for a variety of clinical laboratories in the development of PCR-based methicillin-resistant Staphylococcus aureus (MRSA) active surveillance programs. BD GeneOhm MRSA assay master mix was reconstituted, aliquoted into SmartCycler tubes in 25-l volumes, and frozen at ؊70°C. One hundred percent of archival nasal swab lysates yielded the expected PCR results when incubated in master mix frozen for 1, 2, 3, and 4 weeks. A 98.8% concordance of the final result was observed upon prospective PCR analysis of 320 clinical lysates utilizing freshly reconstituted master mix and 2-week-frozen master mix. Initial unresolved rates generated by frozen master mix and freshly reconstituted master mix differed by 1.6% (P ‫؍‬ 0.16). Of 50 MRSA-positive lysates, the titers of 32 (64%) were determined to the same value upon initial tandem frozen master mix and freshly reconstituted master mix utilization; the titers of an additional 14 were determined to the same value upon repeat testing. Frozen master mix maintains potency for at least 4 weeks, facilitating detection of MRSA from nasal swab lysates, and may decrease the amount of unused reagent up to an average of 33%.While a national surveillance program recently documented a nearly 30% increase in the intensive care unit methicillinresistant Staphylococcus aureus (MRSA) infection rate from 1992 to 2003 (9), equally alarming data have been reported from surveys of hospital emergency departments. Documented purulent skin and soft tissue MRSA infection rates ranging from 15 to 74% and averaging 59% (11) suggest an additional reservoir of the organism that could significantly impact the care of hospitalized patients. These merging sources of MRSA provide impetus for health care entities to institute active surveillance programs, based largely on nasal swab specimen analysis, for detection of MRSA carriers. On a predicted basis, these programs reduce MRSA bacteremia rates by approximately 60% (15,17). While recent data have advocated PCR-based MRSA surveillance programs based on the parameters of time to final result (2), assay sensitivity (2), and decreased incidence of transmission (4), some clinical microbiology laboratories may be apprehensive to implement PCRbased screening due to financial considerations. We hereby characterize and validate a modification of a master mix formulation protocol that not only promotes greater economy in reagent utilization but also allows for effective performance of a commercial MRSA PCR assay.In a study protocol approved by the Wheaton Franciscan Healthcare Institutional Review Board, nasal swabs submitted for active MRSA surveillance were processed to a lysate form for the BD GeneOhm MRSA assay (BD Diagnostics, SainteFoy, Quebec, Canada) per the manufacturer's specifications.Kit-provided vials containing lyophilized master mix were reconstituted with 225 l of kit-provided diluent; 25-l aliquots were distributed into empty SmartCycler reaction tubes that were placed into ...

Modifications of Commercial Toxigenic Clostridium difficile PCR Resulting in Improved Economy and Workflow Efficiency

“…The adoption of C. difficile PCR by smaller laboratories may be further limited by the composition of commercial kits. Previously reported hypothetical matrices for utilization of BD GeneOhm assays have demonstrated that as much as 23 to 45% of prepared master mix could go unused upon adherence to package insert guidelines (10)(11)(12). Our current data extend previous findings from other commercial PCR assays (10,12) indicating that frozen master mix preparation does not compromise the performance of the BD GeneOhm Cdiff assay (Table 2).…”

“…Previously reported hypothetical matrices for utilization of BD GeneOhm assays have demonstrated that as much as 23 to 45% of prepared master mix could go unused upon adherence to package insert guidelines (10)(11)(12). Our current data extend previous findings from other commercial PCR assays (10,12) indicating that frozen master mix preparation does not compromise the performance of the BD GeneOhm Cdiff assay (Table 2). Frozen master mix has been used in other microbial and eukaryotic amplification assays (6,7,22).…”

“…Specimen aliquots were also transferred into kit-provided buffer tubes 1 day prior to lysate generation and stored at 2 to 8°C (overnight aliquot). Freshly reconstituted and frozen master mix were prepared and analyzed as previously described (10,12). tcdB (positive)-and buffer (negative)-based control material was prepared and analyzed analogous to previously described methods (12).…”

Cost-Effective Modification of a Commercial PCR Assay for Detection of Methicillin-Resistant or -SusceptibleStaphylococcus aureusin Positive Blood Cultures

“…These same environmental conditions were employed in the current assessment of BD GeneOhm StaphSR frozen master mix, and a comparable efficacy was observed. Final concordance rates of Ն98.8% were noted in prospective studies of frozen and freshly reconstituted master mix with the BD GeneOhm MRSA (14) and StaphSR assays. Data presented in this study confirm and extend the frozen master mix paradigm.…”

“…We also reported an effective master mix reconstitution and storage paradigm for a commercial MRSA PCR assay (14). Reconstituted BD GeneOhm MRSA master mix was frozen for up to 4 weeks at Ϫ70°C and retained potency, provided that prepared SmartCycler tubes were protected from light and allowed to acclimate in 4°C conditions.…”