Rapid extraction from and direct identification in clinical samples of methicillin-resistant staphylococci using the PCR

Methicillin-resistant staphylococci (MRS) are one of the most common causes of nosocomial infections and bacteremia. Standard bacterial identification and susceptibility testing frequently require as long as 72 h to report re sults, and there may be difficulty in rapidly and accurately identifying me thicillin resistance. The use of the PCR is a rapid and simple process for the amplification of target DNA sequences, which can be used to identify an d test bacteria for antimicrobial resistance. However, many sample preparat ion methods are unsuitable for PCR utilization in the clinical laboratory b ecause they either are not cost-effective, take too long to perform, or do not provide a satisfactory DNA template for PCR. Our goal was to provide sa me-day results to facilitate rapid diagnosis and therapy. In this report, w e describe a rapid method for extraction of bacterial DNA directly from blo od culture bottles that gave quality DNA for PCR in as little as 20 min. We compared this extraction method to the standard QIAGEN method for turnarou nd time (TAT), cost, purity, and use of template in PCR Specific identifica tion of MRS was determined using intragenic primer sets for bacterial and S taphylococcus 16S rRNA and mecA gene sequences. The PCR primer sets were va lidated with 416 isolates of staphylococci, including methicillin-resistant Staphylococcus aureus (n = 106), methicillin-sensitive S. aureus (n = 134) , and coagulase-negative Staphylococcus (n = 176). The total supply cost of our extraction method and PCR was $2.15 per sample with a result TAT of le ss than 4 h. The methods described herein represent a rapid and accurate DN A extraction and PCR-based identification system, which makes the system an ideal candidate for use under austere field conditions and one that may ha ve utility in the clinical laboratory.