title: Rapid extraction from and direct identification in clinical samples of methicillin-resistant staphylococci using the PCR.
abstract: 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 results, and there may be difficulty in rapidly and accurately identifying methicillin 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 and test bacteria for antimicrobial resistance. However, many sample preparation methods are unsuitable for PCR utilization in the clinical laboratory because 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 same-day results to facilitate rapid diagnosis and therapy. In this report, we describe a rapid method for extraction of bacterial DNA directly from blood 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 turnaround time (TAT), cost, purity, and use of template in PCR. Specific identification of MRS was determined using intragenic primer sets for bacterial and Staphylococcus 16S rRNA and mecA gene sequences. The PCR primer sets were validated 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 less than 4 h. The methods described herein represent a rapid and accurate DNA extraction and PCR-based identification system, which makes the system an ideal candidate for use under austere field conditions and one that may have utility in the clinical laboratory.
authors.affiliation: Clinical Investigation Facility, David Grant Medical Center, Travis AFB, California 94535-1800, USA. rjaffe@cbi-biotech.com
authors.forename: R I
authors.initials: RI
authors.lastname: Jaffe
authors.forename: J D
authors.initials: JD
authors.lastname: Lane
authors.forename: S V
authors.initials: SV
authors.lastname: Albury
authors.forename: D M
authors.initials: DM
authors.lastname: Niemeyer
mesh_terms: D015373:Bacterial Typing Techniques; D001769:Blood / Q000382:microbiology*; D003030:Coagulase / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification*; D005798:Genes, Bacterial; D020459:Genes, rRNA; D006801:Humans; D008712:Methicillin / Q000494:pharmacology; D016106:Methicillin Resistance / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D013203:Staphylococcal Infections / Q000097:blood / Q000175:diagnosis / Q000382:microbiology*; D013210:Staphylococcus / Q000145:classification* / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification; D013997:Time Factors
chemical_list: D003030:Coagulase; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D008712:Methicillin