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Journal of Clinical Microbiology, September 2000, p. 3407-3412, Vol. 38, No. 9
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Rapid Extraction from and Direct Identification in Clinical
Samples of Methicillin-Resistant Staphylococci Using the
PCR
Richard I.
Jaffe,1,*
Janae D.
Lane,1
Stephen V.
Albury,1 and
Debra M.
Niemeyer2
Clinical Investigation Facility, David Grant
Medical Center, Travis AFB, California
94535-1800,1 and USAF Force
Protection Battlelab, Lackland AFB, Texas
78236-52552
Received 25 April 2000/Returned for modification 27 May
2000/Accepted 16 June 2000
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.
*
Corresponding author. Present address:
Commonwealth Biotechnologies, Inc., 601 Biotech Dr., Richmond, VA
23235. Phone: (800) 735-9224. Fax: (800) 648-2641. E-mail:
rjaffe{at}cbi-biotech.com.
Journal of Clinical Microbiology, September 2000, p. 3407-3412, Vol. 38, No. 9
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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