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.

Pseudomonas aeruginosa has emerged as one of the most problematic Gram-negative nosocomial pathogens. Bacteremia caused by P. aeruginosa is clinically indistinguishable from other Gram-negative infections although the mortality rate is higher. This microorganism is also inherently resistant to common antibiotics. Standard bacterial identification and susceptibility testing is normally a 48-hour process and difficulty sometimes exists in rapidly and accurately identifying antimicrobial resistance. The Polymerase Chain Reaction (PCR) is a rapid and simple process for the amplification of target DNA sequences. However, many sample preparation methods are unsuitable for the clinical laboratory because they are not cost effective, take too long to perform, or do not provide a good template for PCR. Our goal was to provide same-day results to facilitate rapid diagnosis. In this report, we have utilized our rapid DNA extraction method to generate bacterial DNA direct from clinical samples for PCR. The lower detection level for P. aeruginosa was estimated to be 10 CFU/ml. In addition, we wanted to compare the results of a new rapid-cycle DNA thermocycler that uses continuous fluorescence monitoring with the results of standard thermocycling. We tested 40 clinical isolates of P. aeruginosa and 18 non-P. aeruginosa isolates received in a blinded fashion. Coded data revealed that there was 100% correlation in both the rapid-cycle DNA thermocycling and standard thermocycling when compared to standard clinical laboratory results. In addition, total results turn-around time was less than 1 hour. Specific identification of P. aeruginosa was determined using intragenic primer sets for bacterial 16S rRNA and Pseudomonas outer-membrane lipoprotein gene sequences. The total cost of our extraction method and PCR was $2.22 per sample. The accuracy and rapidness of this DNA-extraction method, with its PCR-based identification system, make it an ideal candidate for use in the clinical laboratory.

A plasmid containing the cloned listeriolysin gene of Listeria monocytogenes was used as a probe to identify Listeria strains by DNA colony hybridization. The probe DNA was labeled with horseradish peroxidase in the presence of glutaraldehyde. After the hybridization and wash procedures, the hybrid molecules were detected by luminescence, which resulted from the oxidation of luminol by a horseradish peroxidase-hydrogen peroxide-coupled reaction. Of the 150 Listeria strains and 16 non-Listena strains examined, the probe hybridized only with L. monocytogenes. The technique was also used to enumerate L. monocytogenes in artificially contaminated foods.