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[DNA fingerprinting by arbitrarily primed polymerase chain reaction (AP-PCR) for methicillin-resistant Staphylococcus
1First Department of Internal Medicine, Kagawa Medical School.
Abstract:
Recently, nosocomial outbreaks of MRSA have become an important social problem in Japan. To examine the routes of transmission of MRSA, the establishment of accurate MRSA typing system is essential. However, more recently, because MRSA strains with type II coagulase have been increasing, it is difficult to discriminate MRSA strains by the coagulase typing method. Under this background, our study was designed to evaluate the clinical significance of DNA fingerprinting by arbitrarily primed polymerase chain reaction (AP-PCR). Several MRSA strains isolated from patients in our department were used in this study. To optimize the condition of AP-PCR, the differences of amplified products by AP-PCR were evaluated according to the following conditions: extracting methods of DNA from MRSA strains, buffer conditions, the temperature of AP-PCR, cycles of AP-PCR, and several primers. As a result, the optimal conditions of AP-PCR were as follows: extracted DNA using the InstaGene kit, amplified DNA by two-step AP-PCR using a M13 reverse primer with a buffer condition of 3.5 mM of magnesium chloride, and a pH of 8.5. The results of AP-PCR correlated well with the results of pulsed-field gel electrophoresis. In conclusion, DNA fingerprinting by AP-PCR seems to be useful in examining the nosocomial MRSA outbreak.
Insights
DNA fingerprinting using arbitrarily primed polymerase chain reaction (AP-PCR) offers a reliable method for tracking Methicillin-resistant Staphylococcus aureus (MRSA) outbreaks. This technique aids in understanding MRSA transmission routes, crucial for infection control.
Area of Science:
- Microbiology
- Molecular Biology
- Infectious Diseases
Context:
- Nosocomial outbreaks of Methicillin-resistant Staphylococcus aureus (MRSA) pose a significant public health challenge in Japan.
- Traditional coagulase typing methods are becoming less effective due to the increasing prevalence of MRSA strains with type II coagulase.
- Accurate MRSA typing systems are essential for investigating transmission routes and controlling hospital-acquired infections.
Purpose:
- To evaluate the clinical significance and optimize the conditions of DNA fingerprinting by arbitrarily primed polymerase chain reaction (AP-PCR) for MRSA strain typing.
- To establish optimal AP-PCR conditions, including DNA extraction methods, buffer compositions, temperature, cycles, and primer selection.
- To compare AP-PCR results with established methods like pulsed-field gel electrophoresis (PFGE).
Summary:
- The study optimized arbitrarily primed polymerase chain reaction (AP-PCR) for Methicillin-resistant Staphylococcus aureus (MRSA) strain typing.
- Optimal conditions involved using the InstaGene kit for DNA extraction, a two-step AP-PCR with an M13 reverse primer, 3.5 mM magnesium chloride, and pH 8.5.
- AP-PCR results demonstrated strong correlation with pulsed-field gel electrophoresis (PFGE), indicating its reliability.
Impact:
- DNA fingerprinting by AP-PCR provides a valuable tool for investigating nosocomial MRSA outbreaks.
- This method can enhance the understanding of MRSA transmission dynamics in healthcare settings.
- Improved MRSA typing contributes to more effective infection prevention and control strategies.