Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing
M. J. Espy,* J. R. Uhl, L. M. Sloan, S. P. Buckwalter, M. F. Jones, E. A. Vetter, J. D. C. Yao, N. L. Wengenack, J. E. Rosenblatt, F. R. Cockerill III, and T. F. Smith
Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota
CLINICAL MICROBIOLOGY REVIEWS, Jan. 2006, p. 165–256
INTRODUCTION
Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose human pathogens (25, 71, 73, 294, 456). This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR and detection methods. Hence, for some time this technology was referred to as rapid-cycle real-time PCR. Other descriptions of real-time PCR in the early literature included homogeneous PCR and kinetic PCR.
M. J. Espy,* J. R. Uhl, L. M. Sloan, S. P. Buckwalter, M. F. Jones, E. A. Vetter, J. D. C. Yao, N. L. Wengenack, J. E. Rosenblatt, F. R. Cockerill III, and T. F. Smith
Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota
CLINICAL MICROBIOLOGY REVIEWS, Jan. 2006, p. 165–256
INTRODUCTION
Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose human pathogens (25, 71, 73, 294, 456). This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR and detection methods. Hence, for some time this technology was referred to as rapid-cycle real-time PCR. Other descriptions of real-time PCR in the early literature included homogeneous PCR and kinetic PCR.
Real-time PCR testing platforms provide equivalent sensitivity and specificity as conventional PCR combined with Southern blot analysis. Since the nucleic acid amplification and detection steps are performed in the same closed vessel, the risk for release of amplified nucleic acids into the environment, and contamination of subsequent analyses, is negligent compared with conventional PCR methods. Real-time PCR instrumentation requires considerably less hands-on time and testing is much simpler to perform than conventional PCR methods. Additionally, accelerated PCR thermocycling and detection of amplified product permits the provision of a test result much sooner for real-time PCR than for conventional PCR. The combination of excellent sensitivity and specificity, low contamination risk, ease of performance and speed, has made real-time PCR technology an appealing alternative to conventional culture-based or immunoassay-based testing methods used in the clinical microbiology for diagnosing many infectious diseases. This review focuses on the application of realtime PCR in the clinical microbiology laboratory.
