Real-Time PCR Technology and Limitations

Real-Time PCR Technology and Its Limitations

• Real-time PCR should be different from single channel PCR to check for inter-channel interference
• Real-time PCR instrument should have a limited number of channels to limit the number of multiplexed targets
• Multiplexing is limited to four target/reporter dyes
• Real-time PCR reagents used in clinical trials are subjected to various performance evaluations during the development or quality control
• The minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines are the most promising among validation guidelines
• Main issues in performance evaluation include analytical sensitivity, analytical specificity, accuracy, repeatability, and reproducibility
• PCR-based techniques have been used clinically and scientifically to detect pathogens qualitatively or quantitatively, to identify mutations in pathogen genes, or to identify the types of pathogens
• Real-time PCR has revolutionized the diagnosis of human pathogenic viruses in clinical laboratories
• Real-time PCR with reverse transcription for the diagnosis of RNA viruses reduces the number of cDNA production steps and provides appropriate sensitivity and specificity within a short period
• Real-time PCR limitations include difficulty in distinguishing live from dead cells and the need for identification in bacterial strains of phenotypic and biochemical characteristics
• Fluorescence of real-time PCR is measured at each cycle and the intensity of the fluorescence signal reflects the instantaneous extent of DNA amplification
• Real-time PCR may be subjective and can produce false-positive and false-negative results based on the height of the threshold and the slope of the amplification curve