Real-time PCR is a second generation PCR platform with significantly improved testing characteristics. Introduced in 1996, it has revolutionized and replaced conventional PCR approaches to quantify DNA and RNA. Today, RT-PCR is the gold standard for quantitative PCR and is rapidly becoming accepted as the method of choice for PCR diagnostics.
Quantification is only one of many advantages of RT-PCR. In contrast to conventional PCR, reaction tubes stay closed after the PCR process is finished. PCR product accumulation is measured in real-time during the amplification process with a fluorescently labeled probe within the PCR reaction tube. The closed-tube detection system reduces PCR product carry-over and risk of false positive signal generation. We also use a second system in our mastermixes which does not allow PCR products to be re-amplified.
Increased Analytical Sensitivity and Specificity
Real-time has shown to be ultra-sensitive; direct comparisons with nested conventional PCR puts it ahead of the conventional approach. Real-time PCR has a lower limit of detection of 5 molecules and has better analytical sensitivity than conventional PCR with eliminated risk of contamination.
For more information about Real-time PCR please refer to the following article. “The Real-time TaqMan PCR and Applications in Veterinary Medicine.” (PDF)