Real-time PCR and Quantitative Real Time PCR have emerged as one of the most powerful molecular techniques for the detection of very small quantities of target DNA specific for infectious agents in clinical and non-clinical laboratory samples. This highly sensitive technique allows the rapid amplification and quantitation of almost any targeted DNA molecules and has several advantages over traditional PCR.
Advantages of Real-time PCR over traditional PCR
Direct visualization of the amplification process
The Real-time PCR system is based on the direct detection and quantitation of a Reporter fluorescent signal that increases proportionally to the number of amplicons generated. Data can therefore be collected at any point of the amplification. Traditional PCR only allows end-point detection.
Precise quantitation possible
Real-time PCR allows quantifying DNA molecules over an extraordinarily wide range (at least 5 log units) – Referemce #1. This technique can detect as little as a two-fold change in yield. In comparison, the agarose gel resolution only allows detection of a minimum of five to ten-fold changes in yield.
Similar or higher sensitivity
Real-time PCR allows the detection of very minute amounts of the targeted DNA sequence (less than five copies of a target DNA molecule in some cases (1).
No post-PCR processing
Traditional PCR requires the use of agarose gel to visualize and analyze the final PCR product. Real-time PCR does not require any post-PCR manipulations, thereby greatly decreasing the analytic turnaround time of each reaction while minimizing the chances for cross contamination in the laboratory (1).
References
- Mark A. Valasek1 and Joyce J. Repa, The power of real-time PCR; Adv Physiol Educ 29: 151–159, 2005.
- Reischl U, Wittwer CT, and Cockerill F. Rapid Cycle Real-time PCR: Methods and Applications; Microbiology and Food Analysis. New York:Springer-Verlag, 2002.
Amplification of serial 10-fold dilutions of Bartonella henselae DNA, starting from 4000 pg down to 4 pg. Bartonella henselae genomic DNA was amplified using Brilliant® II QPCR Master Mix (2X). Reactions were performed on the Stratagene Mx4000® Thermal Cycler. NTC = non-template control
Example of a Real-time PCR amplification plot
