Comparison of fluorometric detection methods for quantitative polymerase chain reaction (PCR)

In this study, we compared the sensitivity of two different detection methods for quantitative polymerase chain reaction (PCR). Various amounts of a 75 mer single-stranded deoxyribonucleic acid (DNA) fragment, which can be used as a DNA label for the immuno-PCR (iPCR) assays, were amplified by PCR. The amount of amplified DNA fragments was determined by the fluorescence (FL) of SYBR Green dye that specifically interacts with double-stranded DNA fragments. In the first selected detection method, real-time PCR, FL measurements were carried out at each thermal cycle, as the DNA was being amplified by PCR. This was achieved using the Applied Biosystems (ABI) Prism 7000 Sequence Detection System and its standard protocol. In the second detection method, referred to as end-point detection, after the PCR amplification was completed, off-line FL measurements were subsequently carried out using a conventional plate reader. In order to achieve the lowest limit of detection (LOD) from the off-line measurement, we have optimized a wide variety of parameters. Our data have indicated the LOD of real-time PCR method was approximately three orders of magnitude lower than the end-point measurement method, with a linear range spanning six orders of magnitude; 10 fmol to 10 zmol of PCR template. The lower LOD of the real-time PCR method could be partly due to the ability to maximize the number of thermal cycles that could be carried out in PCR, without increasing the non-specific amplification of any contaminating DNA. The results of this study can be applied to the development of ultra-sensitive iPCR assays for various disease markers.