![]() ![]() For absolute quantification, data analysis is further complicated by the different sources of DNA from which the samples and standard curves are derived with unique backgrounds and contaminants that can variably affect the activity of Taq polymerase giving misleading results 4. Poorly optimized reactions can result in artifactual Cq values and misinterpreted data that are difficult or even impossible to reproduce 2, 3. Therefore, optimization is critical for each primer pair such that reaction efficiency is consistent between all samples and acceptable (between 90% to 110%) with sample contaminants diluted adequately to assure that all reactions and associated Cq values are within the efficient range of the respective standard curves 1. A stepwise methodology is also described to choose between these complimentary technologies to obtain the best results for any experiment.ĭata from qPCR experiments are taken within each enzymatic reaction curve at the quantification cycle (Cq). We conclude that for sample/target combinations with low levels of nucleic acids (Cq ≥ 29) and/or variable amounts of chemical and protein contaminants, ddPCR technology will produce more precise, reproducible and statistically significant results required for publication quality data. Here, Droplet Digital PCR (ddPCR) and qPCR platforms were directly compared for gene expression analysis using low amounts of purified, synthetic DNA in well characterized samples under identical reaction conditions. The most susceptible, frustrating and often most interesting samples are those containing low abundant targets with small expression differences of 2-fold or lower. ![]() The root cause of poor quality data is typically associated with inadequate dilution of residual protein and chemical contaminants that variably inhibit Taq polymerase and primer annealing. From our findings, we recommend the use of TaqMan qPCR as the preferred method to detect ETBF from clinical stool samples.Quantitative PCR (qPCR) has become the gold standard technique to measure cDNA and gDNA levels but the resulting data can be highly variable, artifactual and non-reproducible without appropriate verification and validation of both samples and primers. For samples that were bft-positive in both fecal and luminal stools, there was no difference in relative abundance between the sites, by any method tested. ![]() TaqMan qPCR and dPCR gave bft copy numbers that were 48-fold and 75-fold higher for the same samples than SYBR qPCR, respectively (p < 0.001). However, SYBR qPCR under-performed compared to TaqMan qPCR and dPCR in detecting bft in clinical stool samples 13/38 samples were reported positive by SYBR, compared to 35 and 36 samples by TaqMan and dPCR, respectively. Bland-Altman analysis found that all three quantitative methods performed comparably in detecting bft from purified bacterial DNA, with the same limits of detection (<1 copy/μl). fragilis toxin (bft) gene from cultured ETBF, and from matched luminal and faecal stool samples from 19 colorectal cancer patients. We compared standard PCR, SYBR green and TaqMan quantitative PCR (qPCR) and digital PCR (dPCR) in detecting the B. However, differences in carriage rates are seen with various testing methods and sampling sites. Gut colonization with enterotoxigenic Bacteroides fragilis (ETBF) appears to be associated with the development of colorectal cancer. ![]()
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