A simple modification of PCR thermal profile applied to evade persisting contamination

Abstract

The polymerase chain reaction (PCR), one of the most commonly applied methods of diagnostics and molecular biology has a frustrating downside known as the false positive signal or contamination. Several solutions to avoid and to eliminate PCR contaminations have been worked out to date but the implementation of these solutions to laboratory practice may be laborious and time consuming. A simple approach to circumvent the problem of persisting PCR contamination is reported. The principle of this approach lies in shortening the steps of denaturation, annealing, and elongation in the PCR thermal cycle. The modification leads to the radical decline of false positive signals obtained for the no-template controls without affecting the detection of target PCR products. In the model experiments presented here, the signal of negative control was shifted by about ten cycles up above those for the examined samples so that it could be neglected. We do not recommend this solution in PCR diagnostics, where the sensitivity of detection is of the highest priority. However, the approach could be useful to pass by the problem of persisting contamination in quantitative PCR, where the range of quantitation is usually much above the limits of detection.Electronic supplementary materialThe online version of this article (doi:10.1007/s13353-015-0336-z) contains supplementary material, which is available to authorized users.

Molecular Identification and Species Richness of Flies (Diptera) and Their Associated Bovidae Hosts at Cattle Farms in Selangor, Malaysia

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DNA decontamination methods for internal quality management in clinical PCR laboratories

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“…Amplicons from the most common sources of contamination are produced at very high copy numbers during PCR and can seriously impact diagnostic analysis because they can be homologous to target molecules and amplified at high efficiency, resulting in carry-over contamination. 3 Contamination of PCR reagents 4 and DNA extraction kits is another major problem when broad-range primers, such as those used for bacterial 16s rDNA, are used for detection in clinical specimens.…”