Investigation on NTC (No-Template Control) Amplification in real time PCR test of Boar DNA sample

Abstract

Amplification in the No-Template Control (NTC) is a critical issue in real-time PCR (qPCR) as it can compromise the validity of results. This study was conducted to investigate the source of an observed signal in the NTC of a qPCR assay designed for boar DNA detection. Methodologies included melt curve analysis to characterize the amplification product and standard curve analysis to assess assay performance. The results from the melt curve analysis definitively confirmed that the product amplified in the NTC was not a primer-dimer, indicating that the signal did not originate from non-specific primer interactions. Furthermore, the primer set clearly demonstrated efficient amplification of the target DNA sequence in positive samples. The overall assay performance was validated by a standard curve, which showed acceptable linearity with a coefficient of determination (R2) of 1. Collectively, these findings suggest that the qPCR assay is robust and specific. The amplification observed in the NTC is attributed to minute DNA contamination rather than methodological flaws like primer-dimer formation, confirming the high sensitivity of the developed test.