Application of bioluminescence assay to assess PCR carryover contamination in forensic DNA laboratories

Abstract

In forensic DNA testing, PCR-based multilocus short tandem repeat (STR) profiling kits, which have high sensitivity and discriminatory power, are generally used to analyze autosomal and Y-chromosomal DNA profiles. Forensic DNA laboratories require strict quality control for DNA testing, as contamination during analyses leads to incorrect interpretation of DNA profiles. Here, we aimed to apply bioluminescence assay to detect and monitor residual PCR products on laboratory work area and equipment surfaces by targeting dATP in the PCR product and allelic ladder marker. Two commercially available bioluminescence assay kits (CheckLite HS Plus and UltraSnap™) were examined for their sensitivity after confirming their reactivity to dATP. In the assay using CheckLite HS Plus, the lower detectable sample volumes were calculated as 10 pl of PCR product of GlobalFiler and PowerPlex Fusion and 1 pl of PCR product of Yfiler Plus and the allelic ladder marker of GlobalFiler, whereas those in the assay using UltraSnap™ were calculated as 1 nl of PCR product and allelic ladder marker. The sample volumes of these kits were lower than those detected through electrophoresis. Thus, the sensitivity of these kits was sufficient to control PCR carryover contamination in the post-PCR areas. Furthermore, residual PCR products in the post-PCR areas were continuously monitored using a bioluminescence assay. The results showed that the bioluminescence values increased after handling PCR samples for electrophoresis and decreased after decontamination. Therefore, we concluded that the bioluminescence assay is useful for assessing PCR carryover contamination in post-PCR processes in forensic DNA laboratories.