Mitigating polymerase chain reaction/amplicon contamination in a high-risk high-burden mycobacterial reference laboratory in a resource-limited setting.

Abstract

Nucleic acid amplification techniques have become important machineries in the diagnosis of several diseases in clinical laboratories. Polymerase chain reaction (PCR) contamination/amplicon contamination leading to false positivity remains a major concern in these laboratories. Prevention of these contaminations in establishing these molecular biology laboratories has been very crucial over the years. Although closed system PCRs have substantial reduction in the PCR contamination rates, the conventional probe-based hybridization methods continue to show occurrence of contamination for various reasons. The study involved checking the crucial parameters as well as the probable candidates of causing the contamination at a high-burden setting. Bringing out the most effective interventions in controlling the PCR contaminations for future endeavors stood as priority. The study explored the efficacies of different sets of interventions contributed to the process of reducing the contaminants. The detection of the contaminating PCR products or amplicons or contaminating organism is done by the genotype MTBDR plus V2 kits (Hains Life Sciences) based on DNA strip technology. The pre- and post-cleaning as well as cleaning of the working surfaces was able to bring down the mean contamination percentage by 36.5%. The combined effect of cleaning the work surfaces, the automated pipetting devices, and the AC machines was able to bring down the mean contamination percentage to 53.5% reducing the contamination rate nearly to 94.6%. Regularly cleaning the work surfaces, the automated pipetting devices, the PCR machine, and the AC machines along with it filters and exposure of UV rays significantly lowers down the mean contamination percentage.