Half Reaction Volume Optimization of Viral Load Real Time PCR: Lessons, Challenges, and Experience in A Resource Limited Setting

Quantitative polymerase chain reaction (qPCR) is a powerful tool for gene expression analysis. However, qPCR is expensive test however, optimizing the assay can be challenging, especially when working with limited amounts of Nucleic acid. This study aimed to evaluate and optimize half reaction qPCR approach for the detection and quantitation of Hepatitis B, C and human CMV. Methods: The reaction efficiency using half volumes of the RT-qPCR assay were evaluated. Comparison and stratification of Ct values between standard and half reactions of Hepatitis B, Hepatitis C and CMV positive samples was evaluated. Results: The qPCR efficiencies of half reaction were 100.9 %, 101.2% and 105.7% of Hepatitis B viral load, Hepatitis C viral load, CMV viral load respectively. The R2 for standard reaction was found to be 0.98, 1 and 1 for all the three PCR assessed as compared to R2 half reactions which was 1. Conclusions: Quantitative polymerase chain reaction (qPCR) is a powerful tool for gene expression analysis. Utilization of half volumes of the RT-qPCR assay was optimized and validated in this article. We explored the benefits and considerations of this optimization strategy in Hepatitis B, Hepatitis C and CMV viral load assays. The use of the RT-qPCR half-reaction proved feasible and economic for the detection of the same.