Comparison of Methods for Extracting DNA From Whole Blood to Quantify Bovine Leukemia Virus Using BLV-CoCoMo Dual qPCR Assay

Abstract

We previously developed a dual-target detection qPCR assay called the BLV-CoCoMo Dual qPCR assay (Daul-CoCoMo assay) for the quantification of bovine leukemia virus (BLV) proviral load (PVL) by improving the original BLV-CoCoMo-qPCR-2 assay (Single-CoCoMo assay). Compared to the Single-CoCoMo assay, the Dual-CoCoMo assay is a simple, user-friendly and cost-effective tool that detects two target genes simultaneously. However, the Dual-CoCoMo assay may have a lower diagnostic sensitivity than the Sinlgle-CoCoMo assay. Here, we aimed to enhance the sensitivity of the Dual-CoCoMo assay by optimizing DNA extraction methods. First the performance of DNA extraction was compared between the conventional Wizard method and a newly selected Pure SPIN method. The average total DNA yields of the Pure SPIN method were higher than the Wizard method. Therefore, PVL was quantified using these DNA samples. In all six samples that were negative by the Wizard+Single-CoCoMo assay, which is the most sensitive conventional method, PVL was quantified by the Pure SPIN+Dual-CoCoMo assay, demonstrating the method is highly effective for quantifying BLV PVL.