Development of Dry and Liquid Duplex Reagent Mix-Based Polymerase Chain Reaction Assays as Novel Tools for the Rapid and Easy Quantification of Bovine Leukemia Virus (BLV) Proviral Loads

Abstract

Bovine leukemia virus (BLV) is prevalent worldwide, causing serious problems in the cattle industry. The BLV proviral load (PVL) is a useful index for estimating disease progression and transmission risk. We previously developed a quantitative real-time PCR (qPCR) assay to measure the PVL using the coordination of common motif (CoCoMo) degenerate primers. Here, we constructed a novel duplex BLV-CoCoMo qPCR assay that can amplify two genes simultaneously using a FAM-labeled MGB probe for the BLV LTR gene and a VIC-labeled MGB probe for the BoLA-DRA gene. This liquid duplex assay maintained its original sensitivity and reproducibility in field samples. Furthermore, we developed a dry duplex assay composed of PCR reagents necessary for the optimized liquid duplex assay. We observed a strong positive correlation between the PVLs measured using the dry and liquid duplex assays. Validation analyses showed that the sensitivity of the dry duplex assay was slightly lower than that of the other methods for the detection of a BLV molecular clone, but it showed similar sensitivity to the singleplex assay and slightly higher sensitivity than the liquid duplex assay for the PVL quantification of 82 field samples. Thus, our liquid and dry duplex assays are useful for measuring the BLV PVL in field samples, similar to the original singleplex assay.

Keywords: bovine leukemia virus (BLV); dry; duplex; liquid; proviral load (PVL); quantitative real-time PCR (qPCR); singleplex.

Figure 1

Flowchart of the experimental procedure in this study. (A) Based on the BLV-CoCoMo-qPCR-2 (Single-CoCoMo) assay, the first step (yellow box) was to construct an MGB probe labeled with FAM dye to detect the BLV LTR gene and an MGB probe labeled with VIC dye to detect the BoLA-DRA gene. This was performed to simultaneously detect the two genes in the same reaction well and to select the optimal qPCR master mix for the novel BLV-CoCoMo Dual qPCR (Liquid Dual-CoCoMo assay) reported in this study. (B) The second step (pink box) was to develop a ready-to-use Dry Dual-CoCoMo assay for simplifying the PCR preparation process. (C) The third step was to evaluate the sensitivity of the Liquid Dual-CoCoMo and Dry Dual-CoCoMo assays using the pBLV-IF2 and field samples by comparing the results with that of the Single-CoCoMo assay. In addition, correlation analysis was also conducted to confirm the relationship between the proviral loads measured by the novel two assays and that measured by the Single-CoCoMo assay.

Figure 2

Comparison of the BLV proviral load (PVL) measured by the BLV-CoCoMo Dual qPCR (Liquid Dual-CoCoMo assay) using two qPCR master mixes and that of the BLV-CoCoMo-qPCR-2 (Single-CoCoMo assay). The PVLs of ten BLV-infected cattle (B1–B10) were amplified in triplicate from those samples in three independent experiments using the Liquid Dual-CoCoMo assay with a THUNDERBIRD Probe qPCR master mix (TBIRD) and GeneAce Probe qPCR mix II (GeneAce) (n = 9). Box plots of S (dark gray), D-T (gray), and D-G (light gray) represent the PVLs measured by the Single-CoCoMo assay, the Liquid Dual-CoCoMo assay with TBIRD, and the Liquid Dual-CoCoMo assay with GeneAce, respectively. The upper bar indicates the maximum value and the lower bar shows the minimum values. The means are denoted by the red circles and the medians are shown as black bars. The p-values were calculated using Dunnett’s test. Asterisks indicate significant differences (* p < 0.05 and *** p < 0.001).

Figure 3

Correlation between the BLV proviral load (PVL) measured by the BLV-CoCoMo Dual qPCR using two different qPCR master mixes and that of BLV-CoCoMo-qPCR-2. The PVLs of ten BLV-infected cattle (B1–B10) were amplified from the corresponding samples in three independent experiments using the Liquid Dual-CoCoMo assay with a THUNDERBIRD Probe qPCR master mix (TBIRD) or a GeneAce Probe qPCR Mix II (GeneAce). The correlation between the mean PVLs measured by the Liquid Dual-CoCoMo assay using TBIRD or GeneAce and that by the Single-CoCoMo assay was evaluated using Pearson’s correlation coefficient (r). The p-values are indicated in the graphs. The dotted line represents the approximate curve.

Figure 4

Appearance of the ready-to-use BLV-CoCoMo Dry Dual qPCR reagent. All the PCR reagents needed for the Liquid Dual-CoCoMo assay containing the CoCoMo primer mix and TaqMan MGB probes for the BLV LTR gene and the BoLA-DRA gene, and the qPCR master mix were dried in one PCR reaction tube. This reagent was produced by Nippon Gene Co., Ltd. (Toyama, Japan).

Figure 5

Correlation between the BLV proviral load (PVL) measured by the Dry Dual-CoCoMo assay and the mean PVL measured by the Liquid Dual-CoCoMo assay. The PVL of ten BLV-infected cattle (B1–B10) were amplified in duplicate using the Dry Dual-CoCoMo assay. The correlation between the PVLs measured by the Dry Dual-CoCoMo assay and the mean PVLs measured by the Liquid Dual-CoCoMo assay was evaluated using Pearson’s correlation coefficient (r). The p-values are indicated in the graphs. The dotted line represents the approximate curve.

Figure 6

Correlation between the BLV proviral loads (PVLs) in BLV-infected cows as measured using the two novel Dual-CoCoMo assays and the Single-CoCoMo assay. The PVLs of 71 BLV-infected cows, including 2 BLV-infected cows with lymphoma, were determined in duplicate using the Liquid Dual-CoCoMo assay and the Dry Dual-CoCoMo assay. The correlation between the mean PVLs measured by the Liquid Dual-CoCoMo assay or the Dry Dual-CoCoMo assay and that measured by the Single-CoCoMo assay was evaluated using Pearson’s correlation coefficient (r). The p-values are indicated in the graphs. The dotted line represents the approximate curve.

Figure 7

Summary of the characteristics of the Single-CoCoMo, Dual-CoCoMo, and Dry Dual CoCoMo assays. Protocols for the Single-CoCoMo, Liquid Dual-CoCoMo, and Dry Dual-CoCoMo assays and values for the following parameters are presented. (A) Limits of detection (detection rate) for the three assays using pBLV-IF2. (B) Assay diagnostic specificity (95% CI) using field samples based on the Single-CoCoMo assay. (C) Assay diagnostic sensitivity (95% CI) using field samples based on the Single-CoCoMo assay. (D) Correlation between the BLV proviral loads of 71 BLV-infected cows as detected using the two novel Dual-CoCoMo assays and the Single-CoCoMo assay. The Pearson correlation coefficient (r) is shown.