3.1. Analytical sensitivity of the VNUA-P54 real-time PCR assay
The limit of detection of the VNUA-P54 assay was analyzed and compared
with the validated p72-based Tignon real-time PCR assay (Tignon et al.,
2011) using ASFV DNA ranging from 0.1 to 10 genome copies. Both assays
were able to detect 10 and 5 ASF genome copies, while the VNUA-P54 assay
detected 10/12 times and the Tignon assay 9/12 times 1 and 2 genome
copies. Both assays failed to detect 0.1 ASFV genome copies
(Supplementary Table S2 and 3). Based on these findings LOD was
calculated for both assays using
https://quodata.de/content/validation-qualitative-pcr-methods-single-laboratory.
The LOD was 2.63 copies for the VNUA-P54 real-time PCR assay and 3.29
copies for the Tignon real-time PCR assay (Table 1).
The sensitivity of the VNUA-P54 assay compared with Tignon real-time PCR
assay was further evaluated using a ten-fold dilution series of the ASFV
strain VNUA/HY/ASF-1/Vietnam/2019. The results showed that both assays
were able to detect ASFV at viral titers of 107,
106, 105, 104, and
103 HAD50/ml. The Ct values obtained
for VNUA P54 assay were 20.78, 24.23, 27.69, 30.92, and 34.39 and Tignon
assay were 22.03, 25.29, 28.58, 32.2, and 35.16, respectively. In
addition, at viral titer 102 HAD50/ml,
VNUA P54 assay could result in Ct value of 37.66 while Tignon assay was
negative. Both assays failed to detect ASFV at viral titer of 10
HAD50/ml. Basing on the Ct values generated from each
known virus titers, a linear relationship was observed between VNUA-P54
and Tignon real-time PCR assays (Figure 1).