1. QUANTITATIVE ANALYSIS OF AFRICAN SWINE FEVER VIRUS BY DIGITAL PCR
Istituto Zooprofilattico Sperimentale della Sardegna
“G. Pegreffi”
4th European Association of Veterinary Laboratory Diagnosticians
Prague 6-9 november 2016
QUANTITATIVE ANALYSIS OF AFRICAN SWINE FEVER VIRUS
BY DIGITAL PCR
M.G. TILOCCA1, S. DEI GIUDICI2, A. LADU2, G. PUGGIONI2, B. VODRET1, A. OGGIANO2.
1Istituto Zooprofilattico Sperimentale della Sardegna, Igiene degli Alimenti, Sassari, Italy. 2Istituto Zooprofilattico Sperimentale della Sardegna, Sanità Animale, Sassari, Italy.
INTRODUCTION:
African Swine Fever (ASF) is an highly lethal hemorrhagic disease affecting pigs. ASF is present in Africa, in several Eastern and Central European countries and in Sardinia (Italy). The molecular diagnosis of ASF virus is carried out by OIE (World Organization of Animal Health) real-time PCRs, King and UPL assays. The basis of digital PCR (dPCR) is to quantify the absolute number of DNA targets, using limiting dilutions, endpoint PCR and Poisson statistics (Figure 1). The employ of dPCR in virus quantification provides higher sensitivity, more accurate data in low viral titer samples and more tolerance to inhibitors. In this work we compare the performance of digital PCR versus real-time PCR methods, King and UPL assays, for the detection of ASF virus.
MATERIALS AND METHODS:
Serially diluted plasmid containing vp72 gene sequence of ASF virus were prepared in seven replicates and analyzed using TaqMan real-time PCR and droplet digital PCR (Bio-Rad) to compare the sensitivity and efficiency of quantification of the two assays. Real-time PCR was carried out using the King and UPL methods. The same real-time PCR primers, probes and template volume were used in the dPCR for both methods. In dPCR, comparative studies were performed to detect the optimal primer/probe concentrations, the annealing temperature for each assay was determined by using thermal gradient.
RESULTS:
Figure 2 and 3 show the results of the annealing temperature and primer/probe concentration optimization for both dPCR assays King (a) and UPL (b). The optimal annealing temperatures were 58°C (King) and 55°C (UPL) while primer/probe concentrations were 0,2/0,8 and 0,2/0,9 µM for King and UPL assay respectively. Respect to the real time PCR conditions, the King dPCR required little modifications, instead both annealing temperature and the primer/probe concentration were modified in the UPL dPCR. Figure 4 and 5 show the linearity and dynamic range of the real time PCRs and dPCR King (a) and UPL (b). The dPCR assays showed a good linearity and dynamic range between 10+4 and 1 copies/µl with a lower variability respect to the real time PCR.
Figure 1) Droplet digital PCR workflow a b a b
Figure 2) dPCR optimization of the annealing temperature using thermal gradient from 65°C to 55°C on 10+4 copies/µl (wells A1- H1) and 10+3 copies/µl (wells A1- H1) of p72 plasmid. King assay (a) and UPL assay (b) .
Figure 4) Amplification plot of Real Time PCR King (a) and UPL (b) on serial p72 plasmid dilutions from to 1 copies/µl. a a b b
Figure 3) dPCR primers and probe optimization on 10+4 e 10+3 copies/µl of p72 plasmid. King assay (a): 0,2/0,8 µM vs 0,2/0,9 µM probe/primers concentration; UPL assay (b): 01/04µM vs 0,2/0,9 µM probe/primers concentration.
Figure 5) Linearity of dPCR King (a) and UPL (b) on serial p72 plasmid dilutions from to 1 copies/µl.
DISCUSSION AND CONCLUSIONS:
The dPCR approach enable to re-examined existing validated protocols in order to quantify low viral titer samples. The results obtained showed that a properly optimized dPCR can be as sensitive as real time PCR, allowing the absolute quantification without the need of calibration curve.