Quantitative PCR to determine the titer of infectious activity of the canine hepatitis virus

Abstract

This article presents data on the development and validation of a method for the indirect determination of the titer of infectivity of canine infectious hepatitis virus of genotype CAV-1 in raw materials for culture vaccines by real-time polymerase chain reaction using the Cq quantification cycle, including the following steps: eluting DNA of canine infectious hepatitis virus genotype CAV-1; performing amplification of a specific fragment orf 16 of canine infectious hepatitis virus genotype CAV-1 DNA using the original specific forward and reverse primers, as well as a molecular probe labeled with fluorescent dye FAM and luminescence quencher RTQ-1: CAV-1-T-F-primer with 5′-CGTAATGGGGAAACCTAGGGG-3′ design, CAV-1-T-R-primer with 5′-TCTGTGTTGTTTCTGTCTTGG-3′ design, and CAV-1-T-Pb-probe with 5′-FAM- CCAATCATCATCTCAACTCAACTAAATGCCGTG-RTQ1-3′ design; calculation of Cq quantification cycle from real-time PCR data; determination of the titer of infectivity of canine infectious hepatitis virus of genotype CAV-1 using a logarithmic function expressed as the equation lg TCAV-1 = -0.2979 × Ct + 9.2595 with an approximation reliability of 0.9941 and amplification efficiency of 99.38%. The analysis time is reduced to 3 h, and the analytical sensitivity is at least 1.0 lg TCD50/cm3.