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E3S Web of Conferences, a Scopus, UGC CARE and DOAJ indexed journal (SCImago Journal Rank indicator: 0.182), published the following paper of the staff members of the FGBI “ARRIAH” (Vladimir):
Doronin M. I., Mikhalishin D. V., Shishkova A. A., Galkina T. S., Shishkov A. V., Malygin M. P. Quantitative PCR to determine the titer of infectious activity of the canine hepatitis virus. E3S Web of Conferences. 2024; 486:01011. DOI: 10.1051/e3sconf/202448601011
The paper presents the results of the development and validation of a method for indirect determination of infectivity titer of genotype CAV-1 infectious canine hepatitis virus in culture vaccine production seed with real-time polymerase chain reaction using quantification cycle (Cq), which includes the following steps:
- elution of DNA of infectious canine hepatitis virus (CAV-1);
- amplification of specific ORF 16 fragment of infectious canine hepatitis virus (CAV-1) DNA using original specific forward and reverse primers, as well as a molecular probe labeled with FAM fluorescent dye and RTQ-1 fluorescence quencher: CAV-1-T-F-primer (primer sequence 5′-CGTAATGGGGAAACCTAGGGG-3′), CAV-1-T-R-primer (primer sequence 5′-TCTGTGTTGTTTCTGTCTTGG-3′), and CAV-1-T-Pb-probe (probe sequence 5′-FAM-CCAATCATCATCTCAACTCAACTAAATGCCGTG-RTQ1-3′);
- calculation of quantification cycle (Cq) based on real-time PCR data;
- determination of infectivity titer of infectious canine hepatitis virus (CAV-1) using a logarithmic function expressed as the equation lg TCAV-1 = –0.2979 × Ct + 9.2595 with approximation reliability of 0.9941 and amplification efficiency of 99.38%.
The developed method for determination of CAV-1 adenovirus infectivity titer makes it possible to reduce the test time to 3 hours; the analytical sensitivity of real-time PCR is at least 1.0 lg TCID50/cm3.
The full text of the paper is available at https://doi.org/10.1051/e3sconf/202448601011