Correlation determination of Ct index and african swine fever virus titer in biological fluids

UDC 619:616.98-076[:578.833.31

Mazlum A., Vlasova N.N., Aronova E.V., Igolkin A.S., Krivonos R.A., Chernykh O.Yu.

Summary. The article is devoted to the determination of the correlation of the Ct index in real-time PCR and the titer values of the virus established in the haemadsorption reaction, as well as the calculation of the number of copies of the genome of the ASF virus in the samples. Other advantages of PCR-RV include fast and efficient way to compare and evaluate quality of samples. In a number of works, where the definition of virus virulence by its content in the blood is shown, it is noted that the rate of change of Ct (respectively, the rate of increase in titer in the blood) in an attenuated virus is lower than that of the virulent. The results of the comparative analysis showed a high stability of correspondence between the values of the determined values of Ct and the virus titer and the number of copies of the genome. According to the results of studies with the Central Scientific Research Institute set, change in the concentration of DNA copies 10 times leads to a change in the Ct value for 3 cycles. At the same time, decrease in virus titer of 1.0 lg GADE-50/cm3 leads to an increase in the Ct value in real-time PCR for 4 cycles. For this, the full-length B646L gene of the ASF virus was amplified using PCR. The identified correlation between the Ct value and the virus titer in the sample allows in laboratory practice the use of PCR-RV for the quantitative determination of the virus content in the sample, and allows for a detailed study of the biological properties of ASF causative agent. The determination of the compliance of the Ct indicators of PCR-RV with the titer of the virus obtained from the blood of an infected animal with ASF isolate Krasnodar 07/17 was carried out. The use of PCR-RV allows you to determine the number of copies of the viral genome in the samples under study, taking into account that the correlation should be studied and optimized for the specific set.

Keywords: African swine fever virus, real-time polymerase chain reaction, haemadsorption reaction, Ct values, CC cell culture, CCM cell culture, African swine fever virus genome, correlation, number of copies, biological properties, quantitative method.

References:

1. Samuylenko A.Ya. et al. Afrikanskaya chuma sviney [African swine fever]. - Moscow, 2006. - pp. 807-817.

2. Bakulov I.A, Makarov V.V. Problemy sovremennoy evolyutsii afrikanskoy chumy sviney [Problems of modern evolution of African swine fever]. - 1990: 122128.

3. Barinskiy I.F., Shubladze A.K. Leykotsitarnyye kul tury v virusologicheskikh issledovaniyakh/AMN SSSR.- M.: Meditsina.- 1980.- 176 s.

4. GOST 28573-90. Svini. Metody laboratornoy diagnostiki afrikanskoy chumy [Pigs. Methods of laboratory diagnosis of African swine fever]. - Standartinform. - Moscow, 2005. - 11 p

5. Instruktsiya k «AmpliPraym DNK-sorb-V - InterLabServis» [Manual for AmpliPrime DNA-Sorb-B - InterLabService]. - http://www.interlabservice.ru/upload/iblock/ff0/Instruktsiya%20DNK-sorb-V_30.10.2012.pdf.

6. Instruktsiya k «test-sistema «AChS» dlya vyyavleniya virusa afrikanskoy chumy sviney metodom polimeraznoy tsepnoy reaktsii» [Instructions to ASF test system for detection of African swine fever virus by polymerase chain reaction method]. - http://www.interlabservice.ru/catalog/reagents/?sid=2119.

7. Instruktsiya k elyutsii PTsR-produkt iz agaroznogo gela - Qiagen [Instructions for elution PCR product from agarose gel - Qiagen]. - http://www.indiana.edu/~lchenlab/protocol_files/agarose_gel_extraction.pdf.

8. Makarov V.V. Afrikanskaya chuma sviney [African swine fever]. - RUDN. - Moscow, 2011. - 268 p.

9. Varentsova A.A. et al. Metodicheskie rekomendatsii po vydeleniyu i titrovaniyu virusa afrikanskoy chumy sviney v kulture kletok kostnogo mozga sviney [Guidelines for isolation and titration of African swine fever virus in pig bone marrow cell culture]. - VNIIZZh. - Vladimir, 2014. - 17 p.

10. Varentsova A.A. et al. Metodicheskie rekomendatsii po vydeleniyu i titrovaniyu virusa afrikanskoy chumy sviney v kulture kletok leykotsitov sviney [Guidelines for isolation and titration of African swine fever virus in cell culture of porcine leukocytes]. - VNIIZZh. - Vladimir, 2014. - 21 p.

11. Elsukova A.A. et al. Metodicheskie rekomendatsii po vyyavleniyu genoma virusa afrikanskoy chumy sviney metodom polimeraznoy tsepnoy reaktsii v rezhime realnogo vremeni [Guidelines for African swine fever virus genome identifying by real-time polymerase chain reaction].- VNIIZZh. - Vladimir, 2015. - 10 p. 12-24. Vide supra.

Author affiliation:

Mazlum Ali, post-graduate student of the reference laboratory for ASF of the Federal Centre of Animal Health; mcrd. Yurjevets, Vladimir, Russia, 600901; e-mail: ali.mazloum6@gmail.com.

Vlasova Natalya N., D.Sc. in Biology, senior scientific researcher of the reference laboratory for ASF of the Federal Centre of Animal Health; mcrd. Yurjevets, Vladimir, Russia, 600901; e-mail: vlasova_nn@arriah.ru.

Aronova Elena V., Ph.D. in Biology, senior scientific researcher of the reference laboratory for ASF of the Federal Centre of Animal Health; mcrd. Yurjevets, Vladimir, Russia, 600901; e-mail: aronova@arriah.ru.

Igolkin Aleksey S., Ph.D. in Veterinary Medicine, Head of the reference laboratory for ASF of the Federal Centre of Animal Health; mcrd. Yurjevets, Vladimir, Russia, 600901; e-mail: igolkin_as@arriah.ru.

Krivonos Roman A., Ph.D. in Veterinary Medicine, Head of the Veterinary Department of Krasnodar region; 36, Rashpilevskaya st., Krasnodar; e-mail: sinkubani@mail.ru.

Responsible for correspondence with the editorial board: Chernykh Oleg Yu., D.Sc. in Veterinary Medicine, director of the Kropotkin territorial veterinary laboratory; 303, Krasnoarmeyskaya st., Kropotkin, 352380; phone: 8-86138-62314; e-mail: gukkvl50@kubanvet.ru.