Vet World   Vol.14   December-2021  Article-5

Development of multiplex reverse transcription-polymerase chain reaction for differentiation of strains of infectious bursal disease virus and primary screening of the virus in Thailand

Background and Aim: A new set of primers (400 base pairs partial of VP2) was designed and used for the infectious bursal disease virus (IBDV) screening test. Using this new primer set, the enzymes MboI and BstNI were unable to differentiate the field and vaccine strains. As a result, a new simple, cheap, and appropriate tool for strain differentiation is required. The objective of this study was to develop the appropriate restriction fragment length polymorphism (RFLP) and multiplex reverse transcription-polymerase chain reaction (RT-PCR) for the differentiation of classic IBDV (cIBDV) strains and very virulent IBDV (vvIBDV) strains in Thailand.

Materials and Methods: Ninety seven bursa of Fabricius from 16 farms were collected from farms in the eastern and central regions of Thailand. RT-PCR screening showed that 82 samples were positive for IBDV and 15 samples were negative. Then, selected samples were sequenced from each farm with a positive test.

Results: The sequencing results showed that samples from six of the farms were vvIBDV and samples from the other six farms were cIBDV. Although the whole genome sequencing was incomplete, both the sequencing results of segment A and segment B showed high similarity between cIBDV and vvIBDV. Restriction enzyme cutting site and primers for multiplex RT-PCR were hard to design. An RT-PCR-RFLP method was developed, but it failed to differentiate IBDV strains. However, the multiplex RT-PCR was able to differentiate cIBDV from vvIBDV. Four primers were used in the multiplex RT-PCR.

Conclusion: These four primers were used together in one reaction at an annealing temperature of 45°C. Therefore, multiplex RT-PCR is a less complicated, cheaper, and less time-consuming method for the differentiation of cIBDV and vvIBDV strains. Keywords: infectious bursal disease virus, multiplex reverse transcription-polymerase chain reaction, restriction fragment length polymorphism, strain, Thailand.

Keywords: infectious bursal disease virus, multiplex reverse transcription-polymerase chain reaction, restriction fragment length polymorphism, strain, Thailand.

How to cite this article: Charoenvisal N (2021) Development of multiplex reverse transcription-polymerase chain reaction for differentiation of strains of infectious bursal disease virus and primary screening of the virus in Thailand, Veterinary World, 14(12): 3105-3110.

Received: 22-07-2021  Accepted: 28-10-2021     Published online: 14-12-2021

Corresponding author: Nataya Charoenvisal   E-mail: nataya.c@chula.ac.th

DOI: 10.14202/vetworld.2021.3105-3110

Copyright: Charoenvisal, et al. This article is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.