A Multiplex PCR Assay for Differential Identification of Wild-type and Vaccine Strains of Mycoplasma gallisepticum

Abstract

Mycoplasma gallisepticum (MG) can cause respiratory disease in chickens and result in serious economic losses in the chicken industry. The use of live vaccines has been a favorable option for the control of MG infection in multi-age commercial layers and broiler breeders. There are three live vaccines, including ts-11, 6/85, and F strain, that have been commonly used in various parts of the world, including South Korea. The definitive diagnosis of the infection, therefore, requires the differentiation of wild-type field strains of MG from the vaccine strains used. Thus, we aimed to develop a novel multiplex PCR assay to discriminate between vaccine strains (ts-11, 6/85, and F strain) and wild-type field strains of MG isolated from infected chickens. We designed four novel primer sets that are each specific to MG species, ts-11, 6/85, and F strain. The multiplex PCR assay using the primer sets differentially identified wild-type and vaccine strains of MG but did not detect other avian bacteria. The detection limit of this assay was 250 fg/μL of genomic DNA of each strain tested. In addition, this assay was applied to 36 MG strains isolated from chickens over the past 20 years in South Korea. As a result, the assay identified 22 wild-type strains and 14 vaccine strains. Consequently, the novel multiplex PCR assay can discriminate between vaccine and wild-type field strains of MG and could be a valuable tool for the diagnosis of MG infection in MG-vaccinated chicken flocks.

Mycoplasma galliscepticum: An overview

“…It was also observed that the positivity rate for MG is higher in the arid inland regions of Iran compared to the coastal humid regions. Kang et al (2023) used multiplex PCR to target four loci (parE, RS03710-rlmB, scpA, and MGF_RS03965) of MG in order to distinguish between three vaccine strains (ts-11, 6/85, and F strains) and wildtype strains of MG. Tan et al (2014) utilized a PCR reaction combined with reverse transcription polymerase chain reaction (RT-PCR) to target the MG 16S rRNA gene and assess the viability of MG. This method helps to detect and to some extent distinguish between living and dead MG.…”