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Development and application of a cycleave dual-probe fluorescence quantitative PCR method for simultaneous detection of Mycoplasma gallisepticum ts-11 vaccine strain and non-ts-11 strains

Authors :
Xue Wang
Nana Sun
Meng Wang
Hui Wang
Yuhan Liu
Hao Shi
Hao Zhu
Peidong Li
Fuyou Zhang
Tianyao Yang
Zhaoyang Li
Chunguo Liu
Source :
Poultry Science, Vol 103, Iss 8, Pp 103907- (2024)
Publication Year :
2024
Publisher :
Elsevier, 2024.

Abstract

ABSTRACT: An attenuated vaccine against the Mycoplasma gallisepticum ts-11 strain has become an effective prevention and control method against MG infection. However, the ts-11 strain is usually difficult to distinguish from the non-ts-11 strain (including field isolates and other vaccine strains (F and 6/85)). Therefore, it is critical to establish a rapid and effective method to distinguish ts-11 strains from non-ts-11 strains. The gene sequences of the ts-11 strain (CP044225.1) and the non-ts-11 strain (including the wild-type (CP006916.3), 6/85 (CP044224.1), and F strains (NC_017503.1) were used to construct a conserved region containing a single point mutation in the potC gene in the ts-11 strain, after which a primer–probe combination method was designed. The primer–probe method was able to accurately and efficiently identify the ts-11 and non-ts-11 strains with minimum detection limits of 2.43 copies/μL and 1.65 copies/μL, respectively. Moreover, it could simultaneously distinguish the ts-11 strain from a non-ts-11 strain, and amplifications of avian influenza virus, infectious bronchitis virus, Newcastle disease virus, fowl adenovirus, infectious laryngotracheitis virus, infectious bursal disease virus, chicken anemia virus, Marek's disease virus, Mycoplasma synoviae, and Ornithobacter rhinotracheale were negative. The detection of clinical samples revealed that the established dual-probe fluorescence quantitative PCR method could be used to screen for mixed and single infections of the ts-11 strain and non-ts-11 strains effectively, with lower variation coefficients for intra- and interbatch repetition. The established cycleave dual-probe fluorescence quantitative PCR method showed good specificity, sensitivity, and repeatability and provides powerful technical support for the rapid and efficient differential diagnosis of the MG ts-11 strain from non-ts-11 strains.

Details

Language :
English
ISSN :
00325791
Volume :
103
Issue :
8
Database :
Directory of Open Access Journals
Journal :
Poultry Science
Publication Type :
Academic Journal
Accession number :
edsdoj.63ba787ced34bb288064464d0e7257b
Document Type :
article
Full Text :
https://doi.org/10.1016/j.psj.2024.103907