Establishment and application of real-time fluorescent RAA detection method tar-getingspecific gene F57 of Mycobacterium avium subsp.paratuberculosis
10.16303/j.cnki.1005-4545.2025.04.11
- VernacularTitle:以副结核分枝杆菌特异性基因F57为靶标的实时荧光RAA检测方法的建立与应用
- Author:
Ziliang ZHAO
1
;
Suhui ZHANG
;
Jiabei HAN
;
Shaomei LI
;
Liu YANG
;
Lizhi FU
;
Kefei SHEN
Author Information
1. 国家生猪技术创新中心,重庆 402460
- Publication Type:Journal Article
- Keywords:
Mycobacterium paratuberculosis;
real time fluorescence RAA;
F57 gene
- From:
Chinese Journal of Veterinary Science
2025;45(4):699-706
- CountryChina
- Language:Chinese
-
Abstract:
To rapidly and accurately detect Mycobacterium avium subsp.paratuberculosis(MAP),this study designed and screened primers and probes using its specific gene F57 as the detection target,established a recombinant enzyme-mediated isothermal amplification(RAA)fluorescence detection method,and applied this method to detect 116 clinical samples from cattle and sheep.The results showed that using the primer and probe combination B12F/B2R(0.4 μmol/L)+Probe B(0.12 μmol/L),MAP could be detected at a constant temperature of 42 ℃ within 20 min;this de-tection method had no cross-reaction with 11 common pathogens such as Escherichia coli,Clos-tridium,and bovine viral diarrhea in sheep and cattle;the lowest detection limit was 1.0×102 cop-ies/μL;the coefficient of variation was 3.77％—5.29％;24 clinical samples were positive,with a co-incidence rate of 88.89％with GBT27637-2011.In summary,this study established a fluorescent RAA detection method for MAP,which is simple,rapid,highly specific,sensitive,reproducible,and has a high coincidence rate with national standards,making it suitable for clinical detection and epi-demiological studies.
