Rapid visual detection of Mycobacterium avium subsp. paratuberculosis by recombinase polymerase amplification combined with a lateral flow dipstick
10.4142/jvs.2018.19.2.242
- Author:
Guimin ZHAO
1
;
Hongmei WANG
;
Peili HOU
;
Chengqiang HE
;
Hongbin HE
Author Information
1. Key Laboratory of Animal Resistant Biology of Shandong, College of Life Science, Shandong Normal University, Jinan 250014, China. hongbinhe@sdnu.edu.cn
- Publication Type:Original Article
- Keywords:
Mycobacterium avium subsp. paratuberculosis;
isothermal detection;
lateral flow dipstick;
paratuberculosis;
recombinase polymerase amplification
- MeSH:
Animals;
Diagnosis;
DNA;
Enterobacteriaceae;
Enzyme-Linked Immunosorbent Assay;
Genome;
Limit of Detection;
Methods;
Mycobacterium avium subsp. paratuberculosis;
Mycobacterium avium;
Mycobacterium;
Paratuberculosis;
Point-of-Care Testing;
Polymerase Chain Reaction;
Recombinases;
Ruminants;
Sensitivity and Specificity
- From:Journal of Veterinary Science
2018;19(2):242-250
- CountryRepublic of Korea
- Language:English
-
Abstract:
Paratuberculosis (Johne's disease) is a chronic debilitating disease of domestic and wild ruminants. However, widespread point-of-care testing is infrequent due to the lack of a robust method. The isothermal recombinase polymerase amplification (RPA) technique has applied for rapid diagnosis. Herein, RPA combined with a lateral flow dipstick (LFD) assay was developed to estimate DNA from Mycobacterium avium subsp. paratuberculosis. First, analytical specificity and sensitivity of the RPA-nfo primer and probe sets were assessed. The assay successfully detected M. paratuberculosis DNA in 30 min at 39℃ with a detection limit of up to eight copies per reaction, which was equivalent to that of the real-time quantitative polymerase chain reaction (qPCR) assay. The assay was specific, as it did not amplify genomes from five other Mycobacterium spp. or five pathogenic enteric bacteria. Six hundred-twelve clinical samples (320 fecal and 292 serum) were assessed by RPA-LFD, qPCR, and enzyme-linked immunosorbent assay, respectively. The RPA-LFD assay yielded 100% sensitivity, 97.63% specificity, and 98.44% concordance rate with the qPCR results. This is the first report utilizing an RPA-LFD assay to visualize and rapidly detect M. paratuberculosis. Our results show this assay should be a useful method for the diagnosis of paratuberculosis in resource-constrained settings.
