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Rapid and visual detection of Mycobacterium avium subsp. paratuberculosis by recombinase polymerase amplification combined with a lateral flow dipstick
Zhao Guimin1,2,†, Wang Hongmei1,2,†,Hou Peili1,2, He Chengqiang1,2, He Hongbin1,2,*
1College of Life Science, Shandong Normal University, Shandong Province 250014, China
2Ruminant Disease Research Center, Shandong Normal University, Shandong Province 250014, China
Correspondence to: Tel: +86-0531-86180201; E-mail: hongbinhe@sdnu.edu.cn
The first two authors contributed equally to this work.
Received: April 18, 2017; Revised: October 20, 2017; Accepted: November 24, 2017; Published online: December 28, 2017.
Paratuberculosis (Johne’s disease) is a chronic debilitating disease of domestic and wild ruminants. Quick diagnosis could facilitate control; however widespread point-of-care testing is infrequently done due to the lack of robust method. Isothermal recombinase polymerase amplification (RPA) technique has emerged as a novel DNA amplify assay for use in rapid diagnosis. Here, an RPA combined with lateral flow dipstick (LFD) assay was developed to estimate DNA from M.paratuberculosis. First, the specificity and sensitivity of RPA-nfo primer and probe sets were assessed. The assay successfully detected M.paratuberculosis DNA in 30 minutes at 39°C, limit of detection up to eight copies per reaction, which was equivalent with the real-time quantitative PCR (qPCR) assay. The assay was specific, as it did not amplify genomes from five other Mycobacterium and five pathogenic enteric bacteria. Then, 612 clinical samples (320 fecal and 292 serum) were assessed by RPA-LFD, qPCR and ELISA assays respectively, also the established RPA-LFD assay yielded 100% sensitivity, 97.63% specificity, and 98.44% concordance rate with the qPCR. This is the first report utilizing an RPA-LFD assay to visual and rapid detect M.paratuberculosis. Our results show this assay should be a useful method for the diagnosis of paratuberculosis in resource constrained setting.
Keywords: Isothermal detection, Lateral flow dipstick, Mycobacterium avium subsp. paratuberculosis, Paratuberculosis, Recombinase polymerase amplification

© 2017 The Korean Society of Veterinary Science.