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J Vet Sci 2018; 19(2): 242-250  https://doi.org/10.4142/jvs.2018.19.2.242
Rapid visual detection of Mycobacterium avium subsp. paratuberculosis by recombinase polymerase amplification combined with a lateral flow dipstick
Guimin Zhao1,2,†, Hongmei Wang1,2,†, Peili Hou1,2, Chengqiang He1,2, Hongbin He1,2,*
1Key Laboratory of Animal Resistant Biology of Shandong, College of Life Science, and 2Ruminant Disease Research Center, Shandong Normal University, Jinan 250014, China
Correspondence to: Tel: +86-531-86180201; Fax: +86-531-86180201; 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: March 31, 2018.
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°C 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.
Keywords: Mycobacterium avium subsp. paratuberculosis, isothermal detection, lateral flow dipstick, paratuberculosis, recombinase polymerase amplification


© 2018 The Korean Society of Veterinary Science.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.