J Vet Sci.  2018 Mar;19(2):242-250. 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

Affiliations
  • 1Key Laboratory of Animal Resistant Biology of Shandong, College of Life Science, Shandong Normal University, Jinan 250014, China. hongbinhe@sdnu.edu.cn
  • 2Ruminant Disease Research Center, Shandong Normal University, Jinan 250014, China.

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.

Keyword

Mycobacterium avium subsp. paratuberculosis; isothermal detection; lateral flow dipstick; paratuberculosis; recombinase polymerase amplification
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