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Guimin Zhao,Hongmei Wang,Peili Hou,Chengqiang He,Hongbin He 대한수의학회 2018 Journal of Veterinary Science Vol.19 No.2
Paratuberculosis (Johne’s disease) is a chronic debilitating disease of domestic and wild ruminants. However, widespread point-of-care testingis infrequent due to the lack of a robust method. The isothermal recombinase polymerase amplification (RPA) technique has applied for rapiddiagnosis. 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 successfullydetected M. paratuberculosis DNA in 30 min at 39oC with a detection limit of up to eight copies per reaction, which was equivalent to thatof the real-time quantitative polymerase chain reaction (qPCR) assay. The assay was specific, as it did not amplify genomes from five otherMycobacterium spp. or five pathogenic enteric bacteria. Six hundred-twelve clinical samples (320 fecal and 292 serum) were assessed byRPA-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.