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

• Zhao G
• Wang H
• Hou P
• He C
• He H

• KMID: 2407623
• DOI: http://doi.org/10.4142/jvs.2018.19.2.242

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

MeSH Terms

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
DNA
Recombinases

Figure

• 

  Fig. 1 Screening of Mycobacterium paratuberculosis recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) primer and probe. (A) The products of the RPA-nfo reaction were detected by agarose-gel electrophoresis from six primer and probe combinations (F1-LF1-R1, F2-LF1-R1, F3-LF1-R1, F4-LF2-R2, F5-LF2-R2, and F6-LF2-R2; Table 2). (B) ‘a’ shows results of six RPA-nfo reactions on LFD and the DNA template from M. paratuberculosis genomic DNA; and ‘b’ shows negative control (DNase-free water) results for the corresponding combination of primers and probe. Lane 1, F1-R1 (235 bp); Lane 2, F2-R1 (213 bp); Lane 3, F3-R1 (158 bp); Lane 4, F4-R2 (259 bp); Lane 5, F5-R2 (224 bp); Lane 6, F6-R2 (200 bp); M, molecular weight standard (DNA marker 1,000).

• 

  Fig. 2 Optimization of incubation temperature and reaction time for the Mycobacterium paratuberculosis recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) assay. (A) Amplification performance of RPA-LFD assay was effective within the range of 35℃ to 45℃. (B) Determination of optimum reaction time. After 10 min of amplification reaction, the test line was clearly visible on the LFD strip; NC, negative control (DNase-free water).

• 

  Fig. 3 Specificity of the recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) assay. The DNA extracted was detected from six strains Mycobacterium and five strains of other bacterial pathogens causing cattle diarrhea; no cross-reactions were detected. Lane 1, Mycobacterium avium subsp. paratuberculosis; Lane 2, Mycobacterium bovis; Lane 3, Mycobacterium tuberculosis; Lane 4, Mycobacterium avium; Lane 5, Mycobacterium phlei; Lane 6, Mycobacterium smegmatis; Lane 7, Escherichia coli O157:H7; Lane 8, Salmonella typhimurium; Lane 9, Campylobacter jujuni; Lane 10, Clostridium perfringens; Lane 11, Leptospira interrogans; NC, negative control (DNase-free water).

• 

  Fig. 4 Comparison of sensitivities of the recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) and quantitative polymerase chain reaction (qPCR) assays. Molecular sensitivity test results of the two assays obtained by using 10-fold serially diluted DNA as a template. (A) Results by qPCR. (B) Result by RPA-LFD. Lanes 1–8, a gradient dilution Mycobacterium paratuberculosis plasmid standard DNA from 4 × 106 to 4 copies per microliter; NC, negative control (DNase-free water).

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