Polymerase spiral reaction assay for rapid visual detection of Mycobacterium avium subsp. paratuberculosis in fecal samples
Abstract A rapid, sensitive, and specific visual detection assay for Mycobacterium avium subsp. paratuberculosis (MAP) was developed and optimized using a polymerase spiral reaction (PSR) method. A pair of primers was designed targeting MAP specific sequence of IS900 putative transposes (p43) gene,...
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Nature Portfolio
2025-07-01
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| Online Access: | https://doi.org/10.1038/s41598-025-12435-3 |
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| author | Vinod Kumar Singh Vikas Gupta Chayanika Das Amit Kumar Sharad K. Yadav |
| author_facet | Vinod Kumar Singh Vikas Gupta Chayanika Das Amit Kumar Sharad K. Yadav |
| author_sort | Vinod Kumar Singh |
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| description | Abstract A rapid, sensitive, and specific visual detection assay for Mycobacterium avium subsp. paratuberculosis (MAP) was developed and optimized using a polymerase spiral reaction (PSR) method. A pair of primers was designed targeting MAP specific sequence of IS900 putative transposes (p43) gene, and PSR results were assessed using agarose gel electrophoresis and colour change with SYBR Green-I dye. The assay was optimized using water bath and the optimum reaction time and temperature for MAP-PSR were 60 min and 64 °C, respectively. The sign of target amplification can be visualized by naked eyes as SYBR Green-I change its colour due to intercalation with amplified products. The developed assay demonstrated that the primers specifically detected MAP and showed no cross-reaction with other common Mycobacterium. The sensitivity of the PSR assay for MAP detection was 122 fg or ~ 23 copy number of the template. The MAP-PSR assay was also evaluated using 100 clinical samples, and a total 59 samples were found positive. When same samples were tested by conventional PCR, 50 samples were found positive, and all PCR positive clinical samples were found positive by MAP-PSR too. In conclusion the developed MAP–PSR assay is simple, rapid, specific, and sensitive, and thereby very suitable for application and promotion in the field and resource limited laboratories. |
| format | Article |
| id | doaj-art-b1ba1d43a3214e989bc34c79fc6f1e72 |
| institution | Kabale University |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-07-01 |
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| spelling | doaj-art-b1ba1d43a3214e989bc34c79fc6f1e722025-08-20T03:46:07ZengNature PortfolioScientific Reports2045-23222025-07-011511710.1038/s41598-025-12435-3Polymerase spiral reaction assay for rapid visual detection of Mycobacterium avium subsp. paratuberculosis in fecal samplesVinod Kumar Singh0Vikas Gupta1Chayanika Das2Amit Kumar3Sharad K. Yadav4Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, DUVASUCCS-National Institute of Animal HealthDivision of Veterinary Microbiology, ICAR-Indian Veterinary Research InstituteDivision of Animal Biotechnology, College of Biotechnology, SVPUATDepartment of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, DUVASUAbstract A rapid, sensitive, and specific visual detection assay for Mycobacterium avium subsp. paratuberculosis (MAP) was developed and optimized using a polymerase spiral reaction (PSR) method. A pair of primers was designed targeting MAP specific sequence of IS900 putative transposes (p43) gene, and PSR results were assessed using agarose gel electrophoresis and colour change with SYBR Green-I dye. The assay was optimized using water bath and the optimum reaction time and temperature for MAP-PSR were 60 min and 64 °C, respectively. The sign of target amplification can be visualized by naked eyes as SYBR Green-I change its colour due to intercalation with amplified products. The developed assay demonstrated that the primers specifically detected MAP and showed no cross-reaction with other common Mycobacterium. The sensitivity of the PSR assay for MAP detection was 122 fg or ~ 23 copy number of the template. The MAP-PSR assay was also evaluated using 100 clinical samples, and a total 59 samples were found positive. When same samples were tested by conventional PCR, 50 samples were found positive, and all PCR positive clinical samples were found positive by MAP-PSR too. In conclusion the developed MAP–PSR assay is simple, rapid, specific, and sensitive, and thereby very suitable for application and promotion in the field and resource limited laboratories.https://doi.org/10.1038/s41598-025-12435-3Polymerase spiral reaction (PSR) assayIS900 (p43gene)Mycobacterium avium subsp paratuberculosisJohne’s diseaseFaecal samples |
| spellingShingle | Vinod Kumar Singh Vikas Gupta Chayanika Das Amit Kumar Sharad K. Yadav Polymerase spiral reaction assay for rapid visual detection of Mycobacterium avium subsp. paratuberculosis in fecal samples Scientific Reports Polymerase spiral reaction (PSR) assay IS900 (p43gene) Mycobacterium avium subsp paratuberculosis Johne’s disease Faecal samples |
| title | Polymerase spiral reaction assay for rapid visual detection of Mycobacterium avium subsp. paratuberculosis in fecal samples |
| title_full | Polymerase spiral reaction assay for rapid visual detection of Mycobacterium avium subsp. paratuberculosis in fecal samples |
| title_fullStr | Polymerase spiral reaction assay for rapid visual detection of Mycobacterium avium subsp. paratuberculosis in fecal samples |
| title_full_unstemmed | Polymerase spiral reaction assay for rapid visual detection of Mycobacterium avium subsp. paratuberculosis in fecal samples |
| title_short | Polymerase spiral reaction assay for rapid visual detection of Mycobacterium avium subsp. paratuberculosis in fecal samples |
| title_sort | polymerase spiral reaction assay for rapid visual detection of mycobacterium avium subsp paratuberculosis in fecal samples |
| topic | Polymerase spiral reaction (PSR) assay IS900 (p43gene) Mycobacterium avium subsp paratuberculosis Johne’s disease Faecal samples |
| url | https://doi.org/10.1038/s41598-025-12435-3 |
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