An improved visual closed tube Loop mediated isothermal amplification (LAMP) assay for rapid identification of orf virus in sheep and goats
The Orf virus (ORFV) is an epitheliotropic virus causing a highly contagious skin disease mainly in sheep and goats. Several diagnostics including molecular tools like Loop mediated isothermal amplification (LAMP) assay are available to detect ORFV in affected species. However, the carry‑over conta...
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| Format: | Article |
| Language: | English |
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Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise G. Caporale
2022-12-01
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| Series: | Veterinaria Italiana |
| Subjects: | |
| Online Access: | https://www.veterinariaitaliana.izs.it/index.php/VetIt/article/view/2426 |
| _version_ | 1797783992622120960 |
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| author | Gnanavel Venkatesan Anand Kushwaha Amit Kumar D.P. Bora P. Sasikumar |
| author_facet | Gnanavel Venkatesan Anand Kushwaha Amit Kumar D.P. Bora P. Sasikumar |
| author_sort | Gnanavel Venkatesan |
| collection | DOAJ |
| description |
The Orf virus (ORFV) is an epitheliotropic virus causing a highly contagious skin disease mainly in sheep and goats. Several diagnostics including molecular tools like Loop mediated isothermal amplification (LAMP) assay are available to detect ORFV in affected species. However, the carry‑over contamination associated with LAMP as open tube format prevents the assay applicability as point‑of‑care test in field diagnostic settings. In this study, the B2L gene based LAMP assay was optimized in a closed tube format using hydroxynaphthol blue (HNB) and calcein as pre‑addition dyes and it has shown a clear positive and negative signal at 60 °C using 4 and 5 mM concentrations of MgSO4 for these dyes, respectively. This optimized assay that could reveal the result within one hour is highly specific and sensitive with a limit of detection of 12.5 femtogram of viral genomic DNA or ~ 85 virus genome equivalent. This improved method can also prevent the cross‑contamination of LAMP reactions in the laboratory without compromising diagnostic sensitivity and specificity when compared to open tube system. This closed tube LAMP method has potential to act as a simple visual detection assay for the rapid and specific diagnosis of ORFV in sheep and goats.
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| first_indexed | 2024-03-13T00:33:39Z |
| format | Article |
| id | doaj.art-370f3f897b6044cca08db30afec1f4af |
| institution | Directory Open Access Journal |
| issn | 0505-401X 1828-1427 |
| language | English |
| last_indexed | 2024-03-13T00:33:39Z |
| publishDate | 2022-12-01 |
| publisher | Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise G. Caporale |
| record_format | Article |
| series | Veterinaria Italiana |
| spelling | doaj.art-370f3f897b6044cca08db30afec1f4af2023-07-10T07:53:43ZengIstituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise G. CaporaleVeterinaria Italiana0505-401X1828-14272022-12-0158210.12834/VetIt.2426.15340.2An improved visual closed tube Loop mediated isothermal amplification (LAMP) assay for rapid identification of orf virus in sheep and goatsGnanavel Venkatesan0Anand Kushwaha1Amit Kumar2D.P. Bora3P. SasikumarIndian Veterinary Research InstituteDivision of Virology, ICAR-IVRI, Mukteswar 263 138, Nainital, Uttarakhand, Indiaivision of Virology, ICAR-IVRI, Mukteswar 263 138, Nainital, Uttarakhand, IndiaDepartment of Microbiology, College of Veterinary Science, AAU, Khanapara, Guwahati, Assam-781022, India The Orf virus (ORFV) is an epitheliotropic virus causing a highly contagious skin disease mainly in sheep and goats. Several diagnostics including molecular tools like Loop mediated isothermal amplification (LAMP) assay are available to detect ORFV in affected species. However, the carry‑over contamination associated with LAMP as open tube format prevents the assay applicability as point‑of‑care test in field diagnostic settings. In this study, the B2L gene based LAMP assay was optimized in a closed tube format using hydroxynaphthol blue (HNB) and calcein as pre‑addition dyes and it has shown a clear positive and negative signal at 60 °C using 4 and 5 mM concentrations of MgSO4 for these dyes, respectively. This optimized assay that could reveal the result within one hour is highly specific and sensitive with a limit of detection of 12.5 femtogram of viral genomic DNA or ~ 85 virus genome equivalent. This improved method can also prevent the cross‑contamination of LAMP reactions in the laboratory without compromising diagnostic sensitivity and specificity when compared to open tube system. This closed tube LAMP method has potential to act as a simple visual detection assay for the rapid and specific diagnosis of ORFV in sheep and goats. https://www.veterinariaitaliana.izs.it/index.php/VetIt/article/view/2426Orf virusClosed tube LAMPVisual detectionPOCTDiagnosisControl |
| spellingShingle | Gnanavel Venkatesan Anand Kushwaha Amit Kumar D.P. Bora P. Sasikumar An improved visual closed tube Loop mediated isothermal amplification (LAMP) assay for rapid identification of orf virus in sheep and goats Veterinaria Italiana Orf virus Closed tube LAMP Visual detection POCT Diagnosis Control |
| title | An improved visual closed tube Loop mediated isothermal amplification (LAMP) assay for rapid identification of orf virus in sheep and goats |
| title_full | An improved visual closed tube Loop mediated isothermal amplification (LAMP) assay for rapid identification of orf virus in sheep and goats |
| title_fullStr | An improved visual closed tube Loop mediated isothermal amplification (LAMP) assay for rapid identification of orf virus in sheep and goats |
| title_full_unstemmed | An improved visual closed tube Loop mediated isothermal amplification (LAMP) assay for rapid identification of orf virus in sheep and goats |
| title_short | An improved visual closed tube Loop mediated isothermal amplification (LAMP) assay for rapid identification of orf virus in sheep and goats |
| title_sort | improved visual closed tube loop mediated isothermal amplification lamp assay for rapid identification of orf virus in sheep and goats |
| topic | Orf virus Closed tube LAMP Visual detection POCT Diagnosis Control |
| url | https://www.veterinariaitaliana.izs.it/index.php/VetIt/article/view/2426 |
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