A Field-Friendly Loop-Mediated Isothermal Amplification (FF-LAMP) method for rapid detection of Nosema bombycis in silkworm, Bombyx mori
Pebrine is a destructive disease that exhibits horizontal and vertical transmission and therefore it is the only mandatory quarantine item in sericulture. Here, a field-friendly loop-mediated isothermal amplification (FF-LAMP) method has been developed and validated for the rapid detection of Nosem...
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University of Modena and Reggio Emilia
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doaj-43c5ada4cf0e4d02a817f3d56e27fa862021-10-02T19:05:20ZengUniversity of Modena and Reggio EmiliaInvertebrate Survival Journal1824-307X2021-06-01181A Field-Friendly Loop-Mediated Isothermal Amplification (FF-LAMP) method for rapid detection of Nosema bombycis in silkworm, Bombyx moriV Sivaprasad0L Satish1G Mallikarjuna2N Chandrakanth 3A V Mary Josepha4S M Moorthy5Central Sericultural Research and Training Institute, Berhampore - 742 101, West Bengal, IndiaCentral Sericultural Research and Training Institute, Mysuru - 570 008, Karnataka, IndiaCentral Sericultural Research and Training Institute, Mysuru - 570 008, Karnataka, IndiaCentral Sericultural Research and Training Institute, Berhampore - 742 101, West Bengal, IndiaCentral Sericultural Research and Training Institute, Mysuru - 570 008, Karnataka, IndiaCentral Sericultural Research and Training Institute, Mysuru - 570 008, Karnataka, India Pebrine is a destructive disease that exhibits horizontal and vertical transmission and therefore it is the only mandatory quarantine item in sericulture. Here, a field-friendly loop-mediated isothermal amplification (FF-LAMP) method has been developed and validated for the rapid detection of Nosema bombycis, a causative agent of pebrine disease in silkworm, Bombyx mori. FF-LAMP primers were selected and designed for small ribosomal subunit gene and the assay was performed to detect the N. bombycis infection in silkworm. The FF-LAMP reaction was effective at 6 mM MgSO4, 1.4 mM dNTPs at 63 °C. The detection range of LAMP assay was found to be 101 dilutions of N. bombycis spores. Specificity of the primers was tested using DNA isolated from pebrine infected silkworm, pebrine free silkworm and pure N. bombycis by conventional PCR and FF-LAMP assay. Results revealed that the primers were specific to N. bombycis DNA. The FF-LAMP assay was validated in different basic silkworm seed farms with simultaneous microscopic examination of N. bombycis infection. This newly developed method is highly effective, specific, sensitive and rapid in detecting N. bombycis infection, eliminating the DNA purification steps and usage of sophisticated equipment. This method can be used in testing large number of samples making it field friendly method in sericulture industry. https://www.isj.unimore.it/index.php/ISJ/article/view/681Bombyx moriNosema bombycispebrineLoop-Mediated Isothermal AmplificationNCBI |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
V Sivaprasad L Satish G Mallikarjuna N Chandrakanth A V Mary Josepha S M Moorthy |
spellingShingle |
V Sivaprasad L Satish G Mallikarjuna N Chandrakanth A V Mary Josepha S M Moorthy A Field-Friendly Loop-Mediated Isothermal Amplification (FF-LAMP) method for rapid detection of Nosema bombycis in silkworm, Bombyx mori Invertebrate Survival Journal Bombyx mori Nosema bombycis pebrine Loop-Mediated Isothermal Amplification NCBI |
author_facet |
V Sivaprasad L Satish G Mallikarjuna N Chandrakanth A V Mary Josepha S M Moorthy |
author_sort |
V Sivaprasad |
title |
A Field-Friendly Loop-Mediated Isothermal Amplification (FF-LAMP) method for rapid detection of Nosema bombycis in silkworm, Bombyx mori |
title_short |
A Field-Friendly Loop-Mediated Isothermal Amplification (FF-LAMP) method for rapid detection of Nosema bombycis in silkworm, Bombyx mori |
title_full |
A Field-Friendly Loop-Mediated Isothermal Amplification (FF-LAMP) method for rapid detection of Nosema bombycis in silkworm, Bombyx mori |
title_fullStr |
A Field-Friendly Loop-Mediated Isothermal Amplification (FF-LAMP) method for rapid detection of Nosema bombycis in silkworm, Bombyx mori |
title_full_unstemmed |
A Field-Friendly Loop-Mediated Isothermal Amplification (FF-LAMP) method for rapid detection of Nosema bombycis in silkworm, Bombyx mori |
title_sort |
field-friendly loop-mediated isothermal amplification (ff-lamp) method for rapid detection of nosema bombycis in silkworm, bombyx mori |
publisher |
University of Modena and Reggio Emilia |
series |
Invertebrate Survival Journal |
issn |
1824-307X |
publishDate |
2021-06-01 |
description |
Pebrine is a destructive disease that exhibits horizontal and vertical transmission and therefore it is the only mandatory quarantine item in sericulture. Here, a field-friendly loop-mediated isothermal amplification (FF-LAMP) method has been developed and validated for the rapid detection of Nosema bombycis, a causative agent of pebrine disease in silkworm, Bombyx mori. FF-LAMP primers were selected and designed for small ribosomal subunit gene and the assay was performed to detect the N. bombycis infection in silkworm. The FF-LAMP reaction was effective at 6 mM MgSO4, 1.4 mM dNTPs at 63 °C. The detection range of LAMP assay was found to be 101 dilutions of N. bombycis spores. Specificity of the primers was tested using DNA isolated from pebrine infected silkworm, pebrine free silkworm and pure N. bombycis by conventional PCR and FF-LAMP assay. Results revealed that the primers were specific to N. bombycis DNA. The FF-LAMP assay was validated in different basic silkworm seed farms with simultaneous microscopic examination of N. bombycis infection. This newly developed method is highly effective, specific, sensitive and rapid in detecting N. bombycis infection, eliminating the DNA purification steps and usage of sophisticated equipment. This method can be used in testing large number of samples making it field friendly method in sericulture industry.
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topic |
Bombyx mori Nosema bombycis pebrine Loop-Mediated Isothermal Amplification NCBI |
url |
https://www.isj.unimore.it/index.php/ISJ/article/view/681 |
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