Development and evaluation of loop-mediated isothermal amplification for rapid detection of Nosema ceranae in honeybee

© 2016 Asian Pacific Tropical Medicine Press Objective To develop loop-mediated isothermal amplification (LAMP) to detect Nosema ceranae (N. ceranae) in honeybee samples. Methods LAMP primers were designed recognizing six distinct fragments of 16s rRNA gene and LAMP reaction was determined by optimi...

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Main Authors: Vena Chupia, Prapas Patchanee, Patcharin Krutmuang, Surachai Pikulkaew
Format: Journal
Published: 2018
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http://cmuir.cmu.ac.th/jspui/handle/6653943832/55991
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Institution: Chiang Mai University
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spelling th-cmuir.6653943832-559912018-09-05T03:07:27Z Development and evaluation of loop-mediated isothermal amplification for rapid detection of Nosema ceranae in honeybee Vena Chupia Prapas Patchanee Patcharin Krutmuang Surachai Pikulkaew Medicine © 2016 Asian Pacific Tropical Medicine Press Objective To develop loop-mediated isothermal amplification (LAMP) to detect Nosema ceranae (N. ceranae) in honeybee samples. Methods LAMP primers were designed recognizing six distinct fragments of 16s rRNA gene and LAMP reaction was determined by optimizing the concentration of reagents, such as forward inner primer and backward inner primer, deoxynucleoside triphosphate and betaine, time and temperature. Ten-fold serial dilutions of DNA were used to determine the detection limit and accuracy using both LAMP and PCR tests. Results LAMP required 1.2 μmol/L of forward inner primer and backward inner primer primers, 0.2 μmol/L of forward outer primers and backward outer primer, 2 μmol/L of Mg2+, 0.6 mol/L of betaine, 0.6 μmol/L of deoxynucleoside triphosphate, 4.8 IU of Bst DNA polymerase and 30 ng of DNA. The optimal temperature was 63 °C and after a 40-min incubation time, a clearly ladder-like pattern of LAMP product appeared in the gel electrophoresis. LAMP appeared more sensitive than a standard PCR in detection of N. ceranae. Conclusions LAMP gave a good results and it could be an alternative diagnostic tool instead of PCR to detect N. ceranae infection in honeybee. 2018-09-05T03:07:27Z 2018-09-05T03:07:27Z 2016-12-01 Journal 22221808 2-s2.0-84997521865 10.1016/S2222-1808(16)61163-5 https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84997521865&origin=inward http://cmuir.cmu.ac.th/jspui/handle/6653943832/55991
institution Chiang Mai University
building Chiang Mai University Library
country Thailand
collection CMU Intellectual Repository
topic Medicine
spellingShingle Medicine
Vena Chupia
Prapas Patchanee
Patcharin Krutmuang
Surachai Pikulkaew
Development and evaluation of loop-mediated isothermal amplification for rapid detection of Nosema ceranae in honeybee
description © 2016 Asian Pacific Tropical Medicine Press Objective To develop loop-mediated isothermal amplification (LAMP) to detect Nosema ceranae (N. ceranae) in honeybee samples. Methods LAMP primers were designed recognizing six distinct fragments of 16s rRNA gene and LAMP reaction was determined by optimizing the concentration of reagents, such as forward inner primer and backward inner primer, deoxynucleoside triphosphate and betaine, time and temperature. Ten-fold serial dilutions of DNA were used to determine the detection limit and accuracy using both LAMP and PCR tests. Results LAMP required 1.2 μmol/L of forward inner primer and backward inner primer primers, 0.2 μmol/L of forward outer primers and backward outer primer, 2 μmol/L of Mg2+, 0.6 mol/L of betaine, 0.6 μmol/L of deoxynucleoside triphosphate, 4.8 IU of Bst DNA polymerase and 30 ng of DNA. The optimal temperature was 63 °C and after a 40-min incubation time, a clearly ladder-like pattern of LAMP product appeared in the gel electrophoresis. LAMP appeared more sensitive than a standard PCR in detection of N. ceranae. Conclusions LAMP gave a good results and it could be an alternative diagnostic tool instead of PCR to detect N. ceranae infection in honeybee.
format Journal
author Vena Chupia
Prapas Patchanee
Patcharin Krutmuang
Surachai Pikulkaew
author_facet Vena Chupia
Prapas Patchanee
Patcharin Krutmuang
Surachai Pikulkaew
author_sort Vena Chupia
title Development and evaluation of loop-mediated isothermal amplification for rapid detection of Nosema ceranae in honeybee
title_short Development and evaluation of loop-mediated isothermal amplification for rapid detection of Nosema ceranae in honeybee
title_full Development and evaluation of loop-mediated isothermal amplification for rapid detection of Nosema ceranae in honeybee
title_fullStr Development and evaluation of loop-mediated isothermal amplification for rapid detection of Nosema ceranae in honeybee
title_full_unstemmed Development and evaluation of loop-mediated isothermal amplification for rapid detection of Nosema ceranae in honeybee
title_sort development and evaluation of loop-mediated isothermal amplification for rapid detection of nosema ceranae in honeybee
publishDate 2018
url https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84997521865&origin=inward
http://cmuir.cmu.ac.th/jspui/handle/6653943832/55991
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