Improvements to the rapid detection of the marine pathogenic bacterium, vibrio harveyi, using loop-mediated isothermal amplification (LAMP) in combination with SYBR green

The common methods that are presently used to identify Vibrio harveyi include microscopic examination and biochemical, immunological and PCR-based assays. These methods require technical expertise, and can be time-consuming. A rapid method is required for the high-throughput screening of large numbe...

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Main Authors: Ahmad Mukhlis Abdul Rahman, Julian Ransangan, Vijay Kumar Subbiah
Format: Article
Language:English
English
Published: MDPI 2022
Subjects:
Online Access:https://eprints.ums.edu.my/id/eprint/42272/1/ABSTRACT.pdf
https://eprints.ums.edu.my/id/eprint/42272/2/FULL%20TEXT.pdf
https://eprints.ums.edu.my/id/eprint/42272/
https://doi.org/10.3390/microorganisms10122346
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spelling my.ums.eprints.422722024-12-16T03:27:26Z https://eprints.ums.edu.my/id/eprint/42272/ Improvements to the rapid detection of the marine pathogenic bacterium, vibrio harveyi, using loop-mediated isothermal amplification (LAMP) in combination with SYBR green Ahmad Mukhlis Abdul Rahman Julian Ransangan Vijay Kumar Subbiah GC1-1581 Oceanography QR1-502 Microbiology The common methods that are presently used to identify Vibrio harveyi include microscopic examination and biochemical, immunological and PCR-based assays. These methods require technical expertise, and can be time-consuming. A rapid method is required for the high-throughput screening of large number of samples. As such, we have developed a rapid, simple yet sensitive and specific detection method based on the use of the loop-mediated isothermal amplification (LAMP) of DNA. A set of six primers, i.e., two outer, two inner and two loop primers, was designed based on the in silico analysis of a large pool of 39 strains of the toxR gene sequence of V. harveyi. The addition of the loop primers decreased the reaction time of the LAMP by more than half. Furthermore, with the application of SYBR Green, the result can be obtained as quickly as in 10 to 15 min without the need of gel electrophoresis. The specificity of the method primers was then determined by performing LAMP with Vibrio and non-Vibrio samples. LAMP has a greater sensitivity than PCR reaction. The sensitivity of PCR was at 0.6 pg concentration of V. harveyi recombinant plasmid DNA standard, while LAMP was able to detect lower amounts even at 0.6 fg. The development of the LAMP assay will provide a valuable tool for the high-throughput rapid detection of V. harveyi contamination both in laboratories and in the field. MDPI 2022 Article NonPeerReviewed text en https://eprints.ums.edu.my/id/eprint/42272/1/ABSTRACT.pdf text en https://eprints.ums.edu.my/id/eprint/42272/2/FULL%20TEXT.pdf Ahmad Mukhlis Abdul Rahman and Julian Ransangan and Vijay Kumar Subbiah (2022) Improvements to the rapid detection of the marine pathogenic bacterium, vibrio harveyi, using loop-mediated isothermal amplification (LAMP) in combination with SYBR green. Microorganisms, 10. pp. 1-16. https://doi.org/10.3390/microorganisms10122346
institution Universiti Malaysia Sabah
building UMS Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Malaysia Sabah
content_source UMS Institutional Repository
url_provider http://eprints.ums.edu.my/
language English
English
topic GC1-1581 Oceanography
QR1-502 Microbiology
spellingShingle GC1-1581 Oceanography
QR1-502 Microbiology
Ahmad Mukhlis Abdul Rahman
Julian Ransangan
Vijay Kumar Subbiah
Improvements to the rapid detection of the marine pathogenic bacterium, vibrio harveyi, using loop-mediated isothermal amplification (LAMP) in combination with SYBR green
description The common methods that are presently used to identify Vibrio harveyi include microscopic examination and biochemical, immunological and PCR-based assays. These methods require technical expertise, and can be time-consuming. A rapid method is required for the high-throughput screening of large number of samples. As such, we have developed a rapid, simple yet sensitive and specific detection method based on the use of the loop-mediated isothermal amplification (LAMP) of DNA. A set of six primers, i.e., two outer, two inner and two loop primers, was designed based on the in silico analysis of a large pool of 39 strains of the toxR gene sequence of V. harveyi. The addition of the loop primers decreased the reaction time of the LAMP by more than half. Furthermore, with the application of SYBR Green, the result can be obtained as quickly as in 10 to 15 min without the need of gel electrophoresis. The specificity of the method primers was then determined by performing LAMP with Vibrio and non-Vibrio samples. LAMP has a greater sensitivity than PCR reaction. The sensitivity of PCR was at 0.6 pg concentration of V. harveyi recombinant plasmid DNA standard, while LAMP was able to detect lower amounts even at 0.6 fg. The development of the LAMP assay will provide a valuable tool for the high-throughput rapid detection of V. harveyi contamination both in laboratories and in the field.
format Article
author Ahmad Mukhlis Abdul Rahman
Julian Ransangan
Vijay Kumar Subbiah
author_facet Ahmad Mukhlis Abdul Rahman
Julian Ransangan
Vijay Kumar Subbiah
author_sort Ahmad Mukhlis Abdul Rahman
title Improvements to the rapid detection of the marine pathogenic bacterium, vibrio harveyi, using loop-mediated isothermal amplification (LAMP) in combination with SYBR green
title_short Improvements to the rapid detection of the marine pathogenic bacterium, vibrio harveyi, using loop-mediated isothermal amplification (LAMP) in combination with SYBR green
title_full Improvements to the rapid detection of the marine pathogenic bacterium, vibrio harveyi, using loop-mediated isothermal amplification (LAMP) in combination with SYBR green
title_fullStr Improvements to the rapid detection of the marine pathogenic bacterium, vibrio harveyi, using loop-mediated isothermal amplification (LAMP) in combination with SYBR green
title_full_unstemmed Improvements to the rapid detection of the marine pathogenic bacterium, vibrio harveyi, using loop-mediated isothermal amplification (LAMP) in combination with SYBR green
title_sort improvements to the rapid detection of the marine pathogenic bacterium, vibrio harveyi, using loop-mediated isothermal amplification (lamp) in combination with sybr green
publisher MDPI
publishDate 2022
url https://eprints.ums.edu.my/id/eprint/42272/1/ABSTRACT.pdf
https://eprints.ums.edu.my/id/eprint/42272/2/FULL%20TEXT.pdf
https://eprints.ums.edu.my/id/eprint/42272/
https://doi.org/10.3390/microorganisms10122346
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