Ahmad Mukhlis Abdul Rahman (2014) Development of loop-mediated isothermal amplification (lamp) of dna in combination with lateral flow dipstick (lfd) for the rapid detection of vibrio harveyi. Masters thesis, Universiti Malaysia Sabah.
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Abstract
Vibrio harveyi is a marine pathogenic bacterium associated with the outbreak of disease in marine organisms particularly in hatcheries and grow-out ponds by attacking their immune system. Conventional detection of V. harveyi is usually achieved through microscopic examination, biochemical, immunological and/or PCR based methods. However, these methods require skilled manpower, dedicated laboratory space and is usually time-consuming. As such, the objective of this study is to develop a rapid, simple yet sensitive and specific detection method based on the use of loop-mediated isothermal amplification (LAMP) of DNA in combination with lateral flow dipstick (LFD). The samples of V. harveyi strain VHJR7 and VHJR4 were obtained from the Borneo Marine Research Institute, Universiti Malaysia Sabah (UMS), Malaysia. The rapid detection of V. harveyi is accomplished through a set of novel outer, inner and loop primers of the toxR gene unique to V. harveyi. Biotinylated LAMP amplicons were produced by the set of six designed primers that recognized specifically the target sequences of V. harveyi followed by hybridization with an FAM-labeled probe and LFD detection. The addition of two loop primers improves the reaction time of LAMP by more than half. Since LAMP is a non-PCR based method, results can be obtained as quickly as 10-15 minutes. Moreover, with the application of LFD, the result can be obtained without the need for gel electrophoresis, which usually includes the use of the harmful-carcinogen ethidium bromide. For sensitivity test, two sets of V. harveyi recombinant plasmid samples were prepared; Set A: from 60 ng to 0.6 fg, and Set B: from 107 copies to 5 copies, and were subjected to LAMP-LFD. LAMP-LFD was compared with PCR, LAMP-UV analysis and LAMP-SYBR Green. In this study, LAMP-LFD, LAMP-SYBR Green and LAMP-UV analysis are greater in sensitivity than that obtained using conventional PCR reaction. Sensitivity of PCR was only at 0.6 pg and 104 copies while LAMP-LFD, LAMP-SYBR Green and LAMP-UV analysis were able to detect low amounts even at a 0.6 fg and 103 copies. A field test study was carried out to determine the sensitivity of the assay. From the specificity test, LAMP-LFD accurately identified V.harveyi samples only. This method will provide a very useful tool to help aquaculturist, and fish and prawn breeders to rapidly monitor the outbreaks of this pathogen in order to prevent mass mortality of fish and prawn.
| Item Type: | Thesis (Masters) |
|---|---|
| Keyword: | Vibrio harveyi, Marine pathogen, DNA detection, ToxR gene, Rapid detection, Aquaculture |
| Subjects: | Q Science > QH Natural history > QH301-705.5 Biology (General) > QH540-549.5 Ecology |
| Department: | INSTITUTE > Biotechnology Research Institute (BRI) |
| Depositing User: | DG MASNIAH AHMAD - |
| Date Deposited: | 07 Apr 2025 12:11 |
| Last Modified: | 07 Apr 2025 12:11 |
| URI: | https://eprints.ums.edu.my/id/eprint/43376 |
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