Yushahfira Akul (2018) Development and validation of loop-mediated isothermal amplification (LAMP) for the early detection of basal stem rot (BSR) caused by ganoderma boninense. Masters thesis, Universiti Malaysia Sabah.
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Abstract
Ganoderma boninense is a pathogenic plant fungus that is associated with the Basal Stem Rot (BSR) disease frequently found in oil palm plantation. Conventional detection of G. boninense is usually achieved through visible symptoms, culturing on Ganoderma selective media (GSM), ergosterol quantification that is proportional to fungal biomass, immunoassay test or through molecular detection such as Polymerase Chain reaction (PCR). However, all of these methods require skilled personnel, is time consuming, may not specific and maybe also expensive. As such, this study aims to develop a rapid, simple yet sensitive and specific detection method based on the use of loop-mediated isothermal amplification (LAMP) of DNA. The basidiocarps of G. boninense were obtained from Langkon estate in Kota Marudu, cultured on GSM and subcultured onto PDA to obtain pure cultures of G. boninense. Genomic DNA of G. boninense was then isolated, ITS gene amplification using PCR was performed and verified through Sanger sequencing. The rapid detection of G. boninensewas accomplished through a set of novel outer, inner and loop primers of the Manganese Superoxide Dismutase (MnSOD) gene, specific to G. boninense. The optimization of LAMP condition was performed to find the suitable concentration of MgSo4 and the primer ratio of outer and inner primers. The loop primers was added to the LAMP reaction and it was proven that the addition of loop primers able to accelerate the time of LAMP reaction. The signal detection of LAMP products was done in two ways which are in gel electrophoresis viewed under UV light and using SYBR Green I. For sensitivity test, two sets of G. boninense recombinant plasmid samples were prepared; Set A: from 60 ng to 0.6 fg, and Set B: from 107 to 5 copies, and were subjected to PCR, LAMP-UV analysis and LAMPSYBR Green. The finding from the sensitivity test showed the LAMP-UV and LAMPSYBR Green is greater in sensitivity than that obt.ained using conventional PCR. The detection limit of LAMP-SYBR and LAMP-UV analysis was at 0.6 fg and 103 copies of recombinant plasmid while conventional PCR was only at 0.6 pg and 104 copies. The specificity test was performed with genomic DNA of G. boninense and G. australe, and it was negative for G. australe while positive for G. boninense. Another specificity test was done using 10 unknown fruiting bodies and all these samples were subjected to DNA extraction, identificatjon using PCR and LAMP. The LAMP assay was negative for all the samples. The evaluation of LAMP assay on trunk tissue samples to detect G. boninense was tested in 20 genomic DNA samples extracted from infected trunk tissues. The results showed 17 out of 20 genomic DNA samples produced LAMP ladder-like bands. In addition, the other three were negative for LAMP, GSM and ergosterol quantification respectively. This indicates that the LAMP assay was highly sensitive compared to GSM and ergosterol detection. In conclusion, the LAMP assay developed through this study was found to be useful in the detection of G. boninense with high sensitivity and specificity in biological and trunk tissue samples.
| Item Type: | Thesis (Masters) |
|---|---|
| Keyword: | Ganoderma boninense, Basal Stem Rot, Oil palm, LAMP assay, Fungal detection, Isothermal amplification, MnSOD gene, Molecular diagnostics, Trunk tissue |
| Subjects: | S Agriculture > SB Plant culture > SB1-1110 Plant culture |
| Department: | FACULTY > Faculty of Science and Natural Resources |
| Depositing User: | DG MASNIAH AHMAD - |
| Date Deposited: | 21 Apr 2025 11:44 |
| Last Modified: | 21 Apr 2025 11:44 |
| URI: | https://eprints.ums.edu.my/id/eprint/43518 |
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