Reverse transcription loop medicated isothermal amplification(RT-Lamp) for diagnosis of Banana bract mosaic virus in banana (Musa spp.) (Record no. 289767)
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| 000 -LEADER | |
|---|---|
| fixed length control field | 05819nam a22001937a 4500 |
| 082 ## - DEWEY DECIMAL CLASSIFICATION NUMBER | |
| Classification number | 660.6 |
| Item number | MID/RE PG |
| 100 ## - MAIN ENTRY--PERSONAL NAME | |
| Personal name | Midhuna Madhu K |
| 245 ## - TITLE STATEMENT | |
| Title | Reverse transcription loop medicated isothermal amplification(RT-Lamp) for diagnosis of Banana bract mosaic virus in banana (Musa spp.) |
| 260 ## - PUBLICATION, DISTRIBUTION, ETC. (IMPRINT) | |
| Place of publication, distribution, etc | Vellanikkara |
| Name of publisher, distributor, etc | Department of Plant Biotechnology, Centre for Plant Biotechnology and Molecular Biology, College of Agriculture |
| Date of publication, distribution, etc | 2021 |
| 300 ## - PHYSICAL DESCRIPTION | |
| Extent | 47p. |
| 502 ## - DISSERTATION NOTE | |
| Dissertation note | M Sc |
| 520 ## - SUMMARY, ETC. | |
| Abstract | Banana (Musa spp.) is one of the most important and widely cultivated crops<br/>around the world. Banana is cultivated on 0.9 million hectares in India with a <br/>production of 30.8 million tonnes (NHB, 2020). Despite its importance, growers are <br/>not in a position to achieve high productivity due to the presence of various biotic <br/>and abiotic stresses. Viruses are a major concern to banana production causing up to <br/>100 per cent yield reduction. The Banana bract mosaic virus (BBrMV), a ssRNA <br/>virus of the genus Potyvirus is reported to cause 40 per cent yield reduction in <br/>banana. The virus is transmitted through vegetative propagules and insect vectors <br/>like Pentalonia nigronervosa, Aphis gossypi and Rhopalosiphum maidis. Internal <br/>quarantine is necessary to prevent the spread of the virus. We need a platform for <br/>virus indexing, so that farmers can get good quality virus-free planting materials. <br/>The available detection methods for BBrMV are the Enzyme-Linked<br/>Immunosorbent Assay (ELISA) and Reverse Transcriptase Polymerase Chain <br/>Reaction (RT-PCR). The ELISA is time consuming while the RT- PCR needs post <br/>PCR sample handling predisposing to sample cross contamination.<br/> Hence the study entitled “Reverse Transcription Loop Mediated Isothermal <br/>Amplification (RT-LAMP) for diagnosis of Banana bract mosaic virus in banana <br/>(Musa spp.)” was undertaken during the period from 2019 to 2021 at the <br/>Department of Plant Biotechnology, College of Agriculture (CoA), Vellanikkara, <br/>Thrissur, with an objective to develop a rapid and efficient molecular diagnostic <br/>method for BBrMV. The LAMP is an auto-cycling strand displacement isothermal <br/>DNA synthesis method using a strand displacement DNA polymerase. The RTLAMP is a specific, highly sensitive and relatively faster diagnostic method for <br/>RNA viruses. <br/> Initially, leaf samples of BBrMV symptomatic banana plants were collected <br/>from Banana Research station (BRS), Kannara, Kerala. Healthy leaf samples were <br/>collected from asymptomatic plants in the field and from tissue culture plants <br/>produced at CPBMB, CoA, Vellanikkara. Samples from banana plants showing <br/>symptoms of other viruses such Banana bunchy top virus (BBTV), Banana streak <br/>virus (BSV) and Cucumber mosaic virus (CMV) as were also collected from BRS, <br/>Kannara, to validate the results of the study. Three different methods using TRI <br/>reagent, Purelink Plant RNA reagent and RNeasy plant mini kit were tried for <br/>isolation of total RNA. Good quality RNA with intact bands could be obtained with <br/>RNeasy plant mini kit (Qiagen). <br/> For the RT-LAMP reaction, a set of six primers were designed using the <br/>software Primer Explorer version 5.0. Initially, three targets in the BBrMV genome <br/>namely, coat protein gene, replicase gene and movement protein gene were selected <br/>for picking LAMP primers. Due to unavailability of loop primers for BBrMV <br/>replicase gene and movement protein gene, six primers targeting the BBrMV coat <br/>protein gene were selected. All the primers were validated using BLASTN. <br/>Both one-step and two-step RT-LAMP assay was optimized for diagnosis of <br/>BBrMV. For two-step RT-LAMP assay, RNA was converted to cDNA and was used <br/>as template for isothermal amplification. Optimisation of the assay was done by <br/>varying the concentration of the reagents like MgSO4 (4-8 mM), dNTP (1.4-1.6 mM <br/>each), betaine (0.8-1 M) and Bst polymerase (4-8 U). The final optimised reaction <br/>mixture contained 40 ng cDNA, 1.6 mM each dNTP, 0.2 µM each primers F3 and B3, <br/>0.8 µM each primers BrFIP and BrBIP and 0.4 µM each primers BrLF and BrLB, 1 <br/>M betaine, 4 mM MgSO4, 1x Thermopol buffer with 2 mM MgSO4, 4 U Bst<br/>polymerase large fragment and 120 µM HNB in 25 µl reaction volume. Incubation <br/>temperature at 65° C was found optimum. One step RT-LAMP is the method suited <br/>for routine diagnostics as the RNA sample can be directly used as the template for <br/>amplification. One step RT-LAMP reaction mixture contained reverse transcriptase <br/>and RNase inhibitor along with the other components of the LAMP reaction and RNA <br/>was used as the template instead of cDNA. <br/>The positive amplicons showed ladder like bands on agarose gel and showed a <br/>colour change from violet to sky blue in the presence of HNB dye in the reaction <br/>mixture. Molecular typing of RT-LAMP products was done using sequence analysis <br/>and restriction digestion. For restriction profiling, the enzyme Sau3AI having internal <br/>cut site in the RT-LAMP internal primer flanking region was used. The enzyme <br/>having single cut site produced fragments of size 100 bp and 45 bp. The RT-LAMP <br/>assay was validated using 12 BBrMV symptomatic samples, healthy samples and <br/>samples showing symptoms of other banana viruses like BBTV, BSV and CMV. All <br/>the 12 BBrMV symptomatic samples amplified in the RT-LAMP assay while no <br/>amplification was observed for healthy samples and samples showing symptoms of <br/>other viruses. Hence, in the current study, a rapid, sensitive and specific RT-LAMP <br/>based detection method for Banana bract mosaic virus was developed. |
| 650 ## - SUBJECT ADDED ENTRY--TOPICAL TERM | |
| Topical term or geographic name as entry element | Plant Biotechnology |
| 650 ## - SUBJECT ADDED ENTRY--TOPICAL TERM | |
| Topical term or geographic name as entry element | Banana |
| 650 ## - SUBJECT ADDED ENTRY--TOPICAL TERM | |
| Topical term or geographic name as entry element | Bract mosaic virus |
| 700 ## - ADDED ENTRY--PERSONAL NAME | |
| Personal name | Smita Nair (Guide) |
| 856 ## - ELECTRONIC LOCATION AND ACCESS | |
| Uniform Resource Identifier | https://krishikosh.egranth.ac.in/handle/1/5810194749 |
| 942 ## - ADDED ENTRY ELEMENTS (KOHA) | |
| Source of classification or shelving scheme | Dewey Decimal Classification |
| Item type | Theses |
| Not for loan | Collection code | Home library | Current library | Shelving location | Date acquired | Full call number | Barcode | Date last seen | Koha item type |
|---|---|---|---|---|---|---|---|---|---|
| Not For Loan | Reference Book | KAU Central Library, Thrissur | KAU Central Library, Thrissur | Theses | 27/07/2022 | 660.6 MID/RE PG | 175433 | 27/07/2022 | Theses |