Reverse transcription loop medicated isothermal amplification(RT-Lamp) for diagnosis of Banana bract mosaic virus in banana (Musa spp.)

Abstract

Banana (Musa spp.) is one of the most important and widely cultivated crops around the world. Banana is cultivated on 0.9 million hectares in India with a production of 30.8 million tonnes (NHB, 2020). Despite its importance, growers are not in a position to achieve high productivity due to the presence of various biotic and abiotic stresses. Viruses are a major concern to banana production causing up to 100 per cent yield reduction. The Banana bract mosaic virus (BBrMV), a ssRNA virus of the genus Potyvirus is reported to cause 40 per cent yield reduction in banana. The virus is transmitted through vegetative propagules and insect vectors like Pentalonia nigronervosa, Aphis gossypi and Rhopalosiphum maidis. Internal quarantine is necessary to prevent the spread of the virus. We need a platform for virus indexing, so that farmers can get good quality virus-free planting materials. The available detection methods for BBrMV are the Enzyme-Linked Immunosorbent Assay (ELISA) and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). The ELISA is time consuming while the RT- PCR needs post PCR sample handling predisposing to sample cross contamination. Hence the study entitled “Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) for diagnosis of Banana bract mosaic virus in banana (Musa spp.)” was undertaken during the period from 2019 to 2021 at the Department of Plant Biotechnology, College of Agriculture (CoA), Vellanikkara, Thrissur, with an objective to develop a rapid and efficient molecular diagnostic method for BBrMV. The LAMP is an auto-cycling strand displacement isothermal DNA synthesis method using a strand displacement DNA polymerase. The RTLAMP is a specific, highly sensitive and relatively faster diagnostic method for RNA viruses. Initially, leaf samples of BBrMV symptomatic banana plants were collected from Banana Research station (BRS), Kannara, Kerala. Healthy leaf samples were collected from asymptomatic plants in the field and from tissue culture plants produced at CPBMB, CoA, Vellanikkara. Samples from banana plants showing symptoms of other viruses such Banana bunchy top virus (BBTV), Banana streak virus (BSV) and Cucumber mosaic virus (CMV) as were also collected from BRS, Kannara, to validate the results of the study. Three different methods using TRI reagent, Purelink Plant RNA reagent and RNeasy plant mini kit were tried for isolation of total RNA. Good quality RNA with intact bands could be obtained with RNeasy plant mini kit (Qiagen). For the RT-LAMP reaction, a set of six primers were designed using the software Primer Explorer version 5.0. Initially, three targets in the BBrMV genome namely, coat protein gene, replicase gene and movement protein gene were selected for picking LAMP primers. Due to unavailability of loop primers for BBrMV replicase gene and movement protein gene, six primers targeting the BBrMV coat protein gene were selected. All the primers were validated using BLASTN. Both one-step and two-step RT-LAMP assay was optimized for diagnosis of BBrMV. For two-step RT-LAMP assay, RNA was converted to cDNA and was used as template for isothermal amplification. Optimisation of the assay was done by varying the concentration of the reagents like MgSO4 (4-8 mM), dNTP (1.4-1.6 mM each), betaine (0.8-1 M) and Bst polymerase (4-8 U). The final optimised reaction mixture contained 40 ng cDNA, 1.6 mM each dNTP, 0.2 µM each primers F3 and B3, 0.8 µM each primers BrFIP and BrBIP and 0.4 µM each primers BrLF and BrLB, 1 M betaine, 4 mM MgSO4, 1x Thermopol buffer with 2 mM MgSO4, 4 U Bst polymerase large fragment and 120 µM HNB in 25 µl reaction volume. Incubation temperature at 65° C was found optimum. One step RT-LAMP is the method suited for routine diagnostics as the RNA sample can be directly used as the template for amplification. One step RT-LAMP reaction mixture contained reverse transcriptase and RNase inhibitor along with the other components of the LAMP reaction and RNA was used as the template instead of cDNA. The positive amplicons showed ladder like bands on agarose gel and showed a colour change from violet to sky blue in the presence of HNB dye in the reaction mixture. Molecular typing of RT-LAMP products was done using sequence analysis and restriction digestion. For restriction profiling, the enzyme Sau3AI having internal cut site in the RT-LAMP internal primer flanking region was used. The enzyme having single cut site produced fragments of size 100 bp and 45 bp. The RT-LAMP assay was validated using 12 BBrMV symptomatic samples, healthy samples and samples showing symptoms of other banana viruses like BBTV, BSV and CMV. All the 12 BBrMV symptomatic samples amplified in the RT-LAMP assay while no amplification was observed for healthy samples and samples showing symptoms of other viruses. Hence, in the current study, a rapid, sensitive and specific RT-LAMP based detection method for Banana bract mosaic virus was developed.