Detection of Banana bunchy top virus (BBTV) through colorimetric closed tube loop medicated isothermal amplification (Lamp)

Abstract

Banana is an important food and cash crop world wide. India leads in world banana production with 31.5 million tons from 0.9 million ha area. The Banana Bunchy Top Disease caused by Banana bunchy top virus (BBTV) is considered to be the most economically destructive of the viral diseases. Primary transmission of disease is through infected planting material. Virus indexing of suckers and tissue culture plantlets is essential to ensure the availability of virus free planting materials. The available detection methods for the BBTV are ELISA and PCR. The ELISA is time consuming while PCR requires thermal cycling and the post amplification sample handling predisposing to sample cross contamination. A nucleic acid amplification method termed Loop Mediated Isothermal Amplification (LAMP) enables rapid, sensitive and specific amplification of template DNA under isothermal condition using a strand displacing polymerase like Bst DNA polymerase and 4 to 6 primers producing 109 copies of the target in less than 1 h. Combined with simple visual detection of amplicons using the Hydroxy Naphthol Blue (HNB) dye, closed tube detection can be made hence avoiding sample cross contaminations. Hence, the present study entitled “Detection of Banana bunchy top virus through colorimetric closed tube Loop Mediated Isothermal Amplification (LAMP)” was undertaken at the Center for Plant Biotechnology and Molecular Biology, College of Agriculture, Kerala Agriculture University, Thrissur, during the period from 2019 to 2021 with the objective to develop a colorimetric closed tube LAMP assay for rapid and sensitive detection of BBTV. As the initial step, samples were collected from 15 BBTV symptomatic banana plants, two symptomless plants and from plants showing symptoms of other viruses infecting banana such as Banana bract mosaic virus (BBrMV), Banana streak virus (BSV) and Cucumber mosaic virus (CMV). The samples were collected from Banana Research Station, Kannara. One sample was taken from healthy tissue culture raised plant at the Center for Plant Biotechnology and Molecular Biology, College of Agriculture, Kerala Agriculture University, Thrissur. Good quality DNA was obtained with Doyle and Doyle method followed by RNase treatment. For LAMP primer designing, coat protein gene, replicase gene and movement protein gene sequences of BBTV were selected as the target. The LAMP external and internal primers could be picked from all the targets, but loop primers were not available for replicase gene and movement protein gene sequences. Hence, six LAMP primers were designed targeting BBTV coat protein gene using the software Primer Explorer version 5.0. The concentration of different components in the LAMP reaction like dNTPs (0.8 to 1.8 mM), MgSO4 (4 to 8 mM), HNB (120 µM and 150µM) and betaine (0.8 to 1M) was optimized. The final reaction mixture contained 50 ng template DNA, 1.4 mM each dNTP, 0.8 μM each primer BBTVFIP and BBTVBIP, 0.2 μM each primer BBTVF3 and BBTVB3, 0.4 μM each primer BBTVLF and BBTVLB, 0.8 M betaine, 6 mM MgSO4, 1X Thermopol buffer with 2 mM MgSO4, 8U Bst polymerase large fragment and 120 μM HNB dye in 25 μl reaction volume. Isothermal amplification was set at 65 oC and end point detection made using 120 µM HNB dye in the reaction mixture where the BBTV positive samples showed colour change from violet to blue. The LAMP positive amplicons appeared as ladder like bands on agarose gel. Molecular characterization of LAMP amplicon was made with sequencing and restriction analysis. A restriction enzyme having cut site in between the LAMP internal primer flanking region was identified. When the LAMP amplicons were digested with the restriction enzyme Sau3AI having single cut site, fragments of expected size (62 bp and 89 bp) were obtained. The optimized LAMP assay was validated using 15 BBTV symptomatic samples, three healthy samples and samples showing symptoms of BBrMV, CMV and BSV, as observed by the agarose gel profile and the colour change with HNB dye. All the 15 BBTV symptomatic samples showed amplification in the LAMP assay while the healthy samples and the samples showing symptoms of other viruses remained unamplified. Comparison of LAMP assay with conventional PCR in the detection of BBTV was also made. Only 13 samples out of the 15 BBTV symptomatic samples tested positive in the PCR assay, thus showing higher sensitivity of the LAMP method. Moreover, the closed tube visual detection in LAMP avoids the post amplification sample handling. Since LAMP reaction can be carried out on a dry bath or water bath, it is a rapid and simple alternative to the available PCR based detection method for BBTV.