Introduction

Banana (Musa species) is among the most imperative harvest plants developed by people; it has high nutritive value and essential organic product, particularly in tropical zones as far as production and utilization (Safarpour et al. 2017).Banana is the fourth most cultivated fruits in more than 130 countries belonging to Asia, America, Africa, Oceania and the Pacific (FAOStat2014).The most vital sucker borne diseases for Musa planting materials are nematodes, weevils, and infections including Cucumber Mosaic Virus (CMV), Banana Bract Mosaic Virus (BBrMV), Banana Streak Virus (BSV), Banana Bunchy Top Virus (BBTV) and bacterial shrivels (Qazi 2016). In addition, unlike other diseases once infected by the virus, no cure is available; ultimately the virus-free planting materials are the possible option for Banana bunchy top disease (BBTD; Singh et al. 2018). The causative agents of BBTD are BBTV and BBTV-infected vegetative planting materials (Punyarani et al. 2013). The BBTV is transferred in a constant mode through the banana aphid (Pentalonia nigronervosa) (Watanabe 2013). Hence, virus detection in banana planting material/ crop is crucial for proper management of viral diseases and for safe germplasm conservation/ exchanges.

Quantity and quality of extracted DNA matters for efficient molecular applications like polymerase chain reaction (PCR), cloning, sequencing, and restriction digestion (Sharma et al. 2013). Several manufactures provide DNA collection cards like, FTATM cards, Isocode card and Generation Capture System for extraction of quality DNA. Different extraction techniques for plant tissues have been developed, each with its own drawbacks and benefits. (Thomson and Henry 1995; Liu et al.2000; Guo et al. 2005; Chi et al. 2009).To achieve reliable and rapid methods of virus indexing, a rapid method of extraction of DNA is needed. This performance can be achieved by using InstantDNA card that has been developed to fix and store nucleic acids from fresh tissue. InstantDNA card is an efficient paper-based system that reduces the different steps involved in DNA purification, isolation, transfer, and deposition, thus stinging down the time and cost to refine a DNA to the finishing step of obtaining purified DNA. Thus, the present study describes the use of the InstantDNA card for the extraction, storage, and subsequent amplification with conventional PCR for the detection of BBTV.

Materials and method

The symptomic BBTV banana plants were collected from the different regions of Central India and were maintained separately in the green house. DNA collection was done on BBTV symptomic (Fig. 1) plants using the InstantDNA kit containing InstantDNA card. Six disc containing BBTV infected crushed leaf samples were used for extraction of DNA.

Figure 1: BBTV infected plant showing severe symptoms

DNA extraction protocol

Leaf was directly placed in a circle of the InstantDNA card followed by a piece of Parafilm over the leaf (Fig. 2). Then with the help of blunt instrument such as a pestle pressure was applied over the leaf area. The collection process is completed when the sample is seen to the back of the microcard. Then samples were dried completely for further use. After drying sample was proceed for downstream analysis.

Figure 2: (a) InstantDNA card sample, (b) symptomatic leaves are pressed into InstantDNA card

Downstream Analysis

Sample was directly placed on the micro card into the container. After that 150 microliter of distilled water was added to per sample in a 200 microliter PCR tube. The sample was then incubated for five to ten minute at room temperature; after incubation was completed distilled water was poured of. Then 5miroliter of Proteinase K (20 mg/ml) was added to 150 microliter of diluted wash solution, the sample punch was then incubate at at 65°C for 30 minutes. After that another 150 microliter of diluted wash solution was added to the sample punch and incubate at room temperature for 3 minutes, after incubation wash solution was discarded. Then 200 microliter of TE buffer was added; and sample was incubated at room temperature for five minutes. Then finally sample punch was dry at 56-65°C for further use.

DNA amplification by PCR

Briefly, one disc measuring 3 mm each was punched from the InstantDNA card using Instant^TM card punch, 3 mm (Himedia, Inc. India). PCR was done in a total volume of 25 microliter, made up of 2.0 microliter of 10 mM dNTPs, 2.5 microliter of 10x PCR buffer, 1.0 microliter of 10 pmol of each primer (Selvarajan et al.  2015), and 1.0 units of Taq polymerase. PCR amplification was performed in PrimaTm HiMedia thermocycler using primary denaturation profile at 94̊C for 4 min and then followed by 35 denaturation cycles at 94^oC for 1 min, 51^oC for 1 min (annealing), and 72^oC for 2 min each and a final extension at 72°C for 10 min describe by (Selvarajan et al. 2010). PCR products were separated assessed on 1% agarose gels stained with ethidium bromide and viewed under Gel Doc™ XR+ Gel Documentation System (Bio Rad).

Results and Discussion

In this study, a DNA extraction protocol was performed by using InstantDNA kit. The current protocol proved reliable and sufficiently fast to process up to several hundred samples per day. The protocol has been used for extracting DNA form BBTV infected plant and provide good quality DNA. Six discs containing crushed sample were used for extraction of DNA via InstantDNA card out of which 66.66% disc samples show amplification. The DNA obtained from InstantDNA cards flaked with BBTV infection show amplification of a 513 bp product (Fig. 3). InstantDNA kit successfully identified BBTV from infected banana leaf samples. The InstantDNA card is particularly suited for the collection, transportation of nucleic acid and detection of plant diseases. Amplification signals successfully generated indicate that InstantDNA is suitable for sampling and detection directly from plant tissues. Many simplified DNA extraction techniques have been developed for plant tissues by Flinders Technology Associates (FTA®) technology (Mbogori et al.  2006; Siegel et al. 2017).  InstantDNA card Provide a long-term, low-cost, low-risk viral pathogen and plant genomic samples archiving system if it is placed in vacuum packed cabinet.

Figure 3: Gel photograph of PCR amplification of BBTV infected plants by using InstantDNA card, Lane M: 100bp ladder, Lane N: negative control and Lane S1-S5 show amplified and non amplified product. BBTV Infected samples exhibit an amplification of 513 bp.

Conclusion

Results showed that InstantDNA card is a practical, efficient and rapid method for sampling, storage and retrieval of viral pathogens, when working under controlled conditions and in the field. Our research exhibit a simple, and efficient technique that greatly reduces the cost and time required to handle large sample sizes. Moreover, this is the first successful application of InstantDNA card for detection of banana viruses, particularly for BBTV. The evaluated technique will be helpful for commercial laboratories for rapid DNA isolation and virus indexing of banana plants. Further, it may prove suitable for the other DNA viruses.

Conflict of interest

Authors had no conflict of interest.

Acknowledgement

Fellowship to Vikram Singh provided by University Grants Commission, New Delhi in the form of  National Fellowship and Scholarship for Higher Education of ST students (201718-NFST-CHH-00544)

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