Development of a novel polyprobe for simultaneous detection of six viruses infecting stone and pome fruits
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Development of a novel polyprobe for simultaneous detection of six viruses infecting stone and pome fruits Md Salik Noorani1,2 · Jawaid Ahmad Khan2 Received: 8 May 2020 / Accepted: 7 August 2020 © King Abdulaziz City for Science and Technology 2020
Abstract A biotin-labeled, non-isotopic, novel polyprobe was developed for the simultaneous detection of six viruses viz. apple chlorotic leaf spot virus (ACLSV), apple mosaic virus (ApMV), apple stem grooving virus (ASGV), cherry virus A (CVA), prunus necrotic ringspot virus (PNRSV) and plum pox virus (PPV) infecting stone and pome fruit trees through dot-blot hybridization assay. The sensitivity of the polyprobe was checked by serial dilutions of total RNA extracted from the tissues of infected trees. ACLSV was detected up to a dilution of 5 −5, whereas ApMV, ASGV, CVA, PPV and PNRSV up to 5−4. The developed assay was validated following testing a total of 45 symptomatic leaf samples collected from different geographical regions of Jammu and Kashmir (India), and the presence of the viruses was further confirmed by RT-PCR and sequencing. The polyprobe, designed for performing molecular hybridization assay can be developed quickly and avoid the tedious transformation and cloning procedures. Apart from simultaneously detecting viruses in stone and pome fruit trees, it holds great potential for virus indexing programmes to ascertain the supply of virus-free plant materials to the growers. Keywords Biotin-labeled polyprobe · Molecular hybridization · Virus detection · Mixed infection
Introduction Stone (apricot, almond, cherry, nectarine, peach and plum) and pome (apple, pear and quince) are important temperate fruit crops grown worldwide, are significantly consumed as human foods. Like other horticultural crops, they are associated with several virus diseases that affect yield and overall growth and of plants (Barba et al. 2015). Around 21 viruses belonging to 12 genera of 9 families from pome fruits and 25 viruses representing 15 genera of 9 families have been identified from stone fruit trees grown across the world (Umer et al. 2019). During the last couple of decades, plant virologists have tried to develop cost-effective, efficient, and less timeconsuming nucleic acid-based diagnostic assays for the * Md Salik Noorani [email protected] 1
Department of Botany, School of Chemical and Life Sciences, Jamia Hamdard (A Deemed to be University), Hamdard Nagar, New Delhi 110062, India
Plant Virus Laboratory, Department of Biosciences, Jamia Millia Islamia (A Central University), Jamia Nagar, New Delhi 110025, India
2
simultaneous detection of several viruses and virus-like agents from the infected plants (James et al. 2006; Pallas et al. 2018). Though PCR is sensitive enough, it becomes costly and timeconsuming when a large numbers of samples are tested. Due to easy handling and lack of required specialized equipment, nucleic acid-based hybridizations with non-isotopic probes are being used as alternative diagnostics for detecting
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