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Development of Simplified Recombinase Polymerase Amplification Assay for Rapid and Robust Detection of Citrus Yellow Vein Clearing Virus

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Abstract

Citrus is an economically important fruit crop, belongs to family Rutaceae, cultivated commercially in over 130 countries, which holds a leading profitable position in the international market. The most important citrus varieties are mandarins, oranges, lemons, sweet limes, grapefruits and pomelos. Citrus yellow vein clearing virus (CYVCV) is an important graft transmissible plant pathogen known to reduce productivity of citrus fruits due to its predominant association and widespread occurrence. Requirement of fast, reliable, efficient & economical CYVCV indexing assay is a prerequisite for production of healthy planting material. Currently, nucleic acid isolation and thermal cycler-based assay available for CYVCV indexing is a cumbersome lab intensive method. The present study was undertaken to develop and validate reverse transcription-recombinase polymerase amplification (RT-RPA) assay requiring no tedious RNA isolation, separate cDNA synthesis and costlier instrument like thermo-cycler. Optimized RT-RPA assay was able to amplify CYVCV up to 10–7 dilution (equivalent to 0.1 pg/μl) with the prepared templates of both RNA and crude saps and showed higher sensitivity in detection of CYVCV infection in field samples as compared to the conventional RT-PCR. Developed RT-RPA assay showed high specificity without any cross-reaction with other citrus pathogens (Indian citrus ringspot virus, citrus yellow mosaic virus, citrus tristeza virus, citrus exocortis viroid and huanglongbing). RT-RPA using crude leaf sap as template is quite simple, robust, highly sensitive, time and cost effective; therefore, it can be used in resource constrained laboratories as screening tool, for field surveys and on-site testing programs in farms, nurseries and biosecurity. Present study, first time reports the development, optimization and validation of crude sap-based RT-RPA assay for the detection of CYVCV infection in citrus plants namely; Kinnow mandarin, Mosambi and Grape fruit.

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Data Availability

Sequence generated in this study was submitted in NCBI GenBank with accession number OQ427642.

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Acknowledgements

The authors wish to thank the Head of the Division of Plant Pathology and Director, ICAR-Indian Agricultural Research Institute, New Delhi, India for laboratory facilities and encouragement.

Funding

This study was funded by ICAR-National Professor Project entitled “Virome analysis of vegetatively propagated fruit crops for enhancing the productivity through virus prevention program” (Ag.Edn.F.No./27/01/NP/2022-HRD) and Department of Biotechnology, Government of India project entitled “National Certification System for Tissue Culture Raised Plants (NCS-TCP)’ (NO. BT/AB/03/02/2021).

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Author notes

  1. Nitika Gupta and Rakesh Kumar have equally contributed to this work.

Authors and Affiliations

  1. Advanced Centre for Plant Virology, Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi, 110012, India

    Nitika Gupta, Rakesh Kumar, Susheel Kumar Sharma, Nishant Srivastava, Ashwini Kumar & Virendra Kumar Baranwal

  2. ICAR-Indian Institute of Seed Science, Uttar Pradesh, Kushmaur, Mau, India

    Gopi Kishan

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  1. Nitika Gupta
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Contributions

Virendra Kumar Baranwal and Nitika Gupta supervised and conceptualized the whole research project. Nitika Gupta and Susheel Kumar Sharma edited the manuscript. Rakesh Kumar conducted the experiments, Nitika Gupta and Rakesh Kumar have written the manuscript. Gopi Kishan has supported in conduction of lab experiments and manuscript writing. Ashwini Kumar and Nishant Srivastva contributed in survey and sample collection.

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Correspondence to Nitika Gupta or Virendra Kumar Baranwal.

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Gupta, N., Kumar, R., Kishan, G. et al. Development of Simplified Recombinase Polymerase Amplification Assay for Rapid and Robust Detection of Citrus Yellow Vein Clearing Virus. Curr Microbiol 81, 103 (2024). https://doi.org/10.1007/s00284-024-03614-y

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