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Development of a SYBR Green-based RT-qPCR assay for the detection of Indian citrus ringspot virus

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Abstract

The Indian citrus ringspot virus (ICRSV) that causes ringspot disease, especially to ‘Kinnow mandarin’ hampers the sustainability of crop production. Presently, the disease is not amenable for control through host resistance or the introduction of chemicals, hence raising virus-free plants is one of the most effective approaches to manage the disease. Consequently, it is necessary to develop rapid, sensitive, specific, and early diagnostic methods for disease control. In the present study, newly designed primers targeting a 164 bp region of the ICRSV coat protein gene were used to develop and optimize a SYBR Green-based quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay, for the detection of ICRSV. The RT-qPCR assay was evaluated and confirmed using viral RNA extracted from ICRSV infected plants maintained in screen house as well as field samples. The standard curves displayed a dynamic linear range across eight log units of ICRSV-cRNA copy number ranging from 9.48.1 fmol (5.709 × 109) to 0.000948 amol (5.709 × 102), with detection limit of 5.709 × 102 copies per reaction using serial tenfold diluted in vitro transcribed viral cRNA. The developed RT-qPCR is very specific to ICRSV does not react to other citrus pathogens, and approximately 100-fold more sensitive than conventional RT-PCR. Thus, this assay will be useful in laboratories, KVKs, and nurseries for the citrus budwood certification program as well as in plant quarantine stations. To our knowledge, this is the first study of the successful detection of ICRSV by RT-qPCR.

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Availability of data and material

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

This research was funded by the project ICAR-CRP on Vaccines and Diagnostics, Govt. of India F. No.16-11/PP/ICAR-CRP/17-18/06. Authors thank Prof. Swadeshmukul Santra, Department of Chemistry, NanoScience Technology Center, University of Central Florida, Orlando, USA for reviewing this manuscript.

Funding

This research was funded by the project ICAR-CRP on Vaccines and Diagnostics, Govt. of India F. No.16-11/PP/ICAR-CRP/17-18/06.

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Authors and Affiliations

  1. Indian Council of Agricultural Research, Central Citrus Research Institute, Amravati Road, Nagpur, Maharashtra, 440033, India

    Amol D. Kokane, Sunil B. Kokane, Mrugendra G. Gubyad & Dilip Kumar Ghosh

  2. Department of Molecular and Cellular Engineering, Sam Higginbottom University of Agriculture, Technology and Sciences, Allahabad, Uttar Pradesh, 211007, India

    Amol D. Kokane, Kapil Lawrence & Pragati Misra

  3. ICAR, Indian Institute of Horticultural Research, Bangalore, Karnataka, 560089, India

    M. Krishna Reddy

Authors
  1. Amol D. Kokane
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  2. Kapil Lawrence
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  3. Sunil B. Kokane
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  4. Mrugendra G. Gubyad
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  5. Pragati Misra
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  6. M. Krishna Reddy
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  7. Dilip Kumar Ghosh
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Contributions

DK, AK, KL, and PM conceived and designed the experiments. AK, SK, and MG performed the experiments and analyzed the data. DK and AK wrote and edited the manuscript. All authors read and approved the final manuscript.

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Correspondence to Dilip Kumar Ghosh.

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Kokane, A.D., Lawrence, K., Kokane, S.B. et al. Development of a SYBR Green-based RT-qPCR assay for the detection of Indian citrus ringspot virus . 3 Biotech 11, 359 (2021). https://doi.org/10.1007/s13205-021-02903-8

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