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Development of a visual detection method for Potato virus S by reverse transcription loop-mediated isothermal amplification

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Abstract

A reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay was developed to detect the Potato virus S (PVS) in potato. Two sets of six novel primers that recognize the coat protein gene sequence of the PVS were designed and RT-LAMP assay was optimized for the parameters such as different concentrations of primers, MgSO4, betaine, dNTPs, Bst DNA polymerase, temperature and duration. The RT-LAMP was carried out under isothermal conditions without the thermal cycler using PVS infected leaf and tuber samples, LAMP specific primers with amplification at 65 °C for 60 min, and 80 °C for 5 min. The results were assessed by gel electrophoresis and visual observation of colour change using SYBR Green I dye. The detection limit of the developed RT-LAMP assay was determined and compared with a conventional reverse transcription-polymerase chain reaction (RT-PCR). RT-LAMP was found 100 times more sensitive than RT-PCR. The optimized RT-LAMP assay is robust, reliable, sensitive and convenient for the detection of the PVS in infected potato tubers including asymptomatic plants. No cross-reactions were observed with healthy plants and other potato viruses. The assay is economical and can be employed in large scale testing of potato plants against PVS under healthy seed potato production programme.

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Acknowledgements

This study was supported by the Indian Council of Agricultural Research, New Delhi, India.

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Authors and Affiliations

  1. ICAR-Central Potato Research Institute, Shimla, HP, 171 001, India

    Ravinder Kumar, Priyanka Kaundal, Sanjeev Sharma & S. K. Chakrabarti

  2. ICAR-Indian Institute of Spices Research, Marikunnu P.O., Kozhikode, Kerala, 673 012, India

    Jeevalatha Arjunan

Authors
  1. Ravinder Kumar
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  2. Priyanka Kaundal
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  3. Jeevalatha Arjunan
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  4. Sanjeev Sharma
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  5. S. K. Chakrabarti
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Contributions

RK conceptualization. RK and PK did the analytical work of processing samples for RNA extraction, cDNA synthesis, and PCR assays. RK, JA, PK and SS methodology. RK, PK and JA Data analysis. SS validation. RK, SS, and SKC helped in preparing and editing the MS.

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Correspondence to Ravinder Kumar.

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No human subjects were used in the writing of the manuscripts.

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Kumar, R., Kaundal, P., Arjunan, J. et al. Development of a visual detection method for Potato virus S by reverse transcription loop-mediated isothermal amplification. 3 Biotech 10, 213 (2020). https://doi.org/10.1007/s13205-020-02214-4

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  • DOI: https://doi.org/10.1007/s13205-020-02214-4

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