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Reverse transcriptase loop-mediated isothermal amplification and reverse transcriptase recombinase amplification assays for rapid and sensitive detection of cardamom vein clearing virus

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Abstract

In the present study, two isothermal molecular assays viz. reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and reverse transcriptase recombinase amplification (RT-RPA) were developed to detect the cardamom vein clearing virus (CdVCV) infecting cardamom. Assays were optimized for parameters like duration, temperature and concentration of magnesium sulfate, and betaine in the case of RT-LAMP and magnesium acetate in the case of RT-RPA. Detection limits of both assays were determined and compared with conventional RT-PCR and SYBR Green-based real-time RT-PCR. RT-LAMP was found 10,000 times additional sensitive than RT-PCR and one-tenth that of real-time RT-PCR. RT-RPA was found 1000 times additional sensitive than RT-PCR and one-hundredth that of real-time RT-PCR. Both assays were specific, rapid, and sensitive for detecting CdVCV. Compared to real-time RT-PCR, these assays are economical and can be employed in large scale screening of cardamom plants against CdVCV for the selection of virus-free plants.

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Acknowledgement

Authors are thankful to Science and Engineering Research Board (SERB), Government of India for funding (EMR/2016/001135), Head, Division of Crop Protection, Director, ICAR-Indian Institute of Spices Research, Kozhikode, Kerala, India for facilities.

Funding

Science and Engineering Research Board (SERB), Government of India (EMR/2016/001135).

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Authors and Affiliations

  1. Division of Crop Protection, ICAR-Indian Institute of Spices Research, Kozhikode, 67012, Kerala, India

    K. P. Naveen & A. I. Bhat

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  1. K. P. Naveen
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  2. A. I. Bhat
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AIB conceptualized, designed experiments and finalized the manuscript; KPN performed all experiments and wrote the manuscript; both authors have read and approved the final manuscript.

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Correspondence to A. I. Bhat.

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The authors declare that they have no conflict of interest.

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Naveen, K.P., Bhat, A.I. Reverse transcriptase loop-mediated isothermal amplification and reverse transcriptase recombinase amplification assays for rapid and sensitive detection of cardamom vein clearing virus. 3 Biotech 10, 250 (2020). https://doi.org/10.1007/s13205-020-02238-w

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