Development and application of reverse transcription-loop mediated isothermal amplification assay for sensitive detection of groundnut bud necrosis virus infecting potato

Baswaraj Raigond; Shruti Pathania; Gaurav Verma; Pooja Bhardwaj; Tarvinder Kochhar; S K Chakrabarti

doi:10.1016/j.virol.2023.109872

Development and application of reverse transcription-loop mediated isothermal amplification assay for sensitive detection of groundnut bud necrosis virus infecting potato

Virology. 2023 Oct:587:109872. doi: 10.1016/j.virol.2023.109872. Epub 2023 Aug 26.

Authors

Baswaraj Raigond¹, Shruti Pathania², Gaurav Verma², Pooja Bhardwaj², Tarvinder Kochhar², S K Chakrabarti²

Affiliations

¹ Division of Plant Protection, ICAR-Central Potato Research Institute, Shimla, Himachal Pradesh, 171001, India; Center on Rabi Sorghum, ICAR-Indian Institute of Millets Research, Regional Station, Solapur, Maharashtra, 413006, India. Electronic address: Baswaraj.Raigond@icar.gov.in.
² Division of Plant Protection, ICAR-Central Potato Research Institute, Shimla, Himachal Pradesh, 171001, India.

PMID: 37657354
DOI: 10.1016/j.virol.2023.109872

Abstract

Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of groundnut bud necrosis virus (GBNV) causing potato stem necrosis disease. The isothermal temperatures, reaction periods and concentrations of reaction mixture were optimized where, the assay worked well at 65 °C for 50 min, 6 U of WarmStart Bst 2.0 DNA polymerase, 1.4 mM dNTPs and 2.0 mM MgSO_4. The optimized assay proved to be specific to GBNV with no cross reactivity to other viruses infecting potato in India. The specificity of RT-LAMP assay was found to be 100 fold more sensitive than that of RT-PCR. The developed assay was applied for the detection of GBNV from 80 potato leaf samples where 24 samples were found infected which was confirmed by RT-PCR. It was concluded that the RT-LAMP assay developed for detection of GBNV was specific, sensitive and suitable for its use in virus indexing under potato seed production programme.

Keywords: Detection; Groundnut bud necrosis virus; LAMP; Orthotospovirus arachinecrosis; Potato; RT-PCR; Thrips.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Nucleic Acid Amplification Techniques
Reverse Transcription
Sensitivity and Specificity
Solanum tuberosum*
Viruses*

Supplementary concepts

LAMP assay