Journal of
General and Molecular Virology

  • Abbreviation: J. Gen. Mol. Virol.
  • Language: English
  • ISSN: 2141-6648
  • DOI: 10.5897/JGMV
  • Start Year: 2009
  • Published Articles: 37

Full Length Research Paper

Field deployable Reverse Transcriptase–Recombinase Polymerase Amplification (RT-RPA) for detection of Maize chlorotic mottle virus (MCMV)

F. M. Mwatuni
  • F. M. Mwatuni
  • Kenya Plant Health Inspectorate Service, P. O. Box 49421-00100 Nairobi, Kenya.
  • Search for this author on:
  • Google Scholar
M. G. Redinbaugh
  • M. G. Redinbaugh
  • Department of Plant Pathology, Ohio State University, OARDC 1680 Madison Ave, Wooster, OH 44691, USA.
  • Search for this author on:
  • Google Scholar
S. Miller
  • S. Miller
  • Department of Plant Pathology, Ohio State University, OARDC 1680 Madison Ave, Wooster, OH 44691, USA.
  • Search for this author on:
  • Google Scholar
X. Ma
  • X. Ma
  • Department of Plant Pathology, Ohio State University, OARDC 1680 Madison Ave, Wooster, OH 44691, USA.
  • Search for this author on:
  • Google Scholar
B. N. Aggrey
  • B. N. Aggrey
  • Jomo Kenyatta University of Agriculture and Technology, P. O. Box 62000-00100 Nairobi, Kenya.
  • Search for this author on:
  • Google Scholar
L. M. Suresh
  • L. M. Suresh
  • International Maize and Wheat Improvement Center (CIMMYT), P. O. Box 1041 – 00621 Nairobi, Kenya.
  • Search for this author on:
  • Google Scholar


  •  Received: 25 January 2022
  •  Accepted: 05 April 2022
  •  Published: 30 April 2022

Abstract

Diagnosis of maize lethal necrosis (MLN)-causing viruses is key in MLN surveillance programs and in testing seed for zero tolerance of Maize chlorotic mottle virus (MCMV) in seed lots. This is crucial for MLN management in farmers’ fields and in commercial seed fields. A customized MCMV detection assay that is specific, sensitive, affordable, and portable is therefore important for this task. Reverse Transcriptase–Recombinase Polymerase Amplification (RT-RPA) meets those conditions earlier described. RPA is a rapid isothermal nucleic acid amplification and detection platform that is based on patented Recombinase Polymerase Amplification (RPA) technology. In this study, a real time endpoint analysis and field deployable RT-RPA diagnostic method for the detection of MCMV was developed. RPA primer sets with their complementary probes were designed, synthesized and tested through a series of primer set evaluations to determine the most efficient primer sets. The primer sets targeted the MCMV genome at position 2765-2948 bp (MCMV_gp2 replicase gene). The parameters evaluated were sensitivity, specificity and reproducibility for the assay with remarkable results. The assay discriminated against other maize infecting viruses hence specific to MCMV. The assay takes only 20 min and its detection limit of 10-4 is well comparable to RT-PCR and other molecular based detection assays. MCMV was also detected directly from leaf saps without the nucleic acid extraction step hence suitable for on-farm testing. RPA is a relatively inexpensive technique that requires minimal instrumentation. This assay is therefore suitable for the detection of MCMV in field surveys, routine MCMV testing for phytosanitary measures and in the seed certification procedures.

 

Key words: Maize lethal necrosis, maize chlorotic mottle virus, diagnostics, recombinase polymerase amplification.

Abbreviation

CIMMYT, International Maize and Wheat Improvement Center; KEPHIS, Kenya Plant Health Inspectorate Service; MCMV, Maize chlorotic mottle virus; MLN, Maize lethal necrosis; NPPOs, National Plant Protection Organizations; SCMV, Sugarcane mosaic virus; OARDC, Ohio Agricultural Research Development Center.

Copyright © 2024 Author(s) retain the copyright of this article.

This article is published under the terms of the Creative Commons Attribution License 4.0

Back to Vol. 11 No. 1
Back to articles
SUBSCRIBE TO RSS
SUBMIT MANUSCRIPT
CONFERENCES