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Universal Primers for Detection of Novel Plant Capsid-Less Viruses: Papaya Umbra-like Viruses as Example

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PCR Primer Design

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2392))

Abstract

For diagnosis of positive-sense single-stranded RNA viruses, primers are usually raised against the sequence encoding capsid proteins, since structural proteins are more conserved. This chapter focuses on the design of primers for a group of novel viruses lacking a capsid, known as papaya Umbra-like viruses (unassigned genus) associated with Papaya Sticky Disease, which represent a threat to papaya production. Based on sequence alignments of a region encoding the RNA-dependent RNA Polymerase, universal primers to detect all the known viruses from four countries are proposed. The Forward universal primer can be used in combination with clade- and subclade-specific primers for rapid virus identification. We walk the reader through downloading sequences from nucleotide databases, doing sequence alignments and phylogenetic tree construction to identify conserved and variable regions as valid primer targets; we also show how to design and analyze the primers.

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Abbreviations

bp:

Base pair

BLAST:

Basic Local Alignment Search Tool

CDS:

Coding sequence

dsRNA:

Double-stranded RNA

Indels:

Insertion–deletions

kb:

Kilobases

kcal/mol:

Kilocalorie per mole

ML:

Maximum Likelihood

NCBI:

National Center for Biotechnology Information

NGS:

Next-Generation Sequencing

NJ:

Neighbor-Joining

PMeV:

Papaya meleira virus

PMeV-1:

Papaya meleira virus 1

PMeV-2:

Papaya meleira virus 2

PMeV-Mx:

Papaya meleira virus-Mexican variant

PpVQ:

Papaya virus Q

PRSV-P:

Papaya ringspot virus type P

PSD:

Papaya Sticky Disease

PCR:

Polymerase chain reaction

qPCR:

Quantitative PCR

RdRP:

RNA-dependent RNA Polymerase

(+) RNA:

Positive-sense single-stranded RNA

RT-PCR:

Reverse transcription polymerase chain reaction

T m :

Melting temperature

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Acknowledgments

This work was funded by the CONACYT research grant A1-S-19850 to L.L.O.

Author information

Authors and Affiliations

  1. Biotechnology Unit, Yucatan Center for Scientific Research, A.C. (CICY), Merida, Yucatan, México

    Jorge H. Ramirez-Prado

  2. Plant Biochemistry and Molecular Biology Unit, Yucatan Center for Scientific Research, A.C. (CICY), Merida, Yucatan, Mexico

    Luisa A. Lopez-Ochoa

Authors
  1. Jorge H. Ramirez-Prado
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  2. Luisa A. Lopez-Ochoa
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Corresponding author

Correspondence to Luisa A. Lopez-Ochoa .

Editor information

Editors and Affiliations

  1. Department of Biology, California State University, Northridge, Los Angeles, CA, USA

    Chhandak Basu

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Ramirez-Prado, J.H., Lopez-Ochoa, L.A. (2022). Universal Primers for Detection of Novel Plant Capsid-Less Viruses: Papaya Umbra-like Viruses as Example. In: Basu, C. (eds) PCR Primer Design. Methods in Molecular Biology, vol 2392. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1799-1_15

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  • DOI: https://doi.org/10.1007/978-1-0716-1799-1_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1798-4

  • Online ISBN: 978-1-0716-1799-1

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