Immunocapture-Multiplex RT-PCR for the Simultaneous Detection and Identification of Plant Viruses and Their Strains: Study Case, Potato Virus Y (PVY)

Part of the Methods in Molecular Biology book series (MIMB, volume 1302)

Abstract

Immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) is a sensitive, reproducible, and robust method for the detection and identification of RNA viruses. The IC step provides a simple method to isolate virus particles from plant tissue, particularly when inhibitory substances are present, and thus enables subsequent use of RT-PCR amplification for large-scale virus testing and typing. The multiplex format of the PCR is often used for the detection and identification of multiple virus/strain simultaneously to save time, labor, and cost. Potato virus Y (PVY) is one of the most economically important viruses infecting potato worldwide. PVY exists as a complex of at least nine strains and many more unclassified recombinants that vary in their genome structures, phenotypes, and their economic importance. In the current chapter, a detailed protocol of an IC-based, multiplex RT-PCR assay for the detection and identification of various PVY strains is described.

Key words

Immunocapture Potato virus Y RT-PCR Strain Identification 

References

  1. 1.
    Nolasco G, de Bias C, Torres V et al (1993) A method combining immunocapture and PCR amplification in a microtiter plate for the detection of plant viruses and subviral pathogens. J Virol Meth 45:201–218CrossRefGoogle Scholar
  2. 2.
    Mulholland V (2009) Immunocapture-PCR for plant virus detection. In: Burns R (ed) Plant pathology techniques and protocols. Humana Press/Springer, New York, USA, pp 183–192CrossRefGoogle Scholar
  3. 3.
    Menzel W, Jelkmann W, Maiss E (2002) Detection of four apple viruses by multiplex RT-PCR assays with coamplification of plant mRNA as internal control. J Virol Meth 99:81–92CrossRefGoogle Scholar
  4. 4.
    Noorani MS, Awsthi P, Sharma MP et al (2013) Simultaneous detection and identification of four cherry viruses by two step multiplex RT-PCR with an internal control of plant nad5 mRNA. J Virol Meth 193:103–107CrossRefGoogle Scholar
  5. 5.
    Wieczorek P, Obrepalska-Steplowska A (2013) Multiplex RT-PCR reaction for simultaneous detection of Tomato torrado virus and Pepino mosaic virus co-infecting Solanum lycopersicum. J Plant Protect Res 53:289–294CrossRefGoogle Scholar
  6. 6.
    Yao B, Wang G, Ma X et al (2014) Simultaneous detection and differentiation of three viruses in pear plants by a multiplex RT-PCR. J Virol Meth 196:113–119CrossRefGoogle Scholar
  7. 7.
    Karasev AV, Gray SM (2013) Continuous and emerging challenges of Potato virus Y in potato. Annu Rev Phytopathol 51:571–586PubMedCrossRefGoogle Scholar
  8. 8.
    Gray S, De Boer S, Lorenzen J et al (2010) Potato virus Y: an evolving concern for potato crops in the United States and Canada. Plant Dis 94:1384–1397CrossRefGoogle Scholar
  9. 9.
    Chikh Ali M, Maoka T, Natsuaki KT et al (2010) The simultaneous differentiation of Potato virus Y strains including the newly described strain PVYNTN-NW by multiplex PCR assay. J Virol Meth 165:15–20CrossRefGoogle Scholar
  10. 10.
    Chikh Ali M, Gray S, Karasev AV (2013) An improved multiplex IC-RT-PCR assay distinguishes nine strains of Potato virus Y. Plant Dis 97:1370–1374CrossRefGoogle Scholar
  11. 11.
    Boonham N, Walsh K, Preston S et al (2002) The detection of tuber necrotic isolates of Potato virus Y, and the accurate discrimination of PVYO, PVN and PVYC strains using RT-PCR. J Virol Meth 102:103–112CrossRefGoogle Scholar
  12. 12.
    Nie X, Singh RP (2002) A new approach for the simultaneous differentiation of biological and geographical strains of Potato virus Y by uniplex and multiplex RT-PCR. J Virol Meth 104:41–54CrossRefGoogle Scholar
  13. 13.
    Moravec T, Cerovska N, Boonham N (2003) The detection of recombinant, tuber necrosing isolates of Potato virus Y (PVYNTN) using a three-primer PCR based in the coat protein gene. J Virol Meth 109:63–68CrossRefGoogle Scholar
  14. 14.
    Nie X, Singh RP (2003) Specific differentiation of recombinant PVYN:O and PVYNTN isolates by multiplex RT-PCR. J Virol Meth 113:69–77CrossRefGoogle Scholar
  15. 15.
    Glais L, Tribodet M, Kerlan C (2005) Specific detection of the PVYN-W variant of Potato virus Y. J Virol Meth 125:131–136CrossRefGoogle Scholar
  16. 16.
    Rigotti S, Gugerli P (2007) Rapid identification of Potato virus Y strains by one-step triplex RT-PCR. J Virol Meth 140:90–94CrossRefGoogle Scholar
  17. 17.
    Lorenzen JH, Piche LM, Gudmestad NC et al (2006) A multiplex PCR assay to characterize Potato virus Y isolates and identify strain mixtures. Plant Dis 90:935–940CrossRefGoogle Scholar
  18. 18.
    Schubert J, Fomitcheva V, Sztangret-Wisniewski J (2007) Differentiation of Potato virus Y using improved sets of diagnostic PCR- primers. J Virol Meth 140:66–74CrossRefGoogle Scholar
  19. 19.
    Chikh Ali M, Maoka T, Natsuaki KT (2008) The discrimination between Potato virus Y serotypes O and N using a multiplex PCR assay. Trop Agr Dev 52:37–42Google Scholar
  20. 20.
    Karasev AV, Nikolaeva OV, Hu X et al (2010) Serological properties of ordinary and necrotic isolates of Potato virus Y: a case study of PVYN misidentification. Am J Potato Res 87:1–9CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Plant Soil and Entomological SciencesUniversity of IdahoMoscowUSA

Personalised recommendations