Short communication
Improvement of Potato virus Y (PVY) detection and quantitation using PVYN- and PVYO-specific real-time RT-PCR assays

https://doi.org/10.1016/j.jviromet.2006.01.019Get rights and content

Abstract

A Potato virus Y (PVY) single nucleotide polymorphism (A/G2213), recently identified as a molecular determinant of the tobacco leaf necrosis symptom induced by PVYN isolates, has been used as a target to develop two PVY group-specific (PVYN and PVYO) fluorescent (TaqMan®-based) real-time RT-PCR assays. These procedures allow detection, characterisation, and quantitation of a wide range of PVY isolates in samples containing 103–108 viral transcripts. Moreover, the high specificity of these two new assays make the simultaneous detection and the reliable quantitation of PVYN and PVYO isolates in mixed solutions, regardless of the YN/YO ratio, feasible. The high sensitivity (threshold of 103 copies per reaction) and the PVY group specificity of these two new PVY detection tools clearly improve previously published PVY detection tests and offer new opportunities for PVY research programs.

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Acknowledgements

We are grateful to Mathieu Rolland for his help in data acquisition, and to Christopher A. Todd and Sebastien Guyader for critical reading of the manuscript. Thanks to Olivier Lievyns for his assistance during the preparation of the manuscript. This work was supported by the Institut National de la Recherche Agronomique (France) and the Fédération Nationale des Producteurs de Plants de Pomme de Terre (FNPPPT, Paris, France).

References (64)

  • A.J.C. Eun et al.

    Simultaneous quantitation of two orchid viruses by the TaqMan (R) real-time RT-PCR

    J. Virol. Meth.

    (2000)
  • F. Fabre et al.

    Improvement of Barley yellow dwarf virus-PAV detection in single aphids using a fluorescent real time RT-PCR

    J. Virol. Meth.

    (2003)
  • M.H. Ismail

    The use of enzyme-linked immunosorbent assay (ELISA) for quantitative detection of Potato virus Y in potato and other test plants

    Microbiol. Res.

    (1997)
  • E. Jacquot et al.

    A single nucleotide polymorphism-based technique for specific characterization of YO and YN isolates of Potato virus Y (PVY)

    J. Virol. Meth.

    (2005)
  • J. Korimbocus et al.

    Improved detection of Sugarcane yellow leaf virus using a real-time fluorescent (TaqMan) RT-PCR assay

    J. Virol. Meth.

    (2002)
  • P. Lunello et al.

    Ultra-sensitive detection of two garlic Potyviruses using a real-time fluorescent (Taqman®) RT-PCR assay

    J. Virol. Meth.

    (2004)
  • I.M. Mackay

    Real-time PCR in the microbiology laboratory

    Clin. Microbiol. Infect.

    (2004)
  • M. Madani et al.

    Quantitative detection of the potato cyst nematode, Globodera pallida, and the beet cyst nematode, Heterodera schachtii, using Real-Time PCR with SYBR green I dye

    Mol. Cell. Probes

    (2005)
  • X.Z. Nie et al.

    Probable geographical grouping of PVYN and PVYNTN based on sequence variation in P1 and 5′-UTR of PVY genome and methods for differentiating North American PVYNTN

    J. Virol. Meth.

    (2002)
  • X.Z. Nie et al.

    Specific differentiation of recombinant PVYN:O and PVYNTN isolates by multiplex RT-PCR

    J. Virol. Meth.

    (2003)
  • K.M. Ririe et al.

    Product differentiation by analysis of DNA melting curves during the polymerase chain reaction

    Anal. Biochem.

    (1997)
  • K. Walsh et al.

    Detection of different strains of Potato virus Y and their mixed infections using competitive fluorescent RT-PCR

    J. Virol. Meth.

    (2001)
  • M. Welti et al.

    Development of a multiplex real-time quantitative PCR assay to detect Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae in respiratory tract secretions

    Diagn. Microbiol. Infect. Dis.

    (2003)
  • K. Yano et al.

    Expression of mRNAs and proteins for peroxiredoxins in Plasmodium falciparum erythrocytic stage

    Parasitol. Int.

    (2005)
  • K.C. Young et al.

    A reverse-transcription competitive PCR assay based on chemiluminescence hybridization for detection and quantitation of hepatitis C virus RNA

    J. Virol. Meth.

    (2002)
  • B. Balaji et al.

    Barley yellow dwarf virus and Cereal yellow dwarf virus quantitation by real-time polymerase chain reaction in resistant and susceptible plants

    Phytopathology

    (2003)
  • B. Blanco-Urgoiti et al.

    Characterization of potato Potyvirus Y (PVY) isolates from seed potato batches. Situation of the NTN, Wilga and Z isolates

    Eur. J. Plant Pathol.

    (1998)
  • G. Bleve et al.

    Development of reverse transcription (RT)-PCR and real-time RT-PCR assays for rapid detection and quantitation of viable yeasts and molds contaminating yogurts and pasteurized food products

    Appl. Environ. Microbiol.

    (2003)
  • Bokx De, J., Hunttinga, H., 1981. Description of plant viruses. AAB/CMI...
  • N. Boonham et al.

    Virus strain discrimination using recombinant antibodies

    Dis. Mark.

    (2000)
  • A.A. Brunt

    The general-properties of Potyviruses

    Arch. Virol.

    (1992)
  • A.A. Brunt et al.

    Plant Viruses Online: Descriptions and Lists from the VIDE Database

    (1996)
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