Skip to main content

Advertisement

SpringerLink
Log in

Multiplex RT-PCR detection of three common viruses infecting orchids

  • Brief Report
  • Published:
Archives of Virology Aims and scope Submit manuscript

Abstract

A multiplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed for simultaneous detection of three orchid viruses: cymbidium mosaic virus (CymMV), odontoglossum ringspot virus (ORSV), and orchid fleck virus (OFV). Primers were used to amplify nucleocapsid protein gene fragments of 845 bp (ORSV), 505 bp (CymMV) and 160 bp (OFV). A 60-bp amplicon of plant glyceraldehyde-3-phophate dehydrogenase mRNA was included as an internal control against false negatives. The assay was validated against 31 collected plants from six orchid genera and compared with results obtained by transmission electron microscopy (TEM). The RT-PCR assay proved more sensitive than TEM for detection of OFV.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

References

  1. Zettler FW, Ko NJ, Wisler GC, Eliott MS, Wong SM (1990) Viruses of orchids and their control. Plant Dis 74:621–626

    Article  Google Scholar 

  2. Blanchfield AL, Mackenzie AM, Gibbs A, Kondo H, Tamada T, Wilson CR (2001) Identification of Orchid fleck virus by reverse transcriptase-polymerase chain reaction and analysis of isolate relationships. J Phytopathol 149:713–718

    Article  CAS  Google Scholar 

  3. Kim SR, Yoon J-Y, Choi GS, Chang MU, Choi JK, Chung BN (2010) Molecular characterization and survey of the infection rate of Orchid fleck virus in commercial orchids. Plant Pathol J 26:130–138

    Article  CAS  Google Scholar 

  4. Gibbs A, Mackenzie A, Blanchfield A, Cross P, Wilson C, Kitajima E, Nightingale M, Clements M (2000) Viruses of Orchids in Australia; their identification, biology and control. Aust Orch Rev 65:10–21

    Google Scholar 

  5. Hu JS, Ferreira S, Xu MQ, Lu M, Iha M, Pflum E, Wang M (1994) Transmission, movement, and inactivation of cymbidium mosaic and odontoglossum ringspot viruses. Plant Dis 78:633–636

    Article  Google Scholar 

  6. Kondo H, Maeda T, Tamada T (2006) Orchid fleck virus: Brevipalpus californicus transmission, biological properties, and genome structure. Exp Appl Acarology 30(1–3):215–223

    Google Scholar 

  7. Eun AJ, Seoh M, Wong S (2000) Simultaneous quantitation of two orchid viruses by the Taqman real-time RT-PCR. J Virol Methods 87:151–160

    Article  PubMed  CAS  Google Scholar 

  8. Jeong JH, Yan DC, Peak KY (2001) Comparison of ELISA and RT-PCR for efficient diagnosis of cymbidium mosaic virus and odontoglossum ringspot virus in Cymbidium hoosai Mokino. J Kor Soc Hort Sci 42:336–340

    CAS  Google Scholar 

  9. Seoh ML, Wong SM, Zhang L (1998) Simultaneous TD/RT-PCR detection of cymbidium mosaic potexvirus and odontoglossum ringspot tobamovirus with a single pair of primers. J Virol Methods 72:197–204

    Article  PubMed  CAS  Google Scholar 

  10. Yamane K, Oyama K, Iuchi E, Ogawa H, Suzuki T, Natsuaki T (2008) RT-PCR detection of odontoglossum ringspot virus, Cymbidium mosaic virus and Tospoviruses and association of infections with leaf-yellowing symptoms in phalaenopsis. J Phytopathol 156:268–273

    Article  CAS  Google Scholar 

  11. Lee MS, Yang MJ, Hseu YC, Lai GH, Chang WT, Hsu YH, Lin MK (2011) One-step reverse transcription loop-mediated isothermal amplification assay for rapid detection of Cymbidium mosaic virus. J Virol Meth 173:43–48

    Article  CAS  Google Scholar 

  12. Lin HY, Huang CH, Lu SH, Kuo IT, Chau LK (2014) Direct detection of orchid viruses using nanorod-based fibre optic particle plasmon resonance immunosensor. Biosen Bioelectron 51:371–378

    Article  CAS  Google Scholar 

  13. Mumford R, Boonham N, Tomlinson J, Barker I (2006) Advances in molecular phytodiagnostics: new solutions for old problems. Eur J Plant Pathol 116:1–19

    Article  CAS  Google Scholar 

  14. Kubo KS, Stuart RM, Freitas-Astua J, Antonioli-Luizon R, Locali-Fabris EC, Coletta-Filho HD, Machado MA, Kitajima EW (2009) Evaluation of the genetic variability of Orchid fleck virus by single-strand conformational polymorphism analysis and nucelotide sequencing of a fragment from the nucleocpasid gene. Arch Virol 154:1009–1014

    Article  PubMed  CAS  Google Scholar 

  15. Sanchez-Navarro JA, Aparicio F, Herranz MC, Minafra A, Myrta A, Pallas V (2005) Simultaneous detection and identification of eight stone fruit viruses by one-step RT-PCR. Eur J Plant Pathol 111:77–84

    Article  CAS  Google Scholar 

  16. Gambino G, Gribaudo I (2006) Simultaneous detection of nine grapevine viruses by multiplex reverse transcription-polymerase chain reaction with coamplification of a plant RNA as internal control. Phytopathology 96:1223–1229

    Article  PubMed  CAS  Google Scholar 

  17. Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH (1997) Multiplex PCR: critical parameters and step-by-step protocol. BioTechniques 23:504–511

    PubMed  CAS  Google Scholar 

  18. Wei T, Lu G, Clover G (2008) Novel approaches to mitigate primer interaction and eliminate inhibitors in multiplex PCR, demonstrated using an assay for detection of three strawberry viruses. J Virol Meth 151:132–139

    Article  CAS  Google Scholar 

  19. Wei T, Lu G, Clover GRG (2009) A multiplex RT-PCR for the detection of Potato yellow vein virus, Tobacco rattle virus and Tomato infectious chlorosis virus in potato with a plant internal amplification control. Plant Pathol 58:203–209

    Article  CAS  Google Scholar 

  20. Tao Y, Man J, Wu Y (2012) Development of a multiplex polymerase chain reaction for simultaneous detection of wheat viruses and a phytoplasma in China. Arch Virol 157:1261–1267

    Article  PubMed  CAS  Google Scholar 

  21. Hu W-C, Huang C-H, Lee S-C, Wu C-I, Chang Y-C (2009) Detection of four calla potyviruses by multiplex RT-PCR using nad5 mRNA as an internal control. Eur J Plant Pathol 126:43–52

    Article  Google Scholar 

Download references

Acknowledgements

This work was generously supported by the South Australian Cymbidium Growers Association. R.A. would also like to thank Margot White of the Royal Tasmanian Botanic Gardens and Shane Hossel for provision of virus-infected samples and assistance with TEM.

Author information

Authors and Affiliations

  1. Tasmanian Institute of Agriculture, University of Tasmania, New Town Research Laboratories, 13 St. John’s Avenue, New Town, TAS, 7008, Australia

    Raymond N. Ali & Calum R. Wilson

  2. Department of Primary Industry, Parks, Water and Environment, New Town Research Laboratories, 13 St. John’s Avenue, New Town, TAS, 7008, Australia

    Alison L. Dann & Peter A. Cross

Authors
  1. Raymond N. Ali
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alison L. Dann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Peter A. Cross
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Calum R. Wilson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Calum R. Wilson.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 1285 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ali, R.N., Dann, A.L., Cross, P.A. et al. Multiplex RT-PCR detection of three common viruses infecting orchids. Arch Virol 159, 3095–3099 (2014). https://doi.org/10.1007/s00705-014-2161-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00705-014-2161-9

Keywords

Search

Navigation