Skip to main content

Advertisement

SpringerLink
Log in

“Development and comparison of reverse transcription-loop-mediated isothermal amplification assay (RT-LAMP), RT-PCR and real time PCR for detection of Potato spindle tuber viroid in potato”

  • Published:
European Journal of Plant Pathology Aims and scope Submit manuscript

Abstract

Potato spindle tuber viroid (PSTVd) is an important pathogen of potato infecting leaves, tubers as well as true potato seeds. We developed and validated a reverse transcription-loop mediated isothermal amplification (RT-LAMP) platform for visual, rapid, specific, and sensitive detection of PSTVd. The optimization of RT-LAMP reaction conditions and reagents concentrations were carried out with time, temperature, MgSO4, dNTPs, and WarmStart Bst 2.0 polymerase. The assay is very simple and rapid which provide detectable results in less than 50 min by incubating all the reagents at 65 °C. The detection of RT-LAMP results could be accomplished as ladder-like bands in an agarose gel or visualized by naked eyes with inclusion of a SYBR Green I dye. The target specificity of RT-LAMP primers was assessed with an introduction of a simple restriction digestion step after the RT-LAMP assay. The RT-LAMP assay demonstrated absence of any cross-reaction with other, potato viruses. The analytical sensitivity of the assay was greater than RT-PCR either the test template was plasmid or total RNA. The detection of RT-LAMP was validated in potato samples and compared with real time PCR and RT-PCR. The RT-LAMP assay developed in this study has the potential as a beneficial tool in detection of PSTVd in a low-cost laboratory due to its simplicity, rapidness, and application in large samples.

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
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Data availability

The nucleotide data generated during and/or analysed during the current study are available in the NCBI repository, under GenBank Accession no.: MH758760 (https://www.ncbi.nlm.nih.gov/nuccore/MH758760).

References

  • Boonham, N., Gonzalez Perez, L., Mendez, M. S., et al. (2004). Development of a real-time RT-PCR assay for the detection of potato spindle tuber viroid. Journal of Virological Methods, 116, 139–146.

    Article  CAS  Google Scholar 

  • Diener, T. O. (1971). Potato spindle tuber "virus". IV. A replicating, low molecular weight RNA. Virology, 45, 411–428.

    Article  CAS  Google Scholar 

  • Flores, R., Delgado, S., Gas, M. E., et al. (2004). Viroids: The minimum non-coding RNAs with autonomous replication. FEBS Letters, 567, 42–48.

    Article  CAS  Google Scholar 

  • Goto, M., Honda, E., Ogura, A., et al. (2009). Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. Biotechniques, 46, 167–172.

  • Gross, H. J., Domdey, H., Lossow, C., Jank, P., Raba, M., Alberty, H., & Sänger, H. L. (1978). Nucleotide sequence and secondary structure of potato spindle tuber viroid. Nature, 273(5659), 203–208.

    Article  CAS  Google Scholar 

  • Karwitha, M., Feng, Z. K., Shen, Y., Essendi, W., Zhang, W. N., Li, J. Y., & Tao, X. R. (2016). Rapid detection of tomato chlorosis virus from infected plant and whitefly by one-step reverse transcription loop-mediated isothermal amplification. Journal of Phytopathology, 164, 255–263.

    Article  CAS  Google Scholar 

  • Khan, M. S., Timmermann, C., Hoque, M. I., Sarker, R. H., & Mühlbach, H. P. (2009). Detection of Potato spindle tuber viroid (PSTVd) in minute amounts of potato (Solanum tuberosum L.) leaf tissue by hybridization techniques and, together with potato viruses by multiplex RT-PCR. Journal of Plant Diseases and Protection, 116, 97–105.

    Article  CAS  Google Scholar 

  • Lenarčič, R., Morisset, D., Mehle, N., et al. (2013). Fast real-time detection of potato spindle tuber viroid by RT-LAMP. Plant Pathology, 62, 1147–1156.

    Article  Google Scholar 

  • Morris, T. J., & Wright, N. S. (1975). Detection on polyacrylamide gel of a diagnostic nucleic acid from tissue infected with potato spindle tuber viroid. American Potato Journal, 52, 57–63.

    Article  CAS  Google Scholar 

  • Nie, X. (2005). Reverse transcription loop-mediated isothermal amplification of DNA for detection of potato virus Y. Plant Disease, 89, 605–610.

    Article  CAS  Google Scholar 

  • Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., & Hase, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 28(12), E63.

    Article  CAS  Google Scholar 

  • Owens, R. A., & Diener, T. O. (1981). Sensitive and rapid diagnosis of potato spindle tuber viroid disease by nucleic acid hybridization. Science, 213(4508), 670–672.

    Article  CAS  Google Scholar 

  • Pfannenstiel, M. A., & Slack, S. A. (1980). Response of potato cultivars to infection by the potato spindle tuber viroid. Phytopathology, 70, 922–926.

    Article  Google Scholar 

  • Shamloul, A. M., Hadidi, A., Zhu, S. F., Singh, R. P., & Sagredo, B. (1997). Sensitive detection of potato spindle tuber viroid using RT-PCR and identification of a viroid variant naturally infecting Pepino plants. Canadian Journal of Plant Pathology, 19, 89–96.

    Article  Google Scholar 

  • Singh, R. P., & Kurz, K. (1997). RT-PCR analysis of PSTVd aphid transmission in association with PLRV. Canadian Journal of Plant Pathology, 19(4), 418–424.

    Article  Google Scholar 

  • Singh, R. P., Benson, A. P., & Salama, F. M. (1966). Purification and electron microscopy of potato spindle virus. Phytopathology, 56, 901–902.

    Google Scholar 

  • Singh, R. P., Boucher, A., & Seabrook, J. E. A. (1988). Detection of the mild strains of potato spindle tuber viroid from single true potato seed by return electrophoresis. Phytopathology, 8, 663–667.

    Article  Google Scholar 

  • Singh, R. P., Boucher, A., & Somerville, T. H. (1992). Detection of potato spindle tuber viroid in the pollen and various parts of potato plant pollinated with viroid-infected pollen. Plant Disease, 76, 951–953.

    Article  Google Scholar 

  • Tomita, N., Mori, Y., Kanda, H., & Notomi, T. (2008). Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nature Protocol, 3(5), 877–882.

    Article  CAS  Google Scholar 

  • Tsutsumi, N., Yanagisawa, H., Fujiwara, Y., & Ohara, T. (2010). Detection of potato spindle tuber viroid by reverse transcription loop-mediated isothermal amplification. Research Bulletin of the Plant Protection Service Japan, 46, 61–67.

    CAS  Google Scholar 

  • Verma, G., Sharma, S., Raigond, B. et al (2019). Development and application of fluorescent loop mediated isothermal amplification technique to detect Phytophthora infestans from potato tubers targeting ITS-1 region. 3 Biotech, 9, 345.

  • Weidemann, H. L., & Buchta, U. (1998). A simple and rapid method for the detection of potato spindle tuber viroid (PSTVd) by RT-PCR. Potato Research, 41, 1–8.

    Article  CAS  Google Scholar 

Download references

Funding

This work was funded by ICAR, New Delhi, India under “Consortium Research Platform on Vaccines and Diagnostics”, project no. 1009850.

Author information

Authors and Affiliations

  1. Division of Plant Protection, ICAR-Central Potato Research Institute, Shimla, Himachal Pradesh, India

    Gaurav Verma, Baswaraj Raigond, Shruti Pathania, Tarvinder Kochhar & Kailash Naga

Authors
  1. Gaurav Verma
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Baswaraj Raigond
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shruti Pathania
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tarvinder Kochhar
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kailash Naga
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Conceptualization: GV; Data curation: GV, SP; Methodology: GV, BR, KN; Software analysis: GV; Validation: SP, TK; Writing – original draft: GV, BR.

Corresponding author

Correspondence to Gaurav Verma.

Ethics declarations

Conflicts of interest/competing interests

The authors declare that they have no conflict of interest.

Research involving human participants and/or animals

This article does not contain any studies with human participants or animals performed by any of the authors.

Electronic supplementary material

ESM 1

(PDF 241 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Verma, G., Raigond, B., Pathania, S. et al. “Development and comparison of reverse transcription-loop-mediated isothermal amplification assay (RT-LAMP), RT-PCR and real time PCR for detection of Potato spindle tuber viroid in potato”. Eur J Plant Pathol 158, 951–964 (2020). https://doi.org/10.1007/s10658-020-02129-z

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10658-020-02129-z

Keywords

Search

Navigation