Advertisement

Rapid and Sensitive Detection of Citrus tristeza virus Using Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay

  • Dilip Kumar Ghosh
  • Ashish Warghane
  • Kajal Kumar Biswas
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2015)

Abstract

Loop-mediated isothermal amplification (LAMP) is one recently developed gene amplification technique that emerges as a simple and quick diagnostic tool for early detection of nucleic acid targets. The LAMP technique works on the principle of strand displacement activity of Bst polymerase. It contains a set of four specially designed primers, which recognizes six different regions on the target nucleotide sequence. In the LAMP reaction, amplification is carried out in an isothermal conditions (60–65°C) using simple and inexpensive device like water bath or dry bath. Additional benefits of LAMP technique are that final results can be seen directly with naked eyes by adding intercalating dye SYBR Green I in the reaction tube. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is one of the novel techniques used for detection of RNA targets. The technology has been successfully applied for rapid and sensitive detection of Citrus tristeza virus (CTV) by using four oligo-primers, targeting a conserved coat protein gene (CPG) of an Indian CTV isolate. The result of assay is visible in naked eyes easily in the presence of SYBR Green I (100×) or on 1.5% agarose gel electrophoresis. CTV-RT-LAMP could be used away from plant pathology laboratories even in remote location.

Key words

Citrus Tristeza disease RT-LAMP SYBR Green I Bst DNA polymerase 

References

  1. 1.
    Bar-Joseph M, Dawson WO (2008) Citrus tristeza virus. In: Mahy BWJ and Van Regenmortel MHV (eds) Encyclopedia of Virolology, Elsevier, OxfordCrossRefGoogle Scholar
  2. 2.
    Ahlawat Y (2012) Virus Diseases of Citrus and Management. Stadium Press, DelhiGoogle Scholar
  3. 3.
    Ghosh DK, Aglave B, Roy A et al (2009) Molecular cloning, sequencing and phylogenetic analysis of coat protein gene of a biologically distinct Citrus tristeza virus isolate occurring in central India. J Plant Biochem Biotechnol 18:105–108CrossRefGoogle Scholar
  4. 4.
    Ayllon MA, Lopez C, Navas-Castillo J et al (2001) Polymorphism of the 5' terminal region of Citrus tristeza virus (CTV) RNA: Incidence of three sequence types in isolates of different origin and Pathogenicity. Arch Virol 146:27–40CrossRefGoogle Scholar
  5. 5.
    Biswas KK, Tarafdar A, Sharma SK (2012) Complete genome of mandarin decline Citrus tristeza virus of Northeastern Himalayan hill region of India: comparative analyses determine recombinant. Arch Virol 157:579–583CrossRefGoogle Scholar
  6. 6.
    Biswas KK, Palchoudhury S, Sharma SK et al (2018) Analyses of 3’ half genome of citrus tristeza virus reveal existence of distinct virus genotypes in citrus growing regions of India. Virus Dis 29(3).  https://doi.org/10.1007/s13337-018-0456-2CrossRefGoogle Scholar
  7. 7.
    Ward LI, Harper SJ (2012) Loop-mediated isothermal amplification for the detection of plant pathogens. Methods Mol Biol 862:161–170CrossRefGoogle Scholar
  8. 8.
    Rigano LA, Malamud F, Orce IG et al (2014) Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick. BMC Microbiol 14:86CrossRefGoogle Scholar
  9. 9.
    Varga A, James D (2006) Use of reverse transcription loop mediated isothermal amplification for the detection of Plum pox virus. J Virol Methods. 138:184–190CrossRefGoogle Scholar
  10. 10.
    Fukuda S, Takao S, Kuwayama M et al (2006) Rapid detection of norovirus from fecal specimens by real-time reverse transcription-loop-mediated isothermal amplification assay. J Clin Microbiol 44:1376–1381CrossRefGoogle Scholar
  11. 11.
    Dukes JP, King DP, Alexandersen S (2006) Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus. Arch Virol 151:1093–1106CrossRefGoogle Scholar
  12. 12.
    Warghane A, Misra P, Bhose S et al (2017) Development of a simple and rapid reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for sensitive detection of Citrus tristeza virus. J Virol Methods 250:6–10CrossRefGoogle Scholar
  13. 13.
    Kuboki N, Inoue N, Sakura FD et al (2003) Loop-mediated isothermal amplification for detection of African trypanosomes. J Clin Microbiol 41:5517–5524CrossRefGoogle Scholar
  14. 14.
    Mori Y, Nagamine K, Tomita N et al (2001) Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Bioph Res Commun 289:150–154CrossRefGoogle Scholar
  15. 15.
    Wang YJ, Zhou Y, Li Z et al (2013) A RT LAMP Assay for detection of Citrus tristeza virus. Scie agri sincia 46:517–524Google Scholar
  16. 16.
    Notomi T, Okayama H, Masubuchi H et al (2000) Loop mediated isothermal amplification of DNA. Nucleic Acids Res 28(12):E63CrossRefGoogle Scholar
  17. 17.
    Fernandez-Soto P, Mvoulouga PO, Akue JP et al (2014) Development of a highly sensitive loop-mediated isothermal amplification (LAMP) method for the detection of Loaloa. PLoS One 9:e94664CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Dilip Kumar Ghosh
    • 1
  • Ashish Warghane
    • 1
  • Kajal Kumar Biswas
    • 2
  1. 1.Plant Virology LaboratoryICAR-Central Citrus Research InstituteNagpurIndia
  2. 2.Division of Plant Pathology, Advanced Centre for Plant VirologyICAR Indian Agricultural Research InstituteNew DelhiIndia

Personalised recommendations