Development of a Reverse Transcription-Recombinase Polymerase Amplification Assay for Detection of Sugarcane Yellow Leaf Virus
- 60 Downloads
Sugarcane yellow leaf virus (SCYLV, genus Polerovirus) is a harmful agent that causes sugarcane yellow leaf disease. Current methods for SCYLV detection present some limitations. In this study, a novel isothermal amplification assay, namely reverse transcription-recombinase polymerase amplification (RT-RPA), was developed to promote SCYLV detection for disease management. The assay was evaluated in terms of specificity, sensitivity, reliability, temperature limit, and time limit. The developed RT-RPA assay was highly specific and non-cross-reactive with Potato leafroll virus, which is a type of species in Polerovirus. This assay can also detect at least 103-fold diluted cDNA from a SCYLV-infected sugarcane leaf, and its sensitivity is tenfold lower than that of RT-polymerase chain reaction (PCR). The reliability of the proposed assay was examined by detecting field sugarcane samples via RT-RPA and RT-PCR assays. The RT-RPA assay showed the same results as those of RT-PCR assay, indicating that the former was highly reliable for SCYLV detection. Analysis of the temperature and time limits revealed a wide operating temperature range from 27 to 45 °C, which was easily reached, and a rapid assay duration of 20 min. In summary, the developed RT-RPA assay was rapid, specific, and reliable with acceptable sensitivity and easily reachable operating temperature and thus could be a promising tool for SCYLV diagnosis in sugarcane.
KeywordsRecombinase polymerase amplification Sugarcane Sugarcane yellow leaf virus Yellow leaf disease
Loop-mediated isothermal amplification
Nucleic acid sequence-based amplification
Polymerase chain reaction
Potato leafroll virus
Recombinase polymerase amplification
Sugarcane yellow leaf virus
This study was funded by the National Natural Science Foundation of China (31771865) and Sugar Crop Research System (CARS-170301). The authors would like to thank Professor Cheng-Gui Han from China Agricultural University for generously donating PLRV-infected N. benthamiana leaves for the cross-reactivity study.
Shu-Zhen Zhang devised the experiments. Xiao-Yan Feng operated the experiments, analyzed the results, and wrote the manuscript. Lin-Bo Shen collected field sugarcane samples. Wen-Zhi Wang, Jun-Gang Wang, Zheng-Ying Cao, Cui-Lian Feng, and Ting-Ting Zhao revised the manuscript. All authors have read and approved the manuscript.
Compliance with Ethical Standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Ahmad, Y.A., L. Rassaby, M. Royer, Z. Borg, K.S. Braithwaite, T.E. Mirkov, M.S. Irey, X. Perrier, G.R. Smith, and P. Rott. 2006. Yellow leaf of sugarcane is caused by at least three different genotypes of Sugarcane yellow leaf virus, one of which predominates on the Island of Reunion. Archives of Virology 151 (7): 1355–1371.CrossRefPubMedGoogle Scholar
- Amata, R.L., E. Fernandez, D. Filloux, D.P. Martin, P. Rott, and P. Roumagnac. 2015. Prevalence of Sugarcane yellow leaf virus in sugarcane producing regions in Kenya revealed by reverse transcription loop-mediated isothermal amplification method. Plant Disease 100 (2): 260–268.CrossRefGoogle Scholar
- Babu, B., B.K. Washburn, S.H. Miller, K. Poduch, T. Sarigul, G.W. Knox, F.M. Ochoa-Corona, and M.L. Paret. 2016. A rapid assay for detection of Rose rosette virus using reverse transcription-recombinase polymerase amplification using multiple gene targets. Journal of Virological Methods 240: 78–98.CrossRefPubMedGoogle Scholar
- Comstock, J.C., and J.D. Miller. 2004. Yield comparisons: Disease-free tissue-culture versus bud-propagated sugarcane plants and healthy versus yellow leaf infected plants. Journal American Society Sugar Cane Technologists 24: 31–40.Google Scholar
- Comstock, J.C., M.S. Irey, and B.E.L. Lockhart. 1998. Incidence of yellow leaf syndrome in CP cultivars based on polymerase chain reaction and serological techniques. Sugar Cane 4: 21–24.Google Scholar
- Euler, M., Y.J. Wang, D. Heidenreich, P. Patel, O. Strohmeier, S. Hakenberg, M. Niedrig, F.T. Hufert, and M. Weidmann. 2013. Development of a panel of recombinase polymerase amplification assays for detection of biothreat agents. Journal of Clinical Microbiology 51 (4): 1110–1117.CrossRefPubMedPubMedCentralGoogle Scholar
- Grisham, M.P., Y.B. Pan, B.L. Legendre, M.A. Godshall, G. Eggleston, and D.M. Hogarth. 2001. Effect of Sugarcane yellow leaf virus on sugarcane yield and juice quality. Proceedings-International Society of Sugar Cane Technology 24: 434–438.Google Scholar
- Lehrer, A.T., and E. Komor. 2008. Symptom expression of yellow leaf disease in sugarcane cultivars with different degrees of infection by Sugarcane yellow leaf virus. Plant Pathology 57 (1): 178–189.Google Scholar
- Mondal, D., P. Ghosh, M.A.A. Khan, F. Hossain, S. Böhlkenfascher, G. Matlashewski, A. Kroeger, P. Olliaro, and A.A.E. Wahed. 2016. Mobile suitcase laboratory for rapid detection of Leishmania donovani using recombinase polymerase amplification assay. Parasites & Vectors 9 (1): 281–288.CrossRefGoogle Scholar
- Moonan, F., and T.E. Mirkov. 2002. Analyses of genotypic diversity among North, South, and Central American isolates of Sugarcane yellow leaf virus: Evidence for Colombian origins and for intraspecific spatial phylogenetic variation. Journal of Virology 76 (3): 1339–1348.CrossRefPubMedPubMedCentralGoogle Scholar
- Rott, P., T.E. Mirkov, S. Schenck, and J.C. Girard. 2007. Recent advances in research on Sugarcane yellow leaf virus, the causal agent of sugarcane yellow leaf. Proceedings-International Society of Sugar Cane Technology 26: 968–977.Google Scholar
- Xu, D.L., G.H. Zhou, X.P. Ren, Q.H. Shao, and S.H. Wu. 2005. Molecular identification of Sugarcane yellow leaf virus occurred at a sugarcane resources collecting station in Guangdong. Acta Phytopathologica Sinica 35 (5): 466–468. (in Chinese).Google Scholar
- Zhang, S.L., M. Ravelonandro, P. Russell, N. Mcowen, P. Briard, S. Bohannon, and A. Vrient. 2014. Rapid diagnostic detection of plum pox virus in Prunus plants by isothermal AmplifyRP® using reverse transcription-recombinase polymerase amplification. Journal of Virological Methods 207 (3): 114–120.CrossRefPubMedGoogle Scholar
- Zhou, G.H., J.G. Li, D.L. Xu, W.K. Shen, and H.H. Deng. 2006. Occurrence of Sugarcane yellow leaf virus in South China and its transmission by the sugarcane-colonizing aphid, Ceratovacuna lanigera. Scientia Agricultura Sinica 39 (10): 2023–2027. (in Chinese).Google Scholar