Abstract
Rapid, isothermal detection is possible using recombinase polymerase amplification assay (RPA) offering great specificity plus sensitivity. An RPA assay was standardized and evaluated to detect four viroids using RNA from infected grapevine plants. From the sequences of the whole genome of four viroids, four pairs of oligonucleotide primers were created. The four viroids employed in this study included Grapevine yellow speckle viroid 1 and 2 (GYSVd-1; GYSVd-2), Australian grapevine viroid (AGVd) and Hop stunt viroid (HSVd). The primers were tested to function both in RPA in addition to polymerase chain reaction (PCR). Applying the RT-RPA assay, the presence of four viroids was determined in a total of 119 symptomatic and asymptomatic grapevine leaf samples from several 22 genotypes obtained from the various regions of India. The results of RT-RPA were compatible with RT-PCR-based detection utilising pure RNA generated cDNA as a template. GYSVd-2 was effectively detected using crude leaf sap in RPA as well. To our awareness, this is the earliest report of consistent diagnosis of most grapevine viroids infection by RT-RPA using RNA as well as crude leaf sap as a template.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
The complete nucleotide sequences of amplified viroids have been submitted in NCBI and obtained accession ids are: MH476216 (GYSVd-1); MH282875 (GYSVd-2); MH476221 (HSVd); MH476220 (AGVd).
References
Adkar-Purushothama CR, Kanchepalli PR, Yanjarappa SM et al (2014) Detection, distribution, and genetic diversity of Australian grapevine viroid in grapevines in India. Virus Genes 49:304–311. https://doi.org/10.1007/s11262-014-1085-5
Ahmed A, van der Linden H, Hartskeerl R (2014) Development of a recombinase polymerase amplification assay for the detection of pathogenic Leptospira. Int J Environ Res Public Health 11:4953–4964. https://doi.org/10.3390/ijerph110504953
Almasi MA, Aghapour-Ojaghkandi M, Aghaei S (2013) Visual detection of curly top virus by the colorimetric loop-mediated isothermal amplification. J Plant Pathol Microbiol. https://doi.org/10.4172/2157-7471.1000198
Basso MF, Fajardo TVM, Saldarelli P (2017) Grapevine virus diseases: economic impact and current advances in viral prospection and management. Rev Bras Frutic. https://doi.org/10.1590/0100-29452017411
Daher RK, Stewart G, Boissinot M, Bergeron MG (2014) Isothermal recombinase polymerase amplification assay applied to the detection of group B streptococci in vaginal/anal samples. Clin Chem 60:660–666. https://doi.org/10.1373/clinchem.2013.213504
Euler M, Wang Y, Heidenreich D et al (2013) Development of a panel of recombinase polymerase amplification assays for detection of biothreat agents. J Clin Microbiol 51:1110–1117. https://doi.org/10.1128/JCM.02704-12
Hajizadeh M, Navarro B, Bashir NS, Torchetti EM, Di Serio F (2012) Development and validation of a multiplex RT-PCR method for the simultaneous detection of five grapevine viroids. J Virol Methods 179(1):62–69
Hammond RW, Zhang S (2016) Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification. J Virol Methods 236:62–67. https://doi.org/10.1016/j.jviromet.2016.06.013
Ivanov AV, Shmyglya IV, Zherdev AV et al (2020) The challenge for rapid detection of high-structured circular RNA: assay of potato spindle tuber viroid based on recombinase polymerase amplification and lateral flow tests. Plants 9:1369. https://doi.org/10.3390/plants9101369
Jiao J, Kong K, Han J, Song S, Bai T, Song C et al (2021) Field detection of multiple RNA viruses/viroids in apple using a CRISPR/Cas12a-based visual assay. Plant Biotechnol J 19:394–405. https://doi.org/10.1111/pbi.13474
Kapoor R, Srivastava N, Kumar S et al (2017) Development of a recombinase polymerase amplification assay for the diagnosis of banana bunchy top virus in different banana cultivars. Arch Virol 162:2791–2796. https://doi.org/10.1007/s00705-017-3399-9
Kappagantu M, Villamor DE, Bullock JM, Eastwell KC (2017) A rapid isothermal assay for the detection of Hop stunt viroid in hop plants (Humulus lupulus), and its application in disease surveys. J Virol Methods 245:81–85. https://doi.org/10.1016/j.jviromet.2017.04.002
Kersting S, Rausch V, Bier FF, von Nickisch-Rosenegk M (2014) Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens. Microchim Acta 181:1715–1723. https://doi.org/10.1007/s00604-014-1198-5
Kim NK, Lee HJ, Ryu TH et al (2021) Detection of apple scar skin viroid by reverse transcription recombinase polymerase amplification assay. Res Plant Dis 27:79–83. https://doi.org/10.5423/RPD.2021.27.2.79
Kovalskaya N, Hammond RW (2022) Rapid diagnostic detection of tomato apical stunt viroid based on isothermal reverse transcription-recombinase polymerase amplification. J Virol Methods. https://doi.org/10.1016/j.jviromet.2021.114353
Kuan CP, Wu MT, Lu YL, Huang HC (2010) Rapid detection of squash leaf curl virus by loop-mediated isothermal amplification. J Virol Methods 169:61–65. https://doi.org/10.1016/j.jviromet.2010.06.017
Lee HJ, Kim HJ, Lee K, Jeong R-D (2020) Rapid detection of peach latent mosaic viroid by reverse transcription recombinase polymerase amplification. Mol Cell Probes 53:101627. https://doi.org/10.1016/j.mcp.2020.101627
MacKenzie DJ, McLean MA, Mukerji S, Green M (1997) Improved RNA extraction from woody plants for the detection of viral pathogens by reverse transcription-polymerase chain reaction. Plant Dis 81:222–226. https://doi.org/10.1094/PDIS.1997.81.2.222
Mekuria TA, Zhang S, Eastwell KC (2014) Rapid and sensitive detection of Little cherry virus 2 using isothermal reverse transcription-recombinase polymerase amplification. J Virol Methods 205:24–30. https://doi.org/10.1016/j.jviromet.2014.04.015
Piepenburg O, Williams CH, Stemple DL, Armes NA (2006) DNA detection using recombination proteins. PLoS Biol. https://doi.org/10.1371/journal.pbio.0040204
Randles JW (2003) Economic impact of viroid diseases. In: Hadidi A, Flores R, Randles JW, Semancik JS (eds) Viroids. CSIRO Publishing, Collingwood, VIC, pp 3–11
Sahana AB, Adkar-Purushothama CR, Chennappa G et al (2013) First report of grapevine yellow speckle viroid-1 and hop stunt viroid infecting grapevines (Vitis vinifera ) in India. Plant Dis 97:1517–1517. https://doi.org/10.1094/PDIS-05-13-0494-PDN
Senarath KD, Usgodaarachchi RB, Navaratne V et al (2014) Non specific amplification with the LAMP technique in the diagnosis of tuberculosis in Sri Lankan settings. J Tuberc Res 02:168–172. https://doi.org/10.4236/jtr.2014.24021
Sidharthan VK, Sevanthi AM, Jaiswal S, Baranwal VK (2020) Robust Virome profiling and whole genome reconstruction of viruses and Viroids enabled by use of available mRNA and sRNA-Seq datasets in grapevine (Vitis vinifera L.). Front Microbiol. https://doi.org/10.3389/fmicb.2020.01232
Singhal P, Kapoor R, Saritha RK, Baranwal VK (2019a) First report of grapevine yellow speckle viroid-2 infecting grapevine ( Vitis vinifera ) in India. Plant Dis 103:166–166. https://doi.org/10.1094/PDIS-07-18-1219-PDN
Singhal P, Kumar S, Rai R et al (2019b) Characterization of viroids infecting grapevine in India. Indian Phytopathol 72:333–341. https://doi.org/10.1007/s42360-019-00134-9
Singhal P, Nabi SU, Yadav MK, Dubey A (2021a) Mixed infection of plant viruses: diagnostics, interactions and impact on host. J Plant Dis Prot 128:353–368. https://doi.org/10.1007/s41348-020-00384-0
Singhal P, Yadav MK, Nabi SU (2021b) Techniques for Detection of Viroids. In: Singh RK (ed) Innovative Approaches in Diagnosis and Management of Crop Diseases, 1st editio. Apple Academic Press
Srivastava N, Kapoor R, Kumar R et al (2019) Rapid diagnosis of Cucumber mosaic virus in banana plants using a fluorescence-based real-time isothermal reverse transcription-recombinase polymerase amplification assay. J Virol Methods 270:52–58. https://doi.org/10.1016/j.jviromet.2019.04.024
Staub U, Pouvka H, Herrmann JV, Gross HJ (1995) Transmission of grapevine viroids is not likely to occur mechanically by normal pruning. Vitis 34(2):119–123
Szychowski JA, Goheen AC, Semancik JS (1988) Mechanical transmission and rootstock reservoirs as factors in the widespread distribution of viroids in grapevines. Am J Enol Vitis 39:213–216
Szychowski JA, Credi R, Reanwarakorn K, Semancik JS (1998) Population diversity in grapevine yellow speckle viroid-1 and the relationship to disease expression. Virology 248:432–444. https://doi.org/10.1006/viro.1998.9292
Taylor R, Woodham R (1972) Grapevine yellow speckle—a newly recognized graft-transmissible disease of Vitis. Aust J Agric Res 23:447. https://doi.org/10.1071/AR9720447
Wan Chow Wah YF, Symons RH (1999) Transmission of viroids via grape seeds. J Phytopathol 147:285–291. https://doi.org/10.1046/j.1439-0434.1999.147005285.x
Zanoli L, Spoto G (2012) Isothermal amplification methods for the detection of nucleic acids in microfluidic devices. Biosensors 3:18–43. https://doi.org/10.3390/bios3010018
Funding
No funding source available.
Author information
Authors and Affiliations
Contributions
All authors contributed to the study conception and design. Research experiments and analysis were performed by Pankhuri Singhal and was assisted by Damini Diksha and Nishant Srivastava in standardization. Virendra Kumar Baranwal has conceived the work, provided the guidance and did manuscript editing. All the authors have contributed in shaping the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
All authors declare no conflict of interest.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Singhal, P., Diksha, D., Srivastava, N. et al. Development of an isothermal reverse transcriptase-recombinase polymerase amplification assay for the rapid diagnosis of four viroids in different grape cultivars. Indian Phytopathology (2024). https://doi.org/10.1007/s42360-024-00746-w
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1007/s42360-024-00746-w