Development of reverse transcriptase-recombinase polymerase amplification (RT-RPA) assay for rapid detection of large cardamom chirke virus

被引：1
作者：
Malavika P. [1 ]
Bhat A.I. [1 ]
Greeshma M. [1 ]
机构：
[1] ICAR-Indian Institute of Spices Research, Kerala, Kozhikode
来源：
VirusDisease | 2024年 / 35卷 / 2期
关键词：
Diagnosis; RT-PCR; RT-RPA; Sensitivity;
D O I：
10.1007/s13337-024-00861-2
中图分类号：
学科分类号：
摘要：
Large cardamom chirke virus (LCCV) causing chirke disease of large cardamom is a major production constraint of this crop. Rapid and accurate detection of LCCV is important for managing the disease. In the present study an isothermal assay namely, reverse transcriptase-recombinase polymerase amplification (RT-RPA) was developed for the detection of LCCV. Total RNA isolated by two different methods and crude extracts isolated using five different methods as templates were assessed for their ability to detect LCCV. Of these, only the total RNA isolated by both methods gave consistent and repeatable results while all the crude extracts used as templates gave non-specific amplification. RT-RPA was up to 1000 times more sensitive than conventional RT-PCR for the detection of LCCV. The detection limit of RPA was 10 fg when recombinant plasmid was used as the template. The RT-RPA assay was validated using field samples and found suitable for large-scale screening of large cardamom plants against LCCV for the selection of virus-free plants. © The Author(s), under exclusive licence to Indian Virological Society 2024.
引用
收藏
页码：302 / 309
页数：7
相关论文
共 26 条
  • [1] Babu B., Washburn B.K., Miller S.H., Poduch K., Sarigul T., Knox G.W., Et al., A rapid assay for detection of rose rosette virus using reverse transcription-recombinase polymerase amplification using multiple gene targets, J Virol Methods, 240, pp. 78-84, (2017)
  • [2] Berendzen K., Searle I., Ravenscroft D., Koncz C., Batschauer A., Coupland G., Somssich I.E., Ulker B., A rapid and versatile combined DNA/RNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsis thaliana ecotypes Col-0 and Landsberg erecta, Plant Methods, 1, pp. 1-15, (2005)
  • [3] Bhat A.I., Pamitha N.S., Naveen K.P., Biju C.N., Identification and characterization of cardamom vein clearing virus, a novel aphid-transmitted nucleorhabdovirus, Eur J Plant Pathol, 156, pp. 1053-1062, (2020)
  • [4] Bhat A.I., Rao G.P., Characterization of plant viruses: methods and protocols, (2020)
  • [5] Bhat A.I., Aman R., Mahfouz M., Onsite detection of plant viruses using isothermal amplification assays, Plant Biotechnol J, 20, pp. 1859-1873, (2022)
  • [6] Jailani A.A.K., Paret M.L., Development of a multiplex RT-RPA assay for simultaneous detection of three viruses in cucurbits, Mol Plant Pathol, 24, pp. 1443-1450, (2023)
  • [7] Jiao Y., Jiang J., An M., Xia Z., Wu Y., Recombinase polymerase amplification assay for rapid detection of maize chlorotic mottle virus in maize, Arch Virol, 164, pp. 2581-2584, (2019)
  • [8] Jiao Y., Jiang J., Wu Y., Xia Z., Rapid detection of Cucumber green mottle mosaic virus in watermelon through a recombinase polymerase amplification assay, J Virol Methods, 270, pp. 146-149, (2019)
  • [9] Jiao Y., Xu C., Li J., Gu Y., Xia C., Xie Q., Et al., Characterization and a RT-RPA assay for rapid detection of chilli veinal mottle virus (ChiVMV) in tobacco, Virol J, 17, pp. 1-9, (2020)
  • [10] Kim N.K., Kim S.M., Jeong R.D., Reverse transcription recombinase polymerase amplification assay for rapid and sensitive detection of barley yellow dwarf virus in oat, Plant Pathol J, 36, (2020)
123