A simplified crude sap-based detection of cucumber mosaic virus in chilli germplasm through reverse transcription recombinase polymerase amplification assay (RT-RPA).

IF 2.9 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

3 Biotech Pub Date : 2025-10-01 Epub Date: 2025-09-16 DOI:10.1007/s13205-025-04459-3

C H Anusha, A Rajashree, B Parameswari, Vijay Mahanthesh, V Venkataravanappa, P Pranusha, Vinod Kumar Sharma, S K Mangrauthia, V Kavi Sidharthan, B Vidya Sagar, B Bhaskar, L Saravanan, V Celia Chalam

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引用次数: 0

Abstract

Chilli cultivation is under constant threat by many pests and diseases, and about 35 viruses are known to infect chilli globally. Among the viruses, the cucumber mosaic virus (CMV) is the most significant constraint to chilli production worldwide. Detection of CMV in infected plants, reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) methods are employed frequently. These conventional methods have been time consuming, laborious and expensive for detection of samples in low resource laboratories. In order to provide alternatives for the conventional methods currently various isothermal amplification methods have been employed. The present study exhibits the optimisation of the reverse transcription recombinase polymerase amplification (RT-RPA) assay by eliminating the steps of ribonucleic acid extraction, cDNA conversion, and the use of a thermal cycler. The optimized RT-RPA assay successfully detected CMV at concentrations as low as 10^-10 (100 fg) and 10^-9 dilutions of both RNA and crude sap templates, demonstrating high sensitivity comparable to the routine RT-PCR assay. Specificity tests confirmed that the RT-RPA assay did not exhibit cross-reactivity with other common chilli-infecting viruses, such as chilli leaf curl virus (ChiLCV), chilli veinal mottle virus (ChiVMV), capsicum chlorosis virus (CaCV), and groundnut bud necrosis virus (GBNV). Its ability to function efficiently with crude sap as a template further enhances its field applicability, making it a promising tool for early virus detection in low-resource laboratories and onsite diagnostics. The integration of such isothermal amplification techniques could revolutionize plant virus detection and management strategies, ultimately aiding in the mitigation of crop losses and ensuring food security.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-025-04459-3.

查看原文本刊更多论文

逆转录重组酶聚合酶扩增法（RT-RPA）对辣椒种质中黄瓜花叶病毒的简易粗汁液检测。

辣椒种植一直受到许多病虫害的威胁，全球已知约有35种病毒感染辣椒。在这些病毒中，黄瓜花叶病毒（CMV）是全球辣椒生产的最大制约因素。感染植物CMV检测常用逆转录聚合酶链反应（RT-PCR）和酶联免疫吸附试验（ELISA）等方法。在资源匮乏的实验室中，这些传统的检测方法耗时、费力且昂贵。为了提供替代传统方法的方法，目前已经采用了各种等温扩增方法。本研究通过消除核糖核酸提取、cDNA转换和使用热循环器的步骤，展示了逆转录重组酶聚合酶扩增（RT-RPA）测定的优化。优化后的RT-RPA检测方法在RNA和粗液模板浓度低至10-10 （100 fg）和10-9的情况下成功检测到巨细胞病毒，与常规RT-PCR检测方法相比具有较高的灵敏度。特异性试验证实，RT-RPA法与其他常见的辣椒感染病毒，如辣椒卷曲叶病毒（ChiLCV）、辣椒脉斑纹病毒（ChiVMV）、辣椒萎黄病毒（CaCV）和花生芽坏死病毒（GBNV）没有交叉反应性。它能够以粗液为模板有效地发挥作用，进一步增强了其现场适用性，使其成为低资源实验室早期病毒检测和现场诊断的有前途的工具。这种等温扩增技术的整合可以彻底改变植物病毒的检测和管理战略，最终有助于减轻作物损失并确保粮食安全。补充信息：在线版本包含补充资料，下载地址：10.1007/s13205-025-04459-3。

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来源期刊

3 Biotech Agricultural and Biological Sciences-Agricultural and Biological Sciences (miscellaneous)

CiteScore

6.00

自引率

0.00%

发文量

314

期刊介绍： 3 Biotech publishes the results of the latest research related to the study and application of biotechnology to: - Medicine and Biomedical Sciences - Agriculture - The Environment The focus on these three technology sectors recognizes that complete Biotechnology applications often require a combination of techniques. 3 Biotech not only presents the latest developments in biotechnology but also addresses the problems and benefits of integrating a variety of techniques for a particular application. 3 Biotech will appeal to scientists and engineers in both academia and industry focused on the safe and efficient application of Biotechnology to Medicine, Agriculture and the Environment.