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Simplified extraction protocol for plant tissues and reverse transcription RPA assay for quick and reliable diagnosis and its application in resistance screening of chilli veinal mottle virus
Crop Protection ( IF 2.8 ) Pub Date : 2023-05-13 , DOI: 10.1016/j.cropro.2023.106280
Huirem Chandrajini Devi, K. Sarda Devi, Rakesh Sanabam, Neeta Pathaw, Oinam Priyoda Devi, Ng. Taibangnganbi Chanu, Albert Maibam, W. Tampakleima Chanu, Jyotsana Sanasam, Subhra Saikat Roy, Ch. Premabati Devi, A. Ratankumar Singh, Ph. Sobita Devi, Nitika Gupta, Susheel Kumar Sharma

The widespread occurrence and predominant association of chilli veinal mottle virus (ChiVMV) with the valuable chilli landraces of North East Region (NER) of India and associated threat to chilli production necessitated the development of simplified and robust detection procedure. Present study reports a simplified reverse transcription-recombinase polymerase amplification (RT-RPA) method for detection of ChiVMV in crude leaf extract prepared through simplified extraction protocol using NaOH:EDTA (1:1) based tissue lysis, without the need of conventional RNA extraction and sample processing. Developed RT-RPA assay could be performed at an isothermal temperature (38 °C) in 30 min and exhibited no cross-reactivity with other prevalent chilli viruses (potato virus Y, cucumber mosaic virus, large cardamom chirke virus, capsicum chlorosis orthotospovirus, pepper mild mottle virus and chilli leaf curl virus), indicating high specificity. It could detect ChiVMV up to 10−9 dilution (100 ag/μl) of crude leaf extract, 10 ag/μl of RNA of ChiVMV infected leaf tissues and plasmid DNA containing viral gene insert, thus exhibiting sufficient sensitivity which is at par or better than the standard RT-PCR. Developed RT-RPA assay was employed to reveal the widespread occurrence of ChiVMV in NER India with 50.81% (1545 samples tested) of symptomatic and 7.85% (1593 samples tested) of asymptomatic samples tested positive. RT-RPA exhibited higher sensitivity in detection of ChiVMV infection in field samples as compared to the standard RT-PCR which gave false negative in 2.07% of symptomatic and 2.01% of asymptomatic samples. The developed assay was further applied in screening of 49 chilli germplasm for ChiVMV infection. The pooled results of field screening based on the disease score indicated that none of the chilli germplasm had immune or complete resistance, while 29 chilli germplasm (bird's eye chilli: C. frutescens; Morokatekpa: C. annuum, Leihao morok, Morok masingkha; Chingpimorok: Capsicum sp.) exhibited tolerant reaction with a disease score of 1.27–2.00. All the tested bird's eye chilli germplasm showed tolerance to the ChiVMV. The artificial screening to the severe isolate and qRT-PCR based viral titre determination confirmed the results of field screening wherein the germplasm RCMC6, RCMC7, RCMC17, RCMC27 and RCMC42 exhibited either asymptomatic or mild mosaic symptoms and decrease in viral titre at 20 DAI thus categorized as tolerant. The simplified RT-RPA assay developed and validated in the present study can be applied in routine detection of ChiVMV in field samples, disease-free seedling production and resistance screening under resource poor laboratory setup with limited technical expertise. Chilli germplasm with tolerance to ChiVMV identified in the present study can be used as donors in resistance breeding.



中文翻译:

用于快速可靠诊断的植物组织简化提取方案和逆转录 RPA 测定及其在辣椒脉斑驳病毒抗性筛选中的应用

辣椒脉斑驳病毒 (ChiVMV) 与印度东北地区 (NER) 有价值的辣椒地方品种的广泛存在和主要关联以及对辣椒生产的相关威胁使得开发简化和稳健的检测程序成为必要。本研究报告了一种简化的逆转录重组酶聚合酶扩增 (RT-RPA) 方法,用于检测通过使用基于 NaOH:EDTA (1:1) 的组织裂解的简化提取方案制备的粗叶提取物中的 ChiVMV,无需常规 RNA 提取和样品处理。开发的 RT-RPA 测定可以在等温温度 (38 °C) 下进行 30 分钟,并且与其他流行的辣椒病毒(马铃薯病毒 Y、黄瓜花叶病毒、大豆蔻奇尔克病毒、辣椒失绿原孢病毒、辣椒轻斑驳病毒和辣椒卷曲病毒),特异性高。它可以检测多达 10 个 ChiVMV−9粗叶提取物的稀释度 (100 ag/μl)、10 ag/μl 的 ChiVMV 感染叶组织 RNA 和含有病毒基因插入片段的质粒 DNA,因此表现出足够的灵敏度,与标准 RT-PCR 相当或更好。开发的 RT-RPA 测定法用于揭示 ChiVMV 在 NER India 的广泛发生,有 50.81%(测试的 1545 个样本)的有症状样本和 7.85%(测试的 1593 个样本)的无症状样本测试呈阳性。与标准 RT-PCR 相比,RT-RPA 在检测现场样本中的 ChiVMV 感染方面表现出更高的灵敏度,标准 RT-PCR 在 2.07% 的有症状样本和 2.01% 的无症状样本中给出假阴性。所开发的检测方法进一步应用于筛选 49 种辣椒种质的 ChiVMV 感染。C. frutescens ; Morokatekpa:C. annuum,Leihao morok,Morok masingkha;Chingpimorok:辣椒sp.) 表现出耐受性反应,疾病评分为 1.27–2.00。所有测试的鸟眼辣椒种质都显示出对 ChiVMV 的耐受性。对严重分离株的人工筛选和基于 qRT-PCR 的病毒滴度测定证实了田间筛选的结果,其中种质 RCMC6、RCMC7、RCMC17、RCMC27 和 RCMC42 在 20 DAI 时表现出无症状或轻度镶嵌症状和病毒滴度降低,因此分类作为宽容。在本研究中开发和验证的简化 RT-RPA 测定法可应用于田间样品中 ChiVMV 的常规检测、无病苗生产和资源贫乏的实验室设置下的抗性筛选,技术专长有限。本研究鉴定出的对 ChiVMV 具有耐受性的辣椒种质可用作抗性育种的供体。

更新日期:2023-05-13
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