Tristeza is a highly destructive disease of citrus caused by the phloem-limited, flexuous filamentous Citrus tristeza virus (CTV) in the genus Closterovirus and the family Closteroviridae. It has been a major constraint for higher productivity and has destroyed millions of citrus trees globally. CTV is graft transmissible and spread through use of virus infected nursery plants. Therefore, virus detection by using specific and reliable diagnostic tools is very important to mitigate disease outbreaks. Currently, the standard molecular techniques for CTV detection include RT-PCR and RT-qPCR. These diagnostic methods are highly sensitive but time consuming, labor intensive and require sophisticated expensive instruments, thus not suitable for point-of-care use. In the present study, we report the development of a rapid, sensitive, robust, reliable, and highly specific reverse transcription-RPA technique coupled with a lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA). RT-RPA technique was standardized to amplify the coat protein gene of CTV (CTV-p25) and detect double labeled amplicons on a sandwich immunoassay by designing specific labeled primer pair and probe combinations. The optimally performing primer set (CTRPA-F1/CTRPA-R9-Btn) and the corresponding TwistAmp nfo probe (CTRPA-Probe) was optimized for temperature and reaction time using purified cDNA and viral RNA as template. The sensitivity of the developed assay was compared with other detection techniques using in vitro-transcribed RNA. The efficacy and specificity of the assay was evaluated using CTV positive controls, healthy samples, field grown citrus plants of unknown status, and other virus and bacterial pathogens that infect citrus plants. The RT-RPA-LFICA was able to detect ≤ 141 fg of RNA when cDNA used as a template. The assay detected ≤ 0.23 ng/µl of CTV RNA when directly used as template without cross-reactivity with other citrus pathogens. Best results were achieved at the isothermal temperature of 40 °C within 15–20 min. The study demonstrated that RT-RPA-LFICA has potential to become an improved detection technique for end users in bud-wood certification and quarantine programs and a promising platform for rapid point-of-care diagnostics for citrus farmers and small nurseries in low resource settings.

衰退是由韧皮部限制，曲折丝状柑橘的高度破坏性疾病柑橘衰退病毒属植物（CTV）线形病毒和家庭修道院病毒科。它一直是提高生产力的主要限制因素，并在全球范围内摧毁了数百万棵柑橘树。CTV可移植性传播，并通过感染病毒的苗圃植物传播。因此，使用特定且可靠的诊断工具进行病毒检测对于减轻疾病暴发非常重要。当前，用于CTV检测的标准分子技术包括RT-PCR和RT-qPCR。这些诊断方法高度灵敏，但耗时，费力且需要复杂的昂贵仪器，因此不适合用于现场护理。在本研究中，我们报告了快速，灵敏，稳健，可靠且高度特异性的逆转录-RPA技术与侧向流免疫色谱分析（CTV-RT-RPA-LFICA）的结合。通过设计特异性标记的引物对和探针组合，将RT-RPA技术标准化以扩增CTV的外壳蛋白基因（CTV-p25），并在三明治免疫测定中检测双标记的扩增子。使用纯化的cDNA和病毒RNA作为模板，针对温度和反应时间优化了性能最佳的引物组（CTRPA-F1 / CTRPA-R9-Btn）和相应的TwistAmp nfo探针（CTRPA-Probe）。使用体外转录的RNA将开发的检测方法的灵敏度与其他检测技术进行了比较。使用CTV阳性对照，健康样品，未知状态的田间种植的柑橘类植物以及感染柑橘类植物的其他病毒和细菌病原体，评估了测定的功效和特异性。当以cDNA为模板时，RT-RPA-LFICA能够检测到≤141 fg的RNA。该分析检测到≤0。直接用作模板时，CTV RNA为23 ng / µl，与其他柑橘病原体无交叉反应。在15–20分钟内在40°C的等温温度下可获得最佳结果。这项研究表明，RT-RPA-LFICA有望成为芽木认证和检疫计划中最终用户的一种改进的检测技术，并为资源贫乏地区的柑橘农户和小型苗圃提供快速的即时诊断服务平台。