Abstract：
Whiteflies (Bemisia tabaci) are a significant pest of cucurbits and vectors many viruses leading to substantial economic losses. Modern diagnostic tools offer the potential for early detection of viruses in the whiteflies before crop production. One such tool is the multiplex reverse transcriptase quantitative PCR (RT-qPCR) probe-based technique, which can detect multiple targets in a single reaction and simultaneously quantify the levels of each target, with a detection limit of 100 copies per target. In this study, a multiplex RT-qPCR-based detection system capable of identifying one DNA virus and three RNA viruses in whiteflies: cucurbit leaf crumple virus (CuLCrV), cucurbit chlorotic yellows virus (CCYV), cucurbit yellow stunting disorder virus (CYSDV), and squash vein yellowing virus (SqVYV) was developed. To ensure the reliability of the assay, an internal gene control as the fifth target to monitor false-negative results was incorporated. This newly developed molecular diagnostic tool possesses several advantages. It can detect up to five desired targets from a single whitefly RNA sample, even at concentrations as low as 1 ng/µl. To evaluate its sensitivity, we conducted experiments using serially diluted cloned plasmids and in vitro transcribed RNA transcripts of the target viruses. We also assessed the specificity of the assay by including aphid-transmitted viruses and other viruses known to infect cucurbits. The diagnostic method successfully detected all five targets simultaneously and allowed for the quantification of up to 100 copies using a mixture of healthy? RNA and in vitro transcribed RNA. Our aim with this study was to develop a highly specific and sensitive one-step multiplex RT-qPCR system for the simultaneous detection of viruses transmitted by whiteflies in cucurbits. This system offers significant advantages for early detection, enabling prompt control measures to mitigate the further spread of viral infections and reduce yield losses. Additionally, we demonstrated the ability to simultaneously detect mixed viruses (CCYV, CYSDV, CuLCrV, and SqVYV) in individual whiteflies and quantify the number of viral copies carried by each whitefly. The multiplex RT-qPCR assay outperforms currently available techniques for detecting many samples at a given time and can be effectively utilized for early monitoring of plant viruses in individual whiteflies and symptomless plants.
摘要：
粉虱（烟粉虱）是葫芦和载体的重要害虫，许多病毒会导致大量的经济损失。现代诊断工具提供了在作物生产前早期检测粉虱中病毒的潜力。一种这样的工具是基于多重逆转录酶定量PCR（RT-qPCR）探针的技术，它可以在单个反应中检测多个目标，并同时量化每个目标的水平，每个目标的检测限为100个拷贝。在这项研究中,一种基于多重RT-qPCR的检测系统，能够识别粉虱中的一种DNA病毒和三种RNA病毒：葫芦叶皱病毒（CuLCrV），葫芦褪绿黄病病毒（CCYV），葫芦黄色发育障碍病毒（CYSDV），并开发了南瓜脉黄化病毒（SqVYV）。为了确保测定的可靠性，纳入内部基因对照作为监测假阴性结果的第五个目标.这种新开发的分子诊断工具具有几个优点。它可以从单个粉虱RNA样品中检测多达五个目标，即使浓度低至1纳克/微升。为了评估其敏感性，我们使用连续稀释的克隆质粒和靶病毒的体外转录RNA转录本进行了实验。我们还通过包括蚜虫传播的病毒和已知感染葫芦的其他病毒来评估测定的特异性。该诊断方法成功地同时检测了所有五个靶标，并使用健康的？RNA和体外转录的RNA的混合物可以定量多达100个拷贝。我们这项研究的目的是开发一种高度特异性和灵敏的一步多重RT-qPCR系统，用于同时检测由葫芦中的粉虱传播的病毒。该系统为早期检测提供了显着的优势，及时采取控制措施，以减轻病毒感染的进一步传播并减少产量损失。此外,我们证明了同时检测混合病毒的能力(CCYV，CYSDV,CuLCrV,和SqVYV）在单个粉虱中，并量化每个粉虱携带的病毒拷贝数。多重RT-qPCR测定优于当前可用的在给定时间检测许多样品的技术,并且可以有效地用于在单个粉虱和无症状植物中早期监测植物病毒。
