Effect of RNA purification systems on the detection limit of pathogenic viruses in woody fruit crops

病毒是木本果树的主要病原。通过对不含某些病毒的繁殖材料进行认证来控制感染木本水果作物的病毒。这些认证项目中的官方病毒检测方法基于生物指示剂索引和基于血清学的测定。然而，这些测定缺乏灵敏度、特异性并且耗时耗力。开发和实施高度敏感和可靠的诊断方法对于控制和管理病毒和病毒性疾病至关重要。基于核酸的诊断方法因其速度、灵敏度、特异性和通用性而优于传统的生物索引和基于血清学的方法。高纯度病毒RNA的分离是分子诊断的先决条件。然而，这种技术尚未开发用于木本水果作物。本研究的主要目的是基于商业上可用的RNA纯化方法并通过进一步改进这些技术来开发用于从葡萄和桃子中分离RNA的高效方法。在与位于安大略水果作物生产中心的生物技术公司Norgen BioTeck的合作中，我们打算比较来自五个商业来源的RNA纯化系统，并进一步改进RNA纯化。纯化RNA的质量和性能将通过逆转录酶聚合酶链反应（RT-PCR）、真实的时间RT-PCR和深度测序进行检测。建议的研究结果将有利于安大略和加拿大其他省份的农业产业的可持续发展和改善。此外，这将有助于加强Norgen BioTek，一个年轻的，总部设在加拿大的生物技术公司，通过公正和独立的验证其产品和可能的新产品的出现，用于在加拿大和国际上的研究和诊断。这是申请人研究计划和Norgen之间的新伙伴关系，我们希望这将导致长期和富有成效的合作。

Viruses are major pathogens of woody fruit crops. Control of viruses infecting woody fruit crops is achieved through certification of propagating materials free from certain viruses. The official methods of virus testing in these certification programs have been based on biological indicator indexing and serology-based assays. However, these assays lack sensitivity, specificity and are time and labor-consuming. Development and implementation of highly sensitive and reliable diagnostics are crucial for the control and management of viruses and viral diseases. Nucleic acid-based diagnostic methods are advantageous over traditional biological indexing and serology-based methods for their speed, sensitivity, and specificity and versatility. Isolation of highly pure viral RNAs is pre-requisite for molecular diagnosis. However, such a technology has not been developed for woody fruit crops. The main objective of this research is to develop highly effective methodologies for isolating RNA from grapes and peaches based on commercially available RNA purification methods and through further improvement of these technologies. In a partnership with Norgen BioTeck, a biotech company located in the centre of fruit crop production in Ontario, we intend to compare RNA purification systems from five commercial sources and make further refinement for RNA purification. Quality and performance of the purified RNAs will be tested through reverse transcriptase polymerase chain reaction (RT-PCR), real time RT-PCR, and deep sequencing. Outcomes of the proposed research will be beneficial to the sustainability and betterment of the agriculture industries in Ontario and other provinces of Canada. Moreover, it would help strengthen Norgen BioTek, a young, Canada-based biotech company, through unbiased and independent validation of their products and emergence of possible new products for use in research and diagnostics in Canada and the internationally. This is a new partnership between the applicant research program and Norgen, which we hope will lead to long-term and productive collaborations.