中文摘要：柑橘衰退病毒(Citrus tristeza virus,CTV)引起的衰退病是一种严重危害世界柑橘产业的病毒病害，到目前为止还没有有效控制该病毒的措施，但快速且有效的检测病原体显得尤为重要，因此，本文对采用高敏感性、强大且低成本的逆转录环介导等温扩增技术对CTV感染的柑橘植株样本进行检测。检测结果显示：CTV-RT-LAMP技术的标准化研究中的灵敏度比常规RT-PCR一步法检测高出100倍，在单个反应混合物中，最大检测限可达0.0001 ng RNA。CTV-RT-LAMP法是一种简单、灵敏、快速、低成本检测技术，可用于具有有限的设施和资源的病理实验室，甚至柑橘苗圃的远程CTV诊断。根据我们的知识和现有的文献，印度首次验证了RT-LAMP检测CTV的有效性。
外文摘要：Tristeza is a devastating disease of citrus and reported to be present in almost all countries where it is cultivated as a commercial crop. The etiological agent of this disease is Citrus tristeza virus (CTV), a member of the genus Closterovirus with in the family Closteroviridae. The pathogen is restricted to the phloem tissue of the infected citrus plant and has a monopartite ss (+) RNA genome of similar to 20 kb size. Till date, there is no effective control measure available for this virus. Management of tristeza depends on destruction of CTV infected field plants, production of virus-free planting material for new orchard establishment and controlling viruliferous aphid vectors responsible for field spread of the pathogen. Availability of rapid diagnostic assay is essential for rapid and efficient detection of the pathogen. In the present investigation, RT-LAMP (reverse transcription-loop mediated isothermal amplification), a highly sensitive, robust and low cost assay has been developed for rapid detection of CTV in infected citrus plant samples. Based on conserved nucleotide sequences available in GenBank and specific to p25 gene (major coat protein gene) of predominant CTV isolates of India, four primer sets (CTV-F3, CTV-B3, CTV-FIP and CTV-BIP) ware designed and custom synthesized. The amplified LAMP products obtained after maintaining isothermal condition of 65 degrees C for 60 min duration could be visible easily with necked eyes in presence of SYBR Green I (100X). Subsequently, LAMP products were verified by electrophoresis run in 1.5% agarose gel. The RT-LAMP results obtained with known CTV isolates maintained in screen house of CCRI, Nagpur were validated using field samples and thereafter it was further confirmed by conventional RT-PCR (reveres transcription-polymerase chain reaction) assay. The sensitivity of CTV-RT-LAMP protocol standardized in the present study was 100 times more than conventional one step RT-PCR assay. It also has maximum detection limit up to 0.0001 ng RNA in individual reaction mixture. CTV-RT-LAMP assay is a simple, sensitive, rapid and less costly detection technique. This assay could be used for CTV diagnosis in pathology laboratories having limited facility and resources and even by citrus nurseries situated in remote locations. As per our knowledge and available literature, the present study reports first time about the usefulness of RT-LAMP assay for detection of CTV from India.
 
外文关键词：Citrus tristeza virus, RT-LAMP, RT-PCR, Detection, CTV
作者：Warghane, Ashish; Misra, Pragati; Bhose, Sumit; Biswas, Kajal Kumar;Sharma, Ashwani Kumar; Reddy, M. Krishna; Ghosh, Dilip Kumar
作者单位：Ghosh, DK (reprint author), ICAR Res Complex, Cent Citrus Res Inst, Nagpur 440033, Maharashtra, India.
期刊名称：JOURNAL OF VIROLOGICAL METHODS
期刊影响因子：1.693
出版年份：2017
出版刊次：9
点击下载：敏感性柑橘衰退病毒逆转录环介导等温扩增简单快速检测(RT-LAMP)方法的建立