Development and application of a reverse transcription loop-mediated isothermal amplification combined with lateral flow dipstick for rapid and visual detection of Citrus leaf blotch virus in kiwifruit
Development and application of a reverse transcription loop-mediated isothermal amplification combined with lateral flow dipstick for rapid and visual detection of Citrus leaf blotch virus in kiwifruit
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逆转录环介导等温扩增结合侧流试纸快速、可视化检测猕猴桃中柑橘叶斑病病毒的开发和应用
DOI：
10.1016/j.cropro.2021.105555
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发表时间：
2021-01-30
期刊：
影响因子：
2.8
通讯作者：
Xi, Dehui
中科院分区：
文献类型：
--
作者：
Peng, Qiding;Ning, Jiachen;Xi, Dehui
柑橘叶斑病病毒(CLBV)在包括猕猴桃在内的一些园艺作物上普遍存在。四川省是中国西南部猕猴桃主产区之一。为了调查侵染猕猴桃果实上的CLBV的发生情况，建立了一种高效的RT-LAMP和LFD相结合的可视化检测方法。本研究根据CLBV外壳蛋白基因的保守区设计了一套LAMP特异性引物，提取和合成后60min即可完成RT-LAMP-LFD检测。优化的RT-LAMP-LFD方法对CLBV具有较高的特异性和敏感性，其敏感性是常规RT-PCR方法的1000倍。并用该方法对四川省90个猕猴桃样品进行了筛选。结果表明，四川猕猴桃CLBV的发病率为20%。系统发育分析表明，CLBV四川分离物在I类群中分为不同的分支，与柑橘、月季、甜樱桃等寄主植物有一定的遗传距离。总之，本研究为猕猴桃CLBV感染状况的监测提供了一种省时、高效的诊断工具。
Citrus leaf blotch virus (CLBV) commonly occurs in some horticultural crops including kiwifruit. Sichuan Province is one of the major kiwifruit producing areas in southwestern China. In order to investigate the occurrence of CLBV infecting kiwifruit in Sichuan Province, an efficient reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with a lateral flow dipstick (LFD) was established as an effective diagnostic method for visually detecting the occurrence of CLBV. In the present study, a set of LAMP specific primers were designed based on the conserved region of coat protein gene of CLBV and the RT-LAMP-LFD assay could be accomplished in 60 min after extraction and cDNA synthesis. The optimized RT-LAMP-LFD method showed high specificity and sensitivity to CLBV, and displayed 1000 times higher sensitivity than that of conventional RTPCR. Furthermore, 90 kiwifruit samples collected from Sichuan Province were screened by this developed method. The results showed that the incidence of CLBV in kiwifruit was 20% in Sichuan Province. Phylogenetic analysis revealed that all the CLBV Sichuan isolates were divided into different branches in group I and has some genetic distance with other host plants like citrus, rose and sweet cherry. Over all, this study provides a time saving and highly efficient diagnostic tool for monitoring CLBV infection status in kiwifruit.