Multiplex reverse transcription loop-mediated isothermal amplification for the simultaneous detection of CVB and CSVd in chrysanthemum
Multiplex reverse transcription loop-mediated isothermal amplification for the simultaneous detection of CVB and CSVd in chrysanthemum
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多重逆转录环介导等温扩增同时检测菊花中的 CVB 和 CSVd
DOI：
10.1016/j.jviromet.2014.09.008
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发表时间：
2014-12-15
影响因子：
3.1
通讯作者：
Hong, Bo
中科院分区：
文献类型：
--
作者：
Liu, Xing-liang;Zhao, Xi-ting;Hong, Bo
建立了同时检测菊花病毒B（Chrysanthemum Virus，CVB）和菊花矮化类病毒（Chrysanthemum stunt viroid，CSVd）的多重逆转录环介导等温扩增（mRT-LAMP）方法。根据CVB外壳蛋白基因和CSVd全序列设计2对mRT-LAMP引物，并在2对引物上插入限制性内切酶酶切位点。通过组合这两组共8条引物设计mRT-LAMP测定。由于随后的限制性内切酶分析，mRT-LAMP方法区分了CVB和CSVd。就CVB和CSVd的检测限而言，mRT-LAMP方法的灵敏度比经典PCR高10（3）倍。以其他菊花病原菌的RNA为模板进行mRT-LAMP检测，均未得到阳性结果。对北京、河南等地的菊花样品进行了检测，结果表明该方法具有较高的灵敏度和可靠性。该方法也为菊花病毒和类病毒，特别是CVB和CSVd的筛选提供了一种高效、方便、快速的检测手段，并可在一次反应中进行诊断。这些结果表明，新的mRT-LAMP方法可常规用于病毒和类病毒调查。(C)2014爱思唯尔有限公司版权所有。
A multiplex reverse transcription loop-mediated isothermal amplification (mRT-LAMP) assay was developed for the simultaneous detection of Chrysanthemum Virus B (CVB) and Chrysanthemum stunt viroid (CSVd), which are the major viral pathogens of chrysanthemum worldwide. Two sets of mRT-LAMP primers were designed for the coat protein gene of CVB and the complete nucleotide sequence of CSVd, and a restriction enzyme cleavage site was inserted into two pairs of species-specific primers. The mRT-LAMP assay was designed by combining these two sets fora total of eight primers. The mRT-LAMP method distinguished between CVB and CSVd due to the subsequent restriction enzyme analysis. The sensitivity of the mRT-LAMP method was 10(3) times higher than classical PCR regarding the detection limits for CVB and CSVd. No positive results were observed when RNA from other chrysanthemum pathogens were used as mRT-LAMP templates. The method was verified by testing chrysanthemum samples collected from Beijing and Henan Province and showed high reliability and sensitivity. The developed mRT-LAMP assay also offers an efficient, convenient, and rapid tool for screening chrysanthemum virus and viroid, especially CVB and CSVd, and can be diagnosed in a single reaction. These results suggest that the new mRT-LAMP method may be used routinely for virus and viroid surveys. (C) 2014 Elsevier B.V. All rights reserved.