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当前位置: 首页 > 文章 > 甘薯褪绿矮化病毒西非株系RT-LAMP检测方法的建立 中国农业科学 2013,46 (18) 3939-3945
Position: Home > Articles > Detection of Sweet potato chlorotic stunt virus Strain WA by Reverse Transcription Loop-Mediated Isothermal Amplification Reaction Scientia Agricultura Sinica 2013,46 (18) 3939-3945

甘薯褪绿矮化病毒西非株系RT-LAMP检测方法的建立

作  者：
乔奇;张振臣;秦艳红;张德胜;田雨婷;王爽;王永江
单  位：
河南省农业科学院植物保护研究所/河南省农作物病虫害防治重点实验室/农业部华北南部作物有害生物综合治理重点实验室
关键词：
甘薯;甘薯褪绿矮化病毒西非株系;逆转录环介导等温扩增技术;检测
摘  要：
【目的】基于逆转录环介导等温扩增技术(Reverse transcription loop-mediated isothermal amplification,RT-LAMP)建立甘薯褪绿矮化病毒(Sweet potato chlorotic stunt virus,SPCSV)西非株系(West African,WA)的可视化检测方法。【方法】针对SPCSV西非株系(SPCSV-WA)的CP核苷酸序列设计4条引物,以感染SPCSV-WA甘薯叶片总RNA为模板,进行一步法RT-LAMP,在65℃条件下反应1 h。扩增产物利用琼脂糖电泳分析和SYBR green I荧光染料显色判断结果,同时对扩增产物的电泳条带进行基因克隆和测序鉴定。利用常规RT-PCR和建立的RT-LAMP方法对14份甘薯样品进行SPCSV-WA检测,比较2种方法的检测结果。【结果】建立的LAMP方法可特异地对SPCSV-WA进行检测,且最低可检测出101拷贝/μL的模板。RT-LAMP产物的克隆测序表明,感染SPCSV-WA甘薯样品的RT-LAMP产物为SPCSV-WA CP基因序列。14份田间甘薯样品检测表明,RT-LAMP与RT-PCR方法检测结果完全一致。【结论】建立的SPCSV-WA RT-LAMP可视化检测方法,是简便、可靠的SPCSV-WA检测方法。
译  名：
Detection of Sweet potato chlorotic stunt virus Strain WA by Reverse Transcription Loop-Mediated Isothermal Amplification Reaction
作  者：
QIAO Qi;ZHANG Zhen-chen;QIN Yan-hong;ZHANG De-sheng;TIAN Yu-ting;WANG Shuang;WANG Yong-jiang;Institute of Plant Protection, Henan Academy of Agricultural Sciences/Henan Key Laboratory of Crop Pest Control/IPM Key Laboratory in Southern Part of North China for Ministry of Agriculture;
关键词：
Ipomoea batatas;;Sweet potato chlorotic stunt virus strain WA;;RT-LAMP;;detection
摘  要：
【Objective】The objective of this study is to develop a one-step reverse transcription loop-mediated isothermal amplification(RT-LAMP) assay for rapid and sensitive detection of Sweet potato chlorotic stunt virus strain WA(SPCSV-WA).【Method】Four RT-LAMP primers, designed on the basis of the coat protein(CP) gene sequences of SPCSV-WA, and template RNA from infected leaves were used for one-step RT-LAMP which were carried out under isothermal conditions at 65℃ for 1 h.RT-LAMP products were analyzed by electrophoresis in agarose gels followed by staining with SYBR green I. A fragment from the RT-LAMP product was then cloned and sequenced to confirm the spectficity of the assay. The RT-LAMP, and meanwhile compared with the conventional RT-PCR assay, was performed to detect SPCSV-WA from 14 sweet potato samples. 【Result】The RT-LAMP showed a high specificity and the detection limit was 101copies/μL RNA template. A fragment cloned from the RT-LAMP product indicated that the nucleotide sequence cloned was SPCSV-WA CP. Fourteen sweet potato samples testing results of RT-LAMP were consistent with that of RT-PCR. 【Conclusion】 The RT-LAMP described in this study represents a very sensitive, specific and rapid assay for the detection of SPCSV-WA.

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