甘薯褪绿矮化病毒西非株系RT-LAMP检测方法的建立

Detection of Sweet potato chlorotic stunt virus Strain WA by Reverse Transcription Loop-Mediated Isothermal Amplification Reaction

【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。

[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 LA fragment from the RT-LAMP product was then cloned and sequenced to confirm the specificity 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 101 copies/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.