草莓白化伴随病毒RT-LAMP检测方法的建立

Establishment of RT-LAMP detection method for strawberry pallidosis-associated virus

草莓白化伴随病毒(strawberry pallidosis-associated virus,SPaV)能引起草莓白化病,与其他病毒复合侵染后会加重症状。本研究建立了SPaV的反转录环介导等温扩增方法(reverse transcription-loop-mediated isothermal amplification,RT-LAMP),对反应体系的各条件进行评估优化后,检测了RT-LAMP的特异性和灵敏度,且对田间样品进行了测试。结果表明,优化后的反应体系包含2.5μL 10×Isothermal Amplification Buffer、6 mmol/L Mg^(2+)、1 mmol/L dNTPs、1μmol/L SPaV-FIP和SPaV-BIP,0.1μmol/L SPaV-F3和SPaV-B3,0.3μmol/L SPaV-LB,0.4 mol/L Betaine,0.256 U/μL WarmStart Bst 2.0 DNA Polymerase,0.12 U/μL WarmStart RTx Reverse Transcriptase,1μL RNA,反应条件为61℃恒温孵育40 min。采用优化后的反应体系,在特异性检测中,只有携带了病毒SPaV的样品才能发生阳性扩增。灵敏度测试中,RT-LAMP比RT-PCR灵敏度高10倍。田间应用中,RT-LAMP和RT-PCR检测结果一致。本研究建立的SPaV RT-LAMP检测方法操作简便、快速、特异,可供实验室及生产实践中应用,助力脱毒种苗的鉴定和田间病毒病调查。

Strawberry pallidosis-associated virus(SPaV)can cause strawberry pallidosis disease,and the symptoms will worse when SPaV coinfects with other viruses.This study established a reverse transcription-loop-mediated isothermal amplification(RT-LAMP)for SPaV detection.After evaluating and optimizing the conditions of the reaction system,the specificity and sensitivity of this RT-LAMP procedure were mensurated and field samples were tested.The results showed that the optimized reaction system contained 2.5μL 10×Isothermal Amplification Buffer,6 mmol/L Mg ^(2+),1 mmol/L dNTPs,1μmol/L SPaV-FIP and SPaV-BIP,0.1μmol/L SPaV-F3 and SPaV-B3,0.3μmol/L SPaV-LB,0.4 mol/L Betaine,0.256 U/μL WarmStart Bst 2.0 DNA Polymerase,0.12 U/μL WarmStart RTx Reverse Transcriptase,1μL RNA,and incubated at 61℃for 40 min.Only the sample containing the SPaV could be positively amplified by the optimized reaction system.The sensitivity of this RT-LAMP was 10 times higher than that of RT-PCR detection.In the field application,the RT-LAMP detection result was consistent with RT-PCR.The RT-LAMP method developed for SPaV detection in this study is simple,rapid and specific,and can be used in laboratory and field to identify virus-free seedlings and investigate virus diseases.