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本研究旨在建立番茄和辣椒种子携带番茄褐色皱果病毒（Tomato brown rugose fruit virus,ToBRFV）快速、精准的核酸检测技术。选用ToBRFV的MP蛋白基因和CP蛋白基因保守序列作为靶标区域设计了1对特异性引物ToBRFV-F-5506/ToBRFV-R-6186，建立了ToBRFV的RT-PCR检测方法。用ToBRFV阳性样品对该方法的灵敏度和特异性进行了系统分析，最后以疑似ToBRFV感染的实际种子样品进行验证，并与已报道的9对ToBRFV特异性引物对实际种子样品的检测效率进行比较分析。结果表明，采用本研究建立的RT-PCR检测方法检测限值至2.5ng/μL，且与番茄和辣椒的其他主要病毒无交叉反应，特异性良好，对50份疑似ToBRFV感染的番茄和辣椒种子样品的阳性检出率高达40%，要优于已报道的9对ToBRFV特异性引物。本研究建立的番茄褐色皱果病毒RT-PCR检测方法具有较高的灵敏度和良好的特异性，适用于ToBRFV感染的番茄和辣椒种子样品检测。
Abstract：This study aimed to develop a rapid and accurate nucleic acids detection method for tomato brown rugose fruit virus(ToBRFV)in tomato and pepper seeds. One pair of specific primers named“ToBRFV-F-5506”and“ToBRFV-R-6186”targeting the MP and CP genes of ToBRFV,respectively,was designed.Then,a reverse transcription-polymerase chain reaction(RT-PCR)assay was developed. The sensitivity and specificity of the established method were analyzed with ToBRFV-positive samples and actual seed samples.Finally,the detection efficiency of RT-PCR system was evaluated with nine pairs of reported ToBRFV-specific primers. The results showed that the limit of detection of our novel RT-PCR assay was 2.5 ng/μL,and there was no cross-reaction with the other main viruses of tomatoes and peppers. In 50 seed samples with suspected ToBRFV infection,40% were found to be ToBRFV-positive,which was much higher than that for the reported nine pairs of ToBRFV-specific primers. We conclude that the RT-PCR method we developed was specific and sensitive,and could be used for detection of the ToBRFV from samples of tomato and pepper seeds.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.004164
中图分类号:S432.41
引用信息:
[1]李献锋,刘明航,魏霜,等.番茄褐色皱果病毒RT-PCR检测方法的建立与评价[J].病毒学报,2022,38(04):905-914.DOI:10.13242/j.cnki.bingduxuebao.004164.
基金信息:
国家重点研发计划（项目号：2021YFD1400100），题目：外来病虫害高效检测关键技术与装备研发; 海关总署科研项目（项目号：2021HK166），题目：番茄褐色皱果病毒精准检测及监测溯源研究;海关总署科研项目（项目号：2021HK165），题目：进口玉米中检疫性病害新型检测技术及质控关键技术研究~~
2021-09-24
2021
2021-11-11
2022-07-05
2022
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2022-07-06
2022-07-06
2022-07-06