【目的】快速鉴定我国进境植物检疫性害虫南瓜实蝇Bactrocera tau(Walker),防止该虫传入扩散。【方法】基于物种特异性PCR(SS-PCR)技术,选取20种形态近似的常见实蝇,将南瓜实蝇作为阳性对照,提取基因组DNA模板,选用mt DNA COⅠ序列,检...【目的】快速鉴定我国进境植物检疫性害虫南瓜实蝇Bactrocera tau(Walker),防止该虫传入扩散。【方法】基于物种特异性PCR(SS-PCR)技术,选取20种形态近似的常见实蝇,将南瓜实蝇作为阳性对照,提取基因组DNA模板,选用mt DNA COⅠ序列,检查同源系列,设计筛选1对可快速准确鉴定南瓜实蝇的种特异性引物NF404和NR610,并进行PCR检测和验证,建立一种利用种特异性引物的快速鉴定方法。【结果】南瓜实蝇在207 bp的位置扩增出一条清晰且单一的目标条带,其余果实蝇未见条带。将截获的南瓜实蝇与其同亚属的近缘种具条实蝇、瓜实蝇的不同虫态和成虫部分虫体组织的虫样,采用本实验室建立的SS-PCR鉴定方法进行验证,结果一致。【结论】建立的南瓜实蝇的SS-PCR鉴定方法种特异性和稳定性强,能快速鉴定目标种类,解决口岸进境果蔬中截获实蝇幼虫、蛹等不同虫态和残体难于快速鉴定问题。展开更多
运用含有SYBR Green I的Real Time RT-PCR法分析SS基因在一年生三七根、茎、芦头3个部位中转录水平的相对表达差异。统计分析表明SS基因在根中的表达量最高。本研究取得了特异性高、重复性好的结果,标准曲线斜率均在-3.33～-4范围内,扩...运用含有SYBR Green I的Real Time RT-PCR法分析SS基因在一年生三七根、茎、芦头3个部位中转录水平的相对表达差异。统计分析表明SS基因在根中的表达量最高。本研究取得了特异性高、重复性好的结果,标准曲线斜率均在-3.33～-4范围内,扩增效率均在95%～100%之间,熔解曲线分析显示产物特异性的单一峰,为Real Time RT-PCR技术用于三七植物基因的差异表达分析建立了相应的技术平台。展开更多
[Objective]The paper was to establish a rapid identification method of Bactrocera cilifera(Hendel)with species-specific primers(SS-COI).[Method]Using B.cilifera(Hendel)as the positive control,and 19 species of fruit f...[Objective]The paper was to establish a rapid identification method of Bactrocera cilifera(Hendel)with species-specific primers(SS-COI).[Method]Using B.cilifera(Hendel)as the positive control,and 19 species of fruit flies such as B.diaphora(Coquillett)and B.dorsalis(Hendel)as the negative controls,a pair of species-specific primers,YF290 and YR511,were designed and screened for accurate identification of B.cilifera,based on mitochondrial DNA COI sequence.[Result]The PCR products were amplified and detected by electrophoresis.Only a clear and single band was observed at about 222 bp in the positive control,while no bands were found in the other negative controls.[Conclusion]The established rapid identification method with species-specific primers(SS-COI)is of great practical significance for rapid identification of fruit flies intercepted from import and export fruits and vegetables at ports,and for rapid clearance and early warning of import fruits and vegetables at ports.展开更多
文摘【目的】快速鉴定我国进境植物检疫性害虫南瓜实蝇Bactrocera tau(Walker),防止该虫传入扩散。【方法】基于物种特异性PCR(SS-PCR)技术,选取20种形态近似的常见实蝇,将南瓜实蝇作为阳性对照,提取基因组DNA模板,选用mt DNA COⅠ序列,检查同源系列,设计筛选1对可快速准确鉴定南瓜实蝇的种特异性引物NF404和NR610,并进行PCR检测和验证,建立一种利用种特异性引物的快速鉴定方法。【结果】南瓜实蝇在207 bp的位置扩增出一条清晰且单一的目标条带,其余果实蝇未见条带。将截获的南瓜实蝇与其同亚属的近缘种具条实蝇、瓜实蝇的不同虫态和成虫部分虫体组织的虫样,采用本实验室建立的SS-PCR鉴定方法进行验证,结果一致。【结论】建立的南瓜实蝇的SS-PCR鉴定方法种特异性和稳定性强,能快速鉴定目标种类,解决口岸进境果蔬中截获实蝇幼虫、蛹等不同虫态和残体难于快速鉴定问题。
文摘运用含有SYBR Green I的Real Time RT-PCR法分析SS基因在一年生三七根、茎、芦头3个部位中转录水平的相对表达差异。统计分析表明SS基因在根中的表达量最高。本研究取得了特异性高、重复性好的结果,标准曲线斜率均在-3.33～-4范围内,扩增效率均在95%～100%之间,熔解曲线分析显示产物特异性的单一峰,为Real Time RT-PCR技术用于三七植物基因的差异表达分析建立了相应的技术平台。
基金Supported by Natural Science Foundation of Fujian Province (2011J01066, 2012JO1061)。
文摘[Objective]The paper was to establish a rapid identification method of Bactrocera cilifera(Hendel)with species-specific primers(SS-COI).[Method]Using B.cilifera(Hendel)as the positive control,and 19 species of fruit flies such as B.diaphora(Coquillett)and B.dorsalis(Hendel)as the negative controls,a pair of species-specific primers,YF290 and YR511,were designed and screened for accurate identification of B.cilifera,based on mitochondrial DNA COI sequence.[Result]The PCR products were amplified and detected by electrophoresis.Only a clear and single band was observed at about 222 bp in the positive control,while no bands were found in the other negative controls.[Conclusion]The established rapid identification method with species-specific primers(SS-COI)is of great practical significance for rapid identification of fruit flies intercepted from import and export fruits and vegetables at ports,and for rapid clearance and early warning of import fruits and vegetables at ports.
文摘【目的】辣椒实蝇Bactrocera latifrons(Hendel)为我国重要的检疫性有害生物,其寄主范围广泛,危害严重。由于传统鉴定方法受到饲养周期、饲养条件、虫态等因素的限制,使得果蔬进出口贸易通关速度、疫情快速鉴定受到较大的影响,因此迫切需要开发关于实蝇的快速鉴定识别的技术。【方法】本研究基于mt DNA COI序列设计了一对能够准确鉴定辣椒实蝇的种特异性引物FL680和RL1057,选用辣椒实蝇作为阳性对照,选用番石榴实蝇B.correcta(Bezzi)、桔小实蝇B.dorsalis(Hendel)和颜带实蝇B.cilifer(Hendel)等20种实蝇作为阴性对照,进行PCR扩增并将PCR产物进行电泳检测。【结果】仅目标种辣椒实蝇能够扩增出清晰且单一的约378 bp的条带,其余实蝇种类均未出现条带。将本实验建立的种特异性PCR(SS-PCR)鉴定方法应用于实际检疫工作中并得到了验证,表明该方法具有强的种特异性。【结论】本文提出辣椒实蝇快速鉴定识别技术可应用于实蝇的疫情监测和口岸的检疫检测工作。
