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转基因延熟番茄"华番一号"的品系特异性检测方法 总被引:3,自引:0,他引:3
本文使用反向PCR方法克隆了转基因延熟番茄"华番一号"(Bioscein)的外源基因和番茄基因组之间的一段边界序列,并依据此段序列设计了具有品系特异性的引物和荧光探针,以实时荧光PCR技术建立了华番一号的品系鉴定检测方法,扩增片段长108bp,横跨在Nos启动子和番茄基因组之间.以转基因大豆(RR)、转基因玉米(Mon810)、转基因抗草甘膦油菜、转基因棉花(保龄棉)、非转基因番茄、马铃薯、茄子、大椒、大米、小麦、烟草等为试材,证明本方法同其它转基因作物及其它蔬菜无非特异性反应.本方法在检测华番一号番茄时,相对检测灵敏度可达到0.1%,绝对灵敏度达到20个拷贝.由于本方法的PCR扩增产物长度只有108bp,因此该方法也可以用于检测加工产品中的转基因成分,或作为常规PCR定性检测后的确证实验方法. 相似文献
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本文针对大豆内源基因Lectin和转基因大豆DAS81419品系的5′端插入位点序列,设计特异性引物及探针,建立了同时检测转基因大豆DAS81419品系和大豆内源基因Lectin的二重荧光定量PCR方法,运用15种转基因大豆、3种转基因玉米、1种转基因油菜、1种转基因水稻和非转基因大豆对该方法进行了特异性评价,并分析了该方法的灵敏度和稳定性。结果显示,该方法能准确从20种转基因样品和1种非转基因样品中检出靶目标,检测结果与待检样品信息一致,表明本方法具有良好的特异性;灵敏度高达0.01%;并具有良好的重复性。该方法特异性强、灵敏度高、稳定性强,适用于各口岸实验室进行转基因大豆DAS81419的快速、准确的检测。 相似文献
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转基因玉米品系GA21拷贝数百分含量与质量百分含量关系研究 总被引:1,自引:0,他引:1
《植物检疫》2016,(6)
转基因成分含量一般以质量百分含量表示,而转基因成分定量检测只能检测内、外源基因的拷贝数,并以外源和内源基因拷贝数百分比值表示其含量。由于玉米、大米等种子含有三倍体的胚乳,导致样品中转基因成分质量百分含量并不等于其拷贝数百分比值。为深入探究两者之间的关系,本文首先建立了转基因玉米品系GA21二重数字PCR检测方法,并在此基础上,测定了品系GA21 5种不同质量百分含量标准参考物质的外源和内源基因拷贝数百分比。结果表明,该品系不同质量百分含量与其拷贝数百分比值存在线性相关,线性方程为:y=0.2464x+0.1458,两者相关系数达0.9951。本研究提供了一种可将样品拷贝数百分比值直接转化为质量百分含量的新方法,该方法适用于玉米、大米等含胚乳种子及其产品转基因质量百分含量的检测。 相似文献
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多重PCR-DHPLC法检测转基因棉花品系 总被引:1,自引:0,他引:1
根据3种转基因棉花品系的边界序列设计品系检测引物,建立了一种特异性检测转基因棉花品系MON531、MON1445和MON15985的多重PCR-DHPLC方法。以20种不同的转基因及非转基因作物DNA验证该方法的特异性,结果只有MON531、MON15985和MON1445有特异的品系扩增片段峰,而其他转基因和非转基因作物无品系扩增片段峰,表明该方法特异性强。灵敏度实验结果表明3种转基因棉花的检测下限均为1 ng,灵敏度高。建立的方法可用于转基因棉花MON531、MON1445和MON15985品系及含有其成分产品的筛查或定性检测。 相似文献
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应用环介导等温扩增技术建立一种快速灵敏的转基因番木瓜GM YK品系检测方法。针对转基因番木瓜GM YK品系外源基因与内源基因结合处序列设计1组特异性引物,利用SYBR Green荧光PCR仪实时监测扩增过程,对反应体系进行优化。用优化后的反应体系检测LAMP方法的灵敏性、特异性,并对市售番木瓜进行检测。LAMP检测方法反应速度快,在60 min内即可完成扩增,检测结果易于观察,通过肉眼观察染色情况即可分辨,该方法灵敏度高,检测限可达到5.78 pg,并且具有良好的特异性。LAMP检测方法可快速、灵敏、特异的检测转基因番木瓜GM YK品系,并适用于基层和现场检测。 相似文献
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Y. Jaufeerally-Fakim J.C. Autrey Ian Toth M. Daniels A. Dookun 《European journal of plant pathology / European Foundation for Plant Pathology》2002,108(2):121-130
A genomic library was produced by a subtractive hybridisation method using DNA from Xanthomonas albilineans serovar I and X. albilineans serovar II, originating from Mauritius. The cloned fragments were amplified and used as probes for Southern hybridisation. Probe F20 was selected on the basis of the RFLP pattern. Upon hybridisation of X. albilineans DNA with probe F20, strains of serovars I and II were differentiated by their banding profiles. This probe was sequenced and oligonucleotide primers were designed. Fragment number and length polymorphisms were obtained after PCR amplification of X. albilineans DNA using F20A as a single primer. The number and size of bands obtained with this primer was correlated to the serotypes of the strains and to the DNA grouping reported by Alvarez et al. (1996). The two serotypes I and II which exist in Mauritius were differentiated by the PCR using primer F20A. The probe and primer developed provide rapid and precise tools for the differentiation of genetic variants within the species X. albilineans. 相似文献
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植原体TaqMan探针实时荧光PCR检测鉴定方法的建立 总被引:19,自引:1,他引:19
本研究成功建立了植原体分类鉴定和检测的TaqMan探针实时荧光PCR方法,该方法根据植原体16S rDNA保守区设计了1个TaqMan广谱探针和3个植原体组间点突变特异性探针,并对9种植原体和5种细菌以及3个植物样本进行实时荧光PCR。结果表明,用广谱探针可检测到所有植原体产生荧光信号,而细菌不产生荧光信号。当用植原体组间特异性探针检测时,仅能检测到该组植原体产生荧光信号,检测的敏感性比常规的PCR-电泳检测高约100倍、检测速度有较大提高。由于PCR产物是荧光探针检测,本方法特异性强,并可以用组特异探针直接确定植原体种类。实验采用完全闭管检测,降低了污染机会。本研究为其它原核生物、特别是不能培养菌、难培养菌的检测鉴定和分类提供了新方法。 相似文献
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根萤叶甲属(Diabrotica)被我国列为进境植物检疫性有害生物,该属中的玉米根萤叶甲(D.virgifera virgifera)、十一星根萤叶甲(D.undecimpunctata)、巴氏根萤叶甲(D.barberi)是北美的主要农业害虫。为了建立一种快速的分子鉴定方法以鉴定这3种根萤叶甲,本研究从田间采集的成虫样品中获得其部分mtDNA COI序列,与Gen Bank中的相关序列进行比对,设计筛选出3种根萤叶甲的特异性引物和TaqMan探针,并进行实时荧光PCR特异性和灵敏度检测。特异性检测结果表明,用目标种根萤叶甲DNA和其特异性探针和引物进行实时荧光PCR反应时,在30个循环反应内(Ct值30)有近S型扩增曲线出现,同时其他种均无荧光信号增长。此外,灵敏度结果显示,玉米根萤叶甲和巴氏根萤叶甲可检测的最小DNA模板浓度,即实时荧光PCR反应的灵敏度为0.1 ng/μL,十一星根萤叶甲为0.01 ng/μL。 相似文献
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A sensitive method for detecting bamboo mosaic virus (BaMV) and establishment of BaMV-free meristem-tip cultures 总被引:7,自引:0,他引:7
A sensitive method was used to detect bamboo mosaic virus (BaMV) and its associated satellite RNA (satBaMV) by 32 P- and digoxigenin (Dig)-labelled probes synthesized from cDNA clones of BaMV genomic (L probe) and satBaMV (S probe) RNA. Both the 32 P- and Dig-labelled L and S probes could detect as little as 490 pg of BaMV viral RNA by slot- and dot-blot hybridization. In infected leaf extracts, 32 P-labelled L and S probes detected virus at 25-fold higher dilutions than Dig-labelled probes, which were also successfully used to detect BaMV infection in plants derived from meristem-tip culture. However, immunoassays failed to detect BaMV in meristem culture. By dot-blot hybridization assays, 25% of the seedlings were shown to be virus-free. These results suggest that a highly sensitive method for the detection of BaMV infection is required for the establishment of BaMV-free cultures. Meristem-tip culture also provides an efficient method for obtaining virus-free bamboo plants. 相似文献
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马铃薯类病毒(PSTVd)非放射性标记cDNA探针制备及其在检测上的应用 总被引:4,自引:0,他引:4
利用含PSTVd单体克隆的重组质粒pGEM PSTVd,通过PCR扩增技术,用生物素标记制备cDNA探针,进行杂交反应检测PSTVd,其中通过化学颜色反应进行判读灵敏度可达50pg,而用化学发光反应进行判读灵敏度可达5pg,分别是R-PAGE检测灵敏度的26倍和260倍,且2种反应特异性和专化性较强。cDNA核酸斑点杂交反应(NASH)检测PSTVd方法准确、灵敏度高,一次检测样品数量多,且对异地样品检测非常方便,是以往其它检测方法的有效补充。 相似文献
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Nathalie Astruc Jose F. Marcos Guy Macquaire Thierry Candresse Dr. Vicente Pallás 《European journal of plant pathology / European Foundation for Plant Pathology》1996,102(9):837-846
A non-radioactive digoxigenin-labelled RNA probe specific for hop stunt viroid (HSVd) diagnosis has been developed. The high sensitivity and specificity of this RNA probe in dot blot hybridizations to nucleic acids from field samples, allowed the confirmation of the presence of HSVd in apricot (Prunus armeniaca L.) and its detection in two fruit tree species not previously described as hosts of this pathogen, almond (Prunus dulcis Miller) and pomegranate (Punica granatum L.). This result supports and extends the notion of the world wide distribution of HSVd, infecting cultivated fruit trees. HSVd was also found to accumulate to much higher levels in mature apricot fruits than in leaves. Additionally, a sample processing procedure which does not involve the use of organic solvents was demonstrated to render faithful results when used for viroid detection. The combined reliability and facility of use of both this extraction procedure and the non-radioactive probe will benefit agronomic investigations addressing the detection and eradication of HSVd. Other applications of the work described here, as the study of possible causal relations between specific disorders and HSVd infection, are also discussed. 相似文献
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