排序方式： 共有162条查询结果，搜索用时 109 毫秒
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实时荧光定量PCR技术在转基因检测中的应用 总被引：8，自引：5，他引：3
实时荧光定量PCR技术是一种新型的核酸定性、定量检测、分析技术。随着转基因产品大规模商业化,转基因生物的环境安全和食用安全性问题引起了全球范围内广泛的争论和关注。实时监测、定量准确、灵敏度高、反应速度快、重复性好及PCR反应后不需电泳检测等优点,使RQ-PCR逐步成为转基因检测的重要工具。 相似文献
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Seyed Mahyar Mirmajlessi Inmaculada Larena Marika Mänd Evelin Loit 《Acta Agriculturae Scandinavica, Section B - Plant Soil Science》2016,66(7):619-629
Verticillium dahliae Kleb, the cause of Verticillium wilt disease, is a destructive pathogen that leads to severe yield losses in strawberry fields and thus considerable economic damages. Although rapid identification and detection methods are becoming available more, pathogen quantification remains one of the main challenges in the disease management. In this study, a real-time polymerase chain reaction (rtPCR) assay was developed to quantitatively assess V. dahliae abundance directly from affected roots and soil collected from different areas in Estonia. A specific primer pair based on the ribosomal DNA (rDNA) internally transcribed spacer was designed for SYBR Green-based assay. Strawberry plant and soil samples were randomly collected from different areas in Estonia and analyzed for V. dahliae by soil plating technique and rtPCR assay. The assay was specific for V. dahliae so that the minimum detection limit was 0.93?pg?µl?1 of pathogen DNA and the lowest amount of V. dahliae detected in soil was 10.48?pg?µl?1 of target DNA corresponding to one microsclerotia per gram of soil. This technique allowed rapid detection and quantification of the pathogen DNA at the picogram level in soils and even in symptomless plants, facilitating the screening of the pathogen in diverse areas. This is the first study about the rtPCR technique being used successfully to assess populations of V. dahliae with high specificity and sensitivity in Estonia strawberry fields. Results of this research can be useful for growers and agricultural organizations to improve available disease management strategies against Verticillium wilt. 相似文献
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为建立一种快速检测跨膜蛋白39A(TMEM39A)基因的方法,根据GenBank中登录的不同种属TMEM39A基因序列的保守区域,设计并合成1对荧光定量通用性引物,经过条件优化,建立了检测TMEM39A基因的SYBR Green Ⅰ实时荧光定量PCR方法。结果显示:标准品模板在3.937×108~3.937×103拷贝·μL-1范围内呈良好的线性关系,R2可达0.999;该方法特异性较好,检测灵敏度可达3.937×102 拷贝·μL-1;组内和组间变异系数均小于1%,具有较高的重复性和稳定性;利用该方法能够检测出不同细胞中的TMEM39A含量,具有种属通用性。该研究方法的建立为临床上提供了一种TMEM39A的快速检测和定量分析技术。 相似文献
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动物源性食品中猪源性成分检测 总被引：1，自引：0，他引：1
为探讨猪源性成分检测可行的方法,利用SYBR Green I Real time PCR对动物源性食品中猪源性成分进行测定。根据猪mtDNA保守序列设计特异性引物,对牛、羊、猪的动物源性食品进行扩增;克隆目的基因片段,并以此为标准品,优化反应条件和反应体系,提高灵敏度;反复检测不同样品,测试稳定性。结果表明SYBR Green I Real time PCR技术能够特异性的检测到动物源性食品中猪源性成分,引物特异性强、稳定性可靠,灵敏度达到1×10-6 ng/μL,建立了动物源性食品中猪源性成分的快速、精准、经济、便捷的检测方法。 相似文献
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通过对兰州大尾羊尾部、大网膜、肝脏、肾周脂肪组织中的总RNA提取,反转录获得总cDNA样品,结合PCR技术,建立了SYBR荧光定量PCR法检测兰州大尾羊心脏型脂肪酸结合蛋白（H-FABP）基因mRNA在不同组织中相对表达量的试验方法,并进行批内、批间重复性检验。结果表明,H-FABP和18S基因标准曲线回归方程分别为CtH-FABP =-3.24375x+38.34230和Ct18S=-3.33137x+33.4573,相关系数（r2）分别为0.9964和0.9992,扩增效率分别为103%和99%;批内、批间重复性测定的变异系数分别为小于1.8%和10%;说明该方法灵敏度高、特异性强、准确可靠、重复性好,是实时荧光定量PCR检测兰州大尾羊心脏型脂肪酸结合蛋白基因mRNA表达量的有效方法。 相似文献
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Development of A Real-Time PCR Assay for Plasmodiophora brassicae and Its Detection in Soil Samples 总被引：1，自引：0，他引：1
A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA(rDNA) and internal transcribed spacer(ITS).A pair of primers PBF1/PBR1 was designed based on the conservative region of rDNA-ITS of P.brassicae.The positive plasmid pB12 was obtained and used as the template to create standard curve.The specificity,sensitivity,and reproducibility of real-time PCR were evaluated respectively.Naturally and artificially infested soil samples containing different concentrations of P.brassicae were detected.The results demonstrated that standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concentration.The melting curve was specific with the correlation coefficient of 0.995 and that the amplification efficiency was 93.8%.The detection limit of P.brassicae genomic DNA was approximately 40 copies per 25 μL.The sensitivity of the assay was at least 100-fold higher than conventional PCR.Only DNA from P.brassicae could be amplified and detected using this assay,suggesting the highly specific of this assay.The coefficient of variation was less than 3%,indicating the PCR method revealed high reproducibility.The detection limit in soil samples corresponded to 1 000 resting spores g-1soil.Bait plants were used to validate the real-time PCR assay.This developed real-time PCR assay allows for fast and sensitive detection of P.brassicae in soil and should be useful in disease management and pest interception so as to prevent further spread of P.brassicae. 相似文献
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根据GenBank中柱状黄杆菌16SrRNA 序列,设计1对特异性引物,PCR 扩增获得16SrRNA 基因片段,并克隆到pMD-18T载体上作为阳性标准品。通过对SYBR Green I荧光定量PCR反应条件的优化,建立了黄杆菌的SYBR Green I荧光定量PCR 诊断方法,以此为基础研制试剂盒。试剂盒扩增产物的熔解曲线分析只出现1个单特异峰,无引物二聚体,对嗜水气单胞菌、荧光假单胞菌、迟缓爱德华、弗氏柠檬酸杆菌均无阳性信号扩增,重复性好,灵敏度可达12 拷贝/μL。结果表明,研制的柱状黄杆菌SYBR Green I实时荧光定量PCR试剂盒具有特异、灵敏、快速、重复性好等特征,适合于大鲵临床样品的检测。 相似文献