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改良Chelex-100法和CTAB法用于转基因抗草甘膦大豆检测效果的比较 总被引:2,自引:0,他引:2
以转基因抗草甘膦大豆为研究材料,分别采用改良Chelex-100法和常规CTAB法提取基因组DNA,以提取DNA的浓度和纯度,同时以PCR扩增大豆的内源基因(lectin)及外源特异性序列(CaMV35S,nos,Cp4-epsps)的效果对两种方法进行比较和评价.结果表明:虽然改良Chelex-100法DNA提取纯度不高,但是提取效率与常规CTAB法相当,而且改良Chelex-100法能够快速在1 h之内从大豆中提取DNA,所提取的DNA可以直接用于PCR扩增反应,PCR扩增产物电泳条带清晰.因此,改良Chelex-100法可以替代CTAB法提取DNA用于转基因检测,该方法具有经济、简便、快速的特点. 相似文献
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本文以大豆主要过敏蛋白P34基因为靶标,采用TaqMan探针实时荧光PCR方法建立食品过敏原大豆成分的检测方法。实验表明建立的检测方法具有特异性,灵敏度高,检测限为10mg/kg。通过对市售样品的检测,该方法适用于对常见食品种类的检测,但不适用于精加工油脂等精细加工产品的检测。 相似文献
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食品过敏原大豆P34蛋白基因的实时荧光PCR检测 总被引:2,自引:0,他引:2
以大豆主要过敏蛋白P34基因为靶标,采用TaqMan探针实时荧光PCR方法建立食品过敏原大豆成分的检测方法。实验表明建立的检测方法具有特异性,灵敏度高,检测限为10mg/kg。通过对市售样品的检测,该方法适用于对常见食品种类的检测,但不适用于精加工油脂等精细加工产品的检测。 相似文献
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富含多糖甜玉米幼穗RNA提取及高温胁迫基因表达分析 总被引:2,自引:0,他引:2
以改良Trizol试剂法从富含多糖的甜玉米幼穗中提取高质量、完整的总RNA,利用实时荧光定量PCR对高温胁迫下粤甜13雌穗发育的8个差异表达基因进行分析。结果表明,8个基因分为上调表达和下调表达,实时荧光定量PCR和测序法基因表达谱检测的基因上调或下调表达的趋势一致,上调表达基因ZM2G058057和下调表达基因ZM2G059964、ZM2G044670相对表达量较高,可能在高温胁迫影响甜玉米幼雌穗发育过程中起更重要的作用。 相似文献
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为了确定小麦转基因成分PCR和实时荧光PCR方法的定性检测低限,将转基因小麦B73与非转基因小麦(检测外源基因)、小麦与大米(检测内源基因)分剐进行质量分数配比后提取DNA用于测定相对检测低限,再将100%转基因小麦B73的DNA进行浓度稀释用于测定绝对检测低限,并应用已知的NOS、bar、ubiquitin,ui-dA(GUS)外源基因和肌Wx012、GAG56D内源基因的引物和探针时模板DNA分别进行PCR与实时荧光PCR扩增.结果表明,最终确定PCR方法检测小麦转基因成分的相对检测低限为0.1%(质量分数),绝对检测低限为0.5 ng/ⅡL;实时荧光PCR方法的相对检测低限为0.1%(质量分数),绝对检测低限为0.01 ng/μL.所确定的检测低限可满足国家对转基因产品的最低标识要求. 相似文献
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为了优化小麦高通量TILLING突变体扫描所需基因组DNA提取的方法,对利用SDS法、改良SDS法、CTAB法、改良CTAB法、AxyPrep DNA提取试剂盒、Qiagen DNeasy Plant Mini Kit试剂盒以及再生Qiagen硅胶柱方法提取的小麦基因组DNA进行了系统比较。结果表明,尽管7种方法所提取DNA的纯度及琼脂糖凝胶电泳检测结果均良好,但再生Qiagen硅胶柱方法、CTAB法和改良SDS法的DNA浓度显著高于其他方法。根据小麦细胞分裂素氧化酶基因1(TaCKX1)的DNA序列设计引物,以新鲜DNA样品用于PCR扩增大于1 000bp的片段时,后4种方法扩增效果优于前3种方法;DNA在-20℃下保存100d后重新进行PCR扩增时,则只有Qiagen试剂盒和再生Qiagen硅胶柱2种方法提取的DNA能扩增出大于1 000bp的片段。采用本实验室与新西兰坎特伯雷大学合作建立的再生硅胶柱与自配提取液提取小麦基因组DNA,既保证了DNA的高质量,又降低了使用试剂盒的成本,为小麦TILLING库的构建及其他相关试验提供了一种经济有效的DNA提取方法。 相似文献
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为了对基因组编辑产品进行精准定性和定量检测,以水稻SP1 基因的编辑植株为材料,在编辑位点上下
游设计通用引物,在编辑位点处设计基因编辑位点特异性TaqMan探针,建立了编辑位点特异性PCR方法。利用该
方法可准确鉴定特异基因组编辑产品,检测灵敏度达到5~10拷贝,可在实时荧光PCR(qPCR)和微滴数字PCR
(ddPCR)平台上对基因组编辑产品进行定量检测。由于数字PCR的微反应单元可消除野生型DNA对通用引物的
竞争性消耗,与qPCR的定量结果相比,ddPCR定量结果具有更高的定量准确性。 相似文献
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马铃薯Y病毒一步RT-PCR检测试剂盒的研制 总被引:1,自引:0,他引:1
马铃薯Y病毒(Potato virus Y,PVY)对马铃薯的危害最大,可导致马铃薯退化,降低马铃薯产量。解决这一问题的重要途径就是培养脱毒种薯,但是否完全脱毒需要经过检测才能证实。本研究依据PVY CP基因序列设计合成了一对引物PY1、PY2,以带毒样品植物总RNA为模板,在同一个反应中同时加入反转录和PCR反应所需试剂,反应程序中包括反转录和PCR反应所需条件,进行反应扩增,带毒样品扩增得到340 bp的目的条带,而健康对照无此目的条带,从而建立了PVY的一步RT-PCR检测技术,并组装成试剂盒。该试剂盒具有良好的稳定性和特异性,灵敏度可以检测到带毒植物组织下限的6.25μg,高于ELISA(100μg)和NASH(15μg)的灵敏度,虽然和常规方法的灵敏度相同,但更为快速、简便、易于操作,适合脱毒苗和脱毒种薯生产单位做大量样品的检测。 相似文献
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为了建立适合黎药胆木的PsbA-trnH-PCR体系来研究不同地理居群胆木遗传多样性,本研究以植物基因组试剂盒法提取胆木基因组DNA为模板,采用单因素实验和正交试验对PsbA-trnH-PCR过程中的关键影响因素进行优化,并对PsbA-trnH-PCR产物进行测序鉴定。结果表明,最佳PsbA-trnH-PCR反应体系(25μL)为:Taq酶1.0 U,dNTPs 0.4 mmol/L,Mg~(2+)0.75 mmol/L,引物0.15μmol/L,模板20 ng,10×PCR Buffer(不含Mg~(2+))2.5μL;采用该最佳体系对胆木基因组DNA进行PCR扩增,获得扩增产物,经单向测序获得了胆木PsbA-trnH部分序列;并建立了稳定的PsbA-trnH-PCR体系,为胆木的药材鉴别及其遗传多样性研究奠定了基础。 相似文献
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马铃薯块茎青枯病菌潜伏侵染的酶联免疫学检测 总被引:4,自引:0,他引:4
试验采用武汉市场上来源不同产地的 7份马铃薯材料 ,用CIP提供的RS NCM ELISA试剂盒进行了块茎青枯病菌携带情况的检测。试验结果显示 ,应用该试剂检测马铃薯块茎青枯病菌携带情况具有较高的灵敏度 ;检测前样品的富集时间对检测结果具有明显的影响 ,其最佳富集时间为4 8h ;不同来源的马铃薯块茎携带青枯病菌的程度具有显著差异 相似文献
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Faming Wang Xin Cai Chengjiao Zhang Wen Shi Yehu Lu Guowen Song 《Fibers and Polymers》2016,17(1):151-159
The main objective of this study was to evaluate the actual performance of a conceptual body mapping sportswear (BMS) kit while doing different activities in a warm dry condition. Eight subjects participated in this study and each subject underwent two trials. Each trial was composed of 40 min treadmill walking at 5.0 km/h, 10 min resting, 20 min treadmill running at 10.0 km/h and a final 20 min resting. All trials were performed in a chamber where the T a =30±0.5 °C, RH=40±5 % and v air =0.17±0.05 m/s. Human physiological and perceptual responses in CON (i.e., traditional cotton sportswear) and BMS were measured and compared. It was found that both physiological responses (such as core temperature, mean skin temperature, mean torso temperature, mean body temperature and hear rate) and local subjective sensations (e.g., thermal sensation) were improved in BMS during the running phase as well as the second recovery phase. It was thus concluded that the conceptual BMS kit does have advantages over traditional cotton sportswear in terms of improvements on both physiological responses and local subjective sensations. 相似文献
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Detection of Diarrheic Shellfish Poisoning and Azaspiracid Toxins in Moroccan Mussels: Comparison of the LC-MS Method with the Commercial Immunoassay Kit 总被引:1,自引:0,他引:1
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Diarrheic shellfish poisoning (DSP) is a recurrent gastrointestinal illness in Morocco, resulting from consumption of contaminated shellfish. In order to develop a rapid and reliable technique for toxins detection, we have compared the results obtained by a commercial immunoassay-“DSP-Check” kit” with those obtained by LC-MS. Both techniques are capable of detecting the toxins in the whole flesh extract which was subjected to prior alkaline hydrolysis in order to detect simultaneously the esterified and non esterified toxin forms. The LC-MS method was found to be able to detect a high level of okadaic acid (OA), low level of dinophysistoxin-2 (DTX2), and surprisingly, traces of azaspiracids 2 (AZA2) in mussels. This is the first report of a survey carried out for azaspiracid (AZP) contamination of shellfish harvested in the coastal areas of Morocco. The “DSP-Check” kit was found to detect quantitatively DSP toxins in all contaminated samples containing only OA, provided that the parent toxins were within the range of detection and was not in an ester form. A good correlation was observed between the two methods when appropriate dilutions were performed. The immunoassay kit appeared to be more sensitive, specific and faster than LC-MS for determination of DSP in total shellfish extract. 相似文献
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The cause of leaf and fruit severe mosaic disease in purple passion fruit (Passiflora edulis) in Chiang Mai province, Thailand, was identified and virus diagnostic kit was developed. Symptomatic plants collected from diseased mother plants in the nursery at the Royal Pangda research station (RPRS) was used for virus isolation through single lesion transfer in Chenopodium amaranticolor, followed by virus propagation for purification in Nicotiana benthamiana, and back inoculation to purple passion fruit. Electron microscopy of leaves with typical symptoms revealed potyvirus-like flexuous rod particles, ca. 750 nm long and pinwheel inclusion bodies in the cytoplasm of infected cells. The deduced amino acid sequence of the complete coat protein revealed that the isolated virus was a strain of Telosma mosaic virus with which it shared 84% identity. A rabbit polyclonal antiserum was produced against purified virions, and used to develop a gold-labeled immunostrip for rapid virus diagnosis. The test strip could detect the virus in diseased passion fruit sap up to a dilution of 1: 640, and positive test line could be read within 3–5 min. Application of strip test for virus assay in RPRS nursery's mother plants help screen clean stocks. This strip test kit supports RPRS program of producing virus-free planting materials for farmers. 相似文献
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水稻群体成穗率与干物质积累动态关系的模拟研究 总被引:26,自引:0,他引:26
应用两个水稻茎数动态模型(TIL和RGR模型),分析了成穗率与群体干物质积累动态的关系。结果表明:(1)成穗率与最高茎数呈极显著负相关,支持了“降低苗峰是提高成穗率的关键”的结论;(2)最高茎数与穗分化始期干重呈极显著正相关,前期干物质积累过快是苗峰过高的原因之一;(3)成穗率与群体干物质积累动态密切相关。同样的干物质积累量,干物质积累动态不同,其茎数动态相差很大。在抽穗期干物质积累量相近的情况下,穗分化以前干物质积累所占的比例越大,成穗率越低。采用一组独立的实测资料进行分析,也得到了相似的结果。由此可见,优化干物质积累动态是提高成穗率,从而实现高产更高产的基础。 相似文献