Chelex-100快速提取用于转基因检测DNA模板的研究

目的:建立从转基因作物中快速提取DNA的方法.方法 :采用Chelex-100法提取抗草甘膦大豆和非转基因大豆、转基因抗虫玉米Bt176和非转基因玉米中的DNA,使用PCR扩增大豆和玉米的内源基因(Lectin,zSSⅡb)及外源特异性序列(CaMV35S,Bt176)评价提取核酸的质量.结果 :Chelex-100法能够快速在1h之内从大豆和玉米中提取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,转基因抗草甘膦大豆样品和转基因抗虫玉米Bt176检测均出现强阳性结果.结论 :Chelex-100法提取DNA可以作为转基因检测的模板,该方法具有经济、简便、快速的特点,适合于转基因检测工作.     

烟草碱法提取基因组DNA的改进

为能够快速高效的对大量转基因烟草样品进行DNA提取,对碱法提取烟草组织DNA的方法进行了改进。采用0.01mol/L的Na OH溶液为提取液,破碎转基因烟草叶片组织后无需经过加热煮沸中和等步骤,上清液可直接用作PCR反应的模板。结果表明,0.01-0.1 mol/L之间的Na OH溶液制备的DNA模板均能成功扩增,该方法制备的DNA模板不依赖特殊的反应条件,多种品牌的PCR Mix均能成功进行扩增反应。与传统SDS提取法和试剂盒提取法获得的DNA相比,PCR结果没有明显差异。此提取方法在小麦、高粱、水稻、玉米等作物上也取得了很好的实验效果。通过本方法制备的DNA模板,其PCR扩增的产物可直接用来测序分析。该方法使用仪器少,样品消耗少,快速、简便、成本低,没有交叉污染,能够在短时间内处理大量样品,因此能够对大量的转基因烟草样品进行DNA的快速提取和鉴定。     

转水稻粗缩病毒运动蛋白缺陷型基因玉米的二重PCR检测研究

本研究以外源基因（RDV MP-）和玉米内源基因Zein作为PCR扩增对象,旨在建立一种简便有效的转基因玉米及其产品的二重PCR检测技术。转外源基因（RDV MP-）材料的常规PCR检测结果证明外源基因在转基因材料中可稳定遗传;对常规PCR检测的阴性结果材料进行二重PCR检测,扩增结果除进一步证实常规PCR检测的阴性结果结论外,还证明提取的植物总DNA质量符合试验要求,进而从二重PCR检测结果还得出常规PCR检测的阴性结果出错率为1.4%。此方法简单,实用,结果可靠,可适用于转基因植物及产品的检测。     

改良Chelex-100法快速提取转基因农产品DNA

旨在建立一种从转基因农产品中快速提取DNA的方法.分别采用改良Chelex-100法和常规CTAB法提取转基因大豆GTS40-3-2基因组DNA,测其浓度和纯度,PCR扩增其内源基因(Lectin)、启动子(CaMV35S)和品系特异性序列,对两种方法进行比较和评价,并研究两种方法提取的DNA在-20℃下保存一个月内的检测效果,以及改良Chelex-100法在玉米、小麦和水稻等其他转基因农产品的应用效果.结果表明,改良Chelex-100法能够快速在1.5h之内从样品中提取DNA,所提取的DNA直接用于PCR扩增反应,产物电泳条带清晰明亮.两种方法提取的DNA在-20℃下保存一个月内的检测效果未见明显差别.该方法在玉米、小麦和水稻等转基因农产品的应用效果稳定.因此,改良Chelex-100法提取的DNA可以作为PCR扩增模板用于转基因农产品检测.该方法具有经济、简便、快速、安全的特点,适合转基因农产品大规模筛选和鉴别.     

转水稻粗缩病毒运动蛋白缺陷型基因玉米的二重PCR检测

以外源基因(RDV MP-)和玉米内源基因Zein作为PCR扩增对象,旨在建立一种简便有效的转基因玉米及其产品的二重PCR检测技术.转外源基因(RDV MP-)材料的常规PCR检测结果证明外源基因在转基因材料中可稳定遗传;对常规PCR检测的阴性结果材料进行二重PCR检测,扩增结果除进一步证实常规PCR检测的阴性结果结论外,还证明提取的植物总DNA质量符合试验要求,进而从二重PCR检测结果还得出常规PCR检测的阴性结果出错率为1.4%.此方法简单,实用,结果可靠,可适用于转基因植物及产品的检测.     

转基因玉米种子快速筛查方法研究与应用

转基因种子快速筛查技术研究与应用对于加强农业转基因生物安全管理至关重要。将DNA热碱提取法和复合荧光PCR技术相结合,建立起玉米种子转基因成分快速筛查方法。结果显示,该方法不仅能保证检测的准确性和灵敏性,而且还大大降低假阳性发生率,提高转基因筛查的效率,适合大批量样品的快速检测。     

马蹄金基因组DNA的提取方法比较

对马蹄金基因组DNA的提取方法———快速提取法、小量提取法和大量提取法进行了对比分析,结果表明,利用快速提取法可在20 min内快速可靠地从马蹄金(Dichondra repensForst.)组织中提取DNA,与小量提取法和大量提取法获得的DNA用于PCR检测结果一致,可为转基因植物的快速检测提供方法。     

一种适于转基因水稻PCR检测的微量DNA快速提取法

对已报道的小麦基因组DNA快速提取方法的部分步骤进行了简化,在水稻上进行了尝试。结果表明,简化法提取的水稻基因组DNA完整性好,PCR扩增效果与试剂盒提取法无明显的差异,结果稳定可靠;而且整个提取过程操作简单、花费时间少,样品用量少,仅需5-10mg,适用于大规模转基因水稻的PCR检测。     

MDA技术在转基因检测中的研究与应用

PCR技术是转基因植物及其产品检测的主要方法,但是因为其对模板纯度和质量有较高要求,对于低质量模板难以获得稳定的检测结果。本研究利用MDA技术可将低至10 copies的微量DNA样品扩增至可用PCR方法稳定检出;进一步利用MDA技术对转基因玉米有证标准物质Bt11粉末的单颗粒痕量样品释出DNA进行扩增,结合PCR扩增检测,玉米粉末颗粒中内源参照基因的检出率达到70%以上,转基因特异性序列检出与内源参照基因检出的比值,与标准物质标称值基本相符,为建立基于MDA技术转基因植物及其产品粉末颗粒等痕量样品检测方法奠定了基础,同时也为加工产品中复合性状转基因作物的检测提供了潜在的解决方案。     

番茄叶片基因组DNA快速制备技术及其在基于实时荧光定量PCR的转基因检测中的应用

以番茄(Solanum lycopersicum L.cv.Micro-Tom)叶片为试材,建立了一种简便快速制备叶片基因组DNA的方法。2~20 mm2的叶片即可满足制备要求,制备过程只需一种提取试剂、只涉及1次移液和1次离心操作,不涉及沉淀。确定了所制备的DNA用于实时荧光定量PCR的合适用量为0.1~0.2μL(反应总体积为12.5μL),发现过量模板的使用可降低PCR效率且可导致扩增失败。该项DNA快速制备及相适应的实时荧光定量PCR技术已成功应用于番茄转基因植株检测。     

Rapid differentiation and in situ detection of 16 sourdough lactobacillus species by multiplex PCR

A two-step multiplex PCR-based method was designed for the rapid detection of 16 species of lactobacilli known to be commonly present in sourdough. The first step of multiplex PCR was developed with a mixture of group-specific primers, while the second step included three multiplex PCR assays with a mixture of species-specific primers. Primers were derived from sequences that specify the 16S rRNA, the 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene. The primer pairs designed were shown to exclusively amplify the targeted rrn operon fragment of the corresponding species. Due to the reliability of simultaneously identifying Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum, a previously described multiplex PCR method employing recA gene-derived primers was included in the multiplex PCR system. The combination of a newly developed, quick bacterial DNA extraction method from sourdough and this multiplex PCR assay allows the rapid in situ detection of several sourdough-associated lactobacilli, including the recently described species Lactobacillus rossii, and thus represents a very useful alternative to culture-based methodologies.     

Rapid Differentiation and In Situ Detection of 16 Sourdough Lactobacillus Species by Multiplex PCR

A two-step multiplex PCR-based method was designed for the rapid detection of 16 species of lactobacilli known to be commonly present in sourdough. The first step of multiplex PCR was developed with a mixture of group-specific primers, while the second step included three multiplex PCR assays with a mixture of species-specific primers. Primers were derived from sequences that specify the 16S rRNA, the 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene. The primer pairs designed were shown to exclusively amplify the targeted rrn operon fragment of the corresponding species. Due to the reliability of simultaneously identifying Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum, a previously described multiplex PCR method employing recA gene-derived primers was included in the multiplex PCR system. The combination of a newly developed, quick bacterial DNA extraction method from sourdough and this multiplex PCR assay allows the rapid in situ detection of several sourdough-associated lactobacilli, including the recently described species Lactobacillus rossii, and thus represents a very useful alternative to culture-based methodologies.     

Molecular detection of anthrax spores on animal fibres

AIMS: To develop a rapid, specific and sensitive diagnostic test for the detection of the spores of Bacillus anthracis on commercial samples of animal fibres (e.g. wool and cashmere). METHODS AND RESULTS: Extraction of DNA from spores using a mechanical disruption method based on bead beating was evaluated but subsequently abandoned as it compromised the sensitivity of the overall protocol. A multiplex PCR and two nested amplification reactions designed for B. anthracis were developed during this study. CONCLUSIONS: A simple selective incubation step in combination with multiplex PCR was found to be more effective than generic DNA extraction coupled to a sensitive nested amplification reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid diagnostic test could be applied to the analysis of commercial fibre samples for the detection of anthrax as required by health and safety legislation resulting in considerable savings in time and expense.     

Multiplex PCR for simultaneous detection of five virulence hemolysin genes in Vibrio anguillarum

A multiplex PCR was developed for detection of hemolysin-producing Vibrio anguillarum using primers targeting five hemolysin genes (vah1, vah2, vah3, vah4 and vah5). This method was successful in amplifying reactions containing as little as 100 fg of genomic template DNA. The direct detection of V. anguillarum in clinical specimens by this multiplex PCR was also successful in reactions containing as few as 10 bacterial cells. This multiplex PCR method can be a rapid and sensitive method for detecting pathogenic V. anguillarum.     

Evaluation of a PCR detection method for Escherichia coli O157:H7/H- bovine faecal samples

AIMS: Combinations of PCR primer sets were evaluated to establish a multiplex PCR method to specifically detect Escherichia coli O157:H7 genes in bovine faecal samples. METHODS AND RESULTS: A multiplex PCR method combining three primer sets for the E. coli O157:H7 genes rfbE, uidA and E. coli H7 fliC was developed and tested for sensitivity and specificity with pure cultures of 27 E. coli serotype O157 strains, 88 non-O157 E. coli strains, predominantly bovine in origin and five bacterial strains other than E. coli. The PCR method was very specific in the detection of E. coli O157:H7 and O157:H- strains, and the detection limit in seeded bovine faecal samples was <10 CFU g(-1) faeces, following an 18-h enrichment at 37 degrees C, and could be performed using crude DNA extracts as template. CONCLUSIONS: A new multiplex PCR method was developed to detect E. coli O157:H7 and O157:H-, and was shown to be highly specific and sensitive for these strains both in pure culture and in crude DNA extracts prepared from inoculated bovine faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This new multiplex PCR method is suitable for the rapid detection of E. coli O157:H7 and O157:H- genes in ruminant faecal samples.     

禽大肠杆菌、肠炎、鼠伤寒、鸡白痢及鸡伤寒沙门菌多重PCR方法的建立及应用

【背景】大肠杆菌病和沙门菌病是最常见的家禽细菌性疾病,给养禽业造成严重经济损失。另外,禽大肠杆菌和沙门菌也是重要的人畜共患病原菌,可通过禽类及其产品传播给人类,对人类健康造成严重威胁。加强禽大肠杆菌和沙门菌的快速鉴别检测,对养禽业和公共卫生都具有重要意义。【目的】建立禽大肠杆菌、肠炎沙门菌、鼠伤寒沙门菌、鸡白痢沙门菌和鸡伤寒沙门菌的多重PCR检测方法。【方法】通过比较分析确定禽致病性大肠杆菌、肠炎沙门菌、鼠伤寒沙门菌、鸡白痢沙门菌和鸡伤寒沙门菌的特异靶标基因,设计5对特异性引物,通过条件优化建立多重PCR方法,分析该多重PCR方法的特异性、敏感性及可靠性。【结果】该方法能特异性地鉴定禽致病性大肠杆菌、肠炎沙门菌、鼠伤寒沙门菌、鸡白痢沙门菌和鸡伤寒沙门菌,每个PCR反应的最低检出限分别为103 CFU细菌和100 pg基因组DNA。临床分离菌株检测显示,多重PCR与传统血清学方法结果一致。【结论】建立的多重PCR方法能够快速鉴别禽致病性大肠杆菌和不同血清型沙门菌,对禽大肠杆菌病和沙门菌病的流行病学调查及临床检测具有重要意义。     

PCR detection of genetically modified soya and maize in foodstuffs

The detection of genetically modified foodstuffs is becoming both a food sales and legal necessity. This study reports a rapid DNA extraction/PCR-based method for the detection of genetically modified soya (GMS) and maize (GMM) in mixed samples of transgenic and unmodified soybeans and maize kernels, and a variety of processed samples including soya flour, soya protein isolates, extruded defatted soya, acid- and alcohol-precipitated soya concentrates, soya lecithin, maize grits, seasoned corn puffs and salted corn chips. The presence of GMS DNA was determined with two pairs of primers directed towards different GMS target sequences and GMM by one primer pair. In addition, a multiplex PCR reaction which utilises an internal positive control was developed for both genetically modified organisms (GMOs). Results indicated that the methods are sensitive and specific enough to detect GMS down to a level of 0.01% dry weight in single-product PCRs and 0.1% in multiplex PCRs and GMM down to 0.001% dry weight in single-product PCRs and 0.01% in multiplex PCR. The methods are considered to represent a viable route for the commercial detection of GMS and GMM in foodstuffs.     

应用xMAP液念芯片多重快速检测四种病原微生物的研究

目的:建立一种多重、快速、特异性好、灵敏度高的病原微生物检测方法。方法:根据GenBank数据库中的小肠结肠炎耶尔森氏菌、单核细胞增生性李斯特菌、产气荚膜梭菌、鼠疫耶尔森氏菌基因序列,分别针对ail、hly、cpe、3a基因设计4对引物和4条探针。通过重叠PCR扩增各目的基因并构建重组质粒,以该重组质粒DNA为模板,通过多重PCR同时扩增上述4个基因,建立xMAP液态芯片检测技术,在此基础上对标准菌株基因组DNA进行检测并验证该方法的特异性和敏感性。结果:xMAP液态芯片对质粒DNA和标准菌株基因组DNA的检测结果与多重PCR结果一致。该方法能在3.5 h内同时完成对4种病原菌的检测,特异性好,且敏感性要高于PCR方法,灵敏度最高可达200CFU/ml。结论:xMAP液态芯片技术是病原微生物的多重快速检测的新方法,具有很好的应用价值和前景。     

应用xMAP液态芯片多重快速检测四种病原微生物的研究

目的：建立一种多重、快速、特异性好、灵敏度高的病原微生物检测方法。方法：根据GenBank数据库中的小肠结肠炎耶尔森氏菌、单核细胞增生性李斯特菌、产气荚膜梭菌、鼠疫耶尔森氏菌基因序列,分别针对ail、hly、cpe、3a基因设计4对引物和4条探针。通过重叠PCR扩增各目的基因并构建重组质粒,以该重组质粒DNA为模板,通过多重PCR同时扩增上述4个基因,建立xMAP液态芯片检测技术,在此基础上对标准菌株基因组DNA进行检测并验证该方法的特异性和敏感性。结果：xMAP液态芯片对质粒DNA和标准菌株基因组DNA的检测结果与多重PCR结果一致。该方法能在3.5 h内同时完成对4种病原菌的检测,特异性好,且敏感性要高于PCR方法,灵敏度最高可达200CFU/ml。结论：xMAP液态芯片技术是病原微生物的多重快速检测的新方法,具有很好的应用价值和前景。     

Response surface methodology to design a selective co-enrichment broth of Escherichia coli, Salmonella spp. and Staphylococcus aureus for simultaneous detection by multiplex PCR

Escherichia coli, Salmonella spp. and Staphylococcus aureus are frequent co-visitors of contaminated foods to cause food-borne diseases. To achieve rapid detection of three organisms by multiplex PCR, a selective co-enrichment broth was considered to design using response surface methodology (RSM) in this work. NaCl, LiCl and KSCN as selective bacterial inhibitors were selected to optimize their concentrations for a matched composition of bacterial biomass with uniform amplification of three targets. Central composite design was employed to collect the data and fit the responses. Three quadratic polynomial models were derived by computer simulation. A statistical analysis was carried out to explore the effects of the variables on the composition of bacterial biomass and PCR amplification yields. In the end, a novel broth (ESS-3 broth) of NaCl 1.60%, LiCl 0.70%, KSCN 0.10% was formulated to allow co-enrichment of the target pathogens and suppress growth of some non-target pathogens. The simultaneous detection of E. coli, Salmonella spp. and S. aureus was developed on a rapid, convenient and sensitive method consisting of selective co-enrichment in ESS-3 broth, DNA extraction with the boiling method and robust test by multiplex PCR. Our work provided broader application of RSM for the simultaneous detection of other combinations of multiple pathogens.