基于DGGE分析的大鼠粪便及肠道细菌DNA提取方法研究

为获得高质量肠道细菌总DNA用于研究膳食纤维对大鼠肠道菌群的影响,本实验以SD大鼠为实验动物,灌胃番茄皮膳食纤维,收集大鼠粪便及盲肠,-70℃冰箱保存。分别采用Tiangen细菌基因组试剂盒法、蛋白酶K-十六烷基三甲基溴化铵(CTAB)法和溶菌酶法提取粪便和肠道中的细菌总DNA,通过琼脂糖凝胶电泳、细菌通用引物PCR扩增和变性梯度凝胶电泳(DGGE)对提取效果进行观察比较,发现将溶菌酶法进行改进后,可以获得满意效果。改进后的溶菌酶法与另外两种方法比较,具有成本低、时间短、DNA得率高和多样性好等特点;该方法获得的DNA样品适合于用DGGE技术分析大鼠粪便及肠道菌群。     

鱼糜制品细菌菌群多样性的PCR-DGGE方法建立

本文优化了冷藏过程中鱼糜制品DNA提取的前处理方法,并采用5种方法提取,应用分光光度法和琼脂糖凝胶电泳估计DNA的纯度和得率;比较了普通PCR和降落PCR反应的扩增效果,运用DGGE分析了鱼丸在冷藏过程中细菌菌群的多样性,并在割胶回收后克隆、鉴定及测序。试验结果表明:玻璃珠-试剂盒法为最佳的DNA提取方法,梯度PCR扩增较普通PCR扩增产物多且纯度高;随着冷藏时间的延长,鱼丸中的细菌种类和数量呈一定的变化趋势,说明PCR-DGGE技术可用于分析鱼丸在冷藏过程中细菌菌群的变化规律。     

PCR—DGGE法分析婴儿肠道菌群多样性

利用PCR—DGGE技术探究婴儿肠道菌群的多样性,为益生菌在婴儿配方乳粉中的应用提供理论依据。采用细菌的16S rDNA V3区通用引物,以15例食源性腹泻婴儿和15例健康婴儿粪便样本的总DNA为模板,PCR扩增后进行变形梯度凝胶电泳(DGGE)分析。结果表明:30份粪便样本的DGGE图谱均表现为高度多态性。食源性腹泻婴儿肠道菌群多样性与健康组婴儿肠道菌群多样性有明显差异,且食源性腹泻婴儿肠道菌群多样性较低。通过聚类分析和相似度分析,可以得出食源性腹泻婴儿的肠道菌群有高度的相似性,与健康婴儿的肠道菌群有明显的差异。PCR—DGGE技术可以初步分析婴儿肠道菌群的多样性,进而为婴儿配方乳粉的研制提供理论依据。     

吡格列酮对KKAy小鼠肠道菌群的影响

目的 研究2型糖尿病动物模型KKAy小鼠与正常C57BL/6J小鼠肠道微生物的差异和降糖药物吡格列酮对KKAy小鼠肠道菌群结构的影响,阐明宿主基因型对肠道菌群的影响及吡格列酮、肠道菌群和2型糖尿病之间的内在联系,为研究和治疗2型糖尿病提供新思路.方法 将KKAy小鼠分为药物组和模型对照组,C57BL/6J小鼠为正常对照组,定期收集小鼠粪便样品,提取其中微生物的总DNA,PCR扩增16S rDNA V3区,用DGGE(变性梯度凝胶电泳)技术分离扩增片段,结合DGGE图谱的数字化分析和特异条带克隆测序等分子生物学方法分析肠道微生物群落结构.结果 KKAy小鼠与C57BL/6J小鼠的DGGE图谱有明显差异,正常对照组小鼠肠道菌群多样性保持稳定,而模型对照组和药物组肠道菌群的多样性随时间延长逐渐下降,药物组下降趋势略大于模型对照组.主成分分析结果显示,喂药前KKAy小鼠(包括模型对照组和药物组)和C57BL/6J小鼠沿PC1方向分别聚为2类,随着实验的延续,药物组逐渐靠近正常对照组,并且喂药70 d后沿PC1方向,药物组与正常对照组聚在一起,而模型对照组另聚一类.结论 肠道微生物群落结构与宿主的基因型有明显的相关性;吡格列酮抑制某些细菌生长,降低了肠道菌群的多样性,同时能够改善肠道中主要微生物的菌群结构.     

大鼠粪便中细菌基因组DNA提取方法的比较

为获得高质量的肠道细菌基因组DNA,用于研究肠道菌群的多样性,本实验分别采用改良的溶菌酶法和两种试剂盒DNA提取法提取大鼠粪便中的细菌基因组DNA,通过DNA浓度和纯度的测定、PCR-变性梯度凝胶电泳技术(PCR-DGGE技术)对提取效果进行比较,以找到适合于粪便中细菌基因组DNA的提取方法。结果表明,改良的溶菌酶法提取的DNA质量好,纯度高,而且具有菌群多样性丰富等特点,更适合用于肠道微生物菌群后续分子生物学研究。     

用于转基因检测的番木瓜基因组DNA提取方法的比较

本研究以番木瓜果实的不同部位,果皮、果肉和种籽为材料,比较了改进CTAB法和试剂盒法两种提取基因组DNA的方法。分别对植物叶绿体基因rbcL和番木瓜特异基因Papain进行了普通PCR和荧光PCR扩增,检验提取DNA的质量。结果显示改进CTAB法和试剂盒法都能提取得到纯度较高的DNA,适合普通和荧光PCR反应的要求,适用于外源基因的检测。改进CTAB法提取的DNA浓度要高于试剂盒法,而试剂盒法提取DNA所用的时间较短,更为方便,但成本较高。同时材料取样部位最好是果皮和果肉,果皮和果肉为材料提取得到的DNA纯度很高,适用于荧光PCR的要求。     

用DGGE技术分析植物乳杆菌对小鼠肠道菌群失调的影响

采用PCR-DGGE方法研究植物乳杆菌对小鼠肠道失调菌群微生态的影响.选用昆明雌性小鼠建立小鼠抗生素相关性菌群失调模型,灌服植物乳杆菌菌液(109CFU/mL)5d,分离提取小鼠粪便中微生物群落的DNA,通过PCRDGGE技术分析植物乳杆菌调节过程微生物群落结构与多样性的影响.PCR-DGGE图谱显示,随着植物乳杆菌饲喂时间的延长,小鼠肠道菌群丰富度呈现逐渐增加的演替过程.植物乳杆菌灌胃第4d小、鼠肠道菌群的丰富度指数(18)与多样性指数(2.75)与正常小鼠基本一致,DGGE图谱的测序结果表明,植物乳杆菌调节小鼠肠道菌群失调主要表现在两个方面:选择性地增加有益菌如Bacteroides stercoris、Bacteroides uniformis strain等的量;抑制致病菌如Clostridium clostridioforme、Pseudomonas stutzeri等的生长.说明植物乳杆菌对头孢地尼引起的肠道菌群失调具有调整作用;建立PCR-DGGE法监测肠道菌群动态变化的分析方法,为进一步研究益生菌对肠道菌群的作用机制奠定基础.     

食源性腹泻儿童肠道菌群的变化研究

目的研究食源性腹泻儿童肠道菌群的变化,为儿童食源性腹泻的防治提供理论依据。方法以10例食源性腹泻儿童粪便样本及10例健康儿童的粪便样本为研究对象,提取粪便总DNA,利用细菌16S rDNA V3区通用引物进行PCR扩增,扩增后产物进行变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)分析,并用Quantity One软件进行分析。结果食源性腹泻组儿童和对照组健康儿童的肠道菌群多样性存在显著性差异,食源性腹泻组的多样性低于对照组。食源性腹泻组儿童肠道菌群具有较高的相似性并与对照组儿童肠道菌群能聚类到不同簇中。结论食源性腹泻儿童肠道菌群多样性发生明显变化,说明肠道菌群的多样性调控可以应用于食源性腹泻的缓解或治愈。     

豉香型白酒酒饼中细菌PCR-DGGE分析方法的建立

本研究以豉香型白酒酒饼中的细菌类群为对象,针对PCR-DGGE分析方法中DNA提取、PCR扩增、DGGE电泳时间和凝胶染色方法等重要环节开展了相关技术参数的比较和优化。结果显示:试剂盒法、CTAB法、SDS法和SDS-CTAB结合法等四种DNA提取方法中,SDS-CTAB结合法提取的DNA得率最高,蛋白质去除干净,完整性好,虽然步骤稍繁琐,但优于其他方法;经均匀设计法优化后,PCR反应中退火温度为50℃,引物浓度为0.4μmol/L,模板2.5μL(约34 ng)时所扩增出的条带最清晰、产物量最高,对应的DGGE分析中DNA条带的多样度和丰度最佳;以进程法比较了不同时长的DGGE电泳效果,发现在变性剂梯度范围为30%~60%、电压85 V、温度60℃条件下,电泳9 h DGGE胶中的DNA条带分离充分,分布位置适中;同时还发现,利用银染色法对胶进行染色,效果优于Goldview染色法。综合上述因素,初步建立了豉香型白酒酒饼微生物PCR-DGGE技术的分析方法。     

磁珠法提取肉及肉制品基因组DNA的效果研究

磁珠法提取DNA具有可自动化、高通量、操作简单、耗时短和安全无毒等优点。通过测定所提DNA产物的浓度、纯度、得率,荧光定量PCR扩增的Ct值和试剂盒的特异性、敏感性等参数指标,系统研究了几种磁珠试剂盒及方法的提取效果。结果表明,方法经优化后,所选6种试剂盒均适用于鲜肉组织的DNA提取。针对加工样品的DNA提取,建议选择试剂盒2、3、4结合该研究优化方法,其特异性和敏感性均超过90%。研究为建立磁珠DNA提取试剂盒质量评价标准奠定基础,同时为磁珠DNA提取试剂盒研发和磁珠法提取肉及肉制品基因组DNA提取方法的选择提供数据支撑和技术参考。     

金枪鱼制品中几种DNA提取方法的效果比较

金枪鱼种类繁多,市场上以次充好、以假乱真的现象时有发生。基于DNA的检测技术已经被广泛用于金枪鱼制品品种掺假检测。而DNA提取对DNA检测技术的运用至关重要。本研究以28份深加工金枪鱼产品为样本,采用3种DNA提取方法(SDS法以及2种市售试剂盒)进行DNA提取,并对DNA的质量、得率、PCR扩增及方法的可操作性等方面进行比较研究。结果表明,3种方法均可用于大多数金枪鱼制品的DNA提取。与其它2种市售试剂盒相比,SDS法提取的DNA得率较低,但纯度较好,A_(260)/A_(280)比值为1.89,盐残留少,对实时荧光定量PCR的抑制少,且成本相对价格经济,满足金枪鱼制品掺假检测的需求。     

Comparison of DNA Extraction and PCR Setup Methods for Use in High-Throughput DNA Barcoding of Fish Species

DNA barcoding is a sequencing-based method that can be used for the identification of fish species in a regulatory setting. The objective of this study was to compare modified versions of three DNA extraction kits (i.e., Qiagen DNeasy Blood and Tissue Kit, Sigma-Aldrich Extract-N-Amp Kit; and Life Technologies MagMax-96 DNA Multi-Sample Kit) and two polymerase chain reaction (PCR) setup methods (manual vs. automated) for use in DNA barcoding, with a focus on minimizing time, costs, and labor. DNA was extracted from 83 fish products using each of the three kits and the results were compared based on sequencing success and sequencing quality parameters. A subset of 14 fish products was also tested in triplicate to compare PCR setup methods. Initially, reduced sequencing success was observed with the MagMax Kit (88 %) compared to the other two kits (95–96 %); however, after PCR and sequencing were repeated for DNA samples that initially failed, all three methods showed very high sequencing success (98–99 %). Overall, the modified Extract-N-Amp Kit offered the greatest reduction in time and costs, while the DNeasy Blood and Tissue Kit produced sequences with the highest quality and highest initial success rates. Automation of the PCR setup process resulted in slightly greater success (100 %) compared to manual PCR setup (98 %), and reduced the potential for human error that may result from manual pipetting. The results of this study demonstrate the advantages of incorporating rapid and/or automated methods into the DNA barcoding workflow, especially with regard to high-throughput operations.     

Application of DNA extraction kit, 'GM quicker' for detection of genetically modified soybeans

Several DNA extraction methods have been officially introduced to detect genetically modified soybeans, but the choice of DNA extraction kits depend on the nature of the samples, such as grains or processed foods. To overcome this disadvantage, we examined whether the GM quicker kit is available for both grains and processed foods. We compared GM quicker with four approved DNA extraction kits in respect of DNA purity, copy numbers of lectin gene, and working time. We found that the DNA quality of GM quicker was superior to that of the other kits for grains, and the procedure was faster. However, in the case of processed foods, GM quicker was not superior to the other kits. We therefore investigated an unapproved GM quicker 3 kit, which is available for DNA extraction from processed foods, such as tofu and boiled soybeans. The GM quicker 3 kit provided good DNA quality from both grains and processed foods, so we made a minor modification of the GM quicker-based protocol that was suitable for processed foods, using GM quicker and its reagents. The modified method enhanced the performance of GM quicker with processed foods. We believe that GM quicker with the modified protocol is an excellent tool to obtain high-quality DNA from grains and processed foods for detection of genetically modified soybeans.     

食用植物油DNA提取方法的优化

目的建立可从不同种类食用植物油中提取得到高质量的、可用于分子生物学检测的DNA的提取方法。方法使用2种改进十六烷基三甲基溴化铵法(cetyltrimethylammonium bromide,CTAB)和2种商用试剂盒提取6种食用植物油的DNA,通过紫外分光光度计检测所得DNA样品的浓度和纯度。设计特异性引物,并通过普通聚合酶链式反应(polymerase chain reaction,PCR)和实时荧光定量PCR(quantitative real-time PCR,qPCR)检测DNA样品是否可用于分子生物学分析。结果使用改良CTAB法1及2种商业试剂盒无法有效提取得到的6种食用植物油的DNA,样品无法满足分子生物学检测的需要,使用改良CTAB法2提取得到的6种食用植物油DNA纯度和浓度检测结果均为最佳,其提取的橄榄油、菜籽油、花生油DNA样品可用于后续实时荧光定量PCR检测。结论该方法可为橄榄油、菜籽油、花生油DNA提取提供有效手段,为食用植物油检测和研究奠定基础。     

大豆粉转基因成分能力验证样品的检测分析

目的 提高实验室对转基因大豆定性检测的准确性和检测人员专业技术水平, 增强实验室竞争力。方法 通过核酸蛋白仪器分析法和单重实时荧光PCR (simplex real-time fluorescence PCR)法对样品内源基因扩增的循环阈值的影响来比较2种方法, 分析2种DNA提取试剂盒的提取质量。对标准SN/T 1204-2016《植物及其加工产品中转基因成分实时荧光PCR定性检验方法》扩增反应体系中DNA模板量进行优化后, 对样品进行检测。再依据标准SN/T 1202-2010《食品中转基因植物成分定性PCR检测方法》的要求, 对样品进行检测。最后对2个标准的检测结果进行比对。结果 通过内源基因扩增循环阈值的数据确认, 更能准确反映试剂盒提取的DNA是否满足后续外源基因的分析检测要求。2种标准方法对19-N578和19-M913 2个待测样品的检测显示3种外源基因CaMV35S、NOS、CP4-EPSPS均为阴性, 19-N578和19-M913待测样品均为非转基因大豆。结论 本次能力验证获得满意评价。DNA提取质量的评估, 体系中DNA模板量的优化, 检测方法的选择和实验的质量控制都是影响能力验证结果的重要因素。     

DNA Extraction from Olive Oil and PCR Amplification of Microsatellite Markers

ABSTRACT: DNA was extracted from single-cultivar of cold-pressed (virgin) unfiltered and cotton-filtered olive oils that were stored at 4 °C for up to a year using different DNA extraction kits and protocols. DNA was amplified using original and nested primers designed on 6 microsatellites loci of the UDO series. The most consistent results in terms of successful single sequence repeat amplifications were achieved using the Qiagen QIAamp DNA stool extraction kit, slightly modified and applied to oil sample amounts as small as 200 μL without any pretreatment. The kit allowed getting polymerase chain reaction (PCR) amplicons visible on gel and scorable peaks at the automatic sequencer for all 6 markers analyzed. Less consistent results were achieved with other kits, such as the Promega Wizard Magnetic DNA Purification System for Food, the LB Link-Biotech ExtMan 50–100 Evolution, the Qiagen Plant Mini kit, and the standard cetyltrimethyl-ammonium bromide-based DNA extraction protocol. The integration in the protocols of further tools, such as the hexane-based phase separation, the addition of water or NaCl solutions to the oil, the precipitation and the use of the pellet, and others, did not result in any substantial use. PCR amplifications that gave low DNA yields were improved by adopting the nested PCR technique, which uses the product of the 1st PCR as a template for a 2nd PCR carried out by means of internal primers. Conclusions are drawn as to the applicability of the method to trace the identity of single-cultivar virgin olive oils. Further work is required to check the sensitivity of the method in determining the varietal composition of blended oils, especially in detecting alleles from cultivars present in only small amounts.     

植物源性食品DNA提取方法的建立及几种方法比较

以大豆和马铃薯植物源性食品为研究对象,比较了研究所建立的基于磁珠的DNA提取方法、3种商品试剂盒法和传统CTAB法共5种方法提取各种食品基因组DNA的效果;通过琼脂糖凝胶电泳、紫外分析法、定性PCR和实时荧光定量PCR法对所提取的DNA进行检测,比较所得DNA的浓度和纯度,以及对下游检测的适用性。结果表明,与其他几种提取方法相比,研究所建立的方法提取到的DNA浓度稍低,但纯度较高,而且对于豆油和淀粉样品,可很好地提取到基因组片段,满足后续PCR检测要求。     

4种方法提取牛肉干DNA的效果比较

目的探求熟肉干制品最佳的DNA提取方案。方法采用4种不同的方法:CTAB法及3种市售试剂盒法,提取11种不同牛肉干样品的DNA,通过对方法的提取耗时,提取DNA的质量及提取DNA用于实时荧光PCR扩增的效果3方面进行比较。结果 TAKARA试剂盒法提取DNA耗时最短仅需要0.5 h,OMEGA试剂盒法提取的DNA的纯度较好,A 260/A 280比值最接近1.8,Tiangen的深加工食品DNA提取试剂盒法提取的DNA做实时荧光PCR的CT值最小,扩增效果最佳。结论 Tiangen试剂盒法对于熟肉干制品DNA的提取效果更为理想。     

Evaluation and optimisation of five different extraction methods for soy DNA in chocolate and biscuits. Extraction of DNA as a first step in GMO analysis

A method is described to discriminate between genetically modified (GM) and non‐modified foodstuffs by detecting the presence of newly introduced genes at the protein or DNA level. Currently available methods operate almost exclusively at the DNA level and are based on the polymerase chain reaction (PCR). The first and most crucial step in this process is the isolation of DNA. In this study, five different methods for the isolation of DNA from chocolate and biscuits were evaluated, using four commercially available extraction kits and a non‐commercial method for amplification of the soybean‐specific lectin gene. The latter method involves the use of hot‐start Taq polymerase, to prevent the formation of non‐specific amplification products, and an increase in the number of cycles from 35 to 41. The performance of the non‐commercial cetyl trimethylammonium bromide (CTAB)‐based method was the best, taking into consideration the adaptations of the extraction procedure, although this method was more time‐consuming than the others. Chocolate (white, milk and dark) and several biscuits generated positive amplification results using this PCR approach. Copyright © 2004 Society of Chemical Industry     

肉制品中志贺氏菌DNA的快速提取方法

肉制品中致病菌DNA的提取对PCR检测具有重要意义。以污染志贺氏菌的猪肉为材料,比较了沸水浴法、裂解液煮沸法、CTAB/SDS法、改良的碱性异硫氰酸胍法4种提取方法对志贺氏菌DNA的提取效果,并以提取的DNA为模板进行PCR检测。结果表明:改良的碱性异硫氰酸胍法提取志贺氏菌DNA的效果较好,猪肉中志贺氏菌的PCR最低检出限为5×100CFU/g,整个检测时间<8h。经改良的碱性异硫氰酸胍法操作简便、经济省时,可用于肉类的PCR检测。