茶籽油DNA提取方法的比较

针对食用油脂中DNA含量极低、DNA序列片段短、破坏严重的特点,通过实时荧光PCR法检测植物的通用tRNALeu,对茶籽油DNA几种提取方法进行比较,建立了食用油脂中DNA提取新方法。结果证明:较之于文献中常见植物油DNA几种提取方法,用改进方法提取的DNA含量高、纯度高,可以作为PCR反应的模板,为食用油脂进行核酸类生物性检测提供了一种简捷有效的方法。     

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。     

白砂糖DNA提取方法的比较

为了得到高效的白砂糖DNA提取方法,以5种白砂糖为研究对象,从前处理方法、裂解液成分及DNA纯化方法等方面对传统CTAB法进行改良,并比较了CTAB法和改良CTAB法的提取效果,结果表明:改良CTAB法提取的白砂糖DNA浓度都在2.60μg/m L以上,纯度在1.8左右,且通过实时荧光定量PCR能扩增出18Sr DNA基因的对应荧光信号。因此,改良CTAB法能够高效地提取白砂糖的DNA,可为后续的分子检测奠定基础。     

牛奶中大肠杆菌和无乳链球菌的快速检测

大肠杆菌和无乳链球菌是两种导致奶牛乳房炎的重要致病菌,这两种致病菌的检测有助于及时诊断和治疗奶牛乳房炎,从而有效控制牛奶质量.利用16S-23S rDNA间区作为检测靶点,建立牛奶中大肠杆菌和无乳链球菌的快速PCR检测方法,并对该检测方法的灵敏度和特异性进行了确认.结果显示,该方法对于牛奶中大肠杆菌和无乳链球菌的检出限均能够达到1 cfu/mL,并且与其它7种对照菌株均无交叉反应,整个检测过程可在20 h内完成.     

快速提取鸭血基因组DNA方法的研究

目的:以鸭血为原料,筛选出高质量、高效率的DNA提取方法。方法:选用4种不同方法提取样品DNA,并建立实时荧光PCR实验,以此鉴别掺假伪劣产品。通过研究DNA质量浓度、提取时间和扩增效果,选取高质量、高效率的提取方法。结果:苯酚/氯仿抽提法提取浓度高、成本低,但步骤烦琐、耗时长,易接触有毒有害试剂;试剂盒法提取DNA纯度较高,但价格昂贵,不适用于大批量样品;核酸提取仪操作简便,但前处理耗时长,适用于大批量产品的检验检测;细胞裂解法操作简单、快速、成本低,因未经后续纯化,所得到的DNA纯度相对较低,但同样满足PCR扩增的检测要求,适用于大批量监督抽查任务。结论:对于大批量的鸭血源性成分检测推荐使用细胞裂解法,但一旦出现阳性样品需要进行复测则需要使用不同的提取方法,以确保实验的准确性。     

黄酒麦曲微生物总DNA提取方法比较

为得到高质量提取麦曲总DNA的方法,采用SDS法、氯化苄法、CTAB法、超声波法、Soil DNA kit法、SDS高盐法及SDS-CTAB法对黄酒麦曲中总DNA的提取效果进行了比较,通过凝胶电泳、PCR、紫外分光光度计及Real-time PCR对不同方法提取产物进行分析得出7种方法中SDS-CTAB法对于提取麦曲总DNA效果最好,蛋白、多糖及小分子等污染较少,DNA提取的质量浓度达到149.6 ng/μL,相对于SDS法,细菌和真菌模板数分别达到其2.343倍和1.753倍。     

大豆卵磷脂DNA的提取方法及应用

以大豆卵磷脂为研究对象,比较了CTAB法、磁珠试剂盒法以及硅胶膜试剂盒法等3种DNA提取纯化方法。通过大豆内源基因Lectin实时荧光PCR检测,发现CTAB法提取的大豆卵磷脂DNA质量优于其它两种方法,并以CTAB法为基础进行条件优化,进一步提高DNA纯度。采用该方法对6个不同品牌的市售大豆卵磷脂产品进行转基因成分(CAMV35S启动子及NOS终止子)检测,结果证实均不含转基因成分。     

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...     

多重PCR检测原料乳中沙门氏菌、无乳链球菌和金黄色葡萄球菌的研究

目的:建立快速检测沙门氏菌、无乳链球菌和金黄色葡萄球菌的多重PCR方法。方法:根据沙门氏菌(Salmonella)组氨酸转运操纵子基因、无乳链球菌(Streptococcus agalactiae)的STRA-Agl-23-1D基因和金黄色葡萄球菌(Staphylococcus aureus)的耐热核酸酶nuc基因分别设计引物,进行PCR扩增及反应条件的优化,建立这3种菌的多重PCR检测方法。结果:通过建立多重PCR方法,3对引物同时特异性地扩增出495 bp、360 bp和279 bp目的片段;3种菌的检测限:沙门氏菌1.9×102 cfu/mL、无乳链球菌2.0×102 cfu/mL、金黄色葡萄球菌2.9×102 cfu/mL;3种菌DNA含量的(最低)检出限分别为3.86、25.5、3.47pg。结论:本文建立的多重PCR检测方法,简单、快速、灵敏度高,具有很好的应用前景。     

酸乳中乳杆菌和链球菌的实时荧光定量分析

采用实时荧光定量分析了酸乳中的德氏乳杆菌L2和嗜热链球菌S1的数量,德氏乳杆菌L2的活菌数与细胞循环数的相互关系曲线为Y=-2.936logX+34.72(R2=0.998),采用平板计数法获得的样品中L2浓度为8.82×10smL-1,荧光定量的结果是8.78×108mL-1;嗜热链球菌S1活菌数与细胞循环教的相互关系曲线为Y=-3.03910gX+35.45(R2=0.995),采用平板计数法,获得的样品中嗜热链球菌S1浓度1.08×108mL-1,荧光定量的结果是1.06×108mL-1.实时荧光定量的方法适合于酸乳中乳酸菌的快速计数.     

Novel extraction method of genomic DNA suitable for long-fragment amplification from small amounts of milk

Isolation of genomic DNA is a prerequisite for assessment of milk quality. As a source of genomic DNA, milk somatic cells from milking ruminants are practical, animal friendly, and cost-effective sources. Extracting DNA from milk can avoid the stress response caused by blood and tissue sampling of cows. In this study, we optimized a novel DNA extraction method for amplifying long (>1,000 bp) DNA fragments and used it to evaluate the isolation of DNA from small amounts of milk. The techniques used for the separation of milk somatic cell were explored and combined with a sodium dodecyl sulfate (SDS)-phenol method for optimizing DNA extraction from milk. Spectrophotometry was used to determine the concentration and purity of the extracted DNA. Gel electrophoresis and DNA amplification technologies were used for to determine DNA size and quality. The DNA of 112 cows was obtained from milk (samples of 13 ± 1 mL) and the corresponding optical density ratios at 260:280 nm were between 1.65 and 1.75. Concentrations were between 12 and 45 μg/μL and DNA size and quality were acceptable. The specific PCR amplification of 1,019- and 729-bp bovine DNA fragments was successfully carried out. This novel method can be used as a practical, fast, and economical mean for long genomic DNA extraction from a small amount of milk.     

麦芽糊精基因组DNA不同提取方法的比较

采用不同方法对麦芽糊精基因组DNA进行提取,从中选取满足麦芽糊精材料进行分子标记检测的最好的DNA提取方法。以市售麦芽糊精为材料,分别采用CTAB、SDS和异硫氰酸胍3种方法提取基因组DNA,比较不同方法的提取效果。光密度检测结果显示,异硫氰酸胍法的提取量和纯度均优于SDS法和CTAB法;琼脂糖凝胶电泳检测结果显示异硫氰酸胍法提取的基因组DNA谱带清晰、重复性好,SDS法和CTAB法提取的基因DNA观察不到谱带;3种方法提取的麦芽糊精基因组DNA都能够满足PCR分析的需要。结果表明,3种方法均能有效地从麦芽糊精中获得DNA,然而异硫氰酸胍法优于SDS法和CTAB法。     

不同热处理方法对鲤鱼DNA的影响及不同DNA提取方法的比较

目的研究不同加热方法处理后鲤鱼基因组DNA长度和含量的变化,并对4种DNA提取方法进行比较。方法用电磁炉和微波炉将鲤鱼背部肌肉加热煮沸1、2、3 h取出,再分别用酚氯仿法、Wizard试剂盒法、磁珠法和离心柱法提取处理前后鲤鱼基因组DNA,利用琼脂糖凝胶电泳对处理前后的总DNA长度进行比较,再用实时荧光定量PCR法检测所得DNA样品中内参基因β-actin含量的变化。结果琼脂糖凝胶电泳结果显示,处理后DNA片段明显降解。生鱼肉提取β-actin的量为1 070 213.39拷贝/μl,电磁炉处理1、2、3 h的浓度分别为143 309.6、441 350.43和256 994.16拷贝/μl,微波炉处理1、2、3 h的浓度分别为269 121.17、267 371.16和134 649.97拷贝/μl。酚氯仿法、Wizard试剂盒法、离心柱法和磁珠法提取的平均DNA浓度分别为718 944.93、737 945.64、26 751.22和2 934.06拷贝/μl。结论加热煮沸导致DNA降解,因此加工食品中DNA的检测应当选择较短的目标片段。4种DNA提取方法比较,Wizard试剂盒法和酚氯仿法提取的D...     

动物肌肉组织基因组DNA两种提取方法的比较

以猪、牛、羊、鸡等动物新鲜冷冻样品为实验材料,采用SDS法和异硫氰酸胍法提取动物肌肉组织基因组DNA,对提取的DNA进行光密度、琼脂糖凝胶电泳和PCR检测,比较了这两种方法的提取效果。结果表明:采用SDS法和异硫氰酸胍法均能从动物肌肉组织提取到完整的基因组DNA,均可满足PCR等后继分子生物学实验的要求。但是异硫氰酸胍法提取的基因组在纯度和浓度及稳定性方面优于SDS法,且操作简单,耗时短,更利于快速检测。      

Comparison of DNA quality in raw and reconstituted milk during sterilization

Responses to milk sterilization are usually evaluated only in terms of physicochemical properties and microbial safety, thus undervaluing the importance of DNA quality in an authentication process by methods based on PCR. Because DNA is a heat-sensitive molecule, we hypothesized that the heating process may impair the detection or quantification of DNA in raw fresh milk (FM) or reconstituted milk (RM), and that differences in DNA quality might exist between FM and RM. We thus investigated the effects of sterilization on the quality of DNA extracted from FM or RM; differences in DNA quality between FM and RM were also evaluated. The quality of extracted DNA from FM or RM was assessed by the specific detection of FM or RM composition in goat milk mixtures using primers targeting the bovine 12S gene, as well as by monitoring DNA yield, purity, ratio of mitochondrial (mt) to nuclear (n) DNA, and the level of DNA degradation. Polymerase chain reaction readily detected both untreated and heat-treated FM or RM in cow-goat milk mixtures, and gave a good sensitivity threshold (0.1%) under all sterilization conditions. The DNA yield and mtDNA:nDNA ratio of FM and RM varied significantly during the sterilization process. These results demonstrated that the sterilization altered the quantification of DNA in FM or RM during sterilization, but DNA could still be readily detected in sterilized FM or RM by PCR. Furthermore, we noted significant differences in DNA integrity, yield, and mtDNA:nDNA ratio between FM and RM during sterilization, which may have potential as a means to distinguish FM and RM.     

Development of a rapid mitochondrial DNA extraction method for species identification in milk and milk products

Isolation of mitochondrial DNA (mtDNA) from milk offers an effective way to monitor aspects of quality control and traceability to ensure food safety. A few methods of DNA isolation from milk have been reported, but many of them are time consuming and expensive. Here, we report a rapid, simple, and efficient method of mtDNA extraction from raw and processed milk (pasteurized, retorted, and UHT milk) to generate substrate for analysis using any PCR analysis platform. Various techniques used for the separation of mitochondria were explored and combined with a sodium dodecyl sulfate method for mtDNA extraction from raw and processed milk. The optimized protocol supports the efficient amplification of mtDNA independent of sample origin and is sufficiently straightforward to allow its widespread adoption by industry.     

几种提取转基因木瓜DNA的方法比较

目的 比较CTAB法、SDS法及试剂盒法分别提取木瓜果皮、果肉、种籽DNA的效果, 以提高转基因木瓜的检测准确率。方法 对转基因木瓜内源基因Papain及外源基因NPTII、CP进行普通PCR, 同时对基因表达零件CaMV35S启动子和NOS终止子进行荧光PCR, 以检测提取DNA的质量。结果 分析电泳产物发现, SDS法提取木瓜种籽样品的DNA未能适合普通PCR检测的要求, 其他方法均能满足普通PCR要求。荧光PCR结果显示, 3种方法提取的DNA都满足荧光扩增要求。结论 比较3种提取方法及3种木瓜材料, 发现用CTAB法或SDS法以果皮为材料提取DNA质量最佳。