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.     

三种人全血基因组DNA提取方法的比较

目的：比较改良酚一氯仿抽提法、盐析法、试剂盒法从人全血中提取基因组DNA的效果,以期建立一种快速、经济的提取高质量基因组DNA的方法。方法：分别用上述三种方法从人全血中提取基因组DNA,通过紫外分光光度计、琼脂糖凝胶电泳、聚合酶链式反应（PCR）、限制性内切酶酶切检测提取的基因组DNA的产量、纯度和质量。结果：改良酚一氯仿抽提法与试剂盒法提取的基因组DNA相比,DNA的产量有统计学差异,DNA的纯度无统计学差异,但试剂盒法提取的基因组DNA有较明显的降解现象：盐析法与改良酚．氯仿抽提法、试剂盒法相比,基因组DNA的产量和纯度都存在统计学差异,并且基因组DNA聚合酶链式反应（PCR）扩增的稳定性也明显劣于另外两种方法;三种方法提取基因组DNA均能进行限制性内切核酸消化。结论：改良酚一氯仿抽据取法是一种经济、快速、高效、稳定提取人全血基因组DNA的方法,适用于批量临床标本处理。     

一种自制DNA提取剂在微量细胞和单个囊胚上的应用

目前微量DNA的提取方法,不仅操作繁琐耗时,而且所需试剂价格昂贵。为了解决这个问题,本研究旨在开发一种快速、经济的微量DNA提取剂。用自制提取剂快速提取50个、100个、500个、5 000个、50 000个猪胎儿成纤维细胞的基因组DNA。经PCR扩增,电泳结果显示:自制提取剂可有效提取数量低至50个的猪胎儿成纤维细胞。分别采用自制提取剂、酚氯仿以及试剂盒3种方法提取猪单个囊胚基因组DNA,结果显示:酚氯仿方法提取的单个囊胚基因组DNA,经PCR扩增后,并没有检测到目的条带;而自制提取剂提取模板DNA,能直接扩增出清晰的目的条带,与试剂盒扩增效果相当。自制提取剂能够快速、有效地提取少量细胞和单个囊胚的基因组DNA,满足下游分子生物学研究需要,因此,本研究为微量样本基因组DNA提取提供帮助。     

三种提取柱状黄杆菌基因组DNA方法的比较

采用酚氯仿抽提法、CTAB法和SDS-蛋白酶K法分别对鱼类病原菌柱状黄杆菌提取基因组DNA。使用超微量紫外分光光度计和琼脂糖凝胶电泳检测所提取的基因组DNA的产量和质量,并用PCR扩增对DNA进行了评价。结果显示,3种方法均可提取到柱状黄杆菌的基因组DNA,并能有效扩增细菌16S rDNA序列,但CTAB法提取的DNA产量和质量最高,CTAB法可以作为柱状黄杆菌DNA提取以开展分子生物学研究的首选方法。     

香鱼基因组DNA提取方法的优化

为获得高质量的基因组DNA,分别采用传统酚-氯仿法、高盐法、试剂盒法和改进酚氯仿法提取香鱼肌肉基因组DNA。琼脂糖凝胶电泳检测结果表明,改进酚氯仿法提取的基因组DNA电泳条带整齐明亮且无降解。紫外分光度计测定DNA浓度和纯度,结果表明,改进酚氯仿法提取的鱼类基因组DNA浓度约为300μg/mL,A260/A280为1.80-1.86。用改进的酚氯仿法提取的DNA进行AFLP分析,扩增结果稳定,电泳条带清晰。综上所述,改进酚氯仿法能够获得高质量DNA,且可以用于进一步的分子生物学研究。     

利用改进的酚-氯仿法从猪毛囊中提取基因组DNA

为提高从猪毛囊组织中提取基因组DNA的效率, 文章在借鉴从其他组织提取基因组DNA方法的基础上, 对经典的酚-氯仿法的反应体系和步骤进行了改进。利用改进的酚－氯仿抽提法, 从猪的毛囊组织中快速、高效地提取了高质量基因组DNA。利用该方法从1~6根猪毛囊中提取的基因组DNA可满足基于PCR技术的相关分子生物学实验需要。     

一种快速高效提取病原真菌DNA作为PCR模板的方法

真菌rDNA-ITS序列分析适合于较高等级水平的生物群体间的系统分析。真菌DNA的提取采用传统的方法,步骤繁琐,需要较长时间。采用Chelex-100法提取真菌DNA,使用PCR扩增rDNA-ITS序列评价提取核酸的质量。结果显示,该方法具有经济、简便、快速、高效的特点,是一种比较理想的提取真菌基因组DNA作为PCR模板的方法。     

利用不同方法从深加工牦牛肉产品中提取基因组DNA效果的比较

为了从深加工牦牛肉产品中获得片段较大、数量较多的基因组DNA,以用于分子追溯的检测,尝试并系统地比较了异硫氰酸胍法、酚-氯仿抽提法和试剂盒提取法的提取效果。通过定量分析、凝胶电泳和PCR片段大小梯度扩增等手段,对利用3种方法提取的基因组DNA的数量和质量进行了比较。结果显示,利用酚-氯仿抽提法提取的基因组DNA质量不佳,异硫氰酸胍法和试剂盒提取法的提取效果相当,PCR可以扩增出730 bp左右的核外基因细胞色素b,而核内基因只能扩增出300bp左右的核基因DNA片段(内乳铁蛋白基因,271 bp;BoLA-DRB3基因,302 bp)。综合考虑提取时间、费用、数量和质量等各方面的因素,认为异硫氰酸胍法操作简便、快速、廉价、有效,是较好的从动物深加工肉产品中提取基因组DNA的方法。     

Chelex-100快速提取放线菌DNA作为PCR扩增模板

旨在建立有效扩增16S rRNA基因序列的放线菌DNA快速提取的方法。采用Chelex-100法提取放线菌DNA,使用PCR扩增16S rRNA基因序列评价提取核酸的质量。结果显示,Chelex-100法能够在10 min之内从放线菌中快速提取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,符合理论预期结果。因此,Chelex-100法提取放线菌DNA可以作为16S rRNA基因序列PCR扩增的模板,该方法具有经济、简便、快速的特点,适合于放线菌菌株大规模地筛选和分类鉴定。     

两种提取肠道微生物总DNA方法的比较

目的比较肠道微生物总DNA两种提取方法的优缺点。方法采用苯酚-氯仿抽提法和试剂盒法提取肠道微生物总DNA,比较其作为模板扩增细菌16S DNA的优缺点。结果两种方法提取的细菌DNA均能用于后续PCR扩增的模板。酚-氯仿抽提法经济可靠,但提取的DNA量及纯度较低试;剂盒法提取方便,操作简单,质量稳定,易标准化,但成本高。结论两种方法各具优缺点,应根据实验室条件和实验要求进行选择。     

Trichomonas vaginalis detection using real-time TaqMan PCR

1978 women and 93 men, all suspected of having a Trichomonas vaginalis infection, were tested for the presence of T. vaginalis by real-time PCR using the T. vaginalis-specific 2-kb repeated sequence, and by direct microscopy and culture. 40 samples were positive by T. vaginalis real-time PCR and 27 were positive by wet mount microscopy, either direct or after culture. All samples positive by direct microscopy of culture were also positive by real-time PCR. Of the 13 samples which were real-time PCR positive but negative by direct microscopy and culture 11 were confirmed by another T. vaginalis real-time PCR based on the beta tubulin gene. Only 2 samples (0.1%) showed inhibition in the PCR. The prevalence of T. vaginalis infection in the female patients was 1.8%. The sensitivity, specificity, positive and negative predictive values of the real-time PCR were 100%, 99.9%, 95% and 100%, respectively. The same test characteristics for the combined conventional T. vaginalis detection methods (microscopy+culture) were 71%, 100%, 100% and 99%, respectively. Therefore, real-time PCR is the method of choice for the diagnosis of T. vaginalis infection.     

Comparative analysis of three methods from dried blood spots for expeditious DNA extraction from mosquitoes; suitable for PCR based techniques

The objective of this work was to compare the quality, purity and quantity of DNA isolated from dried blood spots (DBS) by three methods (Chelex-100, QIAamp DNA mini kit, and TE (Tris EDTA)-Buffer). Sample collection was performed in six districts in Odisha, India and screened for cases of clinical malaria and dengue and vector density. Mosquito abdomens were spotted on Whatman 3MM (MERCK) Filter paper and dried for 10 min at room temperature. DNA was isolated from DBS using three methods (Chelex-100, QIAamp DNA mini kit, and TE-Buffer), and PCR was used to determine the feeding behaviours of vector mosquitoes. DNA was quantified using a UV-spectrophotometer, and q-PCR was used to determine the target gene copy number to compare the methods. The QIAamp DNA mini kit method was used as the reference method. The yield and purity of DNA extracted with Chelex-100 and TE were 14–72 ng/µl and 1.51–1.85 and 9–50 ng/µl and 1.68–2.1, respectively. DNA extracted using the Chelex-100 method was stored for over 1 month at ? 20 °C and was suitable for later use. The Chelex-100 method had a sensitivity of 99.5% and specificity of 78%. A Bland–Altman plot suggested that the Chelex-100 method was similar to the QIAamp DNA mini kit method for determining the feeding behaviours of vector mosquitoes. The Chelex-100 method is simple, cost-effective, and safe and requires minimal time for DNA extraction from dried blood spots. In malaria and dengue research, detecting the feeding behaviours from mosquito DNA from dried blood spots on filter paper by PCR is an easy, minimally invasive and inexpensive molecular technique that can be performed in remote areas.

     

Specific and sensitive detection of Trichomonas tenax by the polymerase chain reaction

A polymerase chain reaction (PCR) protocol was developed for specific detection of Trichomonas tenax by using a pair of primers designed for its 18S rRNA gene. The detection was specific for T. tenax , since no amplification was detected with DNAs from Trichomonas vaginalis , which belongs to the same genus as T. tenax , in addition to various species of oral protists, fungi and bacteria, and human leukocytes. This method had a detection limit of 100 fg for T. tenax genomic DNA and could detect T. tenax cells in dental plaque at a concentration of as low as 5 cells per PCR mixture. Direct detection from clinical dental plaque samples was also possible ; therefore, the present PCR procedure could provide a simple and rapid detection method of T. tenax in dental plaque.     

四川地区阴道毛滴虫内人型支原体的PCR检测

从临床上分离获得20株阴道毛滴虫虫株,经纯化培养后,提取基因组DNA.以人型支原体16S rDNA序列设计特异性引物,利用PCR技术检测阴道毛滴虫内的人型支原体,结果有13株为人型支原体阳性,感染率为65%,表明阴道毛滴虫与人型支原体的共生关系在中国四川具有普遍性.     

指甲游离缘的核DNA分型研究

为探讨指甲游离缘核DNA分型的可行性, 采集无关个体指甲游离缘样本10份, 分别以不同消化体系, 采用有机法、Chelex-100法,有机法结合Chelex-100法等3种方法提取指甲游离缘核DNA, AmpFlSTR IdentifierTM试剂盒复合扩增, ABI PRISM 3130自动遗传分析仪检测分析。结果显示：与对照血样比较,有机法结合Chelex-100法提取的样本核DNA均获得满意的STR分型, 有机法提取的样本核DNA可以进行STR分型, 但部分样本图谱峰值不均衡, Chelex-100法提取的样本核DNA不能分型或出现较多等位基因缺失。提示指甲游离缘可以进行成功地核DNA的分型, 有机法和有机法结合Chelex-100法提取的DNA质量都可成功检测, 其中以有机法结合Chelex-100法提取DNA的检测成功率最高。     

Chlamydomonas (Chlorophyceae) colony PCR

The ease and effectiveness of colony polymerase chain reaction (PCR) has allowed rapid amplification of DNA fragments and screening of large number of colonies of interest including transformants and mutants with genetic manipulations. Here, we evaluated colony PCR in Chlamydomonas. Individual colonies were treated with 10 mM ethylenediaminetetraacetic acid (EDTA) or Chelex-100 and the resulting clear cell lysate was used for PCR reaction. Either genomic DNA or plasmid DNA incorporated into the genome was equally amplified. We found that the Chelex method is superior to EDTA method in certain cases. This colony PCR technique will bypass the tedious process of isolating genomic DNA for PCR reaction and will make it possible for rapid amplification of genomic DNA fragments as well as rapid large-scale screening of transformants.     

An improved colony PCR procedure for genetic screening of <Emphasis Type="Italic">Chlorella</Emphasis> and related microalgae

A colony PCR technique was applied for both genomic and chloroplast DNA in the green microalgae Chlorella. Of five different lysis buffers, Chelex-100 was superior for DNA extraction, PCR and DNA storage. It also was insensitive to variations in cell density. The conditions established for an improved PCR formulation are applicable for screening of genetically-engineered transformants as well as bioprospecting of natural microalgal isolates. Besides multiple Chlorella species, we also demonstrate the efficacy of Chelex-100 for colony PCR with a number of other microalgal strains, including Chlamydomonas reinhardtii, Dunaliella salina, Nannochloropsis sp., Coccomyxa sp., and Thalassiosira pseudonana.     

Eight-hour PCR-based procedure for the detection of Salmonella in raw oysters

The aim of this study was to evaluate the ability of a nested PCR system to detect Salmonella senftenberg in raw oysters. The specific primers of the PCR were derived from the invA gene sequence, essential for Salmonella invasiveness into epithelial cells. First, for the extraction of DNA, four methods (guanidine isothiocyanate, E.Z.N.A. Mollusc Kit, Chelex-100, and lysis with detergents) were compared. A nested PCR method combined with 3.5 h pre-enrichment in buffered peptone water (BPW) and DNA extraction by the resin Chelex-100 is proposed for the detection of S. senftenberg in oyster samples. The detection limit of the method is less than 0.1 CFU/ml (<1 CFU/g of oyster). This procedure is shown to be an excellent tool for the sensitive detection of S. senftenberg from naturally contaminated oysters, with results being obtained within 8 h.