免疫胶体金法提取环境标本中细菌DNA技术*

将抗DNA单克隆抗体标记在胶体金颗粒上制成免疫胶体金试剂,提取标本中DNA,直接用于PCR检测,从而建立一种简单、快速、高效的免疫胶体金方法提取环境标本中的DNA。结果表明:应用免疫胶体金试剂可有效去除环境标本中PCR抑制剂,浓缩模板,提高PCR检测敏感度3～4个数量级。操作步骤简单,无需使用有机溶剂,避免环境污染,吸附了DNA的免疫胶体金可直接用于PCR扩增。研制了免疫胶体金试剂并确定其最佳反应条件,有效提高PCR技术在检测现场环境标本中的敏感性和实用性。     

FTA-DNA直接提取法在病原真菌分子鉴定中的应用

目的建立并评价FTA-DNA直接提取法在病原真菌分子鉴定中的应用。方法采用whatman FTA-DNA直接提取法从25个不同种属的45株培养的菌株和6例临床标本中提取病原真菌DNA,用于病原真菌的测序鉴定。配制不同浓度的孢子悬液探索该方法的检测限和安全性。结果 45株菌株扩增后均能得到1条清晰的DNA扩增片段,并成功测序。应用该方法亦成功从腹水、胸水、口腔拭子、宫颈拭子来源的临床标本中直接提取DNA并成功鉴定病原真菌。该DNA提取方法联合降落PCR能检测到1.0×103个cell/mL的孢子悬液,1.0×104个cell/mL及以下浓度的孢子悬液可以被FTA卡完全灭活。结论 FTA-DNA直接提取法可快速有效地从培养的菌株及部分临床标本中提取并保存病原真菌DNA,用于病原真菌的测序鉴定。     

一种快速提取葱属植物 DNA 的方法

目的：针对分子育种工作的繁琐性及葱属植物次生代谢物较多的特点,研究一种利用普通试剂及简单仪器高效快速提取葱属植物DNA的方法。方法：对碱处理法稍加改进,在碱性环境中高温裂解细胞,将释放的DNA保存在Tris缓冲液中,用PCR检测提取质量并分析DNA的保存时间。结果：与用试剂盒提取的DNA相比,扩增产物无显著差异,利用此方法提取DNA并分析了13个洋葱品种的细胞质雄性不育类型;保存时间分析显示,DNA在常温下只能保存5d,在4℃或-20℃可至少保存2个月。结论：该方法方便快速、成本低、适于田间操作,得到的DNA可满足分子生物学实验的基本要求,可用于以PCR为基础的分子标记辅助育种。     

植物叶片基因组DNA快速提取方法

为了满足分子生物学研究中大量植株基因需要PCR检测的需求,建立了一种无需研磨、无需有机试剂抽提的快速提取植物叶片基因组DNA的方法。通过比较基因组DNA的浓度及PCR检测结果,获得了最佳提取条件,即约50 mg叶片加入150μL含1%β-巯基乙醇的TE提取液,快速破碎1 min。破碎后的样品4℃13 500×g离心1 min,上清于-20℃冷冻后室温融解,4℃、13 500×g离心1 min,离心后收集的上清溶液即可用于PCR检测。整个提取过程仅需10-12 min,具有样品用量小、操作简单、廉价、高效等优点。使用此方法提取的烟草、水稻、大豆、玉米、油菜和花生的基因组DNA均可用于PCR扩增,并可成功扩增长度为3 244 bp的基因片段。此方法提取的基因组DNA也可用于对未知基因的扩增,获得4条新的花生actin基因序列。     

转基因植物快速检测方法的研究

本试验对转基因植物检测中的DNA提取和PCR扩增程序作了改进。经试验,本研究建立的DNA快速提取法与目前广泛使用的CTAB法相比更为简便,快速和经济,提取的DNA质量主扩增效果无明显差异,可用于多种转基因植物,多种植物组织的DNA提取,利用复合PCR法可在同一反应管中同步检测35N,NOS及CP4－EPSPS基因,明显提高了检测效率。应用本试验建立的DNA快速提取－复合PCR扩增－银染检测技术可在6小时内得出结果,达到了快速,简便,灵敏,可靠的检测目的。     

一种适于微生物多样性分析的青贮饲料总DNA的提取方法

目的:提出一种适于微生物多样性分析的青贮饲料中微生物总DNA的提取方法,并评价其效果.方法:间接法抽提样本的总DNA,通过琼脂糖电泳、紫外吸收及PCR分析DNA质量,DGGE评价提取效果,用PCR扩增目的菌株的特定片段来检测提取方法的灵敏度.结果:两个样本DNA的A260/A280值分别为1.99和1.93,A260/,A230值分别为2.19和1.90,提取的DNA不需纯化便可直接用于16S rRNA基因的扩增,提取方法灵敏度为3cfu/g,DGGE结果表明提取方法可以涵盖样品中的所有微生物.结论:提取的DNA纯度较高,可直接用于下游分子操作,提取方法灵敏度较高,能全面反映样品中的微生物原貌,可用于免培养法研究青贮饲料中的微生物菌群组成.     

一种用于PCR的番茄DNA快速粗提方法

在对植物进行分子生物学研究时需要提取植物基因组DNA并对其进行PCR克隆,目前实验室常用CTAB法提取植物DNA,但其步骤相对繁琐,需要用有机溶剂反复抽提,在样本较多时,耗时较长。NaOH可以提取多种物种DNA,本研究优化了实验室条件下更加快速、经济、安全、高效的提取植物DNA的方式,主要包括以下步骤：利用液氮快速粉碎植物样品;采用碱裂解(0.5 mol/L NaOH)的方式一步提取DNA;提取的DNA利用TE缓冲液进行中和;以中和的粗提DNA作为模板进行PCR反应。本研究利用简单的DNA提取流程并结合PCR检测方式,完成了番茄转化子的快速初步鉴定。同时还证明了该NaOH法可用于番茄不同组织的DNA提取。本研究中整个操作过程步骤简单,耗时短,可在短时间内完成大量植物样品的DNA提取,无需使用氯仿、异丙醇等有毒物质,完成提取时间约为CTAB提取法的1/5,而且提取的不同组织DNA完整性较好,质量可观,满足常规的PCR检测,可用于基因克隆及转化子筛选鉴定,具有一定的利用价值和应用前景。     

高温环境样品总DNA直接和间接提取方法的比较

分别采用两种环境总DNA直接提取法和一种间接提取法从6种温泉菌席样品中提取总DNA,以DNA粗产物的纯度、能否用于后续PCR扩增及PCR-DGGE(变性梯度凝胶电泳)所反映的微生物多样性为评价指标对两类方法进行比较和评价。研究发现,虽然间接提取法效率低下,但对于高温极端环境中生物量较小的样品,间接法能得到有研究价值的、纯度较高的环境样品总DNA,而直接法得到的DNA量小且不适于PCR扩增操作。在使用这2类方法都能得到可用于研究操作的DNA的情况下,间接提取法能更好的体现环境样品中微生物的多样性。     

改良CTAB法提取番石榴叶片总DNA

目的:从番石榴叶片中快速提取高质量的总DNA。方法:改良CTAB法。主要改进之处在于不用液氮,而是直接研磨硅胶干燥样品;用高浓度CTAB、低浓度乙醇与NaCl盐析相结合等方法去除多糖。结果:应用改良后的方法可以快速提取番石榴叶片总DNA,有效去除组织中的多糖、蛋白质,抑制提取过程中的组织褐变。提取的DNA可用于限制性内切酶酶切和PCR扩增。结论:传统CTAB法经过改良,可用于快速提取番石榴高质量DNA。     

痰涂片标本用于结核分枝杆菌耐药性检测的研究

目的 研究抗酸染色结核分枝杆菌(简称结核杆菌)阳性痰涂片标本直接用于耐药性检测的方法。方法 对18株临床分离培养的结核杆菌用利福平进行药敏试验。分别提取菌株DNA和与之对应的痰涂片标本的菌体DNA,用聚合酶链反应(PcR)扩增ropB基因后进行固相杂交和核酸测序检测结核杆菌的耐药性。结果 18株结核杆菌中有12株对利福平耐药。经PCR扩增的ropB片段与探针杂交后,敏感菌株未发现rpoB基因的突变,自耐药菌株提取的DNA中rpoB突变体的检出率为100％(12／12),痰涂片提取DNA的检出率为91．7％(11／12)。所有耐药菌株DNA与痰涂片DNA核酸测序结果相吻合,都有rpoB基因核心区域碱基突变。结论 抗酸染色痰涂片阳性标本可直接用于检测结核杆菌利福平耐药基因rpoB突变体,是一种值得临床实验室推广使用的耐药菌诊断方法。     

改良环介导等温扩增技术快速检测婴儿配方奶粉中的阪崎肠杆菌

[目的]采用改良环介导等温扩增(LAMP)技术,快速检测婴儿配方奶粉中的阪崎肠杆菌.[方法]以阪崎肠杆菌(ATCC29544)的16S-23S rRNA间区序列作为靶序列,设计内、外引物和环引物,通过肉眼观察白色沉淀,判断检测结果.[结果]LAMP检测阪崎肠杆菌的灵敏度为0.101 CFU/mL,人工污染阪崎肠杆菌的婴儿配方奶粉的检出限为1.1 CFU/g.采用试剂盒提取DNA,从样品处理到报告结果,耗时1 h.而对照,PCR检测阪崎肠杆菌的灵敏度为101 CFU/mL,人工污染阪崎肠杆菌的婴儿配方奶粉的检出限为1100 CFU/g.采用同样方法提取DNA,从样品处理到报告结果,耗时3 h.[结论]因此,LAMP检测婴儿配方奶粉中的阪崎肠杆菌灵敏度高,耗时短,方法简便.     

PCR-BASED FLUORESCENT METHOD FOR RAPID DETECTION OF SALMONELLA TYPHIMURIUM IN POULTRY SAMPLES

A DNA binding fluorescence method based on polymerase chain reaction (PCR) products was evaluated for rapid detection of Salmonella Typhimurium in poultry products. Wash water samples of chicken carcasses and ground turkey were inoculated with S. Typhimurium to obtain final concentrations of 10° - 10⁵ CFU/mL. One mL of each sample was used to get the DNA template and 5 μL of the sample template was added into 25 μL of SYBR Green PCR Master Mix and two specific Salmonella ompC gene primers. The negative control was the same except 5 μL of each wash solution was added instead of 5 μL sample template. The reaction was carried out in a thermocycler. Finally, the fluorescence signal of each PCR product was measured using a fluorometer. The PCR products were also confirmed by ethidium bromide agarose gel, and the DNA concentrations of the PCR products were measured by a filter fluorescence photometer. The results showed that when bacterial cells increased from 0 to 2 CFU/mL, the fluorescence signal increased significantly. The PCR-based fluorescence method could detect the target bacteria in minutes after PCR amplification compared to hours by gel electrophoresis and also could be done at an earlier time during PCR amplification. The detection limit of this method for S. Typhimurium in the poultry samples was 2 CFU/mL without any enrichment.     

Comparison of different PCR methods for detection of Brucella spp. in human blood samples

For detection of Brucella species by PCR four DNA extraction methods and four targets were compared using pure culture of Brucella melitensis and the best conditions were applied in clinical samples. It was found that the MagNA Pure LC method was the most efficient and sensitive method showing a positive PCR reaction with DNA extracted from as low as 25 and 100 CFU suspended in one ml blood and one ml water, respectively. Detection of Brucella spp. by conventional PCR was investigated using four different targets. The results indicated that The B4-B5 amplification method was the most sensitive one as it could amplify DNA extracted from as a low as 25 and 100 CFU/ml suspended in one ml water and blood, respectively. Furthermore real-time PCR was able to detect Brucella using DNA extracted from as low as 50 CFU/ml blood and 15 CFU/ml water, respectively. The best and optimum detection conditions were applied to the clinical samples. Evaluation of conventional PCR assays on blood specimens confirmed 72% of the results obtained by conventional blood culture methods with a specificity of 95%, while serum samples had a sensitivity of 54% and specificity of 100%. Real-time PCR was generally found to be more sensitive and specific for detecting Brucella spp. in blood and serum samples compared to conventional PCR. The real-time PCR done on blood specimens confirmed 77.5% of the results obtained by conventional blood culture methods with specificity of 100%, while 60% of serum samples were found to be positive with specificity of 100%. These results suggest that serum and blood analysis by conventional and real time PCR is a convenient and safe method for rapid and accurate diagnosis of brucellosis.     

Sample processing effect on polymerase chain reaction used for identification of<Emphasis Type="Italic">Campylobacter jejuni</Emphasis>

Model samples of Campylobacter jejuni for polymerase chain reaction (PCR) were prepared by rapid and simple procedures consisting of centrifugation, proteinase K treatment, Chelex 100 treatment, and boiling lyses. A PCR based on specific amplification of the variable sequence of 16S rRNA gene was performed using Tth DNA polymerase and the PCR products were visualized by agarose gel electrophoresis. The assay allowed the detection of 10 CFU/mL C. jejuni in the physiological saline and 100 CFU/mL in the basic Park and Sanders broth.     

环介导等温扩增快速检测B群链球菌的临床应用

目的建立采用环介导等温扩增（LAMP）技术从临床标本中快速检测B群链球菌的方法。方法根据美国国家生物信息中心上提交的B群链球菌的cfb基因序列（登陆号X72754）设计特异LAMP引物,以热裂解法提取标本中细菌的DNA,然后以LAMP技术扩增cfb基因来鉴定其是否为B群链球菌。在采用LAMP技术检测B群链球菌的同时,以选择性培养法和PCR技术平行检测相同标本,并将3种方法的检测结果比较。结果在176例阴道拭子和176例直肠拭子中,选择性培养法检测到的B群链球菌例数为49和61、LAMP法检测到的B群链球菌例数为49和59,PCR法检测到的B群链球菌例数为48和58。以选择性培养法为金标准,LAMP法检测B群链球菌的灵敏度和特异度分别为96．7％和100％,PCR法检测B群链球菌的灵敏度和特异度分别为95．1％和100％。LAMP法检测B群链球菌的时间为2h左右,模拟菌株的检测结果显示该方法的最低检测限为10^2CFU／mL。结论该方法灵敏度高,特异性强,操作方便简单,适合从临床标本中快速检测B群链球菌。     

Rapid Microbial Sample Preparation from Blood Using a Novel Concentration Device

Appropriate care for bacteremic patients is dictated by the amount of time needed for an accurate diagnosis. However, the concentration of microbes in the blood is extremely low in these patients (1–100 CFU/mL), traditionally requiring growth (blood culture) or amplification (e.g., PCR) for detection. Current culture-based methods can take a minimum of two days, while faster methods like PCR require a sample free of inhibitors (i.e., blood components). Though commercial kits exist for the removal of blood from these samples, they typically capture only DNA, thereby necessitating the use of blood culture for antimicrobial testing. Here, we report a novel, scaled-up sample preparation protocol carried out in a new microbial concentration device. The process can efficiently lyse 10 mL of bacteremic blood while maintaining the microorganisms’ viability, giving a 30‑μL final output volume. A suite of six microorganisms (Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Haemophilus influenzae, Pseudomonas aeruginosa, and Candida albicans) at a range of clinically relevant concentrations was tested. All of the microorganisms had recoveries greater than 55% at the highest tested concentration of 100 CFU/mL, with three of them having over 70% recovery. At the lowest tested concentration of 3 CFU/mL, two microorganisms had recoveries of ca. 40–50% while the other four gave recoveries greater than 70%. Using a Taqman assay for methicillin-sensitive S. aureus (MSSA)to prove the feasibility of downstream analysis, we show that our microbial pellets are clean enough for PCR amplification. PCR testing of 56 spiked-positive and negative samples gave a specificity of 0.97 and a sensitivity of 0.96, showing that our sample preparation protocol holds great promise for the rapid diagnosis of bacteremia directly from a primary sample.     

Chelex-100法及酚氯仿法提取阴道毛滴虫DNA的比较

目的-比较Chelex-100法和酚氯仿法提取阴道毛滴虫基因组DNA。方法-分别用Chelex-100法和酚氯仿法提取阴道毛滴虫基因组DNA,用PCR法检测DNA提取的有效性。结果两种方法提取的DNA经PCR扩增均有特定的条带。结论-两种方法均能提取阴道毛滴虫DNA。Chelex-100方法简便、省时,较适用于分子生物学研究及临床PCR扩增使用。     

免疫捕获PCR法快速检测金黄色葡萄球菌

旨在建立一种快速检测金黄色葡萄球菌(Staphylococcus aureus,SA)免疫捕获PCR技术,并探讨其灵敏度和特异性。SA特异性抗体包被PCR管以富集待测样品中目标菌,之后在同一PCR管里直接进行免疫捕获PCR,并和直接PCR比较。免疫捕获PCR法可特异性检测2株SA菌株,而无法检测到其它8种常见食源性致病菌,说明该方法对SA具有良好的特异性;该方法对纯菌液而言,检测灵敏度可达到2.35×102CFU/mL,是直接PCR的100倍;对5种食品模拟带菌检测发现,无需增菌培养,其灵敏度可达到2.35×103-2.35×104CFU/mL,是直接PCR的10-100倍。免疫捕获PCR法集免疫学与分子生物学检测技术于一体,具有高特异性、高灵敏度、检测快速、易于操作、成本低廉等诸多优点,是一种适合基层实验室使用的检测技术。     

Pathogen detection by core–shell type aptamer-magnetic preconcentration coupled to real-time PCR

The presence of pathogenic bacteria is a major health risk factor in food samples and the commercial food supply chain is susceptible to bacterial contamination. Thus, rapid and sensitive identification methods are in demand for the food industry. Quantitative polymerase chain reaction (PCR) is one of the reliable specific methods with reasonably fast assay times. However, many constituents in food samples interfere with PCR, resulting in false results and thus hindering the usability of the method. Therefore, we aimed to develop an aptamer-based magnetic separation system as a sample preparation method for subsequent identification and quantification of the contaminant bacteria by real-time PCR. To achieve this goal, magnetic beads were prepared via suspension polymerization and grafted with glycidylmethacrylate (GMA) brushes that were modified into high quantities of amino groups. The magnetic beads were decorated with two different aptamer sequences binding specifically to Escherichia coli or Salmonella typhimurium. The results showed that even 1.0% milk inhibited PCR, but our magnetic affinity system capture of bacteria from 100% milk samples allowed accurate determination of bacterial contamination at less than 2.0 h with limit of detection around 100 CFU/mL for both bacteria in spiked-milk samples.     

新生隐球菌基因组DNA不同抽提方法的比较

目的 DNA是进行分子生物学研究的重要基础。在本研究中,我们建立了2种简单快速抽提基因组DNA的方法并可用作PCR扩增的模板。通过比较4种不同的DNA抽提方法以确定哪种更适合进行下一步的基因分析。方法这4种方法是:玻璃珠法,酶法,3%SDS法和氯化苄法。玻璃珠法是用玻璃珠在混漩器上剧烈振荡破碎细胞壁;3%SDS法是将细胞在含10mmol/LDTT的3%SDS溶液中加热,然后用5mmol/LKAc和异丙醇抽提,DNA的产量通过A260测定。结果 3%SDS溶解法、经典酶法、玻璃珠法和氯化苄法的DNA产量分别为0.4154±0.0367、0.8484±0.0756、1.2636±0.2040、0.4070±0.0339(g/L×108CFU/mL)。结论玻璃珠法是最敏感、重复性好、简单、费用合理的抽提方法 。