通用多重不对称PCR结合寡核苷酸芯片检测7种食源性致病菌

应用通用多重不对称聚合酶链式反应(polymerase chain reaction,PCR)和寡核酸芯片技术建立一种同时检测7种常见食源性致病菌的方法。每种致病菌上游或下游引物5’端连接一段异源的共有序列。以荧光标记的该共有序列作为通用引物,与限制性特异引物经一步多重不对称PCR同时获得所有目标菌的单链标记靶序列,可被芯片上固定的特异性寡核苷酸探针捕获。通过芯片扫描、分析荧光信号完成检测。标准菌株检测结果证实,该方法可特异地检测单一和混合感染的目标菌,基因组DNA的检测灵敏度为0.1~1 pg。95份模拟污染和零售食品样本芯片检测结果与常规的分离与生化鉴定及荧光定量PCR结果一致。建立的寡核苷酸芯片方法可为快速、特异、灵敏及高通量地鉴定食源性致病菌提供一种有效的检测手段。     

Chelex 法结合环介导间接聚合酶链式反应检测肉制品单增李斯特菌

目的：建立肉制品中单增李斯特菌环介导间接聚合酶链式反应检测体系,实现快速检测。方法：采用Chelex法从肉制品中分离模板DNA,根据单增李斯特菌hlyA基因的保守区设计两条探针,将探针标记于报告基因(大豆Lectin基因)的两端,此标记的报告基因与待测靶基因经杂交、缺口补平、环化后,采用反向聚合酶链式反应扩增报告基因,实现对靶基因的检测。结果：该检测体系检出限低于100CFU/g(mL),且与其他食源性致病菌的检测无明显交叉反应,对200份肉制品进行检测,阳性率为2.5%,与传统细菌分离检测结果完全相符。结论：环介导间接聚合酶链式反应可以快速、灵敏、特异检测肉制品中单增李斯特菌污染。     

应用基因芯片技术检测肉及肉制品中5种致病菌

建立一种运用多重聚合酶链式反应(PCR)结合基因芯片技术检测大肠埃希氏菌、沙门氏菌、金黄色葡萄球菌、志贺氏菌和单核细胞增生李斯特菌5种食源性致病菌的快速、准确、灵敏的方法。分别选取编码大肠埃希氏菌的slt基因、沙门氏菌invA基因、金黄色葡萄球菌nuc基因、志贺氏菌ipaH基因和单核细胞增生李斯特菌inlA基因,并以细菌16S rDNA基因作为阳性对照,设计引物和探针,进行多重PCR扩增,产物与含特异性探针的芯片杂交。结果表明：该基因芯片可同时特异性地检测5种致病菌,多重PCR检测灵敏度为20pg,而DNA芯片检测灵敏度可达2pg;用所制备的基因芯片检测实际肉及肉制品样品,准确率高于传统培养法。所建立的基因芯片检测方法特异性好、灵敏度高,可为食源性致病菌的检测提供理想手段。     

巢式PCR快速检测海产品中的副溶血弧菌（英文）

副溶血弧菌是一种世界范围性的食源性致病菌,食用了该菌污染的海产品可导致胃肠炎等疾病。为了建立一种可快速、特异地检测海产品中副溶血弧菌的方法,通过把副溶血弧菌基因组序列和其它不同种类弧菌的基因组序列进行比较分析,筛选出了一个副溶血弧菌特异性的标记基因-VP1331,根据该基因建立了副溶血弧菌的巢式PCR快速检测方法,并评估了其特异性、敏感性和稳定性。实验结果表明,该方法只有在以副溶血弧菌基因组DNA为模板时才能扩增出目的片段,而其它11种弧菌和非弧菌均不能扩增出目的片段。该方法的最低检测限为副溶血弧菌基因组DNA 10 fg、纯培养物6.6 CFU。人工污染实验表明,初始菌液浓度为25.7CFU/100 m L时只需经过2 h的增菌培养即可检出。上述结果表明,VP1331基因可以作为副溶血弧菌种特异性标记,本方法可以用于污染海产品中该菌的检测与鉴定。     

应用基因芯片技术鉴定食源性金黄色葡萄球菌的研究

目的:建立快速鉴定食源性金黄色葡萄球菌的基因芯片技术。方法:采用PCR方法扩增金黄色葡萄球菌16SrRNA基因的DNA片段,序列进行BLAST比较并通过软件设计其特异性探针,采用基因芯片杂交技术鉴定食源性金黄色葡萄球菌样品。结果:对所有样品进行基因芯片杂交技术处理并扫描观察,金黄色葡萄球菌的杂交结果呈阳性,其检测结果与传统方法鉴定结果一致。结论:应用基因芯片鉴定技术可快速、准确地鉴定食源性金黄色葡萄球菌,值得推广应用。     

应用可视芯片技术检测食品中常见致病菌的方法研究

利用特异性引物进行PCR扩增,并利用固定于可视芯片的特异探针与扩增产物进行杂交反应加以鉴定。利用可视芯片技术可以准确的检出沙门氏菌属和金黄色葡萄球、小肠结核肠炎耶尔森氏菌、单核细胞增生李斯特氏菌3种食品中常见致病菌,检测灵敏度可达8.5×101cfu/mL。利用可视芯片准确灵敏使用便捷的优点,建立了食品中常见致病菌鉴定方法,为食源性致病菌检测提供了一项快速有效的方法。     

DNA探针及其在食品工业中的应用

DNA探针实质上是一种具有单一螺旋结构的DNA,其多核苷酸通过碱基配对可以同具有互补顺序的另一条多核苷酸（靶DNA）杂交,用同位素、酶或其他检测方法进行标识,就可以很快发现探针并知道混合在其他微生物中的检测菌。利用这一原理,可以辨别出各种致病菌的DNA。该方法可用于包括食品生产中产生的致病菌和各种谷物、禽畜的疾病检测。     

PCR技术在食源性致病微生物快速检测中的应用

PCR(Polymerase Chain Reaction)即聚合酶链式反应,是一种体外快速扩增基因或DNA序列的方法。食源性致病微生物是影响食品质量和安全的主要因素之一,建立和完善食品中致病微生物快速检测技术具有重要的现实意义。PCR技术以其自身的灵敏度高、特异性强、速度快等优点作为食源性致病微生物检测的关键技术在食品中得到广泛的应用。本文简要介绍PCR技术的基本原理,及其在几种食源性致病微生物如沙门氏菌、金黄色葡萄球菌、单核细胞增生李斯特氏杆菌、致病性大肠杆菌及其他一些有害微生物检测中的应用,并分析了PCR技术在食源性致病菌检测的实际应用中存在的一些问题及对此项技术的展望,这将有力促进我们今后更好地研究应用PCR技术检测食品中食源性致病微生物。     

巢式PCR快速检测海产品中的副溶血弧菌

副溶血弧菌是一种世界范围性的食源性致病菌,食用了该菌污染的海产品可导致胃肠炎等疾病。为了建立一种可快速、特异地检测海产品中副溶血弧菌的方法,通过把副溶血弧菌基因组序列和其它不同种类弧菌的基因组序列进行比较分析,筛选出了一个副溶血弧菌特异性的标记基因-VP1331,根据该基因建立了副溶血弧菌的巢式PCR快速检测方法,并评估了其特异性、敏感性和稳定性。实验结果表明,该方法只有在以副溶血弧菌基因组DNA为模板时才能扩增出目的片段,而其它11种弧菌和非弧菌均不能扩增出目的片段。该方法的最低检测限为副溶血弧菌基因组DNA 10 fg、纯培养物6.6 CFU。人工污染实验表明,初始菌液浓度为25.7 CFU/100 mL时只需经过2 h的增菌培养即可检出。上述结果表明,VP1331基因可以作为副溶血弧菌种特异性标记,本方法可以用于污染海产品中该菌的检测与鉴定。     

副溶血性弧菌毒素编码基因的微量、可视化检测

针对副溶血性弧菌主要毒素编码基因(tdh和trh),基于环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术,以毛细管为反应介质,建立一种微量(5μL)、特异、灵敏、低成本、可视化、快速检测的毛细管LAMP(capillary LAMP,cLAMP)。本研究针对每个靶标基因分别设计6条特异性引物,系统优化Mg2+、dNTPs浓度和Bst DNA聚合酶使用量,以及反应时间及反应温度等参数,确立最适cLAMP反应体系;测定该方法的灵敏度和特异性。结果表明:优化后的5μL cLAMP反应体系为6 mmol/L或8 mmol/L Mg2+,1.44 mmol/L或1.28 mmol/L dNTPs、0.096 U/μL Bst DNA聚合酶、反应温度65℃、反应时间60 min。针对靶基因tdh和trh,c LAMP方法检测副溶血性弧菌基因组DNA的最低检测限分别为3 fg/μL和34 fg/μL,纯培养物的最低检测限分别为9.85×103 CFU/mL和8.25×105 CFU/mL;且与其他6种常见致病菌(霍乱弧菌、创伤弧菌、溶藻弧菌、嗜水气单胞菌、大肠杆菌、金黄色葡萄球菌)均无交叉反应。本研究建立的微量、可视化cLAMP方法为食源性致病菌副溶血性弧菌检测试剂盒的研发提供了技术支撑。     

基于上转换荧光标记和磁分离技术的沙门氏菌DNA检测新方法

利用水热法制备NaYF4:Yb3+Er3+荧光纳米颗粒,表面氨基化修饰后与探针核酸单链共价偶联,形成荧光标记显示探针。再将氨基化的Fe3O4磁性纳米颗粒与捕获核酸单链进行共价偶联,制备磁分离捕获探针。基于DNA杂交互补反应,加入体系中的目标DNA链分别与两端互补的荧光显示探针和磁分离捕获探针形成三明治夹心结构,通过外加磁场收集分离。加入的目标DNA链浓度越大,体系荧光强度越大。结果表明,复合结构的荧光强度与目标DNA链浓度成正比,在0.01~10 pmol/L范围内呈现良好的线性关系,最低检测限达3 fmol/L。     

Establishment and preliminary application of oligonucleotide microarray assay for detection of food-borne toxigenic microorganisms

Rapid, high-throughput and accurate detection and identification of food-borne toxigenic microorganisms is crucial for food safety nowadays. An oligonucleotide microarray was designed and established and was applied to detect common food-borne toxigenic microorganisms in this study. PCR amplification of marker genes and 16S rRNA gene of 14 toxigenic bacteria and fungi using specific primers and oligo probes residing in these genes were employed and designed to fabricate the microarray. Optimization of hybridization conditions was implemented. The optimal conditions for hybridization were 51 °C for 30 min. Furthermore, the ratio of biotin labeled to unlabeled primer for PCR amplification was also optimized to enhance specific hybridization of the microarray. Specificity, sensitivity (710 CFU/mL), and reproductivity assessment confirmed the practicability of the microarray. Finally, this microarray was successfully applied to detect 6 common toxigenic microorganisms from 328 food samples. The established microarray may provide potential for rapid detection and identification of toxigenic microorganisms from foods.     

Development and evaluation of microarray-based whole-genome hybridization for detection of microorganisms within the context of environmental applications

The detection and identification of microorganisms in natural communities is a great challenge to biologists. Microarray-based genomic technology provides a promising high-throughput alternative to traditional microbial characterization. A novel prototype microarray containing whole genomic DNA, termed community genome array (CGA), was constructed and evaluated. Microarray hybridizations at 55 degrees C using 50% formamide permitted the examined bacteria to be distinguished at the species level, while strain-level differentiation was obtained at hybridization temperatures of 65 or 75 degrees C. The detection limit was estimated to be approximately 0.2 ng with genomic DNA from a single pure culture using a reduced hybridization volume (3 microL). Using mixtures of known amounts of DNA or a known number of cells from 14 or 16 different species, respectively, about 5 ng of genomic DNA or 2.5 x 10(5) cells were detected under the hybridization conditions used. In addition, strong linear relationships were observed between hybridization signal intensity and target DNA concentrations for pure cultures, a mixture of DNA templates, and a population of mixed cells (r2 = 0.95-0.98, P < 0.01). Finally, the prototype CGA revealed differences in microbial community composition in soil, river, and marine sediments. The results suggest that CGA hybridization has potential as a specific, sensitive, and quantitative tool for detection and identification of microorganisms in environmental samples.     

Detection of Alicyclobacillus species in fruit juice using a random genomic DNA microarray chip

This study describes a method using a DNA microarray chip to rapidly and simultaneously detect Alicyclobacillus species in orange juice based on the hybridization of genomic DNA with random probes. Three food spoilage bacteria were used in this study: Alicyclobacillus acidocaldarius, Alicyclobacillus acidoterrestris, and Alicyclobacillus cycloheptanicus. The three Alicyclobacillus species were adjusted to 2 × 10(3) CFU/ml and inoculated into pasteurized 100% pure orange juice. Cy5-dCTP labeling was used for reference signals, and Cy3-dCTP was labeled for target genomic DNA. The molar ratio of 1:1 of Cy3-dCTP and Cy5-dCTP was used. DNA microarray chips were fabricated using randomly fragmented DNA of Alicyclobacillus spp. and were hybridized with genomic DNA extracted from Bacillus spp. Genomic DNA extracted from Alicyclobacillus spp. showed a significantly higher hybridization rate compared with DNA of Bacillus spp., thereby distinguishing Alicyclobacillus spp. from Bacillus spp. The results showed that the microarray DNA chip containing randomly fragmented genomic DNA was specific and clearly identified specific food spoilage bacteria. This microarray system is a good tool for rapid and specific detection of thermophilic spoilage bacteria, mainly Alicyclobacillus spp., and is useful and applicable to the fruit juice industry.     

基因芯片技术鉴定食源性金黄色葡萄球菌

为了建立快速鉴定食派陛金黄色葡萄球菌的基因芯片技术，采用PCR方法扩增金黄色葡萄球菌16SrRNA基因的DNA片段．序列进行BLAST比较并通过软件设计其特异性探针，采用基因芯片杂交技术鉴定食源性金黄色葡萄球菌样品。结果：对所有样品进行基因芯片杂交技术处理并扫描观察，金黄色葡萄球菌的杂交结果呈阳性，其检测结果与传统方法鉴定结果一致。结论：应用基因芯片鉴定技术可快速、准确地鉴定食源性金黄色葡萄球菌，值得推广应用。     

应用基因芯片技术检测食源性腹泻致病大肠杆菌

目的基因芯片技术在食源性致病大肠杆菌检测中的建立与应用。方法对菌株进行预处理,通过溴化十六烷基三甲铵(cetyltrime thylammonium ammonium bromide,CTAB)方法提取细菌中的基因组DNA并去除其中的杂质,利用提取的基因组设计引物序列和探针序列,设计完成后对致病大肠杆菌致病大肠杆就基因芯片进行杂交与洗涤,使用扫描仪对处理过的基因芯片进行扫描实现食源性致病大肠杆菌的检测。结果在病原菌按不同程度稀释的情况下,与传统的病原分离鉴定检测方法相比,将基因芯片技术应用在食源性致病大肠杆菌致检测中,能够明显的检测出荧光标记的病原菌。结论该方法的灵敏度更高,可以解决传统的致病大肠杆菌检测方法灵敏度较低,病原处于较低的感染状态时难以检出的问题。     

Development of a DNA microarray chip for the identification of sludge bacteria using an unsequenced random genomic DNA hybridization method

A tool, based upon the DNA microarray chip, for the identification of specific bacteria from activated sludge, using the hybridization of genomic DNA with random probes, is described. This chip was developed using the genomic DNAs from Gordonia amarae, the natural filamentous actinomycete that causes sludge foaming and bulking, as well as a nonfilamentous floc forming bacterium (Zoogloea ramigera) and the skin pathogen Mycobacterium peregrinum without any sequence information. The sets of target probes on amine-coated glass were made from a genomic library, constructed with PCR products derived from randomly fragmented genomic DNAs extracted from pure cultures of the three strains. Initial hybridization results, when pure cultures were employed, showed the specificity of the probes as well as the resolution of the system, demonstrating the capabilities of this system to identify specific bacterial strains. The microarray was also tested for its ability to distinguish specific bacteria from among mixed bacterial communities, such as in sludge, soil, or spiked genomic DNA samples. The results showed that the probes are specific, with only mild cross-hybridization occurring in a small number of cases. Furthermore, the chip clearly discriminated the presence of all three strains when they were present alone or together within mixed samples. Moreover, using the spot intensity and DNA hybridization kinetics, the starting genomic DNA concentrations could be estimated relatively well, which would make it possible to predict the number of specific bacteria present within the test samples. Therefore, the random genomic hybridization approach, i.e., without any sequence information available for the probes, is a practical protocol for the identification of and screening for specific bacteria within any complex bacterial community from the environmental samples, such as in activated sludge, although the possibility of cross-hybridization may still exist.