多频大幅脉冲传感系统监测3种食源性致病菌的生长趋势

【目的】探究智能电子舌伏安法结合特定的统计分析系统是否适用于示踪食品致病性细菌生长情况的快速监测。【方法】利用智能电子舌――多频大幅脉冲传感系统的伏安法,监测液体培养基中3种食源性致病菌的16个时段生长情况所致液体基质变化过程的综合信息;结合主成分分析法对获得的复杂综合信息数据进行统计学分析,按获得的主成分得分图分析检测样品。【结果】监测能力强的电极、频率段分别是：金黄色葡萄球菌的为钨电极的100 Hz、铂电极的1 Hz、银电极的10 Hz和钛电极的10 Hz频率段;大肠杆菌O157:H7的为金电极的100 Hz、铂电极的1 Hz、钛电极的1 Hz和钨电极的100 Hz频率段;枯草芽孢杆菌的为钯电极的1、10和100 Hz 3个频率段。【结论】本试验首次用多频大幅脉冲传感系统伏安法结合主成分分析法,能够有效的监测样品细菌的生长情况,有望成为一种具有多种优点的新型检测细菌生长情况的快速监测系统。     

智舌快速检测副溶血弧菌簇类独立软模式识别的建立

[目的]为探索一种新型食品致病菌的快速检测方法.[方法]用基于多频大幅脉冲伏安法为基元模式的智舌,结合簇类的独立软模式识别方法,检测样本培养物的综合信息并进行类别分析,建立智舌结合主成分分析的副溶血弧菌簇类独立软模式的判别模型.[结果]经过对6个工作电极3个频率段检测分析模型的比较,得到最佳电极及频率段组合:钯电极l ...     

不同温度条件下副溶血性弧菌tdh基因表达及其代谢组响应

【目的】采用RT-PCR的方法分析致病性副溶血性弧菌毒力基因表达,并应用代谢组学的方法研究毒力基因不同表达水平下致病性副溶血性弧菌代谢组的响应。【方法】本文以致病性副溶血性弧菌(Vibrio parahaemolyticus)ATCC33846为材料,分别提取不同温度(4、25和37℃)下菌体总RNA和代谢组。采用相对定量的方法检测副溶血性弧菌tdh基因在不同温度条件下的表达差异,同时应用超高压液相色谱-四级杆飞行时间质谱联用仪(UPLC/Q-TOF-MS)系统为工作平台检测其代谢组。采用主成分分析法(principal component analysis,PCA)比较副溶血性弧菌代谢组轮廓差异,并通过皮尔森和斯皮尔曼相关性分析法分析代谢组与tdh基因表达之间相关性。【结果】结果表明,不同温度条件下tdh基因表达强弱的排列顺序25℃4℃37℃;在tdh基因不同表达水平下发生显著性(P0.05)变化的主要代谢物是有机酸、氨基酸、醇、酮、酯;共得到11种代谢物与tdh基因表达高度相关(相关性系数︱r︱=1,P0.05),其中3种为负相关,8种为正相关,且醇类代谢物与tdh基因表达的正相关性最显著。【结论】本研究发现副溶血性弧菌代谢组与毒力基因表达存在一定的相关性,有望为副溶血性弧菌致病机理的深入探究提供一定的理论支持。     

抗副溶血弧菌海洋真菌HL-3菌株的鉴定及其活性物质的分离

【背景】目前水产养殖中副溶血弧菌等水产致病菌的预防和治疗主要使用的是抗生素,短期内能够起到较好的效果,但是长期使用抗生素不仅使得病原菌的耐药性增加,而且会产生一系列问题。因此,寻找安全有效的抗生素替代品迫在眉睫。【目的】筛选具有抗副溶血弧菌活性的海洋微生物,鉴定其种类并优化其培养条件,并对其活性物质进行初步分离。【方法】利用稀释涂布平板法和平板划线法分离纯化海洋微生物,通过牛津杯法筛选具有抗副溶血弧菌活性的菌株。根据菌株的形态特征和ITS序列分析对活性菌株进行鉴定。通过筛选培养基种类和盐度确定其培养条件。通过半制备高效液相色谱制备化合物,并利用核磁共振波谱数据鉴定化合物的结构。【结果】从海螺、小黄鱼、对虾等11种样品中分离出海洋微生物菌株76株,其中真菌26株。筛选得到一株具有抗副溶血弧菌活性的真菌菌株HL-3,鉴定为黄曲霉。黄曲霉HL-3菌株在改良沙氏培养基条件下,产物化学多样性和抗菌活性较好。黄曲霉HL-3菌株在真菌5号培养基中产物单一且抗菌活性较好,纯化出来的化合物结构被鉴定为曲酸。曲酸对副溶血弧菌的最小抑菌浓度为4.0mg/mL。【结论】实验结果为进一步利用各种色谱手段分离纯化...     

溶藻弧菌的毒力相关基因及其对小鼠的致病力

【目的】通过多重PCR检测和小鼠动物实验,对溶藻弧菌环境分离株的毒力因子进行评估,以期获得较强致病菌株和弱致病菌株之间的差别,并初步探讨该菌毒力因子对小鼠的致病机理。【方法】采用多重PCR体系检测毒力相关基因,我妻氏血平板溶血实验和平板酶活实验检测溶藻弧菌株的溶血素和胞外酶;以昆明小白鼠为实验动物,攻毒方式为灌胃和腹腔注射,根据小鼠的致病症状和死亡情况来分析和对比溶藻弧菌的胞外分泌物以及菌体本身的毒性。【结果】10株溶藻弧菌产淀粉酶、卵磷脂酶的比例为100%,脂肪酶、明胶酶次之(为70%),脲酶均未被检出;神奈川现象阳性菌株率为60%。毒力基因检测的结果显示10株溶藻弧菌中toxR、Collagenase、tlh、FlaA、ompW、AspA、fur这些与毒力有关的基因均有分布,而toxS、trh、tdh、UreR并未检出。10株溶藻弧菌中的VA009对小鼠显示了较强的致病性,能造成腹腔积液,经腹腔注射感染此菌后7 d内死亡率高达80%。【结论】不同的溶藻弧菌对小鼠的致病性存在较大差异,溶藻弧菌菌体本身比胞外分泌物对其毒性的贡献要大,而副溶血弧菌的毒性则由其胞外分泌物起主要作用;比较我们筛选出的强致病菌株与弱致病菌株,其上述毒力基因的分布并没有差别,说明溶藻弧菌可能存在一套与副溶血弧菌不同的独立的毒力基因系统。     

草地贪夜蛾和东方粘虫幼虫在轨迹球上对玉米常见挥发物的趋向反应比较

【目的】探讨外来入侵种草地贪夜蛾Spodoptera frugiperda和本地种东方粘虫Mythimna separata由嗅觉信息激发的爬行反应差异。【方法】采用轨迹球技术测定了草地贪夜蛾和东方粘虫的3龄幼虫对玉米广泛释放的11种气味物质的爬行反应参数(爬行速度、爬行轨迹长度、爬行位移、轨迹直线度、矢量角正弦、逆风定向位移和逆风轨迹直线度)。【结果】主成分分析发现,原始记录到的7个爬行参数可以简化为2个主成分,就能够解释总方差的85.45%。其中,第一主成分和第二主成分的生物学意义分别是“活泼因子”和“定向因子”。整体上看,草地贪夜蛾幼虫的活泼因子得分低于东方粘虫幼虫。在11种供试化合物中,反-β-石竹烯和乙酸香叶酯分别是激活草地贪夜蛾和东方粘虫幼虫最有效的物质;但β-蒎烯和癸醛分别是二者定向因子得分最高的物质。【结论】在所有供试的挥发物气流中,东方粘虫幼虫总是比草地贪夜蛾幼虫更活泼。就定向因子的种间比较来看,草地贪夜蛾幼虫对β-蒎烯和芳樟醇的嗅觉定向偏好性显著强于东方粘虫幼虫,而东方粘虫幼虫对乙酸顺-3-己烯酯的嗅觉定向偏好性显著强于草地贪夜蛾幼虫。     

应用PCR-DGGE和Real-Time PCR分析健康与腹泻獭兔盲肠菌群

【目的】研究獭兔腹泻发生后盲肠菌群结构的变化情况,为微生态防治断奶兔腹泻提供理论基础。【方法】分别提取5只健康和腹泻獭兔盲肠内容物总DNA,应用PCR-DGGE及Real-Time PCR技术比较分析獭兔盲肠中菌群结构的差异。【结果】腹泻獭兔PCR-DGGE图谱条带丰富度、均匀度、多样性指数与健康獭兔相比差异均不显著,但聚类分析和主成分分析(PCA)能够将腹泻组和健康组区分开。Real-Time PCR检测显示,腹泻獭兔盲肠正常菌群普雷沃氏菌、梭菌类群Ⅰ数量与健康獭兔相比没有变化(P0.05);链球菌属、梭菌类群Ⅳ和ⅩⅣa、白色瘤胃球菌、溶纤维丁酸弧菌、普拉梭杆菌等有益微生物数量显著降低(P0.01),而埃希氏大肠杆菌数量却显著升高(P0.05)。【结论】獭兔腹泻发生后盲肠有益微生物数量降低,有害微生物数量升高;菌群结构有差异但不显著(P0.05),腹泻獭兔盲肠菌群结构有复杂化的趋势。     

蛭弧菌BDE-1的生物特性及促进其蛭质体形成的研究

【背景】蛭弧菌是众多海洋益生菌中的一类较新成员,应用前景十分广阔。但由于蛭弧菌特殊的繁殖方式和周期,它的应用效果受寄生宿主特性和生物活性的影响,因而优选寄生宿主,维持或者提高蛭弧菌微生态制剂的应用活性是关键。【目的】筛选出能够裂解枯草芽孢杆菌的蛭弧菌,以增进其益生性能;研究提高蛭弧菌的蛭质体密度,以利于保存。【方法】从海南取回海泥样后,以枯草芽孢杆菌作为宿主菌,通过稀营养肉汤(Dilute nutrient broth,DNB)双层平板法分离获得蛭弧菌,并对目标菌株进行透射电镜形态鉴定和16S rRNA基因序列分析;然后进行生物学特性研究,同时开展氨苄青霉素、吲哚、Ca~(2+)和Mg~(2+)影响蛭质体形成的研究。【结果】分离出一株以枯草芽孢杆菌作为宿主的蛭弧菌并命名为BDE-1,其最适温度、盐度和pH分别为25℃、2.0%和7.0;BDE-1可裂解24株试验菌,占总试验菌株数(28株)的85.7%,其中对试验弧菌(13株)的裂解率达92.3%;吲哚、氨苄青霉素、Ca~(2+)和Mg~(2+)4种因子对BDE-1蛭质体的形成均有促进作用,其中吲哚和Ca~(2+)的促进作用显著。【结论】研究结果不仅为蛭弧菌寄生宿主的优化选择提供了可行性解决思路,而且为维持或提高蛭弧菌微生态制剂的应用活性提供了理论依据。     

傅立叶变换红外光谱技术对两株大肠杆菌的鉴别

【目的】应用傅立叶变换红外光谱(Fourier transform infrared spectroscopy,FT-IR)技术对两株不同来源的大肠杆菌进行鉴别。【方法】用FT-IR技术对两株不同来源的大肠杆菌进行指纹图谱数据采集,用化学计量学分析方法对光谱进行分析。【结果】建立了基于主成分分析(Principal component analysis,PCA)和分级聚类分析(Hierarchical cluster analysis,HCA)两种聚类分析模型,均可将两株大肠杆菌进行成功区分。【结论】傅立叶变换红外光谱分析方法简便、快速、易操作,结果重现性好,可用于区分不同来源的同种细菌。     

溶藻弧菌ZJ-T小RNA srvg23535基因突变株的构建及其功能初探

【背景】小RNA被证实对细菌遗传变异、生长繁殖、致病性等具有重要的调控作用,但是在机会致病菌——溶藻弧菌(Vibrioalginolyticus)中研究较少。本实验室前期通过转录组测序和Northern Blot鉴定得到溶藻弧菌ZJ-T中小RNA srvg23535。【目的】初探小RNA srvg23535对溶藻弧菌生物学特性的影响。【方法】利用同源重组技术构建小RNA srvg23535的缺失突变株,并对野生株和srvg23535突变株的菌落形态、运动性、胞外蛋白酶分泌、H_2O_2和Cu~(2+)的压力感应、铁吸收利用、抗生素抗性以及生长代谢等生物学特性进行比较研究。【结果】通过对溶藻弧菌小RNA srvg23535基因缺失突变株的生物学特性分析表明,小RNA srvg23535缺失后,对溶藻弧菌菌落形态、运动性、胞外蛋白酶分泌、H_2O_2和Cu~(2+)的压力感应、铁吸收利用、抗生素抗性以及多数测定的碳源、氮源代谢的影响不显著;但突变株对D-海藻糖(D-trehalose)的代谢减弱,对果胶(Pectin)、丙氨酸-谷氨酰胺(Ala-Gln)的代谢增强,并可利用丙氨酸-天冬氨酸(Ala-Asp)为氮源。【结论】小RNA srvg23535参与调控溶藻弧菌对D-海藻糖(D-trehalose)、果胶(Pectin)、丙氨酸-谷氨酰胺(Ala-Gln)和丙氨酸-天冬氨酸(Ala-Asp)的代谢,这将为获取srvg23535调控靶标基因,进而阐明其与靶标基因的相互作用机制奠定基础。     

Classification and prediction of rice wines with different marked ages by using a voltammetric electronic tongue

A voltammetric electronic tongue (VE-tongue) was developed to discriminate the difference between Chinese rice wines in this research. Three types of Chinese rice wine with different marked ages (1, 3, and 5 years) were classified by the VE-tongue by principal component analysis (PCA) and cluster analysis (CA). The VE-tongue consisted of six working electrodes (gold, silver, platinum, palladium, tungsten, and titanium) in a standard three-electrode configuration. The multi-frequency large amplitude pulse voltammetry (MLAPV), which consisted of four segments of 1 Hz, 10 Hz, 100 Hz, and 1000 Hz, was applied as the potential waveform. The three types of Chinese rice wine could be classified accurately by PCA and CA, and some interesting regularity is shown in the score plots with the help of PCA. Two regression models, partial least squares (PLS) and back-error propagation-artificial neural network (BP-ANN), were used for wine age prediction. The regression results showed that the marked ages of the three types of Chinese rice wine were successfully predicted using PLS and BP-ANN.     

Microarray detection of food-borne pathogens using specific probes prepared by comparative genomics

In order to design a method for the accurate detection and identification of food-borne pathogens, we used comparative genomics to select 70-mer oligonucleotide probes specific for 11 major food-borne pathogens (10 overlapping probes per pathogen) for use in microarray analysis. We analyzed the hybridization pattern of this constructed microarray with the Cy3-labeled genomic DNA of various food-borne pathogens and other bacteria. Our microarray showed a highly specific hybridization pattern with the genomic DNA of each food-borne pathogen; little unexpected cross-hybridization was observed. Microarray data were analyzed and clustered using the GenePix Pro 6.0 and GeneSpring GX 7.3.1 programs. The analyzed dendrogram revealed the discriminating power of constructed microarray. Each food-borne pathogen clustered according to its hybridization specificity and non-pathogenic species were discriminated from pathogenic species. Our method can be applied to the rapid and accurate detection and identification of food-borne pathogens in the food industry. In addition, this study demonstrates that genome sequence comparison and DNA microarray analysis have a powerful application in epidemiologic and taxonomic studies, as well as in the food safety and biodefense fields.     

基于单碱基延伸标签反应的常见食源性致病菌基因芯片检测方法的建立

针对8种常见的食源性致病菌(金黄色葡萄球菌、副溶血弧菌、单核细胞增生李斯特菌、沙门氏菌、阪崎肠杆菌、志贺氏菌、肠出血性大肠杆菌O157:H7和空肠弯曲杆菌),建立了基于单碱基延伸标签反应原理的基因芯片检测方法。筛选和整合8种食源性致病菌基因组中的特异性序列和相应PCR引物,致病菌靶DNA片段被扩增和纯化作为单碱基延伸标签反应的模板,反应产物在DNA芯片上与探针进行杂交反应,然后通过扫描基片的荧光强度进行判断。实验结果表明,可采用基于单碱基延伸标签反应的基因芯片方法同时特异性检测8种食源性致病菌,基因组DNA多重检测灵敏度可达到0.1pg,以鼠伤寒沙门氏菌为单一检测对象的细菌纯培养物灵敏度可达到5×102CFU/mL。本方法可以快速灵敏地检测食源性致病菌,为食源性疾病的诊断和防治提供了一个有效的方法。     

Vibrios of some deep-water invertebrates

Abstract The gut microbiota of 7 species of deep-water (300–400 m) invertebrates from the Gulf of Mexico was examined. High populations of Vibrio spp. were observed in crustaceans (ranging from 10⁵ to 7 × 10⁶ cells/g gut content) while relatively low populations of Vibrio spp. were found in annelids, the water column, and sediment. Although saprophytic Vibrio species were isolated, Vibrio fluvialis and Vibrio hollisae , potential human pathogens, were isolated from the crustaceans, Pleoticus robustus, Nematocarcinas sp., Plesionika sp., and Munida sp., and Vibrio vulnificus was isolated from Nereis sp. These observations confirm the finding of Ohwada et al. [18] that the gut of deep-water invertebrates has a bacterial flora abundant in Vibrio spp. These results also suggest that some marine invertebrates may serve as reservoirs for certain potential pathogenic Vibrio species.     

环介导等温扩增联合横向流动试纸条可视化检测志贺氏菌

【目的】将环介导等温扩增技术(LAMP)与横向流动试纸条(LFD)联合应用,建立一种可应用于志贺氏菌快速检测的LAMP-LFD技术。【方法】以福氏志贺氏菌的侵袭性质粒抗原H(ipa H)基因为检测靶标设计3对特异性引物(其中上游内引物Sfl-ipa H-FIP由生物素标记),进行LAMP反应;同时设计1条异硫氰酸荧光素(FITC)标记的探针Sfl-ipa H-HP,与获得的LAMP产物进行特异性杂交,杂交产物经LFD完成检测。【结果】优化后的LAMP反应条件为63°C 40 min,加上LFD结果判读共需50 min。LAMP-LFD方法能够特异性检测出福氏志贺氏菌,而对肠炎沙门氏菌等其它4种导致腹泻的致病菌和创伤弧菌等5种常见食物源性致病菌,以及4株不同大肠杆菌的检测结果呈阴性。该方法针对福氏志贺氏菌的检测灵敏度为1.0×10~2 CFU/m L或4 CFU/反应,针对人工污染鲤鱼肠组织的检测灵敏度是5.0×10~2 CFU/m L,是以LAMP外引物Sfl-ipa H-F3/Sfl-ipa H-B3的常规PCR方法的100倍。【结论】建立的LAMP-LFD技术具有操作简单、检测快速准确、检测成本低等优点,有望在志贺氏菌的常规监测和即时检测中被普及使用。     

Detection and Identification of Salmonella enterica,Escherichia coli,and Shigella spp. via PCR-Electrospray Ionization Mass Spectrometry: Isolate Testing and Analysis of Food Samples

An assay to identify the common food-borne pathogens Salmonella, Escherichia coli, Shigella, and Listeria monocytogenes was developed in collaboration with Ibis Biosciences (a division of Abbott Molecular) for the Plex-ID biosensor system, a platform that uses electrospray ionization mass spectroscopy (ESI-MS) to detect the base composition of short PCR amplicons. The new food-borne pathogen (FBP) plate has been experimentally designed using four gene segments for a total of eight amplicon targets. Initial work built a DNA base count database that contains more than 140 Salmonella enterica, 139 E. coli, 11 Shigella, and 36 Listeria patterns and 18 other Enterobacteriaceae organisms. This assay was tested to determine the scope of the assay''s ability to detect and differentiate the enteric pathogens and to improve the reference database associated with the assay. More than 800 bacterial isolates of S. enterica, E. coli, and Shigella species were analyzed. Overall, 100% of S. enterica, 99% of E. coli, and 73% of Shigella spp. were detected using this assay. The assay was also able to identify 30% of the S. enterica serovars to the serovar level. To further characterize the assay, spiked food matrices and food samples collected during regulatory field work were also studied. While analysis of preenrichment media was inconsistent, identification of S. enterica from selective enrichment media resulted in serovar-level identifications for 8 of 10 regulatory samples. The results of this study suggest that this high-throughput method may be useful in clinical and regulatory laboratories testing for these pathogens.     

傅立叶变换红外光谱技术对5种沙门氏菌的快速分类鉴定

[目的]建立沙门氏菌属内鼠伤寒沙门氏菌、肠炎沙门氏菌、猪霍乱沙门氏菌、亚利桑那沙门氏菌、波斯坦沙门氏菌5种菌的傅立叶变换红外(Fourier transform infrared,FT-IR)光谱数据库及FT-IR分类鉴定方法.[方法]应用FT-IR技术对5种沙门氏菌进行指纹图谱数据采集,应用化学计量学分析方法对光谱进行分析.[结果]建立了5种沙门氏菌的标准FT-IR光谱数据库,用于FT-IR技术对5种可疑目标沙门氏菌进行鉴定;建立了基于主成分分析(Principal component analysis,PCA)和分级聚类分析(Hierarchical cluster analysis,HCA)两种聚类分析模型,均可成功将5种沙门氏菌进行区分.[结论]傅立叶变换红外光谱分析方法简便、快速、易操作,结果重现性好,是一种区分5种沙门氏菌的有效方法.