Journal of Rapid Methods and Automation in Microbiology最新文献_第10页

DEVELOPMENT OF A SPOT-TITER CULTURE ASSAY FOR QUANTIFYING BACTERIA AND VIRAL INDICATORS 一种用于定量细菌和病毒指标的斑点滴度培养试验的发展

Journal of Rapid Methods and Automation in Microbiology

Pub Date : 2009-12-02 DOI: 10.1111/j.1745-4581.2009.00182.x

N.K. BECK, K. CALLAHAN, S.P. NAPPIER, H. KIM, M.D. SOBSEY, J.S. MESCHKE

ABSTRACT

The spread-plate and double agar layer (DAL) methods are common for the enumeration of bacteria and viral indicators (bacteriophages). However, they may become cumbersome in large matrix experiments or when the titer of the organism varies by several orders of magnitude. A bacterial spot-titer assay has been available for decades but has not been adapted to bacteriophages and has rarely been applied to the analysis of environmental samples. In this study, a spot-titer culture-based method was investigated for bacteria and bacteriophages. The method involves spot-plating replicate 10-µl volumes of several sample dilutions on a single plate, incubating, and counting colonies or plaques. Parallel assays of laboratory cultures and environmentally isolated organisms show that the spot-titer method is equally straightforward and statistically comparable to the spread-plate and DAL methods (R² = 0.989 for laboratory strains and R² = 0.972 for environmental samples), while more cost- and labor-efficient.

PRACTICAL APPLICATIONS

The spread-plate and double agar layer (DAL) methods currently used for enumeration of bacteria and viral indicators, may become labor- and resource-intensive (culture media, plates, technician time and incubator space) in large matrix experiments, which are often needed in the laboratory to evaluate environmental conditions. The spot-titer method has several advantages over the spread-plate and DAL methods: (1) it requires less time to dispense spots than to spread the microbe; (2) it uses fewer materials (15–20% of the laboratory supplies as the traditional methods); (3) it requires less effort; and (4) since the sample is distributed in distinct spots, colony/plaque counting is faster and less labor intensive. The spot-titer method was found to economize resources without sacrificing accuracy or precision, and is a practical method for routine use in large matrix experiments (e.g., survival or disinfection studies) and enumeration of high-titer environmental samples.

扩展板和双琼脂层(DAL)法是常用的细菌和病毒指标(噬菌体)的计数方法。然而，在大型基质实验中或当生物体的滴度变化几个数量级时，它们可能变得麻烦。细菌滴度测定法已有几十年的历史，但尚未适用于噬菌体，也很少应用于环境样品的分析。本研究研究了一种基于点滴度培养的细菌和噬菌体检测方法。该方法包括在单个平板上复制10 μ l体积的几种样品稀释，孵育并计数菌落或空斑。实验室培养物和环境分离生物的平行分析表明，点滴法与张开板法和DAL法同样简单，在统计上可与之相比(实验室菌株的R2 = 0.989，环境样品的R2 = 0.972)，同时成本和劳动效率更高。目前用于细菌和病毒指标计数的扩展板和双琼脂层(DAL)方法在大型基质实验中可能会成为劳动力和资源密集型(培养基，板，技术人员时间和培养箱空间)，而实验室通常需要这些方法来评估环境条件。与扩散板法和DAL法相比，斑点滴度法有几个优点:(1)分配斑点所需的时间比扩散微生物所需的时间少;(2)耗材少(传统方法为实验室耗材的15-20%);(3)省力;(4)由于样品分布在不同的点，菌落/菌斑计数更快，劳动强度更低。发现现场滴度法在不牺牲准确性或精密度的情况下节省资源，并且是在大型基质实验(例如生存或消毒研究)和高滴度环境样品枚举中常规使用的实用方法。

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引用次数: 42

COMPARATIVE ANALYSIS OF METHODS FOR THE PURIFICATION OF DNA FROM LOW-BIOMASS SAMPLES BASED ON TOTAL YIELD AND CONSERVED MICROBIAL DIVERSITY 基于总产率和微生物多样性的低生物量样品DNA纯化方法的比较分析

Journal of Rapid Methods and Automation in Microbiology

Pub Date : 2009-09-02 DOI: 10.1111/j.1745-4581.2009.00153.x

MYRON T. LA DUC, SHARIFF OSMAN, KASTHURI VENKATESWARAN

ABSTRACT

Despite advances in the specificity and sensitivity of molecular biological technologies, the efficient recovery of DNA from low-biomass samples remains extremely challenging. Optimal methods to purify biomolecules from such environments should (1) achieve the greatest total yield and (2) reflect the true microbial diversity of the sample. These attributes were assessed from five DNA purification regimes: a standard-manual procedure, MoBio Ultraclean and Promega Wizard kits, and an automated Axcyte AutoLyser method with and without bead-beating agitation. A homogenous mixture of known concentrations of nine distinct bacterial lineages isolated from low-biomass environments was prepared and suitable aliquots of subsamples were processed in parallel. DNA products from each of these methods were then subjected to polymerase chain reaction (PCR), quantitative PCR and 16S rRNA clone-library analysis. The Axcyte AutoLyser outperformed all other purification regimes examined. This automated method consistently both yielded the highest concentration of PCR-amplifiable DNA, and reported species composition most consistent with the starting solution.

PRACTICAL APPLICATIONS

This communication carefully examines the effectiveness of common DNA purification regimes as well as an automated method. Comparative analyses convincingly demonstrate that the different methods not only result in different recovery of genomic DNA, but more importantly, different estimations of microbial diversity in the sample. This report will hopefully inspire investigators from various industries (pharmaceutical, ecological, medical, semiconductor, etc.) who find themselves in the initial phases of large-scale studies to devote a significant effort into optimizing sample extraction protocols to achieve the most accurate information.

尽管分子生物学技术在特异性和敏感性方面取得了进步，但从低生物量样品中高效回收DNA仍然极具挑战性。从这种环境中纯化生物分子的最佳方法应该(1)达到最大的总产量(2)反映样品的真实微生物多样性。这些属性是通过五种DNA纯化方案进行评估的:标准手动程序，MoBio Ultraclean和Promega Wizard试剂盒，以及自动Axcyte autotolyser方法(有和没有跳动搅拌)。从低生物量环境中分离出已知浓度的9种不同细菌谱系的均匀混合物，并平行处理合适的等量亚样品。然后对每种方法的DNA产物进行聚合酶链反应(PCR)、定量PCR和16S rRNA克隆文库分析。Axcyte AutoLyser优于所有其他的纯化方法。这种自动化方法一致地产生了最高浓度的pcr扩增DNA，并且报告的物种组成与起始溶液最一致。本通讯仔细检查了常见的DNA纯化制度以及自动化方法的有效性。对比分析令人信服地表明，不同的方法不仅导致基因组DNA的不同恢复，更重要的是，样品中微生物多样性的不同估计。本报告将有望激励各个行业(制药，生态，医疗，半导体等)的研究人员，他们发现自己处于大规模研究的初始阶段，投入大量精力优化样品提取方案，以获得最准确的信息。

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引用次数: 19

A NEW FLUOROGENIC ASSAY FOR MONITORING AND DETERMINING PLANKTONIC AND BIOFILM FORMS OF PSEUDOMONAS AERUGINOSA VIABLE COUNT IN VITRO 一种新的荧光检测方法，用于监测和测定铜绿假单胞菌浮游和生物膜形式的体外活菌数

Journal of Rapid Methods and Automation in Microbiology

Pub Date : 2009-09-02 DOI: 10.1111/j.1745-4581.2009.00156.x

WALID F. ELKHATIB, AYMAN M. NOREDDIN

ABSTRACT

A new method was developed to rapidly monitor the Pseudomonas aeruginosa viable counts using alamar blue (AB). The 96-well microtiter plates were used to perform the assay. This procedure is based on fluorogenic measurement as a result of reduction of nonfluorescent AB to red fluorescent form by the viable cells of P. aeruginosa. The correlation between conventional plate count and fluorogenic AB method was highly satisfactory for quantification of planktonic (R² = 0.9487) and biofilm cells of P. aeruginosa (R² = 0.9296).

PRACTICAL APPLICATIONS

The new fluorogenic method can rapidly monitor Pseudomonas aeruginosa counts in vitro with a high correlation with the conventional plating method. The results indicate that fluorogenic method requires much shorter time (2 h) than the conventional plate count (24 h), is a more cost-effective way, quite amenable to high throughput, and continuous monitoring of P. aeruginosa viability is achievable in the kinetic in vitro models without interference with the cell viability.

建立了一种快速监测铜绿假单胞菌活菌计数的新方法。采用96孔微量滴度板进行测定。这个程序是基于荧光测量的结果，非荧光AB减少到红色荧光形式的绿脓杆菌的活细胞。常规平板计数与荧光AB法对铜绿假单胞菌浮游细胞(R2 = 0.9487)和生物膜细胞(R2 = 0.9296)的定量具有高度满意的相关性。新型荧光法可以快速监测铜绿假单胞菌体外计数，与传统的镀样法具有较高的相关性。结果表明，荧光法所需时间(2 h)比传统的平板计数(24 h)短得多，是一种更经济、更适合高通量的方法，并且在不干扰细胞活力的情况下，可以在体外动力学模型中连续监测铜绿假单胞菌的活力。

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引用次数: 3

A NOVEL METHOD FOR ASSESSMENT OF 16S RRNA GENE COPY NUMBER IN BACTERIAL GENOMES BY PULSED-FIELD GEL ELECTROPHORESIS AND PCR AMPLIFICATION 利用脉冲场凝胶电泳和PCR扩增技术评估细菌基因组中16s rrna基因拷贝数的新方法

Journal of Rapid Methods and Automation in Microbiology

Pub Date : 2009-09-02 DOI: 10.1111/j.1745-4581.2009.00165.x

YONGHUI ZENG, NIANZHI JIAO

ABSTRACT

16S rRNA gene (rrn) copy number in bacterial genomes is indicative of ecological strategies of bacteria and is critical for quantification of bacterial abundance in mixed populations using rrn-based approaches. For accurate assessment of rrn copies, a novel technical strategy by means of pulsed-field gel electrophoresis and polymerase chain reaction amplification analysis was introduced. Experimental and in silico analysis on a test bacterial culture Caulobacter crescentus proved it to be simple, effective, accurate and a good alternative to traditional time-consuming methods.

PRATICAL APPLICATIONS

This method can be used for routine determination of gene copy number in most bacteria whose full genome sequences are not available. Moreover, the pulsed-field gel electrophoresis bands containing a target gene fragment can be determined and therefore constructing an expected fragments oriented genomic library is possible.

细菌基因组中16S rRNA基因(rrn)拷贝数反映了细菌的生态策略，对于使用基于rrn的方法定量混合群体中的细菌丰度至关重要。为了准确地评估rrn拷贝数，介绍了一种新的技术策略，即脉冲场凝胶电泳和聚合酶链反应扩增分析。实验和计算机分析结果表明，该方法简便、有效、准确，可替代耗时的传统方法。本方法可用于大多数无法获得全基因组序列的细菌基因拷贝数的常规测定。此外，可以确定含有目标基因片段的脉冲场凝胶电泳带，从而可以构建预期的片段导向基因组文库。

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引用次数: 1

SUITABILITY OF INTERGENIC SPACER OR INTERNAL TRANSCRIBED SPACER MICROSATELLITE-PRIMED PCR FOR THE IDENTIFICATION OF RHIZOCTONIA SOLANI AND SOME PHYTOFUNGI 基因间间隔段或内转录间隔段微卫星引物PCR鉴定茄枯丝核菌及部分植物真菌的适用性

Journal of Rapid Methods and Automation in Microbiology

Pub Date : 2009-09-02 DOI: 10.1111/j.1745-4581.2009.00178.x

K.A. ABD-ELSALAM, J.-R. GUO, M.A. MOSLEM, A.H. BAHKALI, J.-A. VERREET

ABSTRACT

The intergenic spacer (IGS) region or internal transcribed spacer (ITS) region were used in pair-combinations with microsatellite-primed polymerase chain reaction (MP-PCR) primers to establish whether additional polymorphisms can be yielded. A total of 24 Rhizoctonia solani isolates representing 13 anstomosis groups and 9 different fungal species isolate were recovered from different areas and hosts. Forty different primer combinations were tested for their ability to provide discrete bands and individual isolates' readily interpretable and reproducible IGS/ITS-MP-PCR profiles. Both approaches produced highly reproducible and complex genomic fingerprints, with fragments ranging in size from 100 to 2,000 bp (IGS-MP-PCR) and 50 to 2,000 bp (ITS-MP-PCR). MP-PCR markers yielded more bands than IGS/ITS-MP-PCR because of their higher redundancy in the fungal genome. The number of fragments generated by both techniques varied according to the fungal species and also with the primer combination used. Each primer used could differentiate all of the fungal isolates examined in this study. The profiles generated were identical and reproducible between repeated PCR experiments.

PRACTICAL APPLICATIONS

Combining the intergenic spacer/internal transcribed spacer-microsatellite-primed polymerase chain reaction technique with microsatellite–detection assay allows the rapid and specific detection of Rhizoctonia solani anastomosis groups and different phytopathogenic fungi. The utility of this approach stems from its simplicity and reproducibility, the high number of polymorphisms revealed, the very small amounts of DNA needed, rapidity, and ease of performance. The improved technique will present valuable information on the role of some phytopathogenic fungi and R. solani in agriculturally important plant diseases.

利用基因间间隔区(IGS)或内部转录间隔区(ITS)与微卫星引物聚合酶链反应(MP-PCR)引物进行配对组合，以确定是否可以产生额外的多态性。在不同地区和不同寄主中共分离到24株枯丝核菌，分别属于13个菌群和9个不同的真菌种。测试了40种不同引物组合提供离散条带和单个分离物易于解释和可重复的IGS/ITS-MP-PCR图谱的能力。这两种方法都产生了高度可重复性和复杂的基因组指纹，片段的大小从100到2000 bp (IGS-MP-PCR)和50到2000 bp (ITS-MP-PCR)不等。MP-PCR标记比IGS/ITS-MP-PCR产生更多的条带，因为它们在真菌基因组中具有更高的冗余性。两种技术产生的片段数量根据真菌种类和使用的引物组合而变化。每个引物都可以区分本研究中检测的所有真菌分离物。在重复的PCR实验中产生的基因图谱是相同的，可重复的。将基因间间隔物/内转录间隔物-微卫星引物聚合酶链反应技术与微卫星检测技术相结合，可以快速、特异地检测茄根丝核菌吻合群和不同的植物病原真菌。这种方法的实用性源于它的简单性和可重复性、揭示的多态性数量多、所需的DNA数量非常少、快速和易于执行。改进后的技术将为一些植物病原真菌和茄疫病在重要农业植物病害中的作用提供有价值的信息。

{"title":"SUITABILITY OF INTERGENIC SPACER OR INTERNAL TRANSCRIBED SPACER MICROSATELLITE-PRIMED PCR FOR THE IDENTIFICATION OF RHIZOCTONIA SOLANI AND SOME PHYTOFUNGI","authors":"K.A. ABD-ELSALAM, J.-R. GUO, M.A. MOSLEM, A.H. BAHKALI, J.-A. VERREET","doi":"10.1111/j.1745-4581.2009.00178.x","DOIUrl":"10.1111/j.1745-4581.2009.00178.x","url":null,"abstract":"<div>\u0000 \u0000 <section>\u0000 \u0000 <h3> ABSTRACT</h3>\u0000 \u0000 <p> <i>The intergenic spacer (IGS) region or internal transcribed spacer (ITS) region were used in pair-combinations with microsatellite-primed polymerase chain reaction (MP-PCR) primers to establish whether additional polymorphisms can be yielded. A total of 24</i> Rhizoctonia solani <i>isolates representing 13 anstomosis groups and 9 different fungal species isolate were recovered from different areas and hosts. Forty different primer combinations were tested for their ability to provide discrete bands and individual isolates' readily interpretable and reproducible IGS<b>/</b>ITS-MP-PCR profiles. Both approaches produced highly reproducible and complex genomic fingerprints, with fragments ranging in size from 100 to 2,000 bp (IGS-MP-PCR) and 50 to 2,000 bp (ITS-MP-PCR). MP-PCR markers yielded more bands than IGS<b>/</b>ITS-MP-PCR because of their higher redundancy in the fungal genome. The number of fragments generated by both techniques varied according to the fungal species and also with the primer combination used. Each primer used could differentiate all of the fungal isolates examined in this study. The profiles generated were identical and reproducible between repeated PCR experiments.</i></p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> PRACTICAL APPLICATIONS</h3>\u0000 \u0000 <p>Combining the intergenic spacer/internal transcribed spacer-microsatellite-primed polymerase chain reaction technique with microsatellite–detection assay allows the rapid and specific detection of <i>Rhizoctonia solani</i> anastomosis groups and different phytopathogenic fungi. The utility of this approach stems from its simplicity and reproducibility, the high number of polymorphisms revealed, the very small amounts of DNA needed, rapidity, and ease of performance. The improved technique will present valuable information on the role of some phytopathogenic fungi and <i>R. solani</i> in agriculturally important plant diseases.</p>\u0000 </section>\u0000 </div>","PeriodicalId":50067,"journal":{"name":"Journal of Rapid Methods and Automation in Microbiology","volume":"17 3","pages":"383-397"},"PeriodicalIF":0.0,"publicationDate":"2009-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/j.1745-4581.2009.00178.x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"63426531","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 9

DETERMINATION OF INDICATOR BACTERIA IN PHARMACEUTICAL SAMPLES BY MULTIPLEX PCR 多重PCR法测定药品样品中指示菌

Journal of Rapid Methods and Automation in Microbiology

Pub Date : 2009-09-02 DOI: 10.1111/j.1745-4581.2009.00154.x

S. FARAJNIA, M. HASSAN, S. HALLAJ NEZHADI, L. MOHAMMADNEJAD, M. MILANI, F. LOTFIPOUR

ABSTRACT

Rapid and sensitive detection techniques for indicator pathogens are important in pharmaceutical industry. However, common detection methods rely on bacterial culture in combination with biochemical tests, a process that typically takes 5–6 days to complete. Thus, the aim of this study was to develop a multiplex polymerase chain reaction (mPCR) assay for simultaneous detection and identification of four indicator pathogenic bacteria in a single reaction. Specific primers for indicator bacteria, namely Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosaand Salmonella, were applied to allow simultaneous detection of them, and the sensitivity and specificity of each primer pairs were determined. In the mPCR with mixed DNA samples, specific bands for corresponding bacteria were simultaneously detected. Agarose gel electrophoresis of PCR products revealed 100% specificity of mPCR with single bands in the expected sizes. Low levels of microbial contamination less than 10 cfu per milliliter or gram of product were detected using mPCR assay. The detection of all four indicator pathogenic bacteria were completed in less than 8 h with this novel mPCR method, whereas the conventional United States Pharmacopeia methods and uniplex PCR required 5–6 days and 27 h for completion, respectively. Using mPCR assay, the microbial quality control of nonsterile pharmaceutical products can be performed in a cost-effective and timely manner in pharmaceutical industry.

PRACTICAL APPLICATIONS

Detection of pathogenic indicatiors of Escherichia coli, Staphylococcus aureus, Salmonella and Pseudomonas aeruginosa is one of the mandatory tests in microbial quality of nonsterile pharmaceutical products; therefore, rapid and sensitive detection of the contaminations is of great importance for product release. According to the results of the present study, simultaneous detection of low levels of four major potential pathogenic bacteria in pharmaceutical finished products can be performed using mPCR in a cost-effective and timely manner, and upon these properties of the mPCR assay it could have potential applications in pharmaceutical industry.

快速、灵敏的指示性病原体检测技术在制药行业具有重要意义。然而，常用的检测方法依赖于细菌培养与生化试验相结合，这一过程通常需要5-6天才能完成。因此，本研究的目的是建立一种多重聚合酶链反应(mPCR)方法，用于在单一反应中同时检测和鉴定四种指示致病菌。采用大肠杆菌、金黄色葡萄球菌、铜绿假单胞菌、沙门氏菌等指示菌的特异性引物同时检测，并测定每对引物的敏感性和特异性。在混合DNA样品的mPCR中，同时检测到相应细菌的特异性条带。琼脂糖凝胶电泳结果显示，在预期大小的单条带中，mPCR的特异性为100%。低水平的微生物污染低于10 cfu每毫升或克的产品被检测使用mPCR法。4种指示致病菌的检测均在8 h内完成，而传统的美国药典方法和单链PCR分别需要5 ~ 6 d和27 h。在医药工业中，应用mPCR法对非无菌药品进行微生物质量控制是一种经济、及时的方法。大肠杆菌、金黄色葡萄球菌、沙门氏菌和铜绿假单胞菌的致病指标检测是非无菌药品微生物质量的强制性检测之一;因此，快速、灵敏地检测污染物对产品放行具有重要意义。本研究结果表明，利用mPCR法可以同时检测制药成品中4种主要潜在致病菌的低水平，具有较好的成本效益和及时性，在制药行业具有潜在的应用前景。

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引用次数: 19

DIRECT FLUORESCENT ANTIBODY-DIRECT VIABLE COUNT AND POLYMERASE CHAIN REACTION DETECTION LIMIT FOR THE IDENTIFICATION OF VIBRIO CHOLERAE O1 IN MUSSELS (MYTILUS EDULIS) 直接荧光抗体-直接活菌计数及聚合酶链反应检测贻贝(mytilus edulis)霍乱弧菌o1的检出限

Journal of Rapid Methods and Automation in Microbiology

Pub Date : 2009-09-02 DOI: 10.1111/j.1745-4581.2009.00175.x

S.R. PERESSUTTI, V. JURQUIZA, S. GONZÁLEZ-FRAGA, M. PICHEL, N. BINSZTEIN, M. COSTAGLIOLA

ABSTRACT

Vibrio cholerae O1 is natural to the aquatic environment and can cause gastrointestinal infections when it is consumed from contaminated bivalves. Under unfavorable conditions, this bacterium enters into a viable but nonculturable state. Immunofluorescence and polymerase chain reaction (PCR) methods were a useful alternative for detecting this microorganism without a pre-enrichment step. We investigated the detection limit of the direct fluorescent antibody (DFA)-direct viable count (DVC) and PCR techniques for the identification of V. cholerae O1 in mussel (Mytilus edulis) samples. When 10³ cfu/mL V. cholerae O1 were inoculated in samples, 10²–10³ bacteria mL⁻¹ were determined by immunofluorescence tests and 67% of the samples were positive by PCR assay. No significant difference (T statistic value = 6.5, P = 0.2049) between DFA and DFA-DVC procedures was observed. No presence of endogenous V. cholerae O1 was detected.

PRACTICAL APPLICATIONS

Vibrios are considered the major cause of identifiable illness and death from shellfish consumption. In Argentina, the viable but nonculturable (VBNC) forms of Vibrio cholerae O1 were identified in samples of water and plankton. Because of these facts, it is relevant to research the presence of V. cholerae O1 in aquatic bivalves. Immunofluorescence and polymerase chain reaction methods are a useful alternative to traditional enrichment testing for detecting both culturable and VBNC forms of V. cholerae O1. In this work, it was demonstrated that these methods were sensitive and efficient for detecting V. cholerae O1 in mussels without a pre-enrichment step. Moreover, they can be a useful tool for the rapid detection of this pathogen in the seafood industry.

霍乱弧菌O1在水生环境中是天然存在的，当食用被污染的双壳类动物时可引起胃肠道感染。在不利的条件下，这种细菌进入有活力但不可培养的状态。免疫荧光和聚合酶链反应(PCR)方法是检测这种微生物的有效替代方法，无需预先富集步骤。研究了直接荧光抗体(DFA)-直接活菌计数(DVC)和PCR技术在贻贝(Mytilus edulis)样品中鉴定霍乱弧菌O1的检出限。当接种103cfu /mL霍乱弧菌O1时，免疫荧光法检测出102 ~ 103个细菌mL−1,PCR检测67%的样品呈阳性。DFA与DFA- dvc两种方法间无统计学差异(T统计值= 6.5,P = 0.2049)。未检出内源性霍乱弧菌O1。实际应用弧菌被认为是食用贝类导致可识别疾病和死亡的主要原因。在阿根廷，在水和浮游生物样本中发现了有活力但不可培养的霍乱弧菌O1 (VBNC)形式。因此，研究霍乱弧菌O1在水生双壳类动物中的存在具有重要意义。免疫荧光和聚合酶链反应方法是传统富集检测霍乱弧菌O1的有效替代方法，可用于检测可培养型和VBNC型霍乱弧菌。本研究结果表明，这些方法对检测贻贝中的霍乱弧菌O1是敏感和有效的，无需预先富集步骤。此外，它们可以成为海产品行业快速检测该病原体的有用工具。

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引用次数: 2

PERSISTENCE OF SALMONELLA SPP. ON CHICKEN SKIN AFTER EXPOSURE TO AN ITALIAN MARINADE* 接触意大利腌料后，鸡肉皮肤上的沙门氏菌持续存在*

Journal of Rapid Methods and Automation in Microbiology

Pub Date : 2009-09-02 DOI: 10.1111/j.1745-4581.2009.00176.x

THOMAS P. OSCAR, MANPREET SINGH

ABSTRACT

A series of experiments with chicken skin was undertaken to determine the effect of an Italian marinade on persistence of Salmonella spp. during refrigerated storage and marinating. Chicken skin was inoculated with 0.4 to 3.7 log of multiple antibiotic resistant strains of Salmonella Typhimurium (n = 3), Kentucky (n = 1) or Hadar (n = 1). Chicken skin was then exposed to the Italian marinade for 4 or 24 h at 6C to simulate normal marinating conditions of consumers. The persistence of Salmonella spp. on chicken skin was reduced (P < 0.05) by the Italian marinade with a greater reduction observed at 24 h than at 4 h of marinating. As expected, the persistence during marinating increased as a function of the initial number of Salmonella inoculated. In general, the effect of the Italian marinade on persistence was similar among the five strains of Salmonella tested.

PRACTICAL APPLICATIONS

Marinades contain organic acids (acetic, lactic) that decrease meat pH and help to reduce or eliminate pathogenic bacteria, such as Salmonella, from the product. Inclusion of other ingredients in marinades, such as salts and spices that have antimicrobial properties, helps to further reduce or prevent persistence of Salmonella. Results of this study with an Italian-style marinade indicate that consumers should marinate chicken in the refrigerator for 24 h rather than 4 h to maximize the benefit of the Italian marinade on reducing the risk of exposure to Salmonella that might contaminate and persist on chicken purchased in the retail marketplace.

摘要以鸡皮为实验对象，研究了一种意大利腌料在冷藏和腌制过程中对沙门氏菌持续性的影响。用0.4 ~ 3.7 log的鼠伤寒沙门菌(n = 3)、肯塔基沙门菌(n = 1)和哈达尔沙门菌(n = 1)接种鸡皮。然后将鸡皮暴露在意大利腌料中，在6℃下浸泡4或24小时，以模拟消费者的正常腌制条件。意大利腌料降低了沙门氏菌在鸡皮上的持久性(P < 0.05)，腌制24 h时比腌制4 h时降低幅度更大。正如预期的那样，腌制过程中的持久性随着接种沙门氏菌的初始数量的增加而增加。总的来说，意大利腌料对沙门氏菌持久性的影响在五种测试菌株中是相似的。卤汁中含有有机酸(乙酸、乳酸)，可以降低肉的pH值，并有助于减少或消除产品中的沙门氏菌等致病菌。在腌料中加入其他成分，如具有抗菌特性的盐和香料，有助于进一步减少或防止沙门氏菌的持续存在。意大利式腌料的研究结果表明，消费者应该将鸡肉在冰箱中腌24小时而不是4小时，以最大限度地降低暴露于沙门氏菌的风险，沙门氏菌可能会污染和持续存在于零售市场上购买的鸡肉。

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引用次数: 1

FLUORESCENT MEASUREMENTS OF DNA, RNA AND PROTEINS TO PERFORM COMPARATIVE ANALYSES OF MICROBIAL COMMUNITIES FROM THE ENVIRONMENTS 荧光测量dna, RNA和蛋白质，从环境中执行微生物群落的比较分析

Journal of Rapid Methods and Automation in Microbiology

Pub Date : 2009-09-02 DOI: 10.1111/j.1745-4581.2009.00179.x

M. CARMEN PORTILLO, JUAN M. GONZALEZ

ABSTRACT

Simple and rapid methods for the quantification of DNA, RNA and proteins using specific fluorescent dyes are proposed for the comparison and monitoring of microbial communities from the environment. The purpose of this study was the use of straightforward in situ methods which voided the need for preservation of samples and the risk of potential degradation and quantitative changes during transportation. Aside from this, methods used to obtain information on environmental microbial communities are generally time-consuming and present certain difficulty above all when working on solid substrates such as soils and rocks. New generation fluorescent dyes that bind specifically to DNA, RNA and proteins allow simple and rapid estimates of these biomolecules in crude environmental samples.

PRACTICAL APPLICATIONS

Monitoring the metabolic state of microbial communities on different substrates and environments is a requirement for comparing samples and assessing the participation of microorganisms in a variety of processes. Solid substrates are not easily analyzed by microscopic techniques and they require long processing times and tedious work. Aside from this, only a minor fraction (<1%) of microorganisms in most natural environments can be cultured in standard microbiological media (Ward et al. 1990). Other studies using incorporation of labeled substrates to approach activity rates or biomolecule extractions represent complex and long procedures during environmental studies.In order to evaluate microbial communities in a variety of substrates and environments, rapid and simple methods are proposed by measuring DNA, RNA and/or proteins using specific fluorescent dyes, without a need for prior purification, from crude solid samples.

摘要:本文提出了一种使用特定荧光染料进行DNA、RNA和蛋白质定量的简单快速方法，用于比较和监测环境中的微生物群落。本研究的目的是使用直接的原位方法，避免了样品保存和运输过程中潜在降解和数量变化的风险。除此之外，用于获取环境微生物群落信息的方法通常是耗时的，并且在诸如土壤和岩石等固体基质上工作时存在一定的困难。新一代荧光染料与DNA、RNA和蛋白质特异性结合，可以简单快速地估计原始环境样品中的这些生物分子。监测不同基质和环境下微生物群落的代谢状态是比较样品和评估微生物参与各种过程的必要条件。固体底物不易用显微技术分析，而且需要较长的处理时间和繁琐的工作。除此之外，在大多数自然环境中，只有一小部分(<1%)的微生物可以在标准微生物培养基中培养(Ward等)。1990)。其他使用标记底物的结合来接近活性率或生物分子提取的研究在环境研究中代表了复杂和漫长的过程。为了评估各种底物和环境中的微生物群落，提出了一种快速简便的方法，即使用特定的荧光染料从粗固体样品中测量DNA, RNA和/或蛋白质，而无需事先纯化。

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引用次数: 2

MULTIPLEX DETECTION OF ESCHERICHIA COLI AND SALMONELLA ENTERITIDIS BY USING QUANTUM DOT-LABELED ANTIBODIES 量子点标记抗体多重检测大肠杆菌和肠炎沙门氏菌

Journal of Rapid Methods and Automation in Microbiology

Pub Date : 2009-09-02 DOI: 10.1111/j.1745-4581.2009.00155.x

FAHRIYE CEYDA DUDAK, İSMAİL HAKKI BOYACI

ABSTRACT

In this study, we demonstrated the simultaneous detection of Escherichia coli and Salmonella enteritidis, by coupling immunomagnetic separation (IMS) with quantum dots (QDs) labeling. QDs having different emission wavelengths were conjugated with anti-E. coli and anti-Salmonella antibodies. QD–antibody conjugates were used to label immunomagnetically separated bacteria and the fluorescence intensities were measured for enumerations of both species. The concentrations of primary antibodies used in IMS, the ratio of QDs to antibodies during the conjugation and the concentration of QD–antibody conjugates used in labeling were optimized to enhance the sensitivity of the assay. After labeling bacteria with QDs, the quenching observed between bead–bacteria complex and QDs was eliminated by separating QDs from the complex using sodium dodecyl sulfate solution. The fluorescence intensities due to the capturing of different concentrations of bacteria were measured and the working ranges were found to be 5 × 10² to 5 × 10⁵ cfu/mL for E. coli and 4 × 10² to 4 × 10⁵ cfu/mL for S. enteritidis.

PRACTICAL APPLICATIONS

In this study, antibody-conjugated multicolor quantum dots (QDs) were used for simultaneous detection of Escherichia coli and Salmonella enteritidis. The results of this study indicate that QD labels can be used in multiplex, rapid and selective detection of bacteria with detection limits comparable with those of many novel methods in cases where the assay conditions are optimized. Furthermore, the assay can be modified for the simultaneous detection of more than two species through using QD labels having different emission wavelengths.

在这项研究中，我们建立了免疫磁分离(IMS)和量子点(QDs)标记耦合技术同时检测大肠杆菌和肠炎沙门氏菌的方法。具有不同发射波长的量子点与反e共轭。大肠杆菌和抗沙门氏菌抗体使用qd抗体偶联物标记免疫磁分离细菌，并测量两种细菌计数的荧光强度。优化了IMS中使用的一抗浓度、偶联过程中量子点与抗体的比例以及用于标记的量子点抗体偶联物的浓度，以提高检测的灵敏度。用量子点标记细菌后，用十二烷基硫酸钠溶液分离量子点，消除了珠菌复合物与量子点之间的猝灭现象。测定了捕获不同浓度细菌的荧光强度，发现大肠杆菌的工作范围为5 × 102 ~ 5 × 105 cfu/mL，肠炎沙门氏菌的工作范围为4 × 102 ~ 4 × 105 cfu/mL。在本研究中，抗体共轭多色量子点(QDs)用于同时检测大肠杆菌和肠炎沙门氏菌。本研究结果表明，在优化分析条件的情况下，QD标签可用于细菌的多重、快速和选择性检测，其检出限与许多新方法相当。此外，通过使用具有不同发射波长的QD标签，可以修改该检测方法以同时检测两种以上的物种。

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引用次数: 13