痘病毒科不同病毒属特异的PCR检测方法的建立

目的:建立可准确、快速地鉴别诊断可感染人的不同属痘病毒的特异PCR方法。方法:设计针对正痘病毒属、副痘病毒属和传染性软疣病毒属的多对特异引物,并制备相应的DNA模板,针对不同的模板优化引物与反应条件,分别进行检测筛选,建立病毒属特异的单独与多重PCR方法。结果:单一模板的PCR扩增反应中,正痘病毒的检测敏感性可达101拷贝/μL(引物为OPEaL-F1880/OPEaL-R2057),副痘病毒的检测敏感性可达101拷贝/μL(引物为PP2/PP3),传染性软疣病毒的检测敏感性为100 pg/μL体系(引物为MCV1/MCV2);混合模板的PCR扩增反应中,各属特异的引物均可获得预期大小的特异片段。结论:我们建立的PCR诊断方法,可用于痘病毒科不同病毒属感染的实验室特异快速鉴别诊断。     

Enhanced Detection of Surface-Associated Bacteria in Indoor Environments by Quantitative PCR

Methods for detecting microorganisms on surfaces are needed to locate biocontamination sources and to relate surface and airborne concentrations. Research was conducted in an experimental room to evaluate surface sampling methods and quantitative PCR (QPCR) for enhanced detection of a target biocontaminant present on flooring materials. QPCR and culture analyses were used to quantitate Bacillus subtilis (Bacillus globigii) endospores on vinyl tile, commercial carpet, and new and soiled residential carpet with samples obtained by four surface sampling methods: a swab kit, a sponge swipe, a cotton swab, and a bulk method. The initial data showed that greater overall sensitivity was obtained with the QPCR than with culture analysis; however, the QPCR results for bulk samples from residential carpet were negative. The swab kit and the sponge swipe methods were then tested with two levels of background biological contamination consisting of Penicillium chrysogenum spores. The B. subtilis values obtained by the QPCR method were greater than those obtained by culture analysis. The differences between the QPCR and culture data were significant for the samples obtained with the swab kit for all flooring materials except soiled residential carpet and with the sponge swipe for commercial carpet. The QPCR data showed that there were no significant differences between the swab kit and sponge swipe sampling methods for any of the flooring materials. Inhibition of QPCR due solely to biological contamination of flooring materials was not evident. However, some degree of inhibition was observed with the soiled residential carpet, which may have been caused by the presence of abiotic contaminants, alone or in combination with biological contaminants. The results of this research demonstrate the ability of QPCR to enhance detection and enumeration of biocontaminants on surface materials and provide information concerning the comparability of currently available surface sampling methods.     

Direct Detection by In Situ PCR of the amoA Gene in Biofilm Resulting from a Nitrogen Removal Process

Ammonia oxidation is a rate-limiting step in the biological removal of nitrogen from wastewater. Analysis of microbial communities possessing the amoA gene, which is a small subunit of the gene encoding ammonia monooxygenase, is important for controlling nitrogen removal. In this study, the amoA gene present in Nitrosomonas europaea cells in a pure culture and biofilms in a nitrifying reactor was amplified by in situ PCR. In this procedure, fixed cells were permeabilized with lysozyme and subjected to seminested PCR with a digoxigenin-labeled primer. Then, the amplicon was detected with an alkaline phosphatase-labeled antidigoxigenin antibody and HNPP (2-hydroxy-3-naphthoic acid-2′-phenylanilide phosphate), which was combined with Fast Red TR, and with an Alexa Fluor 488-labeled antidigoxigenin antibody. The amoA gene in the biofilms was detected with an unavoidable nonspecific signal when the former method was used for detection. On the other hand, the amoA gene in the biofilms was detected without a nonspecific signal, and the cells possessing the amoA gene were clearly observed near the surface of the biofilm when Alexa Fluor 488-labeled antidigoxigenin antibody was used for detection. Although functional gene expression was not detected in this study, detection of cells in a biofilm based on their function was demonstrated.     

PCR Methods for Identification and Specific Detection of Probiotic Lactic Acid Bacteria

Probiotics are defined as “microbes improving animal feed.” Three lactic acid bacteria, previously selected as probiotic for pig feeding, were identified by sequencing the variable V₁ region of the 16S rDNA after PCR amplification primed in the flanking constant region. A V_R region showing strong nucleotide differences between the three probiotic and the reference strains was delimited. Oligonucleotides specific for each strain were designed. A specific assay for probiotic detection was developed, based on a PCR reaction with three primers. Received: 7 December 1995 / Accepted: 30 January 1996     

Specific Detection of Arcobacter and Campylobacter Strains in Water and Sewage by PCR and Fluorescent In Situ Hybridization

The aim of this study was to evaluate PCR and fluorescent in situ hybridization (FISH) techniques for detecting Arcobacter and Campylobacter strains in river water and wastewater samples. Both 16S and 23S rRNA sequence data were used to design specific primers and oligonucleotide probes for PCR and FISH analyses, respectively. In order to assess the suitability of the methods, the assays were performed on naturally and artificially contaminated samples and compared with the isolation of cells on selective media. The detection range of PCR and FISH assays varied between 1 cell/ml (after enrichment) to 10³ cells/ml (without enrichment). According to our results, both rRNA-based techniques have the potential to be used as quick and sensitive methods for detection of campylobacters in environmental samples.     

Fluorescence-Based Quasicontinuous and In Situ Monitoring of Biofilm Formation Dynamics in Natural Marine Environments

Analyzing the dynamics of biofilm formation helps to deepen our understanding of surface colonization in natural environments. While methods for screening biofilm formation in the laboratory are well established, studies in marine environments have so far been based upon destructive analysis of individual samples and provide only discontinuous snapshots of biofilm establishment. In order to explore the development of biofilm over time and under various biotic and abiotic conditions, we applied a recently developed optical biofilm sensor to quasicontinuously analyze marine biofilm dynamics in situ. Using this technique in combination with microscope-assisted imaging, we investigated biofilm formation from its beginning to mature multispecies biofilms. In contrast to laboratory studies on biofilm formation, a smooth transition from initial attachment to colony formation and exponential growth could not be observed in the marine environment. Instead, initial attachment was followed by an adaptation phase of low growth and homogeneously distributed solitary bacterial cells. Moreover, we observed a diurnal variation of biofilm signal intensity, suggesting a transient state of biofilm formation of bacteria. Overall, the biofilm formation dynamics could be modeled by three consecutive development stages attributed to initial bacterial attachment, bacterial growth, and attachment and growth of unicellular eukaryotic microorganisms. Additional experiments showed that the presence of seaweed considerably shortened the adaptation phase in comparison with that on control surfaces but yielded similar growth rates. The outlined examples highlight the advantages of a quasicontinuous in situ detection that enabled, for the first time, the exploration of the initial attachment phase and the diurnal variation during biofilm formation in natural ecosystems.     

Fluorescence-Based Bacterial Overlay Method for Simultaneous In Situ Quantification of Surface-Attached Bacteria

For quantification of bacterial adherence to biomaterial surfaces or to other surfaces prone to biofouling, there is a need for methods that allow a comparative analysis of small material specimens. A new method for quantification of surface-attached biotinylated bacteria was established by in situ detection with fluorescence-labeled avidin-D. This method was evaluated utilizing a silicon wafer model system to monitor the influences of surface wettability and roughness on bacterial adhesion. Furthermore, the effects of protein preadsorption from serum, saliva, human serum albumin, and fibronectin were investigated. Streptococcus gordonii, Streptococcus mitis, and Staphylococcus aureus were chosen as model organisms because of their differing adhesion properties and their clinical relevance. To verify the results obtained by this new technique, scanning electron microscopy and agar replica plating were employed. Oxidized and poly(ethylene glycol)-modified silicon wafers were found to be more resistant to bacterial adhesion than wafers coated with hydrocarbon and fluorocarbon moieties. Roughening of the chemically modified surfaces resulted in an overall increase in bacterial attachment. Preadsorption of proteins affected bacterial adherence but did not fully abolish the influence of the original surface chemistry. However, in certain instances, mostly with saliva or serum, masking of the underlying surface chemistry became evident. The new bacterial overlay method allowed a reliable quantification of surface-attached bacteria and could hence be employed for measuring bacterial adherence on material specimens in a variety of applications.     

Strategy for In Situ Detection of Natural Transformation-Based Horizontal Gene Transfer Events

A strategy is described that enables the in situ detection of natural transformation in Acinetobacter baylyi BD413 by the expression of a green fluorescent protein. Microscale detection of bacterial transformants growing on plant tissues was shown by fluorescence microscopy and indicated that cultivation-based selection of transformants on antibiotic-containing agar plates underestimates transformation frequencies.     

Improved Method for Detection of Methanotrophic Bacteria in Forest Soils by PCR

A primer set was designed for the specific detection of methanotrophic bacteria in forest soils by PCR. The primer sequences were derived from highly conservative regions of the pmoA gene, encoding the α-subunit of the particulate methane monooxygenase present in all methanotrophs. In control experiments with genomic DNA from a collection of different type I, II, and X methanotrophs, it could be demonstrated that the new primers were specific for members of the genera Methylosinus, Methylocystis, Methylomonas, Methylobacter, and Methylococcus. To test the suitability of the new primers for the detection of particulate methane monooxygenase (pMMO) containing methanotrophs in environmental samples we used DNA extracts from an acid spruce forest soil. For simple and rapid purification of the DNA extracts, the samples were separated by electrophoresis on a low-melting-point agarose gel. This allowed us to efficiently separate the DNA from coextracted humic acids. The DNA from the melted agarose gel was ready for use in PCR reactions. In PCR reactions with DNA from the Ah soil layer, products of the correct size were amplified by PCR by use of the new primers. By sequencing of cloned PCR products, it could be confirmed that the PCR products represented partial sequences with strong similarity to the pmoA gene. The sequence was most related to the pmoA sequence of a type II methanotroph strain isolated from the Ah layer of the investigated soils. Received: 1 September 2000 / Accepted: 2 October 2000     

Visualization and Enumeration of Bacteria Carrying a Specific Gene Sequence by In Situ Rolling Circle Amplification

Rolling circle amplification (RCA) generates large single-stranded and tandem repeats of target DNA as amplicons. This technique was applied to in situ nucleic acid amplification (in situ RCA) to visualize and count single Escherichia coli cells carrying a specific gene sequence. The method features (i) one short target sequence (35 to 39 bp) that allows specific detection; (ii) maintaining constant fluorescent intensity of positive cells permeabilized extensively after amplicon detection by fluorescence in situ hybridization, which facilitates the detection of target bacteria in various physiological states; and (iii) reliable enumeration of target bacteria by concentration on a gelatin-coated membrane filter. To test our approach, the presence of the following genes were visualized by in situ RCA: green fluorescent protein gene, the ampicillin resistance gene and the replication origin region on multicopy pUC19 plasmid, as well as the single-copy Shiga-like toxin gene on chromosomes inside E. coli cells. Fluorescent antibody staining after in situ RCA also simultaneously identified cells harboring target genes and determined the specificity of in situ RCA. E. coli cells in a nonculturable state from a prolonged incubation were periodically sampled and used for plasmid uptake study. The numbers of cells taking up plasmids determined by in situ RCA was up to 10⁶-fold higher than that measured by selective plating. In addition, in situ RCA allowed the detection of cells taking up plasmids even when colony-forming cells were not detected during the incubation period. By optimizing the cell permeabilization condition for in situ RCA, this method can become a valuable tool for studying free DNA uptake, especially in nonculturable bacteria.     

Development of a Fluorescent In Situ Method for Visualization of Enteric Viruses

Studying the interactions between enteric pathogens and their environment is important to improving our understanding of their persistence and transmission. However, this remains challenging in large part because of difficulties associated with tracking pathogens in their natural environment(s). In this study, we report a fluorescent labeling strategy which was applied to murine norovirus (MNV-1), a human norovirus surrogate, and hepatitis A virus (HAV). Specifically, streptavidin-labeled Quantum dots (Q-Dots) were bound to biotinylated capsids of MNV-1 and HAV (bio-MNV-1 and bio-HAV); the process was confirmed by using a sandwich-type approach in which streptavidin-bound plates were reacted with biotinylated virus followed by a secondary binding to Q-Dots with an emission range of 635 to 675 nm (Q-Dots 655). The assay demonstrated a relative fluorescence of 528 ± 48.1 and 112 ± 8.6 for bio-MNV-1 and control MNV-1, respectively. The biotinylation process did not impact virus infectivity, nor did it interfere with the interactions between the virus and host cells or model produce items. Using fluorescent microscopy, it was possible to visualize both bio-HAV and bio-MNV-1 attached to the surfaces of permissive mammalian cells and green onion tissue. The method provides a powerful tool for the labeling and detection of enteric viruses (and their surrogates) which can be used to track virus behavior in situ.Over the last 2 decades, production and consumption of fresh fruits and vegetables have increased dramatically, with an accompanying rise in the number of produce-associated food-borne disease outbreaks (12). It is clear from review of the CDC Food-borne Disease Outbreak database (1996 to 2006; available at: http://www.cdc.gov/foodborneoutbreaks/outbreak_data.htm) that the human noroviruses (HuNoV) are the leading cause of produce-associated food-borne disease. Although most HuNoV outbreaks are small and limited in scope, vegetable salads and berries are notable vehicles (8, 11, 17, 18). Hepatitis A virus (HAV) is the other human enteric virus of concern in fresh produce, and several high-profile outbreaks have occurred in association with HAV-contaminated strawberries and green onions in the last decade (9, 21).Little is known about the behavior of the enteric viruses in fresh produce items although virus persistence in such products is well documented (reviewed by Papafragkou et al. [14]). It is likely that a complex set of physicochemical interactions governs such persistence, which may be associated with attachment and/or internalization of the virus in the produce item(s). Unfortunately, there are no known means by which to visualize the HuNoV and HAV in situ in order to study such purported virus-surface interactions or other mechanisms by which these viruses persist in fresh produce. In the only study of its kind, Chancellor et al. (5) used fluorescent microspheres as a surrogate to examine the uptake of HAV into green onions. The fluorophores were observed microscopically on the surface and within the green onion tissue over a 60-day period. However, the fluorophores employed in that study ranged from 1 μm to 10 μm in diameter, much larger than a HAV particle (28 to 33 nm) (20), calling into question the relevance of these observations (5).Quantum dots, or Q-Dots, are small, 10- to 20-nm fluorescent semiconductor nanocrystals composed of a cadmium selenide core coated in a zinc sulfide shell. Q-Dots absorb photons over a wide range of wavelengths but have a very narrow range of emission; in addition, the fluorescence emitted by Q-Dots is both bright and photostable. The fluorescent wavelength of the Q-Dot is determined by the size of the nanocrystal, which moves from red to blue as the Q-Dots decrease in size. A wide range of Q-Dots are available with different emission wavelengths, ranging from 525 nm (green) to 800 nm (blue). Properties such as these make Q-Dots excellent candidates for use in virus labeling for study of surface interactions. For example, Kampani et al. (10) described a Q-Dot-based fluorescence assay that was used to study the attachment of human T-cell leukemia virus type 1 to host cells. In this case, the viral envelope was biotinylated, and this step was followed by attachment of streptavidin-coated Q-Dots. The fluorescently labeled viruses could be visualized binding to the appropriate host cell line using fluorescent microscopy, and data were interpreted both qualitatively and quantitatively.As HuNoV cannot be cultivated in vitro and as there is no animal model for propagation, a surrogate must be used to study the environmental behavior of these viruses. Recently, a cultivable murine norovirus (MNV-1) has been isolated, and recent studies indicate that this may be an appropriate surrogate for HuNoV (3). In this paper, we report a method for labeling MNV-1 and HAV with streptavidin-coated Q-Dots, as well as the development of a sandwich-type detection assay. We also demonstrate visualization of the labeled viruses bound to the surface of permissive mammalian cell lines and representative fresh produce items.     

Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization Allows for Enrichment-Independent Detection of Microcolony-Forming Soil Bacteria

Advances in the growth of hitherto unculturable soil bacteria have emphasized the requirement for rapid bacterial identification methods. Due to the slow-growing strategy of microcolony-forming soil bacteria, successful fluorescence in situ hybridization (FISH) requires an rRNA enrichment step for visualization. In this study, catalyzed reporter deposition (CARD)-FISH was employed as an alternative method to rRNA enhancement and was found to be superior to conventional FISH for the detection of microcolonies that are cultivated by using the soil substrate membrane system. CARD-FISH enabled real-time identification of oligophilic microcolony-forming soil bacteria without the requirement for enrichment on complex media and the associated shifts in community composition.     

Specific Detection of Two Divergent Simian Arteriviruses Using RNAscope In Situ Hybridization

Simian hemorrhagic fever (SHF) is an often lethal disease of Asian macaques. Simian hemorrhagic fever virus (SHFV) is one of at least three distinct simian arteriviruses that can cause SHF, but pathogenesis studies using modern methods have been scarce. Even seemingly straightforward studies, such as examining viral tissue and cell tropism in vivo, have been difficult to conduct due to the absence of standardized SHFV-specific reagents. Here we report the establishment of an in situ hybridization assay for the detection of SHFV and distantly related Kibale red colobus virus 1 (KRCV-1) RNA in cell culture. In addition, we detected SHFV RNA in formalin-fixed, paraffin-embedded tissues from an infected rhesus monkey (Macaca mulatta). The assay is easily performed and can clearly distinguish between SHFV and KRCV-1. Thus, if further developed, this assay may be useful during future studies evaluating the mechanisms by which a simian arterivirus with a restricted cell tropism can cause a lethal nonhuman primate disease similar in clinical presentation to human viral hemorrhagic fevers.     

副结核荧光PCR试剂盒研制与应用

采用TaqMan荧光标记探针技术原理, 建立副结核分枝杆菌特异的实时荧光PCR快速检测鉴定方法并组装形成临床诊断试剂盒。试剂盒提供荧光PCR与样品核酸提取试剂, 检测全程包括样品处理可在1 d内完成。特异性试验结果表明, 试剂盒对8株副结核分枝杆菌标准菌株的检测均呈典型阳性反应, 对牛分枝杆菌、结核分枝杆菌等其它12种分枝杆菌标准菌株以及大肠杆菌、肺炎链球菌等多种常见微生物均呈阴性反应。试剂盒检测灵敏度可达单个菌细胞、15个基因拷贝, 比常规PCR检测灵敏度提高100倍。对每份添加50~100个菌细胞的20份阴性牛奶样品进行检测, 均呈阳性反应。重复性试验结果显示, 试剂盒组内变异系数为1.41%, 组间变异系数为2.42%。采用所研制的副结核分枝杆菌荧光PCR试剂盒对来自广东地区7个奶牛场的250份牛奶样品和粪便样品、来自10个养猪场的143份猪血清样品, 以及3批次进口食蟹猴共100份血清样品进行检测, 检出牛奶样品中副结核分枝杆菌阳性率为7.7%, 牛粪样品中阳性率为3.7%, 猪血清样品中阳性率为8.2%, 进口猴血清样品中阳性率为3.0%。     

Use of Quantitative Real-Time PCR for Direct Detection of Serratia marcescens in Marine and Other Aquatic Environments

Serratia marcescens is the etiological agent of acroporid serratiosis, a distinct form of white pox disease in the threatened coral Acropora palmata. The pathogen is commonly found in untreated human waste in the Florida Keys, which may contaminate both nearshore and offshore waters. Currently there is no direct method for detection of this bacterium in the aquatic or reef environment, and culture-based techniques may underestimate its abundance in marine waters. A quantitative real-time PCR assay was developed to detect S. marcescens directly from environmental samples, including marine water, coral mucus, sponge tissue, and wastewater. The assay targeted the luxS gene and was able to distinguish S. marcescens from other Serratia species with a reliable quantitative limit of detection of 10 cell equivalents (CE) per reaction. The method could routinely discern the presence of S. marcescens for as few as 3 CE per reaction, but it could not be reliably quantified at this level. The assay detected environmental S. marcescens in complex sewage influent samples at up to 761 CE ml⁻¹ and in septic system-impacted residential canals in the Florida Keys at up to 4.1 CE ml⁻¹. This detection assay provided rapid quantitative abilities and good sensitivity and specificity, which should offer an important tool for monitoring this ubiquitous pathogen that can potentially impact both human health and coral health.     

餐饮及相关食品中病原菌活菌多重实时荧光PCR检测方法的研究与开发

为了应对餐饮等食品中病原菌快速检测的需求、研究建立病原菌筛查方法,选取痢疾志贺氏菌（Shigella dysenteriae）、金黄色葡萄球菌（Staphylococcus aureus）、副溶血性弧菌（Vibrio parahaemolyticus）、阴沟肠杆菌（Enterobacter cloacae）、产气肠杆菌（Enterobacter aerogenes）、沙门氏菌（Salmonella）、蜡样芽胞杆菌（Bacillus cereus）、大肠埃希氏菌O157∶H7（Escherichia coli O157∶H7）、单核细胞增生李斯特氏菌（Listeria monocytogenes）等9种病原菌开展多重实时荧光PCR方法研究工作。为了节约预增菌时间与提升检测效率,研发了适用于多种病原菌预增菌的通用型培养基,采取高温裂解法提取菌液核酸,利用PMA染料灭活死亡细菌DNA,筛选出活菌DNA,采用多重实时荧光PCR技术检测目标菌,该方法可在16 h内完成检测,对于目标病原菌的检测低限可达103 CFU·mL-1。     

Method for In Situ Measurement of Nitrification in a Stream

A method is described in which the oxidation of NH(3)-N to NO(2)-N and NO(3)-N in a stream was measured in situ by use of an equilibration chamber. The conversion was stoichiometric.     

通用引物PCR检测临床常见致病菌的实验研究

通用引物可一次性扩增18种临床常见致病菌和耐药菌株的DNA,扩增片段长度在220bp左右,18种特异性探针分别与18种标准菌株的PCR扩增产物杂交结果显示探针都具有高度特异性;5种37例经法国梅里埃API细菌鉴定系统确定的临床分离菌株进行杂交鉴定,鉴定结果与分离株一致,表明设计的探针具有高度特异性及准确性。80例临床标本分别用法国梅里埃API细菌鉴定系统及PCR杂交法进行检测,阳性率分别为(52.5%)和(67.5%),表明PCR结合寡核苷酸杂交法比传统的生物学培养法更为灵敏,值得推广。     

应用竞争PCR方法检测亚硝化细菌的研究

选用竞争PCR技术建立了亚硝化细菌的快速定量检测方法.实验以实验室分离、鉴定的一株亚硝化细菌提取的DNA为模板,设计特异性引物,制备特异性内标模板,进行竞争PCR实验;同时采用传统的平板计数法和吖啶橙荧光显微镜计数法两种方法进行细菌计数,与竞争PCR结果比较分析,建立了样品细菌数量与内标模板量的线性方程,并将其应用于海水养殖环境中亚硝化细菌的检测中.该方法具有简化检测过程、缩短细菌检测时间、无须昂贵仪器设备等优点,并克服有些环境微生物难以培养的困难,为将来有益菌在应用中实际存活和繁殖情况的监测提供了有力的技术支撑.