Integrated real-time PCR for detection and monitoring of Legionella pneumophila in water systems

We evaluated a ready-to-use real-time quantitative Legionella pneumophila PCR assay system by testing 136 hot-water-system samples collected from 55 sites as well as 49 cooling tower samples collected from 20 different sites, in parallel with the standard culture method. The PCR assay was reproducible and suitable for routine quantification of L. pneumophila. An acceptable correlation between PCR and culture results was obtained for sanitary hot-water samples but not for cooling tower samples. We also monitored the same L. pneumophila-contaminated cooling tower for 13 months by analyzing 104 serial samples. The culture and PCR results were extremely variable over time, but the curves were similar. The differences between the PCR and culture results did not change over time and were not affected by regular biocide treatment. This ready-to-use PCR assay for L. pneumophila quantification could permit more timely disinfection of cooling towers.     

Minicultures of Mammalian Cells in a New Plastic Plate

A new disposable micro tissue culture plate was developed and tested for use in virological procedures. Miniature mammalian cell cultures (minicultures) were grown in these plates. Each plate contained 96 circular cultures in flat wells (7 mm in diameter). Replicate titrations of a number of viruses were performed in various tissues. Excellent reproducibility was demonstrated. Mean infectivity titers determined by miniculture methods were generally within 0.6 log₁₀/ml of macro tube titrations. Standard tissue culture assay techniques such as hemadsorption, interference titration, and microneutralization were easily carried out with this method and were very reproducible. Development of this noncytotoxic disposable micro tissue culture plate now permits the routine performance of rapid, reliable, and reproducible tissue culture tests at a very significant reduction in cost and labor.     

Simultaneous determination of 33 amino acids and dipeptides in spent cell culture media by gas chromatography-flame ionization detection following liquid and solid phase extraction

A rapid, sensitive and reproducible gas chromatographic method with flame ionization detection is described for the simultaneous identification and quantification of 33 amino acids and dipeptides in spent cell culture media in under seven minutes. The method involves the use of the EZ:faast(Phenomenex) amino acid sample testing kit. Instrumental and assay precision, percent recovery, linear range, limit of detection and peak identity in highly complex cell culture media containing either soy hydrolysate or fetal bovine serum were validated using gas chromatography-flame ionization detector (GC-FID).     

Integrated Real-Time PCR for Detection and Monitoring of Legionella pneumophila in Water Systems

We evaluated a ready-to-use real-time quantitative Legionella pneumophila PCR assay system by testing 136 hot-water-system samples collected from 55 sites as well as 49 cooling tower samples collected from 20 different sites, in parallel with the standard culture method. The PCR assay was reproducible and suitable for routine quantification of L. pneumophila. An acceptable correlation between PCR and culture results was obtained for sanitary hot-water samples but not for cooling tower samples. We also monitored the same L. pneumophila-contaminated cooling tower for 13 months by analyzing 104 serial samples. The culture and PCR results were extremely variable over time, but the curves were similar. The differences between the PCR and culture results did not change over time and were not affected by regular biocide treatment. This ready-to-use PCR assay for L. pneumophila quantification could permit more timely disinfection of cooling towers.     

Development of a high throughput Pseudomonas aeruginosa epithelial cell adhesion assay

Pseudomonas aeruginosa colonizes the lungs of cystic fibrosis and mechanically ventilated patients by binding to specific carbohydrate residues on the surface of lung epithelial cells. Studies have shown that blocking this interaction may have therapeutic effects in vivo. To test compounds that may have an effect on the binding of P. aeruginosa to epithelial cells, we have developed a pseudomonal adhesion assay that is compatible with high throughput technology. This assay utilizes a 96-well culture plate assay and P. aeruginosa strains that have been modified to bioluminesce. This method has proven to be a rapid, sensitive and reproducible system for screening agents that inhibit bacterial adhesion.     

Development and evaluation of a Taqman duplex real-time PCR quantification method for reliable enumeration of Legionella pneumophila in water samples

This study describes the development and evaluation of a specific Legionella pneumophila Taqman duplex real-time PCR (qPCR) for fast and reliable quantification of this human pathogen in suspected man-made water systems. The qPCR assay was 100% specific for all L. pneumophila serogroups 1-15 with a sensitivity of 60 genome units/l and an amplification efficiency of 98%. Amplification inhibitors were detected via an exogenous internal positive control, which was amplified simultaneously with L. pneumophila DNA using its own primer and probe set. Mean recovery rates of the qPCR assay for tap water and cooling circuit water, spiked with a known number L. pneumophila bacteria, were 93.0% and 56.3%, respectively. Additionally, by using the Ultraclean Soil DNA isolation kit, we were able to remove amplification inhibitors ubiquitously present in cooling water. The practical value of our qPCR assay was evaluated through analysis of 30 water samples from showers, taps, eyewash stations, fire sprinklers and recirculation loops with qPCR and traditional culture. In conclusion, the described L. pneumophila Taqman duplex real-time assay proved to be specific, sensitive and reproducible. This makes it a promising method complementing the current time-consuming culture standard method.     

Detection of peptide nucleic acids in tissue extracts of treated animals by gel mobility shift assay

We have developed a sensitive and reproducible gel mobility shift assay to detect PNA oligomers in tissue of treated animals. PNA present in purified tissue extracts of treated animals is hybridized to a 33P-labelled DNA oligomer probe, and analyzed by polyacrylamide gel electrophoresis. The PNA-DNA hybrid migrates more slowly than the DNA probe alone and can be quantified relative to a standard curve. This detection method is useful for detecting PNAs in many different tissues, including brain, heart, kidney, liver, spleen, and serum, as well as cells in culture.     

Quantitative Assessment of Hemadsorption by Myxoviruses: Virus Hemadsorption Assay

The standardization and quantitative evaluation of an assay for myxoviruses, based on the enumeration of individual infected clone 1-5C-4 cells manifesting hemadsorption within 24 h of infection, are described. Hemadsorption was detectable earlier than immunofluorescence in infected cells or hemagglutinins in culture medium. The relationship between virus concentration and cells exhibiting hemadsorption was linear. The assay was highly precise, sensitive, and reproducible.     

短端粒小鼠与正常小鼠间充质干细胞的分离培养比较

目的建立从小鼠微粒骨中获取间充质干细胞（MSC）方法,并观察比较野生型小鼠（WT鼠）和端粒酶基因敲除小鼠（Terc-/-鼠）MSC生物学特性的差异。方法采用体外细胞培养技术,从小鼠自体微骨片中分离纯化WT鼠和Terc-/-鼠的间充质干细胞,通过培养和传代,研究其增殖及生长特征。结果原代培养及传代培养显示,WT鼠自体骨间充质干细胞比Terc-/-鼠具有活跃的增殖倍增能力。结论从鼠的微骨片获取MSC的方法重复性好,细胞纯度和细胞数量优于以往从骨髓获取MSC的方法,Terc-/-鼠MSC生长增殖能力明显弱于WT鼠,其机制的研究有待深入。     

Flow cytometric assay for cytotoxic activity of crude Clostridium perfringens enterotoxin using non-adherent cell FM3A

Abstract The flow cytometric assay method was tested for the cytotoxic activity of Clostridium perfringens enterotoxin (CPE) in culture using mouse mammary carcinoma cell line FM3A stained with propidium iodide (PI). From the results obtained, FM3A cells proved to be susceptible to CPE. A reproducible dose-response curve with FM3A was obtained between crude CPE at 13.9–109 ng/ml and between purified CPE at 40–400 ng/ml, respectively. These findings indicate that non-adherent FM3A is preferable to determine the cytotoxic activity of CPE because it can be used without detachment procedures with trypsinin compared with adherent African monkey kidney cell line (Vero cells). Furthermore, the flow cytometry with non-adherent cell FM3A stained with PI only proved to be a useful method to determine the biological activity of CPE in culture isolates.     

Rapid quantitative serological assay of staphylococcal enterotoxin B

A simple, rapid method, based on the Oudin single diffusion technique, is described for the quantitative assay of staphylococcal enterotoxin B. The method yields reproducible results without close control of such assay variables as temperature, antiserum dilution, and assay time, provided that the ionic strength is maintained above 0.2 n sodium chloride equivalent.     

Screening Actinomycetes for Extracellular Peroxidase Activity

A diverse collection of actinomycete strains were screened for production of extracellular peroxidase activity by adapting a chemiluminescence analysis system developed for horseradish peroxidase-based enzyme-linked immunosorbent assay. Extracellular peroxidase activity was found to be common but quantitatively variable, and this rapid and sensitive screening system permitted identification of a small group of high-producing strains. A range of spectrophotometric assays were compared for the measurement of peroxidase activity in concentrated culture supernatants of two selected thermophilic streptomycetes. Of these, the peroxide-dependent oxidation of 2,4-dichlorophenol was identified as the most robust and reproducible assay for quantitative studies.     

Assay of the high-alkaline proteinase from alkalophilic bacillus PB92 using a chromogenic tripeptide substrate

The chromogenic tripeptide substrate, benzyloxycarbonylglycyl-L-prolyl-L-citrulline p-nitroanilide, is proposed for the assay of the high-alkaline proteinase, HAP-PB92, from an alkalophilic Bacillus. The assay method is sensitive, reproducible, and may be adapted for an automatic system.     

A nonradioactive assay method for determination of enzymatic activity of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)

A sensitive and nonradioactive assay method for activity determination of Rubisco is described. The method is based on thin-layer chromatographic separation of 3-phosphoglycerate (3-PGA) and D-ribulose-1,5-bisphosphate (RuBP). This assay method allows the quantitative determination of Rubisco activity. Rates of carbon dioxide fixation on RuBP determined by this method were comparable to those obtained independently by other methods. This assay method is reproducible and relatively free from interference.     

T cell growth factor: parameters of production and a quantitative microassay for activity.

Several soluble factors have recently been associated with the proliferation and differentiation of thymus-derived lymphocytes. One of these factors present in medium conditioned by T cell mitogen-stimulated lymphocytes has the ability to promote the long-term culture of normal and antigen-specific cytotoxic T cells. We report a method to test for this proliferative stimulus in the form of a sensitive microassay based upon the tritiated-thymidine incorporation of continuous murine tumor-specific cytotoxic T cell lines (CTLL). The microassay requires microliter quantitites of sample fluid and is amenable to quantitative analysis. This highly reproducible, quantitative assay for T cell growth factor (TCGF) has allowed investigation as to the kinetics of TCGF generation and has revealed that T lymphocytes are required for its production. Further investigation has supported the notion that this nonspecies-specific factor is actively removed from tissue culture medium by the proliferation of either T cell mitogen-activated lymphocytes or CTLL.     

Comparison of the Immunofluorescent-Cell Counting and Plaque Methods for the Assay of Vaccinia Virus

The immunofluorescent-cell counting method was compared with the agarand liquid-overlay plaque techniques for the assay of vaccinia virus. In addition to being as quantitative, reproducible, and simple to use as the two plaque techniques, the immunofluorescent assay method was found to be more sensitive, rapid, and specific.     

Modeling lymphangiogenesis in a three-dimensional culture system

A lack of appropriate in vitro models of three-dimensional lymph vessel growth hampers the study of lymphangiogenesis. We developed a lymphatic ring assay--a potent, reproducible and quantifiable three-dimensional culture system for lymphatic endothelial cells that reproduces spreading of endothelial cells from a pre-existing vessel, cell proliferation, migration and differentiation into capillaries. In the assay, mouse thoracic duct fragments are embedded in a collagen gel, leading to the formation of lumen-containing lymphatic capillaries, which we assessed by electron microscopy and immunostaining. We developed a computerized method to quantify the lymphatic network. By applying this model to gene-deficient mice, we found evidence for involvement of the matrix metalloproteinase, MMP-2, in lymphangiogenesis. The lymphatic ring assay bridges the gap between two-dimensional in vitro models and in vivo models of lymphangiogenesis, can be used to exploit the potential of existing transgenic mouse models, and rapidly identify regulators of lymphangiogenesis.     

A real-time PCR assay for detection and quantification of Mycoplasma agalactiae DNA

AIMS: The aim of this study was to develop a rapid, sensitive, specific tool for detection and quantification of Mycoplasma agalactiae DNA in sheep milk samples. METHODS AND RESULTS: A real-time polymerase chain reaction (PCR) assay targeting the membrane-protein 81 gene of M. agalactiae was developed. The assay specifically detected M. agalactiae DNA without cross-amplification of other mycoplasmas and common pathogens of small ruminants. The method was reproducible and highly sensitive, providing precise quantification of M. agalactiae DNA over a range of nine orders of magnitude. Compared with an established PCR assay, the real-time PCR was one-log more sensitive, detecting as few as 10(1) DNA copies per 10 microl of plasmid template and 6.5x10(0) colour changing units of reference strain Ba/2. CONCLUSIONS: The real-time PCR assay is a reliable method for the detection and quantification of M. agalactiae DNA in sheep milk samples. The assay is more sensitive than gel-based PCR protocols and provides quantification of the M. agalactiae DNA contained in milk samples. The assay is also quicker than traditional culture methods (2-3 h compared with at least 1 week). SIGNIFICANCE AND IMPACT OF THE STUDY: The established real-time PCR assay will help study the patterns of shedding of M. agalactiae in milk, aiding pathogenesis and vaccine efficacy studies.     

Quantitative characterization of single‐cell adhesion properties by atomic force microscopy using protein‐functionalized microbeads

A method was developed to characterize the adhesion properties of single cells by using protein‐functionalized atomic force microscopy (AFM) probes. The quantification by force spectroscopy of the mean detachment force between cells and a gelatin‐functionalized colloidal tip reveals differences in cell adhesion properties that are not within reach of a traditional bulk technique, the washing assay. In this latter method, experiments yield semiquantitative and average adhesion properties of a large population of cells. They are also limited to stringent conditions and cannot highlight disparities in adhesion in the subset of adherent cells. In contrast, this AFM‐based method allows for a reproducible and quantitative investigation of the adhesive properties of individual cells in common cell culture conditions and allows for the detection of adhesive subpopulations of cells. These characteristics meet the critical requirements of many fields, such as the study of cancer cell migratory abilities.