Detection of Salmonella enterica serovar typhimurium by using a rapid, array-based immunosensor

The multianalyte array biosensor (MAAB) is a rapid analysis instrument capable of detecting multiple analytes simultaneously. Rapid (15-min), single-analyte sandwich immunoassays were developed for the detection of Salmonella enterica serovar Typhimurium, with a detection limit of 8 x 10(4) CFU/ml; the limit of detection was improved 10-fold by lengthening the assay protocol to 1 h. S. enterica serovar Typhimurium was also detected in the following spiked foodstuffs, with minimal sample preparation: sausage, cantaloupe, whole liquid egg, alfalfa sprouts, and chicken carcass rinse. Cross-reactivity tests were performed with Escherichia coli and Campylobacter jejuni. To determine whether the MAAB has potential as a screening tool for the diagnosis of asymptomatic Salmonella infection of poultry, chicken excretal samples from a private, noncommercial farm and from university poultry facilities were tested. While the private farm excreta gave rise to signals significantly above the buffer blanks, none of the university samples tested positive for S. enterica serovar Typhimurium without spiking; dose-response curves of spiked excretal samples from university-raised poultry gave limits of detection of 8 x 10(3) CFU/g.     

Investigation of a simple amperometric electrode system to rapidly quantify and detect bacteria in foods

A two electrode system mounted as a single probe was developed to measure electrochemically the rate of reduction of a redox mediator (thionine) by bacteria. The system gave a rapid (2 min) bacterial-dependent current above 2.5 x 10(5) cfu/ml with pure cultures of bacteria, but when applied to the measurement of the bacterial contamination in samples of meat and milk it was unable to detect or quantify the contamination reliably. Incubation of samples for a few hours before examination enabled the system to detect bacteria in excess of 10(6) cfu/ml.     

Menadione-catalyzed luminol chemiluminescent assay for the viability of Escherichia coli ATCC 25922.

Escherichia coli ATCC 25922 produced O2- in the presence of menadione, and O2- -dependent luminol chemiluminescence intensity was proportional to colony-forming unit (CFU) in the exponential phase. CFU was determined by using a 96-well plate at a range of 3 X 10(3) to 8 x 10(7) CFU /well (0.1 ml) after a 10-min incubation with menadione, followed by chemiluminescent assay for 5 s. After a 4-hr incubation of E. coli (10(5) CFU/0.1 ml) with menadione and an antimicrobial agent inhibiting the synthesis of peptidoglycan, protein, and DNA, the inhibitory concentration (IC) of the antimicrobial agent determined by menadione-catalyzed luminol chemiluminescent assay was in good agreement with minimal inhibitory concentration (MIC) of the NCCLS (National Committee for Clinical Laboratory Standard) method requiring 18 hr. Menadione-catalyzed luminol chemiluminescent assay is expected to be useful for the rapid determination of cell viability under the conditions of various cell growths and stresses.     

Microcalorimetric studies of hybridoma cells

In the present study, overall metabolism has been estimated in hybridoma cells by microcalorimetric measurement. Heat production rate was found to be 30-50 pW/cell at cell concentrations 0.65-4.5 x 10(5)/ml. High cell concentrations (1 x 10(6) cells/ml) caused unstable power-curves with an initial high peak and a rapid declining phase, whereas low cell concentrations (0.6-4.5 x 10(5) cells/ml) produced steady-state power-curves. Oxygen consumption was found to range between 1.5-6.1 x 10(-5) mol 02/cell/min, corresponding to about 80% of the total metabolic activity. The metabolic inhibitors sodium fluoride (50 nM), sodium azide (160 mM) and rotenone (0.1 mM) caused a reduction in overall cell metabolism of 60, 55 and 40% respectively.     

Role of endogenous opioids in ischemic preconditioning but not in short-term hibernation in pigs

Endogenous opioids are involved in ischemic preconditioning (IP) in several species. Whether or not opioids are important for IP and short-term myocardial hibernation (STMH) in pigs is currently unknown. In 34 enflurane-anesthetized pigs, the left anterior descending coronary artery was flow constantly perfused. Subendocardial blood flow (Endo), infarct size (IS; percent area at risk), and the free energy change of ATP hydrolysis (DeltaG) were determined. After 90-min severe ischemia and 120-min reperfusion, IS averaged 28.3 +/- 5.4% (means +/- SE) (n = 8; Endo: 0.047 +/- 0.009 ml. min(-1) x g(-1)). IP by 10-min ischemia and 15-min reperfusion reduced IS to 9.9 +/- 3.8% (P < 0.05, n = 8; Endo: 0.044 +/- 0.009 ml. min(-1) x g(-1)). After naloxone (1 mg/kg iv followed by 2 microg x kg(-1) x min(-1)), IS averaged 25.8 +/- 7.0% (n = 6; Endo: 0.039 +/- 0.008 ml x min(-1) x g(-1)) without and 24.7 +/- 4.7% (n = 6; Endo: 0.044 +/- 0.006 ml x min(-1) x g(-1)) with IP. At 5-min moderate ischemia in the presence of naloxone, Endo decreased from 0.90 +/- 0.07 to 0.28 +/- 0.03 ml x min(-1) x g(-1)and DeltaG decreased from -58.6 +/- 1.0 to -52.6 +/- 0.4 kJ/mol. Prolongation of ischemia to 90 min did not alter Endo, but DeltaG recovered toward control values (57.7 +/- 1.1 kJ/mol), and the myocardium remained viable. These responses are identical to those of nonnaloxone-treated pigs. Endogenous opioids are involved in IP but not in STMH in pigs.     

Immunomagnetic detection of Bacillus stearothermophilus spores in food and environmental samples.

There are currently no methods for the rapid and sensitive detection of bacterial spores that could be used to direct raw materials containing high spore loads away from products that pose a food safety risk. Existing methods require an overnight incubation, cannot detect spores below 10(5) CFU/ml, or are not specific to particular species. This work describes a method to specifically detect < 10(4) CFU of bacterial spores per ml within 2 h. Polyclonal antibodies to Bacillus stearothermophilus spores were attached to 2.8-micron-diameter magnetic polystyrene beads by using a polythreonine cross-linker via the antibody carbohydrate moiety. A biotin-avidin-amplified sandwich enzyme-linked immunosorbent assay coupled to a fluorescent substrate was used to quantitate captured spores. The concentration of B. stearothermophilus spores in samples was linearly correlated to fluorescent activity (r2 = 0.99) with a lower detection limit of 8 x 10(3) CFU/ml and an upper detection limit of 8 x 10(5) CFU/ml. The detection limits are not fixed and can be changed by varying the immunomagnetic bead concentration. Several food and environmental samples were tested to demonstrate the versatility of the assay.     

A latex agglutination test for the detection of Mycoplasma pneumoniae in respiratory exudates: a comparative study with a commercially available DNA-probe test.

We prepared polyclonal antibody specific to Mycoplasma pneumoniae. Using this antibody, we developed a latex agglutination test (LAT) for detecting the organism in respiratory exudates as rapid diagnosis of M. pneumoniae infection. Further, LAT was compared with DNA-probe test (DP) which was the only commercially available test for the rapid detection of the organism. In LAT, both M. pneumoniae and M. genitalium give positive agglutination, but the titer of M. genitalium was significantly lower than that of M. pneumoniae. The detection limit of LAT was 2 x 10(5) CFU/ml and that of DP was 5 x 10(4) CFU/ml in vitro. It was considered that target molecules in LAT were accumulated in the pharyngeal portion of the patients, because of their long half-life at 37 C. However, ribosomal RNA which was target molecule in DP was destroyed at 37 C much sooner, and the accumulation could not be expected. Actually, positive rate in LAT was higher than that in DP among clinical specimens in which M. pneumoniae was detected by culture method. The procedure of LAT is much easier and more rapid than that of DP in which radioactive isotope is required. LAT could be the choice of test for rapid diagnosis of M. pneumoniae infection.     

Development and validation of a liquid chromatographic method for in vitro mupirocin quantification in both skin layers and percutaneous penetration studies

A simple, rapid, and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method for the measurement of mupirocin concentrations in both skin layers and percutaneous samples has been developed. Mupirocin was extracted from skin layers using PBS-acetonitrile (90:10, v/v). The method is sufficiently sensitive and repeatable to be used in percutaneous penetration studies. The samples were chromatographed on a 250 mm x 4 mm C(8) LiChrospher Select B (5 microm). The mobile phase composition was a mixture of acetonitrile-ammonium acetate 0.05 M (27.5:72.5, v/v) adjusted to pH 6.3 with acetic acid. The analyte was detected at 228 nm and the run time was 11 min. Linearity was confirmed in the concentration range 0.2-20 microg/ml and the limit of detection was 9.5 ng/ml.     

Quantitative and rapid detection of the trichloroethylene-degrading bacterium Methylocystis sp. M in groundwater by real-time PCR

We developed a method based on real-time PCR for the specific and rapid enumeration of a trichloroethylene-degrading methanotroph, Methylocystis sp. M, with the aim of monitoring the strain in groundwater. A primer set designed from the nucleotide sequence of the mmoC gene of a soluble methane monooxygenase (sMMO) gene cluster from Methylocystis sp. M was specific to amplify the DNA region from the strain and no PCR products were amplified with the sMMO gene clusters from six other methanotroph strains. The real-time PCR reliably quantified Methylocystis sp. M over at least five orders of magnitude (5x10(6) to 5x10(2 )cells/PCR tube, or 2x10(8) to 2x10(4 )cells/ml). Five cells of Methylocystis sp. M per PCR tube (2x10(2 )cells/ml) were detectable when the cells were suspended in distilled water. The concomitant presence of other methanotrophs in samples did not affect the reliability of enumeration; and recovery of the cells with a membrane filter enabled us to quantify cells of the strain in groundwater. This quantification procedure was completed within 3 h, including preparation time of environmental samples. We conclude that real-time PCR using the mmoC primer set can be used practically to analyze the behavior of Methylocystis sp. M at bioremediation sites.     

Development of a rapid positive/absent test for coliforms using sensitive bioluminescence assay

We have developed a sensitive bioluminescence assay for beta-galactosidase using a luminescent substrate, D-luciferin-O-beta-galactopyranoside (LuGal). The detection limit for beta-galactosidase was 3 x 10-20 mol per assay, which was approximately 50-fold more sensitive than the test using a fluorescent substrate. This assay was applied to a positive/absent (P/A) test for coliforms. Observations made after 7 h of culture followed by a 10-min enzyme assay using LuGal were comparable to those made after a 22-24-h culture by the current method. Therefore, the LuGal method allows a rapid P/A test for coliforms.     

Quantitative TaqMan PCR for detection of Pneumocystis jiroveci

We developed a quantitative real-time PCR assay for detection and quantification of Pneumocystis jiroveci in bronchoalveolar lavage (BAL) specimens based on primers and probe targeting the gene encoding beta-tubulin. The assay was able to detect 50 DNA copies per ml of a standard plasmid containing the target sequence. The intra- and interassay coefficients of variation were 0.46%-4.27% and 0.05-2.00% over 5 log(10) values. Fifty-seven controls of human, viruses, bacteria and fungi DNA samples were amplified and found negative. Fifty-three BAL samples sent to the laboratory for diagnosis of pneumocystosis were prospectively investigated by real-time PCR and direct microscopic examinations (DME) using Giemsa stain and direct immunofluorescence. All PCR negative samples were negative by microscopy. Among the 24 (45%) BAL found PCR positive, 8 were positive by microscopy (35%). The copy numbers of the target gene were between 4.4 x 10(3) and 2.8 x 10(6) per ml for the microscopically positive samples and between 8 and 9.2 x 10(3) per ml for the microscopically negative samples. In conclusion, we developed a rapid, sensitive and specific real time PCR for the diagnosis and quantification of Pneumocystis jiroveci in BAL samples.     

Rapid identification and enumeration of Saccharomyces cerevisiae cells in wine by real-time PCR

Despite the beneficial role of Saccharomyces cerevisiae in the food industry for food and beverage production, it is able to cause spoilage in wines. We have developed a real-time PCR method to directly detect and quantify this yeast species in wine samples to provide winemakers with a rapid and sensitive method to detect and prevent wine spoilage. Specific primers were designed for S. cerevisiae using the sequence information obtained from a cloned random amplified polymorphic DNA band that differentiated S. cerevisiae from its sibling species Saccharomyces bayanus, Saccharomyces pastorianus, and Saccharomyces paradoxus. The specificity of the primers was demonstrated for typical wine spoilage yeast species. The method was useful for estimating the level of S. cerevisiae directly in sweet wines and red wines without preenrichment when yeast is present in concentrations as low as 3.8 and 5 CFU per ml. This detection limit is in the same order as that obtained from glucose-peptone-yeast growth medium (GPY). Moreover, it was possible to quantify S. cerevisiae in artificially contaminated samples accurately. Limits for accurate quantification in wine were established, from 3.8 x 10(5) to 3.8 CFU/ml in sweet wine and from 5 x 10(6) to 50 CFU/ml in red wine.     

Rapid identification of biological warfare agents using an instrument employing a light addressable potentiometric sensor and a flow-through immunofiltration-enzyme assay system

An instrument employing a light addressable potentiometric sensor and a flow-through immunofiltration-enzyme assay system has been developed for the rapid and specific identification of biological warfare (BW) agents. The system has been designed to assay for up to eight agents simultaneously and provides an indication of the absence or presence of a threat within 15 min. Parameters affecting the mixing of the reagents within the instrument's fluidic lines were investigated and optimized. Measurements of blank samples and samples containing Bacillus subtilis spores in the concentration range of 10(4) to 10(6) cfu/ml indicate the limit of detection (LOD) is 3 x 10(3) cfu/ml for B. subtilus. Although the LOD is higher than that of several technologies currently under development, this instrument offers an immediate interim approach for addressing the need to rapidly detect biological warfare agents in the field.     

The correlation method for rapid monitoring of Escherichia coli in foods

AIMS: A new rapid method was developed to rapidly monitor Escherichia coli counts in foods. MATERIALS AND RESULTS: One ml of modified selective broth with 4-methylumbelliferyl beta-D-glucuronide and 1 ml of food sample were mixed in a sterile test tube and incubated at 37 degrees C. The positive reaction (fluorescence under u.v. light) was monitored at regular 30 min intervals. The positive reaction times in test tubes were compared with actual E. coli numbers from tested samples. The growth of E. coli in test tubes (broth) was much faster than growth on agar. The first experiment was performed to evaluate the rapid correlation method using pure E. coli cultures. The correlation between E. coli counts by the conventional plating method and positive reaction (fluorescence production) times in test tubes was highly agreeable (r(2) = 0 x 95). In the case of low E. coli numbers, such as 2 x 0 log10 cfu ml(-1), the rapid correlation method detected their presence after 10 h incubation. When highly contaminated samples were assayed (8 log10 cfu ml(-1)), the rapid correlation method detected the presence of E. coli after 4 h incubation. In the ground beef experiment, the correlation between fluorescence production time and actual E. coli numbers was also strongly agreeable (r(2) = 0 x 92). CONCLUSIONS: From these results, it is obvious that the new rapid method can rapidly monitor E. coli counts in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicated that the new method saved about 10-14 h incubation time compared to conventional plating methods. The rapid correlation method required much shorter incubation times compared to conventional plating methods for monitoring E. coli.     

Detection of brucella antigens with colloid metal particles as markers for specific antigens

Gold and silver sols were comparatively approved as markers of specific IgG isolated from hyperimmune Brucella antisera for the detection of brucellar antigens. The sensitivity of the test system using gold immunosol proved to be some higher (3.1-9.8 ng/ml of soluble and 2.0 x 10(4)-5.3 x 10(6) CFU/ml of corpuscular brucellar antigens) than that achieved with the use of silver immunosol (5.7-18.4 ng/ml of soluble and 6.1 x 10(4)-8.0 x 10(6) CFU/ml of corpuscular brucellar antigens). At the same time silver sol was a cheaper and more available marker. Both test systems were found to be highly specific. False positive results were observed only with Yersinia enterocolitica O:9 at high concentrations due to the fact that they had common polysaccharide haptens incorporated into lipopolysaccharides of these microorganisms. The proposed test systems with colloid metal particles used as markers of specific antibodies for the detection of Brucella antigens are technologically simple, economic, rapid, highly sensitive and specific. Their use in combination with other serological methods will make the results of analyses more informative, thus improving the quality of laboratory diagnostics of brucellosis.     

Activity of natural fatty acids on erythrocyte osmotic resistance]

The aim of the present study was to assess the influence on the functional characteristics of the erythrocyte membrane of adding in vitro different natural fatty acids to blood taken from normal subjects. Blood samples were collected without stasis from healthy volunteers, anticoagulated with heparin or EDTA and incubated at 37 degrees C for 60 min with the different fatty acids at concentrations ranging between 1 x 10(-4) and 3 x 10(-2) molar. Two ml of blood were used for each test. The treated blood was added to graded solutions of NaCl (0.90-0.20 g/dl) at a ratio of 100 microliters/5 ml of solution. The suspensions were kept at room temperature and centrifuged for 10 min at 2000 g, in order to accelerate the sedimentation of the erythrocytes which had not been broken down and so as to obtain a clear supernatant for spectrophotometry of the dissolved haemoglobin. Readings were compared with those obtained from blood samples which had been completely haemolyzed by suspension in distilled water. Results obtained with the blood samples prepared with the fatty acids were compared with control samples from the same donor, also incubated at 37 degrees C for 60 min. Preincubation of the erythrocyte with butyric or caproic or oleic acid at concentrations ranging between 5 and 20 millimolar, provoked a clear deterioration of the osmotic resistances of the erythrocytes, in proportion to the concentration of the fatty acid used. The osmotic insult was systematically more effective in those samples anticoagulated with EDTA than those treated with heparin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of a novel real-time PCR method for detection of HSV types 1 and 2 DNA using HybProbe chemistry

Herpes simplex viruses types 1 and 2 are members of the Alphaherpesviridae subfamily, as they can infect both skin and nerves and develop latent infection within the dorsal root and trigeminal ganglia. Infections with these viruses are common worldwide and cause wide range of clinical syndromes. Although HSV-1/2 infect healthy children and adults, disease is more severe and extensive in the immunocompromised individuals and/or during neuroinfections. The aim of the study was development of real-time PCR assay for detection and differentiation of herpes simplex viruses type 1 and 2. DNA in clinical samples, using specific dual-channel HybProbe chemistry. The nalytical sensitivity of assay was tested using serial dilutions of HSV-1 and HSV-2 DNA in range between 10 degrees and 10(-5). (4.35 x 10(5)-4.00 x 10(2) copies/ml and 4.18 x 10(5)-3.82 x 10(2) copies/ml, respectively). Thirty four cell line isolates and sixteen clinical samples taken from a group of adult patients with neurological signs were tested for the presence of HSV-1/2 DNA in the LightCycler instrument. Described in-house real-time PCR assay detected herpesviral DNA in all cell line isolates (31 of them were HSV-1 positive; 3 were HSV-2 positive) and in 10 clinical samples (positive only for HSV-1). The conclusion is that developed HybProbe-based real-time PCR test is very reliable and valuable tool for detection and differentiation of HSV-1/2 viremia in different kind of samples. The high level of sensitivity and accuracy provided by this assay is favorable for the quantification of herpes simplex virus 1 and 2 DNA in clinical specimens, especially during low-copy infections.     

Pathogenicity of Beauveria bassiana,Metarhizium anisopliae (Deuteromycotina: Hyphomycetes), and other entomopathogenic fungi against Lygus lineolaris (Hemiptera: Miridae)

The pathogenicity of 32 fungal isolates from the genera of Beauveria, Verticillium, Paecilomyces, Metarhizium, Mariannaea, and Hirsutella to second-instar tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), was tested under laboratory conditions. These isolates originated from various insect hosts and substrates from France, Denmark, Austria, Italy, Turkey, Syria, and the United States. A single exposure concentration (1 x 10(7) conidia/ ml) assay for each isolate was first conducted by immersing the insects in 10 ml of a fungal suspension for 5s. These were followed by concentration-mortality assays on five of the most pathogenic isolates using four test concentrations ranging from 2 x 10(4) to 2 x 10(7) conidia/ml. B. bassiana 726 (Bemisia-passaged GHA strain) was used as a standard for comparison in all of the assays. Among the test isolates, three produced mortality not significantly different from the water control. Mortality ranged from 35 to 98% among the other 29 isolates. The LC50 values of the five most pathogenic isolates ranged from 0.8 to 5.0 x 10(5) conidia/ml. The LT50 values for these isolates ranged from 6.0 to 6.9, 3.1 to 5.1, and 2.5 to 4.0 d for concentrations of 2 x 10(5), 2 x 10(6), and 2 x 10(7) conidia/ml, respectively. Two strains of B. bassiana (ARSEF 1394,5665) and one M anisopliae (ARSEF 3540) were more pathogenic to the nymphs than the standard, having significantly lower LC50 and LT50, values. Our results demonstrated that several genera of entomopathogenic fungi have promise as microbial control agents against L. lineolaris.     

Direct measurement of nitric oxide in headspace gas produced by a chicken macrophage cell line in a closed culture system.

A simple and rapid method was applied for direct measurement of nitric oxide (NO) gas produced by cultured macrophages using a modified chemiluminescence detector, the thermal energy analyzer (TEA). HD11 chicken macrophages (1-3 x 10(6)/ml) were cultured on microcarrier beads (100 mg/ml) in 140 ml air-tight glass jars (5 ml cell suspension per jar) containing 0.5 micrograms/ml of LPS and different concentrations of L-arginine. Headspace gas was sampled at 24 hours of culture via a rubber septum and directly injected into a TEA with a liquid nitrogen trap set at -130 to -140 degrees C. The concentration of NO in the gas sample was quantified using a standard gas mixture of NO (2 microliters/L) in nitrogen. Gas samples from L-arginine-supplemented cultures contained NO (0.028-0.066 pl/microliter), whereas NO was not detected in samples from controls. These results suggest that chicken macrophages synthesize NO gas in a dose-dependent manner relative to L-arginine concentration.     

RAPTOR: optimal protein threading by linear programming

This paper presents a novel linear programming approach to do protein 3-dimensional (3D) structure prediction via threading. Based on the contact map graph of the protein 3D structure template, the protein threading problem is formulated as a large scale integer programming (IP) problem. The IP formulation is then relaxed to a linear programming (LP) problem, and then solved by the canonical branch-and-bound method. The final solution is globally optimal with respect to energy functions. In particular, our energy function includes pairwise interaction preferences and allowing variable gaps which are two key factors in making the protein threading problem NP-hard. A surprising result is that, most of the time, the relaxed linear programs generate integral solutions directly. Our algorithm has been implemented as a software package RAPTOR-RApid Protein Threading by Operation Research technique. Large scale benchmark test for fold recognition shows that RAPTOR significantly outperforms other programs at the fold similarity level. The CAFASP3 evaluation, a blind and public test by the protein structure prediction community, ranks RAPTOR as top 1, among individual prediction servers, in terms of the recognition capability and alignment accuracy for Fold Recognition (FR) family targets. RAPTOR also performs very well in recognizing the hard Homology Modeling (HM) targets. RAPTOR was implemented at the University of Waterloo and it can be accessed at http://www.cs.uwaterloo.ca/~j3xu/RAPTOR_form.htm.