Detecting antigens by quantitative immuno-PCR

The quantitative immuno-PCR (qIPCR) technology combines the advantages of flexible and robust immunoassays with the exponential signal amplification power of PCR. The qIPCR allows one to detect antigens using specific antibodies labeled with double-stranded DNA. The label is used for signal generation by quantitative PCR. Because of the efficiency of nucleic acid amplification, qIPCR typically leads to a 10- to 1,000-fold increase in sensitivity compared to an analogous enzyme-amplified immunoassay. A standard protocol of a qIPCR assay to detect human interleukin 6 (IL-6) using a sandwich immunoassay combined with real-time PCR readout is described here. The protocol includes initial immobilization of the antigen, and coupling of this antigen with antibody-DNA conjugates is then carried out by (a) the stepwise assembly of biotinylated antibody, streptavidin and biotinylated DNA, (b) the use of a biotinylated antibody and an anti-biotin-DNA conjugate or (c) the employment of an anti-IL-6 antibody-DNA conjugate. Following the assembly of signal-generating immunocomplexes, real-time PCR is used to amplify and record the signal. Depending on the coupling strategy, the qIPCR assays require 4-7 h with only about 3 h hands-on-time. The use of qIPCR assays enables the detection of rare biomarkers in complex biological samples that are poorly accessible by conventional immunoassays. Therefore, qIPCR offers novel opportunities for the biomedical analysis of, for instance, neurodegenerative diseases and viral infections as well as new tools for the development of novel pharmaceuticals.     

Detection of rViscumin in plasma samples by immuno-PCR

To allow for pharmacokinetic studies in adjunction with the current clinical developments of the potent cytostatic anti-cancer drug rViscumin, a sandwich immuno-PCR (IPCR) assay was developed for the detection of rViscumin in blood plasma. The IPCR was carried out with a commercially available reagent kit, consisting of pre-assembled rViscumin-specific antibody-DNA conjugates as well as a specific competitor DNA fragment to be amplified by PCR. Various combinations of capture- and detection-antibodies were compared for performance in IPCR. Using the optimized assay, as few as 50 zeptomol (approx. 100 fg/ml) rViscumin (MW 57 kDa) was detectable in standardized human serum samples. The IPCR assay was very selective for rViscumin and in spiking experiments in proband plasma samples, signal recovery rates between 70% and 120% were obtained. The linear sensitivity range of the assay covered more than five orders of magnitude. Repeated measurements of rViscumin resulted in a mean standard deviation value of 14.2%.     

A capillary polymerase chain reaction for Salmonella detection from poultry meat

AIMS: In this study, a capillary polymerase chain reaction (cPCR) was applied for Salmonella detection from poultry meat. METHODS AND RESULTS: Salmonella detection limits of the optimized cPCR were determined with DNA templates from the samples of tetrathionate broth (TTB), Rappaport Vassiliadis broth (RVB) and selenite cystine broth (SCB) artificially contaminated with 10-fold dilutions of 6 x 10(8) CFU ml(-1) of pure Salmonella enterica ssp. enterica serovar Enteritidis 64K stock culture. Detection limits of cPCR from TTB, RVB and SCB were found as 6, 6 x 10(1) and 6 x 10(4) CFU ml(-1), respectively. In addition, detection limits of bacteriology were also determined as 6 CFU ml(-1) with TTB and SCB, and 6 x 10(1) CFU ml(-1) with RVB. A total of 200 samples, consisting of 100 chicken and 100 turkey meat samples, were tested with optimized cPCR and bacteriology. Eight and six per cent of the chicken meat samples were found to harbour Salmonella by cPCR and standard bacteriology, respectively. Of six Salmonella isolates, four belonged to serogroup D, two to serogroup B. CONCLUSIONS: The TTB cultures of both artificially and naturally contaminated samples were found to be superior to those of RVB and SCB cultures in their cPCR results. This cPCR, utilizing template from 18-h TTB primary enrichment broth culture, takes approximately 40 min in the successful detection of Salmonella from poultry meat. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that cPCR from TTB enrichment culture of poultry meat would enable rapid detection of Salmonella in laboratories with low sample throughput and limited budget.     

Detection of Rotavirus from stool samples using a standardized immuno-PCR ("Imperacer") method with end-point and real-time detection

Immuno-PCR (IPCR) has been studied to increase the detection sensitivity of current enzyme-linked immuno-sorbent assays (ELISA) as a novel approach for the early detection of Rotavirus infection, a major source for serious diarrhoea for susceptible risk groups. IPCR utilizes specific antibody-DNA conjugates with subsequent amplification of the marker-DNA. An antibody-DNA conjugate specific for Rotavirus antigen VP6 was synthesized and used in combination with a commercially available Rotavirus-ELISA kit. IPCR was carried out using reagents and protocols of the standardized Imperacer system. Real-time PCR monitoring of the marker-DNA amplification was compared to endpoint quantification of amplified haptene-labeled PCR products, using a microtiterplate-based PCR-ELISA. In spiked calibration samples, as few as 100 virus particles/ml could be clearly detected using the IPCR method and either real-time or end-point quantification compared to about 100,000 virus particles/ml in ELISA. Rotavirus positive and negative stool samples were correctly identified by IPCR with a clear separation even of a 10,000-fold dilution of the positive stool samples from the negative control.     

A real-time immuno-PCR assay for routine ultrasensitive quantification of proteins

A fast and robust assay, based on the combination of the highly sensitive immuno-PCR (IPCR), employing standardized self-assembled DNA-protein conjugates as reagents, and the well-established, reliable, and fast real-time PCR detection by means of the TaqMan principle is introduced in this work. The use of anti-species immunoglobulin reagents allows one for easy adaptation of this assay to basically any existing ELISA application. The use of an internal competitor in the real-time IPCR (rtIPCR) further increases the sensitivity and significance of this assay; 0.1-0.01 amol (500-50 fg/mL) IgG from several species (mouse, rabbit, goat, and human) were detectable using direct, indirect, and sandwich model rtIPCR assays, thereby increasing the detection limit of the analogous ELISA tests about 100- to 1000-fold. The robustness of this method was demonstrated in two typical applications by detecting 40 pg/mL of the novel anti-cancer drug rViscumin in human plasma samples as well as 100 pg/mL of a research antibody in cell culture media. In both cases, a comparable ELISA was 1000-fold less sensitive.     

High sensitivity multianalyte immunoassay using covalent DNA-labeled antibodies and polymerase chain reaction.

A multianalyte immunoassay for simultaneous detection of three analytes (hTSH, hCG and beta-Gal) has been demonstrated using DNA-labeled antibodies and polymerase chain reaction (PCR) for amplification of assay response. The labeled antibodies were prepared by covalently coupling uniquely designed DNA oligonucleotides to each of the analyte-specific monoclonal antibodies. Each of the DNA oligonucleotide labels contained the same primer sequences to facilitate co-amplification by a single primer pair. Assays were performed using a two-antibody sandwich assay format and a mixture of the three DNA-labeled antibodies. Dose-response relationships for each analyte were demonstrated. Analytes were detected at sensitivities exceeding those of conventional enzyme immunoassays by approximately three orders of magnitude. Detection limits for hTSH, beta-Gal and hCG were respectively 1 x 10(-19), 1 x 10(-17) and 1 x 10(-17) mol. Given the enormous amplification afforded by PCR and the existing capability to differentiate DNA based on size or sequence differences, the use of DNA-labeled antibodies could provide the basis for the simultaneous detection of many analytes at sensitivities greater than those of existing antigen detection systems. These findings in concert with previous reports suggest this hybrid technology could provide a new generation of ultra-sensitive multianalyte immunoassays.     

Self-assembly of DNA-streptavidin nanostructures and their use as reagents in immuno-PCR.

The self-assembly of bis-biotinylated double-stranded DNA and the tetravalent biotin-binding protein streptavidin (STV) have been studied by non-denaturing gel electrophoresis and atomic force microscopy (AFM). The rapid self-assembly reproducibly generated populations of individual oligomeric complexes. Most strikingly, the oligomers predominantly contained bivalent STV molecules bridging two adjacent DNA fragments to form linear nanostructures. Trivalent STV branch points occurred with a lower frequency and the presence of tetravalent STV was scarce. However, valency distribution, size and the exchange dynamics of the supramolecular aggregates were highly sensitive to stoichiometric variations in the relative molar coupling ratio of bis-biotinylated DNA and STV. The largest aggregates were obtained from equimolar amounts while excess STV led to the formation of smaller oligomers appearing as fingerprint-like band patterns in electrophoresis. Excess DNA, however, induces a complete breakdown of the oligomers, likely a consequence of the instability of STV conjugates containing more than two biotinylated DNA fragments. It was demonstrated that the oligomers can further be functionalized, for instance by the coupling of biotinylated immunoglobulins. Both pure and also antibody-modified DNA-STV oligomers were used as reagents in immuno-PCR (IPCR), a highly sensitive detection method for proteins and other antigens. Employment of the supramolecular reagents led to an approximately 100-fold enhanced sensitivity compared to the conventional IPCR procedure.     

Simple and sensitive bacterial quantification by a flow-based kinetic exclusion fluorescence immunoassay

A flow-based immunoassay system utilizing secondary-antibody coated microbeads and Cy5-secondary antibody for signal production was successfully developed to quantitate target bacteria with a kinetic exclusion assay (KinExA 3000 Instrument). It directly measured the concentration of unliganded antibody separated from the equilibrated mixture of antibody and bacteria through a 0.2 microm polyethersulfone membrane, enabling it to quantify the concentration of bacteria. The novel method demonstrated the qualities of rapidness, sensitivity, high accuracy and reproducibility, and ease to perform. Detection of Pseudomonas aeruginosa and Staphylococcus aureus was accomplished with low detection limits of 4.10 x 10(6) and 5.20 x l0(4)cells/mL, respectively, with an assay time of less than 15 min. The working ranges for quantification were 4.10 x l0(6) to 1.64 x l0(10)cells/mL for P. aeruginosa, and 5.20 x l0(4) to 1.04 x l0(9)cells/mL for S. aureus. It yielded an assay with at least 10-fold greater sensitivity than ELISA and could correctly assess the concentration of predominant bacterium spiked in the mixture of P. aeruginosa and S. aureus. With this reliable platform, the average amount of antibody bound by one cell in the maximum capability could be further provided: (1.6-2.5) x l0(5) antibodies for one P. aeruginosa cell and (2.2-2.7) x l0(8) antibodies for one S. aureus cell. The KinExA system is flexible to determine different kinds of bacteria conveniently by using anti-mouse IgG as the same immobilizing agent. However, a higher specificity of the antibodies to the target bacteria will be required for the use of this system with higher detection sensitivity.     

Amplification of microsphere-based microarrays using catalyzed reporter deposition

Assay sensitivities using three fluorescent signal generation schemes were evaluated on the Luminex flow cytometer. Following microsphere capture of antigen by immobilized antibodies, bound targets were quantified by use of (1) Cy3-labeled "tracer" antibodies (30min total time), (2) biotinylated tracers followed by streptavidin-R-phycoerythrin (60min total time), or (3) biotinylated tracers followed by avidin-peroxidase conjugates and tyramide signal amplification (TSA; 90min total time). Use of TSA for signal generation in three individual toxin assays improved performance up to 100-fold over Cy3-antibody-based detection, and while streptavidin-R-phycoerythrin provided equivalent sensitivities, TSA produced dramatic increases at low concentrations simplifying positive sample identification. Detection limits for TSA-interrogated assays for ricin, cholera toxin, and staphylococcal enterotoxin B were 64pg/ml, 4pg/ml, and 0.1ng/ml, respectively, using optimized conjugates; analogous detection limits for Cy3-antibody-interrogated assays were 8ng/ml, 1ng/ml, and 1ng/ml, respectively. No improvement was observed in botulinum toxoid A assays when TSA amplification was used. As unique preferences for specific avidin-peroxidase conjugates were observed in the individual assays, improvements in multiplexed assays utilizing a single conjugate were significantly lower (3-10-fold improvements). Furthermore, increases in variability resulted in poorer performance of TSA-interrogated assays for botulinum toxoid, indicating that assay-specific optimization should be performed, especially prior to multiplexing.     

Application of a bioluminescence method for the beer industry: sensitivity of MicroStar-RMDS for detecting beer-spoilage bacteria. Rapid Microbe Detection System

We set up the original operating conditions of the MicroStar-Rapid Microbe Detection System (RMDS) to suppress false positives, which have kept this system from practical. The detection limit of our system was between 6.3 x 10(-16) mol and 3.1 x 10(-16) mol in terms of the amount of ATP, which is approximately equal to the ATP content of one yeast cell or 50 lactic acid bacteria cells. The detection time and the detection count were compared between the RMD method and the conventional plate count method (C.P.C. method) using 23 test samples of beer-spoilage Lactobacillus brevis. Judging from the detection time and detection count, 16-24 hours of cultivation for the RMD method corresponded to 40-96 hours of cultivation for the C.P.C. method. The RMD method reached a useful level for our practical use at the point of sensitivity.     

Molecular characteristics of bovine serum albumin-dextran conjugates

Bovine serum albumin (BSA)-dextran conjugates were prepared by using the Maillard reaction; depending on the ratio of dextran to BSA used, about 0.5-1 mol of dextran could be bound to 1 mol of native BSA. SDS-PAGE patterns revealed that BSA and dextran had been covalently bonded. Structural analyses by fluorescence spectroscopy and circular dichroism indicated that the BSA surface in each conjugate was covered with dextran without any great disruption of the native conformation. The conjugates could be grouped into two fractions on the basis of the weight-average molecular mass measured: the main fraction at 1.95-2.35 x 10(5) g/mol and a less-abundant fraction with aggregates greater than 1.50 x 10(6) g/mol. High-performance size-exclusion chromatography in conjunction with multi-angle laser light scattering detection revealed that the BSA-dextran conjugates prepared by using the Maillard reaction had various molar masses and radii.     

Liquid chromatography with amperometric detection using functionalized multi-wall carbon nanotube modified electrode for the determination of monoamine neurotransmitters and their metabolites

The fabrication and application of a novel electrochemical detection (ED) method with the functionalized multi-wall carbon nanotubes (MWNTs) chemically modified electrode (CME) for liquid chromatography (LC) were described. The electrochemical behaviors of dopamine (DA) and other monoamine neurotransmitters at the CME were investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The results indicated that the CME exhibited efficient electrocatalytic effects on the current responses of monoamine neurotransmitters and their metabolites with high sensitivity, high stability and long-life activity. In LC-ED, DA, norepinephrine (NE), 3-methoxy-4-hydroxyphenylglycol (MHPG), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) had good and stable current responses at the CME. The linear ranges of seven analytes were over four orders of magnitude and the detection limits were 2.5 x 10(-10) mol/l for DA, 2.5 x 10(-10) mol/l for NE, 5.0 x 10(-10) mol/l for MHPG, 3.0 x 10(-10) mol/l for DOPAC, 3.5 x 10(-10) mol/l for 5-HT, 6.0 x 10(-10) mol/l for 5-HIAA, 1.25 x 10(-9) mol/l for HVA. The application of this method coupled with microdialysis sampling for the determination of monoamine neurotransmitters and their metabolites in Parkinsonian patients' cerebrospinal fluid was satisfactory.     

A highly sensitive immuno-PCR assay for detection of H5N1 avian influenza virus

With an aim at detecting the ultra-low concentration of avian influenza virus (AIV), a highly sensitive hybrid assay based on immunology and polymerase chain reaction was developed. The TopYield microtiter plates were coated with ten-fold serial dilutions of H5N1 subtype AIV ranging from 10 EID₅₀ ml⁻¹~10⁻⁴ EID₅₀ ml⁻¹,which was recognized by mouse anti-AIV H5 monoclonal antibody (MAb) that was directly linked with reporter DNA using a heterobifunctional cross-linker. After extensive washing, the reporter DNA including a BamH I-restriction site was released by a specific enzymatic restriction, then transferred to PCR tubes, amplified, and used as the signal for detection of AIV. Under the optimized condition, MAb-based immuno-PCR (IPCR) method could measure 100 μl of AIV H5N1 with 10⁻⁴ EID₅₀ ml⁻¹.To evaluate the sensitivity of IPCR, the same concentration and volume of AIV H5N1 were detected by conventional RT–PCR and sandwich ELISA. The results showed that IPCR had an approximately 1,000-fold improvement over the conventional ELISA, and a 100-fold enhancement compared with RT–PCR in detection sensitivity. To further evaluate the specificity of IPCR for AIV H5 subtype, the tracheal swab specimens, taken from chickens which were infected with H9N2, and the allantoic fluid from eggs inoculated by AIV H3N2, H7N1, H9N2, were detected by IPCR. To mimic clinical samples, pharyngeal–tracheal swab specimens were collected from healthy chickens and spiked with H5N1, H5N2, H5N3 for analysis by immuno-PCR. The results demonstrated that IPCR was a highly sensitive and specific assay for AIV H5, and could be applied to clinical detection for low amount of AIV H5 subtype. This MAb-based immuno-PCR method provided a platform capable of mass screening of clinical samples for AIV H5 subtype and could serve as a model for other immuno-PCR assays.     

Detection and quantification of Listeria monocytogenes by 5'-nuclease polymerase chain reaction targeting the actA gene

AIMS: The aim of this study was to develop a 5'-nuclease polymerase chain reaction (PCR) for the rapid detection and quantification of Listeria monocytogenes. METHODS AND RESULTS: Specific primers and a fluorogenic probe were designed, which target a specific sequence of the actA gene encoding for a protein involved in the actin filament assembly. The PCR system was highly sensitive and specific for L. monocytogenes (inclusivity, 100%; exclusivity, 100%), which was determined using 46 L. monocytogenes and 28 non-L. monocytogenes strains. Detection limits of 10(4) cfu ml(-1) after 35 cycles and 10(2) cfu ml(-1) after 45 cycles were achieved by PCR in both real-time and end-point fluorescence measurement modes. Linear calibration lines were obtained in the range from 10(2) to 10(9) cfu ml(-1) for three L. monocytogenes strains in real-time PCR with 45 cycles. CONCLUSIONS: The developed 5'-nuclease PCR of the actA gene provides a new target for the rapid detection and quantification of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: In conjunction with enrichment or with an appropriate quantitative sample preparation technique, the method is suitable for food safety applications.     

Evaluation of the use of chromogenic and fluorogenic substrates in solid-phase enzyme linked immunosorbent assays (ELISA)

Fluorogenic and chromogenic substrates were used in direct and trapping enzyme-linked immunosorbent assays (ELISA) for the detection of mouse IgG and foot-and-mouth disease virus (FMDV). The detection limits for both antigens were compared using different combinations of enzymes and substrates. Various times and concentrations of chemicals were used to obtain maximum sensitivity for both systems. Similar sensitivities were found using fluorogenic and chromogenic substrates. Tetramethyl benzidine substrate for horse-radish peroxidase enzyme conjugates was found to attain the highest sensitivity levels for chromogenic assays (0.12 ng IgG/ml and 1.0 ng/ml FMDV respectively), after 10 min incubation. Of the two fluorogenic enzyme/substrates studied, B-galactosidase was the most sensitive but required extended incubation times (2-3 h) as compared with chromogenic systems. Special microplates for fluoro-immunoassay (FIA) were compared with conventional microplates and no advantage was found to justify their use. An alkaline phosphatase anti-guinea-pig conjugate was used to confirm the equivalence of fluorogenic and chromogenic substrates in terms of sensitivity. A comparison of the amount of signal generated using various concentrations of enzyme in the absence of antigen was made for two different alkaline phosphatase conjugates to obtain theoretical sensitivity limits. One possible advantage of fluorogenic substrates is that high binding ratio can improve the confidence in discrimination of positive results.     

Protein immobilization on the surface of liposomes via carbodiimide activation in the presence of N-hydroxysulfosuccinimide

A method of the covalent immobilization of proteins on the surface of liposomes, containing 10% (by mol) of N-glutaryl phosphatidylethanolamine, is described. Carboxylic groups of liposomal N-glutaryl phosphatidylethanolamine were activated in the presence of water-soluble carbodiimide and N-hydroxysulfosuccinimide and reacted subsequently with protein amino groups. The liposome-protein conjugates formed contained up to 5 x 10(-4) mol protein/mol lipid. Lectins (RCA1 and WGA) upon immobilization on liposomes retained saccharide specificity and the ability to agglutinate red blood cells. The immobilization of mouse monoclonal IgG in a ratio of 3.5 x 10(-4) mol IgG/mol lipid was achieved. The liposome activation in the absence of N-hydroxysulfosuccinimide resulted in a 2-fold decrease of protein coupling yields.     

Comparison of primers for the detection of Salmonella enterica serovars using real-time PCR

AIMS: To evaluate the specificity and sensitivity of PCR primers for the detection of Salmonella enterica in a real-time PCR assay using pure cultures. METHODS AND RESULTS: Unenriched whole cells in sterile water were used as template for each PCR. SYBR Green dye was used for the nonspecific detection of dsDNA. The real-time PCR detection limits of five previously published primer sets used in conventional PCR applications were not below 3 x 10(3) CFU per reaction (rxn). A new primer set, Sen, was designed, which detected Salm. enterica Newport down to 6 CFU rxn(-1) in one case, and gave an average detection limit of 35 CFU rxn(-1) over three separate runs. CONCLUSIONS: Primers originally designed for end-point PCR did not have adequate specificity or sensitivity compared with those specifically designed for real-time PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: This study emphasizes the importance of evaluating real-time PCR primer sets in pure cultures prior to testing in field samples. This study will benefit other researchers in selecting an appropriate primer set for real-time PCR detection of Salm. enterica.     

Simultaneous voltammetric measurement of ascorbic acid, epinephrine and uric acid at a glassy carbon electrode modified with caffeic acid

A stable electroactive thin film of poly(caffeic acid) has been deposited on the surface of a glassy carbon electrode by potentiostatic technique in an aqueous solution containing caffeic acid. Poly(caffeic acid) was used as a modified electrode for the detection of ascorbic acid (AA), epinephrine (EP), uric acid (UA) and their mixture by cyclic voltammetry. This modified electrode exhibits potent and persistent electron-mediating behavior followed by well-separated oxidation peaks towards AA, EP and UA with activation overpotential. For the ternary mixture containing AA, EP and UA, the three compounds can well separate from each other at the scan rate of 20 mVs(-1) with a potential difference of 156, 132 and 288 mV between AA and EP, EP and UA and AA and UA, respectively, which was large enough to determine AA, EP and UA individually and simultaneously. The catalytic peak current obtained, was linearly dependent on the AA, EP and UA concentrations in the range of 2.0 x 10(-5) to 1.0 x 10(-3) mol l(-1), 2.0 x 10(-6) to 8.0 x 10(-5) mol l(-1) and 5.0 x 10(-6) to 3.0 x 10(-4) mol l(-1), and the detection limits for AA, EP and UA were 7.0 x 10(-6), 2.0 x 10(-7) and 6.0 x 10(-7) mol l(-1), respectively. The modified electrode shows good sensitivity, selectivity and stability, and has been applied to the determination of EP in practical injection samples and that of EP, UA and AA simultaneously with satisfactory results.     

Enumeration of Salmonella and Escherichia coli O157:H7 in ground beef, cattle carcass, hide and faecal samples using direct plating methods

AIM: To develop and validate high throughput methods for the direct enumeration of viable and culturable Salmonella and Escherichia coli O157:H7 in ground beef, carcass, hide and faecal (GCHF) samples from cattle. METHODS AND RESULTS: The hydrophobic grid membrane filtration (HGMF) method and the spiral plate count method (SPCM) were evaluated as rapid tools for the estimation of pathogen load using GCHF samples spiked with known levels of Salmonella serotype Typhimurium. Validation studies showed that for a single determination of each sample type the low end of the detection limits were approx. 2.0 x 10(0) CFU g(-1) for ground beef, 5.0 x 10(-1) CFU (100 cm(2))(-1) for Salmonella and 8.0 x 10(-1) CFU (100 cm(2))(-1) for E. coli O157:H7 on carcasses, 4.0 x 10(1) CFU (100 cm(2))(-1) for hide and 2.0 x 10(2) CFU g(-1) for faecal samples. In addition, ground beef (n = 609), carcass (n = 1520) and hide (n = 3038) samples were collected from beef-processing plants and faecal samples (n = 3190) were collected from feed-lot cattle, and these samples were tested for the presence of Salmonella and E. coli O157:H7 by enrichment and enumeration methods. CONCLUSIONS: The direct enumeration methods described here are amenable to high throughput sample processing and were found to be cost-effective alternatives to other enumeration methods for the estimation of Salmonella and E. coli O157:H7, in samples collected during cattle production and beef processing. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of the methods described here would allow for more routine testing and quantification data collection, providing useful information about the effectiveness of beef processing intervention strategies.     

High-sensitive determination of coenzyme Q(10) in iodinate-beta-cyclodextrin medium by inclusion reaction and catalytic polarography

A novel polarographic method for the determination of coenzyme Q(10) in beta-cyclodextrin (beta-CD) and iodinate system is proposed. The stability of coenzyme Q(10) to light was improved by the formation of coenzyme Q(10)-beta-CD inclusion complex. In addition, the sensitivity for the determination of coenzyme Q(10) was enhanced by both the formation and the polarographic catalytic wave of the inclusion complex in the presence of iodinate. In 0.1 mol/L HAc-NaAc (pH 4.7)-5.0 x 10(-5) mol/L beta-CD-1.2 x 10(-3) mol/L potassium iodinate-ethanol/water (60:40, v/v) medium, coenzyme Q(10)-beta-CD inclusion complex yielded a sensitive association/parallel catalytic wave. The second-order derivative peak current of the catalytic wave was proportional to coenzyme Q(10) concentration in the range of 6.0 x 10(-8)-2.5 x 10(-7) mol/L, and the detection limit was 1.0 x 10(-8) mol/L. The proposed method has high analytical sensitivity and is allowed to determine coenzyme Q(10) under light.