Comparison of primers for the detection of pathogenic Escherichia coli using real-time PCR.

AIMS: To evaluate PCR primers for the detection of pathogenic Escherichia coli in a real-time PCR assay and determine their utility in produce irrigation water testing. METHODS AND RESULTS: Three previously published PCR primer sets and one set designed for this study were tested for their ability to produce amplification products for several pathogenic E. coli serotypes from whole cells as template. Two of the previously published primer sets were chosen for real-time PCR detection limit determination. The coneaeA and PEH detection limit of E. coli O157:H7 was 10(0) and 10(1) CFU rxn(-1) in sterile water respectively. To detect E. coli O157:H7 in sprout irrigation water, the water required dilution due to PCR inhibitors. The detection limit of the coneaeA and PEH was 10(1) and between 10(2) and 10(3) CFU rxn(-1) in diluted sprout irrigation water respectively. CONCLUSIONS: The primer set coneaeA was able to produce an amplification product from each E. coli serotype, except O128:H7 and most sensitive for real-time PCR detection of pathogenic E. coli in diluted sprout irrigation water. SIGNIFICANCE AND IMPACT OF THE STUDY: The necessity of a dissociation analysis to distinguish positive samples from those with fluorescence of random dsDNA generation for real-time PCR in a complex background was established.

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.

Refinement of the pressure assay for milk urea nitrogen.

We report improvements in the application of a pressure-based assay for urea. The assay involved the enzymatic hydrolysis of urea and subsequent measurement of CO2 partial pressure. Effects of the preservative bronopol on the assay and their implications for laboratory applications are discussed. A method of remediating these effects with cysteine is described. A method is also described wherein these additives can be used to prepare standards of known urea concentration in milk. The improved assay can be used to measure urea N in unadulterated milk or in bronopol preserved milk with an accuracy of +/-0.7 mg/dl (0.25 mM) in the range from 0 to 30 mg/dl (0 to 10.7 mM).

Chemical assay of urea for automated sensing in milk.

Results of a new chemical assay for urea involving enzymatic hydrolysis to ammonium and carbonate and subsequent measurement of CO2 partial pressure are presented. The assay is simple to implement in an automated version, and the hardware used is not prone to fouling and damage by raw milk. The assay sensitivity at 24 degrees C is about 0.367 kPa per milligram per deciliter of urea N. The assay has no dependence on milk fat in the sample, and effects of milk proteins and lactose are slight (less than 2% change in sensitivity per change in w/v percent). Observed sensitivities to urea in spiked milk samples were not significantly different from each other or from standards in distilled water or 0.1 M phosphate-buffered saline. The standard error of the assay is about 0.3 mg/dl of urea N (0.1 mM) in standard solution, and about 1 mg/dl of urea N (0.3 mM) in milk in a range of 0 to 30 mg/dl of urea N (0 to 11 mM). The assay holds promise for use in an on-line sensor to measure milk urea N in the milking parlor.

Controlled release of insect sex pheromones from paraffin wax and emulsions.

Paraffin wax and aqueous paraffin emulsions can be used as controlled release carriers for insect sex pheromones for mating disruption of orchard pests. Paraffin can be applied at ambient temperature as an aqueous emulsion, adheres to tree bark or foliage, releases pheromone for an extended period of time, and will slowly erode from bark and biodegrade in soil. Pheromone emulsions can be applied with simple spray equipment. Pheromone release-rates from paraffin were measured in laboratory flow-cell experiments. Pheromone was trapped from an air stream with an adsorbent, eluted periodically, and quantified by gas chromatography. Pheromone release from paraffin was partition-controlled, providing a constant (zero-order) release rate. A typical paraffin emulsion consisted of 30% paraffin, 4% pheromone, 4% soy oil, 1% vitamin E, 2% emulsifier, and the balance water. Soy oil and vitamin E acted as volatility suppressants. A constant release of oriental fruit moth pheromone from paraffin emulsions was observed in the laboratory for more than 100 days at 27 degreesC, with release-rates ranging from 0.4 to 2 mg/day, depending on the concentration and surface area of the dried emulsion. The use of paraffin emulsions is a viable method for direct application of insect pheromones for mating disruption. Sprayable formulations can be designed to release insect pheromones to the environment at a rate necessary for insect control by mating disruption. At temperatures below 38 degreesC, zero-order release was observed. At 38 degreesC and higher, pheromone oxidation occurred. A partition-controlled release mechanism was supported by a zero-order pheromone release-rate, low air/wax partition coefficients, and pheromone solubility in paraffin.

Rapid enzyme immunoassay for measurement of bovine progesterone.

Reproductive management is a primary financial concern of the dairy industry with missed estrus detection one of the major causes of lost income. A rapid enzyme immunoassay (EIA) was developed for on-line measurement of progesterone in bovine milk with a biosensor for detection of estrus. The EIA was developed using covalent binding microtiter wells, monoclonal antibody, horseradish peroxidase, and 3,3',5,5'-tetramethylbenzidine (TMB). The EIA took 8 min and had a dynamic response for progesterone in buffer and milk between 0.2 and 20 ng/ml.

Biosensor for on-line measurement of bovine progesterone during milking.

Reproductive management is a major financial concern of the dairy industry, with missed estrus detection a main cause of lost income. A biosensor was developed for on-line measurement of progesterone in bovine milk and detection of estrus. The biosensor used an enzyme immunoassay format for molecular recognition, which was developed to run in approximately eight minutes. The sensor was designed to operate on-line in a dairy parlor using microinjection pumps and valves for fluid transport, fiber optics and photodiodes for light measurement, and a control computer for sequencing. Calibration showed a dynamic response between 0.1 and 5 ng/ml progesterone in milk. The reusability of the test well was evaluated. Thiocyanate (0.5 M, pH 5.1) quickly regenerated the antibody surface while maintaining antibody activity for 15-20 cycles, but noise from the residual enzyme limited reusability.