Polymerase chain reaction-gene probe detection of microorganisms by using filter-concentrated samples.

To detect low levels of microorganisms in environmental samples by using polymerase chain reaction (PCR)-gene probe detection, samples were concentrated by filtration. Fluoropore (Millipore Corp.) filters were compatible with PCR DNA amplification, whereas various other filters including nitrocellulose and cellulose acetate filters inhibited PCR amplification. By concentrating cells on Fluoropore filters and releasing the DNA by freeze-thaw cycling, PCR DNA amplification could be performed without removing the filter. Concentration with Fluoropore FHLP and FGLP filters permitted the detection of single cells of microorganisms in 100-ml samples by PCR-gene probes.

Detection of Escherichia coli and Shigella spp. in water by using the polymerase chain reaction and gene probes for uid.

A method was developed for the detection of the fecal coliform bacterium Escherichia coli, using the polymerase chain reaction and gene probes, based on amplifying regions of the uid gene that code for beta-glucuronidase, expression of which forms the basis for fecal coliform detection by the commercially available Colilert method. Amplification and gene probe detection of four different regions of uid specifically detected E. coli and Shigella species, including beta-glucuronidase-negative strains of E. coli; no amplification was observed for other coliform and nonenteric bacteria.

Detection of bacterial mRNA using polymerase chain reaction.

A method was developed for the detection of bacterial mRNAs using reverse transcriptase followed by the polymerase chain reaction (PCR) and Southern blot analysis. The method involves brief inhibition of protein synthesis with chloramphenicol, followed by reverse transcription, PCR amplification of cDNA and Southern blot hybridization. Detection of mRNAs by reverse transcription-PCR-Southern blot analysis is orders of magnitude more sensitive than Northern blot hybridization.

Multiplex PCR amplification and immobilized capture probes for detection of bacterial pathogens and indicators in water.

Detection of pathogens (Legionella species) and indicator bacteria (coliform bacteria) was achieved by multiplex (simultaneous) PCR amplification of diagnostic gene sequences and by hybridization to immobilized poly-dT-tailed capture probes using a dot- or slot-blot approach. Complex manipulations of primer concentrations and staggered additions of primers were required in order to achieve equal amplification of multiple genes. Multiplex PCR amplification of two different Legionella genes, one specific for L. pneumophila (mip) and the other for the genus Legionella (5S rRNA), was achieved by staggered amplification. Multiplex PCR amplification using differing amounts of primers specific for lacZ and lamB genes permitted the detection of coliform bacteria and those associated with human faecal contamination, including the indicator bacterial species E. coli and enteric pathogens Salmonella and Shigella. Hybridization of biotin-labelled amplified DNA, in which the biotin was incorporated during PCR amplification from biotinylated-dUTP, to immobilized 400-dT-tailed capture probes permitted specific and sensitive detection of target gene sequences. The sensitivity of colorimetric detection achieved by PCR amplification of target DNA was at a level equivalent to 1-2 bacterial cells, which is the same level of sensitivity obtained with radioactive detection. The simultaneous amplification of several genes and hybridization to immobilized capture probes with colorimetric detection is an effective, efficient and rapid detection method for various human bacterial pathogens.

Evaluation of EMIT methods for the determination of the five major antiepileptic drugs on an automated kinetic analyser.

We have evaluated the performance of enzyme-multipled immunoassay methods for the five major antiepileptic drugs on an automated system, the Perkin-Elmer Model KA-150 Kinetic Analyzer. The precision in the normal duplicate mode was found to be in the range of 6% to 10% for all five tests over a typical working day. All EMIT methods were compared to gas-liquid chromatographic procedures and, in addition, the phenytoin and phenobarbital assays were compared to a liquid-chromatographic method. The phenytoin assay was also compared to RIA and to a manual spectroscopic method. In general, most of the comparison studies resulted in acceptable correlation, although one gas chromatographic method did not correlate very well with the phenytoin and phenobarbital immunoassays.

Comparative determination of phenytoin by spectrophotometry, gas chromatography, liquid chromatography, enzyme immunoassay, and radioimmunoassay.

Sera from patients being treated with phenytoin were analyzed for the drug by spectrophotometry, gas chromatography, radioimmunoassay, enzyme immunoassay, and liquid chromatography. The essay values obtained were intercompared statistically. Enzyme immunoassay and liquid chromatography appear to be attractive alternatives to the more traditional methods of spectrophotometry and gas chromatography. Our radioimmunoassay data correlated poorly with results by the four other methods.

Automated turbidimetry of amylase activity by use of a discrete kinetic analyzer.

We adopted an automated turbidimetric rate method for determining amylase activity to the KA-150 Kinetic Analyzer. In the method, an insoluble amylopectin substrate is used with activity determined by the rate of decrease in turbidity. Run-to-run CV for 59 samples with activities up to 400 units (arbitrary amylase units per 100 ml of sample) was 2.8%. A comparison with a similar method, performed by nephelometry, for 104 sera, showed a correlation coefficient (r) of 0.992, with a slope of 1.02. In an additional comparison with an amyloclastic method, for 52 sera r was 0.997, with a slope of 1.01. Day-to-day precision for control sera with activities near the upper limit of normal (279 and 216 units) averaged 2.5% during two months. Measured and calculated activity were linearly related to well above the upper limit of normal (normal range, 60-200 arbitrary units), showing a deviation from linearity of about 10% at 450 units. Commercial reagents available for the Perkin-Elmer Model 91 Amylase Lipase Analyzer can be used with the KA-150.

Otimum kinetic enzymatic procedures for glucose and triglycerides in plasma and serum.

I describe optimum kinetic procedures for measuring glucose and triglycerides in plasma and serum by enzymatic techniques. Glucose was measured by the hexokinase method at a 1000-fold sample dilution. Absorbance and concentration are linearly related for concentrations up to 4 g/liter. Results are reproducible to about 25 mg/liter (standard error). Comparisons between the kinetic glucose method described here and an automated o-toluidine and a manual end-point method showed no apparent bias between the methods; correlation coefficients were 0.998 and 0.991, respectively. Triglycerides (triacylglycerols) were measured in a fully enzymatic system by measuring free glycerol after hydrolysis with lipase. Absorbance and concentration are linearly related to greater than 2.5 g/liter at a 300-fold sample dilution. Repeatability was about 30 mg/liter (standard error). Compared with a manual method, the correlation coefficient was 0.978, with a slope of 0.93.

Making enzymatic methods optimum for measuring compounds with a kinetic analyzer.

Kinetic enzymatic methods for analysis of substrates can be made optimum for a sensitive photometric analyzer by adjusting the activity of the triggering (catalyzing) enzyme so that the reaction rate is maximum at the time of measurement. tat this optimum activity, the exponential time constant for exhaustion of substrate equals the time between triggering and rate measurement. The scale factor (defined as measured activity divided by sample concentration in the reaction mixture) is the same for all tests. Sensitivity to substrate concentration is predictable from instrumental absorbance uncertainty and molar absorptivity of the absorbing species. These predictions from Michaelis theory were verified experimentally for pyruvate and lactate triggered with lactate dehydrogenase, for glucose triggered with hexokinase, and for triglycerides triggered with glycerol kinase, the reaction rate being measured 30 s after triggering. Sensitivities of 1.5 times 10(-7) mol/liter were achieved. Serum diluted 1000-fold and analyzed for glucose gave a repeatability of 25 mg/liter with linearity to 4.0 g/liter. Samples diluted 300-fold and analyzed for triglycerides gave 30 mg/liter repeatability, with linearity to concentrations exceeding 3.0 g/liter.