Joseph L. DiCesare

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.

Detection of Legionella with polymerase chain reaction and gene probe methods

Methods were developed for the detection of Legionella in environmental water sources, based upon the polymerase chain reaction (PCR) and gene probes. All species of Legionella, including all 15 serogroups of L. pneumophila tested, were detected by PCR amplification of a 104 bp DNA sequence that codes for a region of 5S rRNA followed by radiolabelled oligoprobe hybridization to an internal region of the amplified DNA. Strains of L. pneumophila (all serogroups) were specifically detected based upon amplification of a portion of the coding region of the macrophage infectivity potentiator (mip) gene. Pseudomonas spp. that exhibit antigenic cross-reactivity in serological detection methods did not produce positive signals in the PCR-gene probe method using Southern blot analyses. Single cell, single gene Legionella detection was achieved with the PCR-gene probe methods.

Very-high-speed liquid chromatography : II. Some instrumental factors influencing performance

Abstract Instrumental factors influencing very-high-speed liquid chromatography including injection volume, detector flowcell volume, detector response time and total instrumental bandwidth are discussed. Very-high-speed analyses performed using relatively short columns (100mm) of conventional internal diameter (4.6 mm) packed with small particles (3 μm) result in very high performance as determined by the ability to generate over 400 theoretical plates/sec. Minimizing the total instrumental bandwidth by reducing volumes of the injector, connecting tubes and detector flowcell is necessary along with a very rapid detector response time in order to attain this performance. Limitations in performance on peaks having low capacity factors are apparent due to extra-column effects. The relatively high flow-rates which are utilized result in unexpectedly improved chromatographic performance since extra-column effects due to the flow path are not as great at higher flow-rates as originally expected. The largest contribution to extra-column effects appears to arise from the detector flowcell volume and the detector response time.

Biomarker Discovery and Analysis Platform: Application to Alzheimer's Disease

Peptides and proteins have been associated with many disease states such as cancers, diabetes, neurological and cardiovascular diseases [1-4]. Despite the limited success of a handful of biomarkers, most diseases lack sensitive and specific biomarkers. One of the most successful biomarkers, Prostate Specific Antigen (PSA) has a fairly high false-positive rate and very low clinical sensitivity (~25%).

Development of a microparticle-based on-site immunoassay for the detection of atrazine in soil and water samples.

Atrazine is widely used as a herbicide in agriculture and has been identified as a major groundwater contaminant in the US. Because of the possible hazard associated with its usage, there is a need for an efficient and economic screening method for on-site field testing of atrazine and other s-triazine herbicides in soil and water. We have developed a rapid, on-site test for the detection of atrazine based on the principle of microparticle agglutination inhibition immunoassay. The test detects 50 microg kg(-1) (0.050 ppm) atrazine in soil samples with direct extraction and 1.0 microg L(-1) atrazine in water samples when coupled with solid phase extraction.