Biomedical subjects

J P McNevin

Publications and source records attributed to J P McNevin.

3 recordsLinked to original sources

PubMed2000

Rapid solid-phase immunoassay for detection of methicillin-resistant Staphylococcus aureus using cycling probe technology.

A Cycling Probe Technology (CPT) assay with a lateral-flow device (strip) was developed for the detection of the mecA gene from methicillin-resistant Staphylococcus aureus (MRSA) cultures. The assay uses a mecA probe (DNA-RNA-DNA) labeled with fluorescein at the 5' terminus and biotin at the 3' terminus. The CPT reaction occurs at a constant temperature, which allows the probe to anneal to the target DNA. RNase H cuts the RNA portion of the probe, allowing the cleaved fragments to dissociate from the target DNA, making the target available for further cycling. The strip detection step uses a nitrocellulose membrane with streptavidin and immunoglobulin G antibody impregnated on the surface. In the absence of the mecA gene, the uncut probe is bound to an antifluorescein-gold conjugate and is then captured by the streptavidin to form a test line. In the presence of the mecA gene, the probe is cut and no test line is formed on the strip. A screen of 324 S. aureus clinical isolates by the CPT-strip assay showed a 99.4% sensitivity and a 100% specificity compared to the results of PCR for the detection of the mecA gene. Specificity testing showed that the CPT-strip assay did not exhibit any cross-reactivity with a panel of mecA-negative non-S. aureus isolates. The CPT-strip assay is simple and does not require sophisticated equipment. Furthermore, the assay takes 1.5 h starting from a primary culture to the time to detection of the mecA gene in S. aureus isolates.

Bacterial Proteins↗

PubMed1999

Rapid detection of the mecA gene in methicillin resistant staphylococci using a colorimetric cycling probe technology.

A Cycling Probe Technology (CPT) assay was developed for the detection of the mecA gene from methicillin resistant staphylococcal cultures. The assay is based on a colorimetric enzyme-immuno-assay (EIA) and uses a mecA probe (DNA-RNA-DNA) labeled with fluorescein at the 5'-terminus and biotin at the 3'-terminus. The reaction occurs at a constant temperature that allows the target DNA to anneal to the probe. RNase H cuts the RNA portion, allowing the cut fragments to dissociate from the target, making it available for further cycling. CPT-EIA uses streptavidin-coated microplate wells to capture uncut probe followed by detection with horseradish-peroxidase conjugated anti-fluorescein antibody. The assay was compared to PCR and shown to accurately detect the presence or absence of the mecA gene in 159 staphylococcal clinical isolates. The CPT-EIA assay takes two hours starting from cultured cells compared with the 24-48 h required for detection of methicillin resistance by conventional susceptibility tests.

Bacterial Proteins↗

PubMed1996

Molecular cloning and characterization of the CER2 gene of Arabidopsis thaliana.

Plants with defects in the synthesis of their epicuticular wax layer, eceriferum mutants (cer), are readily detected by the naked eye as bright green glossy plants when compared with the more glaucous normal plants. In a previous report, evidence was presented for the isolation of three lines from the Arabidopsis thaliana transformant collection (BRL5, BRL7 and BRL9) which failed to complement the cer2 mutant isolated previously. The analysis of the chemical composition of the epicuticular wax of these mutants suggests that the cer2 mutant of Arabidopsis is defective in very long chain fatty acid elongation. This paper reports the molecular cloning of the CER2 gene of Arabidopsis through the isolation of plant DNA flanking the site of T-DNA insertion as well as the characterization of the two independent T-DNA insertion mutant alleles, BRL5 and BRL9, of this gene. In the mutant line BRL5, T-DNA was found to be inserted in the second exon of the CER2 gene whereas in BRL9, T-DNA is inserted in the only intron of this gene. Nucleotide sequence analysis suggests that the ORF encodes a 47.3 kDa polypeptide. High levels of CER2 transcripts were detected in stems and flowers. The predicted amino acid sequence of the CER2 gene product reveals little homology with known protein sequences. In accordance with structural characterization of the T-DNA insertion mutants, no evidence of transcripts derived from the CER2 gene was found in either BRL5 or BRL9.

Amino Acid Sequence↗