Biomedical subjects

Bruce Harrison

Publications and source records attributed to Bruce Harrison.

2 recordsLinked to original sources

PubMed2006

Staphylococcus aureus genotyping using novel real-time PCR formats.

One approach to microbial genotyping is to make use of sets of single-nucleotide polymorphisms (SNPs) in combination with binary markers. Here we report the modification and automation of a SNP-plus-binary-marker-based approach to the genotyping of Staphylococcus aureus and its application to 391 S. aureus isolates from southeast Queensland, Australia. The SNPs used were arcC210, tpi243, arcC162, gmk318, pta294, tpi36, tpi241, and pta383. These provide a Simpson's index of diversity (D) of 0.95 with respect to the S. aureus multilocus sequence typing database and define 61 genotypes and the major clonal complexes. The binary markers used were pvl, cna, sdrE, pT181, and pUB110. Two novel real-time PCR formats for interrogating these markers were compared. One of these makes use of "light upon extension" (LUX) primers and biplexed reactions, while the other is a streamlined modification of kinetic PCR using SYBR green. The latter format proved to be more robust. In addition, automated methods for DNA template preparation, reaction setup, and data analysis were developed. A single SNP-based method for ST-93 (Queensland clone) identification was also devised. The genotyping revealed the numerical importance of the "South West Pacific" and "Queensland" community-acquired methicillin-resistant S. aureus (MRSA) clones and the clonal complex 239 "Aus-1/Aus-2" hospital-associated MRSA. There was a strong association between the community-acquired clones and pvl.

Bacterial Typing Techniques↗

PubMed2004

Differential amplification and quantitation of Marek's disease viruses using real-time polymerase chain reaction.

Quantitative real-time PCR (qPCR) assays for the three serotypes of Marek's disease virus (MDV) have been developed. An internal control qPCR assay that detects chicken alpha2 (VI) collagen gene was also developed to allow quantitation of MDV. To reduce costs and time, the assays for MDV1 and the internal control were combined into a duplex assay. The sensitivity, specificity, precision, and reproducibility of each assay are reported. The MDV qPCR assays were specific to their target gene when compared using Australian field and vaccine strains of MDV and 10-100-fold more sensitive than standard PCR. Using DNA from infected spleen tissue, the lower limit of detection of total DNA (viral and host combined) was 0.025 ng for the MDV1 and collagen assays, and 0.25 ng for the HVT and MDV2 assays. All assays were found to be highly reproducible for Ct values, but less so for calculated concentrations. MDV1 and HVT were quantitated in spleen tissue of twenty experimentally infected chickens 7-35 days after infection. The relative abundance of MDV1 exhibited a clear peak at day 14 post-infection, whereas HVT displayed an increasing trend over the 35 days post-infection. The duplex assay was optimized such that it was able to accurately quantitate MDV1 in samples of very high, medium, and very low relative abundance of MDV1. These qPCR assays will be useful for reliable differentiation and quantitation of MDV for a range of research and industry applications.

Animals↗