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Multicenter evaluation of PCR reagents for Bacillus anthracis



Multicenter evaluation of PCR reagents for Bacillus anthracis



Abstracts of the General Meeting of the American Society for Microbiology 102: 145-146



We have developed and optimized PCR reagents for five gene targets of Bacillus anthracis. The genes targeted are protective antigen, lethal factor, edema factor (all px01 genes), CAP A (px02 gene) and a chromosomal marker. Optimization for each of the reagents as wet chemistry resulted in assays that detect 100% of samples containing 100 fg of target DNA (approximately 20 gene copies). Although the assay can detect DNA concentrations down to 10 fg, the rate of detection varies depending on the assay at the lower concentrations. Lyophilization of PCR chemistry offers advantages over use of wet chemistry. It standardizes the reaction mixture over a large set of reagent tubes, extends shelf life, allows single use reagent sets, simplifies PCR set up, reduces contamination of reagent from external sources and provides a highly reproducible reaction. We conducted a multicenter study of three reagents as wet chemistry and dry chemistry for B. anthracis to determine the reproducibility of the assays in other laboratories. Five laboratories participated in the study. AFIP prepared the test panels and wet reagents. The dried reagents were manufactured by Idaho Technologies. Ct values were reproducible with both wet and dried chemistry. Each reagent was tested against a panel of 80 organisms consisting of B. anthracis and other Bacillus species in addition to limiting dilutions of B. anthracis DNA. Reproducibility was very good at high concentrations of DNA and was good at low concentrations of DNA. As expected, the lower the concentration, the greater the variation of the standard deviation of the Ct value.

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Accession: 035349766

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