PCR-based detection of Bacillus anthracis using an integrated microfluidic platform

Abstract

A highly-integrated PCR-based detection system has been developed for the rapid identification of pathogenic bacteria. Nanofabricated fluidic cartridges were used to carry out SYBR Green-based fluorogenic PCR assays for the detection of Bacillus anthracis which incorporated the chromosomal BA813 locus as the target for amplification. Real-time PCR assay conditions and operating parameters were optimised to increase detection sensitivity. Optimisation of the system resulted in the detection of as few as 40 B. anthracis colony forming units (CFU) with an average time to detection of 60 min, inclusive of DNA purification and PCR amplification, and a dynamic range of 40 to 400,000 CFU. Real-time fluorescence curves were analysed using a simplified mathematical method to determine threshold cycle (Ct) values with comparable results to a statistically-based analysis algorithm. These results support the utility of the system for rapid, sensitive detection of B. anthracis as well as potential for quantitative determination of target cell concentration.