PCR-Less DNA co-polymerization detection of Shiga like toxin 1 (stx1) in Escherichia coli O157: H7

摘要

There is a great need for rapid identification of bacterial agents, specifically pathogenic species such as Escherichia coli O157:H7, which is a highly infectious and lethal member of the Shigatoxigenic group of E. coli. In this study, the Shiga like toxin gene (stx1) responsible for the pathogenicity of E. coli was recovered from live samples and detected with a two particle DNA assay from genomic DNA. The two particle system consisted of a magnetic microparticle for separation/recovery and a DNA linked gold nanoparticle (AuNP) for reporting. Oligonucleotide reporters on the AuNP were used for fluorescent readout and the gold nanoparticle was used for direct electrochemical readout. Electrochemical detection successfully detected stx1 gene at 5CFU/mL. Signal amplification via self assembling co-polymerization fluorescent readout was accomplished using the oligonucleotide linked AuNP. The two co-polymerization methods and electrochemical detection were compared against standard end-labeled DNA fluorescence detection of the gold nanoparticles in the system. The self assembling reporter consisted of two oligonucleotide sequences that repeatedly hybridized to each other to form large double stranded structures. Tethered co-polymerization amplification was able to detect the stx1 gene at 10~5CFU/mL (p=0.03) of E. coli in a total assay time of 7h, including DNA extraction. The self assembling nature of the amplification system provides an enzyme free means of rapid signal amplification using inexpensive materials. Amplification can be accomplished with most any small single stranded DNA species providing an amplified readout method to a large number of other DNA based technologies.