Detection of PCR amplicons from bacterial pathogens using microsphere agglutination

For rapid and inexpensive detection of polymerase chain reaction (PCR) amplicons, a novel microsphere agglutination assay has been developed. PCR is carried out using biotinylated forward and reverse primers, and the amplified DNA fragments are able to agglutinate streptavidin-coated microspheres (5.7 microm in diameter). Purification of PCR amplicons is unnecessary when initial primer concentrations are 250 nM. Agglutination can be identified visually within 2 min without any additional equipment or reagents. Using listeriolysin (lisA)-specific biotinylated primers, we have successfully detected and identified Listeria monocytogenes lisA+ cells among Salmonella typhimurium, Staphylococcus aureus, Campylobacter jejuni and Escherichia coli O157:H7 cells. The simplicity of this protocol considerably reduces the time and cost of diagnostic PCR experiments. This procedure is potentially useful for various studies and field applications.