Simultaneous and highly sensitive detection of six different foodborne pathogens by high-throughput suspension array technology.

Abstract: In the present paper, multiplex polymerase chain reaction (PCR) was coupled to a suspension array to construct an assay for simultaneous identification of six kinds of pathogens. The suspension array allows the simultaneous detection of different target sequences in a multiplex and high-throughput format. The assay uses a liquid suspension hybridization format with specific oligonucleotide probes covalently bound to the surface of fluorescent color-coded microspheres. Biotinylated target amplicons, hybridized to their complementary probe sequences, are quantified by adding the conjugate, streptavidin R-phycoerythrin. Six probes derived from the sequence analysis of a specific gene were developed and validated. There was little cross-reaction among the various probes. In the multi-channel method, the detection sensitivity was 1.6 × 10−6 mM. Results of nucleic acid detection assays show detection sensitivity for 20–4 × 103 CFU/ml, which is one to five orders of magnitude higher than those of PCR-agarose method in different bacteria. Aside from single channel capability, the assay allows the simultaneous detection of target genes in a single reaction, with detection limits of 1–10 CFU/ml. The accuracy, speed, flexibility, and sensitivity of the proposed assay are beneficial for the diagnosis of pathogenic diseases. [Copyright &y& Elsevier]