Microarray Detection of Labeled NASBA Products for the Specific Identification of Pathogenic Bacteria using tmRNA as a Target

摘要

This paper describes a diagnostics strategy involving sample preparation, isothermal Nucleic Acid Sequence Based Amplification (NASBA) of bacterial tmRNA, fluorescent labeling, and microarray hybridisation to detect and identify Streptococcus pneumoniae at a concentration of less than one colony forming unit (CFU) per ml of culture. The tmRNA transcript of the bacterial ssrA gene exhibits several properties that make it suitable as a molecular target for nucleic acid diagnostics. Sequence homology at the 5' and 3' ends of the gene facilitates universal amplification of tmRNA from different species, while sequence heterogeneity in the internal portions of the gene permit the design of oligonucleotide probes to identify bacteria at the genus and species level. When combined with the universal amplification of tmRNA and the potential high probe density of microarrays, the method presented here may have application for the high-throughput screening of samples for a multitude of pathogenic bacteria.