In the present study, we attempted to detect pathogenic microbes employing the analyte of inorganic pyrophosphate ion (PPi) generated by nucleic acid reaction. We employed a nucleic acid sequence-based amplification (NASBA) method for amplification of RNA (in 16S rRNA) extracted from E. coli which was used as a substitute for pathogenic microbes. PPi was detected as the product of the NASBA reaction by a chemiluminometric inorganic pyrophosphate ion (CL-PPi) assay which consists of three enzymatic reactions of inorganic pyrophosphatase (IP), pyruvate oxidase (PO), and isoluminol-microperoxidase chemiluminescence. Using our method, the difference between the negative control and positive control (E. coli RNA) was detected.