Direct detection of green fluorescent protein messenger RNA expressed in Escherichia coli by rolling circle amplification

Prevailing conventional microbial detection methods depend largely on microbial cultivation in selective media that requires several days. Polymerase chain reaction (PCR)-based methods, including quantitative reverse transcription PCR, are also rapid and useful methods for identification of target microbes, although for practical use they still suffer from disadvantages such as contamination problems and cost. Here we demonstrate that RNA-primed rolling circle amplification (RPRCA) using phi29 DNA polymerase, a precircularized probe, and SYBR Green II achieved real-time detection of specific messenger RNA (mRNA) from living microbes. The precircularized DNA probe was prepared by intramolecular ligation using CircLigase and treated by exonuclease I to eliminate uncircularized oligonucleotide, thereby significantly reducing potential noise by nonspecific amplified DNA by-products that affect successive RPRCA. When in vitro transcribed green fluorescent protein (GFP) mRNA was used as a primer, RPRCA could specifically detect at least 1 fmol of this mRNA in the presence of a precircularized probe that had a sequence complementary to the 3′ terminus of mRNA without reverse transcription. This method could also detect expressed GFP mRNA present in 10 ng of total RNA isolated from Escherichia coli without DNase treatment. These data suggest that RPRCA has the potential to be a direct, rapid, and convenient method for detecting microbial mRNA.