Rapid Sterility Testing by NAT Method Targeting RNA Instead of DNA

We present a rapid sterility testing method targeting ribosomal RNA (rRNA) to enable sensitive and specific detection of microorganisms. The assay follows a simple three-step protocol: (1) activation of microbial cells with concurrent inactivation of DNA, (2) lysis and RNA extraction using magnetic particles, and (3) detection via one-step real-time reverse transcription PCR (RT-PCR). The total assay time is approximately 7 hours. Validation using six compendial organisms listed in USP <71> demonstrated successful detection at a sensitivity of 10 CFU/mL. The method is also compatible with samples containing mammalian cells, maintaining performance in complex matrices. A key feature of this system is the rigorous degradation and removal of residual DNA, effectively reducing the risk of false-positive results that may arise in conventional DNA-targeting assays. By focusing on rRNA, which reflects microbial viability, this method provides a more accurate sterility assessment within a significantly shorter time frame compared to traditional culture-based tests. The approach offers a promising solution for rapid microbiological testing in pharmaceutical quality control and is designed to align with current regulatory expectations for alternative methods.