Development of next-generation sequencing-based sterility test

The sterility testing methods described in pharmacopoeias require an incubation period of 14 days to obtain analysis results. An alternative method that can significantly shorten the detection time and improve the accuracy is in urgent need to meet the sterility testing requirements of regenerative medicine products with a short shelf life. In this study, we developed the next-generation sequencing-based sterility test (NGSST) based on sequencing and multiple displacement amplification. The NGSST can be finished within 48 hours with five steps including whole genome amplification, sequencing, alignment, sterility testing report, and microorganism identification. We use RPKM ratio to minorize the influence of environmental bacteria and determine its cutoff based AUC curve. The NGSST showed high sensitivity in reporting contaminates at 0.1 CFU in supernatant of biological product or 1 CFU in cell suspension. Furthermore, we identified microorganisms in 5 primary umbilical cord mesenchymal stem cell samples that were tested positive by BacT/ALERTR 3D. Overall, the NGSST can serve as a promising alternative for sterility testing of biological products.