P57

To assess the mycobactericidal effect of different RNA-protective buffers for the storage of suspensions of  Mycobacterium tuberculosis (Mtb) H37Ra. Mtb H37Ra was incubated in either an in-house prepared GTC-TCEP buffer or the commercial RNAlater (Ambion, Inc., USA) and DNA/RNA shield (Zymo Research, USA). The reduction in viability was assessed by comparing the growth of logarithmic dilutions of treated and non-treated samples on Löwenstein-Jensen media. To assess RNA fixation, reverse transcription qPCR (RT-qPCR) assays of two representative mRNA targets were performed on extracts of bacterial suspensions treated with RNAlater for up to nine months. Treatment with RNAlater, for 30 min up to overnight, reduced viability of Mtb H37Ra by max. 2 logs, while of 24 suspensions treated with GTC-TCEP or DNA/RNA shield, 22 showed at least a 4-log viability reduction, meeting a condition in the inactivation guidelines of the European Committee of Standardization. On the other hand, did the RNA content in bacterial suspensions treated with RNAlater increase significantly (alpha level = 0.05) at -20 and 4 °C in the course of six months of storage. GTC-TCEP and DNA/RNA shield inactivate Mtb H37Ra more efficiently than RNAlater. At the same time, the RNA content was not stabilised by RNAlater at -20 and 4 °C. This affects shipping conditions and suggests that the mycobacterial transcriptome can change during storage in RNAlater.