Validation of TRIzol-Based Inactivation Protocol with Failure Scenario Testing for Bacterial Select Agent Surrogates

Validated inactivation procedures are required for the safe handling and downstream analysis of highly pathogenic organisms, particularly those categorized as biological select agents and toxins (BSATs). TRIzol-based extraction methods are widely used for nucleic acid and protein isolation, yet their reliability for bacterial inactivation has not been comprehensively evaluated. In this study, we assessed TRIzol-based extraction methods for sample quality and inactivation reliability across a series of mock failure scenarios using five attenuated bacterial isolates: Francisella tularensis holarctica LVS, Bacillus anthracis Sterne, Yersinia enterocolitica, Mycobacterium marinum, and Burkholderia cepacia. Dilution of TRIzol to induce incomplete cell lysis for the initial extraction step, including 0% TRIzol, consistently inactivated all surrogate organisms, suggesting that downstream precipitation and sample washing reagents, including isopropanol and 70% ethanol, were sufficient to inactivate organisms in the absence of TRIzol. Several protocol failure scenarios were then evaluated to simulate human error by omitting extraction, precipitation, and washing steps individually or in combination for the most resistant organism, B. anthracis Sterne strain. Failure-scenario testing demonstrated that reliable inactivation of B. anthracis required strict adherence to the complete protocol due to the spore-forming ability of B. anthracis. Collectively, this work provides a reference with experimental evidence supporting the use of TRIzol-based extraction as a bacterial inactivation strategy for a wide range of bacterial pathogens.