Herakles Li

Abstract:

Background: Molecular viability testing (MVT) is a novel RT-qPCR based diagnostic assay designed for rapid, sensitive, and specific detection of viable bacterial pathogens. Previous research has found it to be 5- to 10- fold more sensitive than standard DNA-targeted PCR for detecting bacterial pathogens in complex samples. It distinguishes viable from nonviable bacterial cells through ratiometric qPCR analysis of ribosomal RNA precursors (pre-rRNA) after a brief (≤1 generation time) nutritional stimulation. As part of a larger project to validate MVT as a diagnostic tool for pathogens specific to Ventilator Associated Pneumonia (VAP), this thesis explored avenues to increase the versatility of MVT, attempting to simplify, streamline, and multiplex MVT in order to expand upon its potential.

Methods: Experiments were based on affinity purification of nucleic acids utilizing universal oligonucleotide probes to capture pre-rRNA. These probes would capture target pre-rRNA from lysed bacterial cells through hybridization of a complementary sequence on a region of the mature bacterial rRNA. This would allow for novel methods of detection for MVT. Extraction of the target pre-rRNA would occur through the probes attached to a solid surface. We explored 1) construction of a multiplex version of MVT, 2) a novel method for nucleic acid purification and extraction, and 3) non-amplification-based MVT methods.
 

Results: Capture and detection of pre-rRNA was validated with the universal probes for various species at high cell counts but not near the limits of detection for MVT. The method was unsatisfactory for use in multiplexing MVT. Extraction and purification was more consistent and sensitive with traditional commercial kit purification compared to affinity purification. Results of the non-amplification and label-free detection methods were not considered promising.

Conclusion: Affinity purification offered a potential avenue to expand the ways MVT analysis could occur. Probe hybridization was positive but compared to previously reported methods, capture of bacterial pre-rRNA was not as efficient or sensitive. Future testing of affinity purification would require significant optimization to achieve the sensitivity necessary for diagnostic testing. These results guided the project team toward alternative MVT methods for detecting bacteria associated with VAP.