Objective: Selectively detect by molecular means viable (living) bacteria within a sample.
Why it is important: The polymerase chain reaction (PCR) is a fast, sensitive, and specific means to detect disease-causing bacteria in patient and environmental samples. However, PCR cannot distinguish viable bacterial cells from dead cells, or from free DNA or RNA in samples. In order to improve specificity of PCR for viable cells, we have developed assays for specific bacterial RNA molecules termed ribosomal RNA precursors (pre-rRNA). Because the synthesis of pre-rRNA is rapidly triggered by nutritional stimulation, viable bacteria can be distinguished from dead cells and free DNA or RNA by measuring the production of species-specific pre-rRNA in samples that have been briefly stimulated with nutrients.
Abstract: Our methods exploit novel biomarkers of microbial viability and growth termed ribosomal RNA precursors, or pre-rRNA. Molecular viability testing (MVT) uses pre-rRNA detection as a means to improve both the sensitivity and specificity of PCR-based detection of bacteria in samples. Pre-rRNAs are intermediates in ribosomal RNA synthesis. In growing bacterial cells, pre-rRNAs are abundant (hundreds or thousands of copies per cell) and easy to detect. When bacterial growth slows, pre-rRNA pools are actively drained. The pools are rapidly replenished when growth-limited cells are given fresh nutrients. These fluctuations occur consistently in viable cells but are not seen in dead cells or with free nucleic acids. Pre-rRNA sequences are hypervariable, such that pre-rRNA-targeted PCR tests can detect and assess the physiology of individual pathogen species within complex clinical samples.
MVT technology improves both the sensitivity and specificity of PCR for bacterial cells. In MVT a sample is split into two equal aliquots, one of which is nutritionally stimulated. If viable pathogen cells are present in the sample, then pathogen pre-rRNA copy number (quantified by reverse transcriptase – qPCR, or RT-qPCR) rapidly increases in the stimulated aliquot relative to a control (non-stimulated) aliquot. Non-viable cells cannot catalyze this increase.
Partners: AttoDx, UCT/SATVI, Nicholas Walter’s lab (UC Anschutz), Gregory T. Robertson (CSU) , Sean Murphy’s lab, South African Tuberculosis Vaccine Initiative (SATVI).
Funders: NIH, EPA, Novartis, Washington Life Sciences Discovery Fund
Current status: Improving methods to detect viable Mycobacterium tuberculosis (the bacterium that causes tuberculosis) in samples from patients undergoing tuberculosis treatment.
Current Evidence:
Validated on bacteria that cause numerous diseases including tuberculosis, health care associated infections, food and water-borne disease.
Validated on bacterial species such as P. aeruginosa, S. aureus, M. avium, M. tuberculosis, A. hydrophila, E. coli, E. faecalis, H. influenzea.
Publications:
- Molecular viability testing of bacterial pathogens from a complex human sample matrix
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Biosynthetic enhancement of the detection of bacteria by the polymerase chain reaction
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Molecular detection of viable bacterial pathogens in water by ratiometric pre-rRNA analysis
Patents: US9115407B2
Next steps:
Optimize MVT for detection of MTB in sputum and oral swabs
Evaluate MVT’s utility in the context of TB treatment monitoring (UCT/SATVI)
Work with CSU and CU Anschutz collaborators to evaluate steady-state pre-rRNA as a measure of therapeutic drug activity in TB
Who is working on it: Kris Weigel, Alaina Olson, and Lauren Sarkissian lead the Cangelosi lab’s work on pre-rRNA assays, including MVT.