This technology has applications in pharmaceutical and medical equipment manufacturing, and food processing.
Since the Viking missions in the mid- 1970s, traditional culture-based methods have been used for microbial enumeration by various NASA programs. Viable microbes are of particular concern for spacecraft cleanliness, for forward contamination of extraterrestrial bodies (proliferation of microbes), and for crew health/safety (viable pathogenic microbes). However, a “true” estimation of viable microbial population and differentiation from their dead cells using the most sensitive molecular methods is a challenge, because of the stability of DNA from dead cells.
The goal of this research is to evaluate
a rapid and sensitive microbial
detection concept that will selectively
estimate viable microbes. Nucleic acid
amplification approaches such as the
polymerase chain reaction (PCR) have
shown promise for reducing time to
detection for a wide range of applications.
The proposed method is based
on the use of a fluorescent DNA intercalating
agent, propidium monoazide
(PMA), which can only penetrate the
membrane of dead cells. The PMAquenched
reaction mixtures can be
screened, where only the DNA from
live cells will be available for subsequent
PCR reaction and microarray
detection, and be identified as part of
the viable microbial community. An
additional advantage of the proposed
rapid method is that it will detect
viable microbes and differentiate from
dead cells in only a few hours, as
opposed to less comprehensive culturebased
assays, which take days to complete.
This novel combination
approach is called the PMA-Microarray
DNA intercalating agents such as PMA have previously been used to selectively distinguish between viable and dead bacterial cells. Once in the cell, the dye intercalates with the DNA and, upon photolysis under visible light, produces stable DNA adducts. DNA cross-linked in this way is unavailable for PCR. Environmental samples suspected of containing a mixture of live and dead microbial cells/spores will be treated with PMA, and then incubated in the dark. Thereafter, the sample is exposed to visible light for five minutes, so that the DNA from dead cells will be cross-linked. Following this PMA treatment step, the sample is concentrated by centrifugation and washed (to remove excessive PMA) before DNA is extracted. The 16S rRNA gene fragments will be amplified by PCR to screen the total microbial community using PhyloChip DNA microarray analysis. This approach will detect only the viable microbial community since the PMA intercalated DNA from dead cells would be unavailable for PCR amplification. The total detection time including PCR reaction for low biomass samples will be a few hours.
Numerous markets may use this technology. The food industry uses spore detection to validate new alternative food processing technologies, sterility, and quality. Pharmaceutical and medical equipment companies also detect spores as a marker for sterility. This system can be used for validating sterilization processes, water treatment systems, and in various public health and homeland security applications.
In accordance with Public Law 96-517, the contractor has elected to retain title to this invention. Inquiries concerning rights for its commercial use should be addressed to:
• Innovative Technology Assets Management JPL
• Mail Stop 202-233
• 4800 Oak Grove Drive
• Pasadena, CA 91109-8099