A method of discriminating between spore-forming and non-spore-forming bacteria is based on a combination of simultaneous sporulation-specific and non-sporulation-specific quantitative polymerase chain reactions (Q-PCRs). The method was invented partly in response to the observation that for the purposes of preventing or reducing biological contamination affecting many human endeavors, ultimately, only the spore-forming portions of bacterial populations are the ones that are problematic (or, at least, more problematic than are the non-spore-forming portions).
The method includes the use of sporulation-specific primers in the form of designed, optimized deoxyribonucleic acid (DNA) oligonucleotides specific for the bacterial spoIVA gene (see table). [In “spoIVA,” “IV” signifies Roman numeral four and the entire quoted name refers to gene A for the fourth stage of sporulation.] These primers are mixed into a PCR cocktail with a given sample of bacterial cells. A control PCR cocktail into which are mixed universal 16S rRNA primers is also prepared. [“16S rRNA” denotes a ribosomal ribonucleic acid (rRNA) sequence that is common to all organisms.] Following several cycles of heating and cooling according to the PCR protocol to amplify amounts of DNA molecules, the amplification products can be analyzed to determine the types of bacterial cells present within the samples.
If the amplification product is strong, relative to the product of a control PCR sequence, then it is concluded that the bacterial population in the sample consists predominantly of spore-forming cells. If the amplification product is weak or nonexistent, then it is concluded that the bacterial population in the sample consists predominantly or entirely of non-spore-forming cells.
This work was done by Kasthuri Venkateswaran, Myron La Duc, and Tara Stuecker of Caltech for NASA’s Jet Propulsion Laboratory.
NPO-44296
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Discrimination or Spore-Forming Bacilli Using spoIVA
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Overview
The document titled "Discrimination of Spore-Forming Bacilli Using spoIVA" (NPO-44296) from NASA's Jet Propulsion Laboratory addresses the critical issue of bacterial contamination in space exploration. It emphasizes that while significant resources are allocated to planetary protection measures aimed at eliminating all bacterial populations, only a small fraction of these, specifically spore-forming bacteria, pose a real threat.
The primary goal of the research is to develop a more efficient method for identifying and quantifying spore-forming bacteria within a given sample. The document outlines a novel approach using specific DNA oligonucleotides designed to target the spoIVA gene, which is associated with spore formation in bacteria. This method is integrated with standard polymerase chain reaction (PCR) techniques, allowing researchers to distinguish between spore-forming and non-spore-forming bacterial cells.
The process involves mixing the spoIVA-specific primers with a sample of bacterial cells in a PCR cocktail. Through cycles of heating and cooling, the DNA is amplified, and the intensity of the resulting amplification product indicates the presence of spore-forming bacteria. A strong amplification signal suggests a predominance of spore-forming cells, while a weak signal or absence of product indicates a population primarily composed of non-spore-forming bacteria.
This targeted approach not only enhances the accuracy of microbial assessments but also enables spacecraft designers and systems architects to implement more appropriate and cost-effective planetary protection strategies. By understanding the actual percentage of spore-forming bacteria in a contaminant population, resources can be allocated more effectively, potentially saving time and money in the process.
The document concludes by highlighting the novelty of using sporulation-specific primers in quantitative PCR (Q-PCR) to assess the spore-forming capacity of bacterial populations. This advancement represents a significant step forward in microbial risk assessment for space missions, ensuring that only the most relevant bacterial threats are addressed.
For further inquiries or detailed information, the document provides contact details for the Innovative Technology Assets Management at JPL. Overall, this research underscores the importance of precision in microbial monitoring for the safety and success of future space exploration endeavors.
