Mycoplasma (myco) contamination of mammalian cell lines are recognized as a major contributor to the lack of reproducibility in modern biomedical research. Despite the availability of commercially marketed detection systems, the unabated prevalence of myco in research attests to the shortcomings of these protocols. Indeed, all currently marketed myco tests fail to address the actual needs of the consumer, the bench scientist. For a myco detection product to be widely adopted it must: 1) require no additional equipment or infrastructure, 2) integrate seamlessly into a laboratorys daily tissue culture routine with no additional labor burden on the users, and 3) be sensitive, inexpensive, and yield results immediately. To this end, in Phase I of this proposal we will optimize the FastMyco assay to test cell-culture media samples. FastMyco uses breakthrough Recombinase Polymerase Amplification (RPA) coupled to an initial reverse transcriptase (RT) step (RT-RPA), resulting in an all-in-one isothermal alternative to PCR myco detection. In FastMyco, the RT-RPA reaction will target the 16S rRNA of myco, with amplification conducted in the cell-culture incubator at 37°C for approx. 15- 20 minutes. The assay has a potential detection limit of less than 1 CFU/ ml and immediate colorimetric output from an onboard detection system. The thermal stability of the system is a major technical and cost advantage of FastMyco over comparable systems. This will be achieved using cutting-edge lyophilization techniques that will create a multi-layered bead containing all necessary enzymes and reagents, freeze-dried around a core of DNA-detection substrate. The bead dissolves at different rates releasing the detection reagent only after target amplification has begun. In Phase II, we will continue to develop FastMyco for large HTP research organizations but also expand the bead-based detection systems to other human and agricultural pathogens. We will strive to integrate FastMyco into every laboratorys routine cell-culture. The assay would also give regulatory agencies such as NIH and FDA the leverage to require this more rigorous testing protocols to help curb the spread of mycoplasma in research.
Public Health Relevance Statement: SBIR 2020: 6823823214: The development of FastMyco: A novel isothermal colorimetric assay for the rapid detection of mycoplasma contamination. NARRATIVE The cost and extent of mycoplasma contamination is enormous, and prevalent in all sectors of biological, agricultural and medical research. The prevalence of contamination is indicative of the ineffectiveness of current mycoplasma detection protocols. In Phase I research, we will continue to develop FastMyco, the only detection assay that needs no additional time or equipment. FastMyco, uses isothermal amplification of RNA with onboard detection to give the user a result in less than 20 minutes at a fraction (~10%) of the price per sample of other competitor assays. The strength of FastMyco is the ability to be used in any cell culture facility and integrated seamlessly, into the routine cell maintenance activities. In Phase II, FastMyco will be further developed into a generic diagnostic test platform, able to be used in the field or in remote locations to detect selected viral or bacterial pathogens.
Project Terms: Acholeplasma laidlawii; Address; Adopted; Agriculture; Alkalies; amplification detection; aqueous; Arginine; assay development; base; Binding; Biological; Biological Assay; biological research; Biomedical Research; Cell Culture Techniques; Cell Line; Cell Maintenance; Cell membrane; Cell Wall; Color; commercial application; computerized data processing; cost; Coupled; Culture Media; Cytolysis; Decision Making; design; Detection; Detergents; Development; Diagnostic tests; DNA; Dyes; Enzymes; Equipment; Exhibits; experimental study; Fluorescence; Freeze Drying; Gentian Violet; Hand; Human; Incubators; Individual; Infrastructure; innovation; Laboratories; Link; luminescence; Magnesium; Major Groove; Mammalian Cell; Medical Research; Methods; Modernization; Mycoplasma; Mycoplasma hyorhinis; novel; novel strategies; Organism; Output; pathogen; pathogenic bacteria; pathogenic virus; Perforation; Phase; Polymerase; Prevalence; Price; Protocols documentation; prototype; rapid detection; Reaction; Reagent; recombinase; remote location; Reporting; Reproducibility; Research; research facility; Ribosomal RNA; RNA; RNA amplification; RNA-Directed DNA Polymerase; Sampling; Scientist; Ships; Small Business Innovation Research Grant; Sodium Chloride; Sodium Hydroxide; Source; Speed; Spottings; System; Techniques; Temperature; Testing; Time; tissue culture; triphenylmethane; Tube; United States National Institutes of Health; Viola
