Portable pathogen analysis system for detecting waterborne pathogens

A method of detecting a microorganism in a liquid sample, said method comprising:

• a. adding a volume of the liquid sample to a concentration tube, the concentration tube comprises a tube for holding a liquid;
• superabsorbent polymer (SAP) beads;
• and a filter positioned a distance above a bottom end of the tube to exclude the beads from the bottom end of the tube;
• b. incubating the concentration tube for a first length of time;
• c. centrifuging the concentration tube by hand for a second length of time, wherein the SAP beads remain above the filter and concentrate collects in the bottom end of the tube, wherein at least a portion of the microorganisms remain in the concentrate;
• d. introducing at least a portion of the concentrate to a system comprising:
• i. a microfluidic disc comprising:
• 1. an inlet fluidly connected to an inlet chamber, wherein the at least a portion of the concentrate is introduced to the inlet chamber via the inlet;
• 2. a clarification chamber fluidly connected to the inlet chamber via a passive valve;
• 3. a waste chamber fluidly connected to the clarification chamber;
• 4. a reagent chamber for holding nucleic acid amplification reagents, the nucleic acid amplification reagents comprise at least primers specific for a target nucleic acid associated with the microorganism;
• 5. a mixing chamber fluidly connected to the reagent chamber via a siphon valve and fluidly connected to the clarification chamber via a siphon valve, the mixing chamber mixes the nucleic acid amplification reagents and the sample from the clarification chamber;
• 6. a reaction chamber fluidly connected to the mixing chamber;
• and
• 7. an oil chamber for holding oil, wherein oil is introduced to fluid moving from the mixing chamber to the reaction chamber to form water-in-oil droplets, wherein the water-in-oil droplets allow for nucleic acid amplification in the reaction chamber;
• ii. a portable analysis device in which the microfluidic disc is integrated, the portable analysis device functions to heat and cool the microfluidic disc so as to effectively achieve nucleic acid amplification of nucleic acid in the water-in-oil droplets in the reaction chamber;
• and to analyze signals from the water-in-oil droplets to determine a concentration of the target nucleic acid in the sample, the portable analysis device comprises a light source for exciting a fluorescent dye in the water-in-oil droplets, a digital camera for obtaining a digital image of the fluorescent dye;
• and a microprocessor with software for processing the digital image of the fluorescent dye in the water-in-oil droplets;
• and
• iii. a stepper motor operatively coupled to the microfluidic disc, wherein the stepper motor functions to rotate the microfluidic disc;
• e. lysing cells in the inlet chamber via mechanical bead-beating induced by magnetic actuation of the microfluidic disc at a first rotation speed;
• f. increasing rotational speed of the microfluidic disc to a second rotational speed to break the passive valve, wherein cell debris is attenuated in the clarification chamber and DNA is left in supernatant;
• g. combining and rotating at a third rotation speed supernatant from the clarification chamber and nucleic acid amplification reagents from the reagent chamber in the mixing chamber via the siphon valves;
• h. forming water-in-oil droplets from fluid moving from the mixing chamber to the reaction chamber via centrifugal emulsification;
• i. subjecting the water-in-oil droplets to nucleic acid amplification via heating and cooling cycles of the reaction chamber;
• j. exciting the dye in the water-in-oil droplets via the light source;
• k. obtaining a digital image of the water-in-oil droplets with the digital camera;
• and
• l. processes the digital image with the software to calculate a concentration of the microorganism in the sample with Poisson distribution analysis;
• wherein detection of the target nucleic acid associated with the microorganism is indicative of the presence of the microorganism in the liquid sample.