Portable pathogen analysis system for detecting waterborne pathogens

Patent metadata

Field	Value
Publication number	US-10894254-B2
Application number	US-201816153423-A
Country	US
Kind code	B2
Filing date	Oct 5, 2018
Priority date	Oct 5, 2017
Publication date	Jan 19, 2021
Grant date	Jan 19, 2021

Abstract

A portable pathogen analysis system (PPAS) designed to detect microbial pathogens at the point-of-sample collection. The system comprises a concentration tube used for the concentration of microbes in large volumes of water samples using super absorbent polymer (SAP) beads and a hand-powered centrifuge; and a processing component, which functions as a portable lab-on-a-disc droplet digital nucleic acid amplification system, which integrates DNA extraction, nucleic acid amplification, and post-amplicon analysis in a single unit. The present invention provides a fast, cost-effective, and user-friendly solution for microbial water quality analysis in low-resource settings.

First claim

What is claimed is:

1. A portable pathogen analysis system for detecting a microorganism in a liquid sample, said system comprising:
a. a microfluidic disc comprising:
i. an inlet fluidly connected to an inlet chamber, wherein a concentrated sample is introduced to the inlet chamber via the inlet;
ii. a clarification chamber fluidly connected to the inlet chamber via a passive valve;
iii. a waste chamber fluidly connected to the clarification chamber;
iv. 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;
v. 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;
vi. a reaction chamber fluidly connected to the mixing chamber; and
vii. 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;

b. 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; and
c. a stepper motor operatively coupled to the microfluidic disc, wherein the stepper motor functions to rotate the microfluidic disc;
wherein detecting the target nucleic acid associated with the microorganism is indicative of the presence of the microorganism in the liquid sample.

2. The system of claim 1 , wherein the microfluidic disc is disposable.

3. The system of claim 1 , wherein flow of the concentrate and reagents on the microfluidic disc is controlled by rotation speeds and passive valves.

4. The system of claim 1 , wherein cell lysis is achieved in the inlet chamber.

5. The system of claim 4 , wherein cell lysis is achieved via mechanical bead-beating induced by magnetic actuation at a first rotational speed.

6. The system of claim 5 , wherein a second rotational speed can break the passive valve to allow fluid from the inlet chamber to the clarification chamber.

7. The system of claim 1 , wherein nucleic acid amplification is achieved via loop-mediated isothermal amplification (LAMP).

8. The system of claim 1 , wherein the portable analysis device can rotate the microfluidic disc at varying speeds.

9. The system of claim 1 , wherein the portable analysis device comprises a display operatively connected to a computer.

10. The system of claim 1 , wherein the portable analysis device comprises a light source for dye excitation for fluorescence measurements.

11. The system of claim 10 , wherein the portable analysis device comprises a camera for taking digital images of fluorescence of the oil-in-water droplets.

12. The system of claim 11 , wherein the portable analysis device processes the digital images with software to calculate a concentration of the microorganism in the sample with Poisson distribution analysis.

13. The system of claim 1 , further comprising 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.

14. 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 microorga

Assignees

• Univ California
• California Inst Of Techn

Inventors

• Jiang Sunny
• Gowda Hamsa
• Huang Xiao
• Wu Xunyi
• Madou Marc
• Hoffmann Michael R

Classifications

• C12Q1/701

  Specific hybridization probes · CPC title

• B01L3/502738 Primary

  characterised by integrated valves (throttle valves in microfluidic sample containers B01L3/502746) · CPC title

• B01L2400/0688

  surface tension valves, capillary stop, capillary break · CPC title

• B01L2200/0673

  Handling of plugs of fluid surrounded by immiscible fluid · CPC title

• B01L3/502784

  specially adapted for droplet or plug flow, e.g. digital microfluidics · CPC title