Methods, devices, and systems of detecting microorganisms

Abstract

A rapid, sensitive method of separating and detecting microorganisms from a sample potentially containing microorganisms, such as but not limited to bacteria, fungi, yeast, viruses, and the like. The method relies on separation techniques to separate and concentrate the cells from the sample, together with chemical techniques to amplify the amount of detectable signal from low numbers of cells to provide a rapid and sensitive method of detecting microorganisms. This detection method may utilize: a filtration device; a centrifugation device; a system; a swab device; and kit comprising one or more of the devices and components to perform the present method of separating and detecting microorganisms in a sample potentially containing microorganisms. The sample may be a chemical (e.g., household cleaners), cosmetic, personal care, pharmaceutical, or consumable (e.g., food or beverage) good in its raw material, in-process, and/or finished product states that needs to be tested for any contaminating microorganisms prior to shipment to the consumer.

We claim: 
     
         1 . A method of detecting microorganisms in a sample potentially containing microorganisms, comprising:
 a) filtering the sample through a pre-filter for allowing microorganisms to flow through;   b) filtering the pre-filter filtrate through a capture filter for retaining microorganisms;   c) culturing the microorganisms retained on the capture filter;   d) incubating the microorganisms retained on the capture filter with extractant in combination with adenosine diphosphate;   e) filtering the lysed cells through the capture filter;   f) adding a luciferin/luciferase reagent to the capture filtered lysed cells; and   g) detecting microorganisms in the sample using a luminometer.   
     
     
         2 . The method of  claim 1 , wherein the detection assay reagent reacts with the incubated substrate mixture producing a light signal indicates the presence of microorganisms in the sample. 
     
     
         3 . A method of detecting microorganisms in a sample potentially containing microorganisms, comprising:
 a) diluting the sample;   b) centrifuging the sample to form a cell pellet;   c) discarding supernatant of centrifuged sample;   d) washing the cell pellet by resuspending the cell pellet in buffer and repeating steps (b) and (c);   e) culturing cells in growth medium;   centrifuging the cultured cells;   g) discarding the supernatant of the centrifuged cultured cells;   h) incubating the cells with extractant in combination with adenosine diphosphate;   i) adding a luciferin/luciferase reagent to the incubated extractant and adenosine diphosphate sample; and   j) detecting microorganisms in the sample using a luminometer.   
     
     
         4 . The method of  claim 3 , wherein the detection assay reagent reacts with the incubated substrate mixture producing a light signal which indirectly indicates the presence of microorganisms in the sample. 
     
     
         5 . A method of detecting microorganisms in a sample potentially containing microorganisms, comprising:
 a) diluting the sample;   b) centrifuging the sample with beads to form a cell pellet;   c) discarding supernatant of centrifuged sample;   d) washing the cell pellet by resuspending the cell pellet in buffer and repeating steps (b) and (c);   e) culturing cells in growth medium;   f) centrifuging cultured cells and beads;   g) discarding supernatant of centrifuged cultured cells;   h) incubating the cells with extractant in combination with adenosine diphosphate;   i) adding a luciferin/luciferase reagent to the incubated extractant and adenosine diphosphate sample; and   j) detecting microorganisms in the sample using a luminometer.   
     
     
         6 . A filtration device comprising:
 a) a vessel for receiving a sample potentially containing microorganisms;   b) a pre-filter for allowing microorganisms to flow through;   c) a capture filter for retaining microorganisms;   d) an outlet; and   e) a fluid retention element,   
       wherein the vessel is operably connected to the pre-filter, which is operably connected to the capture filter, which is operably connected to an outlet through which filtrate flows. 
     
     
         7 . The device of  claim 6 , wherein the pre-filter is a filter of a double-layer filter or two separate filters. 
     
     
         8 . The device of  claim 7 , wherein the double-layer filter and two separate filters each have filters of sequentially decreasing pore sizes. 
     
     
         9 . The device of  claim 6 , wherein the sample comes in contact with the pre-filter comprising a first filter having a pore size large enough to allow passage of microorganisms and retain non-microbial particles and a second filter having a pore size that is smaller than that of the first filter and large enough to allow passage of microorganisms and retain non-microbial particles. 
     
     
         10 . The device of  claim 7 , wherein the first and second filters of the pre-filter have a pore size ranging from about 5 to about 100 microns. 
     
     
         11 . The device of  claim 6 , wherein the capture filter that retains microorganisms has a pore size ranging from about 0.2 to about 4 microns. 
     
     
         12 . The device of  claim 6 , wherein an apparatus is operably attached to the filtration device to apply positive pressure. 
     
     
         13 . The device of  claim 6 , where an apparatus is operably attached to the outlet to apply negative pressure. 
     
     
         14 . The device of  claim 6 , where the device is further modified to allow for an automated and/or high throughput processing. 
     
     
         15 . A system for determining the presence or absence of microorganisms, comprising:
 a) a filtration device comprising:
 i) a vessel for receiving a sample potentially containing microorganisms; 
 ii) a pre-filter for allowing microorganisms to flow through; 
 iii) a capture filter for retaining microorganisms; 
 iv) an outlet; and 
 v) a fluid retention element; 
   b) a temperature-controlled chamber for culturing the microorganisms; and   c) a luminometer for detecting the presence or absence of microorganisms.   
     
     
         16 . The system of  claim 15 , further comprising an apparatus comprising a positive pressure device and/or a negative pressure device for applying pressure to the filters. 
     
     
         17 . The system of  claim 15 , further comprising a computer for operably connecting and controlling the pipette, the filtration device, the temperature-controlled chamber, and the luminometer, thereby forming an automated system. 
     
     
         18 . A kit, comprising one or more of: a filtration device comprising: a vessel for holding a sample potentially containing microorganisms; a pre-filter for allowing microorganisms to flow through; a capture filter for retaining microorganisms; an outlet; and a fluid retention element;
 and buffers, growth media, filters, extractant reagents, substrate reagents, adenosine diphosphate, detection assay reagents, luciferin/luciferase reagent, vessels or containers for amplifying the microorganisms or performing the detection assays, a manifold, and instructions for using the devices, methods, and detection assays.   
     
     
         19 . A method of detecting microorganisms in a sample potentially containing microorganisms, comprising:
 a) filtering the sample through a pre-filter for allowing microorganisms to flow through;   b) filtering the pre-filter filtrate through a capture filter for retaining microorganisms;   c) culturing the microorganisms retained on the capture filter;   d) incubating the microorganisms retained on the capture filter with extractant in combination with substrate;   e) filtering the lysed cells through the capture filter;   adding a detection assay reagent to the capture filtered lysed cells; and   g) detecting microorganisms in the sample using a detection device.   
     
     
         20 . A method of detecting microorganisms in a sample potentially containing microorganisms, comprising:
 a) diluting the sample;   b) centrifuging the sample, optionally with beads;   c) discarding supernatant of centrifuged sample;   d) washing cell pellet by adding buffer and repeating steps (b) and (c);   e) culturing cells in growth medium;   centrifuging cultured cells;   g) discarding supernatant of centrifuged cultured cells;   h) incubating the cultured cells with extractant in combination with substrate;   i) adding a detection assay reagent to the incubated extractant and substrate sample; and   j) detecting microorganisms in the sample using a detection device.