Flow cytometry-based systems and methods for detecting microbes

Abstract

In various embodiments, the present disclosure describes methods and systems for detecting microbes in a sample. The methods are generally applicable to quantifying the number of target bacteria in a sample counted from a detection region of a flow cytometer histogram. The detection methods can be employed in the presence of other microorganisms and other non-target microbe components to selectively quantify the amount of a target microbe. The methods are advantageous over those presently existing for testing of foodstuffs and diagnostic evaluation in their speed, accuracy and ease of use. Various swab collection devices and kits useful for practicing the present disclosure are also described herein.

What is claimed is: 
     
         1 . A flow cytometry method for detecting target microbes in a sample, said method comprising:
 a) mixing the sample with a plurality of probes to form a tagged sample;
 wherein the plurality of probes comprise at least one first probe and at least one second probe; 
 wherein the at least one first probe targets the target microbes; and 
 wherein each at least one first probe comprises at least one first tag having a first wavelength emission range; 
 wherein each at least one first tag has a first wavelength emission range that is substantially similar to one another; and 
 wherein the at least one second probe targets non-target microbe components of the sample; and 
 wherein each at least one second probe comprises at least one second tag; 
 wherein each at least one second tag has a second wavelength emission range that is different from the first wavelength emission range of the at least one first tag; 
   b) introducing the tagged sample into a flow cytometer; and   c) analyzing the tagged sample in the flow cytometer.   
     
     
         2 . The method of  claim 1 , wherein analyzing comprises:
 detecting the second wavelength emission range of the at least one second tag;   selecting at least one emission wavelength from the second wavelength emission range that overlaps the first wavelength emission range of the at least one first tag; and   detecting the first wavelength emission range of the at least one first tag in a region that overlaps the selected at least one emission wavelength.   
     
     
         3 . The method of  claim 1 , wherein analyzing comprises serial gating comprising a plurality of gates, wherein the first gate comprises FSC and SSC. 
     
     
         4 . The method of  claim 1 , wherein prior to mixing the sample, the method comprises:
 a) treating the sample with at least one oxidant and at least one detergent;   b) de-activating the at least one oxidant after treating the sample.   
     
     
         5 . The method of  claim 1 , wherein prior to mixing the sample, the sample is treated with at least one enzyme before the mixing step. 
     
     
         6 . The method of  claim 1 , wherein at least one probe from the plurality of probes is present at a non-saturating concentration. 
     
     
         7 . The method of  claim 1 , wherein the mixing step takes place in the presence of an additive selected from the group consisting of bovine serum albumin, glycerol or a combination thereof. 
     
     
         8 . The method of  claim 1 , wherein the at least one second tag is selected from the group consisting of quantum dots, phycoerythrin, protein fluorophores, particle fluorophores, phycobiliproteins, fluorescein derivatives, rhodamine, phthalocyanine derivatives, peridinin chlorophyll complex, and coumarin derivatives. 
     
     
         9 . The method of  claim 1 , wherein the non-target microbe components are selected from the group consisting of undesired microorganisms, undesired proteins, cellular debris, auto-fluorescing objects and combinations thereof. 
     
     
         10 . The method of  claim 1 , wherein the plurality of probes are selected from the group consisting of polyclonal antibodies, monoclonal antibodies, peptide nucleic acids, DNA probes, RNA probes, aptamers, small molecules, biomimetic molecules, virulent phage and combinations thereof. 
     
     
         11 . The method of  claim 1 , wherein the at least one second probe is a membrane impermeable DNA dye that penetrates non-viable microorganisms. 
     
     
         12 . The method of  claim 1 , further comprising:
 mixing the sample with at least one untagged probe; wherein the at least one untagged probe targets at least one non-target-microbe component of the sample.   
     
     
         13 . The method of  claim 1 , wherein the plurality of probes are present at a non-saturating concentration. 
     
     
         14 . The method of  claim 1 , further comprising: optimizing a performance of the flow cytometer; wherein optimizing comprises:
 a) increasing a sensitivity of at least one detection channel of the flow cytometer by increasing a gain on the at least one detection channel;   b) assigning a signal threshold for each at least one detection channel;   c) collecting raw data from the flow cytometer for a time range; wherein the time range comprises a plurality of intervals; and wherein the raw data comprises signals and non-signals for each at least one detection channel; and   d) analyzing the raw data from each of the plurality of intervals to provide processed data;   wherein analyzing comprises:   eliminating raw data from each of the plurality of intervals in which the signals do not exceed the assigned signal threshold for each at least one detection channel;   and selecting raw data from each of the plurality of intervals in which the signals do exceed the assigned signal threshold for each at least one detection channel.   
     
     
         15 . The method of  claim 1 , wherein the flow cytometer is standardized against the performance of a second flow cytometer. 
     
     
         16 . The method of  claim 13 , further comprising:
 optimizing a performance of the flow cytometer;   wherein optimizing comprises:   a) increasing a sensitivity of at least one detection channel of the flow cytometer by increasing a gain on the at least one detection channel;   b) assigning a signal threshold for each at least one detection channel;   c) collecting raw data from the flow cytometer for a time range;   wherein the time range comprises a plurality of intervals; and   wherein the raw data comprises signals and non-signals for each at least one detection channel; and   d) analyzing the raw data from each of the plurality of intervals to provide processed data;   wherein analyzing comprises:   eliminating raw data from each of the plurality of intervals in which the signals do not exceed the assigned signal threshold for each at least one detection channel; and   selecting raw data from each of the plurality of intervals in which the signals do exceed the assigned signal threshold for each at least one detection channel.