Methods, compositions, and kits for the detection of bacteria in a sample

Abstract

The present invention includes compositions, kits, and methods useful for the detection of bacteria. These agents and methods are primarily directed to a method of detecting the presence of bacteria in a sample, involving incubating the sample with an agent that binds to bacteria, such as, e.g., an agent specific for peptidoglycan or a component thereof, and then detecting bound bacteria. The invention includes lateral-flow immunoassay methods and devices for assessing the total bacterial load in a liquid sample.

1 . A device for the detection of bacteria, comprising:
 (a) a carrier to which a liquid sample suspected of containing bacteria can be applied;   (b) a first binding agent comprising a peptidoglycan recognition protein or fragment thereof that is mobile in the carrier when in the moist state; and   (c) a second binding agent comprising an agent that is specific for peptidoglycan or a component thereof that is immobilized in a first region of the carrier,   wherein one or both of the binding agents bind peptidoglycan or a component thereof.   
     
     
         2 . The device of  claim 1 , wherein the component is selected from the group consisting of: N-acetylglucosamine, N-acetylmuramic acid, a tetrapeptide side chain of peptidoglycan, a peptide cross link of peptidoglycan and teichoic acid. 
     
     
         3 . The device of  claim 1 , wherein the carrier comprises the following components:
 (a) a liquid sample application wick;   (b) a conjugate pad comprising the first binding agent;   (c) a base pad comprising the second binding agent;   (d) a sample pad comprising a blocking agent; and   (e) an absorbent pad.   
     
     
         4 . The device of  claim 1 , further comprising a control binding agent that is immobilized in a second region of the carrier. 
     
     
         5 . The device of  claim 1 , further comprising a detectable secondary agent that binds the first binding agent. 
     
     
         6 . The device of  claim 5 , wherein the first binding agent is labeled with biotin, and the detectable secondary agent comprises streptavidin. 
     
     
         7 . The device of  claim 6 , wherein the streptavidin is conjugated to colloidal gold. 
     
     
         8 . The device of  claim 1 , wherein the first binding agent is labeled. 
     
     
         9 . The device of  claim 8 , wherein said label is coupled directly or indirectly to said first binding agent. 
     
     
         10 . A kit for the detection of bacteria, comprising a binding agent, wherein said binding agent comprises a peptidoglycan recognition protein or fragment thereof and instructions for use. 
     
     
         11 . A kit for the detection of bacteria, comprising:
 (a) a detection binding agent, wherein said binding agent comprises a peptidoglycan recognition protein or fragment thereof;   (b) a capture binding agent that specifically binds peptidoglycan or a component thereof; and   (c) a carrier to which a liquid sample may be applied, wherein the detection binding agent is labeled.   
     
     
         12 . The kit of  claim 11 , further comprising a detectable secondary agent that binds the labeled detection binding agent. 
     
     
         13 . The kit of  claim 11 , wherein the labeled detection binding agent comprises a direct or indirect label. 
     
     
         14 . The kit of  claim 13 , wherein the label is selected from the group consisting of biotin, latex, gold, fluorescent dyes, radiolabels, and enzymes. 
     
     
         15 . The kit of  claim 12 , wherein the secondary agent comprises streptavidin. 
     
     
         16 . The kit of  claim 15 , wherein the streptavidin is conjugated to colloidal gold. 
     
     
         17 . A method of detecting bacteria in a sample, comprising:
 (a) incubating a sample with a binding agent, wherein said binding agent comprises a peptidoglycan recognition protein or fragment thereof, wherein said binding agent comprises a detection means; and   (b) detecting binding agent bound to the sample, thereby detecting bacteria in the sample.   
     
     
         18 . The method of  claim 17 , wherein the sample is a liquid. 
     
     
         19 . The method of  claim 17 , wherein the sample comprises biological material. 
     
     
         20 . The method of  claim 19 , wherein the biological material is obtained from a wound site. 
     
     
         21 . The method of  claim 19 , wherein the biological material is tissue or biological fluid. 
     
     
         22 . The method of  claim 18 , wherein the liquid is a beverage. 
     
     
         23 . The method of  claim 22 , wherein the beverage is selected from the group consisting of: milk, soy milk, water, beer, wine, juice, cider, and carbonated beverages. 
     
     
         24 . The method of  claim 17 , wherein the detection means is selected from the group consisting of: biotin, latex, colloidal gold, fluorescent dye, and an enzyme. 
     
     
         25 . An analytical test device for detecting the presence of bacteria in a liquid sample, comprising:
 (a) a hollow casing having a liquid sample application aperture and a means permitting observation of a test result; and   (b) a carrier comprising a dry porous material contained within said hollow casing,   said carrier communicating directly or indirectly with the exterior of said hollow casing through said liquid sample application aperture to receive applied liquid sample, said carrier having a capture line observable via said means permitting observation, said carrier, in the dry unused state, containing a labeled agent capable of specifically binding bacteria to form a first complex of said labeled agent and said bacteria, wherein said labeled agent comprises a PGRP or component thereof that is dry on said carrier prior to use and is released and mobile upon application of said applied liquid sample, and   said carrier containing in said capture line a means for specifically binding said first complex, said means for binding being immobilized in said capture line; wherein said binding means binds said first complex to form a second complex, said second complex being observable via said means permitting observation, thereby to indicate the presence of bacteria in said liquid sample.   
     
     
         26 . The device of  claim 25 , wherein said dry porous material is selected from the group consisting of: nitrocellulose and nylon. 
     
     
         27 . An enzyme-linked immunosorbent assay comprising a peptidoglycan recognition protein or fragment thereof and a direct or indirect label.