Rapid peptidoglycan-based assay for detection of bacterial contamination

Abstract

The invention relates to a colorimetric method for detecting bacterial or fungal pathogens by detecting peptidoglycan or (1-3)-β-D-glucan in a sample.

1 - 19 . (canceled) 
     
     
         20 . A reader for detecting peptidoglycan or β-glucan in a sample, wherein the reader comprises:
 (a) a reaction tube for incubating the sample with (i) hemolymph from a lepidopteran insect; and (ii) a phenoloxidase substrate that generates a reaction product, and 3-methyl-2-benzothiazolinone hydrazone or other hydrazone derivative thereof; and, 
 (b) an optical light source for detecting the formation of a colored-reaction product by absorbance, wherein formation of the colored-reaction product indicates the presence of peptidoglycan or β-glucan in the sample, and the absence of the colored-reaction product indicates the absence of peptidoglycan or β-glucan in the sample. 
 
     
     
         21 . The reader according to  claim 20 , wherein the sample is a clinical sample, an environmental sample, an agricultural sample, a manufacturing sample, or a medical product. 
     
     
         22 . The reader according to  claim 21 , wherein the sample is a clinical sample, which is a bodily fluid, tissue specimen, hydration fluid, nutrient fluid, blood, blood product, vaccine, anesthetic, pharmacologically active agent, or an imaging agent. 
     
     
         23 . The reader according to  claim 22 , wherein the sample is selected from the group consisting of urine, a urine product, and cerebrospinal fluid. 
     
     
         24 . The reader according to  claim 20 , wherein the sample is a suspension or a liquid. 
     
     
         25 . The reader according to  claim 24 , wherein the sample is processed by centrifugation. 
     
     
         26 . The reader according to  claim 20 , wherein the phenoloxidase substrate is L-3,4-dihydroxyphenylalanine, dopamine, 3,4-dihydroxyphenyl propionic acid, 3,4-dihydroxyphenyl acetic acid, a dihydroxyphenol, a monophenol, or catechol. 
     
     
         27 . The reader according to  claim 20 , further comprising the step of exposing the sample to an extraction solution at an elevated temperature, prior to incubating the sample with the hemolymph. 
     
     
         28 . The reader according to  claim 27 , wherein the extraction solution is an alkaline extraction solution. 
     
     
         29 . The reader according to  claim 27 , further comprising the step of exposing the sample to a neutralization buffer prior to incubating the sample with the hemolymph from a lepidopteran insect, the phenoloxidase substrate that generates a quinone reaction product, and 3-methyl-2-benzothiazolinone hydrazone or derivative thereof. 
     
     
         30 . The reader according to  claim 29 , wherein the hemolymph from a lepidopteran insect is lyophilized. 
     
     
         31 . The reader according to  claim 27 , wherein the neutralization buffer comprises 3-methyl-2-benzothizolinone hydrazone or other hydrazone derivative thereof. 
     
     
         32 . The reader according to  claim 20 , further comprising the step of exposing the sample to a stop reagent. 
     
     
         33 . The reader according to  claim 32 , wherein the stop reagent is an acid reagent, a phenoloxidase inhibitor, or a strong anionic detergent alone or in combination with a phenoloxidase inhibitor. 
     
     
         34 . A reader for detecting bacteria or bacterial fragments in a sample, the reader comprises:
 (a) a reaction tube for extracting the sample in an extraction solution,   (b) a reaction tube for incubating the sample with hemolymph from a lepidopteran, dopamine, and 3-methyl-2-benzothiazolinone hydrazone or other hydrazone derivative dissolved in neutralization buffer, and   (c) an optical light source for detecting the formation of color, wherein the formation of color indicates the presence of bacteria or bacterial fragments in the sample, and the absence of color indicates the absence of bacteria or bacterial fragments in the sample.   
     
     
         35 . The reader according to  claim 34 , further comprising exposing the sample to a neutralization buffer prior to incubating the sample with the hemolymph, and 3-methyl-2-benzothiazolinone or other hydrazone derivative thereof. 
     
     
         36 . The reader according to  claim 35 , wherein the hemolymph, dopamine, and 3-methyl-2-benzothiazolinone hydrazone or other hydrazone derivative are lyophilized. 
     
     
         37 . The reader according to  claim 20 , wherein the lepidopteran insect is a hornworm. 
     
     
         38 . The reader according to  claim 20 , wherein the sample is prepared by filtration prior to incubating the sample with the hemolymph. 
     
     
         39 . The reader according to  claim 20 , wherein formation of the colored-reaction product indicates the presence of peptidoglycan or β-glucan in the sample and provides a positive diagnosis of urinary tract infections, bacterial meningitis, bacterial infections of the CNS, or bacterial infections. 
     
     
         40 . The reader according to  claim 20 , wherein the absorbance of the colored reaction product indicating the presence of peptidoglycan or β-glucan is monitored continuously over time. 
     
     
         41 . The reader according to  claim 40 , wherein the absorbance of the colored reaction product is monitored continuously over time until a threshold absorbance is reached, indicating the presence of peptidoglycan or β-glucan in the sample. 
     
     
         42 - 43 . (canceled) 
     
     
         44 . The reader according to  claim 20  or  claim 34 , which is a photometer or spectrophotometer.