Methods and Systems for Rapid Detection of Microorganisms Using Infectious Agents

Abstract

Disclosed herein are methods and systems for rapid detection of microorganisms in a sample. A genetically modified bacteriophage is also disclosed which comprises an indicator gene in the late gene region. The specificity of the bacteriophage, such as CBA120, allows detection of a specific microorganism, such as E. coli O157:H7, and an indicator signal may be amplified to optimize assay sensitivity.

We claim: 
     
         1 . A recombinant bacteriophage comprising an indicator gene inserted into a late gene region of the bacteriophage CBA120 genome. 
     
     
         2 . The recombinant bacteriophage of  claim 1 , wherein the recombinant bacteriophage specifically infects  E. coli  O157:H7. 
     
     
         3 . The recombinant bacteriophage of  claim 1 , wherein the indicator gene is codon-optimized and encodes a soluble protein product that generates an intrinsic signal or a soluble enzyme that generates signal upon reaction with substrate. 
     
     
         4 . The recombinant bacteriophage of  claim 3 , further comprising an untranslated region upstream of the codon-optimized indicator gene, wherein the untranslated region includes a bacteriophage late gene promoter and a ribosomal entry site. 
     
     
         5 . A method of preparing a recombinant indicator bacteriophage comprising:
 selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium;   preparing a homologous recombination plasmid/vector comprising an indicator gene;   transforming the homologous recombination plasmid/vector into target pathogenic bacteria;   infecting the transformed target pathogenic bacteria with the selected wild-type bacteriophage, thereby allowing homologous recombination to occur between the plasmid/vector and the bacteriophage genome; and   isolating a particular clone of recombinant bacteriophage.   
     
     
         6 . The method of  claim 5 , wherein preparing a homologous recombination plasmid/vector comprises:
 determining the natural nucleotide sequence in the late region of the genome of the selected bacteriophage;   annotating the genome and identifying the major capsid protein gene of the selected bacteriophage;   designing a sequence for homologous recombination downstream of the major capsid protein gene, wherein the sequence comprises a codon-optimized indicator gene; and   incorporating the sequence designed for homologous recombination into a plasmid/vector.   
     
     
         7 . The method of  claim 6 , wherein designing a sequence further comprises inserting an untranslated region including a phage late gene promoter and ribosomal entry site upstream of the codon-optimized indicator gene. 
     
     
         8 . The method of  claim 5 , wherein the homologous recombination plasmid comprises an untranslated region including a bacteriophage late gene promoter and a ribosomal entry site upstream of the codon-optimized indicator gene. 
     
     
         9 . The method of  claim 5 , wherein the wild-type bacteriophage is CBA120 and the target pathogenic bacterium is  E. coli  O157:H7. 
     
     
         10 . The method of  claim 5 , wherein isolating a particular clone of recombinant bacteriophage comprises a limiting dilution assay for isolating a clone that demonstrates expression of the indicator gene. 
     
     
         11 . A method for detecting  E. coli  O157:H7 in a sample comprising:
 incubating the sample with a recombinant bacteriophage derived from CBA120, and detecting an indicator protein product produced by the recombinant bacteriophage, wherein positive detection of the indicator protein product indicates that  E. coli  O157:H7 is present in the sample.   
     
     
         12 . The method of  claim 11 , wherein the sample is a food, environmental, water, commercial, or clinical sample. 
     
     
         13 . The method of  claim 11 , wherein the method detects as few as 10, 9, 8, 7, 6, 5, 4, 3, 2, or a single bacterium in a sample of a standard size for the food safety industry. 
     
     
         14 . The method of  claim 12 , wherein the sample comprises beef or vegetables. 
     
     
         15 . The method of  claim 11 , wherein the sample is first incubated in conditions favoring growth for an enrichment period of 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, or 2 hours or less. 
     
     
         16 . The method of  claim 11 , wherein the total time to results is less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, or less than 6 hours. 
     
     
         17 . The method of  claim 11 , wherein the ratio of signal to background generated by detecting the indicator is at least 2.0 or at least 2.5. 
     
     
         18 . A kit for detecting  E. coli  O157:H7 comprising a recombinant bacteriophage derived from CBA120. 
     
     
         19 . The kit of  claim 18 , further comprising a substrate for reacting with an indicator to detect the soluble protein product expressed by the recombinant bacteriophage. 
     
     
         20 . A system for detecting  E. coli  O157:H7 comprising a recombinant bacteriophage derived from CBA120.