Methods and Systems for the Rapid Detection of Microorganisms Using Recombinant Infectious Agents to Express an Indicator Subunit

Abstract

Disclosed herein are methods and systems for rapid detection of microorganisms such as bacteria in a sample. A genetically modified bacteriophage is also disclosed which comprises an indicator gene encoding one subunit of an indicator protein. The specificity of the bacteriophage allows detection of a particular bacteria of interest and an indicator signal may be amplified to optimize assay sensitivity.

We claim: 
     
         1 . A recombinant indicator bacteriophage comprising an indicator gene inserted into the bacteriophage genome, wherein the indicator gene encodes a peptide or polypeptide subunit of an indicator protein. 
     
     
         2 . The recombinant bacteriophage of  claim 1  further comprising a protease cut site. 
     
     
         3 . The recombinant bacteriophage of  claim 1 , wherein the peptide or polypeptide subunit is a first subunit and is complementary to a second polypeptide subunit of the indicator protein. 
     
     
         4 . The recombinant bacteriophage of  claim 1 , wherein the indicator protein is a luciferase. 
     
     
         5 . The recombinant bacteriophage of  claim 1 , 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. 
     
     
         6 . A cocktail composition comprising at least two different types of recombinant bacteriophages, wherein at least one of the recombinant bacteriophages comprises an indicator gene according to  claim 1 . 
     
     
         7 . 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.   
     
     
         8 . The method of  claim 7  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. 
 
     
     
         9 . The method of  claim 8 , 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. 
     
     
         10 . The method of  claim 9 , 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. 
     
     
         11 . The method of  claim 9 , wherein isolating a particular clone of recombinant bacteriophage comprises a limiting dilution assay for isolating a clone that demonstrates expression of the indicator gene. 
     
     
         12 . A kit for detecting a particular bacteria of interest comprising a recombinant indicator bacteriophage, comprising an indicator gene encoding a first peptide or polypeptide subunit of an indicator protein. 
     
     
         13 . The kit of  claim 12 , further comprising a detection reagent, wherein the detection reagent comprises
 a second polypeptide subunit of an indicator protein, wherein the second polypeptide subunit reconstitutes with the first peptide or polypeptide subunit to form an indicator protein complex, and   a substrate for reacting with an indicator protein complex to detect the indicator protein complex.   
     
     
         14 . The kit of  claim 12 , further comprising a lysis buffer. 
     
     
         15 . A system for detecting a particular bacteria of interest comprising a recombinant indicator bacteriophage, comprising an indicator gene encoding a peptide or polypeptide subunit of an indicator protein.