Methods and systems for the rapid detection of
Disclosed herein are methods and systems for rapid detection of microorganisms such as Listeria spp. 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 Listeria -specific bacteriophage, allows detection of a specific microorganism, such as Listeria spp. and an indicator signal may be amplified to optimize assay sensitivity.
1 . A recombinant bacteriophage (i) comprising a bacteriophage LPJP1 having a phage genome of at least 200 kbp and (ii) comprising a genetic construct comprising an indicator gene, wherein the recombinant bacteriophage specifically infects Listeria grayi.
2 . The recombinant bacteriophage of claim 1 , wherein the recombinant bacteriophage comprises a wild-type bacteriophage LPJP1.
3 . The recombinant bacteriophage of claim 1 , wherein the recombinant bacteriophage comprises a mutant bacteriophage LPJP1.
4 . The recombinant bacteriophage of claim 1 , wherein bacteriophage has one or more mutations in the tail receptor of the bacteriophage genome.
5 . 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 a substrate.
6 . The recombinant bacteriophage of claim 5 , 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.
7 . A cocktail composition comprising at least one recombinant bacteriophage according to claim 1 .
8 . The cocktail composition of claim 7 , wherein at least one type of recombinant bacteriophage comprises a wild-type bacteriophage LPJP1 or a mutant bacteriophage LPJP1.
9 . The cocktail composition of claim 7 , wherein the cocktail further comprises at least one recombinant bacteriophage capable of infecting Listeria innocua.
10 . The cocktail composition of claim 7 , wherein the cocktail further comprises at least one recombinant bacteriophage capable of infecting serovar 3A of Listeria monocytogenes.
11 . The cocktail composition of claim 7 , wherein the cocktail comprises at least one recombinant bacteriophage comprising one or more mutations in the tail receptor of the bacteriophage genome.
12 . A method of preparing a recombinant indicator bacteriophage comprising:
selecting a wild-type bacteriophage LPJP1 having a phage genome of at least 200 kbp that specifically infects Listeria grayi;
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 wild-type bacteriophage LPJP1, thereby allowing homologous recombination to occur between the plasmid/vector and the bacteriophage genome; and
isolating a particular clone of recombinant bacteriophage comprising the indicator gene.
13 . The method of claim 12 , wherein preparing a homologous recombination plasmid/vector comprises:
determining the natural nucleotide sequence in the late region of the genome of the wild-type bacteriophage LPJP1;
annotating the genome and identifying the major capsid protein gene of the wild-type bacteriophage LPJP1;
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.
14 . The method of claim 12 , wherein isolating a particular clone of recombinant bacteriophage comprises a limiting dilution assay for isolating a clone that demonstrates expression of the indicator gene.
15 . A method for detecting Listeria grayi in a sample comprising:
incubating the sample with a cocktail composition comprising at least the recombinant bacteriophage according to claim 1 and
detecting an indicator protein product produced by the recombinant bacteriophage, wherein positive detection of the indicator protein product indicates that Listeria grayi is present in the sample.
16 . The method of claim 15 , wherein at least one type of recombinant bacteriophage comprises a wild-type bacteriophage LPJP1 or a mutant bacteriophage LPJP1.
17 . The method of claim 15 , wherein the cocktail comprises at least one recombinant bacteriophage comprising one or more mutations in the tail receptor of the bacteriophage genome.
18 . The method of claim 15 , wherein the sample is a food, environmental, water, or commercial sample.
19 . The method of claim 15 , 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.
20 . The method of claim 19 , wherein the food sample comprises meat, fish, vegetables, eggs, dairy products, dried food products, or powdered infant formula.
21 . The method of claim 15 , wherein the sample is first incubated in conditions favoring growth for an enrichment period of less than 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or 2 hours.
22 . The method of claim 15 , wherein the total time to results is less than 28 hours, 27 hours, 26 hours, 25 hours, 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or 2 hours.
23 . A kit for detecting Listeria grayi comprising the recombinant bacteriophage of claim 1 .
24 . The kit of claim 23 further comprising a substrate for reacting with a soluble protein product or a soluble enzyme encoded by the indicator gene to detect the soluble protein product or the soluble enzyme.
25 . A system for detecting Listeria grayi comprising the recombinant bacteriophage of claim 1 .