WO2024254447A1 - Compositions et méthodes pour un diagnostic différentiel d'une infection à salmonella typhimurium chez la volaille - Google Patents

Compositions et méthodes pour un diagnostic différentiel d'une infection à salmonella typhimurium chez la volaille Download PDF

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WO2024254447A1
WO2024254447A1 PCT/US2024/033003 US2024033003W WO2024254447A1 WO 2024254447 A1 WO2024254447 A1 WO 2024254447A1 US 2024033003 W US2024033003 W US 2024033003W WO 2024254447 A1 WO2024254447 A1 WO 2024254447A1
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salmonella typhimurium
seq
primers
salmonella
gene
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Nusrat SHARIAT
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University of Georgia
University of Georgia Research Foundation Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • Salmonella a genus of rod-shaped gram-negative bacteria of the family Enterobacteriaceae, is further divided into two species are Salmonella enterica, comprising six subspecies with over 2600 serotypes, and Salmonella bongori. Salmonella spp. are facultative intracellular pathogens causing localized or systemic infections, in addition to a chronic asymptomatic carrier state. They are of worldwide economic and public health significance as various Salmonella serotypes can infect a variety of animals, poultry and birds, and man. For example, in poultry, fowl typhoid and pullorum disease continue to cause economic losses in those parts of the world where the poultry industries are continuing to intensify and where open sided housing is common.
  • Salmonella enterica serovars Typhimurium S. typhimurium
  • Enteritidis Salmonella enterica serovars Typhimurium and Enteritidis are known to represent the main risk for public health, with cases of gastroenteritis occurring in humans as a result of infection increasing making Salmonella one of the major causes of food-borne illnesses.
  • Salmonella enterica serovars Typhimurium S. typhimurium
  • Enteritidis Salmonella enterica serovars Typhimurium
  • Enteritidis Enteritidis
  • Salmonella infection depends upon several factors including the type of strain associated with the infection and the age and health status of the host, with children, the elderly, and individuals who are immunocompromised most severely affected (Acevedo-Villanueva, KY et al, A Novel Approach against Salmonella: A Review of Polymeric Nanoparticle Vaccines for Broilers and Layers. Vaccines 2021: 1041). Although the infectious dose varies among Salmonella strains and host status, the infective dose for Salmonella can be as low as one cell (Food and Drug Administration. Salmonella species.
  • vaccines against salmonellae have generally fallen into four categories: (i) subunit vaccines, including cell fractions or lysates, intact antigens, fragments thereof, or synthetic analogs of naturally occurring antigens or epitopes; (ii) antiidiotypic antibodies; (iii) killed vaccines which corresponds to the whole killed etiologic agent; or (iv) an avirulent or attenuated derivative of the etiologic agent used as a live vaccine.
  • Poulvac ® ST is a live vaccine composed of a modified-live Salmonella Typhimurium, which is claimed as effective for the vaccination of healthy chickens against Salmonella enteritidis, Salmonella Heidelberg or Salmonella Typhimurium colonization of the internal organs, including the intestines and ceca.
  • Live attenuated Salmonella vaccines have been found to be superior to inactivated preparations owing to: (i) their ability to induce cell-mediated immunity in addition to antibody responses; (ii) effectiveness after single-dose administration; (iii) induction of immune responses at multiple mucosal sites; (iv) low production cost; and (v) their possible use as carriers for the delivery of recombinant antigens to the immune system.
  • the ability of live attenuated Salmonella to colonize the gut-associated lymphoid tissue (GALT; Fever's patches) and the deep tissues following oral administration is the factor that aids stimulation of all arms of the immune response, including mucosal, humoral and cellular immunities.
  • the present invention provides a method to differentiate between wild type (virulent) Salmonella Typhimurium and vaccine associated Salmonella Typhimurium.
  • the method contemplates the use of a PCR assay to distinguish between wild-type and vaccine-associated Salmonella Typhimurium.
  • this invention in one aspect, relates to a diagnostic assay for differentially identifying wild-type virulent Salmonella Typhimurium and vaccine- associated Salmonella Typhimurium in a subject comprising primers for detecting Salmonella Typhimurium target sequences.
  • the subject is a farm animal selected from poultry, pigs, or cattle.
  • the subject is a poultry animal selected from a group consisting of chickens, turkeys, geese, ducks, pheasants, bantam, quail, and pigeons.
  • the subject has been vaccinated against a wild-type virulent Salmonella serotype.
  • the primers detect Salmonella Typhimurium cAMP receptor protein (Crp) gene target sequences within a Salmonella Typhimurium cAMP receptor protein (Crp) gene, the gene having a nucleic acid sequence of SEQ ID NO:1.
  • the primers detect Salmonella Typhimurium adenylate cyclase (CYA) gene target sequences within a Salmonella Typhimurium adenylate cyclase (CYA) gene, the gene having a nucleic acid sequence of SEQ ID NO:2.
  • the Salmonella Typhimurium cAMP receptor protein (Crp) gene target sequence are selected from SEQ ID NO: 3, 4 or 5.
  • the Salmonella Typhimurium adenylate cyclase (CYA) gene target sequence are selected from SEQ ID NO: 6, 7 or 8.
  • the primers for detecting the Salmonella Typhimurium cAMP receptor protein (Crp) gene target sequence are selected from SEQ ID NO: 9, 10, 11, 12, and 13.
  • the primers for detecting the Salmonella Typhimurium adenylate cyclase (CYA) gene target sequence are selected from SEQ ID NO: 14, 15, 16, and 17.
  • the primers detect transposon Tn10 in the Salmonella Typhimurium cAMP receptor protein (Crp) gene and Salmonella Typhimurium adenylate cyclase (CYA) gene target sequences of the vaccine-associated Salmonella serotype.
  • transposon Tn10 in the Crp gene of Salmonella Typhimurium is detected using the forward primer having a nucleotide sequence as set forth in SEQ ID NO.13 and a reverse primer having a nucleotide sequence as set forth in SEQ ID NO.11.
  • the transposon Tn10 in the Crp gene of Salmonella Typhimurium is detected using the forward primer having a nucleotide sequence as set forth in SEQ ID NO.17 and a reverse primer having a nucleotide sequence as set forth in SEQ ID NO.16.
  • the invention relates to a method for differentially diagnosing between wild-type virulent Salmonella Typhimurium and vaccine-associated Salmonella Typhimurium in a subject vaccinated against a Salmonella Typhimurium comprising: (a) extracting sample genomic DNA from the subject; (b) adding samples: adding the sample genomic DNA, a positive control or a negative control into PCR tubes of PCR reaction system respectively to obtain a corresponding sample reaction tube, positive reaction tube or negative reaction tube, wherein the PCR reaction system contains the primers as described above for detecting a Crp or CYA gene; (c) performing PCR reaction: placing the reaction tubes on a PCR instrument, setting circulation parameters, and performing PCR reaction; (d) analyzing results after the PCR reaction is completed; and (e) determining the presence of Tn10 in the sample; wherein the presence of Tn10 indicates the presence vaccine-associated Salmonella Typhimurium.
  • the invention relates to a PCR diagnostic assay kit for differentiating between wild-type virulent Salmonella Typhimurium and vaccine- associated Salmonella Typhimurium in a subject vaccinated against a Salmonella Typhimurium comprising: (a) primers as defor detecting Tn10 in the Crp or CYA genes of Salmonella Typhimurium, and the primers for detecting Tn10 comprising a forward primer having a nucleotide sequence as set forth in SEQ ID NO.13 or 17 and a reverse primer having a nucleotide sequence as set forth in SEQ ID NO.11 or 16; (b) primers for wild type virulent Salmonella Typhimurium, and the primers for detecting wild type virulent Salmonella Typhimurium comprising a forward primer having a nucleotide sequence as set forth in SEQ ID NO.9 or 14 and a reverse primer having a nucleotide sequence as set forth in SEQ ID NO.10 or 15; (c)
  • Figure 1 is a bar graph showing the percentage of Salmonella positive samples collected between 2016 to 2022 by the Unites States Department of Agriculture Food Safety and Inspection Services (USDA-FSIS) from chicken samples.
  • Figures 2A-2B are bar graphs showing the amount of Typhimurium due to the vaccine identified by the Unites States Department of Agriculture Food Safety and Inspection Services (USDA-FSIS) in isolates collected between 2016-2022.
  • Figure 2C is a schematic showing the search of whole genome sequencing data (WGS) for isolates of the live attenuated Salmonella Typhimurium isolates through. a BLAST search for WT and VAC sequences.
  • WT whole genome sequencing data
  • VAC vaccine
  • Figure 3A shows an illustration of the creation of unique DNA sequences to Salmonella Typhimurium CRP wild type (WT) field strains and vaccine (VAC) strains.
  • Figure 3B shows an illustration of the Crp mutant assay.
  • Figure 3C shows a PCR assay comparing the primers to the Salmonella Typhimurium CRP wild type (WT) field strains and the primers to the vaccine (VAC) strains.
  • Figure 4A shows an illustration of the creation of unique DNA sequences to Salmonella Typhimurium CYA wild type (WT) field strains and vaccine (VAC) strains.
  • Figure 4B show a PCR assay comparing the primers to the Salmonella Typhimurium CYA wild type (WT) field strains and the primers to the vaccine (VAC) strains.
  • Figure 5A is a PCR assay showing the screening samples that contain Typhimurium for vaccine strain. Some samples consist of mixed serotypes.
  • Figure 5B is a multiplex PCR assay using primers for crp: combine primers for PCR-WT and PCR-3.
  • MOLECULAR CLONING A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol.304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, et al.
  • oligonucleotide and “polynucleotide” as used interchangeably herein refer to a polymer of greater than one nucleotide in length of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), hybrid RNA/DNA, modified RNA or DNA, or RNA or DNA mimetics.
  • the polynucleotides may be single- or double-stranded.
  • the terms include polynucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as polynucleotides having non- naturally-occurring portions which function similarly.
  • polynucleotide primer refers to a short, single-stranded polynucleotide capable of hybridizing to a complementary sequence in a nucleic acid sample.
  • a primer serves as an initiation point for template-dependent nucleic acid synthesis.
  • Nucleotides are added to a primer by a nucleic acid polymerase in accordance with the sequence of the template nucleic acid strand.
  • a “primer pair” or “primer set” refers to a set of primers including a 5′ upstream primer that hybridizes with the 5′ end of the sequence to be amplified and a 3′ downstream primer that hybridizes with the complementary 3′ end of the sequence to be amplified.
  • the term “forward primer” as used herein, refers to a primer which anneals to the 5′ end of the sequence to be amplified.
  • probe and “polynucleotide probe,” as used herein, refer to a polynucleotide used for detecting the presence of a specific nucleotide sequence in a sample. Probes specifically hybridize to a target nucleotide sequence, or the complementary sequence thereof, and may be single- or double-stranded.
  • annealing and “hybridization” are used interchangeably and mean the base-pairing interaction of one nucleic acid with another nucleic acid that results in formation of a duplex or other higher-ordered structure. The primary interaction is base specific, i.e. A/T and G/C, by Watson/Crick and Hoogsteen-type hydrogen bonding.
  • hybridization conditions or “stringent hybridization conditions” refers to hybridization conditions which can take place under a number of pH, salt and temperature conditions.
  • the pH can vary from 6 to 9, preferably 6.8 to 8.5.
  • the salt concentration can vary from 0.15 M sodium to 0.9 M sodium, and other cations can be used as long as the ionic strength is equivalent to that specified for sodium.
  • the temperature of the hybridization reaction can vary from 30° C. to 80° C., preferably from 45° C. to 70° C.
  • other compounds can be added to a hybridization reaction to promote specific hybridization at lower temperatures, such as at or approaching room temperature. Among the compounds contemplated for lowering the temperature requirements is formamide.
  • a polynucleotide is typically “substantially complementary” to a second polynucleotide if hybridization occurs between the polynucleotide and the second polynucleotide.
  • hybridization or “specific hybridization” refers to hybridization between two polynucleotides under stringent hybridization conditions.
  • the term “specifically hybridize,” as used herein, refers to the ability of a polynucleotide to bind detectably and specifically to a target nucleotide sequence.
  • Polynucleotides, oligonucleotides and fragments thereof specifically hybridize to target nucleotide sequences under hybridization and wash conditions that minimize appreciable amounts of detectable binding to non-specific nucleic acids.
  • High stringency conditions can be used to achieve specific hybridization conditions as is known in the art.
  • hybridization and washing are performed at high stringency according to conventional hybridization procedures and employing one or more washing step in a solution comprising 1-3 ⁇ SSC, 0.1-1% SDS at 50-70° C. for 5-30 minutes.
  • hybridizes under stringent conditions describes conditions for hybridization and washing under which nucleotide sequences having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more base pair matches to each other typically remain hybridized to each other.
  • corresponding to refers to a polynucleotide sequence that is identical to all or a portion of a reference polynucleotide sequence.
  • target sequence or “target nucleotide sequence,” as used herein, refer to a particular nucleic acid sequence in a test sample to which a primer and/or probe is intended to specifically hybridize.
  • a “target sequence” is typically longer than the primer or probe sequence and thus can contain multiple “primer target sequences” and “probe target sequences.”
  • a target sequence may be single or double stranded.
  • primer target sequence refers to a nucleic acid sequence in a test sample to which a primer is intended to specifically hybridize.
  • probe target sequence refers to a nucleic acid sequence in a test sample to which a probe is intended to specifically hybridize.
  • an “amplified target polynucleotide sequence product” or “amplified product” refers to the resulting amplicon from an amplification reaction such as a polymerase chain reaction.
  • the resulting amplicon product arises from hybridization of complementary primers to a target polynucleotide sequence under suitable hybridization conditions and the repeating in a cyclic manner the polymerase chain reaction as catalyzed by DNA polymerase for DNA amplification or RNA polymerase for RNA amplification.
  • the “polymerase chain reaction” or PCR is a an amplification of nucleic acid consisting of an initial denaturation step which separates the strands of a double stranded nucleic acid sample, followed by repetition of (i) an annealing step, which allows amplification primers to anneal specifically to positions flanking a target sequence; (ii) an extension step which extends the primers in a 5′ to 3′ direction thereby forming an amplicon polynucleotide complementary to the target sequence, and (iii) a denaturation step which causes the separation of the amplicon from the target sequence (Mullis et al., eds, The Polymerase Chain Reaction, BirkHauser, Boston, Mass.
  • RNA samples can be converted to DNA/RNA heteroduplexes or to duplex cDNA by methods known to one of skill in the art.
  • amplifying and amplification refers to a broad range of techniques for increasing polynucleotide sequences, either linearly or exponentially. Exemplary amplification techniques include, but are not limited to, PCR or any other method employing a primer extension step.
  • amplification examples include, but are not limited to, ligase detection reaction (LDR) and ligase chain reaction (LCR). Amplification methods may comprise thermal-cycling or may be performed isothermally. In various embodiments, the term “amplification product” or “amplified product” includes products from any number of cycles of amplification reactions.
  • LDR ligase detection reaction
  • LCR ligase chain reaction
  • amplification methods comprise at least one cycle of amplification, for example, but not limited to, the sequential procedures of: hybridizing primers to primer-specific portions of target sequence or amplification products from any number of cycles of an amplification reaction; synthesizing a strand of nucleotides in a template-dependent manner using a polymerase; and denaturing the newly-formed nucleic acid duplex to separate the strands.
  • the cycle may or may not be repeated.
  • the term “real-time analysis” refers to periodic monitoring during PCR. Certain systems such as the Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, Calif.) conduct monitoring during each thermal cycle at a pre- determined or user-defined point. Real-time analysis of PCR with FRET probes measures fluorescent dye signal changes from cycle-to-cycle, preferably minus any internal control signals.
  • the term “sample” is a portion of a larger source.
  • a sample is optionally a solid, gaseous, or fluidic.
  • a sample is illustratively an environmental or biological sample.
  • An environmental sample is illustratively, but not limited to water, sewage, soil, or air.
  • a “biological sample” is as sample obtained from a biological organism, a tissue, cell, cell culture medium, or any medium suitable for mimicking biological conditions.
  • Non-limiting examples include, saliva, gingival secretions, cerebrospinal fluid, gastrointestinal fluid, mucous, urogenital secretions, synovial fluid, blood, serum, plasma, urine, feces, cystic fluid, lymph fluid, ascites, pleural effusion, interstitial fluid, intracellular fluid, ocular fluids, seminal fluid, mammary secretions, and vitreal fluid, and nasal secretions, throat or nasal materials, eggs, or other food. Methods of obtaining a sample are known in the art.
  • a sample is whole blood, plasma, or serum that is obtained by venipuncture and optionally processed to obtain the final sample.
  • the term “medium” refers to any liquid or fluid that may or may not contain one or more bacteria.
  • a medium is illustratively a solid sample that has been suspended, solubilized, or otherwise combined with fluid to form a fluidic sample.
  • Non-limiting examples include buffered saline solution, cell culture medium, acetonitrile, trifluoroacetic acid, combinations thereof, or any other fluid recognized in the art as suitable for combination with bacteria or other cells, or for dilution of a biological sample or amplification product for analysis.
  • the term "attenuated” refers to a microorganisms and/or bacteria strains and/or viruses that have been genetically modified so as to not cause illness in an animal model.
  • the terms “attenuated” and “avirulent” are used interchangeably herein.
  • immunizing amount as used herein is in fact meant an amount that is able to induce an immune response in the animal that receives the pharmaceutical composition/vaccine.
  • the immune response invoked may be a humoral, mucosal, local and/or a cellular immune response.
  • subject refers to avian species including but not limited to chickens, turkeys, geese, ducks, pheasants, bantam, quail, pigeons; however, it is contemplated that a method according to the present invention may also be used as a diagnostic tool in other subjects susceptible to Salmonella Typhimurium infection, such as but not limited to reptiles (turtles, lizards, and snakes); amphibians (frogs and toads); rodents (mice, rats, hamsters, and guinea pigs) and other small mammals (hedgehogs); livestock and farm animals (goats, calves, cows, horses sheep, and pigs); and humans.
  • reptiles turtles, lizards, and snakes
  • amphibians frogs and toads
  • rodents mice, rats, hamsters, and guinea pigs
  • other small mammals hedgehogs
  • livestock and farm animals goats
  • multivalent may be bivalent, trivalent, quadravalent, and the like and refers to a vaccine which has other antigenic components from related and/or unrelated microorganism and/or viruses, or their components and fragments along with the attenuated Salmonella strain.
  • the "multivalent” vaccine could be a combination vaccine having an attenuated Salmonella strain in combination with other antigenic components.
  • II. Compositions A. Salmonella Contamination in Poultry [0065] Salmonella is a genus of the family Enterobacteriaceae, characterized as Gram-negative, facultatively anaerobic, non spore-forming, rod-shaped bacteria, and most strains are motile by flagella.
  • Salmonella has an average genome GC content of 50-52%, which is similar to those of Escherichia coli and Shigella.
  • the genus Salmonella is a pathogenic microorganism that causes infections in livestock as well as in human.
  • Salmonella enterica a species of Salmonella bacterium, has a variety of serovars including Gallinarum, Pullorum, Typhimurium, Enteritidis, Typhi, Choleraesuis, and derby (Bopp C A, Brenner F W, Wells J G, Strokebine N A, Exherichia, Shigella, Salmonella.
  • Murry P R Baron E J, et al eds Manual of Clinical Microbiology.7th ed. Washington D.C.
  • Salmonella Gallinarum and Pullorum are fowl- adapted pathogens
  • Salmonella Typhi is a human-adapted pathogen
  • Salmonella Choleraesuis and Salmonella derby are swine-adapted pathogens
  • Salmonella Enteritis and Salmonella Typhimurium are pathogenic for human and animals.
  • Each serovar causes illness in the respective species, resulting in tremendous damage to farmers or consumers.
  • the present invention provides compositions and methods for the quick and reliable detection of Salmonella, particularly compositions and methods for detecting Salmonella Typhimurium in poultry.
  • Salmonella bacteria including Salmonella Typhimurium (S. Typhimurium).
  • Salmonella Typhimurium S. Typhimurium
  • Newly hatched birds are particularly susceptible to Salmonella infection, and a high mortality rate as a result thereof immediately post-hatching can have serious economic consequences.
  • Live attenuated Salmonella vaccines have been shown to protect chickens (Cooper, et al., Microb. Pathog., 9:255-265, 1990; Hassan and Curtis III, Res. Microbial., 141:839-950, 1990).
  • the avirulent S Importantly, the avirulent S.
  • Typhimurium aroA deletion mutant microorganism having the identifying characteristics of S. Typhimurium STM-1 has been found to be particularly effective in protecting poultry against Salmonella infection and disease.
  • the S. Typhimurium STM-1 microorganism and the construction thereof is described in U.S. Pat. No.6,231,871, which is incorporated herein by reference.
  • the invention further contemplates other live, attenuated Salmonella gene mutants, including deletion mutants, in particular aroA deletion mutants, as immunogens as part of a combination vaccine herein described. [0067] Once an outbreak of salmonellosis by S.
  • Typhimurium occurs in poultry, pigs, and cattle, it is difficult to cure by therapeutic agents, including not only because Salmonella are intracellular and thus antibiotics are less effective at entering but also because of the increase observed in antibiotic resistance.
  • S. Typhimurium can cause infections in human via consumption of contaminated, improperly cooked livestock products (e.g., meat products, poultry products, eggs and by-products). Salmonella illness in human usually involves the prompt onset of headache, fever, abdominal pain, diarrhea, nausea, and vomiting. The symptoms commonly appear within 6-72 hours after the ingestion of the organism, and may persist for as long as 4-7 days or even longer (NSW+HEALTH. 2008.01.14.).
  • the Centers for Disease Control and Prevention, USA estimates Salmonella bacteria cause about 1.35 million infections, 26,500 hospitalizations, and 420 deaths in the United States every year. Food is the source for most of these illnesses (Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases (NCEZID), Division of Foodborne, Waterborne, and Environmental Diseases (DFWED), (2023)).
  • NCEZID National Center for Emerging and Zoonotic Infectious Diseases
  • DFWED Division of Foodborne, Waterborne, and Environmental Diseases
  • Salmonella foodborne illness in human between 1973 and 1984 the implicated food vehicles of transmission were reportedly chicken (5%), beef (19%), pork (7%), dairy products (6%), and turkey (9%).
  • 1974-1984 the bacterial contamination test on broilers during the slaughter process showed 35% or more of Salmonella incidence.
  • Salmonella was isolated in 50.6% of chicken, 68.8% of turkey, 60% of goose, 11.6% of pork, and 1.5% of beef.
  • a survey carried out in 2007 reported that Salmonella was found in 5.5% of raw poultry meat and 1.1% of raw pork.
  • Salmonella Enteritis commonly originated from contaminated egg or poultry meat, and Salmonella Typhimurium from contaminated pork, poultry meat, and beef (Center for Disease Control).
  • Live attenuated Salmonella vaccines are potentially superior to inactivated preparations owing to: (i) their ability to induce cell-mediated immunity in addition to antibody responses; (ii) oral delivery with no risk of needle contamination; (iii) effectiveness after single-dose administration; (iv) induction of immune responses at multiple mucosal sites; (v) low production cost; and (vi) their possible use as carriers for the delivery of recombinant antigens to the immune system. [0071] Few live attenuated Salmonella vaccines are actually on the market because results with attenuated mutant strains have not always been good.
  • Vaccination is an important component in a preharvest program; however, it is well known that a live attenuated Salmonella Typhimirium vaccine persists in vaccinates. Therefore, there is a strong possibility that a significant portion of the chickens identified as contaminated with Salmonella Typhimirium may actually be harboring the vaccine strain rather than the wild type strain. A quick and reliable method useful to differentially diagnose those birds infected with wild type Salmonella from those harboring a vaccine strain, and thus not of concern in a control program, would be a major improvement over current practices. [0075] Thus, the present invention provides compositions and methods for the differentiation of live S. Typhimirium vaccine isolates from wild type or field isolates of S. Typhimirium in poultry. D.
  • Salmonella target sequence refers to a DNA or RNA sequence comprising the nucleic acid sequence of a bacterium of the genus Salmonella. It includes but is not limited to, S. Typhimurium.
  • Exemplary Salmonella nucleic acid sequences that may be targeted for amplification according to the present invention are taught by the following publications: Richards AK, et al., Lett Appl Microbiol.71(2):138-145 (2020); Liu W Q et al., PLoS One,4(2):e4510 (2009); Thomson N R et al., Genome Res, 18(10):1624-37 (2008); Encheva V et al., BMC Microbiol, 18 ;5:42 (2005); McClelland M et al., Nat Genet,36(12):1268-74 (2004); Chiu C H et al., Clin Microbiol Rev,17(2):311-22(2004); Deng W et al., J Bacteriol,185(7):2330-7 (2003); Parkhill J et al., Nature,413(6858):848- 52 (2001); McClelland M et al., Nature, 413(6858):85
  • Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 An exemplary nucleotide sequence of the complete 4,857,432 bp genome of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 is available under Genbank Accession No. NC — 003197 and incorporated herein by reference. 1. Salmonella Typhimurium Cya and Crp genes [0079] Provided herein is a PCR detection method using primers for the cAMP receptor protein (Crp) and adenylate cyclase (CYA) genes. [0080] The nucleic acid sequence for mutant Salmonella enterica subsp.
  • Crp cAMP receptor protein
  • CYA adenylate cyclase
  • enterica serovar Typhimurium strain ST04-MSC-p1-Delta-CRP is: atggtgcttg gcaaaccgca aacagacccg actcttgaat ggttcttgtc tcattgccac attcataagt acccgtcaaa gagcacgctg attcaccagg gtgaaaagc agaaacgctg tactacatcg ttaaaggctc cgtggcagtg ctgatcaaag atgaagaagg gaaagaaatg atcctttctt atctgaatca gggtgatttt attggtgaac tgggcttttgaagaaggc caggaacgca gcgccctgat gaatcccccta atgatttgg tgg taaaatcat
  • the nucleic acid sequence for mutant Salmonella enterica subsp. enterica serovar Typhimurium strain ST04-MSC-p1-Delta-CYA::Tn10 transposon Tn10 IS4 family transposase ISVsa5 gene is: ttgtacctct atattgagac tctgaaacag agactggatg ccataaatca actgcgtgtg gatcgcgcgc ttgctgccat gggacccgct ttacagtct tctgccgaca ttattgcact atcaccatcc actgatgccg ggttaccttg atggtaacgt tcccagcggt attgcttct acacgctgaca ttattgcact atcaccatcc actgatgccg ggttaccttg atggta
  • Cyclic-3'5'-AMP (cAMP) and the cyclic AMP receptor protein are necessary for the transcription of a large number of genes and operons concerned with the transport and breakdown of a large number of catabolites.
  • cAMP Cyclic-3'5'-AMP
  • Evidence has been provided that shows that systems used for transporting fuel/carbon sources are all under positive control by cAMP, as are several amino acid permeases.
  • the cAMP concentration in cells also influences lysogenization by temperate phages, synthesis of fimbriae, synthesis of flagella and synthesis of at least one outer membrane protein.
  • cAMP is present in mammalian cells
  • the concentrations present in macrophages and other cells in which Salmonella can invade and multiply are below the concentration of 0.1 to 1.0 mM cAMP necessary to allow ⁇ cya mutants to exhibit a wild-type phenotype in vitro.
  • the inclusion of the ⁇ crp mutation would essentially abolish any benefit that could accrue from uptake of cAMP in vitro or in vivo by such ⁇ cya mutants.
  • Introduction of the mutations into cya and crp of S. Typhimurium can be accomplished by use of transposons, to transfer the mutations from other Salmonella strains into S. Typhimurium. Transposons can be added to a bacterial chromosome at many points.
  • transposon insertion and deletion have been reviewed in Kleckner et al. (1977), J. Mol. Biol.116:125.
  • the transposon Tn10 which confers resistance to tetracycline (and sensitivity to fusaric acid) can be used to create ⁇ cya and ⁇ crp mutations in a variety of bacterial species, including, for example, E. coli and S. Typhimurium. Methods for the creation and detection of these mutants in S. Typhimurium are described in EPO Pub. No.315,682 and incorporated herein by reference. Utilizing Tn10, these mutations can be transposed into various isolates of S. Typhimurium, preferably those which are highly pathogenic.
  • the microbes can serve as the immunogenic component of a vaccine to induce immunity against the microbe.
  • Cya and Crp primers [0085] Oligonucleotides of the instant invention are set forth in SEQ ID NOs: 3-14. CRP and CYA regions as shown in Table 1 were chosen to search for the vaccine strain from whole genome sequencing (WGS) sequences. They were designed to be short ⁇ 120- 130 nucleotides, so they can be captured from short read sequences..
  • Table 1 Sequences used to search for the vaccine strain from WGS sequences
  • Oligonucleotides of the instant invention may be used as primers for PCR amplification. Preferred primer pairs and their corresponding targets, blocking oligonucleotides, and probes are shown in Table 2.
  • Table 2 PCR primers
  • oligonucleotide primers may also be useful for other nucleic acid amplification methods such as the ligase chain reaction (LCR) (EP 0320308; Carrino et al., J. Microbiol.
  • LCR ligase chain reaction
  • oligonucleotides of the present invention also may be used as hybridization probes. Hybridization using DNA probes has been frequently used for the detection of pathogens in food, clinical and environmental samples, and the methodologies are generally known to one skilled in the art. It is generally recognized that the degree of sensitivity and specificity of probe hybridization is lower than that achieved through the previously described amplification techniques.
  • the nucleic acid probes of the present invention can also possess a detectable label, such as a reporter- quencher combination as are employed in Scorpion probe assays or in 5′-exonuclease detection assays, such as the Taqman® assay.
  • a detectable label such as a reporter- quencher combination as are employed in Scorpion probe assays or in 5′-exonuclease detection assays, such as the Taqman® assay.
  • the 3′ terminal nucleotide of the nucleic acid probe may be rendered incapable of extension by a nucleic acid polymerase in one embodiment of the invention. Such blocking may be carried out, for example by the attachment of a replication inhibitor moiety, such as a reporter or quencher, to the terminal 3′ carbon of the nucleic acid probe by a linking moiety, or by making the 3′-terminal nucleotide a dideoxynucleotide.
  • the 3′ end of the nucleic acid probe may be rendered impervious to the 3′ to 5′ extension activity of a polymerase by incorporating one or more modified internucleotide linkages onto the 3′ end of the oligonucleotide.
  • the 3′ terminal internucleotide linkage must be modified, however, additional internucleotide linkages may be modified.
  • Internucleotide modifications which prevent elongation from the 3′ end of the nucleic acid probe and/or which block the 3′ to 5′ exonuclease activity of the DNA polymerase during PCR may include phosphorothioate linkages, methylphosphonate linkages, boranophosphate linkages, and other similar polymerase- resistant internucleotide linkages.
  • An alternative method to block 3′ extension of the probe is to form an adduct at the 3′ end of the probe using mitomycin C or other like antitumor antibiotics such as described in Basu et al., Biochemistry 32:4708-18 (1993).
  • oligonucleotides of the present invention contain both primer and probe regions, and thus can be employed as a primer-probe complex in an appropriate assay, such as a Scorpion probe assay.
  • primer probe complexes of the instant invention contain a non-amplifiable linker that connects the 3′ terminus of the probe region to the 5′ terminus of the primer region. This non-amplifiable linker stops extension of a complementary strand from proceeding into the probe region of the primer-probe complex.
  • Primer-probe complexes of the present invention can also contain a self-complementary region that allows the primer-probe complex to form a stem-loop structure when the probe is unbound from its target DNA, which may be useful, for example, in bringing the reporter and quencher into sufficiently close proximity to one another to cause the reporter signal to be quenched.
  • Methods of Detecting [0093] Today, most food pathogen testing conducted entails a culture step to enrich the potentially low levels of microorganisms contained in a sample. Following culture of the sample, a portion is removed and tested for the presence of pathogens.
  • Pathogen testing after culture can be done by immunoassays (e.g., bioMerieux's Vidas® automated ELISA platform or SDIX's RapidChek® lateral flow assays) or by PCR-based tests (e.g., DuPont Qualicon's BAX® system, Bio-Rad's iQ-CheckTM system). If a pathogen is present in the starting sample, the culture step can increase the concentration of the pathogen as high as 1.0E8-1.0E9 cfu/mL, so that opening the sample after culture exposes both the user and the environment to a risk of contamination. This exposure inhibits many food producers from conducting pathogen testing on-site, instead choosing to send samples to external laboratories for testing.
  • immunoassays e.g., bioMerieux's Vidas® automated ELISA platform or SDIX's RapidChek® lateral flow assays
  • PCR-based tests e.g., DuPont Qualicon's BAX® system,
  • compositions and methods that uses a S. Typhimurium detection assay developed based on identification of the Salmonella Typhimurium primers. In some embodiments, the assay incorporates primers for detection and differentiation of wild type and vaccine strains of S. Typhimurium.
  • the present disclosure therefore relates to detection and identification of WT and vaccine S. Typhimurium through the use of uses primers for two genes: CRP (cAMP receptor protein) and CYA (adenylate cyclase). These two genes are disrupted by transposons (Tn10) in the vaccine strain.
  • CRP cAMP receptor protein
  • CYA adenylate cyclase
  • Tn10 transposons
  • the present detection method finds utility in detection of Tn10 in isolated samples to differentiate between contamination with WT field strain of S. Typhimurium or the vaccine stain interrupted by the transposon.
  • the present detection method finds utility in detection of S. Typhimurium in any type of sample, for example in appropriate samples for food testing, environmental testing, or human or animal diagnostic testing. While examples of suitable methods for detecting these regions are included herein, it is to be understood that the invention is not limited to the methods described.
  • any suitable method can be employed to detect these DNA regions and subsequently S. Typhimurium in a sample.
  • B. Determining Percent Identity of Nucleic Acid Sequences Provided herein are methods of analyzing nucleic acid sequences to determine percent identity.
  • Salmonella Typhimurium short-read sequences from chicken samples uploaded to NCBI Pathogen Detection by FSIS from 2016-2022 were downloaded and assembled. These were analyzed using BLAST with a sequence unique to field strains, followed by a sequence unique to the vaccine strain.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence).
  • the nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the determination of percent identity between two sequences can also be accomplished using a mathematical algorithm.
  • a non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, PNAS 87:22642268, modified as in Karlin and Altschul, 1993, PNAS. 90:58735877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol.215:403.
  • Gapped BLAST are utilized as described in Altschul et al., 1997, Nucleic Acids Res.25:33893402.
  • PSI BLAST is used to perform an iterated search which detects distant relationships between molecules (Id.).
  • the default parameters of the respective programs e.g., of XBLAST and NBLAST
  • the default parameters of the respective programs are used (see, e.g., the NCBI website).
  • a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:1117. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 is used. [0099] The percent identity between two sequences is determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted. C. Assay Methods [00100] Detection of the presence of S. Typhimurium, may be accomplished in any suitable manner.
  • Preferred methods are primer-directed amplification methods and nucleic acid hybridization methods. These methods may be used to detect S. Typhimurium in a sample that is either a complex matrix or a purified culture, e.g., from an animal, environmental, or food source suspected of contamination.
  • primer-directed amplification methods include thermal cycling methods (e.g., PCR, qPCR and LCR), as well as isothermal methods and strand displacement amplification (SDA).
  • SDA strand displacement amplification
  • PCR is an extremely powerful technique for amplifying specific polynucleotide sequences, including genomic DNA, single-stranded cDNA, and mRNA among others.
  • Various methods of conducting PCR amplification and primer design and construction for PCR amplification will be known to those of skill in the art.
  • New DNA synthesis is then primed by hybridizing primers to the target sequence in the presence of DNA polymerase and excess dNTPs.
  • the primers hybridize to the newly synthesized DNA to produce discreet products with the primer sequences at either end. The products accumulate exponentially with each successive round of amplification.
  • the DNA polymerase used in PCR is a thermostable polymerase. This allows the enzyme to continue functioning after repeated cycles of heating necessary to denature the double-stranded DNA.
  • Polymerases that are useful for PCR include, for example, Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Tma DNA polymerase, Tli DNA polymerase, and Pfu DNA polymerase.
  • AmpliTaq® and AmpliTaq Gold® both available from Applied Biosystems. Many are available with or without a 3- to 5′ proofreading exonuclease activity. See for example, Vent® and Vent®. (exo-) available from New England Biolabs.
  • LCR ligase chain reaction
  • NBSA nucleic acid based sequence amplification
  • the latter two amplification methods include isothermal reactions based on isothermal transcription, which produce both single-stranded RNA (ssRNA) and double-stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • the oligonucleotides and methods according to the instant invention may be used directly with any suitable clinical or environmental samples, without any need for sample preparation. In order to achieve higher sensitivity, and in situations where time is not a limiting factor, it is preferred that the samples be pre-treated and that pre- amplification enrichment is performed.
  • the minimum industry standard for the detection of food-borne bacterial pathogens is a method that will reliably detect the presence of one pathogen cell in 25 g of food matrix as described in Andrews et al., 1984, “Food Sample and Preparation of Sample Homogenate”, Chapter 1 in Bacteriological Analytical Manual, 8th Edition, Revision A, U.S. Food and Drug Administration.
  • enrichment methods and media have been developed to enhance the growth of the target pathogen cell in order to facilitate its detection by biochemical, immunological or nucleic acid hybridization means.
  • Typical enrichment procedures employ media that will enhance the growth and health of the target bacteria and also inhibit the growth of any background or non-target microorganisms present.
  • lysis solution is heated at 37° C. for 20 min followed by protease inactivation at 95° C. for 10 min, and cooled to 4° C. as described in the BAX® System User's Guide, DuPont Nutrition and Health, Wilmington, Del.
  • selective enrichment broth culture is streaked on to a selective a selective, differential agar (e.g. XLT-4, BGS).
  • a selective a selective, differential agar e.g. XLT-4, BGS.
  • Typhimurium in a sample comprises (a) performing PCR amplification using primer pairs listed in Table 2 to produce a PCR amplification result; and (b) detecting the amplification, whereby a positive detection of the amplification indicates the presence of the vaccine strain of S. Typhimurium in the sample.
  • a step of preparing the sample may be carried out prior to performing PCR amplification.
  • the preparing step may comprise at least one of the following processes: (1) bacterial enrichment, (2) separation of bacterial cells from the sample, (3) cell lysis, and (4) total DNA extraction.
  • Amplification Conditions [00111] A skilled person will understand that any generally acceptable PCR conditions may be used for successfully detecting S.
  • Detection/Examination/Analysis [00112] Primer-directed amplification products can be analyzed using various methods. Homogenous detection refers to a preferred method for the detection of amplification products where no separation (such as by gel electrophoresis) of amplification products from template or primers is necessary.
  • Homogeneous detection is typically accomplished by measuring the level of fluorescence of the reaction mixture during or immediately following amplification.
  • heterogeneous detection methods which involve separation of amplification products during or prior to detection, can be employed in the present invention.
  • Homogenous detection may be employed to carry out “real-time” primer- directed nucleic acid amplification and detection, using primer pairs of the instant invention (e.g., “real-time” PCR and “real-time” RT-PCR). Preferred “real-time” methods are set forth in U.S. Pat.
  • One “real-time” detection method is the Scorpion probe assay as set forth in U.S. Pat. No.6,326,145, which is hereby incorporated by reference in its entirety.
  • PCR amplification is performed using a Scorpion probe (either unimolecular or bimolecular) as a primer-probe complex, the Scorpion probe possessing an appropriate reporter-quencher pair to allow the detectable signal of the reporter to be quenched prior to elongation of the primer.
  • each probe can have a different detectable label (e.g., reporter-quencher pair) attached, thus allowing each probe to be detected independently of the other probes.
  • detectable label e.g., reporter-quencher pair
  • Another preferred “real-time” detection method is the 5′-exonuclease detection method, as set forth in U.S. Pat.
  • a modified probe is employed during PCR which binds intermediate to or between the two members of the amplification primer pair.
  • the modified probe possesses a reporter and a quencher and is designed to generate a detectable signal to indicate that it has hybridized with the target nucleic acid sequence during PCR. As long as both the reporter and the quencher are on the probe, the quencher stops the reporter from emitting a detectable signal.
  • the efficiency of quenching is a strong function of the proximity of the reporter and the quencher, i.e., as the two molecules get closer, the quenching efficiency increases.
  • the reporter and the quencher are preferably attached to the probe within a few nucleotides of one another, usually within 30 nucleotides of one another, more preferably with a separation of from about 6 to 16 nucleotides. Typically, this separation is achieved by attaching one member of a reporter- quencher pair to the 5′ end of the probe and the other member to a nucleotide about 6 to 16 nucleotides away.
  • each probe can have a different detectable label (e.g., reporter-quencher pair) attached, thus allowing each probe to be detected independently of the other probes.
  • detectable label e.g., reporter-quencher pair
  • Another preferred method of homogenous detection involves the use of DNA melting curve analysis, particularly with the BAX® System hardware and reagent tablets from DuPont Nutrition and Health. The details of the system are given in U.S. Pat. No. 6,312,930 and PCT Publication Nos. WO 97/11197 and WO 00/66777, each of which is hereby incorporated by reference in its entirety.
  • dsDNA double stranded nucleic acid molecule
  • target amplicon target amplification product
  • a typical PCR cycle involves a denaturing phase where the target dsDNA is melted, a primer annealing phase where the temperature optimal for the primers to bind to the now-single-stranded target, and a chain elongation phase (at a temperature Te) where the temperature is optimal for DNA polymerase to function.
  • Tms should be higher than Te, and Tme should be lower (often substantially lower) than the temperature at which the DNA polymerase is heat-inactivated. Melting characteristics are affected by the intrinsic properties of a given dsDNA molecule, such as deoxynucleotide composition and the length of the dsDNA.
  • Intercalating dyes will bind to double stranded DNA.
  • the dye/dsDNA complex will fluoresce when exposed to the appropriate excitation wavelength of light, which is dye dependent, and the intensity of the fluorescence may be proportionate to concentration of the dsDNA.
  • Methods taking advantage of the use of DNA intercalating dyes to detect and quantify dsDNA are known in the art. Many dyes are known and used in the art for these purposes. The instant methods also take advantage of such relationship.
  • intercalating dyes include, but are not limited to, SYBR Green-I®, ethidium bromide, propidium iodide, TOTO®-1 ⁇ Quinolinium, 1-1′-[1,3- propanediylbis [(dimethyliminio)-3,1-propanediyl]]bis[4-[(3-methyl-2(3H)- benzothiazolylidene) methyl]]-, tetraiodide ⁇ , and YoPro® ⁇ Quinolinium, 4-[(3-methyl- 2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)-propyl]-, diiodide ⁇ .
  • a non-asymmetrical cyanide dye such as SYBR Green-I®, manufactured by Molecular Probes, Inc. (Eugene, Oreg.).
  • Melting curve analysis is achieved by monitoring the change in fluorescence while the temperature is increased. When the temperature reaches the T MS specific for the target amplicon, the dsDNA begins to denature. When the dsDNA denatures, the intercalating dye dissociates from the DNA and fluorescence decreases. Mathematical analysis of the negative of the change of the log of fluorescence divided by the change in temperature plotted against the temperature results in the graphical peak known as a melting curve.
  • the present invention could be operated using a combination of these techniques, such as by having a Scorpion probe directed to one target region and a Taqman® probe directed to a second target region. It should also be understood that the invention is not limited to the above described techniques. Rather, one skilled in the art would recognize that other techniques for detecting amplification as known in the art may also be used. For example, techniques such as PCR-based quantitative sequence detection (QSD) may be performed using nucleic acid probes which, when present in the single-stranded state in solution, are configured such that the reporter and quencher are sufficiently close to substantially quench the reporter's emission.
  • QSD quantitative sequence detection
  • the reporter and quenchers become sufficiently distant from each other. As a result, the quenching is substantially abated causing an increase in the fluorescence emission detected.
  • a variety of other heterogeneous detection methods are known in the art which can be employed in the present invention, including standard non-denaturing gel electrophoresis (e.g., acrylamide or agarose), denaturing gradient gel electrophoresis, and temperature gradient gel electrophoresis.
  • standard non-denaturing gel electrophoresis is a simple and quick method of PCR detection, but may not be suitable for all applications.
  • DGGE Denaturing Gradient Gel Electrophoresis
  • the principle of the separation is based on both fragment length and nucleotide sequence. In fragments that are the same length, a difference as little as one base pair can be detected. This is in contrast to non-denaturing gel electrophoresis, where DNA fragments are separated only by size. This limitation of non-denaturing gel electrophoresis results because the difference in charge density between DNA molecules is near neutral and plays little role in their separation. As the size of the DNA fragment increases, its velocity through the gel decreases. e.
  • replication composition any suitable nucleic acid replication composition (“replication composition”) in any format can be used.
  • a typical replication composition for PCR amplification may comprise, for example, dATP, dCTP, dGTP, dTTP, target specific primers and a suitable polymerase.
  • suitable buffers known in the art may be used (Sambrook, J. et al., supra).
  • typical tabletization reagents may be included such as stabilizers and binding agents. Preferred tabletization technology is set forth in U.S. Pat.
  • a preferred replication composition of the instant invention comprises (a) the primer pair from Table 2 and (b) thermostable DNA polymerase.
  • a more preferred replication composition of the present invention comprises (a) the primer pairs and any corresponding probe or blocking oligonucleotide selected from Table 2, wherein each nucleic acid probe or primer-probe complex employed comprises a detectable label; and (b) thermostable DNA polymerase.
  • the detectable label comprises a reporter capable of emitting a detectable signal and a quencher capable of substantially quenching the reporter and preventing the emission of the detectable signal when the reporter and quencher are in sufficiently close proximity to one another.
  • a preferred kit of the instant invention comprises any one of the above replication compositions.
  • a preferred tablet of the instant invention comprises any one of the above replication compositions. More preferably, a kit of the instant invention comprises the foregoing preferred tablet.
  • an internal positive control can be included in the reaction.
  • the internal positive control can include control template nucleic acids (e.g. DNA or RNA), control primers, and control nucleic acid probe.
  • control may be amplified using a single primer; (ii) the amount of the control amplification product is independent of any target DNA or RNA contained in the sample; (iii) the control DNA can be tableted with other amplification reagents for ease of use and high degree of reproducibility in both manual and automated test procedures; (iv) the control can be used with homogeneous detection, i.e., without separation of product DNA from reactants; and (v) the internal control has a melting profile that is distinct from other potential amplification products in the reaction and/or a detectable label on the control nucleic acid that is distinct from the detectable label on the nucleic acid probe directed to the target.
  • Control DNA will be of appropriate size and base composition to permit amplification in a primer-directed amplification reaction.
  • the control template DNA sequence may be obtained from the S. enteritidis or S. Typhimurium genome, or from another source, but must be reproducibly amplified under the same conditions that permit the amplification of the target amplification product.
  • the control reaction is useful to validate the amplification reaction. Amplification of the control DNA occurs within the same reaction tube as the sample that is being tested, and therefore indicates a successful amplification reaction when samples are target negative, i.e. no target amplification product is produced. In order to achieve significant validation of the amplification reaction, a suitable number of copies of the control DNA template must be included in each amplification reaction.
  • Replication compositions may be modified depending on whether they are designed to be used to amplify target DNA or the control DNA.
  • Replication compositions that will amplify the target DNA may include (i) a polymerase (generally thermostable), (ii) a primer pair capable of hybridizing to the target DNA and (iii) necessary buffers for the amplification reaction to proceed.
  • Replication compositions that will amplify the control DNA may include (i) a polymerase (generally thermostable) (ii) the control DNA; (iii) at least one primer capable of hybridizing to the control DNA; and (iv) necessary buffers for the amplification reaction to proceed.
  • the replication composition for either target DNA or control DNA amplification can contain a nucleic acid probe, preferably possessing a detectable label.
  • nucleic Acid Hybridization Methods [00140]
  • nucleic acid hybridization assay methods can be employed in the present invention for detection of S. Typhimurium.
  • the basic components of a nucleic acid hybridization test include probe(s), a sample suspected of containing S. Typhimurium, and a specific hybridization method.
  • probe(s) length can vary from as few as five bases to the full length of the S. Typhimurium diagnostic sequence and will depend upon the specific test to be done. Only part of the probe molecule need be complementary to the nucleic acid sequence to be detected. In addition, the complementarity between the probe(s) and the target sequence(s) need not be perfect. Hybridization does occur between imperfectly complementary molecules with the result that a certain fraction of the bases in the hybridized region(s) are not paired with the proper complementary base. [00141]
  • the sample may or may not contain S. Typhimurium.
  • the sample may take a variety of forms, however will generally be extracted from an animal, environmental or food source suspected of contamination. In a preferred embodiment, the sample will be extracted from a chicken.
  • the DNA may be detected directly but most preferably, the sample nucleic acid must be made available to contact the probe before any hybridization of probe(s) and target molecule(s) can occur. Thus the organism's DNA is preferably free from the cell and placed under the proper conditions before hybridization can occur. Methods of in-solution hybridization necessitate the purification of the DNA in order to be able to obtain hybridization of the sample DNA with the probe(s).
  • Hybridization assays may be conducted directly on cell lysates, without the need to extract the nucleic acids. This eliminates several steps from the sample-handling process and speeds up the assay. To perform such assays on crude cell lysates, a chaotropic agent is typically added to the cell lysates prepared as described above.
  • the chaotropic agent stabilizes nucleic acids by inhibiting nuclease activity. Furthermore, the chaotropic agent allows sensitive and stringent hybridization of short oligonucleotide probes to DNA at room temperature (Van Ness & Chen, Nucleic Acids Res.19:5143-51 (1991)).
  • Suitable chaotropic agents include guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate, potassium iodide, and cesium trifluoroacetate, among others.
  • the chaotropic agent will be present at a final concentration of about 3 M.
  • a variety of methods are known to one of skill in the art (e.g., phenol- chloroform extraction, IsoQuick extraction (MicroProbe Corp., Bothell, Wash.), and others).
  • Pre-hybridization purification is particularly useful for standard filter hybridization assays.
  • purification facilitates measures to increase the assay sensitivity by incorporating in vitro RNA amplification methods such as self-sustained sequence replication (see for example Fahy et al., In PCR Methods and Applications, Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.
  • probe and sample nucleic acids must be in contact for a long enough time that any possible hybridization between the probe and sample nucleic acid may occur.
  • concentration of probe or target in the mixture will determine the time necessary for hybridization to occur. The higher the probe or target concentration, the shorter the hybridization incubation time needed.
  • Various hybridization solutions can be employed. Typically, these comprise from about 20 to 60% volume, preferably 30%, of a polar organic solvent.
  • a common hybridization solution employs about 30-50% v/v formamide, about 0.15 to 1M sodium chloride, about 0.05 to 0.1M buffers, such as sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6-9), about 0.05 to 0.2% detergent, such as sodium dodecylsulfate, or between 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kilodaltons), polyvinylpyrrolidone (about 250-500 kdal), and serum albumin.
  • unlabeled carrier nucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g., calf thymus or salmon sperm DNA, or yeast RNA), and optionally from about 0.5 to 2% wt/vol glycine.
  • Other additives may also be included, such as volume exclusion agents which include a variety of polar water-soluble or swellable agents (e.g., polyethylene glycol), anionic polymers (e.g., polyacrylate or polymethylacrylate), and anionic saccharidic polymers (e.g., dextran sulfate).
  • volume exclusion agents which include a variety of polar water-soluble or swellable agents (e.g., polyethylene glycol), anionic polymers (e.g., polyacrylate or polymethylacrylate), and anionic saccharidic polymers (e.g., dextran sulfate).
  • Nucleic acid hybridization is adaptable to a variety of assay
  • sandwich assay format One of the most suitable is the sandwich assay format.
  • the sandwich assay is particularly adaptable to hybridization under non-denaturing conditions.
  • a primary component of a sandwich-type assay is a solid support.
  • the solid support has adsorbed to it or covalently coupled to it immobilized nucleic acid probe that is unlabeled and complementary to one portion of the DNA sequence.
  • the sandwich assay may be encompassed in an assay kit. This kit would include a first component for the collection of samples suspected of contamination and buffers for the disbursement and lysis of the sample.
  • a second component would include media in either dry or liquid form for the hybridization of target and probe polynucleotides, as well as for the removal of undesirable and nonduplexed forms by washing.
  • a third component includes a solid support (dipstick) upon which is fixed (or to which is conjugated) unlabeled nucleic acid probe(s) that is (are) complementary to one or more of the sequences disclosed herein.
  • a fourth component would contain labeled probe that is complementary to a second and different region of the same DNA strand to which the immobilized, unlabeled nucleic acid probe of the third component is hybridized.
  • polynucleotide sequences disclosed herein or derivations thereof may be used as 3′ blocked detection probes in either a homogeneous or heterogeneous assay format.
  • a probe generated from these sequences may be 3′ blocked or non-participatory and will not be extended by, or participate in, a nucleic acid amplification reaction.
  • the probe incorporates a label that can serve as a reactive ligand that acts as a point of attachment for the immobilization of the probe/analyte hybrid or as a reporter to produce detectable signal.
  • genomic or cDNA isolated from a sample suspected of S. Typhimurium contamination is amplified by standard primer-directed amplification protocols in the presence of an excess of the 3′ blocked detection probe(s) to produce amplification products. Because the probe(s) is 3′ blocked, it does not participate or interfere with the amplification of the target.
  • the detection probe(s) anneals to the relevant portion of the amplified DNA and the annealed complex is then captured on a support through the reactive ligand.
  • a ligand labeled dNTP with the label probe in the replication composition to facilitate immobilization of the PCR reaction product on a support and then detection of the immobilized product by means of the labeled probe reagent.
  • a biotin, digoxigenin, or digoxin labeled dNTP could be added to PCR reaction composition.
  • the biotin, digoxigenin, or digoxin incorporated in the PCR product could then be immobilized respectively on to a strepavidin, anti-dixogin or antidigoxigenin antibody support.
  • the immobilized PCR product could then be detected by the presence of the probe label.
  • Figure 5 shows that lanes U2, U3, U6 are positive for vaccine strain. Lanes U2 and U3 also contain other Salmonella serotypes. Lanes U1, U4, and U5 did not contain vaccine strain.
  • Example 3 Testing multiplex PCR using primers for crp [00164] Results [00165] PCR primers were mixed to run a multiplex PCR for crp and tested on individual colonies belonging to vaccine strain or to wild type Typhimurium. PCR parameters were as shown above. FIG.6 shows that the primers for the vaccine strain were only positive for the vaccine strain, while the WT primers were only positive for the WT strain. [00166] Live attenuated Salmonella vaccines are a critical pre-harvest tool for Salmonella control and are widely used in industry.

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Abstract

L'invention concerne un dosage de diagnostic par PCR, des méthodes et un kit pour effectuer une différenciation entre Salmonella Typhimurium de type sauvage et Salmonella Typhimurium associé à un vaccin chez un sujet vacciné contre Salmonella Typhimurium.
PCT/US2024/033003 2023-06-07 2024-06-07 Compositions et méthodes pour un diagnostic différentiel d'une infection à salmonella typhimurium chez la volaille Ceased WO2024254447A1 (fr)

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WO1992008486A1 (fr) * 1990-11-09 1992-05-29 Washington University Microbes avirulents et leurs applications: salmonella typhi
CN101130076A (zh) * 2007-06-18 2008-02-27 北京神洲天才科技发展有限公司 一种活菌多价疫苗的制备方法
CN102676419A (zh) * 2011-12-06 2012-09-19 河南科技大学 一种伤寒沙门氏菌基因缺失菌株及由其制备的疫苗和应用

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Publication number Priority date Publication date Assignee Title
WO1992008486A1 (fr) * 1990-11-09 1992-05-29 Washington University Microbes avirulents et leurs applications: salmonella typhi
CN101130076A (zh) * 2007-06-18 2008-02-27 北京神洲天才科技发展有限公司 一种活菌多价疫苗的制备方法
CN102676419A (zh) * 2011-12-06 2012-09-19 河南科技大学 一种伤寒沙门氏菌基因缺失菌株及由其制备的疫苗和应用

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Title
CEYSSENS ET AL.: "A molecular assay for rapidly distinguishing the AviPro SALMONELLA VAC T vaccine strain from wild-type field isolates", JOURNAL OF MICROBIOLOGICAL METHODS, vol. 184, no. 106190, 22 March 2021 (2021-03-22), pages 1 - 4, XP086541024, DOI: 10.1016/j.mimet.2021.106190 *
MCWHORTER ANDREA R., CHOUSALKAR KAPIL K.: "A Long-Term Efficacy Trial of a Live, Attenuated Salmonella Typhimurium Vaccine in Layer Hens", FRONTIERS IN MICROBIOLOGY, FRONTIERS MEDIA, LAUSANNE, vol. 9, Lausanne , XP093249904, ISSN: 1664-302X, DOI: 10.3389/fmicb.2018.01380 *
RACCOURSIER MAURICE, SICELOFF AMY, SHARIAT NIKKI: "In silico and PCR Screening for a Live Attenuated Salmonella Typhimurium Vaccine Strain", AVIAN DISEASES, AMERICAN ASSOCIATION OF AVIAN PATHOLOGISTS, INC., US, vol. 68, no. 1, 9 April 2024 (2024-04-09), US , pages 18 - 24, XP009559438, ISSN: 1938-4351, DOI: 10.1637/aviandiseases-D-23-00051 *

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