EP3430159A1 - Schnelles verfahren zum nachweis von salmonella-lebendimpfstoff-stämmen - Google Patents

Schnelles verfahren zum nachweis von salmonella-lebendimpfstoff-stämmen

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Publication number
EP3430159A1
EP3430159A1 EP16716006.8A EP16716006A EP3430159A1 EP 3430159 A1 EP3430159 A1 EP 3430159A1 EP 16716006 A EP16716006 A EP 16716006A EP 3430159 A1 EP3430159 A1 EP 3430159A1
Authority
EP
European Patent Office
Prior art keywords
hours
cfu
minutes
enrichment
live vaccine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP16716006.8A
Other languages
English (en)
French (fr)
Inventor
Cristina Perez GIRONA
Pablo Lozano SÁNCHEZ
Gemma Freixes PROUS
Sergio Martinez MONTEQUÍN
Anna Pallares LLEO
Bruno Teixeira DIAS
Katia Uliaque CUGAT
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Imicroq SL
Original Assignee
Imicroq SL
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Filing date
Publication date
Application filed by Imicroq SL filed Critical Imicroq SL
Publication of EP3430159A1 publication Critical patent/EP3430159A1/de
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/045—Culture media therefor
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/255—Salmonella (G)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Salmonella can contaminate food or food animals during production, processing and preparation. Similarly, Salmonella can contaminate water sources or seafood harvested from such contaminated waters. Human exposure to Salmonella can cause illness, most often gastroenteritis, but also potentially more serious diseases such as salmonellosis and hepatitis A. Exposure to Salmonella can occur either by direct contact with, or ingestion of, contaminated foods or water or indirectly based on cross-contamination. Even though the United States has one of the safest food supplies in the world, there are still millions of cases of foodborne illness each year.
  • Salmonella assays that increase sensitiviety and specificity as well as decrease the time involved to perform these assays have been developed by experts in applied microbiology and microbiological analysis. Although there is no universally accepted definition for these so-called rapid assays, these methods are simply performed faster than the traditional ones, or are easier to implement, or are more sensitive and specific. Despite the name, currently available rapid assays for Salmonella detection still require several days to complete the assay and determine whether a food or water supply is contaminated with a Salmonella live vaccine bacteria.
  • the main obstacles associated with reducing the time neded to conduct Salmonella detection assays is the balance between the sensitivity, specificity, and lower limit of detection of the assay on one hand and having enough Salmonella present to detect its presence above contaminating microorganisms and background noise.
  • Salmonella live vaccine strains have been created in order to elicit an immune response that creates protective antibodies agaisnt pathogenic Salmonella strains (or Salmonella field strains) tha cause infections. Unlike the Salmonella field strains, the Salmonella live vaccine strains are non-pathogenic. Plating and genotyping-based assays have been developed to detect the presence of Salmonella live vaccine strains. However, these assys are not only time consuming and, in the case of genotyping-based assays, require sophisticated equipment.
  • the present specification discloses a rapid method for the detection of Salmonella live vaccine bacteria which provides high sensitivity and specificity, a lower limit of detection, yet can be performed more quickly then currently avaialble methods.
  • Aspects of the present specification disclose a method of detecting a Salmonella live vaccine strain in a sample.
  • the method disclosed herein comprises: a) incubation of the sample in a first liquid pre- enrichment comprising 2 g/L to 6 g/L of a peptone, 0.5 g/L to 4.5 g/L Bile Salts, 0.5 g/L to 4.5 g/L Meat Extract, 0.5 g/L to 4.5 g/L of a first growth inhibiting agent, 0.5 g/L to 4.5 g/L of a second growth inhibiting agent, 0.001 g/L to 0.008 g/L of a third growth inhibiting agent, and 0.001 g/L to 0.008 g/L of a fourth growth inhibiting agent, wherein the incubation at about 34°C to about 40°C for about 5 hours to about 10 hours; b) incubating an aliquot of first pre-enrichment media from step (a) in a liquid pre
  • analysis kit comprising a pre-enrichment media and an enrichment media as disclosed herein.
  • the analysis kit may further comprise detection solution.
  • the analysis kit may further comprise an immunopurification reagent system.
  • the analysis kit may further comprise a label or an insert providing instructions on how to use the kit.
  • FIG. 1 shows a flow diagram of a method disclosed herein.
  • FIG. 2 shows a graph of the proportionality between different densities of bacterial population and the signal obtained chronoamperometric.
  • FIG. 3 shows a graph of the proportionality between the registered electrochemical signal and progress in a particular bacterial load proliferation.
  • the present specification discloses a method of detecting a Salmonella live vaccine in a sample.
  • the method comprises a pre-enrichment step and an enrichment step that use selective pre-growth and growth media.
  • the combination of these steps, together with specific time and temperature conditions, progressively increases the population of the Salmonella live vaccine bacteria as well as an effective reduces the populations of unwanted organisms and/or other background noise which interferes with the detection of the Salmonella live vaccine bacteria.
  • the selective increase of the Salmonella live vaccine bacteria population as well as the effectively removing unwanted organisms and/or other background noise allows for a more sensitive and precise detection of the Salmonella live vaccine bacteria then currently available methods.
  • the method disclosed herein allows for a more rapid detection of a Salmonella live vaccine bacteria since it may be completed in about eight hours to about 30 hours; current Salmonella live vaccine bacteria detection methods require about 2 days to about 5 days to complete.
  • a method disclsoed herein is outlined in FIG. 1.
  • a Salmonella live vaccine can be a Salmonella live vaccine bacteriaic strain or a non-Salmonella live vaccine bacteriaic strain (also referred to a Salmonella live vaccine strain).
  • a Salmonella live vaccine bacteriaic Salmonella bacterial strain is one that causes or facillitates a disease, infection or other adverse effect in a mammal.
  • a Salmonella live vaccine bacteriaic Salmonella bacterial strain is considered to be an abnormal flora.
  • a non-Salmonella live vaccine bacteriaic Salmonella bacterial strain is one that is considered harmless to a mammal because no appreciable disease, infection or other adverse effect is associated with the presence of the non-Salmonella live vaccine bacteriaic Salmonella bacterial strain in the mammal.
  • a Salmonella non-Salmonella live vaccine bacteriaic bacterial strain is considered to be a normal flora. Examples of a non-Salmonella live vaccine bacteriaic Salmonella bacterial strain or a Salmonella live vaccine bacterial strain incude, without limitation, a VacT Salmonella live vaccine bacteria strain and VacE Salmonella live vaccine bacterial strain.
  • a Salmonella strain can be a pathogenic strain (also referred to a field strain) or a non-pathogenic strain (also referred to a live vaccine strain).
  • a pathogenic Salmonella strain is one that causes or facillitates a disease, infection or other adverse effect in a mammal.
  • a Salmonella field strain is considered to be an abnormal or infectous flora.
  • a Salmonella live vaccine strain is typically an attenuated bacterial strain used to elicit an immune response that creates protective antibodies in an individual, such as poltry, livestock or humans, agaisnt pathogenic Salmonella strains (or Salmonella field strains) that cause infections.
  • a Salmonella live vaccine strain is one that is considered harmless to a mammal because no appreciable disease, infection or other adverse effect is associated with the presence of the Salmonella live vaccine strain in the mammal.
  • a Salmonella live vaccine strain is considered to be a benign or beneficial flora.
  • a Salmonella non-pathogenic bacterial strain or a Salmonella live vaccine bacterial strain includes, without limitation, a VacT Salmonella live vaccine strain and VacE Salmonella live vaccine strain.
  • a sample encompasses but is not limited to a purified Salmonella live vaccine bacteria, a partially purified Salmonella live vaccine bacteria, a cell, a crude cell lysate, a partially purified cell lysate, a crude culture media, a partially purified culture media, a raw foodstuff, a partially- cooked foodstuff, a cooked foodstuff, a processed foodstuff; a dairy foodstuff, a beverage, an animal feed, a fecal sample, a vegetative sample, a soil sample, a water sample, a pond sediment, a human tissue sample, a raw livestock tissue sample, a processed livestock tissue sample, such as, e.g., leather.
  • a pre-enrichment step comprises incubating a sample disclosed herein in a pre-enrichment media for a defined time and at a defined temperature.
  • a pre-enrichment media also referred to as a pre-enrichment culture media is a buffered culture media that provides the nutrients necessary to sustain low-growth of the Salmonella live vaccine bacteria.
  • a pre-enrichment media may also contain components which reduce or inhibit the growth of contaminating bacteria or other microorganisms.
  • a pre-enrichment media comprises a low growth nutrient component, a surfactant, and optionally a growth inhibiting agent and/or a growth enhancing agent.
  • a method disclosed herein may use a first pre-enrichment media and a second pre-enrichment media.
  • a first and second pre-enrichment media may comprises the same low growth nutrient component, surfactant, and optionally growth inhibiting agent and/or growth enhancing agent, i.e., the first and second pre-enrichment media will be of identical compositon.
  • a first and second pre-enrichment media may comprises the same low growth nutrient component, surfactant, and optionally growth inhibiting agent and/or growth enhancing agent but in differing amount from one another.
  • a first pre-enrichment media may comprises different low growth nutrient component, surfactant, and optionally growth inhibiting agent and/or growth enhancing agent reltive to a second pre-enrichment media.
  • a pre-enrichment media typically comprises a low growth nutrient component used as a source of proteins, amino acids and nitrogen.
  • a single low growth nutrient component may comprise a pre- enrichment media disclosed herein, or a plurality of low growth nutrient components may comprise a pre- enrichment media disclosed herein.
  • a non-limiting example of a low growth nutrient component is a peptone, such as, e.g. , a peptide from an animal source and a peptone from a plant source.
  • a peptone from an animal source includes, without limitation, an acid casein peptone, a bacteriological peptone, a beef extract powder, a casein peptone, a casein cc peptone, a gelatin peptone, a meat peptone, a polypeptone proteose peptone, and a proteose peptone No 3.
  • a peptone from a plant source includes, without limitation, a malt extract, a soya peptone, and a yeast extract.
  • a low growth nutrient component may be used at a concentration of, e.g. , about 1 g/L, about 2 g/L, about 3 g/L, about 4 g/L, about 5 g/L, about 6 g/L, about 7 g/L, about 8 g/L, about 9 g/L, about 10 g/L, about 1 1 g/L, about 12 g/L, about 13 g/L, about 14 g/L, or about 15 g/L.
  • a low growth nutrient component may be used at a concentration of, e.g., at least 1 g/L, at least 2 g/L, at least 3 g/L, at least 4 g/L, at least 5 g/L, at least 6 g/L, at least 7 g/L, at least 8 g/L, at least 9 g/L, at least 10 g/L, at least 11 g/L, at least 12 g/L, at least 13 g/L, at least 14 g/L, or at least 15 g/L.
  • a low growth nutrient component may be used at a concentration of, e.g.
  • a low growth nutrient component may be used at a concentration of between, e.g., about 1 g/L to 2 g/L, about 1 g/L to 3 g/L, about 1 g/L to 4 g/L, about 1 g/L to 5 g/L, about 1 g/L to 6 g/L, about 1 g/L to 7 g/L, about 1 g/L to 8 g/L, about 1 g/L to 9 g/L, about 1 g/L to
  • 5 g/L to 9 g/L about 5 g/L to 10 g/L, about 5 g/L to 1 1 g/L, about 5 g/L to 12 g/L, about 5 g/L to 13 g/L, about 5 g/L to 14 g/L, about 5 g/L to 15 g/L, about 6 g/L to 7 g/L, about 6 g/L to 8 g/L, about 6 g/L to 9 g/L, about 6 g/L to 10 g/L, about 6 g/L to 11 g/L, about 6 g/L to 12 g/L, about 6 g/L to 13 g/L, about 6 g/L to 14 g/L, about 6 g/L to 15 g/L, about 7 g/L to 8 g/L, about 7 g/L to 9 g/L, about 7 g/L to 10 g/L, about 7 g/L to 10
  • a pre-enrichment media may also comprises a surfactant.
  • Surfactants are compounds that lower the surface tension of a liquid, allowing easier spreading, and lowering of the interfacial tension between two liquids, or between a liquid and a solid. Either a single surfactant may comprise a pre-enrichment media disclosed herein, or a plurality of surfactants may comprise a pre-enrichment media disclosed herein.
  • a surfactant disclosed herein provides bacteriostatic and bactericide action that retards or prevents growth of unwanted organisms contained in a sample disclosed herein.
  • a surfactant may retard or prevent growth of unwanted bacterial cells by disrupting the mechanisms of action of adsorption on the surface, interfering with the osmotic balance, preventing the intake of nutrients, denaturing proteins, inhibiting enzyme activity, and/or damaging the cell membrane.
  • Useful surfactants include, without limitation, ionic surfactants, zwitterionic (amphoteric) surfactants, non-ionic surfactants, or any combination therein.
  • the surfactant used in a method disclosed herein can be varied as appropriate by one skilled in the art and generally depends, in part, on the particular pre-enrichment media being used, the Salmonella live vaccine bacteria being detected, and/or the particular unwanted bacteria being removed.
  • Ionic surfactants include anionic surfactants based on permanent (sulfate, sulfonate, phosphate) or pH dependent (carboxylate) anions.
  • Anionic surfactants include, without limitation, alkyl sulfates like ammonium lauryl sulfate and sodium lauryl sulfate (SDS); alkyl ether sulfates like sodium laureth sulfate and sodium myreth sulfate; docusates like dioctyl sodium sulfosuccinate; tetradecyl sodium dodecyl sulfate; 7-ethyl-2-methyl-4-undecyl sodium sulphate; octadecyl sulphate; sulfonate fluorosurfactants like perfluorooctanesulfonate (PFOS) and perfluorobutanesulfonate; alkyl benzene sulfonates; alkyl aryl ether phosphates; alkyl ether phosphates; alkyl carboxylates like fatty acid salts and sodium stearate; sodium la
  • Ionic surfactants also include cationic surfactants based on permanent or pH dependent cations.
  • Cationic surfactants include, without limitation, alkyltrimethylammonium salts like cetyl trimethylammonium bromide (CTAB) and cetyl trimethylammonium chloride (CTAC); cetylpyridinium chloride (CPC); polyethoxylated tallow amine (POEA); benzalkonium chloride (BAC); benzethonium chloride (BZT); 5- Bromo-5-nitro-1 ,3-dioxane; dimethyldioctadecylammonium chloride; and dioctadecyldimethylammonium bromide (DODAB), as well as pH-dependent primary, secondary or tertiary amines like surfactants where the primary amines become positively charged at pH greater than 10, or the secondary amines become charged at pH less than 4, like octenidine dihydrochloride.
  • Zwitterionic surfactants are based on primary, secondary or tertiary amines or quaternary ammonium cation with a sulfonate, a carboxylate, or a phosphate.
  • Zwitterionic surfactants include, without limitation, 3-[(3-Cholamidopropyl)dimethylammonio]-1 -propanesulfonate (CHAPS); sultaines like cocamidopropyl hydroxysultaine; betaines like cocamidopropyl betaine; or lecithins.
  • Non-ionic surfactants are less denaturing and as such are useful to solubilize membrane proteins and lipids while retaining protein-protein interactions.
  • Non-limiting examples of surfactants include polyoxyethylene glycol sorbitan alkyl esters like polysorbate 20 sorbitan monooleate (TWEEN ® 20), polysorbate 40 sorbitan monooleate (TWEEN ® 40), polysorbate 60 sorbitan monooleate (TWEEN ® 60), polysorbate 61 sorbitan monooleate (TWEEN ® 61 ), polysorbate 65 sorbitan monooleate (TWEEN ® 65), polysorbate 80 sorbitan monooleate (TWEEN ® 80), and polysorbate 81 sorbitan monooleate (TWEEN ® 81 ); poloxamers (polyethylene-polypropylene copolymers), like Poloxamer 124 (PLURONIC ® L44), Poloxamer 181 (P
  • surfactants useful in the methods disclosed herein can be found in, e.g., Winslow, et al., Methods and Compositions for Simultaneously Isolating Hemoglobin from Red Blood Cells and Inactivating Viruses, U.S. 2008/0138790; Pharmaceutical Dosage Forms and Drug Delivery Systems (Howard C. Ansel et al., eds., Lippincott Williams & Wilkins Publishers, 7 th ed. 1999); Remington: The Science and Practice of Pharmacy (Alfonso R. Gennaro ed., Lippincott, Williams & Wilkins, 20 th ed.
  • a surfactant may be used at a concentration of, e.g., about 0.01 % (v/v), about 0.05% (v/v), about 0.075% (v/v), about 0.1 % (v/v), about 0.2% (v/v), about 0.3% (v/v), about 0.4% (v/v), about 0.5% (v/v), about 0.6% (v/v), about 0.7% (v/v), about 0.8% (v/v), about 0.9% (v/v), about 1.0% (v/v), about 2.0% (v/v), about 3.0% (v/v), about 4.0% (v/v), about 5.0% (v/v), about 6.0% (v/v), about 7.0% (v/v), about 8.0% (v/v), about 9.0% (v/v), or about 10.0% (v/v).
  • a surfactant may be used at a concentration of, e.g., at least 0.01 % (v/v), at least 0.05% (v/v), at least 0.075% (v/v), at least 0.1 % (v/v), at least 0.25% (v/v), at least 0.5% (v/v), at least 0.75% (v/v), at least 1.0% (v/v), at least 2.5% (v/v), at least 5.0% (v/v), at least 7.5% (v/v), or at least 10.0% (v/v).
  • a surfactant may be used at a concentration of, e.g.
  • a surfactant may be used at a concentration of between, e.g., about 0.01 % (v/v) to about 0.05% (v/v), about 0.01 % (v/v) to about 0.1 % (v/v), about 0.01 % (v/v) to about 0.5% (v/v), about 0.01 % (v/v) to about 1.0% (v/v), about 0.01 % (v/v) to about 2.0% (v/v), about 0.01 % (v/v) to about 3.0% (v/v), about 0.01 % (v/v) to about 4.0% (v/v), about 0.01 % (v/v) to about 5.0% (v/v), about 0.05% (v/v) to about 0.1 % (v/v), about 0.05% (v/v) to about 0.5% (v/v), about 0.05% (v/v) to about 1.0% (v/v), about 0.05% (v/v) to about 0.05% (v/v) to about 1.
  • the surfactant may be used at a concentration of, e.g., about 0.001 g/L, about 0.002 g/L, about 0.003 g/L, about 0.004 g/L, about 0.005 g/L, about 0.006 g/L, about 0.007 g/L, about 0.008 g/L, about 0.009 g/L, about 0.01 g/L, about 0.02 g/L, about 0.03 g/L, about 0.04 g/L, about 0.05 g/L, about 0.06 g/L, about 0.07 g/L, about 0.08 g/L, about 0.09 g/L, or about 0.1 g/L.
  • the surfactant may be used at a concentration of, e.g., at least 0.001 g/L, at least 0.002 g/L, at least 0.003 g/L, at least 0.004 g/L, at least 0.005 g/L, at least 0.006 g/L, at least 0.007 g/L, at least 0.008 g/L, at least 0.009 g/L, at least 0.01 g/L, at least 0.02 g/L, at least 0.03 g/L, at least 0.04 g/L, at least 0.05 g/L, at least 0.06 g/L, at least 0.07 g/L, at least 0.08 g/L, at least 0.09 g/L, or at least 0.1 g/L.
  • the surfactant may be used at a concentration of, e.g., at most 0.001 g/L, at most 0.002 g/L, at most 0.003 g/L, at most 0.004 g/L, at most 0.005 g/L, at most 0.006 g/L, at most 0.007 g/L, at most 0.008 g/L, at most 0.009 g/L, at most 0.01 g/L, at most 0.02 g/L, at most 0.03 g/L, at most 0.04 g/L, at most 0.05 g/L, at most 0.06 g/L, at most 0.07 g/L, at most 0.08 g/L, at most 0.09 g/L, or at most 0.1 g/L.
  • the surfactant may be used at a concentration of, e.g. , about 0.001 g/L to about 0.005 g/L, about 0.001 g/L to about 0.006 g/L, about 0.001 g/L to about 0.007 g/L, about 0.001 g/L to about 0.008 g/L, about 0.001 g/L to about 0.009 g/L, about 0.001 g/L to about 0.01 g/L, about 0.001 g/L to about 0.02 g/L, about 0.001 g/L to about 0.03 g/L, about 0.001 g/L to about 0.04 g/L, about 0.001 g/L to about 0.05 g/L, about 0.001 g/L to about 0.06 g/L, about 0.001 g/L to about 0.07 g/L, about 0.001 g/L to about 0.08 g
  • a pre-enrichment media may optionally comprises a growth inhibiting agent.
  • a growth inhibiting agent typically comprises a component that reduces or inhibits the growth of contaminating bacteria or other contaminating microorganisms.
  • a growth inhibiting agent may not affect the growth of a Salmonella live vaccine bacteria of interest or affects it to a lesser extent that the contaminating bacteria or other contaminating microorganisms.
  • Either a single growth inhibiting agent may comprise a pre- enrichment media disclosed herein, or a plurality of growth inhibiting agents may comprise a pre-enrichment media disclosed herein.
  • Non-limiting examples of a growth inhibiting agent include an anti-microbial compound, an iodine compound, a magneusium compound, and a triarylmethane dye.
  • An anti-microbial compound is one that is antagonistic to the growth of a microorganism. These compounds can be divided into two broad categories of microbicidal agents which kill a microorganism and microbiostatic agents which slow down or stall the growth of a microorganism.
  • An anti-microbial compound is commonly classified based on its mechanism of action, chemical structure, or spectrum of activity. Most target bacterial functions or growth processes. Those that target the bacterial cell wall (penicillins and cephalosporins) or the cell membrane (polymyxins), or interfere with essential bacterial enzymes (rifamycins, lipiarmycins, quinolones, and sulfonamides) have bactericidal activities.
  • target protein synthesis (macrolides, lincosamides and tetracyclines) are usually bacteriostatic (with the exception of bactericidal aminoglycosides). Further categorization is based on their target specificity. "Narrow- spectrum” antibacterial antibiotics target specific types of bacteria, such as Gram-negative or Gram-positive bacteria, whereas broad-spectrum antibiotics affect a wide range of bacteria.
  • An anti-microbial compound includes, without limitation, an aminocoumarin, an aminoglycoside, an ansamycin, a carbacephem, a carbapenen, a cephalosporin, a cyclic lipopeptide, a glycopeptide, a glycylcycline, a lincosamide, a lipiamycin, a lipopeptide, a macrolide, a monobactam, a nitrofuran, an oxazolidonome, a penicillin, a qunolones, a sulfonamide, and a tetracycline.
  • an aminocoumarin include Novobiocin, Albamycin, Coumermycin and Clorobiocin.
  • An iodine compound combines with amino acids like tyrosine or histidine by a simple chemical reaction that denatures protiens having these amino acids exposed to the extra-cellular environment.
  • Non- limiting examples of an iodine compound include iodine and potassium iodine.
  • magnesium is magnesium chloride and magnesium sulfate.
  • a triarylmethane dye is a group of synthetic organic compounds containing triphenylmethane backbones that produce an intense, pH-dependent color. Triarylmethane dyes can be grouped into families according to the nature of the substituents on the aryl groups. Methyl violet dyes have dimethylamino groups at the p-positions of two aryl groups and include, without limitation, methyl violet 2B, methyl violet 6B, and methyl violet 10B.
  • Fuchsine dyes have amine (NH2 or NHMe) functional groups at the p-positions of each aryl group and include, without limitation, pararosaniline, fuchsine, new fuchsine, fuchsin basic violet, and fuchine acid.
  • Phenol dyes have hydroxyl groups at the p positions of each aryl group and include, without limitation, phenol red, chlorophenol red, and cresol red.
  • Malachite green dyes are related to the methyl violet dyes, except that they contain one phenyl (CeHs) group and include, without limitation, malachite green and brilliant green.
  • a triarylmethane dye includes, without limitation, aluminon, aniline Blue WS, aurin, aurintricarboxylic acid, brilliant blue FCF, brilliant green, bromocresol green, bromocresol purple, bromophenol blue, bromopyrogallol red, bromothymol blue, bromsulphthalein, chlorophenol red, coomassie brilliant blue, cresol red, crystal violet, crystal violet lactone, ethyl green, fast green FCF, fluoran, fuchsine, fuchsine acid, gentian, green S, light green SF yellowish, malachite green, methyl blue, methyl violet, new fuchsine, pararosaniline, patent blue V, phenol red, phenolphthalein, rose bengal, thymolphthalein, victoria blue BO, water blue, xylene cyanol, and xylenol orange.
  • a growth inhibiting agent may be used at a concentration of, e.g. , about 0.01 % (v/v), about 0.05% (v/v), about 0.075% (v/v), about 0.1 % (v/v), about 0.2% (v/v), about 0.3% (v/v), about 0.4% (v/v), about 0.5% (v/v), about 0.6% (v/v), about 0.7% (v/v), about 0.8% (v/v), about 0.9% (v/v), about 1.0% (v/v), about 2.0% (v/v), about 3.0% (v/v), about 4.0% (v/v), about 5.0% (v/v), about 6.0% (v/v), about 7.0% (v/v), about 8.0% (v/v), about 9.0% (v/v), or about 10.0% (v/v).
  • a growth inhibiting agent may be used at a concentration of, e.g., at least 0.01 % (v/v), at least 0.05% (v/v), at least 0.075% (v/v), at least 0.1 % (v/v), at least 0.25% (v/v), at least 0.5% (v/v), at least 0.75% (v/v), at least 1.0% (v/v), at least 2.5% (v/v), at least 5.0% (v/v), at least 7.5% (v/v), or at least 10.0% (v/v).
  • a growth inhibiting agent may be used at a concentration of, e.g., at most 0.01 % (v/v), at most 0.05% (v/v), at most 0.075% (v/v), at most 0.1 % (v/v), at most 0.25% (v/v), at most 0.5% (v/v), at most 0.75% (v/v), at most 1.0% (v/v), at most 2.5% (v/v), at most 5.0% (v/v), at most 7.5% (v/v), or at most 10.0% (v/v).
  • a growth inhibiting agent may be used at a concentration of between, e.g., about 0.01 % (v/v) to about 0.05% (v/v), about 0.01 % (v/v) to about 0.1 % (v/v), about 0.01 % (v/v) to about 0.5% (v/v), about 0.01 % (v/v) to about 1.0% (v/v), about 0.01 % (v/v) to about 2.0% (v/v), about 0.01 % (v/v) to about 3.0% (v/v), about 0.01 % (v/v) to about 4.0% (v/v), about 0.01 % (v/v) to about 5.0% (v/v), about 0.05% (v/v) to about 0.1 % (v/v), about 0.05% (v/v) to about 0.5% (v/v), about 0.05% (v/v) to about 1.0% (v/v), about 0.05% (v/v) to about 0.05% (v/v) to about
  • a pre-enrichment media may optionally comprises a growth enhancing agent.
  • a growth enhancing agent promotes rapid growth of a Salmonella live vaccine bacteria by reducing the lag phase in culture media, thereby reactivating dormant Salmonella live vaccine bacteria.
  • Non-limiting examples of a growth enhancing agent include a siderophore.
  • a siderophore is a high- affinity iron chelating compound that acts to sequester and solubilize the iron. These compounds are important to a Salmonella live vaccine bacteria for its acquisition of iron in order to maintain cellular respiration and DNA synthesis. This is because under most culture environment, the amount of free iron (approximately 1 x 10 ⁇ 9 ) is below the concentration required by most bacterial Salmonella live vaccine bacterias for growth.
  • Non-limiting examples of a siderophore include Aerobactin, Alcaligin, Azotobactin, Bacillibactin, Desferrioxamine B, Desferrioxamine E, Enterobactin, Ferrichrome, Ferrioxiamina-B, Ferrioxiamina-E, Fusarinine C, Mycobactin, Ornibactin, Petrobactin, Pyoverdine, Pyochelin, Salmochelin, Staphyloferring A, Vibriobactin, and Yersiniabactin.
  • a growth enhancing agent may be used at a concentration of, e.g., about 0.01 ⁇ , about 0.05 ⁇ , about 0.075 ⁇ , about 0.1 ⁇ , about 0.2 ⁇ , about 0.3 ⁇ , about 0.4 ⁇ , about 0.5 ⁇ , about 0.6 ⁇ , about 0.7 ⁇ , about 0.8 ⁇ , about 0.9 ⁇ , about 1.0 ⁇ , about 2.0 ⁇ , about 3.0 ⁇ , about 4.0 ⁇ , about 5.0 ⁇ , about 6.0 ⁇ , about 7.0 ⁇ , about 8.0 ⁇ , about 9.0 ⁇ , or about 10.0% (v/v).
  • a growth enhancing agent may be used at a concentration of, e.g. , at least 0.01 ⁇ , at least 0.05 ⁇ , at least 0.075 ⁇ , at least 0.1 ⁇ , at least 0.25 ⁇ , at least 0.5 ⁇ , at least 0.75 ⁇ , at least 1.0 ⁇ , at least 2.5 ⁇ , at least 5.0 ⁇ , at least 7.5 ⁇ , or at least 10.0% (v/v).
  • a growth enhancing agent may be used at a concentration of, e.g., at most 0.01 ⁇ , at most 0.05 ⁇ , at most 0.075 ⁇ , at most 0.1 ⁇ , at most 0.25 ⁇ , at most 0.5 ⁇ , at most 0.75 ⁇ , at most 1.0 ⁇ , at most 2.5 ⁇ , at most 5.0 ⁇ , at most 7.5 ⁇ , or at most 10.0 ⁇
  • a growth enhancing agent may be used at a concentration of between, e.g., about 0.01 % (v/v) to about 0.05 ⁇ , about 0.01 % (v/v) to about 0.1 ⁇ , about 0.01 % (v/v) to about 0.5 ⁇ , about 0.01 % (v/v) to about 1.0 ⁇ , about 0.01 % (v/v) to about 2.0 ⁇ , about 0.01 % (v/v) to about 3.0 ⁇ , about 0.01 % (v/v) to about 4.0 ⁇ , about 0.01 % (v/v) to about 5.0 ⁇ , about 0.05% (v/v) to about 0.1 ⁇ , about 0.05% (v/v) to about 0.5 ⁇ , about 0.05% (v/v) to about 1.0 ⁇ , about 0.05% (v/v) to about 2.0 ⁇ , about 0.05% (v/v) to about 3.0 ⁇ , about 0.05%
  • a pre-enrichment media comprises 2 g/L to 6 g/L of a peptone, 0.5 g/L to 4.5 g/L Bile Salts, 0.5 g/L to 4.5 g/L Meat Extract, 0.5 g/L to 4.5 g/L of a first growth inhibiting agent, 0.5 g/L to 4.5 g/L of a second growth inhibiting agent, 0.001 g/L to 0.008 g/L of a third growth inhibiting agent, and 0.001 g/L to 0.008 g/L of a fourth growth inhibiting agent.
  • a pre-enrichment media comprises 2 g/L to 6 g/L of a Caseine peptone, 0.5 g/L to 4.5 g/L of Bile Salts, 0.5 g/L to 4.5 g/L of Meat Extract, 0.5 g/L to 4.5 g/L of a first iodine compound, 0.5 g/L to 4.5 g/L of a second iodine compound, 0.001 g/L to 0.008 g/L of an aminocoumarin antibiotic, and 0.001 g/L to 0.008 g/L of a triarylmethane dye.
  • a pre-enrichment media comprises 2 g/L to 6 g/L of a Caseine peptone, 0.5 g/L to 4.5 g/L of Bile Salts, 0.5 g/L to 4.5 g/L of Meat Extract, 0.5 g/L to 4.5 g/L of a Iodine, 0.5 g/L to 4.5 g/L of a Potassium Iodide, 0.001 g/L to 0.008 g/L of Novobiocin, and 0.001 g/L to 0.008 g/L of Brilliant Green.
  • a pre-enrichment media comprises 3 g/L to 5 g/L of a peptone, 1.5 g/L to 3.5 g/L Bile Salts, 1 g/L to 3 g/L Meat Extract, 1 g/L to 3 g/L of a first growth inhibiting agent, 1 g/L to 3 g/L of a second growth inhibiting agent, 0.002 g/L to 0.006 g/L of a third growth inhibiting agent, and 0.002 g/L to 0.006 g/L of a fourth growth inhibiting agent.
  • a pre-enrichment media comprises 3 g/L to 5 g/L of a Caseine peptone, 1.5 g/L to 3.5 g/L of Bile Salts, 1 g/L to 3 g/L of Meat Extract, 1 g/L to 3 g/L of a first iodine compound, 1 g/L to 3 g/L of a second iodine compound, 0.002 g/L to 0.006 g/L of an aminocoumarin antibiotic, and 0.002 g/L to 0.006 g/L of a triarylmethane dye.
  • a pre-enrichment media comprises 3 g/L to 5 g/L of a Caseine peptone, 1.5 g/L to 3.5 g/L of Bile Salts, 1 g/L to 3 g/L g/L of Meat Extract, 1 g/L to 3 g/L of a Iodine, 1 g/L to 3 g/L of a Potassium Iodide, 0.002 g/L to 0.006 g/L of Novobiocin, and 0.002 g/L to 0.006 g/L of Brilliant Green.
  • a pre-enrichment media comprises 4 g/L to 4.6 g/L of a peptone, 2.1 g/L to 2.7 g/L Bile Salts, 1.8 g/L to 2.4 g/L Meat Extract, 1.7 g/L to 2.3 g/L of a first growth inhibiting agent, 1.7 g/L to 2.3 g/L of a second growth inhibiting agent, 0.003 g/L to 0.005 g/L of a third growth inhibiting agent, and 0.003 g/L to 0.005 g/L of a fourth growth inhibiting agent.
  • a pre-enrichment media comprises 4 g/L to 4.6 g/L of a Caseine peptone, 2.1 g/L to 2.7 g/L of Bile Salts, 1.8 g/L to 2.4 g/L of Meat Extract, 1.7 g/L to 2.3 g/L of a first iodine compound, 1.7 g/L to 2.3 g/L of a second iodine compound, 0.003 g/L to 0.005 g/L of an aminocoumarin antibiotic, and 0.003 g/L to 0.005 g/L of a triarylmethane dye.
  • a pre-enrichment media comprises 4 g/L to 4.6 g/L of a Caseine peptone, 2.1 g/L to 2.7 g/L of Bile Salts, 1.8 g/L to 2.4 g/L of Meat Extract, 1.7 g/L to 2.3 g/L of a Iodine, 1.7 g/L to 2.3 g/L of a Potassium Iodide, 0.003 g/L to 0.005 g/L of Novobiocin, and 0.003 g/L to 0.005 g/L of Brilliant Green.
  • a pre-enrichment media comprises 4.3 g/L of a peptone, 2.4 g/L Bile Salts, 2.1 g/L Meat Extract, 2 g/L of a first growth inhibiting agent, 2 g/L of a second growth inhibiting agent, 0.004 g/L of a third growth inhibiting agent, and 0.004 g/L of a fourth growth inhibiting agent.
  • a pre-enrichment media comprises 4.3 g/L of a Caseine peptone, 2.4 g/L of Bile Salts, 2.1 g/L of Meat Extract, 2 g/L of a first iodine compound, 2 g/L of a second iodine compound, 0.004 g/L of an aminocoumarin antibiotic, and 0.004 g/L of a triarylmethane dye.
  • a pre-enrichment media comprises 4.3 g/L of a Caseine peptone, 2.4 g/L of Bile Salts, 2.1 g/L of Meat Extract, 2 g/L of a Iodine, 2 g/L of a Potassium Iodide, 0.004 g/L of Novobiocin, and 0.004 g/L of Brilliant Green.
  • a pre-enrichment media further comprises salts.
  • a pre-enrichment media further comprises NaCI, CaC03 and Na2S203.
  • a pre-enrichment media further comprises 0.5 g/L to 2.6 g/L NaCI, 18.0 g/L to 20.6 g/L CaC03 and 13.9 g/L to 16.5 g/L a2S203.
  • a pre-enrichment media further comprises 1.0 g/L to 1.6 g/L NaCI, 19.0 g/L to 19.6 g/L CaCOs and 14.9 g/L to 15.5 g/L Na 2 S20 3 .
  • a pre-enrichment media further comprises 1.3 g/L NaCI, 19.3 g/L CaCOs and 15.2 g/L Na 2 S20 3 .
  • aspects of the present specification disclose, in part, incubation of a sample in a pre-enrichment media.
  • Incubation of a sample is performed under temperature and time parameters that facilitate the growth of the Salmonella live vaccine bacteria in the sample, retard the growth of an unwanted organism in the sample, and/or establish conditions that otherwise increase the population of the Salmonella live vaccine bacteria in the sample and/or retard the growth of an unwanted organism in the sample.
  • Incubation of pre-enrichment media may be performed under constant rotation, reversal or agitation.
  • a temperature used to incubate of a sample in a pre-enrichment media may be, e.g., about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, or about 42°C.
  • a temperature used to incubate of a sample in a pre- enrichment media may be, e.g., at least 25°C, at least 26°C, at least 27°C, at least 28°C, at least 29°C, at least 30°C, at least 31 °C, at least 32°C, at least 33°C, at least 34°C, at least 35°C, at least 36°C, at least 37°C, at least 38°C, at least 39°C, at least 40°C, at least 41°C, or at least 42°C.
  • a temperature used to incubate of a sample in a pre-enrichment media may be, e.g.
  • a temperature used to incubate of a sample in a pre-enrichment media may be, e.g.
  • a temperature used to incubate of a sample in a pre-enrichment media may be, e.g., about 34°C to about 39°C, about 34°C to about 40°C, about 35°C to about 45°C, about 36°C to about 44°C, about 36°C to about 43°C, about 37°C to about 42°C, about 34°C to about 45°C or about 39°C to about 45°C.
  • a time used to incubate of a sample in a pre-enrichment media may be, e.g., about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about
  • a time used to incubate of a sample in a pre-enrichment media may be, e.g. , at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 1 1 hours, at least
  • a time used to incubate of a sample in a pre- enrichment media may be, e.g., at most 4 hours, at most 5 hours, at most 6 hours, at most 7 hours, at most 8 hours, at most 9 hours, at most 10 hours, at most 1 1 hours, at most 12 hours, at most 13 hours, at most 14 hours, at most 15 hours, at most 16 hours, at most 17 hours, at most 18 hours, at most 19 hours, at most 20 hours, at most 21 hours, at most 22 hours, at most 23 hours, or at most 24 hours.
  • a time used to incubate of a sample in a pre-enrichment media may be, e.g.
  • a time used to incubate of a sample in a pre-enrichment media may be, e.g., about 1 hour to about 2 hours, about 1 hour to about 3 hours, about 1 hour to about 4 hours, about 1 hour to about 5 hours, about 1 hour to about 6 hours, about 1 hour to about 7 hours, about 2 hour to about 3 hours, about 2 hour to about 4 hours, about 2 hour to about 5 hours, about 2 hour to about 6 hours, about 2 hour to about 7 hours, about 3 hour to about 4 hours, about 3 hour to about 5 hours, about 3 hour to about 6 hours, about 3 hour to about 7 hours, about 4 hours to about 1 1 hours, about 5 hours to about 10 hours, about 6 hours to about 9 hours, about 7 hours to about 8 hours, about 5 hours to about 10 hours, about 6 hours to about 9 hours, about 5 hours to about 1 1 hours, about 6 hours to about 10 hours, about 7 hours to about 9 hours, about 4 hours to about 10 hours, about 5 hours to about 1 1 hours, about 6 hours to about 10 hours, about 7 hours to
  • a sample in a pre-enrichment media may be incubated at a temperature of, e.g. , about 34°C to about 39°C, about 34°C to about 40°C, about 35°C to about 45°C, about 36°C to about 44°C, about 36°C to about 43°C, about 37°C to about 42°C, about 35°C to about 39°C, about 34°C to about 45°C or about 39°C to about 45°C for a time of, e.g., about 1 hour to about 2 hours, about 1 hour to about 3 hours, about 1 hour to about 4 hours, about 1 hour to about 5 hours, about 1 hour to about 6 hours.
  • Incubation of pre-enrichment media may be performed under constant rotation, reversal or agitation.
  • a sample in a pre-enrichment media may be incubated at a temperature of, e.g., about 34°C to about 39°C, about 34°C to about 40°C, about 35°C to about 45°C, about 36°C to about 44°C, about 36°C to about 43°C, about 37°C to about 42°C, about 35°C to about 39°C, about 34°C to about 45°C or about 39°C to about 45°C for a time of, e.g., about 4 hours to about 1 1 hours, about 5 hours to about 10 hours, about 6 hours to about 9 hours, about 7 hours to about 8 hours, about 5 hours to about 10 hours, about 6 hours to about 9 hours, about 5 hours to about 1 1 hours, about 6 hours to about
  • Incubation of pre-enrichment media may be performed under constant rotation, reversal or agitation.
  • a sample in a pre-enrichment media may be incubated at a temperature of, e.g., about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41 °C, or about 42°C for a time of, e.g.
  • Incubation of pre- enrichment media may be performed under constant rotation, reversal or agitation.
  • a sample in a pre-enrichment media may be incubated at a temperature of, e.g., at least 25°C, at least 26°C, at least 27°C, at least 28°C, at least 29°C, at least 30°C, at least 31 °C, at least 32°C, at least 33°C, at least 34°C, at least 35°C, at least 36°C, at least 37°C, at least 38°C, at least 39°C, at least 40°C, at least 41 °C, or at least 42°C for a time of, e.g., at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 1 1 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least
  • a sample in a pre-enrichment media may be incubated at a temperature of, e.g. , at most 25°C, at most 26°C, at most 27°C, at most 28°C, at most 29°C, at most 30°C, at most 31°C, at most 32°C, at most 33°C, at most 34°C, at most 35°C, at most 36°C, at most 37°C, at most 38°C, at most 39°C, at most 40°C, at most 41 °C, or at most 42°C for a time of, e.g., at most 4 hours, at most 5 hours, at most 6 hours, at most 7 hours, at most 8 hours, at most 9 hours, at most 10 hours, at most
  • Incubation of pre-enrichment media may be performed under constant rotation, reversal or agitation.
  • a sample in a pre-enrichment media may be incubated at a temperature of, e.g. , about 25°C to about 29°C, about 26°C to about 30°C, about 27°C to about 31 °C, about 28°C to about 32°C, about 29°C to about 33°C, about 30°C to about 34°C, about 31 °C to about 35°C, about 32°C to about 36°C, about 33°C to about 37°C, about 34°C to about 38°C, about 35°C to about 39°C, about 36°C to about 40°C, about 37°C to about 41 °C, about 38°C to about 42°C, about 39°C to about 43°C, or about 40°C to about 44°C for a time of, e.g.
  • Incubation of pre-enrichment media may be performed under constant rotation, reversal or agitation.
  • a sample in a pre-enrichment media may be incubated at a temperature of, e.g., about 25°C to about 29°C, about 26°C to about 30°C, about 27°C to about 31 °C, about 28°C to about 32°C, about 29°C to about 33°C, about 30°C to about 34°C, about 31 °C to about 35°C, about 32°C to about 36°C, about 33°C to about 37°C, about 34°C to about 38°C, about 35°C to about 39°C, about 36°C to about 40°C, about 37°C to about 41 °C, about 38°C to about 42°C, about 39°C to about 43°C, or about 40°C to about 44°C for a time of, e.g.
  • a method for the detection of a Salmonella live vaccine bacteria in a sample comprises the step of incubation of the sample in an enrichment media. After a pre-enrichment media step disclosed herein is completed, an aliquot of the pre-enrichment media is transferred to an enrichment media for subsequent growth of the Salmonella live vaccine bacteria.
  • An enrichment step comprises incubating an aliquot of the pre-enrichment media in an enrichment media for a defined time and at a defined temperature.
  • an aliquot volume of pre-enrichment media transferred to an enrichment media may be, e.g., about 1/50, about 1/75, about 1/100, about 1/125, about 1/150, about 1/175, about 1/200, about 1/225, about 1/250, about 1/275, about 1/300, about 1/325, about 1/350, about 1/375, about 1/400, about 1/425, about 1/450, about 1/475, about 1/500, about 1/525, about 1/550, about 1/575, about 1/600, about 1/625, about 1/650, about 1/675, about 1/700, about 1/725, about 1/750, about 1/775, about 1/800, about 1/825, about 1/850, about 1/875, about 1/900,
  • an aliquot volume of pre-enrichment media transferred to an enrichment media may be, e.g., at least 1/50, at least 1/75, at least 1/100, at least 1/125, at least 1/150, at least 1/175, at least 1/200, at least 1/225, at least 1/250, at least 1/275, at least 1/300, at least 1/325, at least 1/350, at least 1/375, at least 1/400, at least 1/425, at least 1/450, at least 1/475, at least 1/500, at least 1/525, at least 1/550, at least 1/575, at least 1/600, at least 1/625, at least 1/650, at least 1/675, at least 1/700, at least 1/725, at least 1/750, at least 1/775, at least 1/800, at least 1/825, at least 1/850, at least 1/875, at least 1/900, at least 1/925, at least 1/950, at least 1/975, or at least 1/1/1/1
  • an aliquot volume of pre-enrichment media transferred to an enrichment media may be, e.g. , at most 1/50, at most 1/75, at most 1/100, at most 1/125, at most 1/150, at most 1/175, at most 1/200, at most 1/225, at most 1/250, at most 1/275, at most 1/300, at most 1/325, at most 1/350, at most 1/375, at most 1/400, at most 1/425, at most 1/450, at most 1/475, at most 1/500, at most 1/525, at most 1/550, at most 1/575, at most 1/600, at most 1/625, at most 1/650, at most 1/675, at most 1/700, at most 1/725, at most 1/750, at most 1/775, at most 1/800, at most 1/825, at most 1/850, at most 1/875, at most 1/900, at most 1/925, at most 1/950, at most 1/975, or at most
  • an aliquot volume of pre-enrichment media transferred to an enrichment media may be, e.g., about 1/5 to about 1/100, about 1/5 to about 1/150, about 1/5 to about 1/200, about 1/5 to about 1/250, about 1/5 to about 1/300, about 1/5 to about 1/350, about 1/5 to about 1/400, about 1/5 to about 1/450, about 1/5 to about 1/500, about 1/5 to about 1/550, about 1/5 to about 1/600, about 1/5 to about 1/650, about 1/5 to about 1/700, about 1/5 to about 1/750, about 1/5 to about 1/800, about 1/5 to about 1/850, about 1/5 to about 1/900, about 1/5 to about 1/950, about 1/5 to about 1/1 ,000, about 1/10 to about 1/100, about 1/10 to about 1/150, about 1/10 to about 1/200, about 1/10 to about 1/250, about 1/10 to about 1/300, about 1/10 to about 1/350
  • a ratio of pre-enrichment media to enrichment media may be, e.g., about 1:5, about 1:10, about 1:25, about 1:50, about 1:75, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, about 1:425, about 1:450, about 1:475, about 1:500, about 1:525, about 1:550, about 1:575, about 1:600, about 1:625, about 1:650, about 1:675, about 1:700, about 1:725, about 1:750, about 1:775, about 1:800, about 1:825, about 1:850, about 1:875, about 1:900, about 1:925, about 1:950, about 1:975, or about 1:1,000.
  • a ratio of pre-enrichment media to enrichment media may be, e.g., at least 1:5, at least 1:10, at least 1:25, at least 1:50, at least 1:75, at least 1:100, at least 1:125, at least 1:150, at least 1:175, at least 1:200, at least 1:225, at least 1:250, at least 1:275, at least 1:300, at least 1:325, at least 1:350, at least 1:375, at least 1:400, at least 1:425, at least 1:450, at least 1:475, at least 1:500, at least 1:525, at least 1:550, at least 1:575, at least 1:600, at least 1:625, at least 1:650, at least 1:675, at least 1:700, at least 1:725, at least 1:750, at least 1:775, at least 1:800, at least 1:825, at least 1:850, at least 1 :875, at least 1 :900
  • a ratio of pre-enrichment media to enrichment media may be, e.g., at most 1:5, at most 1:10, at most 1:25, at most 1:50, at most 1:75, at most 1:100, at most 1:125, at most 1:150, at most 1:175, at most 1:200, at most 1:225, at most 1:250, at most 1:275, at most 1:300, at most 1:325, at most 1:350, at most 1:375, at most 1:400, at most 1:425, at most 1:450, at most 1:475, at most 1:500, at most 1:525, at most 1:550, at most 1:575, at most 1:600, at most 1:625, at most 1:650, at most 1:675, at most 1:700, at most 1:725, at most 1:750, at most 1:775, at most 1:800, at most 1 :825, at most 1 :850, at most 1 :875, at most 1
  • a ratio of pre-enrichment media to enrichment media may be, e.g., about 1:5 to about 1:100, about 1:5 to about 1:150, about 1:5 to about 1:200, about 1:5 to about 1:250, about 1:5 to about 1:300, about 1:5 to about 1:350, about 1:5 to about 1:400, about 1:5 to about 1:450, about 1:5 to about 1:500, about 1:5 to about 1:550, about 1:5 to about 1:600, about 1:5 to about 1:650, about 1:5 to about 1:700, about 1:5 to about 1:750, about 1:5 to about 1 * 800, about 1:5 to about 1:850, about 1:5 to about 1:900, about 1:5 to about 1:950, about 1:5 to about 1:1,000, about 1:10 to about 1:100, about 1:10 to about 1:150, about 1:10 to about 1:200, about
  • An enrichment media also referred to as an enrichment culture media is a buffered culture media that provides the nutrients necessary to sustain high-growth of the Salmonella live vaccine bacteria. This is typically done by tailoring the media particularly conducive to the growth of the Salmonella live vaccine bacteria such as, e.g., considering survivable osmotic pressure ranges, survivable pH ranges, resistance to selective compounds, minimal nutritional requirements.
  • Non-limiting examples of an enrichment media include a Rappaport Vassiliadis Soya Medium (RVS), a McConkey Broth, a Fraser Broth, a Soy-Triptone Broth (TSB), a Reinforce Clostridium Broth, a Campylobacter Thioglycolate Medium, a Nitrate Broth, a Triple Sugar Iron Broth (TSI), a Sodium Hippurate Broth, a Selenite Cystine Broth, a GN Broth, a Todd Hewitt Broth, a Malt Extract Broth, an Azide Dextrose Broth, and a Hektoen Broth.
  • RVS Rappaport Vassiliadis Soya Medium
  • McConkey Broth a Fraser Broth
  • Soy-Triptone Broth a Reinforce Clostridium Broth
  • Campylobacter Thioglycolate Medium a Nitrate Broth
  • TSI Triple Sugar Iron Broth
  • the enrichment media may be a Rappaport Vassiliadis Soya Medium, a Selenite Cystine Broth, or a GN Broth.
  • An enrichment media typically comprises a high growth nutrient component used as a source of proteins, amino acids and nitrogen.
  • a high growth nutrient component may comprise an enrichment media disclosed herein, or a plurality of high growth nutrient components may comprise an enrichment media disclosed herein.
  • a non-limiting example of a high growth nutrient component is a peptone as disclosed herein.
  • a high growth nutrient component may be used at a concentration of, e.g. , about 1 g/L, about 2 g/L, about 3 g/L, about 4 g/L, about 5 g/L, about 6 g/L, about 7 g/L, about 8 g/L, about 9 g/L, about 10 g/L, about 11 g/L, about 12 g/L, about 13 g/L, about 14 g/L, or about 15 g/L.
  • a high growth nutrient component may be used at a concentration of, e.g., at least 1 g/L, at least 2 g/L, at least 3 g/L, at least 4 g/L, at least 5 g/L, at least 6 g/L, at least 7 g/L, at least 8 g/L, at least 9 g/L, at least 10 g/L, at least 11 g/L, at least 12 g/L, at least 13 g/L, at least 14 g/L, or at least 15 g/L.
  • a high growth nutrient component may be used at a concentration of, e.g., at most 1 g/L, at most 2 g/L, at most 3 g/L, at most 4 g/L, at most 5 g/L, at most 6 g/L, at most 7 g/L, at most 8 g/L, at most
  • a high growth nutrient component may be used at a concentration of between, e.g. , about 1 g/L to 2 g/L, about 1 g/L to 3 g/L, about 1 g/L to 4 g/L, about 1 g/L to 5 g/L, about 1 g/L to 6 g/L, about 1 g/L to 7 g/L, about 1 g/L to 8 g/L, about 1 g/L to 9 g/L, about 1 g/L to
  • 5 g/L to 9 g/L about 5 g/L to 10 g/L, about 5 g/L to 1 1 g/L, about 5 g/L to 12 g/L, about 5 g/L to 13 g/L, about 5 g/L to 14 g/L, about 5 g/L to 15 g/L, about 6 g/L to 7 g/L, about 6 g/L to 8 g/L, about 6 g/L to 9 g/L, about 6 g/L to 10 g/L, about 6 g/L to 11 g/L, about 6 g/L to 12 g/L, about 6 g/L to 13 g/L, about 6 g/L to 14 g/L, about 6 g/L to 15 g/L, about 7 g/L to 8 g/L, about 7 g/L to 9 g/L, about 7 g/L to 10 g/L, about 7 g/L to 1
  • An enrichment media typically comprises a growth promoting agent.
  • a growth promoting agent is an iron containing compound that can be used by a Salmonella live vaccine bacteria of interest as an iron source.
  • a growth promoting agent is ammonium ferric citrate.
  • ammonium ferric citrate may be used at a concentration of, e.g. , about 0.01 mg/mL, about 0.02 mg/mL, about 0.03 mg/mL, about 0.04 mg/mL, about 0.05 mg/mL, about 0.06 mg/mL, about 0.07 mg/mL, about 0.08 mg/mL, about 0.09 mg/mL, about 0.1 mg/mL, about 0.2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL, about 0.5 mg/mL, about 0.6 mg/mL, about 0.7 mg/mL, about 0.8 mg/mL, about 0.9 mg/mL, about 1.0 mg/mL, about 2.0 mg/mL, about 3.0 mg/mL, about 4.0 mg/mL, about 5.0 mg/mL, about 6.0 mg/mL
  • ammonium ferric citrate may be used at a concentration of, e.g., at least 0.01 mg/mL, at least 0.02 mg/mL, at least 0.03 mg/mL, at least 0.04 mg/mL, at least 0.05 mg/mL, at least 0.06 mg/mL, at least 0.07 mg/mL, at least 0.08 mg/mL, at least 0.09 mg/mL, at least 0.1 mg/mL, at least 0.2 mg/mL, at least 0.3 mg/mL, at least 0.4 mg/mL, at least 0.5 mg/mL, at least 0.6 mg/mL, at least 0.7 mg/mL, at least 0.8 mg/mL, at least 0.9 mg/mL, at least 1.0 mg/mL, at least 2.0 mg/mL, at least 3.0 mg/mL, at least 4.0 mg/mL, at least 5.0 mg/mL, at least 6.0 mg/mL, at least 7.0 mg/mL, at least
  • ammonium ferric citrate may be used at a concentration of, e.g. , at most 0.01 mg/mL, at most 0.02 mg/mL, at most 0.03 mg/mL, at most 0.04 mg/mL, at most 0.05 mg/mL, at most 0.06 mg/mL, at most 0.07 mg/mL, at most 0.08 mg/mL, at most 0.09 mg/mL, at most 0.1 mg/mL, at most 0.2 mg/mL, at most 0.3 mg/mL, at most 0.4 mg/mL, at most 0.5 mg/mL, at most 0.6 mg/mL, at most 0.7 mg/mL, at most 0.8 mg/mL, at most 0.9 mg/mL, at most 1.0 mg/mL, at most 2.0 mg/mL, at most 3.0 mg/mL, at most 4.0 mg/mL, at most 5.0 mg/mL, at most 6.0 mg/mL, at most 7.0 mg/mL,
  • ammonium ferric citrate may be used at a concentration of, e.g., about 0.01 mg/mL to about 0.05 mg/mL about 0.01 mg/mL to about 0.1 mg/mL, about 0.01 mg/mL to about 0.5 mg/mL, about 0.05 mg/mL to about 0.1 mg/mL, about 0.05 mg/mL to about 0.5 mg/mL, about 0.05 mg/mL to about 1.0 mg/mL, about 0.05 mg/mL to about 5.0 mg/mL, about 0.1 mg/mL to about 0.5 mg/mL, about 0.1 mg/mL to about 1.0 mg/mL, about 0.1 mg/mL to about 5.0 mg/mL, about 0.1 mg/mL to about 10 mg/mL, about 0.1 mg/mL to about 15 mg/mL, about 0.5 mg/mL to about 1.0 mg/mL, about 0.5 mg/mL to about 5.0 mg/mL,
  • An enrichment media typically comprises a growth enhancing agent as disclosed herein in the concentration ranges disclosed herein.
  • an enrichment media comprises 6 g/L to 10 g/L of a peptone, 3 g/L to 7 g/L Bile Salts, 2 g/L to 6 g/L Meat Extract, 2 g/L to 6 g/L of a first growth inhibiting agent, 2 g/L to 6 g/L of a second growth inhibiting agent, 0.001 g/L to 0.008 g/L of a third growth inhibiting agent, and 0.001 g/L to 0.008 g/L of a fourth growth inhibiting agent.
  • an enrichment media comprises 6 g/L to 10 g/L of a Caseine peptone, 3 g/L to 7 g/L of Bile Salts, 2 g/L to 6 g/L of Meat Extract, 2 g/L to 6 g/L of a first iodine compound, 2 g/L to 6 g/L of a second iodine compound, 0.001 g/L to 0.008 g/L of an aminocoumarin antibiotic, and 0.001 g/L to 0.008 g/L of a triarylmethane dye.
  • an enrichment media comprises 6 g/L to 10 g/L of a Caseine peptone, 3 g/L to 7 g/L of Bile Salts, 2 g/L to 6 g/L of Meat Extract, 2 g/L to 6 g/L of a Iodine, 2 g/L to 6 g/L of a Potassium Iodide, 0.001 g/L to 0.008 g/L of Novobiocin, and 0.001 g/L to 0.008 g/L of Brilliant Green.
  • an enrichment media comprises 7.5 g/L to 8.5 g/L of a peptone, 4 g/L to 6 g/L Bile Salts, 3 g/L to 5 g/L Meat Extract, 3 g/L to 5 g/L of a first growth inhibiting agent, 3 g/L to 5 g/L of a second growth inhibiting agent, 0.002 g/L to 0.006 g/L of a third growth inhibiting agent, and 0.002 g/L to 0.006 g/L of a fourth growth inhibiting agent.
  • an enrichment media comprises 7.5 g/L to 8.5 g/L of a Caseine peptone, 4 g/L to 6 g/L of Bile Salts, 3 g/L to 5 g/L of Meat Extract, 3 g/L to 5 g/L of a first iodine compound, 3 g/L to 5 g/L of a second iodine compound, 0.002 g/L to 0.006 g/L of an aminocoumarin antibiotic, and 0.002 g/L to 0.006 g/L of a triarylmethane dye.
  • an enrichment media comprises 7.5 g/L to 8.5 g/L of a Caseine peptone, 4 g/L to 6 g/L of Bile Salts, 3 g/L to 5 g/L g/L of Meat Extract, 3 g/L to 5 g/L of a Iodine, 1 g/L to 3 g/L of a Potassium Iodide, 0.002 g/L to 0.006 g/L of Novobiocin, and 0.002 g/L to 0.006 g/L of Brilliant Green.
  • an enrichment media comprises 8.3 g/L to 8.9 g/L of a peptone, 4.4 g/L to 5.0 g/L Bile Salts, 4.0 g/L to 4.6 g/L Meat Extract, 3.7 g/L to 4.3 g/L of a first growth inhibiting agent, 3.7 g/L to 4.3 g/L of a second growth inhibiting agent, 0.003 g/L to 0.005 g/L of a third growth inhibiting agent, and 0.003 g/L to 0.005 g/L of a fourth growth inhibiting agent.
  • an enrichment media comprises 8.3 g/L to 8.9 g/L of a Caseine peptone, 4.4 g/L to 5.0 g/L of Bile Salts, 4.0 g/L to 4.6 g/L of Meat Extract, 3.7 g/L to 4.3 g/L of a first iodine compound, 3.7 g/L to 4.3 g/L of a second iodine compound, 0.003 g/L to 0.005 g/L of an aminocoumarin antibiotic, and 0.003 g/L to 0.005 g/L of a triarylmethane dye.
  • an enrichment media comprises 8.3 g/L to 8.9 g/L of a Caseine peptone, 4.4 g/L to 5.0 g/L of Bile Salts, 4.0 g/L to 4.6 g/L of Meat Extract, 3.7 g/L to 4.3 g/L of a Iodine, 3.7 g/L to 4.3 g/L of a Potassium Iodide, 0.003 g/L to 0.005 g/L of Novobiocin, and 0.003 g/L to 0.005 g/L of Brilliant Green.
  • a pre-enrichment media comprises 8.6 g/L of a peptone, 4.7 g/L Bile Salts, 4.3 g/L Meat Extract, 4 g/L of a first growth inhibiting agent, 4 g/L of a second growth inhibiting agent, 0.004 g/L of a third growth inhibiting agent, and 0.004 g/L of a fourth growth inhibiting agent.
  • a pre-enrichment media comprises 8.6 g/L of a Caseine peptone, 4.7 g/L of Bile Salts, 4.3 g/L of Meat Extract, 4 g/L of a first iodine compound, 4 g/L of a second iodine compound, 0.004 g/L of an aminocoumarin antibiotic, and 0.004 g/L of a triarylmethane dye.
  • an enrichment media comprises 8.6 g/L of a Caseine peptone, 4.7 g/L of Bile Salts, 4.3 g/L of Meat Extract, 4 g/L of a Iodine, 4 g/L of a Potassium Iodide, 0.004 g/L of Novobiocin, and 0.004 g/L of Brilliant Green.
  • a pre-enrichment media further comprises salts.
  • an enrichment media further comprises NaCI, CaCCb and Na2S203.
  • an enrichment media further comprises 1 .3 g/L to 3.9 g/L NaCI, 37.4 g/L to 40.0 g/L CaC03 and 29.2 g/L to 31.8 g/L a2S203.
  • an enrichment media further comprises 2.3 g/L to 2.9 g/L NaCI, 38.4 g/L to 39.0 g/L CaCOs and 30.2 g/L to 30.8 g/L Na 2 S20 3 .
  • an enrichment media further comprises 2.6 g/L NaCI, 38.7 g/L CaCCb and 30.5 g/L Na 2 S20 3 .
  • aspects of the present specification disclose, in part, incubation of an aliquiot of pre-enrichment media in an enrichment media.
  • Incubation of an aliquiot of pre-enrichment media is performed under temperature and time parameters that facilitate the growth of the Salmonella live vaccine bacteria in the sample, retard the growth of an unwanted organism in the sample, and/or establish conditions that otherwise increase the population of the Salmonella live vaccine bacteria in the sample and/or retard the growth of an unwanted organism in the sample.
  • Incubation of enrichment media may be performed under constant rotation, reversal or agitation.
  • a temperature used to incubate an aliquiot of pre-enrichment media in an enrichment media may be, e.g., about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21 °C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C,
  • a temperature used to incubate an aliquiot of pre-enrichment media in an enrichment media may be, e.g. , at least 15°C, at least 16°C, at least 17°C, at least 18°C, at least 19°C, at least 20°C, at least 21 °C, at least 22°C, at least 23°C, at least 24°C, at least 25°C, at least 26°C, at least 27°C, at least 28°C, at least 29°C, at least 30°C, at least 31 °C, at least 32°C, at least 33°C, at least 34°C, at least 35°C, at least 36°C, at least 37°C, at least 38°C, at least 39°C, at least 40°C, at least 41°C, at least 42°C, at least 43°C, at least 44°C, at least 45°C, at least 46°C, at least 47°C, at least 48°C, at least 49°C
  • a temperature used to incubate an aliquiot of pre-enrichment media in an enrichment media may be, e.g. , at most 15°C, at most 16°C, at most 17°C, at most 18°C, at most 19°C, at most 20°C, at most 21°C, at most 22°C, at most 23°C, at most 24°C, at most 25°C, at most 26°C, at most 27°C, at most 28°C, at most 29°C, at most 30°C, at most 31 °C, at most 32°C, at most 33°C, at most 34°C, at most 35°C, at most 36°C, at most 37°C, at most 38°C, at most 39°C, at most 40°C, at most 41 °C, at most 42°C, at most 43°C, at most 44°C, at most 45°C, at most 46°C, at most 47°C, at most 48°C, at most 49°
  • a temperature used to incubate an aliquiot of pre-enrichment media in an enrichment media may be, e.g. , about 15°C to about 19°C, about 16°C to about 20°C, about 17°C to about 21 °C, about 18°C to about 22°C, about 19°C to about 23°C, about 20°C to about 24°C, about 21°C to about 25°C, about 22°C to about 26°C, about 23°C to about 27°C, about 24°C to about 28°C, about 25°C to about 29°C, about 26°C to about 30°C, about 27°C to about 31 °C, about 28°C to about 32°C, about 29°C to about 33°C, about 30°C to about 34°C, about 31°C to about 35°C, about 32°C to about 36°C, about 33°C to about 37°C, about 34°C to about 38°C, about 35°C to about 39°
  • a temperature used to incubate of a sample in a pre-enrichment media may be, e.g., about 34°C to about 39°C, about 34°C to about 40°C, about 35°C to about 45°C, about 36°C to about 44°C, about 36°C to about 43°C, about 37°C to about 42°C, about 34°C to about 45°C or about 39°C to about 45°C.
  • a time used to incubate an aliquiot of pre-enrichment media in an enrichment media may be, e.g., about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours.
  • a time used to incubate an aliquiot of pre-enrichment media in an enrichment media may be, e.g., at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 1 1 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 7 hours, at least 18 hours, at least 9 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours.
  • a time used to incubate an aliquiot of pre-enrichment media in an enrichment media may be, e.g., at most 2 hours, at most 3 hours, at most 4 hours, at most 5 hours, at most 6 hours, at most 7 hours, at most 8 hours, at most 9 hours, at most 10 hours, at most 1 1 hours, at most 12 hours, at most 13 hours, at most 14 hours, at most 15 hours, at most 16 hours, at most 17 hours, at most 18 hours, at most 19 hours, at most 20 hours, at most 21 hours, at most 22 hours, at most 23 hours, or at most 24 hours.
  • a time used to incubate an aliquiot of pre-enrichment media in an enrichment media may be, e.g., about 2 hours to about 4 hours, about 3 hours to about 5 hours, about 4 hours to about 6 hours, about 5 hours to about 7 hours, about 6 hours to about 8 hours, about 7 hours to about 9 hours, about 8 hours to about 10 hours, about 9 hours to about 1 1 hours, about 10 hours to about 12 hours, about 11 hours to about 13 hours, about 12 hours to about 14 hours, about 13 hours to about 15 hours, about 14 hours to about 16 hours, about 15 hours to about 17 hours, about 16 hours to about 18 hours, about 17 hours to about 19 hours, about 18 hours to about 20 hours, about 19 hours to about 21 hours, about 20 hours to about 22 hours, about 21 hours to about 23 hours, about 22 hours to about 24 hours, about 23 hours to about 25 hours, or about 24 hours to about 26 hours.
  • a time used to incubate an aliquiot of pre-enrichment media in an enrichment media may be, e.g., about 12 hours to about 20 hours, about 13 hours to about 19 hours, about 14 hours to about 18 hours, about 15 hours to about 17 hours, about 12 hours to about 22 hours, about 13 hours to about 21 hours, about 14 hours to about 20 hours, about 15 hours to about 19 hours, about 16 hours to about 18 hours, about 13 hours to about 23 hours, about 14 hours to about 22 hours, about 15 hours to about 21 hours, about 16 hours to about 20 hours or about 17 hours to about 19 hours.
  • an aliquiot of pre-enrichment media in an enrichment media may be incubated at a temperature of, e.g., about 34°C to about 39°C, about 34°C to about 40°C, about 35°C to about 45°C, about 36°C to about 44°C, about 36°C to about 43°C, about 37°C to about 42°C, about 35°C to about 39°C, about 34°C to about 45°C or about 39°C to about 45°C for a time of, e.g., about 12 hours to about 20 hours, about 13 hours to about 19 hours, about 14 hours to about 18 hours, about 15 hours to about 17 hours, about 12 hours to about 22 hours, about 13 hours to about 21 hours, about 14 hours to about 20 hours, about 15 hours to about 19 hours, about 16 hours to about 18 hours, about 13 hours to about 23 hours, about 14 hours to about 22 hours, about 15 hours to about 21 hours, about 16 hours to about 20 hours or about 17 hours to about
  • an aliquiot of pre-enrichment media in an enrichment media may be incubated at a temperature of, e.g. , about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, or about 50°C for a time of, e.g.
  • Incubation of the enrichment media may be under constant rotation, reversal or agitation.
  • an aliquiot of pre-enrichment media in an enrichment media may be incubated at a temperature of, e.g. , at least 15°C, at least 16°C, at least 17°C, at least 18°C, at least 19°C, at least 20°C, at least 21°C, at least 22°C, at least 23°C, at least 24°C, at least 25°C, at least 26°C, at least 27°C, at least 28°C, at least 29°C, at least 30°C, at least 31°C, at least 32°C, at least 33°C, at least 34°C, at least 35°C, at least 36°C, at least 37°C, at least 38°C, at least 39°C, at least 40°C, at least 41 °C, at least 42°C, at least 43°C, at least 44°C, at least 45°C, at least 46°C, at least 47°C, at least 48°C, at least 49
  • Incubation of the enrichment media may be under constant rotation, reversal or agitation.
  • an aliquiot of pre-enrichment media in an enrichment media may be incubated at a temperature of, e.g. , at most 15°C, at most 16°C, at most 17°C, at most 18°C, at most 19°C, at most 20°C, at most 21°C, at most 22°C, at most 23°C, at most 24°C, at most 25°C, at most 26°C, at most 27°C, at most 28°C, at most 29°C, at most 30°C, at most 31 °C, at most 32°C, at most 33°C, at most 34°C, at most 35°C, at most 36°C, at most 37°C, at most 38°C, at most 39°C, at most 40°C, at most 41 °C, at most 42°C, at most 43°C, at most 44°C, at most 45°C, at most 46°C, at most 47°C, at most 48°C, at a temperature of, e.g.
  • an aliquiot of pre-enrichment media in an enrichment media may be incubated at a temperature of, e.g. , about 15°C to about 19°C, about 16°C to about 20°C, about 17°C to about 21 °C, about 18°C to about 22°C, about 19°C to about 23°C, about 20°C to about 24°C, about 21°C to about 25°C, about 22°C to about 26°C, about 23°C to about 27°C, about 24°C to about 28°C, about 25°C to about 29°C, about 26°C to about 30°C, about 27°C to about 31 °C, about 28°C to about 32°C, about 29°C to about 33°C, about 30°C to about 34°C, about 31 °C to about 35°C, about 32°C to about 36°C, about 33°C to about 37°C, about 34°C to about 38°C, about 35°C
  • a Salmonella live vaccine bacteria may be subsequently purified after one or more incubation steps.
  • a Salmonella live vaccine bacteria may be purified after incubation in an enrichment media and/or after incubation in a second pre-enrichment media.
  • Purification of a Salmonella live vaccine bacteria includes capture of the Salmonella live vaccine bacteria to a more concentrated form and/or removal contaminating microorganism, impurities, and debris.
  • a purification step disclosed herein increases detection of a Salmonella live vaccine bacteria by increasing the concentration of Salmonella live vaccine bacteria and/or decreasing contaminants, thereby increasing the level of detectable signal measured in a subsequent detection step.
  • Common purification procedures used to capture a Salmonella live vaccine bacteria and/or remove contaminating microorganism, impurities, and debris include affinity chromatography, ion-exchange chromatography, size exclusion chromatography, hydrophobic-interaction chromatography, ceramic hydroxyapatite chromatography, reverse-phase HPLC, gel filtration, precipitation, immuno-precipitation, diafiltration, chromatofocusing.
  • a Salmonella live vaccine bacteria is purified after an inclubation step using an immuno-precipitation using antibodies or aptamers for a Salmonella live vaccine bacteria of interest.
  • Immunoprecipitation is the technique of precipitating an antigen out of solution using an antibody or aptamer that specifically binds to that antigen. Immunoprecipitation requires that the antibody be coupled to a solid substrate at some point in the procedure. This is typically done using standard coupling procedures known in the art. Examples of solid substrates include agarose particles and magnetic particles.
  • the immuno-precipitation method employs antibodies or aptamers for a Salmonella live vaccine bacteria linked to magnetic particles.
  • any concentration of magnetic particles linked with an antibody or apatmer may be used during its incubation during a purification step, with the proviso that the concentration is useful to practice the methods disclosed herein.
  • a concentration of magnetic particles linked with an antibody or apatmer used during a purification step may be, e.g.
  • a concentration of magnetic particles linked with an antibody or apatmer used during a purification step may be, e.g.
  • a concentration of magnetic particles linked with an antibody or apatmer used during a purification step may be, e.g.
  • At most 1 x 10" immunmagnetic particles/mL of media comprising the Salmonella live vaccine bacteria of interest at most 1 x 10 5 immunmagnetic particles/mL of media comprising the Salmonella live vaccine bacteria of interest, at most 1 x 10 6 immunmagnetic particles/mL of media comprising the Salmonella live vaccine bacteria of interest, at most 1 x 10 7 immunmagnetic particles/mL of media comprising the Salmonella live vaccine bacteria of interest, or at most 1 x 10 8 immunmagnetic particles/mL of media comprising the Salmonella live vaccine bacteria of interest.
  • a concentration of magnetic particles linked with an antibody or apatmer used during a purification step may be, e.g., about 1 x 10 4 to about 1 x 10 5 immunmagnetic particles/mL of media comprising the Salmonella live vaccine bacteria of interest, about 1 x 10 4 to about 1 x 10 6 immunmagnetic particles/mL of media comprising the Salmonella live vaccine bacteria of interest, about 1 x 10 4 to about 1 x 10 7 immunmagnetic particles/mL of media comprising the Salmonella live vaccine bacteria of interest, about 1 x 10 4 to about 1 x 10 8 immunmagnetic particles/mL of media comprising the Salmonella live vaccine bacteria of interest, about 1 x 10 5 to about 1 x 10 6 immunmagnetic particles/mL of media comprising the Salmonella live vaccine bacteria of interest, about 1 x 10 5 to about 1 x 10 7 immunmagnetic particles/mL of media comprising the Salmonella live vaccine bacteria of interest.
  • aspects of the present specification disclose, in part, incubation of immunmagnetic particles with media comprising the Salmonella live vaccine bacteria of interest.
  • Incubation of immunmagnetic particles with media comprising the Salmonella live vaccine bacteria of interest is performed under temperature and time parameters that facilitate binding of the Salmonella live vaccine bacteria to the magnetic particles linked with an antibody or apatmer for the Salmonella live vaccine bacteria of interest.
  • Incubation of immunmagnetic particles with media comprising the Salmonella live vaccine bacteria of interest may be performed under agitation/rotation.
  • a temperature used to incubate immunmagnetic particles with media comprising the Salmonella live vaccine bacteria of interest may be, e.g., about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C
  • a temperature used to incubate immunmagnetic particles with media comprising the Salmonella live vaccine bacteria of interest may be, e.g., at least 15°C, at least 16°C, at least 17°C, at least 18°C, at least 19°C, at least 20°C, at least 21 °C, at least 22°C, at least 23°C, at least 24°C, at least 25°C, at least 26°C, at least 27°C, at least 28°C, at least 29°C, at least 30°C, at least 31°C, at least 32°C, at least 33°C, at least 34°C, at least 35°C, at least 36°C, at least 37°C, at least 38°C, at least 39°C, at least 40°C, at least 41 °C, at least 42°C, at least 43°C, at least 44°C, at least 45°C, at least 46°C, at least 47°C, at least 48°C, at least 49°C
  • a temperature used to incubate immunmagnetic particles with media comprising the Salmonella live vaccine bacteria of interest may be, e.g., at most 5°C, at most 16°C, at most 17°C, at most 18°C, at most 19°C, at most 20°C, at most 21°C, at most 22°C, at most 23°C, at most 24°C, at most 25°C, at most 26°C, at most 27°C, at most 28°C, at most 29°C, at most 30°C, at most 31 °C, at most 32°C, at most 33°C, at most 34°C, at most 35°C, at most 36°C, at most 37°C, at most 38°C, at most 39°C, at most 40°C, at most 41°C, at most 42°C, at most 43°C, at most 44°C, at most 45°C, at most 46°C, at most 47°C, at most 48°C, at most 49°C
  • a temperature used to incubate immunmagnetic particles with media comprising the Salmonella live vaccine bacteria of interest may be, e.g., about 15°C to about 19°C, about 16°C to about 20°C, about 17°C to about 21°C, about 18°C to about 22°C, about 19°C to about 23°C, about 20°C to about 24°C, about 21 °C to about 25°C, about 22°C to about 26°C, about 23°C to about 27°C, about 24°C to about 28°C, about 25°C to about 29°C, about 26°C to about 30°C, about 27°C to about 31°C, about 28°C to about 32°C, about 29°C to about 33°C, about 30°C to about 34°C, about 31 °C to about 35°C, about 32°C to about 36°C, about 33°C to about 37°C, about 34°C to about 38°C, about 35°C to about 39°
  • a temperature used to incubate immunmagnetic particles with media comprising the Salmonella live vaccine bacteria of interest may be, e.g., about 34°C to about 39°C, about 34°C to about 40°C, about 35°C to about 45°C, about 36°C to about 44°C, about 36°C to about 43°C, about 37°C to about 42°C, about 34°C to about 45°C or about 39°C to about 45°C.
  • a time used to incubate immunmagnetic particles with media comprising the Salmonella live vaccine bacteria of interest may be, e.g., about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 1 10 minutes, about 120 minutes, about 130 minutes, about 140 minutes, or about 150 minutes.
  • a time used to incubate immunmagnetic particles with media comprising the Salmonella live vaccine bacteria of interest may be, e.g. , at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 1 10 minutes, at least 120 minutes, at least 130 minutes, at least 140 minutes, or at least 150 minutes.
  • a time used to incubate immunmagnetic particles with media comprising the Salmonella live vaccine bacteria of interest may be, e.g.
  • a time used to incubate immunmagnetic particles with media comprising the Salmonella live vaccine bacteria of interest may be, e.g. , about 5 minutes to about 20 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 40 minutes, about 5 minutes to about 50 minutes, about 5 minutes to about 60 minutes, about 5 minutes to about 70 minutes, about 5 minutes to about 80 minutes, about 5 minutes to about 90 minutes, about 5 minutes to about 100 minutes, about 5 minutes to about 1 10 minutes, about 5 minutes to about 120 minutes, about 5 minutes to about 130 minutes, about 5 minutes to about 140 minutes, about 5 minutes to about 150 minutes, about 10 minutes to about 20 minutes, about 10 minutes to about 30 minutes, about 10 minutes to about 40 minutes, about 10 minutes to about 50 minutes, about 10 minutes to about 60 minutes, about 10 minutes to about 70 minutes, about 10 minutes to about 80 minutes, about 10 minutes to about 90 minutes, about 10 minutes to about 100 minutes, about 10 minutes to about 1 10 minutes, about 10 minutes to about 120 minutes, about 10 minutes
  • immunmagnetic particles may be incubated with media comprising the Salmonella live vaccine bacteria of interest at a temperature of, e.g. , about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41 °C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, or about 50°C for a time of, e.g.
  • immunmagnetic particles may be incubated with media comprising the Salmonella live vaccine bacteria of interest at a temperature of, e.g. , at least 15°C, at least 16°C, at least 17°C, at least 18°C, at least 19°C, at least 20°C, at least 21°C, at least 22°C, at least 23°C, at least 24°C, at least 25°C, at least 26°C, at least 27°C, at least 28°C, at least 29°C, at least 30°C, at least 31 °C, at least 32°C, at least 33°C, at least 34°C, at least 35°C, at least 36°C, at least 37°C, at least 38°C, at least 39°C, at least 40°C, at least 41°C, at least 42°C, at least 43°C, at least 44°C, at least 45°C, at least 46°C, at least 47°C, at least 48°C, at least
  • immunmagnetic particles may be incubated with media comprising the Salmonella live vaccine bacteria of interest at a temperature of, e.g. , at most 15°C, at most 16°C, at most 17°C, at most 18°C, at most 19°C, at most 20°C, at most 21°C, at most 22°C, at most 23°C, at most 24°C, at most 25°C, at most 26°C, at most 27°C, at most 28°C, at most 29°C, at most 30°C, at most 31 °C, at most 32°C, at most 33°C, at most 34°C, at most 35°C, at most 36°C, at most 37°C, at most 38°C, at most 39°C, at most 40°C, at most 41 °C, at most 42°C, at most 43°C, at most 44°C, at most 45°C, at most 46°C, at most 47°C, at most 48°C,
  • immunmagnetic particles may be incubated with media comprising the Salmonella live vaccine bacteria of interest at a temperature of, e.g., about 15°C to about 19°C, about 16°C to about 20°C, about 17°C to about 21 °C, about 18°C to about 22°C, about 19°C to about 23°C, about 20°C to about 24°C, about 21°C to about 25°C, about 22°C to about 26°C, about 23°C to about 27°C, about 24°C to about 28°C, about 25°C to about 29°C, about 26°C to about 30°C, about 27°C to about 31°C, about 28°C to about 32°C, about 29°C to about 33°C, about 30°C to about 34°C, about 31 °C to about 35°C, about 32°C to about 36°C, about 33°C to about 37°C, about 34°C to about 38°C, about 35°C to
  • the Salmonella live vaccine bacteria-bound immunoparticles may be isolated from the media.
  • immunoparticles may be isolated from the media using a magnetic separator which concentrates the immunoparticles in a specific location allowing for the media to be removed.
  • immunoparticles may be isolated from the media by centrifuged to concentrate the immunoparticles, thereby allowing for the media to be removed.
  • the isolated Salmonella live vaccine bacteria-bound immunoparticles may be washed one or more times using a buffered solution. Subsequent isolation of the immunoparticles may be accomplished using a magnetic separator or centrifugation and removal of the buffered solution.
  • a purification step disclosed herein is optional.
  • a method of detecting a Salmonella live vaccine bacteria disclosed herein does not comprise a purification of a Salmonella live vaccine bacteria from a pre-enrichment media, an enrichment media, or both a pre- enrichment media and an enrichment media.
  • a method of detecting a Salmonella live vaccine bacteria disclosed herein does not comprise a purification of a Salmonella live vaccine bacteria from an enrichment media, a second pre-enrichment media, or both an enrichment media and a second pre-enrichment media.
  • a method of detecting a Salmonella live vaccine bacteria disclosed herein does not comprise a purification of a Salmonella live vaccine bacteria from a first pre- enrichment media.
  • presence or absence of a Salmonella live vaccine bacteria of interest may be determined by qualitatively or quantitatively measuring the amount of Salmonella live vaccine bacteria contained within the media.
  • detection presence or absence of a Salmonella live vaccine bacteria of interest occurs without the need of a purification step disclosed herein.
  • detection presence or absence of a Salmonella live vaccine bacteria of interest occurs after completion of a purification step disclosed herein.
  • media comprising a Salmonella live vaccine bacteria of interest may be processed to remove debris and other contaminants without any Salmonella live vaccine bacteria purification.
  • media comprising a Salmonella live vaccine bacteria of interest is centrifuged to remove debris.
  • a secondary antibody to boast a detection signal is optional.
  • a method of detecting a Salmonella live vaccine bacteria does not comprise use of a secondary antibody to boast a detection signal.
  • a method disclosed herein may qualitatively or quantitatively detect Salmonella live vaccine bacteria having a concentration in a sample disclosed herein of, e.g. , about 1 x 10 " 5 cfu/mL, about 1 x 10' 4 cfu/mL, about 1 x 10 ⁇ 3 cfu/mL, about 1 x 10 "2 cfu/mL, about 1 x 10 "1 cfu/mL, about x 10° cfu/mL, about 1 x 10 1 cfu/mL, about 1 x 10 2 cfu/mL, about 1 x 10 3 cfu/mL, about 1 x 10 4 cfu/mL, about 1 x 10 5 cfu/mL, about 1 x 10 s cfu/mL, about 1 x 10 7 cfu/mL, about 1 x 10 s cfu/mL, about 1 x
  • a method disclosed herein may qualitatively or quantitatively detect Salmonella live vaccine bacteria having a concentration in a sample disclosed herein of, e.g. , at most 1 x 10 5 cfu/mL, at most 1 x 10 "4 cfu/mL, at most 1 x 10 -3 cfu/mL, at most 1 x 10 "2 cfu/mL, at most 1 x 10 -1 cfu/mL, at most 1 x 10° cfu/mL, at most 1 x 10 1 cfu/mL, at most 1 x 10 2 cfu/mL, at most 1 x 10 3 cfu/mL, at most 1 x 10 4 cfu/mL, at most 1 x 10 5 cfu/mL, at most 1 x 10 6 cfu/mL, at most 1 x 10 7 cfu/mL, at most 1 x 10 s c
  • a method disclosed herein may qualitatively or quantitatively detect Salmonella live vaccine bacteria having a concentration after a pre-enrichment step of, e.g. , about 1 x 1 Cr 5 cfu/mL, about 1 x 10 -4 cfu/mL, about 1 x 10 ⁇ 3 cfu/mL, about 1 x 10 "2 cfu/mL, about 1 x 10 '1 cfu/mL, about 1 x 10° cfu/mL, about 1 x 10 1 cfu/mL, about 1 x 10 2 cfu/mL, about 1 x 10 3 cfu/mL, about 1 x 10 4 cfu/mL, about 1 x 10 5 cfu/mL, about 1 x 10 6 cfu/mL, about 1 x 10 7 cfu/mL, about 1 x 10 8 cfu/mL, about 1
  • a method disclosed herein may qualitatively or quantitatively detect Salmonella live vaccine bacteria having a concentration after a pre-enrichment step of, e.g., at least 1 x 10 -5 cfu/mL, at least 1 x 10 "4 cfu/mL, at least 1 x 10 -3 cfu/mL, at least 1 x 10 2 cfu/mL, at least 1 x 10 "1 cfu/mL, at least 1 x 10° cfu/mL, at least 1 x 10 1 cfu/mL, at least 1 x 10 2 cfu/mL, at least 1 x 10 3 cfu/mL, at least 1 x 10 4 cfu/mL, at least 1 x 10 5 cfu/mL, at least 1 x 10 6 cfu/mL, at least 1 x 10 7 cfu/mL, at least 1 x 10 s c
  • a method disclosed herein may qualitatively or quantitatively detect Salmonella live vaccine bacteria having a concentration after a pre-enrichment step of, e.g., at most 1 x 10 5 cfu/mL, at most 1 x 10- 4 cfu/mL, at most 1 x 10 "3 cfu/mL, at most 1 x 10 "2 cfu/mL, at most 1 x 10 "1 cfu/mL, at most 1 x 10° cfu/mL, at most 1 x 10 1 cfu/mL, at most 1 x 10 2 cfu/mL, at most 1 x 10 3 cfu/mL, at most 1 x 10 4 cfu/mL, at most 1 x 10 5 cfu/mL, at most 1 x 10 6 cfu/mL, at most 1 x 10 7 cfu/mL, at most 1 x 10 8 cf
  • a method disclosed herein may qualitatively or quantitatively detect Salmonella live vaccine bacteria having a concentration after a pre-enrichment step of, e.g. , about 1 x 10 -5 cfu/mL to about about 1 x 10 4 cfu/mL, about 1 x 10 ⁇ 5 cfu/mL to about about 1 x 10 3 cfu/mL, about 1 x 10 "5 cfu/mL to about about 1 x 10 2 cfu/mL, about 1 x 1 Cr 5 cfu/mL to about about 1 x 10 "1 cfu/mL, about 1 x 10 -5 cfu/mL to about about 1 x 10° cfu/mL, about 1 x 10 5 cfu/mL to about about 1 x 10 1 cfu/ml_, about 1 x 10 5 cfu/mL to about 1 x 10 2
  • a method disclosed herein may qualitatively or quantitatively detect Salmonella live vaccine bacteria having a concentration after an enrichment step of, e.g. , about 1 x 10 ⁇ 5 cfu/mL, about 1 x 10 -4 cfu/mL, about 1 x 10 "3 cfu/mL, about 1 x 1 Cr 2 cfu/mL, about 1 x 10 ⁇ 1 cfu/mL, about 1 x 10° cfu/mL, about 1 x 10 1 cfu/mL, about 1 x 10 2 cfu/mL, about 1 x 10 3 cfu/mL, about 1 x 10 4 cfu/mL, about 1 x 10 5 cfu/mL, about 1 x 10 6 cfu/mL, about 1 x 10 7 cfu/mL, about 1 x 10 8 cfu/mL, about 1 x 10 9
  • a method disclosed herein may qualitatively or quantitatively detect Salmonella live vaccine bacteria having a concentration after an enrichment step of, e.g., at least 1 x 10 5 cfu/mL, at least 1 x 10 4 cfu/mL, at least 1 x 10 3 cfu/mL, at least 1 x 10 "2 cfu/mL, at least 1 x 10 "1 cfu/mL, at least 1 x 10° cfu/mL, at least 1 x 10 cfu/mL, at least 1 x 10 2 cfu/mL, at least 1 x 10 3 cfu/mL, at least 1 x 10 4 cfu/mL, at least 1 x 10 5 cfu/mL, at least 1 x 10 6 cfu/mL, at least 1 x 10 7 cfu/mL, at least 1 x 10 8 cfu/mL, at least
  • a method disclosed herein may qualitatively or quantitatively detect Salmonella live vaccine bacteria having a concentration after an enrichment step of, e.g., about 1 x 1 CH 5 cfu/mL to about about 1 x 10 4 cfu/mL, about 1 x 10 5 cfu/mL to about about 1 x 10 "3 cfu/mL, about 1 x 10 "5 cfu/mL to about about 1 x 10 2 cfu/mL, about 1 x 10 5 cfu/mL to about about 1 x 10 cfu/mL, about 1 x 10 "5 cfu/mL to about about 1 x 10° cfu/mL, about 1 x 10 '5 cfu/mL to about about 1 x 10 1 cfu/mL, about 1 x 10 5 cfu/mL to about about 1 x 10 2 cfu/mL, about
  • nucleic acid-based detection method includes DNA-based detection methods and RNA-based detection methods.
  • DNA-based detection methods include, without limitation, like Southern blot analysis, PCR-based assays, sequence analysis, immuno-based detection assays, and hybridization assays using FRET, polarization or other fluorescent, chemiluminescent or bioluminescent detection methods.
  • RNA-based detection methods include, without limitation, like Northern blot analysis, RT-PCR-based assays, RNA sequencing, immuno-based detection assays, and hybridization assays using FRET, polarization or other fluorescent, chemiluminescent or bioluminescent detection methods.
  • detection presence or absence of a Salmonella live vaccine bacteria of interest occurs using a protein-based detection method.
  • protein-based detection methods include gel-based detection methods, immuno-based detection methods and protein-interaction-based methods.
  • Gel-based detection methods include, without limitation polyacrylamide gel electrophoresis and SDS-PAGE.
  • Immuno-based detection methods include, without limitation, Western blot analysis, ELISA, and immunoprecipitation.
  • Protein-interaction-based methods include, without limitation, protein-protein interaction-based assays, protein-DNA interaction-based assays, and protein-RNA interaction-based assays.
  • detection presence or absence of a Salmonella live vaccine bacteria of interest occurs using an activity-based detection method.
  • activity-based detection methods include enzymatic activity assays and assays based on protein function.
  • An enzymatic activity assays typically involves incubating an aliquot of media containing or potentially containing a Salmonella live vaccine bacteria of interest in a buffered solution containing a suitable substrate. If the desired enzyme is present in the aliquot, then is will catalyze the conversion of the substrate into a product. Measuring either the loss of substrate or the formation of product can then be either qualitatively or quantitatively correlated to the amount of enzyme present and hence the amount of Salmonella live vaccine bacteria.
  • assays based on protein function measure the amount of function present in the sample and extrapolate the amount of Salmonella live vaccine bacteria based on this measurement.
  • detection presence or absence of a Salmonella live vaccine bacteria of interest occurs using a growth-based detection method.
  • growth-based detection method include plating assays measuring colony formation with or without growth selective agents and spectrophotometer assays measuring cell density.
  • a plating assay typically involves plating an aliquot of media onto an agar plate comprising nutrients to sustain growth of the Salmonella live vaccine bacteria of interest. The inoculated agar plates are then incubated for a specified temperature and time and growth of Salmonella live vaccine bacteria colonies assessed. In some embodiments, the agar plates are incubated at about 25°C to about 42°C for about 12 hours to about 48 hours.
  • the agar plates are incubated at about 37°C for about 14 hours to about 16 hours. This assessment may be qualitative by simply assessing the presence or absence of Salmonella live vaccine bacteria colonies, or quantitative, where the number of Salmonella live vaccine bacteria colonies are counted.
  • an agar plate may contain a chromogenic compound that stains or otherwise provides a visual signal that identifies the colony as a Salmonella live vaccine bacteria colony.
  • an agar plate may contain a compound that selects for Salmonella live vaccine bacteria colony growth by either supplying a compound that facilitates or is required for Salmonella live vaccine bacteria growth or inhibits the growth of contaminating microorganisms.
  • a spectrophotometer assays measuring cell density typically involves measuring the cell density of an aliquot taken from media using a spectrophotometer at a specific wavelength.
  • detection presence or absence of a Salmonella live vaccine bacteria of interest occurs using a sensor-based detection method.
  • Sensors can be classified according to the type of energy being transfer, such as, e.g., thermal, electromagnetic, mechanical, and electrochemical.
  • a sensor-based detection method is a biosensor-based detection method.
  • a biosensor is a type of analytical device incorporating a biological material, a biologically derived material or a biomimic intimately associated with or integrated within a physicochemical transducer or transducing microsystem.
  • a biosensor converts the modification of the physical or chemical properties of a biomatrix (e.g., enzyme, antibodies, receptors, organelles, microorganisms) into an electric or other kinds of signal whose amplitude depends on the concentration of defined analytes in the solution.
  • Non-limiting examples of a biosensor include an enzymatic biosensor, a DNA sensor, and an immunosensor.
  • Enzyme-based biosensor require the immobilization of an enzyme onto an electrode surface for the quantification of an analyte.
  • DNA-based biosensor require the immobilization of non- complimentary DNA strand of a target sequence onto an electrode surface for the quantification of an analyte.
  • Immuno-based biosensor require the immobilization of an antibody onto an electrode surface for the quantification of an analyte.
  • a biosensor comprises a biorecognition element, a signal transducer, and a detector.
  • the biorecognition element includes antibodies, peptides, nucleic acids, or enzymes and is the portion of the sensor that initially binds to or interacts with the analyte (e.g., a Salmonella live vaccine bacteria). In many cases this is associated with a conformational change, substrate cleavage, or enzymatic reaction that transduces the biorecognition event into a signal that may be detected via several modalities.
  • a biosensor comprises two electrodes (reference and a working electrodes) or three electrodes (reference, working, and counter electrode).
  • a reference electrode includes liquid and solid-state reference electrodes and is an electrode having a known stable potential to which all other electrode potentials are referenced.
  • a reference electrode can be manufactured by film deposition, electroplating and screen printing.
  • Non-limiting examples of a reference electrode include silver-silver chloride electrode, calomel electrode, hydrogen electrode, mercury-mercury oxide electrode, mercury-mercurous sulfate electrode, copper-copper sulfate electrode, and palladium hydride electrode.
  • a biosensor typically uses the power of electrochemical techniques for biological processes by quantitatively producing an electrical signal that relates to the concentration of a biological analyte.
  • electrochemical biosensors can be classified as potentiometric, amperometric, voltametric and impedimetric/conductimetric based upon the analytical principles of operation.
  • Potentiometric electrochemical sensors measure an equilibrium potential difference between a working electrode and a reference electrode at zero current flow.
  • Voltammetric electrochemical sensors measure the current as a function of varying potential is applied between a working electrode and a reference electrode.
  • Amperometric electrochemical sensors measure the current as a function of constant potential is applied between a working electrode and a reference electrode.
  • Impedimetric/conductimetric electrochemical sensors measure the changes of electrical properties between a working electrode and a reference electrode. Electrochemical detection using a biosensor may be a direct measurement of the change in electrical properties or an indirect measurement using an auxiliary reaction which involves a marking (redox active) compound for signal generation.
  • a detection solution typically includes a buffered solution comprises magnesium chloride, p-aminofenil phosphate, and glucose.
  • a detection solution may comprise a surfactant as disclosed herein.
  • Any buffer may be used, with the proviso that the resulting buffered solution is useful to practice the methods disclosed herein.
  • a buffered solution can be varied as appropriate by one skilled in the art and generally depends, in part, on the pH value desired for the mobile phase, the protein being eluted, and the conductivity values being employed.
  • aspects of this embodiment may optionally include, e.g., 2-(N-morpholino) ethanesulfonic acid (MES), N-(2-acetamido)iminodiacetic acid (ADA), dimethylarsinic acid (Cacodylate), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), N-(2-Acetamido)-2- aminoethanesulfonic acid (ACES), cholamine chloride, N,N'-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-(N-morpholino) propanesulfonic acid (MOPS), 2- ⁇ [tris(hydroxymethyl)methyl]amino ⁇ ethanesulfonic acid (TES), N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid) (HEPES), piperazine- N,N'-bis(2-hydroxypropanesulfonic acid
  • Non-limiting examples of how to make and use specific buffers are described in, e.g., MOLECULAR CLONING, A LABORATORY MANUAL (Joseph Sambrook & David W. Russell eds., Cold Spring Harbor Laboratory Press, 3rd ed. 2001 ) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Frederick M. Ausubel et al., eds. John Wiley & Sons, 2004).
  • a concentration of magnesium chloride used in a detection solution may be, e.g., about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM.
  • a concentration of magnesium chloride used in a detection solution may be, e.g., at least 5 mM, at least 10 mM, at least 15 mM, at least 20 mM, at least 25 mM, at least 30 mM, at least 35 mM, at least 40 mM, at least 45 mM, at least 50 mM, at least 55 mM, at least 60 mM, at least 65 mM, at least 70 mM, at least 75 mM, at least 80 mM, at least 85 mM, at least 90 mM, at least 95 mM, or at least 100 mM.
  • a concentration of magnesium chloride used in a detection solution may be, e.g. , at most 5 mM, at most 10 mM, at most 15 mM, at most 20 mM, at most 25 mM, at most 30 mM, at most 35 mM, at most 40 mM, at most 45 mM, at most 50 mM, at most 55 mM, at most 60 mM, at most 65 mM, at most 70 mM, at most 75 mM, at most 80 mM, at most 85 mM, at most 90 mM, at most 95 mM, or at most 100 mM.
  • a concentration of magnesium chloride used in a detection solution may be, e.g., about 5 mM to about 10 mM, about 5 mM to about 20 mM, about 5 mM to about 30 mM, about 5 mM to about 40 mM, about 5 mM to about 50 mM, about 5 mM to about 60 mM, about 5 mM to about 70 mM, about 5 mM to about 80 mM, about 5 mM to about 90 mM, about 5 mM to about 100 mM, about 10 mM to about 20 mM, about 10 mM to about 30 mM, about 10 mM to about 40 mM, about 10 mM to about 50 mM, about 10 mM to about 60 mM, about 10 mM to about 70 mM, about 10 mM to about 80 mM, about 10 mM to about 90 mM, about 10 mM to about 100 mM, about 10 m
  • concentration of p-aminofenil phosphate may be used in a detection solution, with the proviso that the concentration is useful to practice the methods disclosed herein.
  • a concentration of p-aminofenil phosphate used in a detection solution may be, e.g.
  • a concentration of p- aminofenil phosphate used in a detection solution may be, e.g., at least 0.1 mM, at least 0.2 mM, at least 0.3 mM, at least 0.4 mM, at least 0.5 mM, at least 0.6 mM, at least 0.7 mM, at least 0.8 mM, at least 0.9 mM, at least 1.0 mM, at least 1.1 mM, at least 1.2 mM, at least 1.3 mM, at least 1.4 mM, at least 1.5 mM, at least 1.6 mM, at least 1.7 mM, at least 1.8 mM, at least 1.9 mM, at least 2.0 mM, at least 2.1 mM, at least 2.2 mM, at least 2.3 mM, at least 2.4 mM, or at least 2.5 mM.
  • a concentration of p-aminofenil phosphate used in a detection solution may be, e.g. , at most 0.1 mM, at most 0.2 mM, at most 0.3 mM, at most 0.4 mM, at most 0.5 mM, at most 0.6 mM, at most 0.7 mM, at most 0.8 mM, at most 0.9 mM, at most 1.0 mM, at most 1.1 mM, at most 1.2 mM, at most 1.3 mM, at most 1.4 mM, at most 1.5 mM, at most 1.6 mM, at most 1.7 mM, at most 1.8 mM, at most 1.9 mM, at most 2.0 mM, at most 2.1 mM, at most 2.2 mM, at most 2.3 mM, at most 2.4 mM, or at most 2.5 mM.
  • a concentration of p-aminofenil phosphate used in a detection solution may be, e.g. , about 0.1 mM to about 0.5 mM, about 0.1 mM to about 1.0 mM, about 0.1 mM to about 1.5 mM, about 0.1 mM to about 2.0 mM, about 0.1 mM to about 2.5 mM, about 0.3 mM to about 0.5 mM, about 0.3 mM to about 1.0 mM, about 0.3 mM to about 1.5 mM, about 0.3 mM to about 2.0 mM, about 0.3 mM to about 2.5 mM, about 0.5 mM to about 1.0 mM, about 0.5 mM to about 1.5 mM, about 0.5 mM to about 2.0 mM, about 0.5 mM to about 2.5 mM, about 0.7 mM to about 1.0 mM, about 0.7 mM to about 1.5 mM, about 0.7 mM to about 1.5
  • a concentration of glucose used in a detection solution may be, e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM.
  • a concentration of glucose used in a detection solution may be, e.g., at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 15 mM, at least 20 mM, at least 25 mM, at least 30 mM, at least 35 mM, at least 40 mM, at least 45 mM, or at least 50 mM.
  • a concentration of glucose used in a detection solution may be, e.g., at most 1 mM, at most 2 mM, at most 3 mM, at most 4 mM, at most 5 mM, at most 6 mM, at most 7 mM, at most 8 mM, at most 9 mM, at most 10 mM, at most 15 mM, at most 20 mM, at most 25 mM, at most 30 mM, at most 35 mM, at most 40 mM, at most 45 mM, or at most 50 mM.
  • a concentration of glucose used in a detection solution may be, e.g., about 1 mM to about 10 mM, about 1 mM to about 15 mM, about 1 mM to about 20 mM, about 1 mM to about 30 mM, about 1 mM to about 40 mM, about 1 mM to about 50 mM, about 5 mM to about 10 mM, about 5 mM to about 15 mM, about 5 mM to about 20 mM, about 5 mM to about 30 mM, about 5 mM to about 40 mM, about 5 mM to about 50 mM, about 10 mM to about 15 mM, about 10 mM to about 20 mM, about 10 mM to about 30 mM, about 10 mM to about 40 mM, or about 10 mM to about 50 mM.
  • a detection solution comprises between 0.1 % (v/v) and 10% (v/v) detergent B-Per, or a surfactant of polyoxyethylene glycol sorbitan alkyl esters family (TWEEN ® family) or polyoxyethylene glycol octylphenol ethers family (TRITON ® family).
  • a detection solution may be 200 mM Phosphate buffer (pH 5.5) detection solution.
  • 200 mM Phosphate buffer (pH 5.5) detection solution may comprise 0.0021 g/mL Sodium Hydrogen Phosphate, 0.0289 g/mL Sodium Dihydrogen Phosphate, 0.0020 g/mL Magnesium chloride, 0.0018 g/mL Glucose, and 0.0002 g/mL p-aminophenyl phosphate.
  • a detection solution may be 200 mM Phosphate buffer (pH 5.7) detection solution.
  • 200 mM Phosphate buffer (pH 5.7) detection solution may comprise 0.0174 g/mL Potassium Hydrogen Phosphate, 0.0204 g/mL Potassium Phosphate (Phtalate), 0.0020 g/mL Magnesium chloride, 0.0018 g/mL Glucose, and 0.0002 g/mL p-aminophenyl phosphate.
  • a detection solution may be 200 mM Phosphate buffer (pH 7.0) detection solution.
  • 200 mM Phosphate buffer (pH 7.0) detection solution may comprise 0.0156 g/mL Sodium Hydrogen Phosphate, 0.0142 g/mL Sodium Dihydrogen Phosphate, 0.0020 g/mL Magnesium chloride, 0.0018 g/mL Glucose, and 0.0002 g/mL p-aminophenyl phosphate.
  • a detection solution may be 160 mM Phosphate-citrate buffer (pH 5.5) detection solution.
  • 160 mM Phosphate-citrate buffer (pH 5.5) detection solution may comprise 0.0161 g/mL Sodium Hydrogen Phosphate, 0.0090 g/mL Citric Acid, 0.0020 g/mL Magnesium chloride, 0.0018 g/mL Glucose, and 0.0002 g/mL p-aminophenyl phosphate.
  • a detection solution may be 200 mM Acetate buffer (pH 5.7) detection solution.
  • 200 mM Acetate buffer (pH 5.7) detection solution may comprise 1.15 ⁇ Acetic Acid, 0.0015 g/mL Sodium Acetate, 0.0020 g/mL Magnesium chloride, 0.0018 g/mL Glucose, and 0.0002 g/mL p-aminophenyl phosphate.
  • a detection solution may be 200 mM Tris buffer (pH 9.8) detection solution.
  • 200 mM Tris buffer (pH 9.8) detection solution may comprise 0.0243 g/mL TRIS, 0.0020 g/mL Magnesium chloride, 0.0018 g/mL Glucose, and 0.0002 g/mL p- aminophenyl phosphate.
  • aspects of the present specification disclose, in part, incubation of a detection solution. Incubation of a detection solution is performed under temperature and time parameters that facilitate detection of a signal. Incubation a detection solution may be performed under agitation/rotation.
  • a temperature used to incubate a detection solution may be, e.g., about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21 °C, about 22°C, about 23°C, about 24°C, or about 25°C.
  • a temperature used to incubate a detection solution may be, e.g., at least 15°C, at least 16°C, at least 17°C, at least 18°C, at least 19°C, at least 20°C, at least 21°C, at least 22°C, at least 23°C, at least 24°C, or at least 25°C.
  • a temperature used to incubate a detection solution may be, e.g., at least 15°C, at least 16°C, at least 17°C, at least 18°C, at least 19°C, at least 20°C, at least 21°C, at least 22°C, at least 23°C, at least 24°C, or at least 25°C.
  • a temperature used to incubate a detection solution may be, e.g.
  • a temperature used to incubate a detection solution may be, e.g., about 15°C to about 19°C, about 16°C to about 20°C, about 17°C to about 21 °C, about 18°C to about 22°C, about 19°C to about 23°C, about 20°C to about 24°C, about 21 °C to about 25°C, about 22°C to about 26°C, about 23°C to about 27°C, about 24°C to about 28°C, or about 25°C to about 29°C.
  • a temperature used to incubate a detection solution may be, e.g.
  • a time used to incubate a detection solution may be, e.g., about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 1 10 minutes, about 120 minutes, about 130 minutes, about 140 minutes, or about 150 minutes.
  • a time used to incubate a detection solution may be, e.g., at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 1 10 minutes, at least 120 minutes, at least 130 minutes, at least 140 minutes, or at least 150 minutes.
  • a time used to a detection solution may be, e.g.
  • a time used to a detection solution may be, e.g., about 5 minutes to about 20 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 40 minutes, about 5 minutes to about 50 minutes, about 5 minutes to about 60 minutes, about 5 minutes to about 70 minutes, about 5 minutes to about 80 minutes, about 5 minutes to about 90 minutes, about 5 minutes to about 100 minutes, about 5 minutes to about 1 10 minutes, about 5 minutes to about 120 minutes, about 5 minutes to about 130 minutes, about 5 minutes to about 140 minutes, about 5 minutes to about 150 minutes, about 10 minutes to about 20 minutes, about 10 minutes to about 30 minutes, about 10 minutes to about 40 minutes, about 10 minutes to about 50 minutes, about 10 minutes to about 60 minutes, about 10 minutes to about 70 minutes, about 10 minutes to about 80 minutes, about 10 minutes to about 90 minutes, about 10 minutes to about 100 minutes, about 10 minutes to about 1 10 minutes, about 10 minutes to about 120 minutes, about 10 minutes to about 130 minutes, about 10 minutes to about 140 minutes, about 10 minutes to about 150 minutes
  • a detection solution may be incubated at a temperature of, e.g. , about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21 °C, about 22°C, about 23°C, about 24°C, or about 25°C for a time of, e.g., about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 1 10 minutes, about 120 minutes, about 130 minutes, about 140 minutes, or about 150 minutes.
  • a detection solution may be incubated at a temperature of, e.g. , at least 15°C, at least 16°C, at least 17°C, at least 18°C, at least 19°C, at least 20°C, at least 21 °C, at least 22°C, at least 23°C, at least 24°C, or at least 25°C for a time of, e.g., at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 1 10 minutes, at least 120 minutes, at least 130 minutes, at least 140 minutes, or at least 150 minutes.
  • a detection solution may be incubated at a temperature of, e.g., at most 15°C, at most 16°C, at most 17°C, at most 18°C, at most 19°C, at most 20°C, at most 21°C, at most 22°C, at most 23°C, at most 24°C, or at most 25°C, for a time of, e.g., at most 5 minutes, at most 10 minutes, at most 15 minutes, at most 20 minutes, at most 30 minutes, at most 40 minutes, at most 50 minutes, at most 60 minutes, at most 70 minutes, at most 80 minutes, at most 90 minutes, at most 100 minutes, at most 1 10 minutes, at most 120 minutes, at most 130 minutes, at most 140 minutes, or at most 150 minutes.
  • a detection solution may be incubated at a temperature of, e.g., about 5°C to about 19°C, about 16°C to about 20°C, about 17°C to about 21 °C, about 18°C to about 22°C, about 19°C to about 23°C, about 20°C to about 24°C, about 21 °C to about 25°C, about 22°C to about 26°C, about 23°C to about 27°C, about 24°C to about 28°C, or about 25°C to about 29°C for a time of, e.g., about 5 minutes to about 20 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 40 minutes, about 5 minutes to about 50 minutes, about 5 minutes to about 60 minutes, about 5 minutes to about 70 minutes, about 5 minutes to about 80 minutes, about 5 minutes to about 90 minutes, about 5 minutes to about 100 minutes, about 5 minutes to about 1 10 minutes, about 5 minutes to about 120 minutes, about 5 minutes to about 130 minutes, about 5
  • a volume of an aliquot to be analyzed for a detectable electrochemical signal may be, e.g., about 1 ⁇ _, about 2 ⁇ _, about 3 ⁇ _, about 4 ⁇ _, about 5 ⁇ _, about 6 ⁇ _, about 7 ⁇ _, about 8 ⁇ _, about 9 ⁇ ., about 10 ⁇ _, about 1 1 ⁇ _, about 12 ⁇ , about 13 ⁇ _, about
  • a volume of an aliquot to be analyzed for a detectable electrochemical signal may be, e.g., at least 1 ⁇ _, at least 2 ⁇ , at least 3 ⁇ _, at least 4 ⁇ _, at least 5 ⁇ ., at least 6 ⁇ ., at least 7 ⁇ , at least 8 ⁇ ., at least 9 ⁇ _, at least 10 ⁇ _, at least 1 1 ⁇ _, at least 12 ⁇ _, at least 13 ⁇ _, at least 14 ⁇ _, at least
  • a volume of an aliquot to be analyzed for a detectable electrochemical signal may be, e.g., at most 1 ⁇ _, at most 2 ⁇ _, at most 3 ⁇ _, at most 4 ⁇ _, at most 5 ⁇ !_, at most 6 ⁇ _, at most 7 pL, at most 8 pL, at most 9 ⁇ _, at most 10 ⁇ _, at most 1 1 ⁇ _, at most 12 ⁇ _, at most 13 ⁇ _, at most 14 ⁇ _, at most 15 ⁇ _, at most 16 ⁇ _, at most 17 ⁇ _, at most 18 ⁇ -, at most 19 ⁇ , or at most 20 ⁇ _.
  • a volume of an aliquot to be analyzed for a detectable electrochemical signal may be, e.g., about 1 ⁇ . to about 5 ⁇ _, about 1 ⁇ JL to about 10 ⁇ _, about 1 ⁇ _ to about 15 ⁇ , about 1 ⁇ _ to about 20 ⁇ _, 2 ⁇ _ to about 5 ⁇ , about 2 ⁇ . to about 10 pL, about 2 ⁇ .
  • An electrochemical signal can be analyzed using an instrument which is capable of measuring and/or analyzing potentiometric, voltammetric, amperometric and/or impedance/conductance parameters.
  • instrumentation is operated using computer-controlled software.
  • PalmSens3 a potentiostat, galvanostat, and impedance analyzer and PSTrace, its accompanying software (PalmSens BV, Utrecht, Netherlands).
  • a method disclosed herein can be performed to completion in, e.g., about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, or about 36 hours.
  • a method comprising a pre- enrichment step disclosed herein and an enrichment step disclosed herein can be performed to completion in, e.g., about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, or about 36 hours.
  • a method comprising a pre-enrichment step disclosed herein, an enrichment step disclosed herein and a detection step disclosed herein can be performed to completion in, e.g., about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, or about 36 hours.
  • a method comprising a pre-enrichment step disclosed herein, an enrichment step disclosed herein, a purification step disclosed herein, and a detection step disclosed herein can be performed to completion in, e.g., about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, or about 36 hours.
  • a method disclosed herein can be performed to completion in, e.g. , less than 18 hours, less than 20 hours, less than 22 hours, less than 24 hours, less than 26 hours, less than 28 hours, less than 30 hours, less than 32 hours, less than 34 hours, or less than 36 hours.
  • a method comprising a pre-enrichment step disclosed herein and an enrichment step disclosed herein can be performed to completion in, e.g., less than 18 hours, less than 20 hours, less than 22 hours, less than 24 hours, less than 26 hours, less than 28 hours, less than 30 hours, less than 32 hours, less than 34 hours, or less than 36 hours.
  • a method comprising a pre-enrichment step disclosed herein, an enrichment step disclosed herein and a detection step disclosed herein can be performed to completion in, e.g., less than 18 hours, less than 20 hours, less than 22 hours, less than 24 hours, less than 26 hours, less than 28 hours, less than 30 hours, less than 32 hours, less than 34 hours, or less than 36 hours.
  • a method comprising a pre-enrichment step disclosed herein, an enrichment step disclosed herein, a purification step disclosed herein, and a detection step disclosed herein can be performed to completion in, e.g., less than 18 hours, less than 20 hours, less than 22 hours, less than 24 hours, less than 26 hours, less than 28 hours, less than 30 hours, less than 32 hours, less than 34 hours, or less than 36 hours.
  • a method comprising a first pre-enrichment step disclosed herein, an enrichment step disclosed herein and a second pre-enrichment step disclosed herein can be performed to completion in, e.g., about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, or about 36 hours.
  • a method comprising a first pre-enrichment step disclosed herein, an enrichment step disclosed herein, a second pre-enrichment step disclosed herein and a detection step disclosed herein can be performed to completion in, e.g.
  • a method comprising a first pre-enrichment step disclosed herein, an enrichment step disclosed herein, a purification step disclosed herein, a second pre-enrichment step disclosed herein, and a detection step disclosed herein can be performed to completion in, e.g. , about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, or about 36 hours.
  • a method comprising a first pre-enrichment step disclosed herein, an enrichment step disclosed herein, a purification step disclosed herein, a second pre-enrichment step disclosed herein, a purification step disclosed herein and a detection step disclosed herein can be performed to completion in, e.g. , about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, or about 36 hours.
  • a method comprising a first pre-enrichment step disclosed herein, an enrichment step disclosed herein and a second pre-enrichment step disclosed herein can be performed to completion in, e.g. , less than 18 hours, less than 20 hours, less than 22 hours, less than 24 hours, less than 26 hours, less than 28 hours, less than 30 hours, less than 32 hours, less than 34 hours, or less than 36 hours.
  • a method comprising a first pre-enrichment step disclosed herein, an enrichment step disclosed herein, a second pre-enrichment step disclosed herein and a detection step disclosed herein can be performed to completion in, e.g., less than 18 hours, less than 20 hours, less than 22 hours, less than 24 hours, less than 26 hours, less than 28 hours, less than 30 hours, less than 32 hours, less than 34 hours, or less than 36 hours.
  • a method comprising a first pre- enrichment step disclosed herein, an enrichment step disclosed herein, a purification step disclosed herein, a second pre-enrichment step disclosed herein and a detection step disclosed herein can be performed to completion in, e.g.
  • a method comprising a first pre-enrichment step disclosed herein, an enrichment step disclosed herein, a purification step disclosed herein, a second pre-enrichment step disclosed herein, a purification step disclosed herein and a detection step disclosed herein can be performed to completion in, e.g. , less than 18 hours, less than 20 hours, less than 22 hours, less than 24 hours, less than 26 hours, less than 28 hours, less than 30 hours, less than 32 hours, less than 34 hours, or less than 36 hours.
  • a method disclosed herein can be performed to completion in, e.g., about 18 hours to about 20 hours, about 18 hours to about 22 hours, about 18 hours to about 24 hours, about 18 hours to about 26 hours, about 18 hours to about 28 hours, about 18 hours to about 30 hours, about 18 hours to about 32 hours, about 18 hours to about 34 hours, about 18 hours to about 36 hours, about 20 hours to about 22 hours, about 20 hours to about 24 hours, about 20 hours to about 26 hours, about 20 hours to about 28 hours, about 20 hours to about 30 hours, about 20 hours to about 32 hours, about 20 hours to about 34 hours, about 20 hours to about 36 hours, about 22 hours to about 24 hours, about 22 hours to about 26 hours, about 22 hours to about 28 hours, about 22 hours to about 30 hours, about 22 hours to about 32 hours, about 22 hours to about 34 hours, about 22 hours to about 36 hours, about 24 hours to about 26 hours, about 24 hours to about 28 hours, about 24 hours to about 30 hours, about 24 hours to about 26 hours, about 24 hours to about 28 hours, about 24 hours
  • a method comprising a pre-enrichment step disclosed herein and an enrichment step disclosed herein can be performed to completion in, e.g. , about 18 hours to about 20 hours, about 18 hours to about 22 hours, about 18 hours to about 24 hours, about 18 hours to about 26 hours, about 18 hours to about 28 hours, about 18 hours to about 30 hours, about 18 hours to about 32 hours, about 18 hours to about 34 hours, about 18 hours to about 36 hours, about 20 hours to about 22 hours, about 20 hours to about 24 hours, about 20 hours to about 26 hours, about 20 hours to about 28 hours, about 20 hours to about 30 hours, about 20 hours to about 32 hours, about 20 hours to about 34 hours, about 20 hours to about 36 hours, about 22 hours to about 24 hours, about 22 hours to about 26 hours, about 22 hours to about 28 hours, about 22 hours to about 30 hours, about 22 hours to about 32 hours, about 22 hours to about 34 hours, about 22 hours to about 36 hours, about 24 hours to about 26 hours, about 22 hours to about 28 hours, about 22 hours to about 30 hours,
  • a method comprising a pre-enrichment step disclosed herein, an enrichment step disclosed herein and a detection step disclosed herein can be performed to completion in, e.g. , about 18 hours to about 20 hours, about 18 hours to about 22 hours, about 18 hours to about 24 hours, about 18 hours to about 26 hours, about 18 hours to about 28 hours, about 18 hours to about 30 hours, about 18 hours to about 32 hours, about 18 hours to about 34 hours, about 18 hours to about 36 hours, about 20 hours to about 22 hours, about 20 hours to about 24 hours, about 20 hours to about 26 hours, about 20 hours to about 28 hours, about 20 hours to about 30 hours, about 20 hours to about 32 hours, about 20 hours to about 34 hours, about 20 hours to about 36 hours, about 22 hours to about 24 hours, about 22 hours to about 26 hours, about 22 hours to about 28 hours, about 22 hours to about 30 hours, about 22 hours to about 32 hours, about 22 hours to about 34 hours, about 22 hours to about 36 hours, about 24 hours to about 26 hours, about 22 hours to about 28 hours,
  • a method comprising a pre-enrichment step disclosed herein, an enrichment step disclosed herein, a purification step disclosed herien, and a detection step disclosed herein can be performed to completion in, e.g. , about 18 hours to about 20 hours, about 18 hours to about 22 hours, about 18 hours to about 24 hours, about 18 hours to about 26 hours, about 18 hours to about 28 hours, about 18 hours to about 30 hours, about 18 hours to about 32 hours, about 18 hours to about 34 hours, about 18 hours to about 36 hours, about 20 hours to about 22 hours, about 20 hours to about 24 hours, about 20 hours to about 26 hours, about 20 hours to about 28 hours, about 20 hours to about 30 hours, about 20 hours to about 32 hours, about 20 hours to about 34 hours, about 20 hours to about 36 hours, about 22 hours to about 24 hours, about 22 hours to about 26 hours, about 22 hours to about 28 hours, about 22 hours to about 30 hours, about 22 hours to about 32 hours, about 22 hours to about 34 hours, about 22 hours to about 32 hours, about 22 hours to about
  • a method comprising a first pre-enrichment step disclosed herein, an enrichment step disclosed herein and a second pre-enrichment step disclosed herein can be performed to completion in, e.g. , about 18 hours to about 20 hours, about 18 hours to about 22 hours, about 18 hours to about 24 hours, about 18 hours to about 26 hours, about 18 hours to about 28 hours, about 18 hours to about 30 hours, about 18 hours to about 32 hours, about 18 hours to about 34 hours, about 18 hours to about 36 hours, about 20 hours to about 22 hours, about 20 hours to about 24 hours, about 20 hours to about 26 hours, about 20 hours to about 28 hours, about 20 hours to about 30 hours, about 20 hours to about 32 hours, about 20 hours to about 34 hours, about 20 hours to about 36 hours, about 22 hours to about 24 hours, about 22 hours to about 26 hours, about 22 hours to about 28 hours, about 22 hours to about 30 hours, about 22 hours to about 32 hours, about 22 hours to about 34 hours, about 22 hours to about 36 hours, about 22 hours to about 24 hours, about 22
  • a method comprising a first pre-enrichment step disclosed herein, an enrichment step disclosed herein, a second pre-enrichment step disclosed herein and a detection step disclosed herein can be performed to completion in, e.g., about 18 hours to about 20 hours, about 18 hours to about 22 hours, about 18 hours to about 24 hours, about 18 hours to about 26 hours, about 18 hours to about 28 hours, about 18 hours to about 30 hours, about 18 hours to about 32 hours, about 18 hours to about 34 hours, about 18 hours to about 36 hours, about 20 hours to about 22 hours, about 20 hours to about 24 hours, about 20 hours to about 26 hours, about 20 hours to about 28 hours, about 20 hours to about 30 hours, about 20 hours to about 32 hours, about 20 hours to about 34 hours, about 20 hours to about 36 hours, about 22 hours to about 24 hours, about 22 hours to about 26 hours, about 22 hours to about 28 hours, about 22 hours to about 30 hours, about 22 hours to about 32 hours, about 22 hours to about 34 hours, about 22 hours to about 32 hours, about 22 hours
  • a method comprising a first pre-enrichment step disclosed herein, an enrichment step disclosed herein, a purification step disclosed herien, a second pre-enrichment step disclosed herein and a detection step disclosed herein can be performed to completion in, e.g.
  • a method comprising a first pre-enrichment step disclosed herein, an enrichment step disclosed herein, a purification step disclosed herien, a second pre-enrichment step disclosed herein, a purification step disclosed herien and a detection step disclosed herein can be performed to completion in, e.g., about 18 hours to about 20 hours, about 18 hours to about 22 hours, about 18 hours to about 24 hours, about 18 hours to about 26 hours, about 18 hours to about 28 hours, about 18 hours to about 30 hours, about 18 hours to about 32 hours, about 18 hours to about 34 hours, about 18 hours to about 36 hours, about 20 hours to about 22 hours, about 20 hours to about 24 hours, about 20 hours to about 26 hours, about 20 hours to about 28 hours, about 20 hours to about 30 hours, about 20 hours to about 32 hours, about 20 hours to about 34 hours, about 20 hours to about 36 hours, about 22 hours to about 24 hours, about 22 hours to about 26 hours, about 22 hours to about 28 hours, about 22 hours to about 30 hours,
  • aspects of the present specification disclose, in part, a Salmonella live vaccine bacteria analysis kit to carry out the method of detecting a Salmonella live vaccine bacteria disclosed herein.
  • An analysis kit disclosed herein contains components necessary for the detection of the Salmonella live vaccine bacteria of interest.
  • an analysis kit disclosed herein typically comprises a pre-enrichment media and an enrichment media. In some embodiment, an analysis kit disclosed herein typically comprises a pre- enrichment media, an enrichment media, and a detection solution. In some embodiment, an analysis kit disclosed herein typically comprises a pre-enrichment media, an enrichment media, a detection solution and an electrochemical biosensor. [149] In some embodiment, an analysis kit disclosed herein typically comprises a first and second pre- enrichment media and an enrichment media. In some embodiment, an analysis kit disclosed herein typically comprises a first and second pre-enrichment media, an enrichment media, and a detection solution. In some embodiment, an analysis kit disclosed herein typically comprises a first and second pre- enrichment media, an enrichment media, a detection solution and an electrochemical biosensor.
  • an analysis kit disclosed herein typically comprises a pre-enrichment media, an enrichment media, and immunmagnetic particles capable of binding to a Salmonella live vaccine bacteria of interest.
  • an analysis kit disclosed herein typically comprises a pre- enrichment media, an enrichment media, immunmagnetic particles capable of binding to a Salmonella live vaccine bacteria of interest, and a magnetic source used to capture the immunoparticles.
  • an analysis kit disclosed herein typically comprises a pre-enrichment media, an enrichment media, immunmagnetic particles capable of binding to a Salmonella live vaccine bacteria of interest, and a detection solution.
  • an analysis kit disclosed herein typically comprises a pre- enrichment media, an enrichment media, immunmagnetic particles capable of binding to a Salmonella live vaccine bacteria of interest, a magnetic source used to capture the immunoparticles, and a detection solution.
  • an analysis kit disclosed herein typically comprises a pre-enrichment media, an enrichment media, immunmagnetic particles capable of binding to a Salmonella live vaccine bacteria of interest, a detection solution, and an electrochemical biosensor.
  • an analysis kit disclosed herein typically comprises a pre-enrichment media, an enrichment media, immunmagnetic particles capable of binding to a Salmonella live vaccine bacteria of interest, a detection solution, a magnetic source used to capture the immunoparticles, and an electrochemical biosensor.
  • an analysis kit disclosed herein typically comprises a first and second pre- enrichment media, an enrichment media, and immunmagnetic particles capable of binding to a Salmonella live vaccine bacteria of interest.
  • an analysis kit disclosed herein typically comprises a first and second pre-enrichment media, an enrichment media, immunmagnetic particles capable of binding to a Salmonella live vaccine bacteria of interest, and a magnetic source used to capture the immunoparticles.
  • an analysis kit disclosed herein typically comprises a first and second pre-enrichment media, an enrichment media, immunmagnetic particles capable of binding to a Salmonella live vaccine bacteria of interest, and a detection solution.
  • an analysis kit disclosed herein typically comprises a first and second pre-enrichment media, an enrichment media, immunmagnetic particles capable of binding to a Salmonella live vaccine bacteria of interest, a magnetic source used to capture the immunoparticles, and a detection solution.
  • an analysis kit disclosed herein typically comprises a first and second pre-enrichment media, an enrichment media, immunmagnetic particles capable of binding to a Salmonella live vaccine bacteria of interest, a detection solution, and an electrochemical biosensor.
  • an analysis kit disclosed herein typically comprises a first and second pre-enrichment media, an enrichment media, immunmagnetic particles capable of binding to a Salmonella live vaccine bacteria of interest, a detection solution, a magnetic source used to capture the immunoparticles, and an electrochemical biosensor.
  • An analysis kit disclosed herein may further comprise an instrument which is capable of measuring and/or analyzing potentiometric, voltammetric, amperometric and/or impedance/conductance parameters.
  • An analysis kit disclosed herein may further comprise a suitable container, for example, a vessel, vials, tubes, mini- or microfuge tubes, test tube, flask, bottle, syringe or other container. Where an additional component or agent is provided, the kit can contain one or more additional containers into which this agent or component may be placed.
  • An analysis kit disclosed herein will also typically include a means for containing the agent (e.g. a vessel), composition and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
  • An analysis kit disclosed herein may further comprise a labels or inserts.
  • Labels or inserts include "printed matter," e.g., paper or cardboard, or separate or affixed to a component, a kit or packing material (e.g., a box), or attached to an ampule, tube or vial containing a kit component.
  • Labels or inserts can additionally include a computer readable medium, such as a disk (e.g., hard disk, flash memory), optical disk such as CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic/optical storage media, FLASH media or memory type cards.
  • Labels or inserts may include identifying information of one or more components therein, amounts used for one or more components therein, step by step instructions of how to perform a method of detecting a Salmonella live vaccine bacteria of interest.
  • Labels or inserts can include information identifying manufacturer information, lot numbers, manufacturer location and date and patent information.
  • a method of detecting a Salmonella live vaccine strain in a sample comprising the steps: a) incubation of the sample in a first liquid pre-enrichment media, the first pre-enrichment media comprising 2 g/L to 6 g/L of a peptone, 0.5 g/L to 4.5 g/L Bile Salts, 0.5 g/L to 4.5 g/L Meat Extract, 0.5 g/L to 4.5 g/L of a first growth inhibiting agent, 0.5 g/L to 4.5 g/L of a second growth inhibiting agent, 0.001 g/L to 0.008 g/L of a third growth inhibiting agent, and 0.001 g/L to 0.008 g/L of a fourth growth inhibiting agent, wherein the incubation at about 34°C to about 40°C for about 5 hours to about 10 hours; b) incubating an aliquot of first pre-enrichment media from step (a) in a liquid enrichment
  • step (a), step (b) and/or step (d) is a peptone from an animal source or a peptone from a plant source.
  • the peptone from an animal source is an acid casein peptone, a bacteriological peptone, a beef extract powder, a casein peptone, a casein cc peptone, a gelatin peptone, a meat peptone, a polypeptone proteose peptone, or a proteose peptone No 3.
  • the peptone from a plant source is a malt extract, a soya peptone, or a yeast extract.
  • step (a), step (b) and/or step (d) is a first iodine compound.
  • step (a), step (b) and/or step (d) is a second iodine compound.
  • step (a), step (b) and/or step (d) is an aminocoumarin antibiotic.
  • aminocoumarin antibiotic is Novobiocin, Albamycin, Coumermycin or Clorobiocin.
  • step (a), step (b) and/or step (d) is a triarylmethane dye.
  • the triarylmethane dye is a methyl violet dye, a fuchsine dye, a phenol dye, or a malachite green dye.
  • methyl violet dye is methyl violet 2B, methyl violet 6B, or methyl violet 10B.
  • fuchsine dye is pararosaniline, fuchsine, new fuchsine, fuchsin basic violet, or fuchine acid.
  • phenol dye is phenol red, chlorophenol red, or cresol red.
  • the triarylmethane dye is aluminon, aniline Blue WS, aurin, aurintricarboxylic acid, brilliant blue FCF, brilliant green, bromocresol green, bromocresol purple, bromophenol blue, bromopyrogallol red, bromothymol blue, bromsulphthalein, chlorophenol red, coomassie brilliant blue, cresol red, crystal violet, crystal violet lactone, ethyl green, fast green FCF, fluoran, fuchsine, fuchsine acid, fuchsin basic violet, gentian, green S, light green SF yellowish, malachite green, methyl blue, methyl violet, new fuchsine, pararosaniline, patent blue V, phenol red, phenolphthalein, rose bengal, thymolphthalein, victoria blue BO, water blue, xylene cyanol, or xylenol orange.
  • step (a) and/or step (d) is 3 g/L to 5 g/L, 4 g/L to 4.6 g/L, 4.2 g/L to 4.4 g/L or 4.3 g/L.
  • step (a) and/or step (d) is .5 g/L to 3.5 g/L, 2.1 g/L to 2.7 g/L, 2.3 g/L to 2.5 g/L or 2.4 g/L.
  • step (a) and/or step (d) is 1 g/L to 3 g/L, 1.8 g/L to 2.4 g/L, 2.0 g/L to 2.2 g/L or 2.1 g/L.
  • step (a) and/or step (d) is 1 g/L to 3 g/L, 1.7 g/L to 2.3 g/L, 1.9 g/L to 2.1 g/L or 2 g/L.
  • step (a) and/or step (d) is 1 g/L to 3 g/L, 1.7 g/L to 2.3 g/L, 1.9 g/L to 2.1 g/L or 2 g/L.
  • step (a) and/or step (d) are 0.002 g/L to 0.006 g/L, 0.003 g/L to 0.005 g/L or 0.004 g/L.
  • step (a) and/or step (d) are 0.002 g/L to 0.006 g/L, 0.003 g/L to 0.005 g/L or 0.004 g/L.
  • step (a) and/or step (d) further comprise salts.
  • the salts comprise NaCI, CaCC and/or Na2S203.
  • step (b) The method according to any one of embodiments 1-29, wherein the amount of peptone in step (b) is 7.5 g/L to 8.5 g/L, 8.3 g/L to 8.9 g/L, 8.5 g/L to 8.7 g/L or 8.6 g/L.
  • step (b) The method according to any one of embodiments 1 -30, wherein the amount of Bile Salts in step (b) is 4 g/L to 6 g/L, 4.4 g/L to 5.0 g/L, 4.6 g/L to 4.8 g/L or 4.7 g/L.
  • (b) is 3 g/L to 5 g/L, 4.0 g/L to 4.6 g/L, 4.2 g/L to 4.4 g/L or 4.3 g/L.
  • step (b) The method according to any one of embodiments 1 -32, wherein the amount of first growth inhibiting agent in step (b) is 3 g/L to 5 g/L, 3.7 g/L to 4.3 g/L, 3.9 g/L to 4.1 g/L or 4 g/L.
  • step (b) The method according to any one of embodiments 1 -33, wherein the amount of second growth inhibiting agent in step (b) is 3 g/L to 5 g/L, 3.7 g/L to 4.3 g/L, 3.9 g/L to 4.1 g/L or 4 g/L.
  • step (b) The method according to any one of embodiments 1 -34, wherein the amount of third growth inhibiting agent in step (b) is 0.002 g/L to 0.006 g/L, 0.003 g/L to 0.005 g/L or 0.004 g/L.
  • step (b) The method according to any one of embodiments 1 -35, wherein the amount of fourth growth inhibiting agent in step (b) is 0.002 g/L to 0.006 g/L, 0.003 g/L to 0.005 g/L or 0.004 g/L.
  • step (b) further comprise salts.
  • the salts comprise NaCI, CaC03 and/or Na2S203.
  • the method according to embodiment 38, wherein the NaCI is in an amount of .3 g/L to 3.9 g/L, 2.3 g/L to 2.9 g/L, 2.5 g/L to 2.7 g/L or 2.6 g/L.
  • first pre-enrichment media in step (a), the enrichment media in step (b) and/or the second pre-enrichment media step (d) further comprises a growth enhancing agent.
  • the growth enhancing agent is a siderophore.
  • the siderophore is Aerobactin, Alcaligin, Azotobactin, Bacillibactin, Desferrioxamine B, Desferrioxamine E, Enterobactin, Ferrichrome, Ferrioxiamina-B, Ferrioxiamina-E, Fusarinine C, Mycobactin, Omibactin, Petrobactin, Pyoverdine, Pyochelin, Salmochelin, Staphyloferring A, Vibriobactin, or Yersiniabactin.
  • step (a) The method according to any one of embodiments 1-44, wherein the incubation in step (a) at about 35°C to about 39°C.
  • step (a) wherein the incubation in step (a) at about 36°C to about 38°C.
  • step (a) The method according to any one of embodiments 1 -46, wherein the incubation in step (a) is for about 6 hours to about 9 hours.
  • step (a) wherein the incubation in step (a) is for about 7 hours to about 8 hours.
  • step (b) The method according to any one of embodiments 1 -48, wherein the aliquot of first pre-enrichment media in step (b) is about 1/5 to about 1/500 a volume of an enrichment media used in step (b).
  • step (b) The method according to any one of embodiments 1-49, wherein the incubation in step (b) at about 35°C to about 44°C.
  • step (b) wherein the incubation in step (b) at about 36°C to about 43°C.
  • step (b) The method according to embodiment 51 , wherein the incubation in step (b) at about 37°C to about 42°C.
  • step (b) The method according to any one of embodiments 1 -52, wherein the incubation in step (b) is for about 15 hours to about 19 hours.
  • step (b) wherein the incubation in step (b) is for about 16 hours to about 18 hours.
  • step (d) The method according to any one of embodiments 1 -56, wherein the incubation in step (d) at about 35°C to about 44°C.
  • step (d) wherein the incubation in step (d) at about 36°C to about 43°C.
  • step (d) the incubation in step (d) at about 37°C to about 42°C.
  • step (d) is for about 2 hours to about 6 hours.
  • step (d) is for about 3 hours to about 5 hours.
  • detection step (e) is performed using a sensor-based detection method, a nucleic acid-based detection method, a protein-based detection method, an activity-based detection method, or a growth-based detection method.
  • the electrochemical detection method comprises an enzymatic biosensor, a DNA sensor, or an immunosensor.
  • nucleic acid-based detection method comprises a DNA-based detection method or an RNA-based detection method.
  • the DNA-based detection method comprises a Southern blot analysis, a PCR-based assay, a sequence analysis, an immuno-based detection assay, or a hybridization assays using FRET, polarization or other fluorescent, chemiluminescent or bioiluminescent detection.
  • PCR-based assay comprises a real-time PCR- based assay.
  • RNA-based detection method comprises a Northern blot analysis, a RT-PCR-based assay, a RNA sequence analysis, an immuno-based detection assay, or a hybridization assays using FRET, polarization or other fluorescent, chemiluminescent or bioiluminescent detection.
  • the protein-based detection method is a gel-based detection method, an immuno-based detection method or a protein-interaction-based method.
  • the immuno-based detection method comprises a Western blot analysis, an ELISA, or an immunoprecipitation assay.
  • the activity-based detection method comprises an enzymatic activity assay or an assay based on protein function.
  • the growth-based detection method comprises a plating assays measuring colony formation or spectrophotometer assays measuring cell density.
  • a Salmonella live vaccine strain analysis kit comprising a pre-enrichment media and an enrichment media.
  • a Salmonella live vaccine strain analysis kit comprising a pre-enrichment media as defined in any one of embodiments 1 -29 or 42-44 and an enrichment media as defined in any one of embodiments 1-17 or 30-44.
  • the analysis kit according to any one of embodiments 78-81 , further comprising immunmagnetic particles capable of binding to a Salmonella live vaccine strain of interest.
  • the analysis kit according to any one of embodiments 78-83, further comprising an instrument which is capable of measuring and/or analyzing potentiometric, voltammetric, amperometric and/or impedance/conductance parameters.
  • Enzyme-based electrochemical sensors were manufactured using a screen printing process by depositing a series of layers of different material on a substrate material. Initially, a layer of compound paste comprising silver is deposited directly onto a polyester substrate (MYLAR ® A (500 gauge); DuPont E. I. De Nemours & Co., Wilmington, DE). A layer of silver/silver chloride, which functions as both the counter electrode and reference electrode, is then deposited over the compound paste on one side to form a wall. The third layer comprised carbon graphite (Gwent Electronic Materials Ltd., Pontypool, UK) which functions as the working electrode is deposited on the other side to form an oppossing wall.
  • a channel is thus formed inbetween the silver/silver chloride wall and the carbon graphite wall.
  • a layer of insulating paste (Gwent Electronic Materials Ltd., Pontypool, UK) is then deposited over the silver/silver chloride and carbon graphite layers and a pressure-sensitive adhesive (KIWO, Inc., Seabrook, TX) is then deposited over this insulating paste layer.
  • a sensor is then modified by adding a biological layer within the channel by incubating the sensor in an atmosphere saturated with 5% glutaraldehyde to 5% for 5 minutes at room temperature in the presence of an enzyme solution comprising glucose dehydrogenase (GDH), which is then dried for 30 minutes at 37°C.
  • GDH glucose dehydrogenase
  • Presence of the Salmonella bacteria was detected by measuring the electrochemical signal produced as a result of a redox reaction associated with the conversion of glucose to gluconic acid.
  • an aliquot of the enriched bacteria culture was added to a detection solution comprising p-aminophenyl phosphate (PAPH) and glucose.
  • PAPH p-aminophenyl phosphate
  • alfanaphtylphosphate may be used instead of PAPH.
  • a sensor as described above was inserted into this solution and electrochemical signals were detected amperometrically under an applied potential of about 200 mV using a PalmSens3 (a potentiostat, galvanostat, and impedance analyzer) and its accompanying software PSTrace (PalmSens BV, Utrecht, Netherlands).
  • an electrochemical signal is as follows. Salmonella bacteria detected using this procedure synthesize the enzyme alkaline phosphatase (ALP), which is subsequently released into the culture media. Addition of an enriched bacteria culture comprising ALP results in the hydrolysis PAPH into PAP. GDH, immobilized in the biological layer of the sensor, catalyzes the conversion of glucose into gluconic acid, which is associated with a redox reaction that oxidizes PAP into PIQ. The electrochemical signals detected by the sensor occurs when it measures the electrons generated when PIQ is reduced back to PAP.
  • ALP alkaline phosphatase
  • Each detection solution was then mixed with a 1 mL solution of phospho-buffered saline comprising bacteria (Salmonella typhimurium) at one of the following concentrations: 1 x 10 2 cfu/mL, 1 x 10 3 cfu/mL, 1 x 10 4 cfu/mL, 1 x 10 5 cfu/mL, 1 x 10 6 cfu/mL, 1 x 10 7 cfu/mL, or 1 x 10 8 cfu/mL.
  • the detection solution was then incubated at 37°C for 30 minutes.
  • the electrochemical signal for each solution was detected amperometrically under an applied potential of about 200 mV using a PalmSens3 as described above.
  • a bacterial concentration of about 1 x 10 4 cfu/mL produced a detectable signal of about 0.5 ⁇ .
  • a linear concentration- response curve was observed in a range of about 1 x 10 5 cfu/mL to about 1 x 10 7 cfu/mL.
  • the electrochemical signal for each solution was detected amperometrically under an applied potential of about 200 mV using a PalmSens3 as described above. As shown in FIG. 3, a detectable signal of about 0.5 ⁇ was detected after 4 hours of incubation. A linear time-response curve was observed in a range of about 6 hours to about 8 hours. These results indicate that and average anodic current of >0.5 ⁇ is indicative of the presence of bacteria.
  • the enrichment culture for both concentrations were incubated for about 5 to about 7 hours at 41 ,5°C with circular stirring. After this enrichment step, 20 pL of a solution 3.0 x 10 8 anti-Salmonella immunomagnetic particles/mL (DYNABEADS ® Anti-Salmonella, Life Technologies, Inc., Carlsbad, CA) was added to the enrichment cultures and incubated for 30 minutes at room temperature with agitation circular. After this incubation time, the enrichment cultures were contacted with a magnet for 3 minutes in order to localize the anti-Salmonella immunomagnetic particles within the container tube and the supernatant was extracted using 1 mL micropipette.
  • a solution 3.0 x 10 8 anti-Salmonella immunomagnetic particles/mL (DYNABEADS ® Anti-Salmonella, Life Technologies, Inc., Carlsbad, CA) was added to the enrichment cultures and incubated for 30 minutes at room temperature with agitation circular. After this incuba
  • the supernatant was then added to 1 mL of 100 mM phosphate buffer, mixed for 10 seconds, contacted with a magnet for 3 minutes and the washed supernatant was extracted using 1 mL micropipette.
  • the processed supernatant was then tested to detect the presence of the bacteria using two different assays: 1 ) a plating assay; and 2) an electrochemical detection assay disclosed herein.
  • the enrichment cultures were contacted with a magnet for about 1 minute to about 3 minutes in order to localize the anti-Salmonella immunomagnetic particles within the container tube.
  • 1.0 mL of pre-enrichment culture media (Table 2) was added to the anti-Salmonella immunomagnetic particles and incubated at about 36°C to about 38°C for about 2 hours to about 3 hours with or without circular stirring.
  • the pre-enrichment cultures were contacted with a magnet for about 1 minute to about 3 minutes in order to localize the anti-Salmonella immunomagnetic particles within the container tube.
  • a a 50 pL detection solution comprising 185 mM sodium phosphate buffer (pH 5.7), 10 mM magnesium chloride, 1.0 mM 4-aminophenyl-Phosphate and 10 mM glucose was added to the 1.0 mL of 100 mM phosphate buffer containing the anti-Salmonella immunomagnetic particles. The detection solution was then incubated at 37°C for about 30 to about 75 minutes.
  • the electrochemical signal for each solution was detected amperometrically under an applied potential of about 200 mV verse Ag/AgCI for 30 seconds using a PalmSens3 (a potentiostat, galvanostat, and impedance analyzer) and its accompanying software PSTrace (PalmSens BV, Utrecht, Netherlands).
  • PalmSens3 a potentiostat, galvanostat, and impedance analyzer
  • PSTrace PalmSens BV, Utrecht, Netherlands
  • the incubated pre-enrichment culture media was incubated in an enrichment media, purified, incubated in a pre-enrichment media, purified as described in Example 4.
  • detection of the presence of the bacteria using a plating assay and an electrochemical detection assay disclosed herein were performed as described above in Example 4.
  • the meaning of the open-ended transitional phrase “comprising” is being defined as encompassing all the specifically recited elements, limitations, steps and/or features as well as any optional, additional unspecified ones.
  • the meaning of the closed-ended transitional phrase “consisting of” is being defined as only including those elements, limitations, steps and/or features specifically recited in the claim whereas the meaning of the closed-ended transitional phrase “consisting essentially of is being defined as only including those elements, limitations, steps and/or features specifically recited in the claim and those elements, limitations, steps and/or features that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.
  • the open-ended transitional phrase “comprising” includes within its meaning, as a limiting case, claimed subject matter specified by the closed-ended transitional phrases “consisting of” or “consisting essentially of.”
  • claimed subject matter specified by the closed-ended transitional phrases “consisting of” or “consisting essentially of.”
  • embodiments described herein or so claimed with the phrase “comprising” are expressly or inherently unambiguously described, enabled and supported herein for the phrases “consisting essentially of and “consisting of.”

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