WO2020205491A1 - Séquences et leur utilisation pour la détection et la caractérisation du genre cronobacter - Google Patents

Séquences et leur utilisation pour la détection et la caractérisation du genre cronobacter Download PDF

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WO2020205491A1
WO2020205491A1 PCT/US2020/025161 US2020025161W WO2020205491A1 WO 2020205491 A1 WO2020205491 A1 WO 2020205491A1 US 2020025161 W US2020025161 W US 2020025161W WO 2020205491 A1 WO2020205491 A1 WO 2020205491A1
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seq
combination
primer
probe
sample
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PCT/US2020/025161
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English (en)
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Sai Siddarth KALBURGE
Indira Padmalayam
Martin EASTER
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Hygiena, Llc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the field relates to methods for detection and characterization of bacteria belonging to the genus Cronobacter based on the presence of nucleic acid sequences, including PCR-based methods for detection, and to oligonucleotide molecules and reagents and kits useful therefor.
  • Cronobacter consists of seven species of gram-negative, rod-shaped bacteria.
  • the 7 species include Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter universalis, Cronobacter muytjensii, Cronobacter turicensis, Cronobacter condimentii and Cronobacter dublinensis.
  • the species C. dublinensis consists of 3 sub-species including C. dublinensis subsp. dublinensis, C. dublinensis subsp.
  • opportunistic pathogens that rarely cause infectious disease in healthy adults but can cause severe and sometimes fatal infections in neonates and immune-compromised individuals.
  • the bacteria can cause severe neurological impairments including hydrocephalus, quadriplegia and developmental delays in infants.
  • the Cronobacter species has been isolated from various plant and animal foods and products. However, the only food source that has been epidemiologically linked to disease outbreaks is powdered infant formula.
  • the World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO) concluded that Cronobacter and Salmonella contaminations of powdered infant formula were responsible for infections and illness in infants. Even low levels of contamination of Cronobacter in powdered infant formula are considered to be a risk factor.
  • Cronobacter Monitoring and Controlling Cronobacter to ensure the safety of powdered infant formula and other foods is therefore of critical importance.
  • One aspect is for a method for detecting the presence of the genus Cronobacter in a sample, said sample comprising nucleic acids, said method comprising:
  • reaction mixture comprising:
  • a primer pair comprising a 5' forward primer of SEQ ID NO:1 , SEQ ID NO:2, or a combination thereof and a 3' reverse primer of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or a combination thereof;
  • a primer pair comprising a 5' forward primer of SEQ ID NO:8, SEQ ID NO:9, or a combination thereof and a 3' reverse primer of SEQ ID NO:10, SEQ ID NO:1 1 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or a combination thereof;
  • SEQ ID NO:18 or a combination thereof and a 3' reverse primer of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 , or a combination thereof;
  • a primer pair comprising a 5' forward primer of SEQ ID NO:22,
  • SEQ ID NO:31 SEQ ID NO:32, SEQ ID NO:33, or a combination thereof and a 3' reverse primer of SEQ ID NO:34, SEQ ID NO:35, or a combination thereof; or
  • step (b) performing PCR amplification of said nucleic acids of said sample using the reaction mixture of step (a);
  • step (c) detecting the amplification of step (b), whereby a positive detection of amplification indicates the presence of the genus Cronobacter in the sample.
  • Another aspect is for an isolated polynucleotide comprising SEQ ID NO:1 , 2, 3,
  • a further aspect is for a replication composition for use in performance of PCR, comprising:
  • reaction mixture comprising:
  • a primer pair comprising a 5' forward primer of SEQ ID NO:1 , SEQ ID NO:2, or a combination thereof and a 3' reverse primer of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or a combination thereof;
  • a primer pair comprising a 5' forward primer of SEQ ID NO:8, SEQ ID NO:9, or a combination thereof and a 3' reverse primer of SEQ ID NO:10, SEQ ID NO:1 1 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or a combination thereof;
  • SEQ ID NO:18 or a combination thereof and a 3' reverse primer of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 , or a combination thereof;
  • SEQ ID NO:31 SEQ ID NO:32, SEQ ID NO:33, or a combination thereof and a 3' reverse primer of SEQ ID NO:34, SEQ ID NO:35, or a combination thereof; or
  • nucleic acid probe comprises SEQ ID NO: 36, 37, 38, 39, 40, 41 , 42, 43,
  • thermostable DNA polymerase thermostable DNA polymerase
  • An additional aspect is for a kit for detection of the genus Cronobacter, inclusive of all types of species, in a sample, comprising the aforementioned replication composition.
  • a further aspect is for a tablet comprising the aforementioned replication composition.
  • SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, and 35 are primer sequences for use in the detection of bacteria belonging to the genus
  • SEQ ID NOs: 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, and 47 are probe sequences for use in the detection of bacteria belonging to the genus
  • the probe is 5'-labeled with a fluorescent dye and 3'- termini is composed of a quencher dye.
  • the 3' termini are comprised of one of the primers listed above, a suitable linker moiety, such as a spacer consisting of an 18 atom polyethylene glycol and a quencher dye.
  • the term“about” or“approximately” means within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,
  • PCR Polymerase chain reaction
  • isolated refers to materials, such as nucleic acid molecules and/or proteins, which are substantially free or otherwise removed from components that normally accompany or interact with the materials in a naturally occurring environment. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant
  • polynucleotides and chemically synthesized polynucleotides.
  • polynucleotide “polynucleotide sequence”,“nucleic acid sequence”, and“nucleic acid fragment” are used interchangeably herein. These terms encompass nucleotide sequences and the like.
  • a polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural, or altered nucleotide bases.
  • a polynucleotide may also consist of nucleotide sequences joined at the 3' end of one nucleotide sequence to the 5' end of another nucleotide sequence by a linker such as a 3 or 6 carbon (propanediol or hexanediol, respectively) moiety, or a linker arm of either 3 or 6 polyethylene glycol subunits (triethylene glycol or hexaethylene glycol, respectively) or an 18 atom polyethylene glycol (Spacer 18). Any suitable linkers or spacers that are known in the art will work for this application.
  • a polynucleotide in the form of a polymer of DNA may be comprised of one or more strands of cDNA, genomic DNA, synthetic DNA, or mixtures thereof.
  • amplification product refers to nucleic acid fragments produced during a primer-directed amplification reaction.
  • Typical methods of primer-directed amplification include polymerase chain reaction (PCR), ligase chain reaction (LCR), or strand displacement amplification (SDA).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • the replication composition may comprise the components for nucleic acid replication, for example: nucleotide triphosphates, two (or more) primers with appropriate sequences, thermostable polymerase, buffers, solutes, and proteins.
  • the nucleic acid replication compositions may comprise, for example: a thermostable ligase (e.g ., Thermus aquaticus ligase), two sets of adjacent thermostable ligase (e.g ., Thermus aquaticus ligase), two sets of adjacent thermostable ligase (e.g ., Thermus aquaticus ligase), two sets of adjacent thermostable ligase (e.g ., Thermus aquaticus ligase), two sets of adjacent
  • oligonucleotides (wherein one member of each set is complementary to each of the target strands), Tris-HCI buffer, KCI, EDTA, NAD, dithiothreitol, and salmon sperm DNA. See, for example, Tabor et al., Proc. Natl. Acad. Sci. U.S.A. 82:1074-1078 (1985).
  • primer refers to an oligonucleotide (synthetic or occurring naturally) that is capable of acting as a point of initiation of nucleic acid synthesis or replication along a complementary strand when placed under conditions in which synthesis of a complementary strand is catalyzed by a polymerase.
  • a primer can further contain a detectable label, for example a 5' end label.
  • probe refers to an oligonucleotide (synthetic or occurring naturally) that is complementary (though not necessarily fully complementary) to a polynucleotide of interest and forms a duplexed structure by hybridization with at least one strand of the polynucleotide of interest.
  • a probe or primer-probe complex can further contain a detectable label.
  • a probe can either be an independent entity or complexed with or otherwise attached to a primer, such as where a probe is connected via its 3' terminus to a primer’s 5' terminus through a linker, which may be a nucleotide or non-nucleotide linker and which may be a non-amplifiable linker, such as a hexaethylene glycol (HEG) or 18-carbon linker.
  • a“primer-probe complex” One example of such a primer-probe complex can be found in U.S. Patent No.
  • label and“detectable label” refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, semiconductor nanocrystals, ligands (e.g., biotin, avidin, streptavidin, or haptens), and the like.
  • a detectable label can also include a combination of a reporter and a quencher.
  • reporter refers to a substance or a portion thereof which is capable of exhibiting a detectable signal, which signal can be suppressed by a quencher.
  • the detectable signal of the reporter is, e.g., fluorescence in the detectable range.
  • quencher refers to a substance or portion thereof which is capable of
  • quenching and“fluorescence energy transfer” refer to the process whereby, when a reporter and a quencher are in close proximity, and the reporter is excited by an energy source, a substantial portion of the energy of the excited state nonradiatively transfers to the quencher where it either dissipates nonradiatively or is emitted at a different emission wavelength than that of the reporter.
  • the reporter may be selected from fluorescent organic dyes modified with a suitable linking group for attachment to the oligonucleotide, such as to the terminal 3' carbon or terminal 5' carbon.
  • the quencher may also be selected from organic dyes, which may or may not be fluorescent, depending on the
  • the absorption band of the quencher should at least substantially overlap the fluorescent emission band of the reporter to optimize the quenching.
  • Non-fluorescent quenchers or dark quenchers typically function by absorbing energy from excited reporters, but do not release the energy radiatively.
  • reporter-quencher pairs for particular probes may be undertaken in accordance with known techniques. Fluorescent and dark quenchers and their relevant optical properties from which exemplary reporter-quencher pairs may be selected are listed and described, for example, in Berlman, Handbook of
  • modifying reporters and quenchers for covalent attachment via common reactive groups may be found, for example, in Haugland, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes of Eugene, OR, 1992, the content of which is incorporated herein by reference.
  • reporter-quencher pairs may be selected from xanthene dyes including fluoresceins and rhodamine dyes. Many suitable forms of these compounds are available commercially with substituents on the phenyl groups, which can be used as the site for bonding or as the bonding functionality for attachment to an oligonucleotide.
  • fluorescent compounds for use as reporters are the naphthylamines, having an amino group in the alpha or beta position.
  • naphthylamino compounds 1 -dimethylaminonaphthyl-5 sulfonate, 1 - anilino-8-naphthalene sulfonate and 2-p-touidinyl-6-naphthalene sulfonate.
  • Other dyes include 3-phenyl-7-isocyanatocoumarin; acridines such as 9-isothiocyanatoacridine; N- (p-(2-benzoxazolyl)phenyl)maleimide; benzoxadiazoles; stilbenes; pyrenes and the like.
  • the reporters and quenchers are selected from
  • fluorescein and rhodamine dyes are well known in the art.
  • Suitable examples of quenchers may be selected from 6-carboxy-tetramethyl- rhodamine, 4-(4-dimethylaminophenylazo) benzoic acid (DABYL),
  • TAMRA tetramethylrhodamine
  • BHQ-0TM BHQ-1TM
  • BHQ-2TM BHQ-3TM
  • QSY-7TM QSY-9TM
  • QSY-21TM QSY-35TM
  • ThermoFisher Scientific Wired, MA
  • Suitable examples of reporters may be selected from dyes such as SYBR green,
  • 6-carboxyfluorescein (6-FAM), tetrachloro-6-carboxyfluorescein (TET), 2,7-dimethoxy- 4,5-dichloro-6-carboxyfluorescein, hexachloro-6-carboxyfluorescein (HEX), 6-carboxy- 2',4,7,7'-tetrachlorofluorescein (6-TETTM available from ThermoFisher Scientific
  • a probe which contains a reporter and a quencher is a probe that is to be used in a 5'-exonuclease assay, such as the Taqman® real-time PCR technique.
  • the oligonucleotide probe will have a sufficient number of phosphodiester linkages adjacent to its 5' end so that the 5' to 3' nuclease activity employed can efficiently degrade the bound probe to separate the reporters and quenchers.
  • a probe which contains a reporter and a quencher is a Scorpion probe in either a unimolecular or bimolecular conformation.
  • a probe portion of the primer-probe complex is flanked by self
  • a reporter is typically attached at or near one of the self-complementary regions, such as at the 5' terminus of the Scorpion probe, and a quencher is attached at or near the other self-complementary region, such as at the 3' end of the complementary sequence and adjacent to the non-amplifiable linker, such that the quencher is in sufficiently close proximity to the reporter to cause quenching when the probe is in its stem-loop conformation.
  • the reporter is typically attached to the probe region of the Scorpion probe, such as at the 5' terminus of the Scorpion probe, while the quencher is attached to the blocking oligonucleotide, such as at the 3' terminus of the blocking oligonucleotide, such that the quencher is in sufficiently close proximity to the reporter to cause quenching when the probe is unbound from its target DNA and is instead hybridized to the blocking oligonucleotide.
  • probe which contains a reporter and quencher is a Molecular Beacon type probe, which contains a probe region flanked by self-complementary regions that allow the probe to form a stem-loop structure when unbound from the probe’s target sequence.
  • probes typically have a reporter attached at or near one terminus and a quencher attached at or near the other terminus such that the quencher is in sufficiently close proximity to the reporter to cause quenching when the probe is in its unbound, and thus stem-loop, form.
  • replication inhibitor moiety refers to any atom, molecule or chemical group that is attached to the 3' terminal hydroxyl group of an oligonucleotide that will block the initiation of chain extension for replication of a nucleic acid strand. Examples include, but are not limited to: 3'-deoxynucleotides (e.g., cordycepin),
  • non parti cipatory refers to the lack of participation of a probe or primer in a reaction for the amplification of a nucleic acid molecule.
  • a non-participatory probe or primer is one that will not serve as a substrate for, or be extended by, a DNA or RNA polymerase.
  • A“non-participatory probe” is inherently incapable of being chain extended by a polymerase. It may or may not have a replication inhibitor moiety.
  • a nucleic acid molecule is“hybridizable” to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength.
  • Hybridization and washing conditions are well known and exemplified, for example, in Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989), particularly Chapter 1 1 and Table 1 1.1 therein (entirely incorporated herein by reference).
  • the conditions of temperature and ionic strength determine the“stringency” of the hybridization.
  • low stringency hybridization conditions corresponding to a Tm of 55°C, can be used, e.g., 5X SSC, 0.1 % SDS, 0.25% milk, and no formamide; or 30% formamide, 5X SSC, 0.5% SDS.
  • Moderate stringency hybridization conditions correspond to a higher Tm, e.g., 40% formamide, with 5X or 6X SSC.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although, depending on the stringency of the hybridization, mismatches between bases are possible.
  • the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T for hybrids of nucleic acids having those sequences.
  • the relative stability (corresponding to higher T ) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA.
  • equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-9.51 ).
  • the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-1 1.8).
  • the length for a hybridizable nucleic acid is at least about 10 nucleotides.
  • a minimum length for a hybridizable nucleic acid is at least about 1 1 nucleotides, at least about 12 nucleotides, at least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 21 nucleotides, at least about 22 nucleotides, at least about 23 nucleotides, at least about 24 nucleotides, at least about 25
  • nucleotides at least about 26 nucleotides, at least about 27 nucleotides, at least about 28 nucleotides, at least about 29 nucleotides, at least 30 nucleotides, or more.
  • the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.
  • Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by, e.g., Sambrook et al. (supra); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc and Wiley-lnterscience (1987).
  • Applicants have solved the stated problem through a method that sequences upstream of and within a genome region encoding for a Gamma-glutamyl-gamma- aminobutyrate hydrolase, the amplification of which is only detectable in bacteria belonging to the genus Cronobacter.
  • the present detection method finds utility in detection of bacteria belonging to the genus Cronobacter in any type of sample, for example in appropriate samples for food testing, environmental testing, or human or animal diagnostic testing. While examples of suitable methods for detecting these regions are included herein, it is to be understood that the present disclosure is not limited to the methods described. Rather any suitable method can be employed to detect these DNA regions and subsequently the genus Cronobacter itself.
  • Oligonucleotides of the instant disclosure are set forth in SEQ ID NOs: 1 -54.
  • Oligonucleotides of the instant disclosure may be used as primers for PCR amplification. Exemplary primer pairs and their corresponding probes are shown in Table 1.
  • oligonucleotide primers may also be useful for other nucleic acid amplification methods such as the ligase chain reaction (LCR) (Backman et al., 1989, EP 0 320 308; Carrino et al., 1995, J. Microbiol. Methods 23: 3-20); nucleic acid sequence-based amplification (NASBA), (Carrino et al., 1995, supra); and self- sustained sequence replication (3SR) and 'Q replicase amplification' (Pfeffer et al., 1995 Veterinary Res. Comm. 19: 375-407).
  • LCR ligase chain reaction
  • NASBA nucleic acid sequence-based amplification
  • 3SR self- sustained sequence replication
  • 'Q replicase amplification' Pfeffer et al., 1995 Veterinary Res. Comm. 19: 375-407.
  • the oligonucleotide primers of the present disclosure can also contain a detectable label, for example a 5' end label.
  • hybridization probes can be SEQ ID NO: 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, and 47.
  • Hybridization using DNA probes has been frequently used for the detection of pathogens in food, clinical and environmental samples, and the methodologies are generally known to one skilled in the art. It is generally recognized that the degree of sensitivity and specificity of probe hybridization is lower than that achieved through the previously described amplification techniques.
  • the nucleic acid probes of the present disclosure can also possess a detectable label, such as a reporter-quencher combination as are employed in 5’-exonuclease detection assays, such as the Taqman® assay or in Scorpion probe assays.
  • a detectable label such as a reporter-quencher combination as are employed in 5’-exonuclease detection assays, such as the Taqman® assay or in Scorpion probe assays.
  • the 3' terminal nucleotide of the nucleic acid probe may be rendered incapable of extension by a nucleic acid polymerase in some embodiments. Such blocking may be carried out, for example, by the attachment of a replication inhibitor moiety, such as a reporter or quencher, to the terminal 3' carbon of the nucleic acid probe by a linking moiety, or by making the 3'-terminal nucleotide a dideoxynucleotide.
  • the 3' end of the nucleic acid probe may be rendered impervious to the 3' to 5' extension activity of a polymerase by incorporating one or more modified internucleotide linkages onto the 3' end of the oligonucleotide. Minimally, the 3' terminal internucleotide linkage must be modified; however, additional internucleotide linkages may be modified.
  • Internucleotide modifications which prevent elongation from the 3' end of the nucleic acid probe and/or which block the 3' to 5' exonuclease activity of the DNA polymerase during PCR may include phosphorothioate linkages, methylphosphonate linkages, boranophosphate linkages, and other similar polymerase-resistant internucleotide linkages.
  • An alternative method to block 3' extension of the probe is to form an adduct at the 3' end of the probe using mitomycin C or other like antitumor antibiotics such as described in Basu et al., Biochemistry 32:4708-4718, 1993.
  • mitomycin C or other like antitumor antibiotics such as described in Basu et al., Biochemistry 32:4708-4718, 1993.
  • a nucleic acid probe sequence can also optionally be employed with the primer sequence pairs of the present disclosure in an amplification based detection technique, such as in the 3'-exonuclease assay.
  • exemplary primer/probe combinations are indicated in Table 1.
  • Detection of the presence of the genus Cronobacter itself may be accomplished in any suitable manner.
  • methods are primer-directed
  • amplification methods and nucleic acid hybridization methods may be used to detect the genus Cronobacter in a sample that is either a complex matrix or a purified culture, e.g., from an animal, environmental, or food source suspected of contamination.
  • Some embodiments comprise (1 ) culturing a complex sample mixture in a non- selective growth media to resuscitate the target bacteria, (2) releasing total target bacterial DNA, and (3) subjecting the total DNA to an amplification protocol with a primer pair disclosed herein and optionally with a nucleic acid probe comprising a detectable label.
  • primer-directed nucleic acid amplification methods are known in the art which can be employed in the present detection methods, including thermal cycling methods (e.g., PCR, RT-PCR, and LCR), as well as isothermal methods and strand displacement amplification (SDA).
  • the primer-directed amplification assay method is PCR.
  • the primer pairs listed in Table 1 may be used as primers for use in primer-directed nucleic acid amplification for the detection of SEQ ID NOs: 1 -3 and subsequently detection and identification of the genus Cronobacter.
  • the oligonucleotides and methods according to the instant disclosure may be used directly with any suitable clinical or environmental samples, without any need for sample preparation.
  • the samples can be pre-treated and pre-amplification enrichment can be performed.
  • the minimum industry standard for the detection of food-borne bacterial pathogens is a method that will reliably detect the presence of one pathogen cell in 25 g of food matrix as described in Andrews et al., 1984,“Food Sample and Preparation of Sample Homogenate”, Chapter 1 in Bacteriological Analytical Manual, 8th Edition, Revision A, Association of Official Analytical Chemists, Arlington, VA.
  • enrichment methods and media have been developed to enhance the growth of the target pathogen cell in order to facilitate its detection by biochemical, immunological or nucleic acid hybridization means.
  • Typical enrichment procedures employ media that will enhance the growth and health of the target bacteria and also inhibit the growth of any background or non-target microorganisms present.
  • the FDA has set forth a protocol for enrichment of samples of powdered infant formula to be tested for pathogenic Cronobacter ⁇ U.S. Food and Drug
  • Selective media have been developed for a variety of bacterial pathogens and one of skill in the art will know to select a medium appropriate for the particular organism to be enriched, e.g. the genus Cronobacter.
  • a general discussion and recipes of non-selective media are described in the FDA Bacteriological Analytical Manual. (1998) published and distributed by the Association of Analytical Chemists, Suite 400, 2200 Wilson Blvd, Arlington, VA 22201 -3301.
  • a sample of the complex mixtures is removed for further analysis. This sampling procedure may be accomplished by a variety of means well known to those skilled in the art.
  • 5 pi of the enrichment culture is removed and added to 200 mI of lysis solution containing protease.
  • the lysis solution is heated at 37°C for 20 min followed by protease inactivation at 95°C for 10 min as described in the BAX® System User’s Guide, Hygiena, LLC (Camarillo, CA).
  • Some embodiments are directed to methods for detecting the presence of the genus Cronobacter in a sample comprising (a) performing PCR amplification using primer pairs listed in Table 1 to produce a PCR amplification result; and (b) detecting the amplification, whereby a positive detection of the amplification indicates the presence of bacteria belonging to the genus Cronobacter in the sample.
  • a step of preparing the sample may be carried out prior to performing PCR amplification.
  • the preparing step may comprise at least one of the following processes: (1 ) bacterial enrichment, (2) separation of bacterial cells from the sample, (3) cell lysis, and (4) total DNA extraction.
  • any generally acceptable PCR conditions may be used for successfully detecting bacteria belonging to the genus Cronobacter using the oligonucleotides of the instant disclosure, and depending on the sample to be tested and other laboratory conditions, routine optimization for the PCR conditions may be necessary to achieve optimal sensitivity and specificity. Optimally, they achieve PCR amplification results from all of the intended specific targets while giving no PCR results for other, non-target species.
  • Primer-directed amplification products can be analyzed using various methods.
  • “Homogenous detection” refers to a method for the detection of amplification products where no separation (such as by gel electrophoresis) of amplification products from template or primers is necessary. Homogeneous detection is typically accomplished by measuring the level of fluorescence of the reaction mixture during or immediately following amplification.
  • heterogeneous detection methods which involve separation of amplification products during or prior to detection, can be employed in the present methods.
  • Homogenous detection may be employed to carry out“real-time” primer-directed nucleic acid amplification and detection, using primer pairs of the instant disclosure (e.g.,“real-time” PCR and“real-time” RT-PCR).
  • Exemplary“real-time” methods are set forth in U.S. Patent Nos. 6,171 ,785, 5,994,056, 6,326,145, 5,804,375, 5,538,848, 5,487,972, and 5,210,015, each of which is hereby incorporated by reference in its entirety.
  • the“real-time” detection method is the 5'-exonuclease detection method, as set forth in U.S. Patent Nos.
  • a modified probe is employed during PCR which binds intermediate to or between the two members of the amplification primer pair.
  • the modified probe possesses a reporter and a quencher and is designed to generate a detectable signal to indicate that it has hybridized with the target nucleic acid sequence during PCR. As long as both the reporter and the quencher are on the probe, the quencher stops the reporter from emitting a detectable signal.
  • the intrinsic 5' to 3' nuclease activity of the polymerase degrades the probe, separating the reporter from the quencher, and enabling the detectable signal to be emitted.
  • the amount of detectable signal generated during the amplification cycle is proportional to the amount of product generated in each cycle.
  • the efficiency of quenching is a strong function of the proximity of the reporter and the quencher, i.e., as the two molecules get closer, the quenching efficiency increases.
  • the reporter and the quencher are, in some embodiments, attached to the probe within a few nucleotides of one another, usually within 30 nucleotides of one another, and in some embodiments with a separation of from about 6 to 16 nucleotides. Typically, this separation is achieved by attaching one member of a reporter-quencher pair to the 5' end of the probe and the other member to a nucleotide about 6 to 16 nucleotides away.
  • amplification and detection is performed using labeled Taqman® probes.
  • SEQ ID NOs: 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, and 47 can possess a CAL Fluor 610 reporter attached at the 5' terminus and a BHQ2 quencher attached at the 3' terminus.
  • Scorpion probe assay Another“real-time” detection method is the Scorpion probe assay as set forth in U.S. Patent No. 6,326,145, which is hereby incorporated by reference in its entirety.
  • PCR amplification is performed using a Scorpion probe (either unimolecular or bimolecular) as a primer-probe complex, the Scorpion probe possessing an appropriate reporter-quencher pair to allow the detectable signal of the reporter to be quenched prior to elongation of the primer. Post-elongation, the quenching effect is eliminated, and the amount of signal present is quantitated.
  • a Scorpion probe either unimolecular or bimolecular
  • SEQ ID NOs: 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, and 47 can possess a CAL Fluor 610 reporter attached at the 5’ terminus and an internal Spacer 18 and an internal BHQ2 quencher.
  • each probe can have a different detectable label (e.g., reporter-quencher pair) attached, thus allowing each probe to be detected independently of the other probes.
  • Another method of homogenous detection involves the use of DNA melting curve analysis, particularly with the BAX® System hardware and reagent tablets from Hygiena, LLC (Camarillo, CA). The details of the system are given in U.S. Patent No. 6,312,930 and PCT Publication Nos. WO 97/1 1 197 and WO 00/66777, each of which is hereby incorporated by reference in its entirety.
  • dsDNA double stranded nucleic acid molecule
  • target amplicon amplification product during each amplification cycle at selected time points.
  • TMS melting start at a temperature
  • TME completes at another temperature
  • a typical PCR cycle involves a denaturing phase where the target dsDNA is melted, a primer annealing phase where the temperature optimal for the primers to bind to the now-single-stranded target, and a chain elongation phase (at a temperature TE) where the temperature is optimal for DNA polymerase to function.
  • TMS should be higher than T E , and TME should be lower (often substantially lower) than the temperature at which the DNA polymerase is heat-inactivated. Melting characteristics are effected by the intrinsic properties of a given dsDNA molecule, such as deoxynucleotide composition and the length of the dsDNA.
  • Intercalating dyes will bind to double stranded DNA.
  • the dye/dsDNA complex will fluoresce when exposed to the appropriate excitation wavelength of light, which is dye dependent, and the intensity of the fluorescence may be proportionate to concentration of the dsDNA.
  • Methods taking advantage of the use of DNA intercalating dyes to detect and quantify dsDNA are known in the art. Many dyes are known and used in the art for these purposes. The instant methods also take advantage of such relationship.
  • intercalating dyes include, but are not limited to, SYBR® Green-I, ethidium bromide, propidium iodide, TOTO®-1 ⁇ Quinolinium, 1 -1 '-[1 ,3- propanediylbis [(dimethyliminio)-3,1 -propanediyl]]bis[4-[(3-methyl-2(3H)- benzothiazolylidene) methyl]]-, tetraiodide ⁇ , and YoPro® ⁇ Quinolinium, 4-[(3-methyl- 2(3H)-benzoxazolylidene)methyl]-1 -[3-(trimethylammonio)-propyl]-, diiodide ⁇ .
  • the intercalating dye is a non-asymmetrical cyanide dye such as SYBR® Green-I, manufactured by Molecular Probes, Inc. (Eugene, OR).
  • Melting curve analysis is achieved by monitoring the change in fluorescence while the temperature is increased. When the temperature reaches the TMS specific for the target amplicon, the dsDNA begins to denature. When the dsDNA denatures, the intercalating dye dissociates from the DNA and fluorescence decreases. Mathematical analysis of the negative of the change of the log of fluorescence divided by the change in temperature plotted against the temperature results in the graphical peak known as a melting curve.
  • the present methods could be operated using a combination of these techniques, such as by having a Scorpion probe directed to one target region and a Taqman® probe directed to a second target region. It should also be understood that the present disclosure is not limited to the above described techniques. Rather, one skilled in the art would recognize that other techniques for detecting amplification as known in the art may also be used. For example, techniques such as PCR-based quantitative sequence detection (QSD) may be performed using nucleic acid probes which, when present in the single-stranded state in solution, are configured such that the reporter and quencher are sufficiently close to substantially quench the reporter's emission.
  • QSD quantitative sequence detection
  • the reporter and quenchers become sufficiently distant from each other. As a result, the quenching is substantially abated causing an increase in the fluorescence emission detected.
  • heterogeneous detection methods include standard non-denaturing gel electrophoresis (e.g., acrylamide or agarose), denaturing gradient gel electrophoresis, and temperature gradient gel electrophoresis.
  • standard non-denaturing gel electrophoresis e.g., acrylamide or agarose
  • denaturing gradient gel electrophoresis denaturing gradient gel electrophoresis
  • temperature gradient gel electrophoresis e.g., temperature gradient gel electrophoresis.
  • Standard non-denaturing gel electrophoresis is a simple and quick method of PCR detection but may not be suitable for all applications.
  • DGGE Denaturing Gradient Gel Electrophoresis
  • electrophoresis results because the difference in charge density between DNA molecules is near neutral and plays little role in their separation. As the size of the DNA fragment increases, its velocity through the gel decreases.
  • DGGE is primarily used to separate DNA fragments of the same size based on their denaturing profiles and sequence.
  • two strands of a DNA molecule separate, or melt, when heat or a chemical denaturant is applied.
  • the denaturation of a DNA duplex is influenced by two factors: 1 ) the hydrogen bonds formed between complimentary base pairs (since GC rich regions melt at higher denaturing conditions than regions that are AT rich); and 2) the attraction between neighboring bases of the same strand, or“stacking”. Consequently, a DNA molecule may have several melting domains with each of their individual characteristic denaturing conditions determined by their nucleotide sequence.
  • DGGE exploits the fact that otherwise identical DNA molecules having the same length and DNA sequence, with the exception of only one nucleotide within a specific denaturing domain, will denature at different temperatures or Tm.
  • ds double-stranded
  • the dsDNA fragment will travel faster than a denatured single-stranded (ss) DNA fragment, since the branched structure of the single-stranded moiety of the molecule becomes entangled in the gel matrix.
  • the dsDNA fragment will completely dissociate and mobility of the molecule through the gel is retarded at the denaturant concentration at which the particular low denaturing domains of the DNA strand dissociate.
  • the electrophoresis is conducted at a constant temperature (around 60°C) and chemical denaturants are used at concentrations that will result in 100% of the DNA molecules being denatured (i.e., 40% formamide and 7M urea).
  • This variable denaturing gradient is created using a gradient maker, such that the composition of each DGGE gel gradually changes from 0% denaturant up to 100% denaturant.
  • gradients containing a reduced range of denaturant e.g., 35% to 60%
  • DGGE Temperature Gradient Gel Electrophoresis
  • TGGE Temperature Gradient Gel Electrophoresis
  • This method makes use of a temperature gradient to induce the conformational change of dsDNA to ssDNA to separate fragments of equal size with different sequences.
  • DNA fragments with different nucleotide sequences will become immobile at different positions in the gel.
  • Variations in primer design can be used to advantage in increasing the usefulness of DGGE for characterization and identification of the PCR products. These methods and principles of using primer design variations are described in PCR Technology Principles and Applications, Henry A. Erlich Ed., M. Stockton Press, NY, pages 71 to 88 (1988).
  • the level of fluorescence can be measured using a laser fluorometer such as, for example, an ABI Prism Model 7500 Fast Sequence Detector.
  • a laser fluorometer such as, for example, an ABI Prism Model 7500 Fast Sequence Detector.
  • similar detection systems for measuring the level of fluorescence in a sample are included in the present disclosure.
  • replication composition in any format can be used.
  • a typical replication composition for PCR amplification may comprise, for example, dATP, dCTP, dGTP, dTTP, target specific primers and a suitable polymerase.
  • replication composition is in liquid form
  • suitable buffers known in the art may be used (Sambrook, J. et al., supra).
  • the replication composition is contained in a tablet form
  • typical tabletization reagents may be included such as stabilizers and binding agents.
  • Exemplary tabletization technology is set forth in U.S. Patent Nos. 4,762,857 and 4,678,812, each of which is hereby incorporated by reference in its entirety.
  • a replication composition disclosed herein can comprise (a) at least one primer pair selected from Table 1 , and (b) thermostable DNA
  • Another replication composition can comprise (a) at least two primer pairs selected from Table 1 , each directed toward a different target DNA region; and (b) thermostable DNA polymerase.
  • a replication composition disclosed herein can comprise (a) at least two primer pairs and any corresponding probe or blocking oligonucleotide selected from Table 1 , wherein each nucleic acid probe or primer-probe complex employed comprises a detectable label; and (b) thermostable DNA polymerase.
  • the detectable label can comprise a reporter capable of emitting a detectable signal and a quencher capable of substantially quenching the reporter and preventing the emission of the detectable signal when the reporter and quencher are in sufficiently close proximity to one another.
  • a kit of the instant disclosure can comprise any one of the above replication compositions.
  • a tablet can comprise any one of the above replication compositions.
  • a kit of the instant disclosure can comprise the foregoing tablet.
  • an internal positive control can be included in the reaction.
  • the internal positive control can include control template nucleic acids (e.g. DNA or RNA), control primers, and control nucleic acid probe.
  • control template nucleic acids e.g. DNA or RNA
  • control primers e.g. DNA or RNA
  • control nucleic acid probe e.g. DNA or RNA
  • the advantages of an internal positive control contained within a PCR reaction have been previously described (U.S. Patent No. 6,312,930 and PCT Application No.
  • WO 97/1 1197 each of which is hereby incorporated by reference in its entirety), and include: (i) the control may be amplified using a single primer; (ii) the amount of the control amplification product is independent of any target DNA or RNA contained in the sample; (iii) the control DNA can be tableted with other amplification reagents for ease of use and high degree of reproducibility in both manual and automated test procedures; (iv) the control can be used with homogeneous detection, i.e., without separation of product DNA from reactants; and (v) the internal control has a melting profile that is distinct from other potential amplification products in the reaction and/or a detectable label on the control nucleic acid that is distinct from the detectable label on the nucleic acid probe directed to the target.
  • Control DNA will be of appropriate size and base composition to permit amplification in a primer-directed amplification reaction.
  • the control template DNA sequence may be obtained from the Cronobacter genome, or from another source, but must be reproducibly amplified under the same conditions that permit the amplification of the target amplification product.
  • Exemplary control sequences include, for example, those found in SV40.
  • the concentration range of SV40 when used, can about 10 1 to about 10 7 , about 10 2 to about 10 7 , about 10 3 to about 10 7 , about 10 4 to about 10 7 , about 10 5 to about 10 7 , about 10 6 to about 10 7 , about 10 1 to about 10 6 , about 10 2 to about 10 6 , about 10 3 to about 10 6 , about 10 4 to about 10 6 , about 10 5 to about 10 6 , about 10 1 to about 10 5 , about 10 2 to about 10 5 , about 10 3 to about 10 5 , about 10 4 to about 10 5 , about 10 1 to about 10 4 , about 10 2 to about 10 4 , about 10 3 to about 10 4 , about 10 1 to about 10 3 , about 10 2 to about 10 3 , or about 10 1 to about 10 2 copies per PCR reaction.
  • control reaction is useful to validate the amplification reaction.
  • Amplification of the control DNA occurs within the same reaction tube as the sample that is being tested, and therefore indicates a successful amplification reaction when samples are target negative, i.e. no target amplification product is produced.
  • a suitable number of copies of the control DNA template must be included in each amplification reaction.
  • the negative control replication composition will contain the same reagents as the replication composition but without the polymerase.
  • the primary function of such a control is to monitor spurious background fluorescence in a homogeneous format when the method employs a fluorescent means of detection.
  • Replication compositions may be modified depending on whether they are designed to be used to amplify target DNA or the control DNA.
  • compositions that will amplify the target DNA may include (i) a polymerase (generally thermostable), (ii) a primer pair capable of hybridizing to the target DNA and (iii) necessary buffers for the amplification reaction to proceed.
  • Replication compositions that will amplify the control DNA may include (i) a polymerase (generally
  • thermostable (ii) the control DNA; (iii) at least one primer capable of hybridizing to the control DNA; and (iv) necessary buffers for the amplification reaction to proceed.
  • the replication composition for either target DNA or control DNA amplification can contain a nucleic acid probe, and can possess a detectable label.
  • nucleic acid hybridization assay methods can be employed in the present disclosure for detection of bacteria belonging to the genus Cronobacter.
  • the basic components of a nucleic acid hybridization test include a probe, a sample suspected of containing bacteria belonging to the genus Cronobacter, and a specific hybridization method.
  • the probe length can vary from as few as 5 bases to the full length of the Cronobacter diagnostic sequence and will depend upon the specific test to be done. Only part of the probe molecule need be complementary to the nucleic acid sequence to be detected. In addition, the complementarity between the probe and the target sequence need not be perfect. Hybridization does occur between imperfectly complementary molecules with the result that a certain fraction of the bases in the hybridized region are not paired with the proper complementary base.
  • Probes particularly useful in nucleic acid hybridization methods are any of SEQ ID NOs:1 -54, or sequences derived therefrom.
  • the sample may or may not contain bacteria belonging to the genus
  • Cronobacter The sample may take a variety of forms; however, the sample will generally be extracted from an animal, environmental or food source suspected of contamination.
  • the DNA may be detected directly but, in some embodiments, the sample nucleic acid must be made available to contact the probe before any
  • hybridization of probe and target molecule can occur.
  • the organism s DNA is, in some embodiments, free from the cell and placed under the proper conditions before hybridization can occur.
  • Methods of in-solution hybridization necessitate the
  • hybridization assays may be conducted directly on cell lysates, without the need to extract the nucleic acids. This eliminates several steps from the sample-handling process and speeds up the assay.
  • a chaotropic agent is typically added to the cell lysates prepared as described above.
  • the chaotropic agent stabilizes nucleic acids by inhibiting nuclease activity.
  • the chaotropic agent allows sensitive and stringent hybridization of short oligonucleotide probes to DNA at room temperature (Van Ness and Chen, Nucl. Acids Res. 19:5143-5151 (1991 )).
  • Suitable chaotropic agents include guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate, lithium
  • the chaotropic agent will be present at a final concentration of about 3M. If desired, one can add formamide to the hybridization mixture, typically 30-50% (v/v).
  • Pre-hybridization purification is particularly useful for standard filter hybridization assays. Furthermore, purification facilitates measures to increase the assay sensitivity by incorporating in vitro RNA amplification methods such as self- sustained sequence replication (see for example Fahy et al., In PCR Methods and Applications, Cold Spring Flarbor Laboratory: Cold Spring Flarbor, NY (1991 ), pp. 25- 33) or reverse transcriptase PCR (Kawasaki, In PCR Protocols: A Guide to Methods and Applications, M. A. Innis et al., Eds., (1990), pp. 21 -27).
  • in vitro RNA amplification methods such as self- sustained sequence replication (see for example Fahy et al., In PCR Methods and Applications, Cold Spring Flarbor Laboratory: Cold Spring Flarbor, NY (1991 ), pp. 25- 33) or reverse transcriptase PCR (Kawasaki, In PCR Protocols: A Guide to Methods and Applications, M. A. Inn
  • the DNA can be detected by any of a variety of methods. Flowever, the most useful embodiments have at least some characteristics of speed, convenience, sensitivity, and specificity.
  • Hybridization methods are well known in the art.
  • the probe and sample must be mixed under conditions which will permit nucleic acid hybridization. This involves contacting the probe and sample in the presence of an inorganic or organic salt under the proper concentration and temperature conditions.
  • the probe and sample nucleic acids must be in contact for a long enough time that any possible hybridization between the probe and sample nucleic acid may occur.
  • concentration of probe or target in the mixture will determine the time necessary for hybridization to occur. The higher the probe or target concentration, the shorter the hybridization incubation time needed.
  • hybridization solutions can be employed. Typically, these comprise from about 20 to about 60%, about 25 to about 60%, about 30 to about 60%, about 35 to about 60%, about 40 to about 60%, about 45 to about 60%, about 50 to about 60%, about 55 to about 60%, about 20 to about 55%, about 25 to about 55%, about 30 to about 55%, about 35 to about 55%, about 40 to about 55%, about 45 to about 55%, about 50 to about 55%, about 20 to about 50%, about 25 to about 50%, about 30 to about 50%, about 35 to about 50%, about 40 to about 50%, about 45 to about 50%, about 20 to about 45%, about 25 to about 45%, about 30 to about 45%, about 35 to about 45%, about 40 to about 45%, about 20 to about 40%, about 25 to about 40%, about 30 to about 40%, about 35 to about 40%, about 20 to about 35%, about 25 to about 35%, about 30 to about 35%, about 20 to about 30%, about 25 to about 30%, or about 20 to about 25% volume of a polar organic solvent.
  • the hybridization solution comprises about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% polar organic solvent.
  • a common hybridization solution employs about 30 to about 50%, about 35 to about 50%, about 40 to about 50%, about 45 to about 50%, about 30 to about 45%, about 35 to about 45%, about 30 to about 40%, about 35 to about 40%, or about 30 to about 35% v/v formamide; about 0.15 to about 1 M, about 0.25 to about 1 M, about 0.5 to about 1 M, about 0.75 to about 1 M, about 0.15 to about 0.75 M, about 0.25 to about 0.75 M, about 0.5 to about 0.75 M, about 0.15 to about 0.5 M, about 0.25 to about 0.5 M, or about 0.15 to about 0.25 M sodium chloride; about 0.05 to about 0.1 M, about 0.06 to about 0.1 M, about 0.07 to about 0.1 M, about 0.08 to about 0.1 M, about 0.09 to about 0.1 M, about 0.05 to
  • dodecylsulfate and, in some embodiments, between about 0.5 to about 20 mM, about 1 to about 20 mM, about 5 to about 20 mM, about 10 to about 20 mM, about 15 to about 20 mM, about 0.5 to about 15 mM, about 1 to about 15 mM, about 5 to about 15 mM, about 10 to about 15 mM, about 0.5 to about 10 mM, about 1 to about 10 mM, about 5 to about 10 mM, about 0.5 to about 5 mM, about 1 to about 5 mM, or about 0.5 to about 1 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kilodaltons), polyvinylpyrrolidone (about 250-500 kdal), and serum albumin.
  • FICOLL Pharmacia Inc.
  • unlabeled carrier nucleic acids from about 0.1 to about 5 mg/mL, about 0.5 to about 5 mg/mL, about 1 to about 5 mg/mL, about 2 to about 5 mg/mL, about 3 to about 5 mg/mL, about 4 to about 5 mg/mL, about 0.1 to about 4 mg/mL, about 0.5 to about 4 mg/mL, about 1 to about 4 mg/mL, about 2 to about 4 mg/mL, about 3 to about 4 mg/mL, about 0.1 to about 3 mg/mL, about 0.5 to about 3 mg/mL, about 1 to about 3 mg/mL, about 2 to about 3 mg/mL, about 0.1 to about 2 mg/mL, about 0.5 to about 2 mg/mL, about 1 to about 2 mg/mL, about 0.1 to about 1 mg/mL, about 0.5 to about 1 mg/mL, or about 0.1 to about 0.5 mg/mL;
  • fragmented nucleic DNA e.g., calf thymus or salmon sperm DNA, or yeast RNA
  • fragmented nucleic DNA e.g., calf thymus or salmon sperm DNA, or yeast RNA
  • 0.5 to about 2% wt/vol about 1 to about 2% wt/vol, about 1.5 to about 2% wt/vol, about 0.5 to about 1.5% wt/vol, about 1 to about 1.5% wt/vol, or about 0.5 to about 1 % wt/vol glycine.
  • volume exclusion agents which include a variety of polar water-soluble or swellable agents (e.g., polyethylene glycol), anionic polymers (e.g., polyacrylate or polymethylacrylate), and anionic saccharidic polymers (e.g., dextran sulfate).
  • polar water-soluble or swellable agents e.g., polyethylene glycol
  • anionic polymers e.g., polyacrylate or polymethylacrylate
  • anionic saccharidic polymers e.g., dextran sulfate
  • Nucleic acid hybridization is adaptable to a variety of assay formats. One of the most suitable is the sandwich assay format. The sandwich assay is particularly adaptable to hybridization under non-denaturing conditions.
  • a primary component of a sandwich-type assay is a solid support. The solid support has adsorbed to it or covalently coupled to it immobilized nucleic acid probe that is unlabeled and
  • the sandwich assay may be encompassed in an assay kit.
  • This kit would include a first component for the collection of samples suspected of contamination and buffers for the disbursement and lysis of the sample.
  • a second component would include media in either dry or liquid form for the hybridization of target and probe polynucleotides, as well as for the removal of undesirable and nonduplexed forms by washing.
  • a third component would include a solid support (dipstick) upon which is fixed (or to which is conjugated) unlabeled nucleic acid probe(s) that is (are)
  • a fourth component would contain labeled probe that is complementary to a second and different region of the same DNA strand to which the immobilized, unlabeled nucleic acid probe of the third component is hybridized.
  • SEQ ID NOs: 1 -47 or derivations thereof may be used as 3' blocked detection probes in either a homogeneous or heterogeneous assay format.
  • a probe generated from these sequences may be 3' blocked or non- participatory and will not be extended by, or participate in, a nucleic acid amplification reaction.
  • the probe incorporates a label that can serve as a reactive ligand that acts as a point of attachment for the immobilization of the probe/analyte hybrid or as a reporter to produce detectable signal.
  • genomic or cDNA isolated from a sample suspected of Cronobacter contamination is amplified by standard primer-directed amplification protocols in the presence of an excess of the 3' blocked detection probe to produce amplification products. Because the probe is 3' blocked, it does not participate or interfere with the amplification of the target. After the final amplification cycle, the detection probe anneals to the relevant portion of the amplified DNA and the annealed complex is then captured on a support through the reactive ligand.
  • a ligand labeled dNTP with the label probe in the replication composition to facilitate immobilization of the PCR reaction product on a support and then detection of the immobilized product by means of the labeled probe reagent.
  • a biotin, digoxigenin, or digoxin labeled dNTP could be added to PCR reaction composition.
  • the biotin, digoxigenin, or digoxin incorporated in the PCR product could then be immobilized respectively on to a strepavidin, anti-dixogin or antidigoxigenin antibody support.
  • the immobilized PCR product could then be detected by the presence of the probe label.
  • the medium used to grow the pathogenic Cronobacter strains and comparative non-target strains was Brain Heart Infusion broth (BHI) obtained from BBL (Becton- Dickenson). Samples of pathogenic Cronobacter strains were obtained from cultures grown overnight in BHI broth to approximately 10 9 cfu/ml. Samples of the comparative non-target strains were enriched in BHI at approximately 10 9 cfu/ml.
  • BHI Brain Heart Infusion broth
  • Material tested was either food enrichment (powdered infant formula prepared as described in the BAX® system user guide for the BAX® E. sakazakii assay) or overnight cultures of bacteria isolates belonging to the genus Cronobacter grown at 37 °C in BHI media. 5 pi of the material to be tested was added to 200 mI of BAX® lysis reagent (Hygiena, LLC (Camarillo, CA)). The mixture was incubated at 37 °C for 20 minutes, then further incubated at 95 °C for 10 minutes, and finally cooled to 4 °C.
  • the reagents that were used in the PCR amplification reaction were custom made lyophilized reagent tablets that contained deoxynucleotides (Roche Diagnostics, Indianapolis, Ind., USA), BSA and surfact-amps (Sigma-Aldrich, ST. Louis, MO., USA). Additionally, the reaction included Go Taq DNA Polymerase (Promega, Madison, Wis., USA) and PCR buffer (Hygiena, LLC (Camarillo, CA)).
  • Amplification and testing was performed on the BAX® Q7 machine (Hygiena, LLC (Camarillo, CA)). The thermal cycling conditions were: 2 minutes at 94 °C, followed by 43 cycles of 94 °C for 10 seconds and 63 °C for 40 seconds, with the fluorescent signal captured during the 63 °C step at each cycle.

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Abstract

La présente invention concerne un procédé rapide de détection et de caractérisation du genre Cronobacter sur la base de la présence de séquences d'acide nucléique, en particulier, un procédé à base de PCR pour la détection, et des molécules d'oligonucléotides et des réactifs et des kits utiles pour ceux-ci. Ce procédé peut être utilisé pour détecter le genre Cronobacter dans un échantillon d'aliment ou d'eau, tel qu'un enrichissement de préparation pour nourrisson en poudre. La présente invention concerne en outre des compositions de réplication et des kits pour la mise en œuvre du procédé de la présente invention.
PCT/US2020/025161 2019-04-05 2020-03-27 Séquences et leur utilisation pour la détection et la caractérisation du genre cronobacter WO2020205491A1 (fr)

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CN112899378A (zh) * 2020-12-30 2021-06-04 广东省微生物研究所（广东省微生物分析检测中心） 含有特异性分子靶标的克罗诺杆菌标准菌株及其检测和应用
CN117887873A (zh) * 2024-03-14 2024-04-16 国家食品安全风险评估中心 一种用于特异性检测克罗诺杆菌属的引物探针组合及检测方法

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CN112899378A (zh) * 2020-12-30 2021-06-04 广东省微生物研究所（广东省微生物分析检测中心） 含有特异性分子靶标的克罗诺杆菌标准菌株及其检测和应用
CN112899378B (zh) * 2020-12-30 2022-05-20 广东省微生物研究所（广东省微生物分析检测中心） 含有特异性分子靶标的克罗诺杆菌标准菌株及其检测和应用
CN117887873A (zh) * 2024-03-14 2024-04-16 国家食品安全风险评估中心 一种用于特异性检测克罗诺杆菌属的引物探针组合及检测方法
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