WO2013081202A1 - Oligonucléotide pour la détection d'o157:h7 d'e. coli et l'utilisation de celui-ci - Google Patents

Oligonucléotide pour la détection d'o157:h7 d'e. coli et l'utilisation de celui-ci Download PDF

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WO2013081202A1
WO2013081202A1 PCT/KR2011/009138 KR2011009138W WO2013081202A1 WO 2013081202 A1 WO2013081202 A1 WO 2013081202A1 KR 2011009138 W KR2011009138 W KR 2011009138W WO 2013081202 A1 WO2013081202 A1 WO 2013081202A1
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seq
probe
nucleic acid
primer
coli
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리준
쳉윈덴
에이 옵다이크제이슨
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Hanwha Vision Co Ltd
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Samsung Techwin Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • E. coli O157 H7 oligonucleotides suitable for detection oligonucleotide and the oligonucleotide with a nucleotide
  • E. coli O157: H7 Since E. coli O157: H7 was first known in 1982 as the cause of the onset of hemorrhagic colitis, it has become widespread worldwide, with thousands of infected in Japan and more than 20,000 infected and 250 killed in the United States each year. It is one of the food poisoning bacteria. In addition, E. coli O157: H7 is known as a major cause of hemorrhagic colitis along with the bacteria of the genus Campylobacter , Salmonella , and Shigella . Since E. coli O157: H7 is mainly transmitted through food such as meat, dairy products, and drinking water, a method for quickly and economically confirming the presence of E. coli O157: H7 in a sample such as food is required.
  • a common method for detecting E. coli O157: H7 is to incubate the sample in selective medium, isolate the bacteria suspected of E. coli O157: H7, and then identify it by biochemical or immunological methods. Immunological methods using antibodies can detect bacteria with high accuracy. However, a large amount of sample is required and an antibody is required for each diagnosis.
  • oligonucleotides are provided that can be suitably used to accurately, sensitively and rapidly detect E. coli 0157: H7 species.
  • the oligonucleotide may be a first primer comprising the sequence of SEQ ID NO: 16, 3, 4, or 6; It may be a second primer comprising the sequence of SEQ ID NO: 7, 8, 9, 10, or 11.
  • the oligonucleotide may comprise the sequence of SEQ ID NO: 17 or 18.
  • the first primer may have a sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6.
  • the probe may have a sequence of SEQ ID NO: 12, 13, or 14.
  • a composition comprising an oligonucleotide that can be suitably used to accurately, sensitively and rapidly detect E. coli 0157: H7 species.
  • the composition may comprise a first primer comprising a sequence of SEQ ID NO: 16, 3, 4, or 6; A second primer comprising the sequence of SEQ ID NO: 7, 8, 9, 10, or 11 may be included.
  • the composition may comprise the sequence of SEQ ID NO: 17 or 18.
  • the first primer may have a sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6.
  • the probe may have a sequence of SEQ ID NO: 12, 13, or 14.
  • kits for detecting E. coli 0157: H7 species is provided.
  • the E. coli 0157: H7 species detection kit may comprise a first primer comprising a sequence of SEQ ID NO: 16, 3, 4, or 6; A second primer comprising the sequence of SEQ ID NO: 7, 8, 9, 10, or 11 may be included.
  • the kit may further comprise a probe consisting of a DNA sequence and an RNA sequence.
  • the kit may comprise the sequence of SEQ ID NO: 17 or 18.
  • the first primer may be one of SEQ ID NO: 1, 2, 3, 4, 5, or 6.
  • the probe may be one of SEQ ID NO: 12, 13, or 14.
  • first primers E. coli Of O157: H7 It can be used to sample the target nucleic acid or fragment thereof.
  • the combination may be as follows, but is not limited thereto.
  • the kit for detection of E. coli 0157: H7 species may comprise one of the following oligonucleotides:
  • a first primer comprising at least 10 or 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 1 and a second primer comprising at least 10 or 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 7
  • a probe having a primer set and a nucleotide sequence of any one of SEQ ID NOs: 12 to 14;
  • a first primer comprising at least 10 or 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 1 and a second primer comprising at least 10 or 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 8
  • a probe having a primer set and a nucleotide sequence of any one of SEQ ID NOs: 12 to 14;
  • a first primer comprising at least 10 or 15 contiguous nucleotides selected from the nucleotide sequence of SEQ ID NO: 1 and a second primer comprising at least 10 or 15 contiguous nucleotides selected from the nucleotide sequence of SEQ ID NO: 10;
  • a probe having a primer set and a nucleotide sequence of any one of SEQ ID NOs: 12 to 14;
  • a first primer comprising at least 10 or 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 2 and a second primer comprising at least 10 or 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 7
  • a probe having a primer set and a nucleotide sequence of any one of SEQ ID NOs: 12 to 14;
  • a first primer comprising at least 10 or 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 2 and a second primer comprising at least 10 or 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 10;
  • a probe having a primer set and a nucleotide sequence of any one of SEQ ID NOs: 12 to 14;
  • a first primer comprising at least 10 or 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 3 and a second primer comprising at least 10 or 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 7
  • a probe having a primer set and a nucleotide sequence of any one of SEQ ID NOs: 12 to 14;
  • a first primer comprising at least 10 or 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 3 and a second primer comprising at least 10 or 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 10;
  • a probe having a primer set and a nucleotide sequence of any one of SEQ ID NOs: 12 to 14;
  • a first primer comprising at least 10 or 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 4 and a second primer comprising at least 10 or 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 11;
  • a probe having a primer set and a nucleotide sequence of any one of SEQ ID NOs: 12 to 14;
  • a first primer comprising at least 10 or 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 5 and a second primer comprising at least 10 or 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 7
  • a probe having a primer set and a nucleotide sequence of any one of SEQ ID NOs: 12 to 14;
  • a first primer comprising 10 or 15 consecutive nucleotides selected from the nucleotide sequences of SEQ ID NO: 5 and a second primer comprising 10 or 15 consecutive nucleotides selected from the nucleotide sequences of SEQ ID NO: 10
  • a probe having a primer set and a nucleotide sequence of any one of SEQ ID NOs: 12 to 14; or
  • a first primer comprising at least 10 or 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 6 and a second primer comprising at least 10 or 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO: 9
  • a probe having a primer set and a nucleotide sequence of any one of SEQ ID NOs: 12 to 14.
  • a method of detecting E. coli 0157: H7 species from a sample is provided.
  • the method comprises (a) amplifying a target nucleic acid of E. coli 0157: H7 in a sample to increase the copy number of the target nucleic acid, wherein the amplification comprises the nucleotide sequence of SEQ ID NO: 16, 3, 4, or 6 Hybridizing with a target nucleic acid in the sample to obtain a hybridized product of the target nucleic acid and the primer, wherein the first primer and the second primer comprising the nucleotide sequence of SEQ ID NO: 7, 8, 9, 10 or 11 are obtained.
  • nucleic acid polymerase to expand the first and second primers of the hybridized product to produce an extended primer product; (b) hybridizing the target nucleic acid with one or more probe oligonucleotides that can hybridize with the target nucleic acid to obtain a hybridization product of the target nucleic acid: probe oligonucleotide, wherein the probe comprises a DNA sequence and an RNA sequence and is detected Possible markers are linked; (c) contacting the hybridized product of the target nucleic acid : probe with RNase H to cleave the probe to cause separation of the probe fragment from the target nucleic acid; And (d) detecting the detectable label.
  • the first primer may have a sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6.
  • the probe oligonucleotide may have an oligonucleotide of SEQ ID NO: 17 or 18.
  • the probe oligonucleotide may be one of the oligonucleotides of SEQ ID NO: 12, 13, 14.
  • the probe oligonucleotide can be labeled with a detectable label, such as, for example, a fluorescence resonance energy transfer (FRET) pair.
  • FRET fluorescence resonance energy transfer
  • a method of detecting a target RNA sequence of E. coli 0157: H7 species from a sample comprises the steps of (a) reverse transcripting E. coli 0157: H7 target RNA in the presence of reverse transcriptase activity and reverse amplification primers to generate target cDNA of the target RNA; (b) amplifying the target cDNA of E.
  • coli 0157: H7 in the sample to increase the copy number of the target nucleic acid, wherein the amplification comprises a first sequence comprising the nucleotide sequence of SEQ ID NO: 16, 3, 4, or 6
  • a second primer comprising a primer and a nucleotide sequence of SEQ ID NOs: 7, 8, 9, 10 or 11 is hybridized with a target nucleic acid in the sample to obtain a hybridized product of the target nucleic acid and the primer, and template-dependent nucleic acid polymerase Using to extend the first and second primers of the hybridized product to produce an extended primer product; (c) hybridizing the target nucleic acid with at least one probe oligonucleotide substantially complementary to the target cDNA to obtain a hybridization product of a target nucleic acid: probe oligonucleotide, wherein the probe comprises a DNA sequence and an RNA sequence, and detects Possible markers are linked; (d) cleaving the probe by contacting RNase H with the hybridized product of the
  • the first primer may have a sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6.
  • the probe oligonucleotide may have an oligonucleotide of SEQ ID NO: 17 or 18.
  • the probe oligonucleotide may be one of the oligonucleotides of SEQ ID NO: 12, 13, 14.
  • the probe oligonucleotide can be labeled with a detectable label, such as, for example, a FRET pair.
  • amplification refers to any process for increasing the copy number of a nucleotide sequence.
  • Amplification of nucleic acids refers to the process by which nucleotides (eg, DNA or RNA) are incorporated into a nucleic acid.
  • nucleotide refers to a combination of base-sugar-phosphate. Nucleotides are, for example, monoliths of nucleic acids such as DNA or RNA.
  • nucleotide includes ribonucleoside triphosphates such as rATP, rCTP, rGTP, or rUTP, and deoxy-ribonucleoside triphosphates such as dATP, dCTP, dGTP, or dTTP.
  • nucleoside refers to a combination of base-sugars, ie a combination lacking phosphate in nucleotides.
  • nucleoside and nucleotide can be used interchangeably in the art.
  • the nucleotide deoxyuridine, dUTP is deoxynucleoside triphosphate.
  • it acts as a DNA monomer, such as dUMP or deoxyuridine monophosphate, and is inserted into DNA. In this regard, even if no dUTP moiety is present in the DNA output, the dUTP may be considered inserted.
  • PCR polymerase chain reaction
  • the term “polymerase chain reaction (PCR)” generally refers to an amplification method that increases the copy number of the target nucleic acid (s) in a sample. The process is described in detail in US Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188, the contents of which are incorporated herein by reference.
  • the sample may comprise a single nucleic acid or multiple nucleic acids.
  • PCR comprises two or more expandable primer nucleic acids in a reaction mixture comprising the target nucleic acid (s). The primer is complementary to the opposite strand of the double-stranded target sequence.
  • the reaction mixture is subjected to thermal cycling in the presence of nucleic acid polymerase and nucleic acid monomers such as dNTP and / or rNTP to amplify the target nucleic acid by extension of the primer.
  • the temperature change cycling includes the following procedure; Annealing and hybridizing the primer and the target nucleic acid; Expanding said primers using nucleic acid polymerase; And denaturing the hybridized primer extension product and target nucleic acid.
  • RT-PCR refers to a single strand of DNA prior to multiple cycles of DNA-dependent DNA polymerase primer expansion using RNA template and reverse transcriptase, or enzymes with reverse transcriptase activity. PCR to generate cDNA molecules.
  • multiplex PCR generally refers to a PCR that produces, by two or more kinds of primer sets, two or more amplified target products in a single reaction.
  • nucleic acid means a polymer comprising two or more nucleotides.
  • nucleic acid is used interchangeably with the terms “polynucleotide” or “oligonucleotide” herein.
  • Nucleic acids include DNA and RNA. The structure of the nucleic acid may be double-stranded and / or single-stranded.
  • nucleic acid analog means a nucleic acid comprising one or more nucleotide analogues and / or one or more phosphate ester analogs and / or one or more pentose sugar analogs.
  • nucleic acid analogs include nucleic acids in which phosphate ester and / or sugar phosphate ester bonds have been replaced by bonds of the same kind, such as N- (2-aminoethyl) -glycine amide and other amides.
  • Nucleic acid analogs may be nucleic acids comprising one or more nucleotide analogues and / or one or more phosphate ester analogs and / or one or more pentose sugar analogs and may form a double helix by hybridization.
  • annealing and “hybridization” can be used interchangeably, meaning that one nucleic acid and another nucleic acid interact with base-pairs to form a double, triple or higher order structure.
  • the primary interaction is the reaction of A / T and G / C by base-specific, eg, hydrogen bonding of Watson / Click and Hoogsteen-type.
  • base-stacking and hydrophobic interactions can also contribute to the stability of the double helix.
  • nucleotide is a deoxyribonucleotide or ribonucleotide that exists in single- or double-stranded form, and is interpreted to include analogs of nucleotides unless otherwise specified.
  • primer refers to a single strand of single strand that can act as a starting point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) in suitable buffers at suitable temperatures. Means oligonucleotides. Suitable lengths of primers are typically 15-30 nucleotides, depending on various factors, such as temperature and the use of the primer. Short primers may generally require lower temperatures to form a hybridization complex that is sufficiently stable with the template.
  • forward primer and reverse primer” refer to primers that bind to the 3 'end and the 5' end, respectively, of a predetermined portion of the template to be amplified by the polymerase chain reaction.
  • the sequence of the primer does not need to have a sequence that is completely complementary to some sequences of the template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing with the template to perform the primer-specific function. Therefore, the primer set according to one embodiment does not need to have a sequence that is perfectly complementary to the nucleotide sequence that is a template, and it is interpreted that it is sufficient to have sufficient complementarity within a range capable of hybridizing to the sequence and acting as a primer.
  • the design of such primers can be easily carried out by those skilled in the art with reference to the nucleotide sequence of the polynucleotide to be a template, for example, by using a primer design program (for example, PRIMER 3 program).
  • the primer according to one embodiment is hybridized or annealed to one site of the template to form a double chain structure.
  • Conditions for nucleic acid hybridization suitable for forming such double chain structures are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization, A Practical Approach , IRL Press, Washington, DC (1985).
  • the primer may include at least 10 or 15 contiguous nucleotides in the base sequence of any one of SEQ ID NOs 1 to 12, wherein the primer is a base of any one of SEQ ID NOs 1 to 12.
  • the primer may be a nucleotide having a sequence.
  • the primer may be a nucleotide having any one of the nucleotide sequences of SEQ ID NO: 3, 6-12 or 16. In one embodiment the primer may be any one of SEQ ID NOs: 1-12.
  • probe refers to a nucleic acid having a sequence complementary to a target nucleic acid sequence and capable of hybridizing with the target nucleic acid to form a double helix.
  • the probe sequence may be completely complementary to the target nucleic acid sequence.
  • the probe can be labeled such that the target nucleic acid can be detected simultaneously with PCR.
  • target nucleic acid or “target sequence” includes the full length of a target nucleic acid or fragment thereof that can be amplified and / or detected.
  • the target nucleic acid may be between two primers used for amplification.
  • hybrid oligonucleotide refers to an oligonucleotide molecule comprising DNA and RNA moieties in a single molecule.
  • the hybrid oligonucleotide includes one or more DNA portions and one or more RNA portions, such as, for example, DNA-RNA, RNA-DNA, or DNA-RNA-DNA oligonucleotides.
  • the oligonucleotide set for detecting E. coli 0157: H7 comprises (i) a first primer having an oligonucleotide of SEQ ID NO: 16, 3, 4, or 6 (ii) SEQ ID NO: 7, 8, 9 A second primer having an oligonucleotide of 10, or 11;
  • the oligonucleotide set may further comprise a probe selected from SEQ ID NOs: 17 or 18.
  • the first primer may be any one of SEQ ID NO: 1, 2, 3, 4, 5, or 6.
  • the probe may be any one of SEQ ID NO: 12, 13, or 14.
  • these oligonucleotides may have a sequence of at least 10, or at least 15 consecutive SEQ ID NOs: 1-12.
  • Primer pairs of the first and second primers are specific for a portion of the I segment of E. coli 0157: H7.
  • the I segment is at positions 312001-315400 of the E. coli 0157: H7 genome (GenBank: AE005174.2.).
  • the sequence of this I fragment is set forth in SEQ ID NO: 15.
  • the probe may have a DNA-RNA-DNA hybrid structure.
  • the probe may be a nucleic acid or a nucleic acid analog.
  • the probe may be a protected nucleic acid.
  • the DNA or RNA portion of the probe may be partially methylated to prevent degradation by RNA-specific enzymes such as, for example, RNase H.
  • the probe can be modified.
  • the base portion of the probe can be partially or fully methylated. Such modifications can inhibit enzyme or chemical degradation.
  • the —OH group at the 5 ′ end or 3 ′ end of the nucleic acid probe may be blocked.
  • the 3 ′ terminal OH group of the nucleic acid probe may be blocked, making expansion impossible by template-dependent nucleic acid polymerase.
  • the probe may have a detectable label.
  • the detectable label can be any chemical moiety that can be detected by any method known in the art.
  • detectable labels can include any moiety that can be detected by spectroscopy, photochemistry or by biochemical, immunochemical or chemical means. Suitable methods of labeling nucleic acid probes may be selected depending on the type of label and the location and probe of the label.
  • labels include enzymes, enzyme substrates, isotopes, fluorescent dies, chromophores, chemiluminescent labels, electrochemical luminescent labels, ligands with specific binding partners, and reactions with one another. And other labels capable of increasing, modifying or decreasing the strength of the detection signal. These labels can withstand temperature changes during the temperature change cycling of PCR.
  • the detectable label can be a fluorescence resonance energy transfer (FRET) pair.
  • the detectable label is a FRET pair comprising a fluorescent donor and a fluorescent acceptor separated by an appropriate distance, and the fluorescence emission of the donor is quenched by the acceptor. However, when the donor-acceptor pair is separated by cleavage, the fluorescence emission of the donor is increased.
  • a donor chromophore may transfer energy to the acceptor chromophore in an excited state. This shift is always non-radiative and occurs through dipole-dipole coupling. Any process that sufficiently increases the distance between the chromophores will reduce the FRET efficiency so that the release of donor chromophores can be detected radioactively.
  • donor chromophores examples include FAM, TAMRA, VIC, JOE, Cy3, Cy5, and Texas Red. Acceptor chromophores are chosen such that their excitation spectrum overlaps the emission spectrum of the donor. An example of such a pair is FAM-TAMRA. In addition, examples of such detectable labels are non-fluorescent acceptors capable of quenching a wide range of donors. Other examples of suitable donor-acceptor FRET pairs are well known in the art.
  • the probe may be present in dissolved or free form in solution.
  • the probe can be immobilized on a solid support. Different probes may be attached to the solid support and used to simultaneously detect different target sequences in the sample. Reporter molecules with different fluorescence wavelengths can be used for different probes, and thus can be separated and detected by allowing different probes to hybridize.
  • solid supports for immobilization of the oligonucleotide probes include polystyrene, avidin coated polystyrene bead cellulose, nylon, acrylamide gel and activated dextran, controlled pore glass (CPG), glass Plates and high cross-linked polystyrene. These solid supports are widely used in hybridization and diagnostic studies because of their chemical stability, ease of functionalization and defined surface area. Solid supports such as controlled pore glass (500 A, 1000 A) and non-swelling high cross-linked polystyrene (1000 A) are particularly preferred in terms of being compatible with oligonucleotide synthesis.
  • the probe can be attached to the solid support in various ways.
  • the probe can be attached to the solid support by having a 3 'or 5' terminal nucleotide of the probe attached to the solid support.
  • the probe may be attached to the solid support by a linker that provides separation of the probe from the solid support.
  • the linker may be most preferably 30 atoms or more in length, and more preferably 50 atoms or more in length.
  • Hybridization of the probe immobilized on the solid support generally requires separation of at least 30 atoms, more preferably at least 50 atoms from the solid support.
  • the linker generally comprises a spacer located between the linker and the 3 ′ nucleoside.
  • the linker arm is generally attached to the 3'-OH of the 3 'nucleoside, with an ester bond that can be cleaved with a basic reagent to release the oligonucleotide from the solid support. .
  • linkers are known in the art that can be used to attach the probes to a solid support.
  • the linker may be in the form of any compound that does not severely interfere with the hybridization of the target sequence with the probe attached to the solid support.
  • the linker may be in the form of a homopolymeric oligonucleotide so that the linker can be easily added by automated synthesis.
  • polymers such as functionalized polyethylene glycols can be used as the linker. Such polymers are preferred over single polymerizable oligonucleotides because they do not seriously interfere with the hybridization of the probes attached to the solid support.
  • polyethylene glycol is more desirable because it is commercially available, is soluble in organic and water soluble media, can be easily functionalized, and is completely stable under oligonucleotide synthesis and post-synthesis conditions. Do.
  • the bond between the solid support, the linker and the probe should preferably not be cleaved during the removal of base protecting groups under high temperature base conditions.
  • Examples of preferred bonds include carbamate and amide bonds. Immobilization of probes is well known in the art and one skilled in the art will readily be able to determine the immobilization conditions.
  • the hybridization probe may be bound to a solid support.
  • the oligonucleotide probe is contacted with a nucleic acid sample under conditions suitable for hybridization.
  • the fluorescent label is quenched by the acceptor.
  • the fluorescent label is separated from the quencher and the fluorescence emission is increased.
  • the immobilization of the hybridized probe to the solid support allows the target sequence to which the probe hybridizes to be easily separated from the sample.
  • the isolated target sequence can be separated from the solid support and used in experiments by methods well known in the art as required by the investigator (eg, purification, amplification, etc.).
  • the oligonucleotides according to one embodiment can be used for amplification and detection of target nucleic acids.
  • the amplification may involve extending the primers using template-dependent polymerase, resulting in the formation of PCR fragments or amplicons.
  • the amplification may be Polymerase Chain Reaction or Ligase Chain Reaction, Self-Sustained Sequence Replication, Strand Displacement Amplification, Transcriptional Amplification System, Q-Beta Replicase, Nucleic Acid Sequence Based Amplification (NASBA), Cleavage Fragment Length Polymorphism, Isothermal and Chimeric Primer-initiated Amplification may be performed using an amplification method such as Amplification of Nucleic Acid, Ramification-extension Amplification Method or other suitable nucleic acid amplification method.
  • the amplification may include simultaneous real time detection of the target nucleic acid.
  • PCR fragment refers to a polynucleotide molecule (or collectively several molecules) produced upon amplification of a specific target nucleic acid. Such PCR fragments generally, but not exclusively, refer to DNA PCR fragments. PCR fragments may be single-stranded or double-stranded, or mixtures thereof in any concentration ratio. PCR fragments may be 100-500 nucleotides or more in length.
  • buffers are compounds added to modify the stability and / or activity of one or more elements of the amplification reaction by modulating the amplification reaction.
  • the buffer reagent of the present invention is compatible with PCR amplification and RNase H cleavage activity.
  • buffers include, but are not limited to, HEPES ((4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid), MOPS (3- (N-morpholino) -propanesulfonic acid), and acetate or phosphate containing buffers, and the like.
  • PCR buffers may also generally comprise about 70 mM KCl and about 1.5 mM or more MgCl 2 , and about 50-200 mM of each of the dATP, dCTP, dGTP and dTTP. - May include additives for effectively optimizing PCR or PCR reactions.
  • Additives are compounds added to modify the stability and / or activity of one or more elements of the composition.
  • the composition is an amplification reaction composition.
  • the additive inactivates the contaminated enzyme, stabilizes the folding of the protein, and / or reduces the aggregation of the protein.
  • additives examples include betaine, formamide, KCl, CaCl 2 , MgOAc, MgCl 2 , NaCl, NH 4 OAc, NaI, Na (CO 3 ) 2 , LiCl, MnOAc, NMP, trehalose ( trehalose), dimethyl sulfoxide (“DMSO”), glycerol, ethylene glycol, dithiothreitol (“DTT”), pyrophosphatase (the inorganic pyrophosphatase from Thermoplasma acidophilum (“TAP”)) ), Bovine serum albumin ("BSA”), propylene glycol, glycineamide, CHES, Percoll, aurintricarboxylic acid, Tween 20, Tween 21, Tween 40, Tween 60, Tween 85, Brij 30, NP-40, Triton X-100, CHAPS, CHAPSO, Mackernium, LDAO (
  • Coli SSB RecA, nicking endonuclease, 7-deazaG, dUTP, anionic surfactant, cationic surfactant, non-ionic surfactant, zwitte rgent, sterols, osmolytes, cations, and other chemicals, proteins, or cofactors that can alter the efficiency of amplification.
  • two or more additives may be included in the amplification reaction. If the additive does not interfere with the activity of RNase H, it may be optionally added to increase the selectivity of primer annealing.
  • thermoostable as applied to an enzyme, maintains biological activity at increased temperatures (eg, 55 ° C. or higher), or repeats heating and cooling It refers to an enzyme that can maintain biological activity in the course of the cycle.
  • Thermostable polynucleotide polymerases are found to be particularly used in amplification reactions.
  • thermostable polymerases are thermophilic bacteria Thermus aquaticus (Taq polymerase), Thermus thermophilus (Tth polymerase), Thermococcus litoralis (Tli or VENT polymerase), Pyrococcus furiosus (Pfu or DEEPVENT polymerase), Pyrococcus woosii (Pwo polymerase) and polymerases isolated from other Pyrococcus species, Bacillus stearothermophilus (Bst polymerase), Sulfolobus acidocaldarius (Sac polymerase), Thermoplasma acidophilum (Tac polymerase), Thermus rubber (Tru polymerase), Thermus brockianus (DYNAZYME polymerase) Thermotoga neapolitana (Tne polymerase), Thermotoga neapolitana (Tne polymerase), Thermus brockianus (DYNAZYME polymerase) Thermotog
  • the PCR reaction may comprise one or more thermostable polymerase enzymes having complementary features that lead to efficient amplification of the target sequence.
  • nucleotide polymerase which has high processivity (the ability to copy larger nucleotide segments), has other nucleotides with proofreading ability (the ability to correct errors while stretching the target nucleic acid sequence).
  • the thermostable polymerase may be used in the form of a wild type.
  • the polymerase may be modified to include mutations that comprise fragments of the enzyme or which provide advantageous features for enhancing the PCR reaction.
  • the heat stable polymerase may be Taq polymerase. Many variants of Taq polymerase with increased properties are known, which are AmpliTaq, AmpliTaq Stoffel fragments, SuperTaq, SuperTaq plus, LA Taq, LApro Taq, and EX Taq.
  • RNA sequence a template for mRNA sequence (s) for amplification by PCR.
  • s mRNA sequence
  • the method takes advantage of the high sensitivity and specificity of the PCR process and is widely used for the detection and quantification of RNA.
  • the reverse transcriptase-PCR process performed as an end-point or real time assay, involves two steps of separate molecular synthesis: (i) synthesis of cDNA from an RNA template; And (ii) replication of the newly synthesized cDNA via PCR amplification.
  • a number of protocols have been developed, taking into account the three basic steps of the process: (a) denaturation of RNA and hybridization of reverse primers; (b) synthesis of cDNA; And (c) PCR amplification.
  • reverse transcriptase-PCR reverse transcriptase-PCR
  • reverse transcriptase-PCR reverse transcriptase-PCR
  • dNTP deoxyribonucleoside triphosphate
  • the annealing of the reverse primer is a separate step before the addition of the enzyme and then added to a single reaction vessel.
  • reverse transcriptase activity is a component of thermostable Tth DNA polymerase. Annealing and cDNA synthesis are performed in the presence of Mn 2+ , and then PCR is performed in the presence of Mg 2+ after Mn 2+ is removed by chelating reagent.
  • the “continuous” method eg, one step reverse transcriptase-PCR
  • Continuous reverse transcriptase-PCR is described as a single enzyme system using the thermostable Taq DNA polymerase and Tth polymerase's reverse transcriptase activity and a two enzyme system using AMV reverse transcriptase and Taq DNA polymerase with a starting temperature of 65 ° C. do.
  • the RNA denaturation step is omitted.
  • RNA: DNA hybrids can be the substrate of RNase H, the presence of RNase H in the reaction buffer will result in unwanted degradation of the RNA: DNA hybrid formed in the first step of the process. There are two main ways to overcome this problem.
  • RNase H is physically separate from the rest of the reverse transcription reaction by using a wax-like membrane that can melt during the high temperatures of the beginning of the DNA denaturation step.
  • the second method is to modify RNase H to inactivate at reverse transcription temperature which is generally 45-55 ° C.
  • Several methods are known in the art, including reacting RNase H with an antibody or including reversible chemical modifications. The various RNases H used in the disclosed method will be described in detail later.
  • RNAse H enzymes that can be used in the present invention are disclosed in US Patent Application No. 2009/0325169 to Walder et al., which is incorporated herein by reference.
  • One step reverse transcriptase-PCR offers several advantages over unlinked reverse transcriptase-PCR.
  • One step reverse transcriptase-PCR is the handling of reaction mixture reagents and nucleic acid products compared to unlinked reverse transcriptase-PCR (e.g., opening a reaction tube for the addition of components or enzymes between two reactions). Are less demanding, thus less labor intensity and less time required.
  • One step reverse transcriptase-PCR also requires less sample and can reduce the risk of contamination.
  • the sensitivity and specificity of one-step reverse transcriptase-PCR has proven to be suitable for studying the expression level of one or several genes in the detection of a given sample or pathogenic RNA. In general, this process limits the use of gene-specific primers to initiate cDNA synthesis.
  • RNA copy numbers with high sensitivity. This is accomplished during the amplification process by a fluorescent double-labeled hybridization probe technique, such as a 5 'fluorescence generating nuclease assay (eg TaqMan) or endonuclease assay (eg CataCleave), which will be described below. It is possible by detecting reverse transcriptase-PCR products through fluorescence monitoring and measurement of PCR products.
  • a fluorescent double-labeled hybridization probe technique such as a 5 'fluorescence generating nuclease assay (eg TaqMan) or endonuclease assay (eg CataCleave), which will be described below. It is possible by detecting reverse transcriptase-PCR products through fluorescence monitoring and measurement of PCR products.
  • Detection of post-amplified amplicons is time consuming and labor intensive.
  • Real time methods were developed to monitor amplification during the PCR process. These methods generally use a fluorescently labeled probe that binds to newly synthesized DNA or a die that increases in fluorescence emission when intercalated to two strands of DNA.
  • the probe is generally designed so that donor emission can be extinguished by fluorescence resonance energy transfer (FRET) between two chromophores in the absence of a target.
  • FRET fluorescence resonance energy transfer
  • a donor chromophore transfers energy to an acceptor chromophore in an excited state when the pair of chromophores is located at close range. This transfer always occurs non-radially, through dipole-dipole coupling. Any process that sufficiently increases the distance between chromophores reduces the FRET efficiency, allowing radiological detection of donor chromophore emissions.
  • Common acceptor chromophores include FAM, TAMRA, VIC, JOE, Cy3, Cy5, Texas Red, and the like.
  • acceptor chromophore is chosen so that the emission spectrum and the excitation spectrum of the donor can overlap.
  • this binding pair is FAM-TAMRA.
  • acceptors There may also be non-fluorescent acceptors that quench a wide range of donors.
  • Other examples of suitable donor-acceptor FRET pairs are well known to those skilled in the art.
  • Molecular beacons are single-stranded oligonucleotides, in which the probes are designed to form secondary structures that are close to donor and acceptor chromophores, which can reduce donor release. At the appropriate reaction temperature, the beacons are unstructured and in particular bind to amplicons.
  • TaqMan and CataCleave techniques differ from molecular beacons in that the FRET probes used are cleaved, causing the donor and acceptor chromophores to fall far enough to alter the FRET.
  • the TaqMan technique utilizes a single stranded oligonucleotide probe labeled with a donor chromophore at the 5 'end and an acceptor chromophore at the 3' end.
  • the DNA polymerase used for amplification must have 5 '-> 3' exonuclease activity.
  • TaqMan probes bind to one strand of the amplicon as the primers bind. While the DNA polymerase stretches the primer, the polymerase will eventually encounter the bound TaqMan probe. At this point, the exonuclease activity of the polymerase will degrade the TaqMan probe sequentially starting at the 5 'end.
  • the mononucleotide containing the probe comes out of the reaction buffer.
  • the FRET changes. Release from the donor is monitored to confirm probe cleavage. Because of the action of TaqMan, a particular amplicon can only be detected once every cycle of PCR. Extension of the primer through the TaqMan target site produces a double stranded product and prevents further binding of the TaqMan probe until the amplicon is denatured in the next PCR cycle.
  • CataCleave Another real-time detection method (named CataCleave).
  • the CataCleave technique differs from TaqMan in that cleavage of the probe is performed by secondary enzymes lacking polymerase activity.
  • CataCleave probes have a sequence in the target molecule of an endonuclease such as, for example, a restriction enzyme or an RNase.
  • the CataCleave probe has a chimeric structure, wherein the 5 'and 3' ends of the probe are made of DNA and the cleavage site is made of RNA.
  • the DNA sequence portion of the probe is labeled with a FRET pair at or inside the sock end.
  • the PCR reaction includes an RNase H enzyme that can specifically cleave the RNA sequence portion of the RNA-DNA double strand. After cleavage, both halves of the probe dissociate in the target amplicon at the reaction temperature and disperse into the reaction solution. As the donor and acceptor are separated, the FRET is changed in the same way as the TaqMan probe, and donor emission can be monitored. Cleavage and dissociation will reproduce sites for further CataCleave binding. In this way, a single amplicon can be repeated multiple times as a target or probe cleavage until the primer is stretched through the CataCleave probe binding site.
  • the probe used in the method is a CataCleave probe.
  • suitable CataCleave probes include oligonucleotides comprising one of the sequences of SEQ ID NOs: 17 or 18.
  • the probe is one of the sequences of SEQ ID NOs: 12, 13, or 14.
  • Table 1 SEQ ID NO: order Primer / Probe Name One TTAACGAGCTGTATGTCGTGAGAAT O157-IF 2 AACGAGCTGTATGTCGTGAGAATC O157-I-F1 3 CCCTCCAAATGAAATTCCAACA O157-I-F2 4 GGCTTTGTTGCAAGGCTATG O157-I2-F 5 CGAGCTGTATGTCGTGAGAATC O157-I3-F 6 CAAGCCTATTCAGAGCATGG O157-I4-F 7 ATGGATCATCAAGCTCTAAGAAAGAAC O157-IR 8 AGTGTCGTCTGTATGGATCATCAAG O157-I-R1 9 CCTCAAGCGAAGATGCAAAAT O157-I-R2 10 TGGATCATCAAGCTCTAAGAAAGAAC O157-I-R3 11 GATTGCAACTGCTCATCAGG O157-I2-R 12 ATAGGCTTrGrArArGCAGTGCA, wherein the rG and 9th-12th positions may be
  • X 1 does not exist in the 1st and 2nd positions or T, X 2 does not exist in the 3rd and 4th positions, or A, 2nd X 3 does not exist Or is C 17 ATAGGCTTGAAGCAGTGCAX 1 , wherein X 1 is absent or at least three consecutive nucleotides at positions 9-14 are ribonucleotides. 18 TCAGAGCATGGAAATAAAACTT, wherein three or more consecutive nucleotides at positions 10-14 are ribonucleotides.
  • Probes of SEQ ID NO: 12, 13, 14, 17 or 18 may have a detectable label bound to the 5 'and 3' ends, respectively.
  • the 5 'terminus may be FAM (6-carboxyfluorescein), and the 3' terminus may be coupled to Black Hole Quencher (BHQ) for short wavelength emission.
  • BHQ Black Hole Quencher
  • the kit for detecting E. coli 0157: H7 in a sample comprises the oligonucleotides described above.
  • the kit further comprises a reagent for nucleic acid amplification.
  • the reagent may further comprise one or more selected from the group consisting of one or more dNTPs, rNTPs, nucleic acid polymerases, uracil N-glycosylase (UNG) enzymes, buffers, and cofactors (eg, Mg 2+ ).
  • dNTPs dNTPs
  • rNTPs nucleic acid polymerases
  • UNG uracil N-glycosylase
  • Bus eg, Mg 2+
  • the nucleic acid polymerase may be selected from the group consisting of DNA polymerase, RNA polymerase, and reverse transcriptase.
  • the nucleic acid polymerase may be heat stable.
  • the nucleic acid polymerase may retain its activity, for example, at increased temperatures of 95 ° C. or higher.
  • Thermostable DNA polymerase is isolated from heat-resistant bacteria selected from the group consisting of Thermus aquaticus, Thermus flavus, Thermus ruber, Thermus thermophilus, Bacillus stearothermophilus, Thermus lacteus, Thermus rubens, Thermotoga maritima, Thermococcus littoralis, and Methanothermus fervidus. have.
  • An example of a thermostable DNA polymerase is Taq polymerase.
  • the Taq polymerase is known to have optimal activity at about 70 ° C.
  • the E. coli 0157: H7 detection kit may further include an element capable of specifically cleaving an RNA portion of the DNA-RNA hybrid.
  • the cleavage element may be RNase H.
  • the cleavage element can cleave the RNA portion either specifically or nonspecifically.
  • the specific RNA cleavage element may be RNase HI.
  • the nonspecific RNA cleavage element may be RNase HII.
  • RNase H can hydrolyze RNA in RNA-DNA hybrids.
  • divalent ions eg Mg 2+ , Mn 2+
  • RNase H cleaves RNA 3'-OP bonds to produce 3'-hydroxyl and 5'-phosphate end products.
  • RNase H may be selected from the group consisting of Pyrococcus furiosus RNase HII, Pyrococcus horikoshi RNase HII, Thermococcus litoralis RNase HI, and Thermus thermophilus RNase HI.
  • the RNase H may be heat stable. For example, the RNase H can maintain its activity during denaturation in PCR.
  • the cleavage element may be reversibly modified in the form of a thermostable RNase HII, which is inactivated in the modified form and activated in the unmodified form, wherein the modification is the binding of the RNase HII to the ligand, the intersection of the RNase HII Binding, or chemical reaction of amino acid residues in RNase HII, and the enzymatic activity of the modified RNase HII can be restored by adjusting the pH of the sample comprising the RNase HII or by applying heat.
  • RNA portion of the probe including the DNA sequence and the RNA sequence, is cleaved by the cleavage element and separated. This separation can occur spontaneously by decreasing the melting temperature of the cleaved composite or can be enhanced by factors such as temperature rise. Separate fragments can be detected by several methods known in the art.
  • a method of detecting E. coli 0157: H7 in a sample comprises the following steps: (a) amplifying a target nucleic acid of E. coli 0157: H7 in a sample to increase the copy number of the target nucleic acid As a step, the amplification is carried out in the sample a first primer comprising a nucleotide sequence of SEQ ID NO: 16, 3, 4, or 6 and a second primer comprising a nucleotide sequence of SEQ ID NO: 7, 8, 9, 10 or 11 Hybridizing with a target nucleic acid to obtain a hybridized product of the target nucleic acid and the primers, and extending the first and second primers of the hybridized product using a template-dependent nucleic acid polymerase to produce an extended primer product.
  • the target nucleic acid Comprising; (b) hybridizing the target nucleic acid with one or more probe oligonucleotides that can hybridize with the target nucleic acid to obtain a hybridization product of the target nucleic acid: probe oligonucleotide, wherein the probe comprises a DNA sequence and an RNA sequence and is detected Possible markers are linked; (c) contacting the hybridized product of the target nucleic acid : probe with RNase H to cleave the probe to cause separation of the probe fragment from the target nucleic acid; And (d) detecting the detectable label.
  • the first primer may comprise the sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6.
  • the probe may comprise a sequence of SEQ ID NO: 12, 13, or 14.
  • Amplification of the target sequence in the sample may be performed by a nucleic acid amplification method such as Polymerase Chain Reaction or Ligase Chain Reaction, Self-Sustained Sequence Replication, Strand Displacement Amplification, Transcriptional Amplification System, Q-Beta Replicase, Nucleic Acid Sequence Based Amplification (NASBA), Cleavage Fragment Length Polymorphism, Isothermal and Chimeric Primer-initiated Amplification of Nucleic Acid, Ramification-extension Amplification Method or appropriate amplification methods for amplification of nucleic acids can be performed.
  • a nucleic acid amplification method such as Polymerase Chain Reaction or Ligase Chain Reaction, Self-Sustained Sequence Replication, Strand Displacement Amplification, Transcriptional Amplification System, Q-Beta Replicase, Nucleic Acid Sequence Based Amplification (NASBA), Cleavage Fragment Length Polymorphism, Isothermal and Chimeric Primer-initiated Amplification
  • the method comprises amplifying a target nucleic acid fragment of E. coli 0157: H7, wherein the amplifying step comprises a first primer of SEQ ID NO: 16, 3, 4, or 6 and SEQ ID NO: 7, 8, 9 Hybridizing a second, 10, or 11 second primer to a target nucleic acid in a sample to obtain a hybridized product; And expanding the primers of the hybridized product using template-dependent nucleic acid polymerase to produce an expanded primer product; Hybridizing the target nucleic acid fragment with the probe of SEQ ID NO: 17 or 18 to obtain a hybridized product; Cleaving the probe by contacting RNase H with the product hybridized from the target nucleic acid fragment and the probe to separate the probe fragment from the hybridized product; And detecting the detectable label.
  • the first primer may be one of SEQ ID NO: 1, 2, 3, 4, 5, or 6.
  • the probe may be one of SEQ ID NO: 12, 13, or 14.
  • the method comprises amplifying a target nucleic acid fragment of E. coli 0157: H7, wherein the amplifying step comprises a first primer of SEQ ID NO: 16, 3, 4, or 6 and SEQ ID NO: 7, 8, 9, 10, or 11 Hybridizing a second primer of to a target nucleic acid in a sample to obtain a hybridized product; And expanding the primers of the hybridized product using a template-dependent nucleic acid polymerase to produce an expanded primer product.
  • the first primer may have a sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6.
  • the probe may have a sequence of SEQ ID NO: 12, 13, or 14.
  • the hybridization can be carried out in a liquid medium.
  • Appropriate liquid medium can be selected as needed.
  • the liquid medium can be, for example, water, buffers, or PCR mixtures.
  • buffers may include, but are not limited to, PBS, Tris, MOPS, and Tricine.
  • the hybridization can be performed under conditions that can enhance the binding of the primer and target nucleic acid, for example, can be low temperature and low salt concentration. Conditions for enhancing these hybridizations are well known in the art.
  • the target nucleic acid can be a single-stranded or double-stranded nucleic acid.
  • a double-stranded target nucleic acid can be modified to separate into single strands.
  • the target nucleic acid may be DNA or RNA.
  • Expansion of the primer depending on the template means polymerization, which is well known in the art.
  • the nucleic acid polymerase may be heat stable.
  • Methods of detecting E. coli include hybridizing a target nucleic acid fragment with a probe of SEQ ID NO: 17 or 18 to obtain a hybridized product.
  • the probe the sample probe described above may be used.
  • the probe has a sequence of SEQ ID NO: 12, 13, or 14.
  • the probe may be labeled with a detectable label such as, for example, a visually detectable label.
  • the detectable label is well known in the art and may be appropriately selected.
  • FRET pairs can be used for the purpose of detecting target sequences according to one embodiment of the invention.
  • the hybridization can be carried out in a liquid medium.
  • Appropriate liquid medium can be selected as needed.
  • the liquid medium can be, for example, water, buffers, or PCR mixtures.
  • buffers may include, but are not limited to, PBS, Tris, MOPS, and Tricine.
  • the hybridization can be performed under conditions that can enhance the binding of the primer and target nucleic acid, for example, can be low temperature and low salt concentration. Conditions for enhancing these hybridizations are well known in the art.
  • the target nucleic acid can be a single-stranded or double-stranded nucleic acid.
  • a double-stranded target nucleic acid can be denatured to separate into single strands, as described above.
  • the target nucleic acid may be DNA or RNA.
  • the method of detecting E. coli 0157: H7 includes cleaving the probe by contacting RNase H with the product hybridized with the product from the target nucleic acid fragment and separating the probe fragment from the hybridized product.
  • the hybridized product and RNase H may be contacted with each other in a liquid medium.
  • Appropriate liquid medium can be selected as needed.
  • the liquid medium can be, for example, water, buffers, or PCR mixtures. Examples of buffers may include, but are not limited to, PBS, Tris, MOPS, and Tricine.
  • the hybridization can be performed under conditions that can enhance the binding of the primer and target nucleic acid, for example, can be low temperature and low salt concentration.
  • the RNase H may be RNase HI or RNase HII.
  • RNase H can hydrolyze RNA in RNA-DNA hybrids.
  • divalent ions eg Mg 2+ , Mn 2+
  • RNase H cleaves RNA 3'-OP bonds to produce 3'-hydroxyl and 5'-phosphate end products.
  • RNase H may be selected from the group consisting of Pyrococcus furiosus RNase HII, Pyrococcus horikoshi RNase HII, Thermococcus litoralis RNase HI, and Thermus thermophilus RNase HI.
  • the RNase H may be heat stable. For example, the RNase H can maintain its activity during denaturation in PCR.
  • the cleavage element may be reversibly modified in the form of a thermostable RNase HII, which is inactivated in the modified form and activated in the unmodified form, wherein the modification is the binding of the RNase HII to the ligand, the intersection of the RNase HII Binding, or chemical reaction of amino acid residues in RNase HII, and the enzymatic activity of the modified RNase HII can be restored by adjusting the pH of the sample comprising the RNase HII or by applying heat.
  • the PCR mixture may comprise an RNase H enzyme that can specifically cleave the RNA sequence portion of the RNA-DNA duplex.
  • RNase H enzyme that can specifically cleave the RNA sequence portion of the RNA-DNA duplex.
  • two halves of the probe are separated from the target amplicon at the reaction temperature and diffuse into the reaction buffer.
  • the donor and acceptor are separated, the FRET is reversed and donor release is monitored. Cleavage and dissociation regenerate the sites to which the probe can further bind. In this way, a single amplicon can serve as a plurality of targets for probe cleavage until the primer is extended through the probe binding site.
  • the method for detecting E. coli 0157: H7 comprises the probe nucleic acid fragment.
  • One exemplary method of detecting a target E. coli 0157: H7 sequence is to provide a food sample or surface extract, mix the sample or extract and the growth medium, and incubate the number of E. coli 0157: H7 or Increasing the population (enrichment), crushing the E. coli cells (lysis), and lysate obtained above for amplification and detection of the target E. coli 0157: H7 sequence.
  • Food samples include fish such as salmon, dairy products such as milk and eggs, poultry, fruit juice, ground pork, pork, ground beef or meat such as beef, vegetables such as spinach, or stainless steel, rubber, plastic, and ceramics. It may include the surface of the same natural environment, but is not limited thereto.
  • the limit of detection (LOD) for food contamination is described in terms of the number of colony forming units (CFUs) that can be detected on the surface of 25 g solid or 25 ml liquid food or confined spaces. .
  • colony forming units are a measure of the number of living bacteria.
  • CFU measures live cells.
  • One CFU one bacterial cell grows and forms one colony on agar plates under acceptable conditions.
  • the United States Food Testing Inspection Service defines a minimum LOD of 1 CFU per solid food of 25 g of solid food or 25 mL of liquid food or 1 CFU per surface area.
  • E.g. E. coli Cultures can be grown to specific cell densities by measuring absorbance on a spectrometer . remind Plates in agar medium through 10-fold serial dilutions of the target and count live bacteria. This data is used to generate a standard curve for CFU / volume against smeared cell density. In order for MPN to be meaningful, test samples at different inoculation levels are analyzed.
  • the kit comprises a mixture comprising dATP, dCTP, dGTP and dTTP; DNA polymerase; RNase H II; And a buffer solution.
  • the DNA polymerase may be, for example, a heat stable DNA polymerase obtained from Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis or Pyrococcus furiosus (Pfu).
  • RNase H includes, but is not limited to, thermally stable RNase H enzymes such as, for example, Pyrococcus furiosus RNase H II, Pyrococcus horikoshi RNase H II, Thermococcus litoralis RNase HI or Thermus thermophilus RNase HI.
  • Buffer solutions are compounds that are added to amplification reactions that modify the stability, activity, and / or lifetime of one or more components of the amplification reaction by adjusting the pH of the amplification reaction, and such buffer solutions are well known in the art, For example, it may be, but is not limited to, Tris, Tricine, MOPS, or HEPES.
  • the kit may comprise a dNTP mixture (dATP, dCTP, dGTP, dTTP) and a DNA polymerase cofactor.
  • the primer set and probe may be packaged in one reaction vessel, strip or microplate, and may be packaged by methods known in the art.
  • Another aspect includes obtaining a lysate of E. coli 0157: H7 from a subject sample; Mixing the lysate with the kit to perform real-time PCR; And it provides a method for detecting E. coli O157: H7 comprising the step of confirming the presence of E. coli O157: H7 from the real-time PCR results.
  • the method may include obtaining a lysate of E. coli 0157: H7 from the sample of interest.
  • the detection method according to one embodiment may be applied to a sample that is expected to be contaminated with E. coli 0157: H7.
  • the sample may include, but is not limited to, for example, cultured cells, body fluids such as blood, saliva, and foods such as meat, dairy products, beverages, and the like. Since the lysate contains the DNA of E. coli 0157: H7, it can be used as a template for the real-time PCR reaction in a later step. Methods for obtaining the lysate include, for example, 1 mg / ml proteinase K, 0.3125 mg / ml sodium azide, 0.125% Triton X-100, and 12.5 mM Tris-HCl, pH 8.0 E.
  • the DNA may be extracted from the lysate according to a method known in the art to be used as a template for real-time PCR reaction. Specific methods of extracting DNA from the lysate are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001), which is herein incorporated by reference. Inserted by reference.
  • Mixing the lysate and the kit may include performing a real-time PCR.
  • the E. coli 0157: H7 detection kit can be carried out using conventional real-time PCR methods and apparatus.
  • the real-time PCR method is a method of detecting and quantitating fluorescence appearing in real time every cycle of PCR by the principle of DNA polymerase and FRET using a device in which a thermal cycler and a spectral fluorescent photometer are integrated. This method distinguishes specific amplification products from non-specific amplification products and makes it easy to obtain analysis results in an automated fashion.
  • Devices that can be used in the real-time PCR method include, but are not limited to, AB real-time PCR devices 7900, 7500, 7300, Stratagene Mx3000p, BioRad Chromo 4 and Roche Lightcycler 480 devices.
  • the laser of the real-time PCR device may implement a peak as shown in FIG. 1 by detecting a fluorescent labeling factor labeled on a probe of the amplified PCR product.
  • the real-time PCR reaction can be carried out under conventional conditions known in the art, for example, initial denaturation at 95 ° C. for 10 minutes. And then subjected to denaturation (15 seconds at 95 ° C.), annealing of primers and probes, reaction of RNase HII and elongation (20 seconds at 60 ° C. or 63 ° C.) for a total of 50 to 60 times. Can be done. According to one embodiment, a total of 63 E. coli 0157: H7 can be detected by the method.
  • the E. coli O157: H7 is the presence or absence of the cycle number at which the C p value from the curve appears to detect a fluorescent marker labeling the probe of the PCR product amplified by the real-time PCR process, PCR amplification product was amplified a predetermined amount It can confirm by calculating.
  • the C p value may be determined that the E. coli 0157: H7 is present at 10 to 50, or 15 to 45. Meanwhile, the calculation of the C p value may be automatically performed by a program included in the real time PCR device.
  • Samples that can be tested for the detection of E. coli O175: H7 are, but are not limited to, meat (eg beef including ground beef), vegetables (eg spinach), fruit juice and the like.
  • the detection result can be quickly confirmed in real time even with a small number of samples.
  • the detection result can be quickly confirmed in real time even with a small number of samples.
  • Figure 1a is a result showing the amplification curve by real-time PCR reaction for E. coli O157: H7 using a kit according to one embodiment
  • Figure 1b shows the Cp value determined from the data of Figure 1a.
  • Figure 2 shows the amplification curves by real-time PCR reaction for a total of 63 E. coli O157: H7 species using a kit according to one embodiment.
  • 3A to 3C show amplification curves by real-time PCR reactions on a total of 59 non- E. Coli O157: H7 species, compared to O157: H7 species using a kit according to one embodiment.
  • the kits and methods according to one embodiment show high accuracy.
  • the 59 species were divided into three groups and tested.
  • FIG. 4A and 4B show amplification curves by real-time PCR reactions of E. coli O157: H7 species using various combinations of primers and probes.
  • FIG. 4A shows fluorescence curves and
  • FIG. 4B shows Cp values. Indicates.
  • Example 1 E. coli Prepare primers and probes for real-time detection of O157: H7
  • the primer used for the real-time detection of E. coli 0157: H7 was a primer base sequence capable of amplifying only a part of the I fragment of E. coli 0157: H7.
  • the I segment was at a position of 312001-315400 of the E. coli 0157: H7 genome (GenBank: AE005174.2.).
  • a polynucleotide (1720 bp) of a portion of the I fragment was used in one embodiment and is shown in SEQ ID NO: 15 in Sequence Listing.
  • Table 1 shows the sequences of representative primers designed according to one embodiment.
  • the probe was prepared by catacleave probe (Catacleave TM probe) that can specifically bind to the template target of the PCR to be able to detect the amount of the PCR product to increase in real time in real time PCR.
  • the 5 'end was labeled with 6-carboxyfluorescein (FAM) and the 3' end was labeled with Black Hole Quencher (Integrated DNA Technologies, Coralville, IA).
  • FAM 6-carboxyfluorescein
  • the determined primers and probes were synthesized by the Roche company.
  • probe sequence shows a label linked to the nucleotide.
  • E. coli 0157: H7 Total DNA of E. coli 0157: H7 for use as a template in real-time PCR was extracted by the following method.
  • Appropriately cultured E. coli 0157: H7 was harvested to produce 1 mg / ml proteinase K, 0.3125 mg / ml sodium azide, 0.125% Triton X-100, and 12.5 mM Tris-HCl, pH 8.0 After dilution in 45 ⁇ l lysis solution containing, the samples were incubated at 55 ° C. for 15 minutes, inactivated proteinase K at 95 ° C. for 10 minutes, and then cooled to 4 ° C. The reaction was centrifuged to obtain a supernatant, and then the supernatant itself or DNA extracted from the supernatant was added to the real time PCR reaction according to DNA extraction methods known in the art.
  • the master mix contained 180 ⁇ l of 10 ⁇ I buffer (10 ⁇ I buffer is HEPES-containing buffer (HEPES-KOH, MgCl 2 , KCl, BSA, DMSO)), 20 ⁇ M of forward primer (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4) 72 ⁇ l, 20 ⁇ M reverse primer (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 11) 72 ⁇ L, 25 ⁇ M CataCleave probe (SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14) 14.4 ⁇ l, dNTP mix (2 mM dGTP, dCTP, dATP, dTTP) 72 ⁇ l, 36 ⁇ l Platinum Taq DNA Polymerase (Invitrogen), 14.4 ⁇
  • the reactants were first subjected to uracil DNA N-glycosylase reaction and denaturation for 10 minutes at 37 ° C and 10 minutes at 95 ° C, followed by denaturation at 95 ° C for 15 seconds, primers at 60 ° C or 63 ° C for 20 seconds. And annealing (catalyst) probe, the reaction of RNase HII and elongation (50) was repeated 50 times to perform a real-time PCR reaction. After the repetition process, the mixture was cooled at 40 ° C. for 10 seconds. The reaction was performed using a Roche Lightcycler 480 instrument, and PCR amplification was observed in real time using LightCycler 480 Software v1.5.0.
  • Example 3 using a probe according to one embodiment E. coli Detection of O157: H7
  • Real time PCR was performed using DNA from the maximal density culture (about 2 ⁇ 10 9 cfu / mL) as a template.
  • 3A to 3C show the amplification curves by real-time PCR reactions for each species shown in Tables 6 to 8, respectively.
  • Tables 5a to 5c are the results obtained by calculating the C p values from the amplification curves of FIGS. 3a to 3c.
  • E. coli O157: H7 can be efficiently detected with a smaller amount of samples compared to the conventional methods, and a set of primer sets and probes can be detected. It can be seen that since E. coli O157: H7 of various species can be detected at one time, it can save time and effort in detecting E. coli O157: H7 from a sample.

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Abstract

L'invention concerne un oligonucléotide, une trousse et un procédé de détection d'O157:H7 d'E. coli. L'invention concerne également un procédé de détection d'O157:H7 d'E. coli en temps réel à partir d'un échantillon d'essai par l'utilisation de la trousse. Selon le procédé de détection, le résultat de détection peut être rapidement confirmé en temps réel même à partir d'un échantillon d'essai ayant un faible nombre de copies.
PCT/KR2011/009138 2011-11-29 2011-11-29 Oligonucléotide pour la détection d'o157:h7 d'e. coli et l'utilisation de celui-ci Ceased WO2013081202A1 (fr)

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PCT/KR2011/009138 WO2013081202A1 (fr) 2011-11-29 2011-11-29 Oligonucléotide pour la détection d'o157:h7 d'e. coli et l'utilisation de celui-ci

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/KR2011/009138 WO2013081202A1 (fr) 2011-11-29 2011-11-29 Oligonucléotide pour la détection d'o157:h7 d'e. coli et l'utilisation de celui-ci

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WO2013081202A1 true WO2013081202A1 (fr) 2013-06-06

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107817228A (zh) * 2017-06-30 2018-03-20 四川农业大学 对E.coli O157:H7免酶及免荧光标记的检测方法
WO2022086140A1 (fr) * 2020-10-20 2022-04-28 에스티팜 주식회사 Oligonucléotide pour la synthèse d'arn à coiffe en 5'

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
FORTIN, N. Y. ET AL.: "Use of real-time polymerase chain reaction and molecular beacons for the detection of Escherichia coli 0157:H7", ANALYTICAL BIOCHEMISTRY, vol. 289, no. 2, 15 February 2001 (2001-02-15), pages 281 - 288 *
HARVEY, J. J. ET AL.: "Characterization and applications of CataCleave probe in real-time detection assays", ANALYTICAL BIOCHEMISTRY, vol. 333, no. 2, 15 October 2004 (2004-10-15), pages 246 - 255 *
IBEKWE, A. M. ET AL.: "Detection and quantification of Escherichia coli 0157:H7 in environmental samples by real-time PCR", JOURNAL OF APPLIED MICROBIOLOGY., vol. 94, no. 3, 2003, pages 421 - 431 *
OBERST, R. D. ET AL.: "PCR-based DNA amplification and presumptive detection of Escherichia coli 0157:H7 with an internal fluorogenic probe and the 5' nuclease (Taqman) assay", APPLIED AND ENVIRONMENTAL MICROBIOLOGY., vol. 64, no. 9, September 1998 (1998-09-01), pages 3389 - 3396 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107817228A (zh) * 2017-06-30 2018-03-20 四川农业大学 对E.coli O157:H7免酶及免荧光标记的检测方法
CN107817228B (zh) * 2017-06-30 2022-06-14 四川农业大学 对E.coli O157:H7免酶及免荧光标记的检测方法
WO2022086140A1 (fr) * 2020-10-20 2022-04-28 에스티팜 주식회사 Oligonucléotide pour la synthèse d'arn à coiffe en 5'

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