WO2008004706A1 - Procédé de détection d'une substance cible à l'aide d'un procédé d'amplification de l'acide nucléique, disponible dans des conditions thermostatiques - Google Patents

Procédé de détection d'une substance cible à l'aide d'un procédé d'amplification de l'acide nucléique, disponible dans des conditions thermostatiques Download PDF

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Publication number
WO2008004706A1
WO2008004706A1 PCT/JP2007/063771 JP2007063771W WO2008004706A1 WO 2008004706 A1 WO2008004706 A1 WO 2008004706A1 JP 2007063771 W JP2007063771 W JP 2007063771W WO 2008004706 A1 WO2008004706 A1 WO 2008004706A1
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Prior art keywords
primer
protein
target substance
substance
antibody
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PCT/JP2007/063771
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English (en)
Japanese (ja)
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Yuichiro Ishikawa
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Synthera Technologies Co., Ltd.
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Publication of WO2008004706A1 publication Critical patent/WO2008004706A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex

Definitions

  • the present invention relates to a method for detecting a target substance using a reaction (for example, an antigen-antibody reaction) between a target substance and a substance that can specifically bind to the target substance. Specifically, the present invention relates to a method for labeling the binding substance and detecting the label after binding to a target substance.
  • a reaction for example, an antigen-antibody reaction
  • both of these methods detect the amplified oligonucleic acid chain by amplifying it by PCR. Therefore, complicated temperature control (dissociation, annealing, synthesis) must be repeated precisely and repeatedly in the reaction system, making rapid amplification detection difficult.
  • the problem to be solved by the present invention is that the detection of a target substance using a binding substance labeled with an oligonucleic acid chain can be performed more quickly, accurately and easily at a low cost.
  • the present inventor has intensively studied to solve the above problems. As a result, a complex temperature control is required as an amplification method of labeled oligonucleic acid strands: Instead of the PCR method, we focused on nucleic acid amplification methods (eg, LAMP method and ICAN method) that can be reacted under constant temperature conditions. . Then, as an oligonucleic acid chain used for labeling, an oligonucleic acid chain having a region binding to a primer used in such a nucleic acid amplification method is used, and the oligonucleic acid chain is amplified to detect a target substance.
  • the present invention is as follows.
  • a target substance detection method comprising a binding substance labeled with an oligonucleic acid chain having a region that binds to a primer used in a nucleic acid amplification method capable of reacting under constant temperature conditions, and a target in a test sample
  • the target substance is a plurality of kinds of substances
  • As the binding substance a substance that is labeled so that it can be identified and detected corresponding to the type of the target substance can be used.
  • a plurality of types of target substances can be identified and detected by using at least one primer set as a primer used in the nucleic acid amplification method.
  • nucleic acid amplification method examples include LAMP method and ICAN method.
  • the labeling treatment examples include those in which an oligonucleic acid chain is fixed to the binding substance via at least a portion of the adapter.
  • the adapter may include any protein selected from protein G, protein A and protein L, a fusion protein of at least two proteins selected from protein G, protein A and protein L, protein G and protein. Examples include fusion proteins of at least one protein selected from A and Protein L and other proteins, and combinations thereof.
  • Examples of the target substance include an antigen, and examples of the binding substance include an antibody.
  • a kit for detecting a target substance comprising a binding substance labeled with an oligonucleic acid chain having a region that binds to a primer used in a nucleic acid amplification method capable of reacting under constant temperature conditions.
  • examples of the primer include a primer for LAMP method and a primer for ICAN method.
  • FIG. 1 is a schematic flow diagram showing an embodiment of the detection method of the present invention.
  • FIG. 2A is a schematic flow diagram showing an embodiment of the detection method of the present invention.
  • FIG. 2B is a schematic flow diagram (a continuation of the flow diagram shown in FIG. 2A) showing an embodiment of the detection method of the present invention.
  • FIG. 3 is a schematic diagram showing an example of an embodiment in which an oligonucleic acid chain is cleaved from a labeled binding substance.
  • FIG. 4 is a schematic diagram showing an example of an embodiment in which an oligonucleic acid chain is cleaved from a labeled binding substance.
  • Figure 5 shows an antibody with an oligopeptide nucleic acid strand complexed through a portion of the adapter. It is the schematic which shows one Example which uses and identifies and detects an antigen.
  • FIG. 6 is a chart showing an example of a result of GeneScan analysis performed by DNA sequencer ABI-3100 (also an enlarged view of the chart shown in FIG. 5 (5)).
  • FIG. 7 is a graph showing the results of DNA amplification obtained by the detection method of the present invention.
  • FIG. 8 is a graph showing the results of DNA amplification when the real-time PCR method is used instead of the LAMP method in the detection method of the present invention.
  • Figure 9 is a graph plotting the mean values (ave) shown in Table 3 (below), where the vertical axis represents the Ct value and the horizontal axis represents the logarithmic antigen concentration (pg / mL).
  • amplified fragment 2 5 amplified fragment 2 6: amplified fragment BEST MODE FOR CARRYING OUT THE INVENTION
  • a binding substance labeled with an oligonucleic acid chain having a region that binds to a primer used in a nucleic acid amplification method capable of reacting under constant temperature conditions is brought into contact with a target substance in a test sample, and the binding substance and Process of forming a complex with the target substance (complex formation process),
  • the method of the present invention may further include other steps, and these other steps can be performed using known means and methods.
  • nucleic acid amplification method used in the present invention will be described, then, the outline of the entire detection method of the present invention will be exemplarily described, and then the complex formation process, the amplification process, and the detection process will be described. These will be described in order.
  • an amplification product is obtained by using a nucleic acid amplification method in which an oligonucleic acid chain to be labeled is in a saddle shape and can react under a constant temperature condition.
  • the nucleic acid amplification method is not limited as long as it can amplify the oligo-nucleic acid strand that is in the shape of a cage under a certain reaction temperature condition.
  • LAMP Loop-Mediated Isothermal Amplification
  • ICAN like properly preferred Isothermal and Chimeric primer-initiated Amplincation oi Nucleic acids
  • the LAMP method is characterized in that four types of primers are set for the six regions in the cage-shaped oligonucleic acid strand, and the amplification reaction proceeds at a constant temperature using a strand displacement reaction. It is. In other words, the LAMP method does not require denaturation (dissociation) from double strands to single strands or strict temperature control (it does not depend on the so-called PCR cycle) like the PCR method. The reaction can be continued continuously only by premixing the primer, DNA synthase and substrate, etc., and maintaining the temperature at a constant temperature (approximately 60 to 65 ° C) ("K. NAGAMINE et al., Mol. Cell. Probes, vol. 16 (3), 223-229 (2002) "etc.).
  • the LAMP method has higher amplification efficiency than the PCR method, and can amplify the vertical DNA from 10 9 to 10 10 times in 15 minutes to 1 hour.
  • the amplification product by the LAMP method has a repetitive structure having sequences complementary to each other on the same strand, and a variety of sequences having a length almost the same as the target region in the vertical DNA can be used as a repeat unit. This results in the synthesis of unit amplification products.
  • LAMP primer sets are the 6 different regions (5 ′ end side) in the target region of vertical DNA. F3, F2, Fl, Blc, B2c, B3c) and their complementary regions (B3, B2, Bl, Flc, F2c, F3c in this order from the 5 ′ end) It consists of a combination of specific primers designed based on the sequence.
  • the LAMP primer set is a Forward Inner Primer (hereinafter referred to as ⁇ Finer Primer Primer '') formed by linking nucleic acids of the Flc region and F2 region from the 5 ′ end.
  • BIP Backward Inner Primer
  • F3 primer F3 primer
  • B3 plastic consisting of nucleic acids in the B3 region It consists of four types with Imama.
  • loop primers Loop Primer F and Z or Loop Primer B
  • the loop primer is a primer having a sequence complementary to the base sequence of the single-stranded region formed between the B1 region and the B2 region or between the F1 region and the F2 region.
  • Each of the above-mentioned primers for the LAMP method only needs to have a —OH group that serves as a base for complementary strand synthesis at the 3 ′ end, and its backbone is not necessarily limited to that by a phosphodiester bond.
  • a phosphothioate body with S as the backbone instead of P may be composed of peptide nucleic acids based on peptide bonds.
  • Each LAMP primer can be prepared by chemical synthesis using, for example, an automatic DNA synthesizer.
  • the DNA polymerase that can be used in the LAMP method is not particularly limited as long as it has a strand displacement activity.
  • examples of such enzymes include Bst DNA polymerase (large fragment), Bca (exo-) DNA polymerase, Klenow fragment of E. coli DNA polymerase I, Vent (Exo-) DNA polymerase (from Vent DNA polymerase to ethanuclease) Non-active), DeepVent (Exo-) DNA polymerase (Deep Vent DNA polymerase excluding etanuclease activity), KOD DNA polymerase and the like, preferably Bst DNA polymerase (large fragment).
  • Bst DNA polymerase it is desirable to perform the reaction at around 60-65 ° C, which is the optimum temperature for the reaction.
  • amplification products can be detected by observing (visual confirmation) this white turbidity after completion of the reaction, or measuring the turbidity after the reaction and the turbidity change during the reaction with a suitable measuring instrument. In this way, amplification products can be detected.
  • a spectrophotometer or the like may be used as the measuring instrument, and usually the absorbance at a wavelength of 650 nm may be measured.
  • Amplification is performed in an accelerated and efficient manner, so pre-added to the reaction solution are ethimubu bumumide and SYBR (registered trademark) Green I, which are specifically intercalated into the double-stranded DNA molecule. By doing so, the presence or absence of amplification can be checked carefully, and the amount of amplification can be detected in real time if necessary.
  • the detection method using a labeled nucleic acid (DNA, RNA, PNA, etc.) that specifically recognizes the amplified DNA, or the reaction solution after completion of the reaction is directly subjected to agarose gel electrophoresis. It is also possible to adopt a detection method.
  • the ICAN method is a method that does not depend on the so-called PCH cycle, and is premixed with cages, primers, DNA synthase and substrate, and kept at a constant temperature (about 50 to 65 ° C). It is a method that can continuously and continuously advance the reaction (see “Isogai. E et al. Comp. Immunol. Microbiol. Infect. Dis. 2005 (5-6): 363-370" etc.) .
  • the primer for the ICAN method only needs to have a —OH group that serves as a base for complementary strand synthesis at the 3 ′ end, and its backbone is not necessarily limited to that based on a phosphodiester bond.
  • the phosphothioate body with S (sulfur) as the backbone may be composed of peptide nucleic acids based on peptide bonds.
  • Each ICAN primer can be prepared by chemically synthesizing using, for example, an automatic nucleic acid synthesizer.
  • the DNA polymerase that can be used in the ICAN method is not particularly limited as long as it has a strand displacement activity and a cage-type exchange activity.
  • examples of such an enzyme include Bca (exo-) DNA polymerase, BcaBEST TM DNA polymerase and the like, and preferably Bca (exo-) DNA polymerase.
  • the ICAN method specifically cleaves the RNA strand at the DNA-RNA hybrid site.
  • Another feature is the use of RNase H.
  • the reaction intermediate obtained through strand displacement reaction and cage exchange reaction based on the cage DNA has a DNA-RNA hybrid site consisting of the RNA portion derived from the chimeric primer and its complementary DNA.
  • RNase H the strand displacement reaction and the cage-type exchange reaction proceed again, which becomes a mechanism for obtaining a reaction product and a new reaction intermediate.
  • the ICAN method has a higher amplification efficiency than the PCR method, and can amplify DNA-type DNA 10 6 to 10 8 times in 30 minutes to 1 hour. This amplification efficiency is about 10 times the amount of synthesis compared to the normal PCR method.
  • an antigen and an antibody are described as examples of the target substance and the binding substance, but the same description can be applied to cases where the target substance and the binding substance are other than the antigen and the antibody.
  • a case will be described in which multiple types (two types) of substances are used as target substances, and they are identified and detected using the ICA method (individually detected).
  • the following examples can be referred to as needed.
  • a plurality of types (four types) of antigens 1 are immobilized as target substances on a well plate 2 serving as a support (FIG. 1 (a ))
  • Add oligonucleotide conjugate antibodies (labeled antibodies) 3 and 4 as binding substances for these antigens 1 (Fig. 1 (b)).
  • Antibody 3 is a complex antibody that forms a complex with oligonucleotide chain 5
  • antibody 4 is a complex antibody that forms a complex with oligonucleotide chain 6.
  • Each of antibodies 3 and 4 has a sequence capable of binding a common primer for ICAN method (F primer 9, R primer 10) in the oligonucleotide chains 5 and 6.
  • the length of oligonucleotide strands 5 and 6 is The nucleotide sequences are designed (or selected) so that the lengths of the amplified fragments obtained by the ICAN method are different from each other. Specifically, as shown in FIG. 1 (e), (as shown in 0, the sequence between / 3 is amplified from oligonucleotide chain 5 in antibody 3, and ⁇ ⁇ is amplified from oligonucleotide chain 6 in antibody 4. The sequence between is amplified, and the sequence between ⁇ 3 is shorter.
  • antibodies 3 and 4 are each bound to a specific antigen by antigen-antibody reaction to form antigen-antibody complexes 7 and 8 (FIG. 1 (c)). Antigens that did not form the complex are removed by washing (FIG. 1 (d)).
  • the length of the obtained fragment is identified and detected by electrophoresis using an agarose gel or the like (FIG. 1 (g)).
  • Fig. 1 (g) two different bands corresponding to nucleotide fragments 11 and 12 are detected.
  • the test sample containing 4 types of antigens contained 2 types of antigens as target substances.
  • FIG. 2A As shown in the schematic flow chart of ⁇ , first, multiple types (4 types) of antigens 1 were immobilized as target substances on the well plate 2 as a support (Fig. 2A (a)). As a binding substance to the antibody, add the oligonucleotide complex antibody (labeled antibody) 13, 14 (Fig. 2A (b)).
  • the antibody 13 is a composite antibody that forms a complex with the oligonucleotide chain
  • the antibody 14 is a composite antibody that forms a complex with the oligonucleotide chain 16.
  • Antibodies 13 and 14 both have restriction enzyme sites 1 18 (for example, EcoRI, etc.) in their respective oligonucleotide strands 15 and 16, and have a common ICAN primer (Primer 23, R primer) 24) has a sequence to which it can bind.
  • the nucleotide sequences of the oligonucleotide strands 15 and 16 are designed (or selected) such that the lengths of the amplified fragments obtained by the ICAN method are different from each other. Specifically, as shown in FIG.
  • the sequence between the oligonucleotides 15 in the antibody 13 and the oligonucleotide j3 is amplified, and from the oligonucleotide chain 16 in the antibody 14 V ⁇
  • the sequence between is amplified, and the sequence between O ⁇ is shorter.
  • antibodies 13 and 14 are each bound to a specific antigen by antigen-antibody reaction to form antigen-antibody complexes 19 and 20 (FIG. 2A ( C )). Antigens that did not form the complex are removed by washing (Hl2A (d)).
  • nucleotide fragments 21 and 22 were amplified between 3 and 3
  • nucleotide fragments 25 of different lengths were amplified from each of nucleotide fragments 21 and 22 as amplification products.
  • And 26 fragments amplified between ⁇ are obtained (FIG. 2B (g)).
  • the length of the obtained fragment is identified and detected by electrophoresis using an agarose gel or the like (FIG. 2B (h)).
  • FIG. 2B (h) two different bands corresponding to nucleotide fragments 25 and 26 were detected.
  • the test sample containing 4 types of antigens contained 2 types of antigens as target substances.
  • this step is a step in which the target substance in the test sample is brought into contact with the labeled binding substance to form a complex of the target substance and the binding substance.
  • the bound substance a bound substance labeled with an oligonucleic acid chain having a region bound to a primer used in a nucleic acid amplification method capable of reacting under constant temperature conditions is used.
  • the target substance in the test sample may be fixed to the support, may not be fixed, or may include both.
  • binding substance means a substance that can specifically bind to a specific target substance, and examples thereof include an antibody against an antigen (target substance).
  • the support is not limited as long as it can fix a target substance such as an antigen, and can contact a binding substance (including a solution state) such as an antibody with the target substance.
  • a binding substance including a solution state
  • insoluble materials and shapes are usually used.
  • a support that can be used in an assembly system based on an antigen-antibody reaction is preferable, and specific examples include multi-plastic well plates, plastic beads, latex beads, magnetic beads, plastic tubes, nylon membranes, and nitrocellulose membranes.
  • the target substance to be detected is not limited as long as it is contained in the test sample.
  • various proteins including antibody proteins
  • peptides oligopeptides, polypeptides, etc.
  • polysaccharides oligopeptides, polypeptides, etc.
  • polysaccharides oligopeptides, polypeptides, etc.
  • Glycolipids oligopeptides, polypeptides, etc.
  • nucleic acids DNA and RNA
  • test samples include, but are not limited to, biological components (tissues and blood), foods such as meat and vegetables, soil and river water, and combustion waste.
  • the concentration of the target substance in the test sample is not limited, but according to the method of the present invention, for example, even if the amount of the target substance per test sample is ng order or less, a specific target substance can be clearly detected. It may be less than pg order, or even less than fg order.
  • the substance in the test sample containing the target substance is immobilized on a support and then brought into contact with the labeled binding substance, thereby performing a specific binding reaction between the target substance and the binding substance.
  • the reaction may be performed without being fixed to a support or the like, or a combination thereof may be performed, without limitation.
  • Examples of methods for fixing the target substance to the support include, for example, a method for fixing the target substance on the support surface, a substance that specifically binds to the target substance (such as an antibody) is previously bound to the support surface, and fixed. Then, by binding the target substance to this fixed binding substance, The method of fixing to a support body indirectly etc. are mentioned.
  • the target substance can be selected in advance from among a wide variety of substances in the test sample, so that the detection sensitivity and detection accuracy can be further increased.
  • both the binding substance immobilized on the support and the labeled binding substance used later are antibodies, both antibodies usually recognize the target substance (antigen). Use different epitopes.
  • the target substance When the target substance is immobilized on the support, it is preferable to perform blocking according to a conventional method before contacting with the labeled binding substance. In the case of direct immobilization, it is desirable to perform blocking after immobilization, and in the case of indirect immobilization, after immobilization of the binding substance to the support and before the binding of the target substance.
  • a plurality of types of substances in the test sample may be used as target substances.
  • the number of types of target substances is not particularly limited as long as it is plural (at least two types).
  • a specific target substance is selected. It can be clearly identified and detected, and may be 50 types or more, and may be 100 types or more.
  • the form of the labeled binding substance used in this step is not limited. However, when one kind of substance in the test sample is used as the target substance, one kind of binding substance that can specifically bind to the target substance is used. It is preferable to use the same (same) labeling treatment.
  • a common (one type) labeling process is applied to all of the binding substances that can specifically bind to each of these target substances. Can be used. If there is a binding substance that can specifically bind to all of the multiple types of target substances, one that has been subjected to one type of labeling treatment may be used. As a result, multiple types of target substances can be comprehensively detected.
  • each of the binding substances that can specifically bind to each of these target substances has a different standard for each type. It is also possible to use information that has been subjected to recognition processing (may be a common labeling process among some types). That is, a plurality of types of binding substances labeled so as to be discriminated and detected corresponding to the types of target substances may be used (for example, see FIGS. L (b) to (d)). In this case, multiple types of target substances can be detected comprehensively, and the number of types of detected target substances and the number of types of target substances contained in the test sample can be determined by specifying the types. Identification can be done.
  • “different labeling process” and “labeling process capable of distinguishing and detecting” refer to selection or design of oligonucleic acid chains to be labeled so that the lengths of fragments amplified by a predetermined primer are different from each other.
  • the predetermined primer may be one using one kind of primer set, or may be one using two or more kinds of primer sets, and is not limited.
  • an amplified fragment can be obtained by binding to any type of oligonucleic acid chain to be labeled.
  • the length of the amplified fragment obtained depends on the type of oligonucleic acid chain (label (For example, see Fig. L (e) and (£)).
  • the length of the amplified fragment obtained by using each primer that specifically binds to each type of oligonucleic acid chain to be labeled is It depends on the type of chain.
  • a binding substance to be labeled for example, in addition to an antibody (antibody protein) that can specifically bind to a specific antigen substance, a hybridized substance is used for a specific target gene or nucleic acid molecule.
  • Single-stranded nucleic acids synthetic nucleic acids such as DNA, RNA (mRNA, etc.) and peptide nucleic acids
  • various proteins except antibodies
  • proteins include proteins that can specifically bind to lipids (lectins and the like), antigenic substances that can specifically bind to specific antibodies, and antibodies are preferred.
  • the binding substance is an antibody
  • the labeled antibody may be referred to as “complex antibody”.
  • the binding substance is an antibody
  • monoclonal antibodies having specificity common to a plurality of specific types of antigens can be used. According to the Atsy system using such antibodies, multiple species A class of antigens can be comprehensively detected by a single antibody.
  • “to bring the target substance in the test sample into contact with the labeled binding substance” means that the target substance and the binding substance are brought into direct contact with each other and bonded in a broader sense.
  • the primary binding substance (primary antibody, etc.) is bound to the target substance, and then the labeled binding substance is brought into contact as a binding substance having specificity for the primary binding substance. It also means that the target substance and the labeled binding substance are indirectly bound.
  • the primary binding substance may further be bound with a secondary binding substance, a tertiary binding substance, and a ⁇ -order binding substance. Substances that can specifically bind to ⁇ -order binding substances may be used.
  • ⁇ is preferably 1 to 11, more preferably 1 or 2.
  • an oligonucleic acid chain having a region that binds to a primer (such as a primer for LAMP method and a primer for ICAN method) that can be reacted under constant temperature conditions is used.
  • the region that binds to the primer for the LAMP method is a primer set composed of the FIP, BIP, F3 primer, and B3 primer (Loop Primer F and Loop Primer B as required) described above.
  • the region that binds to the primer for the ICAN method is a region that includes two regions that serve as a basis for designing a primer set composed of the two chimeric primers (F and R primers) described above, and It means a region that can be amplified using this primer set, and the specific nucleic acid sequence is not limited.
  • the oligonucleic acid chain to be labeled has, for example, a region that can be cleaved by a restriction enzyme (see FIG. 2A (e)), a region that has a region that can be cleaved by light irradiation, or can be cleaved by active oxygen. (See Fig. 3).
  • a restriction enzyme see FIG. 2A (e)
  • a region that has a region that can be cleaved by light irradiation or can be cleaved by active oxygen.
  • Oligonucleic acid strands include oligonucleotide strands (oligo DNA strands and oligos A UNA chain (preferably an oligo DNA chain)), an oligopeptide nucleic acid chain (oligo PNA chain), or a mixed chain thereof is preferably mentioned, and an oligonucleotide chain is more preferable. Further, in the present invention, the oligonucleic acid chain includes those containing a part of the oligopeptide chain. When the oligopeptide chain is contained at one end of the oligonucleic acid chain, for example, to facilitate the labeling of the binding substance, or as a cleavage part for later separation from the complex (Fig. 4). The oligopeptide peptide chain can be used.
  • the oligonucleic acid chain may be a natural product or a synthetic product, but is preferably a synthetic product.
  • the length of the oligonucleic acid chain used as a label is not particularly limited, but is preferably, for example, 100 to 5,000 mer, more preferably 100 to 1,000 mer, and still more preferably 100 to 500 mer.
  • the length of the oligonucleic acid chain satisfies the above range, complexing with a binding substance is facilitated, the state after complexing is stabilized, detection sensitivity can be improved, and detection time can be shortened.
  • the labeled binding substance can be prepared, for example, by covalently binding one end of an oligonucleic acid chain used as a label to the binding substance.
  • the oligonucleic acid chain is obtained by chemical or enzymatic treatment (preferably chemical treatment) of, for example, one or more thiol groups, amino groups (substituents), or thiotin (or avidin). It may be introduced. This facilitates complexation with a binding substance, stabilizes the state after complexation, improves the yield of the resulting complex, and increases detection sensitivity and detection effect.
  • the oligonucleic acid chain is labeled on the binding substance.
  • the oligonucleic acid chain with an amino group or thiol group added to the 5 'end is fixed to the binding substance using a divalent crosslinking agent.
  • a divalent crosslinking agent See “E. Hendrickson et al., Nucl. Acids: Res., Vol 23 (3), p522—529 (1995)”) and (ii) preliminarily both oligonucleic acid strand and binding substance biotin.
  • a method of immobilizing the oligonucleic acid chain to the binding substance via avidin by mixing the binding substance and the oligonucleic acid chain and adding avidin is preferable.
  • aviddin generally includes all avidin proteins having a specific binding ability to piotin protein. For example, avidin, streptavidin, neutravidin and the like are preferable. Avi Gin and -eutravidin are more preferred.
  • the oligonucleic acid chain that becomes the labeling moiety can be complexed with the binding substance via at least a portion of the adapter (see FIG. 5 (1)). Oligo Nucleic acid strands are fixed to the binding substance via a portion of the adapter, which can further enhance the structural stability after conjugation, improve the resulting conjugation rate, and improve detection sensitivity and detection effect. Result.
  • the adapter part may be, for example, any protein selected from protein G, protein A and protein L, or at least two types of proteins selected from protein G, protein A and protein L.
  • fusion protein for example, a protein A and protein G fusion protein
  • at least one protein selected from protein G, protein A and protein L and another protein for example, an anti-IgG antibody.
  • an anti-IgG antibody for example, an anti-IgG antibody
  • the method for preparing the complex containing the adapter part is not limited, but (i) First, the oligonucleic acid strand to be labeled is bound to the adapter part, and (ii) the adapter part is then fixed to the binding substance. The method is preferred.
  • a method can be employed in which an oligonucleic acid chain is bound to an adapter part by avidin-modifying a part of the adapter, biotinylating the oligonucleic acid chain, and mixing both.
  • both the adapter part and the oligonucleic acid chain are biotined in advance, and the adapter part and the oligonucleic acid chain are mixed together, and avidin is added, whereby the oligonucleic acid chain is separated from the adapter partly through avidin.
  • avidin modification of the adapter part may be carried out by first binding a linker compound with the adapter part and then binding avidin to the compound.
  • the adapter part used here is protein A, G, or L
  • a linker compound for example, I Sulfosuccinimidyl 4— (N—maleimidomethyl) cyclohexane— 1 — Carboxylate (Sulfo-SMCC) ”and the like can be preferably used.
  • Sulfo-SMGG Sulfosuccinimidyl 4— (N—maleimidomethyl) cyclohexane— 1 — Carboxylate (Sulfo-SMCC)
  • Sulfo-SMCC PIERCE, # 22322, Molecular Weight: 436.37, Spacer Arm Length: 11.6 A
  • Sulfo-SMCC first add the Sulfo-SMCC solution to the protein G solution, and slowly agitate at room temperature to bind Protein G and Su 0-SMCC. Obtain activated protein G. This bond is a bond between protein G and the Sulfo-NHS ester group of Sulfo-SMCC.
  • One or two or more Sulfo-SMCC (preferably two or more, more preferably two) per protein G molecule ⁇ 3 molecules).
  • a protein G / avidin conjugate via Sulfo-SMCC is obtained by mixing a solution of maleimide activated protein G with a separately prepared solution of maleimide activated avidin.
  • the conjugate is preferably one in which one or two or more avidin molecules (preferably two or more molecules, more preferably two to three molecules) are bound to one protein G molecule. The same applies to the case where a conjugate is obtained using protein A, L, or the like instead of protein G in the above.
  • the adapter part / labeling part conjugate obtained in (i) and the binding substance are mixed to form a complex.
  • the adapter part and the binding substance do not have binding reactivity originally, for example, both of them may be piotinated and mixed in the presence of avidin. Techniques can be used to obtain the complex. (4) Complex formation reaction
  • the method and conditions are appropriately determined in consideration of the type and physical properties of the target substance and binding substance. It can be set and is not limited.
  • a labeled binding substance when brought into contact with a target substance immobilized (coated) on a support, generally, a blocking treatment is performed in advance with a known blocking solution, and the well is washed thoroughly with a known washing solution such as PBS. Keep it. After that, an appropriate amount of a solution containing a plurality of labeled binding substances is added, and the target substance and the binding substance are subjected to a binding reaction while stirring at room temperature for 30 to 60 minutes to form a complex of both substances. It is preferable to wash thoroughly.
  • a labeled binding substance when brought into contact with a target substance that is not immobilized on a support, generally, an appropriate pretreatment is performed on the test sample containing the target substance and impurities other than the target substance are introduced. Is preferably removed or reduced.
  • Such exemplification can be preferably applied particularly to a complex formation reaction (antigen-antibody reaction) when the binding substance is an antibody and the target substance is an antigen.
  • the oligonucleic acid chain in the complex obtained in the complex formation step that is, the oligonucleic acid chain that is the labeling portion in the binding substance that has formed the complex is converted into the above-mentioned 1.
  • This is the step of amplification by the predetermined nucleic acid amplification method (LAMP method, ICAN method, etc.) explained in the item.
  • a system comprising the complex obtained in the complex formation step is mixed with a predetermined primer set, DNA synthase, substrate, etc. in advance, and kept at a constant temperature, whereby an oligo in the complex is obtained. Amplify a predetermined region of the nucleic acid strand.
  • the predetermined amplification region may be a part or all of the oligonucleic acid chain.
  • an amplification product having a repetitive structure having mutually complementary sequences on the same strand in a state where fragments of various units are mixed
  • an amplification product having a repetitive structure having mutually complementary sequences on the same strand in a state where fragments of various units are mixed
  • those with different repeat unit chain lengths should also be present in the amplification product. It becomes. Therefore, it may be difficult to detect the identification in the subsequent detection process.
  • the amplification product by the LAMP method is treated with a restriction enzyme. It is preferable to select or design an oligonucleic acid chain and / or a primer for the LAMP method in advance so that the structure can be cleaved for each repeating unit.
  • the above selection or design is performed so that an appropriate restriction enzyme recognition site is contained around the junction with the adjacent structural unit.
  • an amplified fragment having a substantially uniform chain length is obtained for each type of oligonucleic acid chain to be labeled, and identification and detection are facilitated.
  • the cutting for each repeating structural unit may be cutting for each unit, or cutting for every two units or more.
  • an appropriate restriction enzyme is added in advance to the system containing the complex obtained in the complex forming step, and the portion containing the amplification region in the oligonucleic acid chain is removed from the binding substance. It can also be cleaved and isolated, and the isolated oligonucleic acid strand can be amplified as a saddle (see (d) to (g) of FIGS. 2A and 2B). In this case, it is desirable to select or design an oligonucleic acid chain as appropriate so that it can be cleaved at an appropriate site.
  • the oligonucleic acid chain to be labeled has a region that can be cleaved by light irradiation
  • the oligonucleic acid chain is isolated and amplified as a trapezoid by irradiation with light of a predetermined wavelength. Can do.
  • a reagent that produces and releases free radicals such as HRP (horseradish peroxidase) and Fe complexes is added. By generating active oxygen, the oligonucleic acid strand can be isolated (see Fig. 3) and amplified as a cage.
  • This step is a step for detecting the amplification product obtained in the amplification step, as described above.
  • the amplification product can be easily detected by visual turbidity measurement, or may be performed using other listed means or in combination.
  • a plurality of types of amplified fragments having different chain lengths can be identified and detected by various electrophoresis methods (see, for example, FIG. 1 (g)).
  • a DNA sequencer for example, Applied Biosystems, product name: ABI-3100 was used for the obtained amplified fragment.
  • the length of the amplified fragment can be identified and detected (see Fig. 5 (3) to (5) and Fig. 6). 6. Detection kit
  • the kit of the present invention has, as a constituent component, an oligonucleic acid having a region that binds to a primer (a primer for LAMP method, a primer for ICAN method, etc.) used in a nucleic acid amplification method capable of reacting under constant temperature conditions.
  • a primer for LAMP method, a primer for ICAN method, etc.
  • a kit for detecting a target substance containing a binding substance labeled with a chain is as described in the description of the detection method of the present invention.
  • the kit of the present invention can be used effectively for carrying out the detection method of the present invention and is extremely useful.
  • the kit of the present invention may contain other components in addition to the above components.
  • Other components include, for example, primer set, dNTP, DNA polymerase, RNase H, restriction enzyme, various buffers, sterilized water, various reaction vessels (Eppendorf tube, etc.), blocking agent (Bovine Serum Albumin (BSA), Skim milk , Serum components such as Goat serum), and detergents, surfactants, fluorescent reagents (DNA intercalators, etc.), various plates, preservatives such as sodium azide, and experimental operation manuals (instructions)
  • a thermostatic chamber which can be any liquid, gas or solid medium
  • a turbidity measuring device such as a spectrophotometer
  • the 550mer oligonucleotide was prepared by using the primer of SEQ ID NO: 1 (5-MUSTagBio) with 5 as a primer and 3 linked with biotin as a primer. As a primer, PCR was performed using a primer of SEQ ID NO: 2 (3- MUSTag515).
  • PCRs described above were carried out using pcDNA3.1 (manufactured by In vitro) with Pinl as a vertical DNA and Taq polymerase as a polymerase under the following reaction solution composition and reaction conditions.
  • the following monoclonal antibodies were prepared by a conventional method.
  • Piotinylated 12CA5 was mixed with a 550mer oligonucleotide in a 1: 1 molar ratio. Subsequently, NeutrAvidin (manufactured by Piaerce) was added at a molar ratio of 1: 1 to the piotinated antibody and allowed to react at room temperature for 15 minutes. The reaction solution was passed through a 5 mL desalting column, and the target fraction was collected to obtain an oligonucleotide-conjugated antibody.
  • NeutrAvidin manufactured by Piaerce
  • antibody-bound beads were prepared by binding 9E10 (anti-Myc monoclonal antibody) to magnetic beads (manufactured by BioLabs) having a particle size of ⁇ as a support.
  • 9E10 anti-Myc monoclonal antibody
  • magnetic beads manufactured by BioLabs
  • a synthetic peptide (antigen solution) of HA-GST-Myc was used as the antigen.
  • Amount of antigen 200 pg 40 pg 8 pg 1.6 pg 320 fg 64 fg 12.8 fg 2.4 fg were thoroughly washed with PBST three times, an appropriate amount of a solution containing the oligonucleotide-conjugated antibody was added, and the reaction was carried out with shaking at room temperature for 30 minutes. After that, wash well 3 times with PBST, add the EcoRI enzyme solution prepared with EcoRI buffer solution to the residue obtained by centrifugation, and react at 37 ° C for 2 hours to obtain the oligonucleotide in the oligonucleotide conjugate antibody. The strand was broken. Thereafter, amplification was performed using the supernatant obtained by centrifugation.
  • a primer of SEQ ID NO: 3 (MUSTag FIP), a primer of 4 (MUSTag BIP), a primer of SEQ ID NO: 5 (MUSTag F3) and a primer of 6 (MUSTag B3) are added (ie, SEQ ID NOs: 3 to 6).
  • Amplification reaction was performed by the LAMP method under the following reaction solution composition and reaction conditions.
  • Loopamp DNA amplification kit (Eiken Chemical Co., Ltd.) was used for the reaction solution.
  • Sterilized water appropriate amount (about 2.3 wL)
  • reaction time has been greatly shortened.
  • the reaction time when using the LAMP method can be further reduced to about 1/3 by using a loop primer (Loop-F and Loop-B) as a LAMP primer set.
  • anti-IL-1 ⁇ anti-IL-8
  • anti-EGF monoclonal antibody R & D
  • the antibody binding plate was prepared by mixing and binding.
  • antigen IL-1 protein, IL-8, and EGF recombinant protein (manufactured by R & D) were used.
  • antigen solutions serially diluted to an antigen amount of 200 pg to 2.4 were prepared, and each was added to an antibody-binding plate and incubated at room temperature for 60 minutes.
  • Table 2 shows the amount of antigen in each well (reaction systems A to H).
  • the antigen-sensitized tool is thoroughly washed 3 times with PBST, and an appropriate amount of a solution containing three kinds of oligonucleotide-conjugated antibodies (prepared from anti-IL-1 «, anti-IL-8 and anti-EGF polyclonal antibodies) is added. And reacted at room temperature for 60 minutes. Thereafter, the plate was further washed three times with PBST, added with an EcoRI enzyme solution prepared with an EcoRI buffer solution, and reacted at 37 ° C for 15 minutes to cleave the oligonucleotide chain in the oligonucleotide-conjugated antibody. Thereafter, amplification was performed using the supernatant obtained by centrifugation.
  • the primer of SEQ ID NO: 7 (5- MUSTag- Forw3), the primer of SEQ ID NO: 8 (3- MUSTag_GEX), the probe of SEQ ID NO: 9 (TaqMan Probe # 1), the probe of SEQ ID NO: 10 (TaqMan Probe # 2) and the probe of SEQ ID NO: 11
  • TaqMan Probe # 3 (ie, the primer of SEQ ID NO: 7-8 and the probe of SEQ ID NO: 9-: 11) are added, and the Q- An amplification reaction by the PGR method was performed.
  • As a reaction solution TaqMan Universal PCR master mix (manufactured by ABI) was used.
  • Each of the probes below has a FAM fluorescent dye added to the 5 'side and a BHQ dye added to the 3' side.
  • ATCAGCCTCGACTGTGCCTTC (Distribution U number 10)
  • a g cone on the right side of the table represents the antigen concentration (0 to 200 pg / mL).
  • the detection sensitivity of each antigen was as follows.
  • IL-1 8 pg / ml
  • a target substance contained in a test sample can be detected more quickly, accurately, and at a lower cost than conventional biochemical methods using PCR (such as immuno-PCR).
  • An easy target substance detection method can be provided. According to this detection method, in particular, early diagnosis of stroke and myocardial infarction (which leads to reduction of sequelae), identification of pathogens in infectious diseases, and disseminated intravascular coagulation syndrome (
  • DIG disseminated intravascular coagulation
  • the amplification rate is very high in the LAMP method and the ICAN method as nucleic acid amplification methods that can be reacted under constant temperature conditions. Therefore, when the detection method of the present invention is performed using these methods, the reaction solution becomes turbid. The presence or absence of amplification can be easily discriminated only by measuring the intensity or by visual inspection (confirmation of cloudiness). For this reason, the target substance can be detected without using a special measuring instrument after the reaction or performing a separate operation for confirming the amplification. Therefore, the detection method of the present invention enables rapid detection on the spot where the test sample is collected, and food pollutants (residual agricultural chemicals, bacteria, etc.) or environmental pollution that particularly require such a method. It is extremely useful in the field of substance inspection (dioxin, PCB, etc.).

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Abstract

L'invention concerne un procédé permettant de détecter une substance cible par lequel la substance cible peut être plus rapidement, plus précisément et plus facilement détectée à un faible coût par l'utilisation d'une substance de liaison ayant été marquée par une chaîne d'acide oligonucléique. À savoir, l'invention concerne un procédé pour détecter une substance cible caractérisé en ce qu'il comporte l'étape de mise en contact, dans un échantillon à tester, de la substance cible et d'une substance de liaison marquée par une chaîne d'acide oligonucléique ayant une région capable de se lier à une amorce à utiliser dans un procédé d'amplification d'acide nucléique disponible dans des conditions thermostatiques, pour former ainsi un complexe de la substance cible avec la substance de liaison ; l'étape d'amplification de la chaîne d'acide oligonucléique dans le complexe par le procédé d'amplification d'acide nucléique tel que décrit ci-dessus ; et l'étape de détection du produit de l'amplification.
PCT/JP2007/063771 2006-07-05 2007-07-04 Procédé de détection d'une substance cible à l'aide d'un procédé d'amplification de l'acide nucléique, disponible dans des conditions thermostatiques WO2008004706A1 (fr)

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Cited By (2)

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CN113267529A (zh) * 2021-05-12 2021-08-17 江西师范大学 一种温度型生物传感器及利用温度型生物传感器检测目标适体的方法
CN116699126A (zh) * 2023-06-13 2023-09-05 深圳市博卡生物技术有限公司 一种抗体偶联的微球复合物的封闭剂

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WO2000056877A1 (fr) * 1999-03-19 2000-09-28 Takara Shuzo Co., Ltd. Procede d'amplification d'une sequence d'acide nucleique

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WO2000056877A1 (fr) * 1999-03-19 2000-09-28 Takara Shuzo Co., Ltd. Procede d'amplification d'une sequence d'acide nucleique

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113267529A (zh) * 2021-05-12 2021-08-17 江西师范大学 一种温度型生物传感器及利用温度型生物传感器检测目标适体的方法
CN116699126A (zh) * 2023-06-13 2023-09-05 深圳市博卡生物技术有限公司 一种抗体偶联的微球复合物的封闭剂

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