WO2008004706A1

WO2008004706A1 - Method of detecting target substance using nucleic acid amplification method available under thermostatic conditions

Info

Publication number: WO2008004706A1
Application number: PCT/JP2007/063771
Authority: WO
Inventors: Yuichiro Ishikawa
Original assignee: Synthera Technologies Co., Ltd.
Priority date: 2006-07-05
Filing date: 2007-07-04
Publication date: 2008-01-10
Also published as: JP2009219355A

Abstract

It is intended to provide a method of detecting a target substance whereby the target substance can be more quickly and accurately and easily detected at a low cost by using a binding substance having been labeled with an oligonucleic acid chain. Namely, a method of detecting a target substance characterized by comprising: the step of contacting a binding substance, which has been labeled with an oligonucleic acid chain having a region capable of binding to a primer to be used in a nucleic acid amplification method available under thermostatic conditions, with the target substance in a test sample to thereby form a complex of the target substance with the binding substance; the step of amplifying the oligonucleic acid chain in the complex by the nucleic acid amplification method as described above; and the step of detecting the amplification product.

Description

Description Method of detection of target substance using nucleic acid amplification method capable of reaction under isothermal conditions Technical Field

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. Background art

Establishment of monoclonal antibody production technology makes it possible to obtain antibodies that can react specifically with specific antigens, and research and development of and improvements in antigen detection systems that utilize the specificity of antigen-antibody reactions. It has become indispensable in all fields such as the spread and clinical fields. In general, such an assembly system is one in which a specific label is applied to an antibody to be reacted with an antigen, and the label is detected after the reaction to detect antigen. Various studies have been conducted for the purpose of developing new label types and detection methods in order to further increase detection sensitivity. Among them, immuno-PCR methods (see, for example, “T. Sano et al., Science, vol. 258, 120-122 (1992)”) and Double Determinant Immuno-PCR methods (see, for example) For example, “Kozo Imai, Asako Suzuki, Yuji Hinoda, Detection method of trace antigen using Imnmno-PCR, Protein nucleic acid enzyme, Yodosha, 1996, Vol.41, No.5, p.614-617” etc. Are well known).

However, 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.

Especially in the field of clinical testing, especially in the field of emergency medicine, extremely rapid and accurate diagnosis is required. For example, promptness is important in determining whether a patient who has lost consciousness has a myocardial infarction, a cerebral infarction, or another disease. However, The biochemical diagnosis by the previous method is not practical because it takes too much time and time. For this reason, rapid diagnosis is currently performed using large-scale and expensive devices such as CT scans, or based on doctors' rules of thumb. In addition, when amplifying by PCR, an apparatus such as a thermal cycler is usually used to accurately control the temperature, but it is still expensive and is not easily portable. For this reason, diagnosis using such devices has to rely on the inspection center or the examination room of a large hospital, and cannot be easily diagnosed in small clinics or emergency vehicles. Disclosure of the invention

Therefore, 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. To provide a method for detecting a target substance. It is another object of the present invention to provide a detection kit that can be used in such a detection method.

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. Thus, the inventors have found that the above-described problems can be solved and completed the present invention. That is, the present invention is as follows.

(1) 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 A step of contacting a substance to form a complex of a target substance and a binding substance, a step of amplifying an oligonucleic acid chain in the complex by the nucleic acid amplification method, and a step of detecting an amplification product, Method.

In the detection method of the present invention, for example, 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. In this case, 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.

Examples of the nucleic acid amplification method include LAMP method and ICAN method.

Examples of the labeling treatment include those in which an oligonucleic acid chain is fixed to the binding substance via at least a portion of the adapter. In addition, 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. (2) 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.

In the kit of the present invention, examples of the primer include a primer for LAMP method and a primer for ICAN method. Brief Description of Drawings

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). Explanation of symbols

1: Antigen 2 well plate

3: Labeled antibody 4 Labeled antibody

5: nucleotide chain 6 nucleotide chain

7: Antigen-antibody complex 8 Antigen-antibody complex

9: F primer 1 0: R primer

1 1: Amplified fragment 1 2: Amplified fragment

1 3: Labeled antibody 1 4: Labeled antibody

1 5: Nucleotide chain 1 6: Nucleotide chain

1 7: Restriction enzyme site 1 8: Restriction enzyme site

1 9: antigen-antibody complex 2 0: antigen-antibody complex

2 1: nucleotide fragment 2 2: nucleotide fragment

2 3: F primer 2 4: R primer

2 5: amplified fragment 2 6: amplified fragment BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail. The scope of the present invention is not limited to these explanations, and modifications other than the following examples can be made as appropriate without departing from the spirit of the present invention. This specification includes the entire specification of Japanese Patent Application No. 2 0 0 6-1 8 5 8 6 9 which is the basis for claiming priority of the present application. In addition, all publications cited in this specification, for example, prior art documents, and publications, patent publications, and other patent documents, are incorporated herein by reference. The method of the present invention comprises:

A method for detecting a target substance,

(X) 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),

(ii) a step of amplifying the oligonucleic acid chain in the complex by the nucleic acid amplification method (amplification step), and

(iii) Amplification product detection process (detection process)

It is characterized by including. The method of the present invention may further include other steps, and these other steps can be performed using known means and methods.

In the following, first, the 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.

1. Nucleic acid amplification method

In the present invention, as will be described in detail later, 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.

Here, 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. For example, LAMP (Loop-Mediated Isothermal Amplification ) method and ICAN (like properly preferred Isothermal and Chimeric primer-initiated Amplincation oi Nucleic acids) method, such as the power ^s. (1) LAMP method

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.).

In addition, 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.

In addition, as mentioned above, because four types of primers including six regions in vertical DNA are designed and used at the same time (up to six when using loop primers), specificity for vertical DNA The possibility of non-specific amplification is greatly reduced. Therefore, the target substance can be detected accurately.

Here, the above-mentioned 4 types (up to 6 types) of LAMP primers (hereinafter sometimes referred to as “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. Specifically, 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.

It may be abbreviated as “FIP”. ), And Backward Inner Primer (hereinafter abbreviated as “: BIP”), which is formed by linking the Blc region and B2 region nucleic acids from the 5 ′ end side, and consisting of the F3 region nucleic acid: F3 primer, B3 plastic consisting of nucleic acids in the B3 region It consists of four types with Imama. If necessary, loop primers (Loop Primer F and Z or Loop Primer B) may be designed and used to: amplify DNA and detect amplification products. 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. For example, 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). When using Bst DNA polymerase, it is desirable to perform the reaction at around 60-65 ° C, which is the optimum temperature for the reaction.

Known techniques can be applied to the detection of amplification products by the LAMP method, and are not limited. In the LAMP method, a large amount of substrate is consumed by nucleic acid synthesis, and pyrophosphate, a by-product, reacts with coexisting magnesium to become pyrophosphoric acid magnesium, which becomes cloudy enough to be confirmed with the naked eye. Therefore, the amplification product 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. Note that 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. In addition, according to the LAMP method 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. In addition, 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. (2) ICAN method

Like the LAMP 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.) .

In the ICAN method, two types of primers (F primer and H primer) are used as in the PCR method, but as this primer, a chimeric primer consisting of a DNA part (5 'side) and an RNA part (3' side). There is a special point in using. 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 ™ 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. Here, when the RNA strand of this hybrid site is cleaved by 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.

Known techniques can be applied to the detection of amplification products by the ICAN method, and there is no limitation. Specifically, a detection method similar to the LAMP method described above can be employed.

2. Overview of the detection method of the present invention

In the following examples, 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. In the following example, 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). However, for example, when one type of substance is used as a target substance, or when multiple types of substances are used as target substances but are comprehensively detected without being identified, or they are detected using the LAMP method, etc. For all other embodiments included in the present invention, such as cases, the following examples can be referred to as needed.

In the first embodiment, as shown in the schematic flow diagram of FIG. 1, first, 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, and 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 γ δ is amplified from oligonucleotide chain 6 in antibody 4. The sequence between is amplified, and the sequence between α3 is shorter.

Next, 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)).

After that, an amplification reaction was performed (Fig. 1 (e)), and each of the oligonucleotide strands 5 and 6 in the antibodies 3 and 4 forming the antigen-antibody complex 7 and 8 was different in length as an amplification product. Nucleotide fragments 1 1 (fragment amplified between the fragments) and 12 (fragment amplified between γδ) are obtained (FIG. 1 ()).

The length of the obtained fragment is identified and detected by electrophoresis using an agarose gel or the like (FIG. 1 (g)). In Fig. 1 (g), two different bands corresponding to nucleotide fragments 11 and 12 are detected. As a result, it can be confirmed that the test sample containing 4 types of antigens contained 2 types of antigens as target substances.

In the second embodiment, FIG. As shown in the schematic flow chart of Β, 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 15, and 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. 2B 0, (g), 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 δ The sequence between is amplified, and the sequence between Oβ is shorter.

Next, 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)).

Thereafter, a restriction enzyme that cleaves restriction enzyme sites 17 and 18 is added and reacted to form a nucleotide fragment from each of oligonucleotide chains 15 and 16 in antibodies 13 and 14 that form antigen-antibody complex 19, 20 21 and 22 are obtained (Fig. 2A (e)) _α

After that, amplification reaction was carried out using nucleotide fragments 21 and 22 as the vertical shape (Fig. 2 (£)), and nucleotide fragments 25 of different lengths (amplified between 3 and 3) were amplified from each of nucleotide fragments 21 and 22 as amplification products. And 26 (fragments amplified between γδ) 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)). In FIG. 2B (h), two different bands corresponding to nucleotide fragments 25 and 26 were detected. As a result, it can be confirmed that the test sample containing 4 types of antigens contained 2 types of antigens as target substances.

3. Complex formation process

As described above, 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. As 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. Here, the target substance in the test sample may be fixed to the support, may not be fixed, or may include both.

The “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).

(1) Support 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. As such a support, insoluble materials and shapes are usually used. For example, 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.

(2) Target substance

The target substance to be detected is not limited as long as it is contained in the test sample. For example, various proteins (including antibody proteins), peptides (oligopeptides, polypeptides, etc.), polysaccharides, Glycolipids, various nucleic acids (DNA and RNA

), And other low-molecular chemical compounds, biological components, and the like, but substances having immunogenicity (that is, those capable of producing antibodies or those in which antibodies already exist) are preferred.

Examples of 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.

In this step, 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. In the latter fixing method, 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. In the latter immobilization method, when 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.

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.

In the present invention, a plurality of types of substances in the test sample may be used as target substances. In this case, the number of types of target substances is not particularly limited as long as it is plural (at least two types). However, according to the method of the present invention, for example, even if there are 10 types or more, 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.

(3) Binding substance

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.

On the other hand, when multiple types of substances in the test sample are used as target substances, 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.

In addition, when multiple types of substances are used as target substances, 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. In the above, “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. Means that 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. When a single primer set is used, 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 (£)). When two or more primer sets are used, 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.

Here, as 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) that can specifically bind to specific target substances, specific sugars Examples 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. When the binding substance is an antibody, the labeled antibody may be referred to as “complex antibody”.

When the binding substance is an antibody, it is generally preferable to use a monoclonal antibody having specificity for each specific single type of antigen (target substance). For example, 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.

In this step, “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. In this indirect binding, the primary binding substance may further be bound with a secondary binding substance, a tertiary binding substance, and a η-order binding substance. Substances that can specifically bind to η-order binding substances may be used. In addition, η is preferably 1 to 11, more preferably 1 or 2.

As the oligonucleic acid chain used for the labeling treatment of the binding substance, 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. .

Here, for example, 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. Means a region that includes 6 regions that serve as a base for designing and can be amplified using this primer set, and the specific nucleic acid sequence is not limited. 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). In this case, prior to the amplification reaction, the oligonucleic acid chain can be separated and isolated from the complex with the binding substance to form a cage, and a more efficient amplification reaction can be performed.

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. When 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. In this case, 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. Specifically, the oligonucleic acid chain is labeled on the binding substance. Specifically, 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. (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. Preferably, 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. Among them, “avidin” 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.

In the present invention, 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. It may be a fusion protein (for example, a protein A and protein G fusion protein), or at least one protein selected from protein G, protein A and protein L and another protein (for example, an anti-IgG antibody). Or any combination thereof. These adapter proteins are preferred because they can be easily and stably bound to the antibody, particularly when the binding substance is an antibody molecule.

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.

Specifically, in (i), 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. Alternatively, 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. It is also possible to adopt a method of coupling to the In the former method, 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. When the adapter part used here is protein A, G, or L, as a linker compound, for example, I Sulfosuccinimidyl 4— (N—maleimidomethyl) cyclohexane— 1 — Carboxylate (Sulfo-SMCC) ”and the like can be preferably used. Molecular structure of Sulfo-SMGG

aleimide group

Commercially available Sulfo-SMCC (PIERCE, # 22322, Molecular Weight: 436.37, Spacer Arm Length: 11.6 A) can be used. When using 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). Thereafter, 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.

Next, in the above (ii), when the adapter part and the binding substance have binding reactivity, the adapter part / labeling part conjugate obtained in (i) and the binding substance are mixed to form a complex. Can be obtained. In addition, if 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

Regarding the reaction of bringing the binding substance and target substance into contact with each other to form a complex of the binding substance and the target substance, 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.

For example, when a labeled binding substance is 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. In addition, when a labeled binding substance is 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.

4. Amplification process

In this step, as described above, 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. Specifically, 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.

In particular, when amplification is performed by the LAMP method, 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) is obtained. When using multiple types of binding substances that have been subjected to different labeling treatments as described in section 3. (3) above, 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. In such a case, 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. For example, when cleaving by restriction enzyme treatment, 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. As a result, 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. Note that the cutting for each repeating structural unit may be cutting for each unit, or cutting for every two units or more.

In the present invention, in carrying out this step, 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. Similarly, when 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. In addition, if the oligonucleic acid chain to be labeled has a region that can be cleaved by active oxygen, 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.

5. Detection process

This step is a step for detecting the amplification product obtained in the amplification step, as described above.

As described in the item 1 above, the amplification product can be easily detected by visual turbidity measurement, or may be performed using other listed means or in combination. In particular, 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)). Alternatively, using a fluorescently labeled primer, a DNA sequencer (for example, Applied Biosystems, product name: ABI-3100) was used for the obtained amplified fragment. By identifying the peak position and its height by analysis with GeneScan software, the length of the amplified fragment can be identified and detected (see Fig. 5 (3) to (5) and Fig. 6). 6. Detection kit

As described above, 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 kit for detecting a target substance containing a binding substance labeled with a chain. Here, the details of the labeled binding substance are 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) Depending on the conditions, a thermostatic chamber (which can be any liquid, gas or solid medium), a turbidity measuring device (such as a spectrophotometer), etc. may be mentioned. Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Example 1

Preparation of oligonucleotide conjugate antibodies>

(1) Preparation of oligonucleotide chain

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).

Each of the PCRs described above was 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.

"Primer"

5- MUSTagBio:

Biotin-GGGAATTCGCGGACGAGGAGAAGCTGCCGCCCGGCTGG (No. 1) 3-MUSTag515:

AGCTTGACGGGGAAAGCCGG (allocation lj number 2)

Vertical DNA (lOOM / μ /): IML

Taq polymerase: 2.5unit

5, Primer (20μΜ): 2μL

3 'primer (20μΜ): 2 ^ L

dNTP (2.5 mM each): 8 μL

10 x Buffer: 10

Sterilized water: appropriate amount (about 77wL)

Total: 100 μL

A total of 35 cycles under the cycle conditions of 1 cycle of “thermal denaturation at 95 ° C for 1 minute · dissociation → annealing for 1 minute at 55 ° C → synthesis' extension for 30 seconds at 72 ° C”. The amplification product after each PCR was purified to a single oligonucleotide by purifying the supernatant obtained by centrifugation after PCR with MnElute PCR Purification spin column (Qiagen). (2) Preparation of pyotinylated antibody

The following monoclonal antibodies were prepared by a conventional method.

12CA5 (anti-HA monoclonal antibody)

Next, the 12CA5 antibody and Sulfo-NHS-LC-Biotin (manufactured by Pierce) were mixed so that the molar specific power was 20 and reacted at room temperature for 30 minutes. The reaction solution was passed through a 5 mL desalting column, and the target antibody fraction was recovered to obtain a biotinylated antibody.

(3) Preparation of oligonucleotide conjugate antibody

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. Example 2

Detection using complex antibodies>

Using the complex antibody obtained in Example 1, “antigen-antibody reaction” and “label detection” were performed as follows.

In accordance with a conventional antigen-antibody reaction sandwich method, antibody-bound beads were prepared by binding 9E10 (anti-Myc monoclonal antibody) to magnetic beads (manufactured by BioLabs) having a particle size of Ιμηι as a support. A synthetic peptide (antigen solution) of HA-GST-Myc was used as the antigen.

First, 9E10-bound beads were placed in a microtube, and each antigen solution diluted to an antigen amount of 200 pg to 2.4 fg per tube was added thereto, followed by incubation at room temperature for 30 minutes. Table 1 shows the amount of antigen in each tube (reaction systems A to B). Reaction system ABCDEFGH

Amount of antigen 200 pg 40 pg 8 pg 1.6 pg 320 fg 64 fg 12.8 fg 2.4 fg These 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.

That is, 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. In addition, Loopamp DNA amplification kit (Eiken Chemical Co., Ltd.) was used for the reaction solution.

"Primer"

MUSTagFIP:

CGCGTGGGGATACCCCCTAA-ATGCGGTGGGCTCTATGG (Guide (I number 3) MUSTag BIP:

GGTGTGGTGGTTACGCGCAG-AGGGAAGAAAGCGAAAGGAG (SEQ ID NO: 4) MUSTag F3:

TGGGAAGACAATAGCAGGCA (SEQ ID NO: 5)

MUSTag B3:

CGAACGTGGCGAGAAAGG (SEQ ID NO: 6) 7063771

Vertical DNA (Supernatant after centrifugation): 2μL

Bst DNA polymerase: lnL

Primer ϊΊΡ (20μΜ): 2βL

Primer ΒΙΡ (20 ζΜ): 2 zL

Primer F3 (5 M): lμ

Primer F3 (5 ^ M): 1 / L

2 X Buffer: 12.5 / z L

SYBR- Green (x2 concentration solution): 1.25 ML

Sterilized water: appropriate amount (about 2.3 wL)

A

Whisper B + I. 25

Incubate at 63 ° C for 90 minutes. The reaction was performed using an MX-3005p real-time PCR device (Stratagene), and the SYRR-Green fluorescence that fluctuates with the synthesis of the DNA strand was measured every minute after the reaction was started, and DNA amplification was observed. . The results are shown in Fig. 7.

At the same time, for comparison of reactivity, the primer of SEQ ID NO: 7 (MUSATag-Forw3) and the primer of SEQ ID NO: 8 (MUSTag-GEX) were added to the supernatant after the same centrifugation, and the following reaction solution composition and reaction were performed. PCR was performed under conditions. The reaction solution was Brilliant SYBR green Q-PCR master mix kit (Stratagene). Figure 8 shows the result. "Primer"

5-MUSTag-Forw3:

TGCATCTAGAGGGCCCTATTCTATA (SEQ ID NO: 7)

3-MUSTag-GEX:

GGCAAGCCACGTTTGGTG (SEQ ID NO: 8) <Reaction solution composition>

Vertical DNA (Supernatant after centrifugation): 2 μ

2 X Buffer: 12.5

5 'primer

5-MUSTag-Forw3 (20 Μ)

3 'primer

3-MUSTag-GEX (20 μΜ):

Reierence Dye: 0.375 // L

-Bacteria water: appropriate amount (about 6.3 L)

Total 25 u L

First heat denaturation at 95 ° C for 10 minutes, then heat denaturation at 95 ° C for 30 seconds. Dissociation → annealing at 60 ° C for 1 minute → synthesis at 72 ° C for 1 minute. 40 cycles in total. As a result of the above, as shown in FIG. 7 and FIG. 8, in both reaction systems, peaks indicating the presence of oligonucleotide fragments obtained by EcoRI treatment were observed depending on the antigen concentration. Real-time: Antigen amounts of 64 fg or less that were detected using the PCR method could not be detected using the LAMP method. On the other hand, when the LAMP method was used, the reaction time required for detection of 320 fg of the minimum detection concentration was 76 minutes, which was about 1 minute per cycle. Compared to the real-time PCR method, which requires more than 3 minutes per cycle, it can be said that the 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. Example 3

Detection using multiple complex antibodies simultaneously>

In accordance with the conventional method of antigen-antibody reaction sandwich method, three types of antibodies (anti-IL-1α, anti-IL-8, and anti-EGF monoclonal antibody: R & D) were applied to a 96-well immunoplate (manufactured by nunc) as a support. The antibody binding plate was prepared by mixing and binding. As the antigen, IL-1 protein, IL-8, and EGF recombinant protein (manufactured by R & D) were used.

First, 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). Table 2

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.

That is, 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.

"Primer"

5-MUSTag-Forw3:

TGCATCTAGAGGGCCCTATTCTATA (SEQ ID NO: 7)

3-MUSTag-GEX:

GGCAAGCCACGTTTGGTG (layout IJ number 8) 《Probe》

Each of the probes below has a FAM fluorescent dye added to the 5 'side and a BHQ dye added to the 3' side.

TaqMan Probe # 1:

CCTTCTAGTTGCCAGCCATCTGTT (SEQ ID NO: 9)

TaqMan Probe # 2:

ATCAGCCTCGACTGTGCCTTC (Distribution U number 10)

TaqMan Probe # 3:

ACCGACAATTGCATGAAGAACTC (SEQ ID NO: 11)

Vertical DNA (Supernatant after centrifugation): 2 ^ L

2xBuffer: 12.5

5 'primer

5-MUSTag-Forw3 (20 M): 2 ^ L

3 'primer

3-MUSTag-GEX (20 μΜ): 2 / it

Reierence Dye: 0.375 / L

TaqMan Probe # 1 (20 μM): 1 / i L

TaqMan Probe # 2 (20 μM): 1 / i L

TaqMan Probe # 3 (20 μM): 1 μL

Sterile water:

total

First cycle of heat denaturation at 95 ° C for 10 minutes, followed by one cycle of "thermal denaturation at 95 ° C for 30 seconds · dissociation → annealing at 60 ° C for 1 minute → synthesis at 72 ° C for 1 minute · extension" 40 cycles in total. As a result, as shown in Table 3 and FIG. 9, peaks indicating the presence of oligonucleotide fragments obtained by EcoRI treatment were observed depending on the antigen concentration.

Table 3

Ct raw value Ag cone

[Upper table] pg / mL

200 40 8 1.6 0.32 0.064 0.013

0

[Table below]

[Upper table] The numbers in the table indicate Gt values (Threshold cycle).

[Table below] Gt values shown in the table above are averaged (ave) and standard error (SD) fc% CV is

The calculated result is shown.

Note) “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.

EGF: 1.6 pg / mL

IL-8: 0.32 pg / mL

IL-1: 8 pg / ml

The above results indicate that the detection method of this example is clearly more sensitive than ordinary ELISA and can simultaneously detect three types of antigens (site force-in), which is impossible with conventional methods. Show. Industrial applicability

According to the present invention, 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 (

In clinical fields such as diagnosis of disseminated intravascular coagulation (DIG), examination or diagnosis efficiency, evaluation efficiency, etc. can be dramatically improved, which is extremely useful.

In addition, 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.).

Since the detection kit of the present invention can be used in the detection method of the present invention, it is extremely useful. Sequence listing free text

Sequence number 1: Synthetic DNA

Sequence number 2: Synthetic DNA

Sequence number 3: Synthetic DNA

Sequence number 4: Synthetic DNA

Sequence number 5: Synthetic DNA

Sequence number 6: Synthetic DNA

Sequence number 7: Synthetic DNA

Sequence number 8: Synthetic DNA

Sequence number 9: Synthetic DNA

Sequence number 10: Synthetic DNA

Sequence number 11: Synthetic DNA

Claims

The scope of the claims

1. A method for detecting a target substance,

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 to obtain a target substance and a binding substance. Forming a composite of

A step of amplifying the oligonucleic acid chain in the complex by the nucleic acid amplification method, and a step of detecting the amplification product

Including the method.

2. The method according to claim 1, wherein the target substance is a plurality of types of substances, and the binding substance is labeled so that it can be identified and detected corresponding to the type of the target substance.

3. The method according to claim 2, wherein at least one primer set is used as a primer used in the nucleic acid amplification method.

4. The method according to any one of claims 1 to 3, wherein the nucleic acid amplification method is a LAMP method or an ICAN method.

5. The method according to any one of claims 1 to 4, wherein in the labeling treatment, an oligonucleic acid chain is fixed to a binding substance through at least a part of an adapter.

6. The adapter part is selected from protein G, protein A and protein, protein G, protein A and protein

Claim 5 which is a fusion protein of at least two types of proteins selected from L, a fusion protein of at least one type of protein selected from Protein G, Protein A and Protein L and other proteins, or a combination thereof. The method described.

7. The method according to any one of claims 1 to 6, wherein the target substance is an antigen and the binding substance is an antibody.

8. A target substance detection kit 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.

The kit according to claim 8, wherein the primer is a primer for LAMP method or a primer for ICAN method.