JP4268944B2 - Nucleic acid detection or quantification method - Google Patents

Description

  The present invention relates to the fields of nucleic acid detection or quantification methods and genetic diagnosis techniques in samples. More specifically, the present invention relates to a qualitative or quantitative method for visually determining the presence of a target nucleic acid in a simple, rapid and specific manner and a kit based thereon.

  In recent years, polymerase chain reaction (PCR) (Patent Document 1) and ligase chain reaction (LCR) (Patent Document 2, Patent) are effective means for highly sensitive detection and analysis of a small amount of nucleic acid present in various samples. Document 3), strand displacement amplification (SDA) (Patent Document 4, Patent Document 5), NASBA (Non-Patent Document 1, Patent Document 6) and TMA (Patent Document 7) have been developed and used. Yes.

  As a method for detecting such an amplification product, gel electrophoresis typified by agarose gel is generally used, and the presence or absence of the target nucleic acid is determined from the molecular size of the amplification product separated by electrophoresis. In recent years, capillary gel electrophoresis technology that realizes higher-speed electrophoresis has been developed, and trace analysis can be performed in a shorter time. For detection by electrophoresis, an intercalator-type insertion-binding dye represented by ethidium bromide is used. An intercalator is inserted and bonded with high affinity to a nucleic acid, and its fluorescence intensity increases. Since detection by an intercalator detects all nucleic acids present, non-specific products are often detected, making determination difficult. In addition, since some intercalators are carcinogenic in nature, it is necessary to pay sufficient attention to handling and disposal.

  As a more specific method for detecting and identifying a nucleic acid sequence, there is a nucleic acid hybridization method using an oligonucleotide probe having a sequence complementary to a target nucleic acid. In the nucleic acid hybridization method, in order to hybridize with a single-stranded oligonucleotide probe, when the target nucleic acid is a double-stranded DNA, a denaturation treatment with heat or alkali is required in advance. Among the nucleic acid amplification methods described above, the amplification product obtained by NASBA or TMA is a single-stranded RNA complementary to the template, and thus can be hybridized with a complementary oligonucleotide probe without denaturation treatment. However, these known nucleic acid hybridization methods such as dot blot and sandwich hybridization are complicated and require a long time. Recently, a so-called real-time detection method has been developed in which a molecular beacon (Non-patent Document 2) based on fluorescence resonance energy transfer (RET) is used to monitor a product generation state during an amplification reaction. However, this requires two types of probes labeled with fluorescent materials having different excitation wavelengths and absorption wavelengths, and expensive dedicated amplification and detection devices.

  As a simpler method for quantifying a nucleic acid amplification product, there is a method of measuring the amount of nucleic acid amplified by an agglutination reaction of a polymer carrier (Patent Document 8). In this method, the degree of aggregation of the polymer carrier is optically quantified by the intensity of scattered light, absorbance or transmitted light, and an automatic analyzer must be used for the optical quantification. Necessary.

As a method for detecting a target nucleic acid which is excellent in operability and is quick and simple, there is a method based on chromatography (Patent Document 9). In this method, the step of extracting a gene from a cell, a virus or a bacterium, the step of fragmenting an arbitrarily extracted gene, and the step of detecting include an arbitrarily extracted gene or a fragment thereof on a single gene detection device. This is a gene detection method that is continuously performed by moving a liquid sample by capillary action. This document describes that it is possible to determine the presence or absence of a target gene and further identify its type, but there is no mention of quantitative measurement. In addition, a detection method that does not involve an amplification reaction is a simple gene detection method, but it is difficult to detect without an amplification reaction in terms of detection sensitivity when detecting a small amount of nucleic acid such as the presence or absence of pathogenic microorganisms. It was expected to present.
JP 61-274597 A WO89 / 12696 JP-A-2-2934 U.S. Pat. No. 5,270,184 U.S. Pat. No. 5,455,166 Japanese Patent No. 2650159 Japanese Patent No. 3241717 JP-A-9-168400 JP 2001-157598 A Nature, 350: 91, 1991. Nature Biotech, 14: 303, 1996.

  In genetic testing and diagnosis in clinical settings, it is required that the method be simple, rapid, sensitive, and highly specific, and that reagents and equipment costs be low. The object of the present invention is to reduce the time and the number of steps required for the detection step of the amplification product while taking advantage of the nucleic acid hybridization method with high specificity, and it is simpler, quicker and more specific without using a special device. It is another object of the present invention to provide a detection or quantification method excellent in visual judgment and a target nucleic acid detection or quantification kit suitably used in the method.

  In addition, in nucleic acid amplification testing, there is always a concern that the amplification reaction may not proceed due to various factors, and false negatives are always a concern. It is to provide a method for detecting the two simultaneously.

As a result of intensive studies to solve the above problems, the present inventors have conceived that the above object can be achieved by amplifying a single-stranded nucleic acid from a target nucleic acid, and moving and detecting it by capillary action. The invention has been reached.
That is, the present invention is as follows.
1. A method for specifically detecting or quantifying a target nucleic acid in a sample, the step of amplifying as a single-stranded nucleic acid from a target nucleic acid arbitrarily extracted from a sample, and detecting the amplification product by chromatography And a method for detecting or quantifying a nucleic acid, comprising a step of evaluating the detected image by visual determination.
2. The method of 1 above, wherein the amplification step involves amplification of the target nucleic acid and an internal standard nucleic acid that can be competitively or non-competitively co-amplified with the target nucleic acid.
3. The method according to 1 or 2 above, wherein the amplification is performed by NASBA or TMA.
4). The detection is performed using the first oligonucleotide probe complementary to the amplification product bound to the membrane and the complementary second oligonucleotide probe labeled with a colored polymer carrier. Method 3.
5. 5. The method according to any one of 1 to 4 above, which uses an assay device based on nucleic acid chromatography.
6). One that is amplified competitively or non-competitively as an internal standard nucleic acid in order to determine not only whether the sample and the colored polymer carrier-labeled oligonucleotide probe are developed correctly or not, but also whether the nucleic acid amplification reaction is correct or false negative of the nucleic acid extraction operation 6. The method according to any one of 1 to 5, wherein a strand nucleic acid is co-detected.
7). The above-mentioned 1-6, wherein the target nucleic acid amount in the sample is visually determined from the contrast of the color development amount with the amplification product derived from a known amount of the internal standard nucleic acid competitively or non-competitively amplified with the target nucleic acid. The method for quantifying a nucleic acid according to any one of the above.
8). 8. Detection or quantification of a nucleic acid according to any one of 1 to 7 above, wherein two or more different target nucleic acids present in a sample are simultaneously detected or quantified by combining a plurality of colored polymer carriers and capture polynucleotides. Method.
9. 9. A target nucleic acid detection kit comprising an amplification primer, a detection probe, a colored polymer carrier, a porous membrane, and a developing water-absorbing substrate useful for performing the method according to any one of 1 to 8 above.

  According to the present invention, single or two or more kinds of nucleic acids present in a sample can be detected simply, rapidly and specifically under visual judgment. Furthermore, when an internal standard nucleic acid that is co-amplified during the amplification reaction is used, it is possible to correctly determine whether the amplification and the detection reaction are correct from the detection result, as well as the detection result of the sample and the polymer carrier-labeled oligonucleotide. In addition, single-stranded nucleic acid amplified competitively or non-competitively as an internal standard nucleic acid can be co-detected, and the amount of target nucleic acid can also be measured visually based on the color development ratio with the target nucleic acid. . By co-amplification and co-detection of an internal standard nucleic acid in the present invention, it becomes possible to detect a target nucleic acid in a sample having various backgrounds such as an amplification inhibitor. In addition, by using the NASBA (or TMA) method that specifically amplifies RNA instead of DNA, it is possible to detect or quantify only living organisms without detecting dead bacteria that are false positive groups. is there. Furthermore, by co-amplifying the internal standard nucleic acid in the amplification step, not only false negatives can be avoided, but also visual detection determination can be made even when the target nucleic acid is in a very small amount. By including a known amount of the internal standard nucleic acid, visual quantification is possible by comparing the color development amount with the internal standard nucleic acid. Furthermore, it is possible to provide a gene detection method excellent in operability that does not require any special dedicated equipment in the detection step.

  In a preferred embodiment of the present invention, the target single-stranded nucleic acid obtained by the amplification reaction is hybridized by chromatography on a membrane bound with a first oligonucleotide probe complementary to the amplification product, and then the binding. The presence of the target nucleic acid can be detected or quantified by hybridizing a second oligonucleotide probe complementary to the target nucleic acid labeled with a colored polymer carrier to the product and developing the color. Furthermore, the correctness of the amplification and detection reaction can be determined from the detection result of the amplification product derived from the internal standard nucleic acid.

Hereinafter, the present invention will be specifically described. In this specification, nucleic acid chromatography means chromatography using nucleic acids.
First, target nucleic acid is extracted from a sample. The sample is not particularly limited as long as it contains the target nucleic acid. For example, cultured cell (fungus) strains, peripheral blood, microorganisms such as bacteria and viruses, and the like can be used. In the present invention, the extraction method is not particularly limited, and can be performed using a commercially available extraction kit. For example, simple method (EDTA-SDS-Phenol-Ethanol), lithium chloride-urea method, protease K-deoxyribonuclease method, phenol SDS method, guanidine thiocyanate cesium chloride method, guanidine thiocyanate trifluoroacetic acid cesium acetate method, acid guanidine thiocyanate phenol chloroform Method (AGPC method), vanadyl ribonucleoside compound method, magnetic silica method and the like.

The target nucleic acid thus obtained is amplified as a single-stranded nucleic acid. As the amplification method, the NASBA method or TMA method for producing a single-stranded nucleic acid as an amplification product is preferable, and the step of performing a denaturation treatment of the amplification product can be omitted, and the detection operation can be immediately performed. Of course, the nucleic acid amplification method of the present invention is not limited to the NASBA method or the TMA method as long as it produces a single-stranded nucleic acid.
In the NASBA method, RNA is mainly used as a target nucleic acid as a template, single-stranded RNA complementary to the template RNA is generated as an amplification product, and a thermal cycler for complicated temperature control is unnecessary.

The NASBA method generally comprises the following [Step 1] to [Step 9]. [Step 1] A target nucleic acid in a sample (in the presence of an appropriate buffer, a first primer having a sequence complementary to the target nucleic acid sequence (RNA) in the sample and a promoter sequence of RNA polymerase on the 5 ′ end side thereof) RNA), DNA is synthesized using dNTPs as a material by reverse transcriptase, [Step 2] Only RNA of the RNA / DNA hybrid is decomposed by ribonuclease H to obtain single-stranded DNA (second template), [Step 3] Next, a second primer having a sequence complementary to the target nucleic acid sequence (DNA) is hybridized to the single-stranded DNA (second template), and an extension reaction is performed with DNA polymerase using dNTPs as a material. Double-stranded DNA is obtained. [Step 4] Using RNA polymerase that recognizes the promoter sequence, a large number of RNA copies (third template) are synthesized using NTPs as a material. [Step 5] Using the obtained RNA (third template) as a template, synthesis of an RNA / DNA hybrid with a second primer using reverse transcriptase, [Step 6] Single-stranded DNA that degrades only RNA by ribonuclease H Synthesis of (fourth template), [Step 7] Synthesis of double-stranded DNA with the first primer using DNA polymerase using the obtained single-stranded DNA (fourth template) as a template, [Step 8] RNA polymerase Is used to synthesize a large number of RNA copies (third template) from the double-stranded DNA. [Step 9] By repeating these steps (Steps 5 to 8), the nucleic acid in the sample can be amplified.
On the other hand, the TMA method is in principle the same method as the NASBA method, but using two types of primers specific to the target RNA and two types of enzymes (reverse transcriptase and RNA polymerase) to complement the template RNA. Single stranded RNA is produced isothermally.

  In the nucleic acid amplification reaction by the NASBA method or the like, a nucleic acid that can be competitively amplified by a primer for a target RNA is preferably added as an internal standard nucleic acid. Specifically, a synthetic RNA obtained by modifying a part of the target nucleic acid is prepared, competitively amplified with the target RNA, and detected on a membrane in which a complementary oligonucleotide probe is bound to the modified site. Alternatively, the nucleic acid coexisting in the sample may be co-amplified, but in this case, a dedicated amplification primer different from the target nucleic acid is required. Specifically, nucleic acids containing housekeeping genes such as β-actin and GAPDH and synthetic RNA are non-competitively co-amplified and detected on a membrane in which an oligonucleotide probe complementary to the amplified product is bound. It is desirable that these internal standard nucleic acids are in a certain amount above the necessary amount that can be always amplified and detected regardless of the presence or absence of the target nucleic acid. Specifically, the amount of the internal standard nucleic acid is more preferably set to be equal to or less than the expected target nucleic acid amount so that the amplification efficiency of the target nucleic acid is given priority.

The amplified nucleic acid is detected based on nucleic acid chromatography. A capture oligonucleotide probe is bound in advance to an identifiable position on the porous membrane, and a sample, colored latex, or the like is moved (developed) from a site different from the capture oligonucleotide probe position by capillary action. Only target single-stranded nucleic acids that have specifically bound (hybridized) to the capture and labeled oligonucleotide probes are identified at the capture oligonucleotide probe binding position. When using an internal standard nucleic acid, the target nucleic acid and the internal standard nucleic acid may be detected separately, but a co-detection method in which detection is simultaneously performed using the same membrane is preferable.
Specifically, the determination of the result in detection is desirably performed as follows. If the target nucleic acid amount is equal to, or less than or equal to the expected target nucleic acid amount, compared to the color development amount of the internal standard nucleic acid that is co-amplified competitively or non-competitively and is co-detected Judgment is made visually. For example, when an internal standard nucleic acid that can be competitively amplified with the target nucleic acid is used, the color development amount of the amplification product obtained from the target nucleic acid is equivalent to the color development amount equivalent to the amplification product derived from the control internal standard nucleic acid. The amount of target nucleic acid is determined to be larger than that of the internal standard nucleic acid when the color of the internal standard nucleic acid as a control is slightly recognized or not, and only the color of the target nucleic acid is developed. On the other hand, when only the color of the internal standard nucleic acid as a control is developed or the color of the target nucleic acid is very small, it is determined that the amount of the target nucleic acid is small.

  Further, by combining a plurality of colored polymer carriers and capture polynucleotides, two or more different target nucleic acids present in a sample can be simultaneously detected or quantified. For example, when two different target nucleic acids (E and F) are to be detected simultaneously, complementary oligonucleotide probes complementary to each target nucleic acid are immobilized on different positions on the membrane, and each An oligonucleotide probe complementary to a target nucleic acid is bound to a red (for target nucleic acid E) or blue (for target nucleic acid F) polymer carrier, and the mixture is applied to a latex-bound oligonucleotide probe coating pad and dried. Install on test strip. A NASBA product containing two types of target nucleic acids is applied to the test strip, and detection by chromatography is performed. On the membrane to which each target nucleic acid is bound, a colored line of oligonucleotide-labeled latex having each complementary sequence is obtained via the NASBA product. Of course, it is not limited to two types.

The porous membrane is not particularly limited as long as it can bind the oligonucleotide probe for capture, and examples thereof include nitrocellulose and nylon. Various binding methods including physical binding can be selected. A binding activity modified nylon membrane is desirable.
As the colored polymer carrier, polystyrene latex colored with pigments such as blue and red is preferable, and those having a carboxyl group as a functional group are particularly useful for labeling oligonucleotide probes. When identifying a plurality of nucleic acids, visibility is improved by combining latexes of different colors. The size of the colored polymer carrier particles is selected from a particle size smaller than the membrane pore diameter, and usually a diameter of 500 nm or less is used.

The capture oligonucleotide probe and the label oligonucleotide probe are single-stranded nucleic acids having a sequence complementary to a single-stranded nucleic acid (amplification product) to be detected. When the membrane or latex to be bound is chemically modified, it is desirable to have a functional group that reacts with it. For example, 5 ′ or 3 for a membrane or latex modified with a carboxyl group 'Synthetic oligonucleotides with aminated ends are preferred.
The form of binding of the oligonucleotide probe for capture to the membrane is not particularly limited. For example, it binds as a line or a circular spot. Furthermore, in order to simultaneously capture the target nucleic acid and the amplification product derived from the internal standard nucleic acid, it is preferable to bind each capture oligonucleotide probe to different positions on the same membrane.

The latex-labeled oligonucleotide is applied to a holding member such as a glass fiber cloth, dried, and placed on one end of the membrane. The latex in the holding part is dissolved and leached on the membrane by the liquid sample or the developing solution, and reaches the capture oligonucleotide binding position. At this time, if a single-stranded nucleic acid to be detected is present in the sample, colored latex accumulates on the capture oligonucleotide binding position via the single-stranded nucleic acid, and visual determination is possible.
Detection is preferably performed by nucleic acid chromatography using an amplification product containing a large amount of single-stranded nucleic acid. In particular, the nucleic acid obtained by NASBA or TMA is a single-stranded RNA and can be developed as it is without the need for a denaturation operation before detection. The detection device is preferably an assay device based on nucleic acid chromatography.

FIG. 1 is a schematic diagram showing an outline of a configuration example of a detection apparatus according to the present invention. A pad 5 on which a latex-bound oligonucleotide probe is applied and dried is placed on the upstream end side of the membrane 3 on which the capture oligonucleotide probe is immobilized. The development pad 6 is installed at a portion that does not overlap the membrane on the pad. “Upstream” means the end side of the membrane on which the development pad is disposed. An absorbent pad 4 is installed on the downstream end side of the membrane to form a test strip. In the case of the amplification product by the NASBA method, it is immediately applied to the sample well on the strip without performing denaturation treatment, and detection by chromatography is performed. Amplification products and reagents move from the upstream side to the downstream side by capillary action. Reference numeral 1 denotes a target nucleic acid detection part (capture oligonucleotide binding part), and reference numeral 2 denotes an internal standard nucleic acid detection part (capture oligonucleotide binding part).
FIG. 2 is a schematic diagram showing a determination example when the target nucleic acid is quantitatively detected according to the present invention. In FIG. 2, 7 is a target nucleic acid, and 8 is an internal standard nucleic acid detection pattern. (A) in the amplification product has no target nucleic acid, (B) has a target nucleic acid amount less than the internal standard nucleic acid amount, (C) has a target nucleic acid amount and an internal standard nucleic acid amount at the same level, (D) target nucleic acid The amount is determined to be greater than the amount of the internal standard nucleic acid.

In the present invention, the target of detection or quantification is mainly related to genes of pathogenic microorganisms (eg, bacteria, viruses, protozoa, etc.) that are expected to be involved or involved in infectious diseases, or cancer, therapeutic drugs, etc. Genes that are expected to be involved and genes that change expression due to these diseases (for example, oncogenes, tumor suppressor genes, drug resistance genes, etc.) can be mentioned as targets of measurement (amplification or detection), but are particularly limited to these. It is not a thing.
In addition, an amplification primer, a detection probe, a colored polymer carrier, a porous membrane, and a development water-absorbing substrate useful for carrying out the method of the present invention can be provided as a target nucleic acid detection kit. The water-absorbing base material for development is a latex-labeled oligonucleotide coating pad, a development pad (part for introducing a development liquid component), and an absorption pad on the opposite side across the membrane (part that promotes development by the absorption capacity of the aqueous liquid) Etc.) and a material excellent in water absorption is preferable, and examples thereof include glass fiber cloth and cellulose fiber.

Examples are given below to illustrate the specifications of the materials and methods of the present invention and will be described in more detail. However, the present invention is not limited to these examples.
Example 1
A NASBA amplification reaction was performed using total RNA extracted from Extragen (manufactured by Kainos) from a cultured strain of methicillin-resistant Staphylococcus aureus (MRSA), one of the major causative bacteria for intractable nosocomial infections. The product was immediately detected by nucleic acid chromatography without pretreatment.
(1) Preparation of Latex-Binding Oligonucleotide Probe Carboxyl group-containing polystyrene latex (solid content 10% (w / w), manufactured by Bangs) and amino group-containing oligonucleotide probe (SEQ ID NO: 5, amino group at the 3 ′ end for synthesis) Are mixed in MES (2-Morpholinoethanesulfonic acid, monohydrate) (Dojindo Laboratories) buffer solution to which the required amount of water-soluble carbodiimide has been added, combined, and then combined with monoethanolamine (Wako Pure Chemical Industries, Ltd.) The product was further reacted. After centrifuging the reaction solution, the supernatant was removed, and an aqueous solution was added to the resulting precipitate and the washing operation was repeated. After washing, the suspension was resuspended in a buffer containing HEPES (2- [4- (2-Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid) (manufactured by Saikyo Kasei Co., Ltd.) and stored at 4 ° C. until use.
(2) Immobilization of oligonucleotide A carboxyl group-modified nylon membrane (manufactured by Pall) was treated with water-soluble carbodiimide and washed with deionized water. An amino group-containing oligonucleotide probe having a sequence complementary to the single-stranded nucleic acid to be detected (SEQ ID NO: 4, introducing an amino group at the 5 ′ end during synthesis) was bound to the activated membrane, and air-dried for 15 minutes. did. Thereafter, the membrane was treated with a Tris-based buffer, and after the remaining active groups were erased, the membrane was washed with deionized water and air-dried.

(3) MRSA based on NASBA method
Amplification of RNA NASOS lyophilized reagent from Kinos was dissolved in 100 μL of a primer solution containing 0.4 μM each of primer-P1 (SEQ ID NO: 1), P2 (SEQ ID NO: 2), and P3 (SEQ ID NO: 3). 5 μL each was dispensed into a 5 mL Eppendorf tube. 2.5 μL each of water or total RNA derived from 10 ³ CFU equivalent of cultured strain was added, heated at 65 ° C. for 5 minutes, and then kept at 41 ° C. for 5 minutes. 2.5 μL of the enzyme solution was added, tapped lightly, and reacted at 41 ° C. for 60 minutes.
(4) Detection by Nucleic Acid Chromatography As a detection nucleic acid chromatography apparatus, a strip comprising the following components as shown in FIG. 1 was prepared.
A pad (manufactured by Millipore) with a latex-bound oligonucleotide probe applied and dried was placed on the upstream end side of the membrane on which the capture oligonucleotide probe was immobilized. A development pad (Millipore) was installed at a portion that did not overlap the membrane on the pad. Furthermore, an absorption pad (Millipore) was installed on the downstream end side of the membrane to obtain a test strip. The NASBA product was immediately applied to the sample well on the strip without denaturation treatment and detected by chromatography. Amplification products and reagents move from the upstream side to the downstream side by capillary action. When water was used as the specimen, no line was detected. On the other hand, a colored line specific to the target nucleic acid was detected in the amplification product of total RNA derived from the cultured strain. Immediately after the amplification, the target nucleic acid could be specifically detected in a short time of 5 to 15 minutes by performing nucleic acid chromatography analysis without any denaturation operation.
FIG. 3- (A) shows MRSA detection results by the nucleic acid chromatography of the present invention. 9 in FIG. 3 is a target nucleic acid coloring line obtained by NASBA amplification using the total RNA extracted from the MRSA culture strain as a template.

Example 2
Using total RNA extracted from magnetic protozoa Cryptosporidium parvum (hereinafter referred to as C. parvum) that causes water-borne infections as a template, adding synthetic RNA as an internal standard nucleic acid during NASBA amplification reaction, competitive amplification is performed, and after completion of amplification The product was immediately detected by nucleic acid chromatography without pretreatment.
(1) Preparation of Latex-Binding Oligonucleotide Probe A carboxyl group-containing polystyrene latex (solid content: 4% (w / w), manufactured by Duke) was used in the same manner as in Example 1. The 3 ′ terminal amino group-containing oligonucleotide probe having the target nucleic acid complementary sequence is SEQ ID NO: 9.
(2) Immobilization of oligonucleotide A 5′-terminal amino group-containing oligonucleotide probe (SEQ ID NO: 8, 10) having a complementary sequence to the target nucleic acid and the internal standard nucleic acid was used in the same manner as in Example 1.
(3) Competitive
Amplification of C. parvum RNA using NASBA method NASBA lyophilized reagent from Kinos was dissolved in 100 μL of a primer solution containing 0.4 μM each of primers P4 (SEQ ID NO: 6) and P5 (SEQ ID NO: 7). 5 μL each was dispensed into a 5 mL Eppendorf tube. After adding 1.25 μL of internal standard RNA equivalent to 100 molecules to all tubes, add 1.25 μL each of water or total RNA derived from 10 oocysts, heat at 65 ° C. for 5 minutes, and then incubate at 41 ° C. for 5 minutes. . After adding 2.5 μL of enzyme solution and tapping lightly, the mixture was reacted at 41 ° C. for 30 minutes.
(4) Detection by nucleic acid chromatography The NASBA product was immediately applied to the sample well on the strip without denaturation treatment, and detection by chromatography was performed. When water was used as a specimen, only the internal standard nucleic acid detection line was detected in blue. On the other hand, in the amplified product of total RNA derived from 10 oocysts, not only the detection line of the internal standard nucleic acid but also the second colored line specific to the target nucleic acid was detected. Immediately after the amplification, the target nucleic acid could be specifically detected in a short time of 5 to 15 minutes by performing nucleic acid chromatography analysis without any denaturation operation. In addition, by confirming the detection of the co-amplified internal standard nucleic acid, it was possible to make a correct determination without any abnormality in the amplification and detection reactions.
FIG. 3- (B) shows the results of C. parvum detection by nucleic acid chromatography of the present invention. 3 in FIG. 3 is a target nucleic acid coloring line obtained by NASBA amplification using total RNA extracted from 10 oocysts of C. parvum as a template. 3 of FIG. 3 is a colored line of NASBA amplification product of 100 molecules of internal standard nucleic acid that is competitively amplified with the target nucleic acid.

Example 3
Synthetic RNA that becomes the internal standard nucleic acid during the NASBA amplification reaction, using total RNA extracted from the peripheral blood nucleated cells of normal human multidrug resistance gene 1 (hereinafter referred to as MDR1), one of the drug resistance factors, by the AGPC method. In addition, competitive amplification was performed, and detection by nucleic acid chromatography was performed immediately without pretreatment of the product after completion of amplification.
(1) Preparation of Latex-Binding Oligonucleotide Probe A carboxyl group-containing polystyrene latex (solid content 4% (w / w), manufactured by Duke) was used in the same manner as in Example 1. A 3 ′ terminal amino group-containing oligonucleotide probe having a target nucleic acid complementary sequence is SEQ ID NO: 14.
(2) Immobilization of oligonucleotides 5′-terminal amino group-containing oligonucleotide probes (SEQ ID NOs: 10 and 13) having complementary sequences to the target nucleic acid and the internal standard nucleic acid were used in the same manner as in Example 1.
(3) Competitive
Amplification of MDR1 RNA using NASBA method NASBA lyophilized reagent from Kinos was dissolved in 100 μL of a primer solution containing 0.4 μM of each of primers P6 (SEQ ID NO: 11) and P7 (SEQ ID NO: 12), and 0.5 mL Eppendorf was added. Dispense 5 μL each into a tube. After adding 1.25 μL of 10000 molecules of internal standard RNA to all tubes, add 1.25 μL of water or RNA corresponding to 0.1 μg of total RNA derived from peripheral blood nucleated cells of healthy humans, and heat at 65 ° C. for 5 minutes. Then, it was kept at 41 ° C. for 5 minutes. After adding 2.5 μL of enzyme solution and tapping lightly, the mixture was reacted at 41 ° C. for 30 minutes.
(4) Detection by nucleic acid chromatography The NASBA product was immediately applied to the sample well on the strip without denaturation treatment, and detection by chromatography was performed. A specific colored line was shown for each of the same target and internal standard RNA as in Example 2.

The nucleic acid detection or quantification method of the present invention is a method of detecting a target nucleic acid in a sample by sandwich hybridization method by chromatography, and simultaneously detecting two or more kinds of nucleic acids at the same time, thereby amplifying and detecting. The main feature is that the correctness of the reaction can be determined and quantified in one test. Therefore, according to the present invention, a sample or reagent containing a single-stranded nucleic acid amplified from a target nucleic acid on the porous membrane used in the present invention without performing a complicated and time-consuming operation of the sandwich hybridization method. Can be detected simply or rapidly and specifically to detect or quantify the presence of the target nucleic acid in the sample. Further, the target nucleic acid amount can be visually determined based on the contrast with the color development amount of the internal standard nucleic acid that can be co-amplified as a control.
In addition, by providing an amplification primer, a detection probe, a colored polymer carrier, a porous membrane, and a water-absorbing substrate for development as a kit, nucleic acid can be easily detected and quantified without requiring a high level technique.

It is a schematic diagram which shows the outline of the structural example of the detection apparatus by this invention. It is a schematic diagram which shows the example of determination at the time of carrying out quantitative detection of the target nucleic acid by this invention. It is the figure which showed the specific detection of the nucleic acid amplification product by the nucleic acid chromatography of an Example using the detection apparatus of this invention. (A) MRSA detection as sample: amplification and detection of total RNA derived from cultured strain, (B) detection of C. parvum: amplification and detection of total RNA derived from 10 oocysts and 100 molecules of internal standard nucleic acid It is.

Explanation of symbols

1 Target nucleic acid detection part (capture oligonucleotide binding part)
2 Internal standard nucleic acid detection part (capture oligonucleotide binding part)
3 Membrane 4 Absorption pad 5 Latex-bonded oligonucleotide probe coating pad 6 Deployment pad

Claims (8)

A method for specifically detecting or quantifying a target nucleic acid in a sample as a single-stranded nucleic acid using a primer not bound to a hapten or peptide from a target nucleic acid arbitrarily extracted from the sample Amplifying , hybridizing the amplified product with a first oligonucleotide probe complementary to the amplified product bound to the membrane and a complementary second oligonucleotide probe labeled with a colored polymer carrier, and detecting A method for detecting or quantifying a nucleic acid comprising a step of evaluating the detected image by visual judgment. The method of claim 1, wherein the amplification step involves amplification of a target nucleic acid and an internal standard nucleic acid that can be competitively co-amplified with the target nucleic acid.   The method according to claim 1 or 2, wherein the amplification is performed by NASBA or TMA.   The method according to claim 1, wherein an assay device based on nucleic acid chromatography is used. In order to determine the correctness of the nucleic acid amplification reaction or the false negative of the nucleic acid extraction operation as well as whether the sample and the colored polymer carrier-labeled oligonucleotide probe are developed correctly or not, a single-stranded nucleic acid that is competitively amplified is used as an internal standard nucleic acid. The method according to any one of claims 1 to 4, wherein the method is detected. 6. The target nucleic acid amount in a sample is visually determined from a contrast of a color development amount with a target nucleic acid and an amplification product derived from a known amount of an internal standard nucleic acid competitively amplified. A method for quantifying a nucleic acid according to item 1.   The nucleic acid detection according to any one of claims 1 to 6, wherein two or more different target nucleic acids present in a sample are simultaneously detected or quantified by combining a plurality of colored polymer carriers and capture polynucleotides. Quantitation method. An amplification primer not bound to a hapten or peptide useful for performing the method of any one of claims 1 to 7, a detection probe, a colored polymer carrier, a porous membrane, and a water-absorbing substrate for development. Target nucleic acid detection kit.

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