JPH07163399A - Method for determining nucleic acid by pcr, method for measuring number of microorganism or cell, number of specific gene and number of specific gene copy by pcr and measuring kit used for these methods - Google Patents

Abstract

PURPOSE:To simply and accurately determine a nucleic acid useful for measuring the number of microorganisms by reacting a reaction solution of PCR with a coloring matter compound which is not fluorescent in liberation and is fluorescent by reaction with a duplex nucleic acid and measuring the intensity of the obtained fluorescence. CONSTITUTION:A nucleic acid specimen is subjected to PCR in the presence of a primer required for amplification of a specific sequence region of the target nucleic acid. In the case of the target nucleic acid existing in the specimen, an amplification product comprising the amplified duplex is reacted with a coloring matter compound of formula I [a group of formula II (X is O, S, Se or Te) is pyrylium (analog) ring; R<1> and R<2> are H, a halogen, a (substituted) lower alkyl, etc.; R<3> is a (substituted) aryl, etc.] such as 2,4-bis(N,N-dimethylaminophenyl)-6-methylpyrylium salt which is not fluorescent in liberation and is fluorescent by reaction with a duplex nucleic acid to give fluorescence. The intensity of the fluorescence is measured and the amount of the target nucleic acid in the nucleic acid specimen is obtained to determine the nucleic acid.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for quantifying a nucleic acid, a method for quantifying a nucleic acid, wherein a PCR amplification product is detected with a dye compound which does not emit fluorescence when released and reacts with a double-stranded nucleic acid to emit fluorescence. The present invention relates to a method for measuring the number of cells, a specific gene number and a specific gene copy number, and a measurement kit used for these methods. This measuring method can be used for measuring the number of bacteria to be measured in various solutions or soil.

[0002]

2. Description of the Related Art The PCR method is a method for enzymatically amplifying a specific sequence defined by two kinds of primers as a template (template), and has also come to be used for detecting nucleic acids. Is coming. That is, by selecting a specific sequence in the target nucleic acid as a detection target, preparing a primer necessary for amplification of this specific sequence, performing PCR using the target nucleic acid as a template, and detecting the amplified specific sequence, the target Nucleic acid can be detected. PC like this
In the method for detecting a nucleic acid using R, a specific sequence that is a characteristic part of a detection target is amplified and detected, and since the amplification rate is also large, even if the amount of the target nucleic acid in the sample is very small, It becomes possible to detect. For example, it is possible to obtain an amplification factor of about 1,000,000 times in a reaction of several hours,
If one molecule of nucleic acid is present, it may be possible to detect this nucleic acid by using the PCR method. This amplification reaction proceeds only when there is a complementary sequence between the template nucleic acid and the two kinds of primers, and an amplification product cannot be obtained when there is no complementary sequence.

The use of the PCR method has dramatically improved the detection sensitivity of nucleic acids, and has recently come to be used for the detection of nucleic acids in various fields in place of the nucleic acid detection method using the hybridization method. In particular, it has been widely used for identification of pathogens in the field of clinical examination of infectious diseases caused by viruses, bacteria, etc., analysis of genes in genetic diseases, detection of genetic markers in diagnosis of cancer, etc.

Another application of the PCR method is MPN (M
ost Probable Number) method (H.
O. Halvorson and N.M. R. Ziegl
er, J.I. Bacteriol. , 25 , 101 (19
33)) and the measurement of the number of specific bacteria. The MPN method, also called the most probable value method or the dilution frequency method, inoculates a large number of test dilutions of several stages into a large number of test tubes in a fixed amount, and after culturing for a sufficient period, the presence of bacterial growth Is a method of determining the number of bacteria and measuring the number of bacteria by statistical processing (“Soil Microbial Experiment Method”, Yokendo, No. 45).
page). This method requires a lot of labor and laboratory equipment,
In particular, bacteria such as soil microorganisms have a problem of long culture time. Furthermore, in this MPN method, since the total number of bacteria of all the bacteria that grow in the medium used is measured, it is impossible to measure the number of specific bacteria when a plurality of bacteria are mixed in the sample. Therefore, DNA is used for detection as a means for detecting microorganisms without culturing D
Attempts have been made to increase it at the NA level. As a means therefor, a method in which the MPN method and the PCR method are combined is considered. The method in which this PCR method is combined with the MPN method is to extract DNA from a sample containing the measurement target bacterium, dilute the sample in several steps, and amplify the nucleic acid sequence specific to the measurement target bacterium. This is a method of performing PCR using the two types of primers described above, detecting the obtained amplification product, and determining the number of bacteria to be measured from the amount.

That is, in the PCR method, a picogram amount of D
Microgram quantities of the target DNA can be obtained from the NA material in a short time. Therefore, the cloned DN was cloned using the PCR method.
A. A large amount of genomic DNA can be rapidly amplified in vivo , and has been applied to PCR cloning and various detections. In the PCR method, a target double-stranded DNA is heat-denatured to be a single strand, and then each single strand serving as a template is annealed with a primer, and a complementary position is obtained using a DNA polymerase starting from the binding position of the primer. Synthesize chains. Therefore, in principle, only one template DNA needs to be present. Genome DN as template DNA
Regarding the case of handling A, the length of even one template DNA greatly differs depending on the type of living body to be detected. For example, human genomic DNA is 10 ³ times as long as E. coli. In other words, the same picogram amount of Genome D
Even though it is NA, it can be said that the number of DNAs contained in it varies depending on the species.

[0006] Therefore, such a quantitative problem is solved by DNA
MPN-P is a method of capturing and detecting as a problem of the number of
The CR method. In this method, the template DNA is serially diluted, and finally diluted to the presence or absence of one in each reaction system, and then amplified by PCR to detect this,
This is a method of deterministically determining the number of template DNAs in a sample from the results. Therefore, this MPN-PCR method
It uses the principle of the MPN method for measuring the number of bacteria.
In the PN method, the bacterium is used as a template DNA, or the bacterium is propagated by P
It can be said that it is replaced with CR. That is, MP
In the N method, a sample solution containing a bacterium is diluted stepwise until one bacterium exists or not, and then this is proliferated and detected by culturing, and from the obtained results, the sample solution is definitively established. The MPN-PCR method replaces the bacteria in this process with the template DNA and the culture with PCR. Therefore, MPN-P
It is not the amount of template DNA that matters in the CR method,
The number of molecules (number).

An example of a concrete operation of the conventional MPN-PCR method will be described below. First, a sample DNA extracted from a sample containing microorganisms is diluted stepwise by 10 times (for example, a dilution ratio of 1, 10 ^-1 , 10 ^-2 ...
・ Prepare 10 ^-9 ). The dilution rate of 10 ⁻ⁿ means that the sample concentration is diluted to 1/10 ⁿ . PCR was performed for each of the diluted solutions (10 in total),
Amplification of the template DNA as a detection target is performed. After completion of PCR, each reaction solution is subjected to agarose gel electrophoresis, and the amplification product is detected as the presence or absence of a band. At this time, no band of the amplification product can be seen at a certain dilution ratio. For example, in the case until the sample subjected to 10 ^-5 dilution amplification product was observed, and this dilution rate, sample dilutions before and after, i.e. ^{10-4, 10-5,} dilution of 10 ^-6 PCR is performed again on each of the 5 samples (15 samples in total) of the diluted solution of 1. and the presence or absence of the amplification product is examined again by agarose gel electrophoresis. The results, the number of samples amplification product was observed in five samples at each dilution, MPN table to post later (J. Bacteriol., 25, 10
1 (1933), page 400) to determine the number that is deterministically obtained in advance. For example, at 10 ^-4 dilution all samples are positive out of 5 samples (amplification products are detected) At 10 ^-5 dilution 3 out of 5 samples are positive 10
^{At -6} dilution, when 1 out of 5 samples is positive, the numerical value (1.1) is obtained when P ₁ , P ₂ and P _{3 in the} MPN table are 5, ₃ and ₁ , respectively. A value obtained by multiplying this value by the reciprocal of 10 ^−5, which is the limit of the dilution rate at which the amplification product was recognized in the first PCR, is the number of template DNAs in the sample DNA (1.
1 × 10 ⁵ ).

If one target DNA exists for each individual such as a microorganism, the MPN-
The number of template DNAs obtained by the PCR method is the number of individuals such as microorganisms contained in the sample used for preparing the sample DNA.
When a genomic gene or the like is used as a target gene, some copies may exist depending on the gene. In the case of one copy, the number of DNAs directly becomes the number of individuals as described above, but when there are a plurality of copies, it is necessary to determine the correlation with the number of individuals. Furthermore, since the extraction efficiency of DNA is also involved, it is desirable to create a calibration curve when obtaining an accurate value. This method can also be used for quantification when only a specific gene in a cell is amplified and there are multiple copies, such as cancer cells.

[0009]

The detection of the amplification product obtained by the PCR method is performed by, for example, developing the PCR reaction mixture by gel electrophoresis to detect the amplification product and non-detection target components such as template nucleic acid and primers. In the separated state, the band of the amplification product has been identified by the molecular weight or the like of the amplification product by fluorescent staining, and then the fluorescence intensity of the band has been measured.

However, in the detection of nucleic acid using the PCR method, the reaction solution contains a template nucleic acid and a large excess of primers, etc., and depending on the type of the nucleic acid sequence to be amplified, the non-detection target component and the amplification target component are amplified. In many cases, the separation operation by gel electrophoresis with the product is difficult. In addition, when the number of samples increases, complicated gel electrophoresis requires a great deal of labor and time, which is an obstacle to efficient detection operation. Particularly, in gene analysis in clinical practice, it is required that many specimens can be efficiently processed in a shorter time, and conventional methods have not been able to sufficiently meet such requirements.

On the other hand, in the application of the PCR method to the MPN method, in order to estimate the number of bacteria more accurately, firstly, usually 1
A method has been adopted in which an experiment is performed with 0-step dilution, and a re-experiment is performed with a 3-step dilution series that includes the observed dilution of the amplification product. Furthermore, since this method is a method for obtaining the number of bacteria based on probability theory, the number of samples required for stochastic determination of the number of bacteria is required. In contrast, a minimum of 5-10 sample reactions is usually performed. As a result, this method requires a laborious task of performing at least about 25 samples per sample by PCR and then evaluating by gel electrophoresis, and requires a long time to obtain the results. Is required.

[0012] As described above, in the detection of nucleic acids and the measurement of the number of bacteria using the PCR method, it is often the case that complicated and time-consuming work is required for the detection and quantification of the amplification product after the amplification reaction by PCR. This has been an obstacle to efficient processing of multiple samples. Therefore, in the detection or quantification method using PCR, it has been strongly desired to establish a method capable of simply and accurately detecting a PCR amplification product.

The most complicated task in detecting PCR amplification products is the separation by gel electrophoresis of the amplification products and the primers added in the reaction solution in large excess. Various methods have been studied as methods for omitting this separation work. For example, a method using a so-called fluorescent intercalator, which shows a fluorescence increase when reacted with a double-stranded nucleic acid, has been attracting attention. The method using this fluorescent intercalator is a method of reacting a double-stranded nucleic acid amplified by PCR with a fluorescent intercalator, measuring the increase in fluorescence intensity generated at that time, and detecting an amplification product. . In this method, theoretically, even if the intercalator reacts with the primer (single strand), fluorescence does not increase, so that there is an advantage that a complicated separation operation between the amplification product and the primer can be omitted.

For example, JP-A-5-237000 discloses ethidium bromide, acridine orange, bisbentimide, diaminophenyl indole, actinomycin, thiazole range, chromomycin, and their known fluorescent intercalators. A method of using a dye such as a derivative for detecting a PCR amplification product is disclosed. Among these dyes, the increase in fluorescence during the reaction with the double-stranded nucleic acid is about 50 times when excited by ultraviolet light and about 20 times when excited by visible light, compared with the free state (the state in which the double-stranded nucleic acid does not react). Relatively large ethidium bromide may be mentioned as preferred.

However, this Japanese Laid-Open Patent Publication No. 5-23700.
Each of the dyes listed in Japanese Patent Publication No. 0-0. 0 0 0 0 0 0 0 emits fluorescence even when it is released, and the measured fluorescence intensity also includes the fluorescence intensity of the double-stranded nucleic acid and the unreacted dye. It is not the fluorescence intensity itself derived from the reaction between the nucleic acid and the dye. Therefore, it is necessary to subtract the fluorescence intensity derived from the unreacted dye from the measured value as a blank. That is, with this method, it is not possible to simply determine the presence or absence and the amount of the amplification product from the fluorescence intensity measurement result, and an operation of subtracting the blank value is required. In addition, if the amount of the amplification product produced is very small, there may be a case where a sufficient difference from the blank value does not occur and detection with high sensitivity may not be possible.

YOYO-1 (Nucleic Acid)
s Research, 20 (11), 2803-28.
No. 12 (1992) is non-fluorescent when released, and causes an increase in fluorescence (about 3000 times) when it is inserted into a double-stranded nucleic acid. If this can be used, the problem of high blank in the above measurement can be solved. However, this dye is not practical because it easily decomposes at room temperature. As will be described later, in the detection of PCR amplification products with this dye, higher-order structures generated by the reaction between the primers are also detected,
There is a drawback that an accurate quantification cannot be performed.

From this point of view, the above-mentioned Japanese Patent Laid-Open No. 5-2 is used.
In Japanese Patent No. 37000, P of a target nucleic acid to be detected is
In order to quantify the initial concentration before the CR reaction, amplification is performed at the number of cycles at which sufficient fluorescence intensity is obtained with respect to the blank value. That is, when the initial concentration of the target nucleic acid is low, the cycle number becomes large, and when the initial concentration of the target nucleic acid is high, the cycle number becomes small. Therefore,
In the method for quantifying the target nucleic acid in the patent publication, PCR is used.
The change in fluorescence intensity during the reaction is monitored, and the initial concentration of the target nucleic acid is determined from the number of cycles at the time when the fluorescence intensity changes rapidly.

However, such a quantitative method is
There is a problem that a complicated operation of monitoring the fluorescence intensity in each cycle of CR reaction is required. In addition, it may be difficult to judge the point where the fluorescence intensity changes,
It can be said that there is still a problem to be improved in terms of efficiently quantifying a sensitive target nucleic acid.

Further, in the detection of the PCR amplification product with a dye such as ethidium bromide, theoretically, the primer and the dye do not react with each other and the fluorescence does not increase.
It should be possible to detect the amplification product even when the amplification product and the primer are mixed. However, according to the studies by the inventors of the present invention, a reaction between the primers occurs during PCR to generate a mass of nucleic acid (a higher-order structure having a three-dimensional structure), which is also caused by ethidium bromide. It turned out to be detected. Therefore, if the amount of the amplification product is overwhelmingly large with respect to the higher-order structure, there is no problem, but if not, the ratio of the fluorescence increase due to the higher-order structure to the actually measured fluorescence increase is large. Therefore, it becomes impossible to perform accurate quantification. Moreover, this amount is not always constant and decreases when the amount of the amplification product is overwhelmingly large, but tends to occur in large amounts when the amount of the amplification product is small or the amplification product does not occur. However, it cannot be easily corrected by comparison with a blank or the like.

The presence of this higher-order structure is such that, for example, when a reaction mixture obtained by carrying out a PCR reaction in a system in which no template nucleic acid is added is developed by gel electrophoresis, it is clouded with ethidium bromide in the low molecular weight region. This can be confirmed by the fact that the portion stained in the pattern appears.

The problem of measurement error due to this higher-order structure is a problem caused by the PCR reaction itself, and tends to become more prominent as the number of amplification cycles increases when the initial concentration of the target nucleic acid is low. There is.

Therefore, the above-mentioned conventional method for quantifying an amplification product is still insufficient for more sensitive quantification.

Further, also in the method for measuring the number of bacteria in which MPN method is combined with PCR, the formation of higher-order structures due to the reaction between the primers as described above is unavoidable at the stage of performing PCR, resulting in a large measurement error. The same is true.

An object of the present invention is to provide a method for detecting a nucleic acid which simplifies the process of detecting a PCR amplification product and enables accurate quantification. Another object of the present invention is to detect the number of microorganisms or cells to be detected using a simple and accurate method for detecting a PCR amplification product, the number of specific genes contained therein,
Alternatively, it is to provide a method for measuring the copy number of a specific gene. A further object of the present invention is to provide a measuring kit used in these methods.

[0025]

The above object can be achieved by the present invention described below. The nucleic acid quantification method of the present invention is a method in which PCR is performed on a nucleic acid sample in the presence of a primer necessary for amplification of a specific sequence region of a target nucleic acid, and two nucleic acids are amplified when the target nucleic acid is present in the sample. A target nucleic acid in the nucleic acid sample is obtained by measuring the intensity of fluorescence obtained by reacting a dye compound that does not emit fluorescence when released with a double-stranded nucleic acid It is characterized by determining the amount of. A kit that can be used in this method for quantifying nucleic acids has a reactor in which a necessary amount of a dye compound that does not emit fluorescence when released and that emits fluorescence by reacting with a double-stranded nucleic acid is arranged in a reaction region for PCR. It is characterized by Further, in this kit, a necessary amount of primers necessary for PCR amplification of a specific sequence region of a target nucleic acid may be arranged in the reaction region. In addition, this kit for nucleic acid measurement has a reaction region for PCR and a reagent region isolated from the reaction region, and does not emit fluorescence when released into the reagent region and reacts with a double-stranded nucleic acid. It may be characterized in that a necessary amount of a dye compound that emits fluorescence is arranged, and the dye compound in the reagent region is provided so as to be added in the reaction region, and in this case also, the reaction region It may be configured such that a necessary amount of a primer necessary for PCR amplification of a specific sequence region of the target nucleic acid is further arranged therein.

On the other hand, the method of measuring the number of microorganisms or cells, the number of specific genes, or the copy number of specific genes of the present invention was obtained by the process of extracting nucleic acid from a sample containing the microorganisms or cells to be detected. Process for preparing serially diluted samples of nucleic acid extract and PC for amplifying sequences specific to the microorganism or cell to be detected for each diluted sample
Fluorescence obtained by reacting a process of performing R and an amplification product composed of the double-stranded nucleic acid obtained by the PCR with a dye compound that does not emit fluorescence when released and that emits fluorescence by reacting with the double-stranded nucleic acid Is measured and the fluorescence is obtained, and the number of microorganisms or cells to be detected, the number of specific genes, or the copy number of specific genes is determined from the dilution ratio of the diluted sample. A kit that can be used for this measurement method is a reactor in which a necessary amount of a dye compound that emits fluorescence by reacting with a double-stranded nucleic acid is arranged in each reaction area of many PCR reaction areas And using these reaction regions, it is possible to form a serial dilution of a nucleic acid extract extracted from a sample containing a microorganism or cell to be detected, and at each dilution, a sequence unique to the microorganism or cell to be detected is formed. It is characterized in that PCR for amplification can be performed. In each reaction region of this kit, a primer for PCR amplification of a sequence unique to the microorganism or cell to be detected may be further arranged. These inventions are
As described later, it can also be applied to the MPN-PCR method.

The dye compound used in the present invention does not emit fluorescence when released, but emits fluorescence when it reacts with a double-stranded nucleic acid. The reaction of the dye compound with the double-stranded nucleic acid means that the dye compound is specifically inserted and bound in the groove or the like of the stable double-helical structure of the double-stranded nucleic acid. The compound becomes fluorescent when irradiated with excitation light. Therefore, when it is free, that is, when it exists alone, it does not emit fluorescence even when irradiated with excitation light.

This dye compound does not easily enter a short double-stranded portion that cannot form a stable double-helical structure formed between primers, and even if it enters, fluorescence is generated except for a stable double-helical structure. It does not emit. Therefore, as this dye compound, a compound that specifically enters into the main groove or the sub groove of the double helix structure and binds to it and emits fluorescence in that state can be used. By using the dye compound having such characteristics, the amplification product can be added to the PCR reaction solution directly (that is, without performing the operation of separating the amplification product from the primer or the template nucleic acid (template)). Only can be accurately detected. The fluorescence of the dye compound in the free state may be so low that it can be ignored.

Examples of the dye compound include DAPI
(4 ', 6-Diamino-2-Phenylind
ole Dihydrochloride), Hoec
hst33258 (trade name), Hoechst3334
2 and pyrylium compound salts represented by the following general formula [I] or pyrylium analog compound salts, and the like.

[0030]

[Chemical 21] In the above general formula [I],

[0031]

[Chemical formula 22] Represents a heterocycle, and X is O, S, Se or Te. Examples of the heterocycle include 5- and 6-membered rings such as a pyrylium ring and a pyrylium-like ring.

R ¹ and R ² are each independently a hydrogen atom, halogen atom, sulfonate group, amino group, styryl group, nitro group, hydroxyl group, carboxyl group, cyano group, substituted or unsubstituted lower alkyl group, substituted Alternatively, it represents an unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group, or a substituted or unsubstituted cycloalkyl group.

R³Is -A or -LA. L
Is -L¹-, -L²-L³-Or-L ^Four-L^Five-L⁶-In
Yes, L¹~ L⁶Are each independently-(CH = CH)
-, Divalent derived from a substituted or unsubstituted aryl group
Group, a substituted or unsubstituted lower alkylene group or -C
H = R^Four-(R^FourRepresents a ring structure having an oxo group)
Forget 2 derived from a substituted or unsubstituted aryl group
Examples of the valent group include a phenylene group and the like.
Yes, it can be attached at any of the ortho, meta, or para positions
May be The lower alkylene group has a carbon number of 1 to
To list 4 straight or branched alkylene groups
And a substituent thereof is, for example, represented by -LA.
Groups may be mentioned. Ring structure with oxo group
A heterocyclic ring, an aromatic ring, an aliphatic ring, etc.
Examples thereof include those having a so group.

As -L-, the following general formula [II],
The groups represented by [III], [IV], [V] or [VI] can be mentioned as preferable ones.

[0035]

[Chemical formula 23] (In the above general formula [II], Z represents a hydrogen atom or a substituted or unsubstituted lower alkyl group, and n is 0, 1 or 2
Is. In addition, as the substituent when Z is an alkyl group, the group defined by the above-mentioned -LA can be mentioned, for example.

[0036]

[Chemical formula 24] (In the above general formula [III], n is 0, 1 or 2, and Φ represents a substituted or unsubstituted o-, m- or p-phenylene group.)

[0037]

[Chemical 25] (In the above general formula [IV], Φ is a substituted or unsubstituted o
Represents a-, m- or p-phenylene group. )

[0038]

[Chemical formula 26]

[0039]

[Chemical 27] As the substituent of the phenylene group in the above general formula, those exemplified above can be mentioned.

A in R ³ of the general formula [I] is a substituted or unsubstituted aryl group, —CH═R ⁵ (R ⁵ is a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group or a substituted or unsubstituted cycloalkyl group Represents an unsubstituted aromatic ring). As the heterocycle of R ⁵ ,

[0041]

[Chemical 28] (M and N each independently represent an oxygen atom, a sulfur atom or a nitrogen atom, and Y ⁻ represents an anion) and the like.
For example, a substituted or unsubstituted aryl group etc. can be mentioned. The substituted or unsubstituted cycloalkyl group may be saturated or unsaturated, for example,

[0042]

[Chemical 29] Groups derived from those capable of forming a resonance system such as Moreover, an azulene ring can be mentioned as a substituted or unsubstituted aromatic ring. Examples of the substituent of these groups include a lower alkyl group and a substituted or unsubstituted aryl group.

The hydrogen atom bonded to the carbon atom to which R ¹ , R ² and R ^{3 of the} pyrylium ring containing X or its analogous ring is not bonded is a halogen atom, a sulfonate group, an amino group, a styryl group or a nitro group. It may be substituted with a group, a hydrosil group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group or a substituted or unsubstituted lower aralkyl group.

Y ⁻ represents an anion, and examples of the anion include BF ₄ ⁻ , perchlorate ion, HO ₃ SCH ₂ CO
O ⁻ , or halogen ion such as chlorine ion, bromine ion, iodine ion, or fluorine ion, or a compound having an anion function such as aliphatic hydrocarbon or aromatic sulfonate, and further Zn, Ni, Cu, Pt , C
Examples thereof include complex ions of transition metals such as o and Pd.

When the group further substituted by each of the above-mentioned various substituents is a halogen atom, examples of the halogen atom include Cl, Br and I. The lower alkyl group may be linear or branched, and the carbon number thereof is preferably about 1 to 4. Examples of the aryl group include a phenyl group and the like. Examples of the substituent of the aryl group and the phenylene group include an amino group substituted with a lower alkyl group (lower alkylamino group). The lower alkylamino group is preferably one in which a dimethylamino group, a diethylamino group or the like is substituted at the para position.
Examples of the lower aralkyl group include a lower alkyl group substituted with the above substituted or unsubstituted aryl group.

Among the compounds of the general formula [I], those in which the heterocycle containing X is substituted with two or more substituted or unsubstituted aryl groups are preferable. For example, when the heterocycle containing X is a 6-membered ring, such compounds include (1) the 2-position and 4-position of the 6-membered ring containing X are substituted with a substituted or unsubstituted aryl group, and the 3-position, 5 that one of the position and 6-position is substituted by R ^3, (2) 3-position and 5-position of the 6-membered ring is substituted with a substituted or unsubstituted aryl group, 2-position, 4-position及6-position of One of which is substituted with R ³ , (3) the 2- and 6-positions of the 6-membered ring are substituted with a substituted or unsubstituted aryl group, and any of the 3-, 4- and 5-positions is R ³ And the like, and the like. Introducing a substituted or unsubstituted aryl group at such a position is
It is preferable for obtaining good properties as an intercalator for nucleic acid base pairs. Furthermore, it is more preferable that the heterocycle containing X is substituted with two or more of the substituted or unsubstituted aryl groups so that these substitution positions are not adjacent to each other.

Specific examples of the compound represented by the general formula [I] include:
For example, those shown in Table 1 below can be mentioned. Furthermore, since the intensity of fluorescence emitted when it is inserted into a double-stranded nucleic acid is extremely high, a particularly preferable compound is the following general formula [VII]

[0048]

[Chemical 30] (In the general formula [VII], X represents O or S,
Y ^- is represented by an anion) 2,4-bis (N, N-dimethylaminophenyl) -6-methyl pyrylium salt or 2,4-bis, (N, N-dimethylaminophenyl) -6- Mention may be made of methylthiopyrylium salts.

Since the dye compound used in the present invention specifically emits fluorescence by reacting with a stable double-helical structure, it has a higher-order structure caused by the reaction between the primers as described above. Even if it is taken in by something
Since the double-stranded portion of this higher-order structure does not have a stable double-helical structure, it does not emit fluorescence.

Further, the compound represented by the general formula [I] is
Since it is specifically inserted into the double helix structure at a ratio of 1 to 20 to 30 base pairs, it has the advantage of being less likely to be incorporated into the higher-order structure derived from the primer as described above, and further has a sequence to be amplified. Even when incorporated into a double-stranded nucleic acid fragment having a double-helix structure having, the incorporation density is significantly lower than that of ethidium bromide and the melting temperature of the double-stranded nucleic acid due to incorporation of the compound of general formula [I] ( The increase in Tm) is very small and has little effect on PCR. Also,
Since the uptake density is low, even when the reaction solution is developed by gel electrophoresis after PCR to confirm the length of the amplification product, the uptake of the compound has almost no effect on the mobility of the double-stranded nucleic acid. , Accurate measurement is possible.

On the other hand, conventional fluorescent intercalators such as ethidium bromide, acridine orange and YOYO-1 are inserted into a double-stranded nucleic acid at a ratio of one to several base pairs (about 2 to 5 base pairs). Therefore, when these dyes are used, they are incorporated into the double-stranded portion of the higher-order structure formed from the primer as described above to cause an undesired increase in fluorescence. Even when incorporated into a double-stranded nucleic acid, it has a great influence on the Tm of the double-stranded nucleic acid and the mobility of gel electrophoresis.

In order to make effective use of the above-mentioned advantages of the dye compound used in the present invention and perform more accurate quantification,
It is preferable that the amplification product form a stable double helix structure. Therefore, in the present invention, for example, the amplification product length is 10
It can be preferably used when the number of base pairs is 0 or more, and is 300
More preferably, it is about base pairs. The upper limit of the amplification product is not particularly limited, but the rate at which nucleotides are incorporated by PCR is said to be 35 to 100 nucleotides per second when the reaction is performed at 72 ° C, for example. These depend on the reaction conditions such as the pH of the reaction, the salt concentration, and what kind of base sequence the target nucleic acid has. Therefore, when the reaction time of each cycle in PCR is 1 minute, 2000 base pairs or less is preferable. In addition, if the amplification product is too long, byproducts are likely to occur, so 10
00 base pairs or less is more preferable ("PCR Proto
cols ”, Edited by Michael
A. Innis, David H. Gelfand,
John J. Sninsky and Thomas
sJ. White, 1990 by Acade
mic Press, Inc. (San Diego
o, Calfornia 92101)).

As the primer used for PCR, one that can define a specific sequence region to be amplified in the target nucleic acid is used as desired. The length is required to have a length that is necessary for recognizing a specific sequence at the end of a specific sequence region as an amplification target in a template nucleic acid, but is not unnecessarily long. I won't. This is because if the length is too long, the double-stranded portion is likely to be partially formed between the primers. Considering these points, the length of the primer is about 30 bases or less,
It is preferably about 28 bases or less. The lower limit of the primer length is not particularly limited, but is set to a length that allows recognition of the end of the specific sequence region as an amplification target, for example, 14
The number of base pairs is 18 or more, preferably 18 or more.

In the nucleic acid quantification method of the present invention, first, the sequence region characteristic of the target nucleic acid to be quantified is specified.
Next, PCR is carried out using a primer required for amplification of the specified sequence region (specific sequence region) and a target nucleic acid used as a template, and the obtained amplification product has a dye compound having the characteristics described above. Are reacted and the intensity of fluorescence generated upon irradiation with excitation light is measured. The fluorescence intensity obtained at this time is proportional to the initial concentration of the template (concentration before PCR) if the concentration of each reaction component is set in an appropriate range. It is possible to quantify the target nucleic acid in the unknown sample by performing the above-mentioned PCR and fluorescence intensity measurement on the unknown sample.

In the reaction system in which the optimum conditions have already been determined, the quantification using the calibration curve as described above is simple, but the reaction conditions, especially the amount of target nucleic acid, the amount of primer, the reaction temperature, etc. If the system is different and the setting of the reaction conditions for creating the calibration curve is unknown, the complicated work of finding the conditions for writing the calibration curve is required.

The conditions for writing such a calibration curve have not yet been set.
However, the nucleic acid of unknown samples is gradually diluted
The target nucleic acid fragment is detected by PCR followed by PCR.
By using the MPN-PCR method to obtain the
The approximate amount of acid can be easily grabbed, which allows
It is possible to easily obtain the line creation conditions. For example,
In the specific example described in the section of the prior art, the mold D in the sample is
NA number is 1.1 × 10 ^FiveIt was an individual. At this time, 10^{ー 5}Rare
Although 3 out of 5 samples were positive in the
10^-4If the concentration is higher than the dilution, all 5 samples are positive.
Should be sex. In the example in this prior art section,
Dilution of template DNA depending on the presence or absence of amplification products in the first experiment
Limit the concentration area that includes the limit, and
In addition, the study by the MPN method was added in detail. this
Is the presence or absence of a band in agarose gel electrophoresis.
Can only detect ON / OFF, or DNA
Quantitative measurement with fluorescence
Because I can't get it. On the other hand, the MPN described later
According to the method of the present invention using the PCR method, for example,
An instrument that can set multiple reaction areas such as black plate
For amplification product formation for all dilution series
It becomes possible. For example, in the method of the present invention,
The sample of the specific example described in
10 when processed above^{ー 5}Positive and negative wells at dilution
There are 10 wells^-4When the concentration is higher than the dilution
Between samples (samples with the same dilution ratio) at each dilution
The same level of amplification is performed in
Therefore, the same level of fluorescence should be obtained. Where each
In 5 samples of the same dilution ratio in the dilution ratio
Obtain the average value of the fluorescence intensity, and use the dilution factor on the horizontal axis to determine the fluorescence intensity.
When plotted on the vertical axis, the value obtained is on a straight line. That
The longer the linear region, the better the PCR conditions.
If the deviation is significant, the primer amount etc.
It means that it needs to be reviewed.

Here, if a linear relationship is obtained between the fluorescence intensity and the dilution rate, the number of template DNAs has already been determined in this MPN-PCR method, and this is used as a calibration curve for fluorescence. From the strength, the number and amount of template DNA (number × molecular weight) can be quantified.

As described above, according to the method of the present invention, the fluorescence obtained only by the reaction between the amplification product and the dye compound by adding the dye compound to the reaction solution is directly measured. That is, it is not necessary to separate the amplification product from the primer or template after PCR, and even if a higher-order structure is formed by the reaction between the primers, the influence on the measurement result due to it can be avoided. Therefore, the fluorescence obtained by adding the dye compound to the reaction solution can be directly used for the calculation of the quantitative value, and the accurate quantitative determination can be performed. For ethidium bromide, which emits fluorescence even in a free state, the quantitative value is obtained based on the value obtained by subtracting the blank from the measured value of the fluorescence intensity. However, the blank value often changes depending on the measurement condition, and thus a complicated operation of setting the quantitative operation condition in consideration of the relative relationship of the blank value to the actual measurement value for each measurement condition is required. On the other hand, in the method of the present invention, since the dye compound does not fluoresce in the free state or emits negligible negligible fluorescence, it is necessary to consider a blank as in ethidium bromide. Instead, the complicated work of setting the quantitative operation conditions in consideration of the blank value becomes unnecessary. Further, it is not necessary to monitor the fluorescence intensity in the PCR process as in JP-A-5-237000.

Furthermore, in the present invention, if a microplate is used for detection, the amount of the reagent can be reduced by transferring the reaction solution from the reaction container after PCR to the microplate and measuring the fluorescence with the fluorescence detector for the microplate. Also, it is convenient because the measurement time is short. If the PCR reaction itself is performed on a microplate, a simpler quantitative operation can be performed.

Therefore, for example, the required amount of the dye compound used in the present invention is previously placed in each reaction region of a reactor having a large number of wells as reaction regions such as a microplate by a method such as evaporation and drying. The product can be used as a kit for quantifying nucleic acid. This constitution is possible because the dye compound is stable even at room temperature.

Further, a reagent region isolated from the reaction region for PCR is provided, and the dye compound used in the present invention is arranged in this reagent region so that it can be added to the reaction region when performing PCR. Good. When the dye compound is arranged in the reagent area, it can be arranged as a solution. When this compound is water-soluble, it may be placed by dissolving it in a suitable buffer or the like, and when it is soluble in an organic solvent, it may be placed by dissolving it in an appropriate solvent. it can.

In addition, buffers, nucleotides, enzymes and the like necessary for PCR may be placed in this reagent region together with this dye compound. When a dye compound is insoluble in water when an aqueous solution containing all of them is prepared, the solvent used for dissolving the dye compound is preferably 1% or less with respect to the aqueous solution. The arrangement in which the reagent for PCR and the dye compound are arranged together in this reagent region has a significantly lower uptake density than ethidium bromide even when the dye compound is incorporated into the double-stranded nucleic acid fragment of the amplification product. The melting temperature (Tm) rises very little due to
This is possible because the influence on the reaction efficiency is extremely small.

FIG. 9 shows an example of the structure of a kit having such a reactor. In this reactor, a primer 3 is placed in a suitable container 1 capable of forming a reaction region 5 by a method such as coating, and further, a suitable wall material, for example, a liquid-tight container such as paper coated with paraffin. An enzyme for PCR, nucleotides,
Pack a solution containing the required amount of buffer, etc.
It is arranged inside to form a reagent area (FIG. 9).
(See (A) and (B)). To perform PCR using this, when the template nucleic acid solution is injected into the reaction region 5 by an appropriate means such as Pipetman, the container 2 constituting the reagent region is destroyed and the components in the container 2 also react. Move to area 5,
PCR can be performed by mixing these (FIG. 9).
(See (C)). The dye compound used to detect the amplification product is
It may be added to the reaction region after PCR, or may be placed in the reaction region 5 in advance. Alternatively, this dye compound may be contained in the solution in the reagent region 2.

Further, a plurality of reagent areas may be provided and each component to be arranged in the reagent area may be distributed and arranged in the plurality of reagent areas.

The dye compound may be added in advance in the reaction region in which PCR is carried out as described above, or P
It may be added in the reaction region after CR. Even when the dye compound is added to the reaction region after the PCR, a reagent region in which a necessary amount of the dye compound is arranged may be provided separately from the reaction region, and the dye compound may be added from the reagent region to the reaction region after the PCR. It should be noted that the compound of the general formula [I] which is insoluble in water does not dissolve in the reaction solution during PCR even if it is placed in the reaction area, and an appropriate solvent is added to the reaction area after PCR. This makes it possible to react the compound with the amplification product. Therefore, by using the compound of the general formula [I], it is possible to surely eliminate the influence on the PCR because the compound is water-insoluble, although the PCR by the compound is small. Examples of such a solvent include acetonitrile, ethanol, dimethyl sulfoxide (DMSO) and the like. If a predetermined amount of the primer is placed in advance in the reaction area of this kit by coating or by arranging it as a powder separately in the reactor, the operation of PCR in the reaction area can be improved. Can be simplified.

On the other hand, P by the dye compound in the present invention
The detection of the CR amplification product can be suitably used for a method (MPN-PCR method) in which PCR is applied to the MPN method. By using this method, the number of individuals such as microorganisms, animal cells, human cells, and plant cells (the number of cells, the number of bacteria, the number of hyphae, etc.) can be measured, and the number of specific genes they have or the number of copies of specific genes can be measured. Can be measured, and these operations can be greatly simplified.

As an example of the operation of this MPN-PCR method, the case of measuring the number of individual microorganisms to be detected (the number of bacteria) from soil will be described below.

First, the soil sample is subjected to a treatment for extracting the nucleic acid of the microorganism contained in the soil sample. For example, add phosphate buffer (1 ml) to sample soil (1 g),
Vortex twice for 20 seconds each. Furthermore, 10%
Add 1/10 volume of SDS, vortex again, and
Incubate at 0 ° C for 1 hour to lyse bacteria in soil. The soil is removed by centrifugation and the nucleic acid fraction is collected as a supernatant.
1/5 volume of 7.5 M sodium acetate is added, mixed and left at 4 ° C. for 5 minutes. The supernatant is collected (1 ml), 4/5 volume of isopropanol is added, and the nucleic acid fraction is collected by centrifugation. Dissolve this in 0.1 ml TE buffer, then R
DNA is collected by Nase treatment.

A part of the recovered DNA was diluted 100 times and further a part thereof was diluted to obtain a dilution series of 10 ^-1 to 10 -10.
^-Create up to ⁸ . These are arranged in the block of sample A of the microplate shown in FIG. In other words, 10 times the DNA solution (already diluted 100 times) recovered from soil
To ^-1 dilution solution A1 to A5, 10 ^-2 dilution solution B1~B
Disposed 5, 10 ^{@ 8} dilutions until is disposed H1 to H5, are arranged in the same manner for each dilution. Here, a reaction solution containing reagents necessary for PCR (nucleotide, primer salt, Taq polymerase, etc.) is added to all the wells including the blank, and PCR is performed. After the reaction, the fluorescence intensity of each well is measured using the above-described dye compound, and for example, a well having a fluorescence intensity of 2 times or more that of the blank is regarded as positive, and the dilution limit at which the positive appears is determined. For example, at 10 ⁻⁴ , 10 ⁻⁵ and 10 ⁻⁶ , the number of positive samples in 5 samples in each dilution (the number of positive samples /
Number of test samples at each dilution) is 5/5 at 10 ^-4 dilution,
^10-5 When 0/5 by 3 / 5,10 ^-6 dilution with dilution, dilution limit positive appears is 10 ^-5. Therefore, the MPN table (J. Bacteriol., 25 , 10) showing the number of positive samples (5, 3, 0) at these dilution ratios is shown below.
1 (1933), page 400, "III. MPN (Mo
st Probe Number) table, 10-fold dilution in the case of 5 replicates ", partially quoted from" P ₁ , P ₂ , P _3.
Then, 0.79 is obtained as a numerical value when P ₁ = 5, P ₂ = 3, and P ₃ = 0. This is the statistically determined number of template DNAs contained in this sample. Therefore, by multiplying this by the reciprocal of the above limiting dilution ratio, and further considering the dilution rate of the first sample, the number of template DNAs contained in the soil is 0.79 × 10 ⁵ ×
10 ² = 0.79 × 10 ⁷ . If this template DNA is unique to a microorganism as a detection target in soil, and if there is one for each microorganism,
The number of template DNAs obtained here directly becomes the number of microorganisms (the number of bacteria) in the soil sample (1 g). Of course, in this case, in the dilution series where all wells emit fluorescence,
Since the fluorescence amount reflects the number of template DNAs, a calibration curve of the fluorescence amount (fluorescence intensity) and the number of template DNAs (the number of microorganisms) can be created based on this.

[0070]

[Table 1] In Table 1, those shown in parentheses are highly reliable ones (established L is 0.05 or more. If a code not shown in parentheses appears, reconfirm the experimental method. The MPN table quoted as Table 1 is a part of the MPN table of the above-mentioned document, and when the result concerning the part other than this part is obtained, the MPN table of the above-mentioned document is appropriately used.
Refer to the corresponding part of the table. Further, since this document has MPN tables of various conditions, it is possible to use them according to desired test conditions and the like.

It should be noted that the above description of the prior art section has been made.
MPN combining PCR and agarose gel electrophoresis
-In the PCR method, the first step to find the dilution limit at which positive appears
In-depth PCR and closer examination of the obtained dilution limit
2nd time to find the specific number from the MPN table
PCR is being performed. For example, the soil sample (1
Similarly recover DNA from g) and do not dilute as above
In 10^-1-10 ^-9Prepare serial dilutions up to dilution,
It is applied to the conventional method, and the first P
Perform CR and detect the presence of amplified products by agarose gel electrophoresis.
Is detected, 10^-7It becomes positive until dilution. Where 1
0^-610,^-710,^-85 suns each for 3 dilutions of
Perform PCR again for each pull, and
10 is the number of positive samples^-65/5 by dilution, 1
0^-73/5, 10 by dilution^-8It becomes 0/5 by dilution, and
Similarly, a value of 0.79 was obtained from the MPN table.
A value (0.
79 x 10⁷) Is the number of template DNAs in the soil. Shi
However, this method involves operations involving PCR and electrophoresis.
Since the work is repeated twice, the operation is very complicated. this
On the other hand, in the method of the invention, the result is directly obtained by one PCR.
Can be obtained, and the electrophoresis operation can be omitted.
It Therefore, the measurement operation can be greatly simplified.

The detection of the amplification product in the PCR by this MPN-PCR method by the dye compound described above can be carried out by each operation and its condition used in the above-mentioned method for quantifying the PCR amplification product, or the kit or the like.

Since this MPN-PCR method requires simultaneous processing of multi-step diluted samples for statistical processing, an instrument having a large number of reaction regions (wells) such as a microplate is used. It is preferable.
Even when the PCR reaction is carried out in another container, it is preferable that the detection of the amplification product with the dye compound is carried out with an instrument such as a microplate.

The method for measuring the number of template DNAs is, for example, measurement of the number of bacteria to be measured in various solutions or soil, measurement of the number of microorganisms in various samples, measurement of the number of cells in various samples,
It can be effectively used to measure the number of cells in various tissues, or the number of specific genes in various biological samples and the copy number thereof. For example, the concentration of the template DNA is generally estimated in gram amount from the measurement of light absorption intensity and the like, but the copy number (the number of specific genes) is a problem as in the case of cancer cells, for example. In many cases. In such a case, the method of the present invention is effective. When knowing the copy number of a specific gene in a tissue by the method of the present invention, the number is determined by the method described above for all common genes, then the number of specific genes such as oncogenes is determined, and normal cells and By obtaining the ratio of the common gene to the specific gene for cancer cells, the copy number of the specific gene can be obtained regardless of the amount of cells from which nucleic acid is extracted.

This method is also useful for determining whether or not cancer cells are present in a certain human tissue. For example, the number of specific genes common to cells constituting a certain tissue is obtained by this method, and then the number of specific genes of cancer cells to be detected is obtained by this method for the same tissue sample.

[0076]

【Example】

Reference Example 1 100 ml of acetic anhydride and 30 ml of concentrated sulfuric acid were mixed while being cooled, and the obtained mixed liquid was heated for 3 hours while being kept at 80 ° C. in a water bath. 20 ml of acetic anhydride, p
-Dimethylaminoacetophenone (30 ml) was added at room temperature, the temperature was then raised to 45 ° C, and the mixture was stirred for 24 hours for reaction. To this reaction solution, an equal amount of ethanol was added, cooled, and an aqueous potassium iodide solution was further added to precipitate crude crystals. The crude crystals were collected by filtration and recrystallized from a mixed system of ethanol / ether (volume ratio, 1: 4) to give 2
Green crystals of -methyl-4,6-bis- (4-N, N-dimethylaminophenyl) pyrylium iodide (Compound 1 in Table 2 (where Y is I)) were obtained. Analysis result of the obtained compound 1 (Y: I) Melting point: 254-257 ° C. UV / visible (CH ₃ CN ε × 10 ⁻⁴ ) λ max: 444
nm (2.43), 550 nm (8.24) NMR ( ¹ H, DMSO) δ ppm: 8.3737 (1 H,
s), 8.2729 (1H, d, J = 9.0 Hz),
8.1795 (1H, d, J = 9.0Hz), 7.88
64 (1H, s), 6.9117 (4H, t, J _AB = J
_BC = 9.77), 3.1829 (6H, s), 3.13
40 (6H, s), 2.6809 (3H, s) FAB mass m / z 333 IR (KBr) νcm ^-1 : 1645, 1610 (s
h), 1580 (s), 1490 (s), 1270, 1
200,1160 Further, 2-methyl-4,6-as described above except that an aqueous solution of perchloric acid is used instead of the aqueous solution of potassium iodide.
A perchlorate salt of bis- (4-N, N-dimethylaminophenyl) pyrylium (Compound 1 (Y: CIO ₄ )) was obtained.

Reference Example 2 20 g of sodium sulfide nonahydrate was dissolved in ion-exchanged water so that the total amount was 50 ml. After 7 g of sodium hydrogen carbonate was added to and dissolved in this solution, 50 ml of ethanol was further added under ice cooling, and the mixture was stirred at room temperature for 30 minutes. Precipitated sodium carbonate was filtered off, washed with 25 ml of ethanol, and the filtrate and the washing solution were combined to obtain about 125 ml of a solution of sodium hydrosulfide in water and ethanol.

Next, 2-methyl-4,6 obtained in Reference Example 1
-Bis- (4-N, N-dimethylaminophenyl) pyrylium iodide 0.92 g was added to 20 ml DMS.
5 ml of the previously prepared water / ethanol solution of sodium hydrosulfide was added to the resulting solution, and the mixture was stirred at room temperature for 5 minutes. After stirring, 0.75 ml of hydroiodic acid was added, and the mixture was further stirred for 5 minutes. Then, after performing dichloromethane extraction and silica gel column purification according to a conventional method, recrystallization was performed with an ethanol / ether mixed solution (volume ratio, 1: 4) to obtain 0.7 g of 2-methyl-4,6-bis-. (4-
Crystals of N, N-dimethylaminophenyl) thiopyrylium iodide (Compound 2 in Table 2 (wherein Y is I)) were obtained. Analysis result of the obtained compound 2 (Y: I) Melting point: 246 to 248 ° C. UV / visible (CH ₃ CN ε × 10 ⁻⁴ ) λ max: 495
nm (2.50), 587 nm (4.95) NMR ( ¹ H, DMSO) δ ppm: 8.5679 (1 H,
s), 8.4323 (1H, s), 8.2436 (2)
H, d, J = 9.27 Hz), 7.9786 (2H,
d, J = 9.28), 6.8959 (4H, t, J _AB =
J _BC = 9.28), 3.1756 (6H, s), 3.1.
157 (6H, s), 2.8323 (3H, s) FAB mass m / z 349 IR (KBr) νcm ⁻¹ : 1600 (s), 1560
(S), 1460 (s), 1430 (s), 1370
(S), 1260 (s), 1160 (s) Furthermore, 2-methyl-4,6-bis- was prepared in the same manner as above except that an aqueous solution of perchloric acid was used instead of hydroiodic acid.
A perchlorate of (4-N, N-dimethylaminophenyl) thiopyrylium (Compound 2 (Y: CIO ₄ )) was obtained. Reference Example 3 Compounds 3 to 55 shown in Table 2 below were prepared.
In Table 2, Φ is a p-phenylene group:

[0079]

[Chemical 31] Or represents a phenyl group.

[0080]

[Table 2]

[0081]

[Table 3]

[0082]

[Table 4]

[0083]

[Table 5]

[0084]

[Table 6]

[0085]

[Table 7]

[0086]

[Table 8]

[0087]

[Table 9]

[0088]

[Table 10]

[0089]

[Table 11]

[0090]

[Table 12]

[0091]

[Table 13]

[0092]

[Table 14]

[0093]

[Table 15]

[0094]

[Table 16]

[0095]

[Table 17]

[0096]

[Table 18]

[0097]

[Table 19]

[0098]

[Table 20] In addition, these compounds were synthesized by the following known methods. The specific reaction procedure was in accordance with a conventional method.

Compound 7 was prepared according to the method described by W. Foerst et al.
of Preparative Organic Chemistry ", Acad. Press (1
964) and the compound [i]

[0100]

[Chemical 32] After synthesizing

[0101]

[Chemical 33] The compound obtained by reacting with (p-N, N-dimethylaminobenzaldehyde) was further reacted with a desired anion. Compound 17 is the same as compound [i]

[0102]

[Chemical 34] The compound obtained by reacting with (p-diethylaminostyrylbenzaldehyde) was further reacted with a desired anion. In addition, compound [ii] is reacted with sodium hydrosulfide to give compound [ii]

[0103]

[Chemical 35] After obtaining, the compound [ii] was synthesized in the same manner as the compounds 7 and 17 to synthesize the compounds 8 and 18.

R. Wizinger et al., Helv. Chim. Acta, 39 , 2
According to the method described in 17 (1956), the route shown below from acetophenone and acetaldehyde

[0105]

[Chemical 36] Via compound [iii]

[0106]

[Chemical 37] Was synthesized. Using this compound [iii] as a raw material, p-
The compound obtained from the reaction with dimethylaminobenzaldehyde is further reacted with a desired anion to give compound 5.
Compound 15 was similarly prepared from the reaction with p-diethylaminostyrylbenzaldehyde, and Compound 9 was similarly prepared from the reaction with p-dimethylaminocinnamaldehyde.

[0107]

[Chemical 38] Compound 11 was obtained in the same manner from the reaction with. Alternatively, the compound [iv] can be obtained by reacting the compound [iii] with sodium hydrosulfide.

[0108]

[Chemical Formula 39] Compounds 6, 16, 10 and 12 are obtained in the same manner as in the case of the synthesis of Compounds 5, 15, 9 and 11 except that the compound [iv] is used instead of the compound [iii]. It was

Further, after obtaining the cation part of the compound 3 in the same manner as in the synthesis of the compound [iii] except that p-dimethylaminobenzaldehyde was used as the starting material, acetaldehyde was reacted with sodium hydrosulfide. The compound thus obtained was further reacted with a desired anion to obtain Compound 4. Further, in the same manner, the compound [v] was obtained from p-methylbenzaldeh and acetophenone.

[0110]

[Chemical 40] And then reacting it with sodium hydrosulfide to give compound [vi]

[0111]

[Chemical 41] Got Further, the compounds [v] and [vi] are each added by p
Compound 13 obtained by reacting with dimethylaminobenzaldehyde and further reacting the obtained compound with a desired anion.
And 14 were obtained.

The compounds 19, 20 and 21 are prepared by combining the compound [i] or [ii] with the cation moiety of the compound 1 or 2.

[0113]

[Chemical 42] Was obtained by further reacting the obtained compound with a desired anion. Compounds 22, 23 and 24 are compounds [i] or [ii] and the cation moiety of compound 1 or 2,

[0114]

[Chemical 43] Was obtained by further reacting the obtained compound with a desired anion. Compounds 25 and 26 are compounds [i] or [ii]

[0115]

[Chemical 44] Was obtained by further reacting the obtained compound with a desired anion. Compounds 27, 28 and 29 are prepared by reacting compound [i] or [ii] with the cation moiety of compound 1 or 2 with ethyl orthoformate [HC (OC ₂ H ₅ ) ₃ ] It was synthesized by reacting the desired anion. Compounds 30, 31, and 32 are compounds [iii] or [iv], [iii] and [i].
In the same manner as in v], a compound [iii] synthesized from p-dimethylaminoacetophenone and a dimethylamino derivative of [iv] are reacted with ethyl orthoformate, and the obtained compound is further reacted with a desired anion. Was synthesized by.

Compounds 33 to 55 were respectively synthesized by the following reactions. Synthesis of compound 33

[0117]

[Chemical formula 45] Synthesis of compound 34

[0118]

[Chemical formula 46] Synthesis of compound 35

[0119]

[Chemical 47] Synthesis of compound 36

[0120]

[Chemical 48] Synthesis of compound 37

[0121]

[Chemical 49] Synthesis of compound 38

[0122]

[Chemical 50] Synthesis of compound 39

[0123]

[Chemical 51] Compound 40 is used as a raw material in the synthesis of Compound 36.

[0124]

[Chemical 52] From

[0125]

[Chemical 53] It was changed to and synthesized.

Compound 41 was used as a starting material in the synthesis of compound 37.

[0127]

[Chemical 54] From

[0128]

[Chemical 55] It was changed to and synthesized.

Compound 42 was used as a starting material in the synthesis of Compound 38.

[0130]

[Chemical 56] From

[0131]

[Chemical 57] It was changed to and synthesized.

Compound 43 was used as a starting material in the synthesis of Compound 39.

[0133]

[Chemical 58] From

[0134]

[Chemical 59] It was changed to and synthesized. Synthesis of compound 44

[0135]

[Chemical 60] Synthesis of compound 45

[0136]

[Chemical formula 61] Synthesis of compound 46

[0137]

[Chemical formula 62] Synthesis of compound 47

[0138]

[Chemical formula 63] Compound 48 is used as a raw material in the synthesis of Compound 44.

[0139]

[Chemical 64] From

[0140]

[Chemical 65] It was changed to and synthesized. Compound 49 is used as a raw material in the synthesis of Compound 45.

[0141]

[Chemical formula 66] From

[0142]

[Chemical formula 67] It was changed to and synthesized. Compound 50 is used as a raw material in the synthesis of Compound 46.

[0143]

[Chemical 68] From

[0144]

[Chemical 69] It was changed to and synthesized. Compound 51 is used as a raw material in the synthesis of Compound 47.

[0145]

[Chemical 70] From

[0146]

[Chemical 71] It was changed to and synthesized. Synthesis of compound 52

[0147]

[Chemical 72] Synthesis of compound 53

[0148]

[Chemical formula 73] Synthesis of compound 54

[0149]

[Chemical 74] Synthesis of compound 55

[0150]

[Chemical 75] Reference Example 4 Compound I obtained in Reference Example 1 was dissolved in 10 mM phosphate buffer containing 10% acetonitrile, and the final concentration of Compound 1 was adjusted to 3 × 10 ⁻⁵ M to ^prepare Sample I.
An absorption spectrum of a part of this sample I was measured by a conventional method using a spectrophotometer.

Next, Salmon sparm DNA (manufactured by Sigma) was dissolved in TE buffer (10 mM Tris-1 mM EDTA) and purified by phenol extraction. In order to facilitate handling, this purified product was further digested with the restriction enzyme EcoRI, and the obtained treatment liquid was used as a DNA solution. Mix a portion of this DNA solution with a portion of Sample I to give a final DNA concentration of 5
Sample II was prepared by adjusting the concentration to 0 μg / ml and further adjusting the final concentration of Compound 1 to 3 × 10 ⁻⁵ M. The absorption spectrum of a part of this sample II was measured by a conventional method. As a result, the absorption peak of the compound was shifted to the long wavelength side by 20 to 30 nm due to the interaction with DNA. This is a typical intercalator property.

When the fluorescence spectrum of a part of Sample I was measured by a conventional method, a weak peak was detected near 650 nm when excited at 550 nm in the absence of DNA. Next, when the fluorescence spectrum was similarly measured using a part of Sample II, Sample II
Among them, when excited at the same wavelength, a peak of fluorescence intensity about 100 times that in the absence of DNA was detected near 650 nm. The above results indicate that Compound 1 is a strong intercalator.

Next, using the previously prepared DNA solution, Compound 1, and 10 mM phosphate buffer containing 10% acetonitrile, Compound 1 was contained at a concentration of 5 × 10 ⁻⁶ M, and the DNA concentration was Different solutions were prepared. The fluorescence intensity of each obtained solution was measured by a conventional method to determine the change in fluorescence intensity with respect to the DNA concentration. The fluorescence intensity increased as the DNA concentration increased, and the maximum was about 400 times higher than the fluorescence intensity in the absence of DNA. As the excitation light,
An Xe lamp was used as a light source and ultraviolet light was removed by a 480 nm low-cut filter. Further, with respect to the compounds in Table 2, the same measurement of fluorescence intensity as above was performed. A typical example thereof is shown in Table 3.

[0154]

[Table 21] Example 1 [Method for quantifying nucleic acid using 2,4-bis (N, N-dimethylaminophenyl) -6-methylpyrylium salt] 16 of Pseudomonas aeruginosa
S ribosomal RNA gene (hereinafter 16Sr
PCR was performed targeting the RNA gene (abbreviated as RNA gene), and the amplification product was detected using 2,4-bis (N, N-dimethylaminophenyl) -6-methylpyrylium salt.

First, P. All DA of aeruginosa
N was prepared as follows. 2 ml of cells cultured overnight in 2 × YT medium were collected by centrifugation and suspended in 0.5 ml of 0.1 M sodium phosphate buffer (pH 8.0). 0.05% of 10% SDS solution was added to the obtained suspension.
After adding ml and mixing thoroughly, the mixture was kept at 70 ° C. for 1 hour. This suspension was stirred with a vortex mixer to completely lyse the cells. An equal amount of phenol / chloroform was added to this lysate, and after mixing and centrifugation, the upper layer was separated and twice the amount of ethanol was added to collect DNA as a precipitate. This was mixed with 100 μl of TE buffer (pH 8.
It was dissolved in 0) and used as a template DNA for PCR.

The following two PCR primers were used. Primer 1 5'AGAGTTTTGATCATGGC
TCAG 3 '(SEQ ID NO: 1) Primer 2 5'AACCCAACATCTCACG
ACAC 3 ′ (SEQ ID NO: 2) These primers are DNA synt manufactured by ABI.
It was synthesized using hesizer381A. The reagents and methods necessary for the synthesis were based on the protocol of ABI. PCR was performed under the following conditions using these template DNAs and primers. PCR conditions: Reaction solution composition (total volume 50 μl) 5 μl 10 × buffer 5 μl dNTPs Primers 1 and 2 10 pmol Taq DNA polymerase 0.5 unit template DNA 500 pg, 100 pg, 10 pg, 1 pg, 100 f, respectively
g or 10 fg of each component, and sterilized water was added to bring the total volume to 50 μl. Thin-Walled Gene
The reaction was carried out in an Amp tube (total volume 0.5 ml, manufactured by Takara Shuzo). A sample having the same composition as above except that it did not contain the template DNA was prepared as a blank. Here, 10 × buffer and dNTPs attached to the polymerase were used as they were. Reaction cycle 92 ° C., 5 minutes pre-incubation 92 ° C., 45 seconds / 55 ° C., 60 seconds / 72 ° C., 90 seconds 3
0 cycle Finally, incubation at 72 ° C. for 5 minutes Here, the PCR device is G of Perkin-Elmer.
The ene PCR System 9600 was used. The detection of the amplification product was performed as follows. 96-well microtiter plate (Falcon manufactured by Becton-Dickinson)
TE buffer (10 mM Tris-HCl (p) was added to the assay plate 3911 (U bottom) so that the total amount became 50 μl.
H8.0) -1mM EDTA) 2x, 10x, 20
Diluted twice (see FIG. 1). Next, 150μ in each well
1 μl of a g / ml solution of 2,4-bis (N, N-dimethylaminophenyl) -6-methylpyrylium iodide in acetonitrile was added and mixed sufficiently by pipetting. This microplate was set on a Millipore fluorescence device (Cytoflow 2350), and was placed on the excitation light side.
Irradiation of excitation light and measurement of fluorescence were carried out using filters that transmit light of 0 nm and 645 nm on the radiant light side, respectively. The relationship between the measured fluorescence intensity (645 nm) at each dilution of the resulting reaction solution and the amount of template DNA is shown in FIG. Further, no fluorescence was observed in the blank sample. The results of FIG. 2 show that the fluorescence intensity increases in proportion to the amount of template DNA in each diluted sample, and the amount of template DAN can be quantified from the fluorescence intensity. When the template DNA was 100 fg or 10 fg, no PCR amplification product was detected.

The same operation as described above was performed, and each reaction solution was added without adding 2,4-bis (N, N-dimethylaminophenyl) -6-methylpyrylium salt to the reaction solution in each well. 5 μl aliquot, agarose gel electrophoresis,
Stain with ethidium bromide (hereinafter referred to as EB),
The amount of PCR amplification product was examined by the intensity of the band. As a result, template DAN was 500 pg, 100 pg, 10 pg
However, a clear band was slightly observed at 1 pg, and a PCR amplification product was not observed at 100 fg and 10 fg. Further, the fluorescence intensity was saturated at 500 pg, which was almost the same as 100 pg. Further, in addition to the band of the PCR amplification product on the gel, fluorescence considered to be a higher-order structure which is a reaction product between the primers was observed in the low molecular weight region.

Comparative Example 1 [Detection of PCR amplification product using EB] 2,4-bis (N, N-dimethylaminophenyl) -6
-The same operation as in Example 1 was performed except that EB and the following operating conditions were used instead of the methylpyrylium salt. E
As a solution of B at 250 μg / ml, 1 μl thereof was added to each well, mixed sufficiently by pipetting, and left at room temperature for 5 minutes. 48 on the excitation light side of the Millipore fluorescence device
Fluorescence intensity was measured by setting a filter that transmits light of 5 nm and 620 nm on the radiant light side. As a result, all reaction solutions in each well including the blank sample well fluoresced red. Moreover, there was no difference between the fluorescence intensity of the sample containing the template DAN and the fluorescence intensity of the sample containing no template DAN, and quantification was impossible. This is because in the sample (blank) containing no template DNA, a higher-order structure causing fluorescence increase of EB was generated in the reaction between the primers.

Comparative Example 2 [Detection of PCR amplification product using YOYO-1] 2,4-bis (N, N-dimethylaminophenyl) -6
Instead of the methylpyrylium salt, YOYO-1 (Mo
The same operation as in Example 1 was performed, except that the following operating conditions were used and the following conditions were used. YOY
For O-1, 1 mM solution was diluted 120 times, and 1 μl
Was added to each well, mixed well by pipetting, and left at room temperature for 5 minutes. A fluorescence intensity was measured by setting a filter for transmitting 485 nm on the excitation light side and a filter for transmitting 530 nm on the emission light side of the Millipore fluorescence device. The obtained results are shown in FIG. In the results of FIG. 3, an increase in fluorescence intensity was observed in proportion to the amount of template DAN, but the rate of increase was low. Further, in the blank, a fluorescence intensity similar to that of the sample containing the template DAN was observed. The fluorescence in this blank is also considered to be due to the higher-order structure which is a reaction product between the primers. Therefore, in the quantitative determination using this dye, the difference from the blank is low and the template D
Since the rate of increase in fluorescence intensity proportional to the amount of NA is also low, it can be seen that the quantification is not good.

From the above results of Example 1 and Comparative Examples 1 and 2, 2,4-bis (N, N-dimethylaminophenyl)
It was found that the quantification by fluorescence using -6-methylpyrylium iodide enables accurate quantification without being affected by the higher-order structure which is a reaction product between the primers. This result reflects the result of EB staining of each reaction solution of Example 1 in agarose gel electrophoresis. The agarose gel staining with this EB showed 5
Fluorescence is saturated at 00 pg and 100 pg, so that the amount of amplified product is apparently the same, but EB is detected in detection with 2,4-bis (N, N-dimethylaminophenyl) -6-methylpyrylium salt. Even in the sample in which the fluorescence was saturated, the fluorescence intensity was not saturated and the quantification was possible.

Example 2 [PC using microplate]
Measurement of the number of bacteria in soil by the R method] DNA from soil mixed with an overnight culture solution (2 × YT medium) of Escherichia coli JM109 strain
Are recovered by a conventional method, and E. PCR targeting the 16S rRNA gene of E. coli was performed, and PCR-MPN
The number of bacteria was measured by the method.

E. coli is about 10 ⁷ per 1 g of soil,
The cells were added so that the number of bacteria was about 10 ⁶ , about 10 ⁵ , and about 10 ⁴ (Samples A, B, C, and D shown in FIG. 4, respectively) and the mixture was stirred. 1 g of each soil containing Escherichia coli was suspended in 0.5 ml of 0.1 M sodium phosphate buffer (pH 8.0). To this suspension, 0.05 ml of a 10% SDS solution was added, thoroughly mixed, and then kept at 70 ° C. for 1 hour. This suspension was stirred with a vortex mixer to completely lyse the cells. An equal amount of phenol / chloroform was added to this lysate, and after mixing and centrifugation, the upper layer was separated and twice the amount of ethanol was added to collect DNA as a precipitate. This was dissolved in 100 μl of TE buffer (pH 8.0) and
It was used as R template DNA.

PCR primers were E. 16 of E. coli
The following two genes were used for the SrRNA gene. Primer 1 5'AGAGTTTTGATCCTGGC
TCAG 3 ′ (SEQ ID NO: 3) Primer 2 5 ′ AACCCAAACATTCCACG
ACAC 3 '(SEQ ID NO: 4) These primers are DNA synt manufactured by ABI.
It was synthesized using hesizer381A. The reagents and methods necessary for the synthesis were based on the protocol of ABI.

PCR was carried out under the same conditions as in Example 1 using these template DNA and primers. PCR
Is a 96-well microtiter plate (Bect
It was performed in each well of Falcon assay plate 3911 (U bottom) manufactured by on-Dickinson. Also,
In this microplate, each well is preliminarily prepared in Example 1.
1 μl of a solution of 2,4-bis (N, N-dimethylaminophenyl) -6-methylpyrylium iodide in acetonitrile having the same concentration as above was applied and dried and stored. As the template DNA, a 10-fold dilution series was created stepwise in 8 columns in the vertical row as shown in FIG. 4, and the same sample was prepared in the 5 horizontal rows, and PCR reaction was performed. As a PCR device,
MJ Research Inc. Model PT
C-100-96 was used.

After completion of the PCR reaction, each cell of the microplate was
Add 5 μl of acetonitrile to the well and stir well
Then, it was left for 5 minutes. After that, Millipore fluorescence device
Trouble 2350) and set the same filter as in Example 1.
Fluorescence was measured using a Luther. As a result, as shown in Figure 5.
The results were obtained. Here, more than twice the fluorescence of the blank
Those having the above were considered to have formed PCR amplification products
(Well filled with diagonal lines). This result is calculated by MPN method.
Calculated by applying to the table used, each soil
The number of Escherichia coli obtained from 4.9 × 10⁶, 7.9 ×
10^Five2.2 x 10 ^Five, 7.9 × 10³Became.

Comparative Example 3 E. coli prepared in Example 2 was used. E. coli contained in each soil sample mixed with different concentrations of E. coli. the number of E. coli,
E. The plate count method was used to determine the number of bacteria from the number of colonies appearing after inoculating a culture plate for E. coli with a predetermined amount of a sample and culturing. The results obtained,
Looking at the correlation with the results obtained in Example 2, they were in very good agreement as shown in FIG. From the results of Example 2 and Comparative Example 3 described above, according to the method for measuring the number of bacteria using PCR of the present invention, the number of bacteria can be measured by a simple operation, and the obtained results are also obtained by the conventional method. It was in good agreement with the plate count method.

Example 3 [PC using microplate]
Quantification of template DNA by R method] In each well of a microplate similar to that used in Example 2, 2,4-bis (N,
1 μl of a solution of N-dimethylaminophenyl) -6-methylpyrylium iodide in acetonitrile and a primer (2 kinds, 10 pmole each) were applied and dried and stored. On the other hand, E. coli DNA was extracted from 2 ml of the overnight culture of E. coli by the same method as in Example 2,
It was diluted 100 times to obtain a template DNA solution.

Next, a dilution series of this template DNA solution was prepared in the same manner as in Example 2, and 1 μl was added to the microplate in the same manner as in FIG. Next, Ampli Wax PC
RGem 100 (manufactured by Perkin-Elmer) 1
A reaction solution for PCR containing individual cells and the following components was added to each well.

5 μl 10 × buffer 5 μl dNTPs Taq DNA polymerase 0.5 unit Further, sterilized water was added to each well to make the total volume 50 μl, and PCR was performed. Here, the PCR device is MJ
Research Inc. Model PTC-1
00-96 was used. The PCR reaction conditions are the same as in Example 1.

After PCR, 5 μl of acetonitrile was added to each well of the microplate after PCR, stirred well, and left for 5 minutes. After that, Millipore fluorescence device (Site Flow 23
50), and evaluated using the same filter as in Example 1. As a result, the result as shown in FIG. 7 was obtained. Here, the one having fluorescence twice or more that of the blank was regarded as the PCR amplification product, and was shaded. When this result was calculated by applying it to a table used in the MPN method, it was found that the number of molecules of the template DNA was 3.5 × 10 ⁷ . Considering that 100-fold dilution is performed first, it can be said that the number of molecules of the template DNA is 3.5 × 10 ⁹ .

Comparative Example 4 The E. coli used in Example 3 was used. When the number of bacteria in 2 ml of E. coli culture was determined by the colony count plate method, 4
The number is × 10 ⁹ , which is almost the same as the result obtained in Example 3.

Dilution of template DNA and M from Examples 2 and 3
The desired concentration range for detection by the PN method can be known, and the concentration of the sample can be set. As a result, it becomes possible to set a calibration curve for obtaining the number of individuals, the number of copies, and the amount of template DNA.

Example 4 [Detection Kit of Cancer-Specific Genes by PCR] (1) Extraction of mRNA Samples of 2 mm square each were collected from a patient by biopsy from two tissues suspected of having colorectal cancer. A and B). MRNA was extracted from this tissue by a conventional method (Neochemistry Chemistry Experimental Course 2, Nucleic Acid I, page 48). That is, 2 ml of D solution [4 M guanidine thiocyanate; 25 mM sodium citrate (pH 7.0); 0.5% sodium N-lauroyl sarcosinate; and 0.1 M 2-mercaptoethanol] for each tissue.
And homogenized quickly. Homogenization was performed in a sterile tube with Polytron for 10 seconds 3 times. 0.2 to this
ml of 2M sodium acetate (pH 4), 2 ml of water saturated phenol, 0.4 ml of chloroform-isopentyl alcohol (49: 1, v / v) were added. Each reagent was added and well stirred. Vortex for 10 seconds (Vor
tex) After shaking with a mixer, the mixture was cooled in ice for 15 minutes. After centrifugation at 4 ° C. and 10,000 × g for 20 minutes, an equal amount of isopropropyl alcohol was added to the aqueous layer, and the mixture was left at −20 ° C. overnight. After centrifugation at 10,000 xg for 20 minutes at 4 ° C, D solution was added to the precipitate at a ratio of 0.6 ml and dissolved at 50 ° C. After adding an equal amount of isopropyl alcohol and cooling at -20 ° C for 1 hour, the mixture was centrifuged at 4 ° C and 10000 xg for 20 minutes, and the precipitate was suspended in 75% ethanol, and then at 4 ° C and 1000 ° C.
Centrifuge again at 0xg for 20 minutes, dry the precipitate and dry the crude RNA.
Fraction. The crude RNA fraction was heated at 65 ° C. for 5 minutes, rapidly cooled to room temperature, and added to an equal volume of twice the concentration of TNEL buffer [20].
mM Tris-HCl buffer (pH 7.6); 0.5 M sodium chloride; 1 mM EDTA and 0.1% sodium N-lauroyl sarcosinate]. The crude RNA fraction was applied to an oligo (dT) -cellulose column (Pharmacia) equilibrated with TNEL buffer, and then mRNA was extracted with an extraction solution (TNEL buffer minus 0.5 M sodium chloride). It eluted.

(2) Preparation of cDNA To prepare double-stranded cDNA from the above mRNA, Pha was used.
Time Saver ^™ cDNA from rmcia
The Synthesis Kit was used. (3) Structure of quantification kit As a primer for detecting colorectal cancer, an oligonucleotide having the following sequence was used. Primer 1: 5'GACTCTGGAGGTGAGAA
TCATA 3 '(SEQ ID NO: 5) Primer 2: 5'ATCCAATCACCCACAT
GCATT 3 '(SEQ ID NO: 6) 10 pmol of each of the solutions of the primers 1 and 2 was placed on the bottom of the Eppendorf as the container 1 shown in FIG.
And the container 2 was made of paper coated with paraffin, and the following components were packed in the paper. dNTP: 5 μl Taq polymerase: 0.5 unit 150 μg / ml of 2,4-bis (N, N-dimethylaminophenyl) -6-methylpyrylium iodide in acetonitrile: 1 μl 10 × buffer ..5 μl distilled water ... 39 μl (freezing storage) This container 2 was set in the container 1 as shown in FIG. 9 (B) to obtain a kit. Next, the cDNAs obtained from the respective tissues A and B by the above-mentioned operation
Serial dilution of sample (1 (no dilution), 1/2, 1/1
0, 1/50, 1/100, 1/1000, 1/100
00 dilution) was prepared, and the undiluted sample and each diluted solution were individually injected into the kit having the above-mentioned constitution at the bottom portion of the container 1 by Pipetman. At that time, the paraffin-coated paper in the container 2 was torn. After the injection of cDNA, each container was centrifuged to transfer the components in the container 2 to the reaction region 5 with the primer. After confirming the complete transition,
The container 2 containing the reagent was removed from the container 1, and the Ampli Wax PCR Gen 100 (Pe
rkin-Elmer) one each. (4) PCR reaction and detection

PCR was carried out under the same conditions as in Example 1,
Further, the fluorescence intensity was measured without diluting the sample in each container. The results for organization A are shown in FIG. Organization A
Shows that the target gene is amplified. Further, it can be seen that amplification products are quantitatively produced corresponding to the dilution rate of the template DNA. On the other hand, in tissue B, no fluorescence was observed. (5) Analysis by Electrophoresis The cDNA from the sample after detection was recovered by ethanol precipitation and then examined by agarose gel electrophoresis. The amount of cDNA used as a template from Tissue A was 1
(Undiluted), 317 up to 1/2 to 1/1000 dilution
A band was observed in the bp portion, but no band was observed in the case of 1 / 10,000 dilution, reaction with the primer alone without addition of cDNA, and from tissue B.

Comparative Example 5 The tissues A and B collected in Example 4 were evaluated by the conventional culture method. Proliferation was observed in the tissue A, and it was determined to be a malignant cancer. No growth was observed in Tissue B, and it was determined to be a benign polyp.

[0177]

INDUSTRIAL APPLICABILITY According to the present invention, since the dye compound that specifically reacts with the double-stranded nucleic acid is used for detecting the PCR amplification product, it is possible to perform various detections using PCR, quantification, or operations for counting the number of bacteria Great simplification and accurate measurement and quantification are possible.

[0178]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 20 Sequence type: Nucleic acid Number of strands: 1 Strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics: PCR primer sequence AGAGTTTGAT CATGGCTCAG 20

SEQ ID NO: 2 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics: PCR primer Sequence AACCCAACAT CTCACGACAC 20

SEQ ID NO: 3 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics: PCR primer Sequence AGAGTTTGAT CCTGGCTCAG 20

SEQ ID NO: 4 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics: PCR primer Sequence AACCCAACAT CTCACGACAC 20

SEQ ID NO: 5 Sequence length: 21 Sequence type: Nucleic acid Number of strands: 1 strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics: PCR primer sequence GACTCTGGAG TGAGAATCAT A twenty one

SEQ ID NO: 6 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics: PCR primer sequence ATCCAATCAC CCACATGCAT T twenty one

[Brief description of drawings]

FIG. 1 is a diagram showing the arrangement of each sample prepared in Example 1 on a microplate.

FIG. 2 is a diagram showing the relationship between the fluorescence intensity and the amount of template DNA (template) obtained in Example 1.

FIG. 3 is a graph showing the relationship between the fluorescence intensity and the amount of template DNA (template) obtained in Comparative Example 2.

FIG. 4 is a diagram showing the arrangement of each sample prepared in Example 2 on a microplate.

FIG. 5 is a diagram showing the state of PCR amplification product formation in each sample prepared in a microplate in Example 2, in which the shaded area indicates wells whose fluorescence intensity is twice or more that of the blank.

FIG. 6 is a diagram showing the correlation between the numbers of bacteria obtained in Example 2 and Comparative Example 3.

FIG. 7 is a diagram showing positions of wells showing fluorescence intensity twice or more that of the blank in Example 3.

FIG. 8 is a view showing a result of a sample from a tissue A in Example 4.

FIG. 9 is a diagram showing the structure and method of using an example of the kit for detecting nucleic acid of the present invention.

[Explanation of symbols]

 1 container 2 container constituting reagent area 3 primer 4 pipette tip 5 reaction area

 ─────────────────────────────────────────────────── (72) Inventor Masahiro Kawaguchi 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon Inc. (72) Inventor Yoshinori Tomita 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon Incorporated (72) Inventor Ken Miyazaki 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon Inc.

Claims (60)

[Claims] 1. A process of performing PCR on a nucleic acid sample in the presence of a primer necessary for amplifying a specific sequence region of a target nucleic acid, and a double-stranded nucleic acid amplified when the target nucleic acid is present in the sample. The process of measuring the fluorescence intensity obtained when a dye compound that does not emit fluorescence when released is reacted with a double-stranded nucleic acid and reacts with a double-stranded nucleic acid, and the measured fluorescence intensity A method for quantifying nucleic acid, comprising determining the amount of target nucleic acid in the nucleic acid sample. 2. The dye compound is represented by the following general formula [I]: (In the above general formula [I], Represents a heterocycle, X is O, S, Se or Te, represents a pyrylium ring or a pyrylium-like ring, and R ¹ and R ² are each independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, R represents a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group or a substituted or unsubstituted cycloalkyl group, R ³ is -A or -L-a, L ^{is, -L 1 -, - L 2} -L 3 - or -L ⁴ -L ⁵ -L ⁶ -
And L ^{1 to} L ⁶ are each independently-(CH = C
H) -, 2-valent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted lower alkylene group, or -CH = R ⁴ - (R ⁴ represents a ring structure having an oxo group)
Represents a substituted or unsubstituted aryl group, -CH = R
⁵ (R ⁵ represents a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group or a substituted or unsubstituted aromatic ring), and R ¹ , R ^{2 of} the pyrylium ring containing X or a ring similar thereto,
The hydrogen atom bonded to the carbon atom to which R ³ is not bonded is a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, It may be substituted with a substituted or unsubstituted aryl group or a substituted or unsubstituted lower aralkyl group, and Y ⁻ represents an anion. The method for quantifying a nucleic acid according to claim 1, which is a compound represented by the formula (1). 3. A compound represented by the general formula [I] is
2,4-bis (N, N-dimethylaminophenyl) -6
-Methylpyrylium salt, or 2,4-bis (N, N-
The method for quantifying a nucleic acid according to claim 2, which is a dimethylaminophenyl) -6-methylthiopyrylium salt. 4. The amplification is carried out by carrying out PCR in a reaction system containing the dye compound and an aqueous medium, wherein the dye compound is insoluble in water, and then adding a solvent to the reaction system to dissolve the dye compound. The method for quantifying a nucleic acid according to any one of claims 1 to 3, which comprises reacting with a product. 5. The method for quantifying a nucleic acid according to claim 1, wherein the amplification product has a chain length of 100 base pairs or more. 6. The method for quantifying a nucleic acid according to claim 1, wherein the primer has a chain length of 30 bases or less. 7. A nucleic acid characterized by having a reactor in which a necessary amount of a dye compound which does not emit fluorescence when released and which emits fluorescence when reacted with a double-stranded nucleic acid is arranged in a reaction region for PCR. Quantitative kit. 8. The kit for nucleic acid quantification according to claim 7, wherein a necessary amount of a primer necessary for PCR amplification of a specific sequence region of a target nucleic acid is further arranged in the reaction region. 9. The dye compound is represented by the following general formula [I]: (In the above general formula [I], Represents a heterocycle, X is O, S, Se or Te, represents a pyrylium ring or a pyrylium-like ring, and R ¹ and R ² are each independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, R represents a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group or a substituted or unsubstituted cycloalkyl group, R ³ is -A or -L-a, L ^{is, -L 1 -, - L 2} -L 3 - or -L ⁴ -L ⁵ -L ⁶ -
And L ^{1 to} L ⁶ are each independently-(CH = C
H) -, 2-valent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted lower alkylene group, or -CH = R ⁴ - (R ⁴ represents a ring structure having an oxo group)
Represents a substituted or unsubstituted aryl group, -CH = R
⁵ (R ⁵ represents a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group or a substituted or unsubstituted aromatic ring), and R ¹ , R ^{2 of} the pyrylium ring containing X or a ring similar thereto,
The hydrogen atom bonded to the carbon atom to which R ³ is not bonded is a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, It may be substituted with a substituted or unsubstituted aryl group or a substituted or unsubstituted lower aralkyl group, and Y ⁻ represents an anion. The nucleic acid quantification kit according to claim 7 or 8, which is a compound represented by the formula (4). 10. A compound represented by the general formula [I] is
2,4-bis (N, N-dimethylaminophenyl) -6
-Methylpyrylium salt, or 2,4-bis (N, N-
The kit for quantifying nucleic acid according to claim 9, which is a dimethylaminophenyl) -6-methylthiopyrylium salt. 11. The method according to claim 7, wherein the dye compound is insoluble in water and further has a solvent for dissolving the dye compound in a reaction solution after PCR in the reaction region to react with an amplification product. A kit for quantifying nucleic acid according to the item Crab. 12. A dye having a reaction region for PCR and a reagent region isolated from the reaction region, which does not emit fluorescence when released into the reagent region and emits fluorescence by reacting with a double-stranded nucleic acid. A kit for quantifying a nucleic acid, wherein a required amount of a compound is arranged and a dye compound in the reagent area is provided so as to be added in the reaction area. 13. The nucleic acid quantification kit according to claim 12, wherein the reagent region also contains a reagent for PCR. 14. The kit for nucleic acid quantification according to claim 13, wherein a reagent region is divided into a plurality of regions, and a reagent region containing the dye compound and a reagent region containing a PCR reagent are provided. 15. The kit for quantifying nucleic acid according to claim 12, further comprising a necessary amount of a primer necessary for PCR amplification of a specific sequence region of a target nucleic acid in the reaction region. 16. The dye compound is represented by the following general formula [I]: (In the above general formula [I], Represents a heterocycle, X is O, S, Se or Te, represents a pyrylium ring or a pyrylium-like ring, and R ¹ and R ² are each independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, R represents a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group or a substituted or unsubstituted cycloalkyl group, R ³ is -A or -L-a, L ^{is, -L 1 -, - L 2} -L 3 - or -L ⁴ -L ⁵ -L ⁶ -
And L ^{1 to} L ⁶ are each independently-(CH = C
H) -, 2-valent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted lower alkylene group, or -CH = R ⁴ - (R ⁴ represents a ring structure having an oxo group)
Represents a substituted or unsubstituted aryl group, -CH = R
⁵ (R ⁵ represents a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group or a substituted or unsubstituted aromatic ring), and R ¹ , R ^{2 of} the pyrylium ring containing X or a ring similar thereto,
The hydrogen atom bonded to the carbon atom to which R ³ is not bonded is a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, It may be substituted with a substituted or unsubstituted aryl group or a substituted or unsubstituted lower aralkyl group, and Y ⁻ represents an anion. The nucleic acid quantification kit according to any one of claims 12 to 15, which is a compound represented by the formula (4). 17. A compound represented by the general formula [I] is
2,4-bis (N, N-dimethylaminophenyl) -6
-Methylpyrylium salt, or 2,4-bis (N, N-
The kit for nucleic acid quantification according to claim 16, which is a dimethylaminophenyl) -6-methylthiopyrylium salt. 18. The method according to claim 12, wherein the dye compound is water-insoluble, and further comprises a solvent for dissolving the dye compound in a reaction solution after PCR in the reaction region to react with an amplification product. A kit for quantifying nucleic acid according to the item Crab. 19. The P according to claim 12, wherein a solution of the dye compound is arranged in a reagent area.
A kit for quantifying CR amplification products. 20. A process of extracting a nucleic acid from a sample containing a microorganism or a cell to be detected, a step of preparing a stepwise diluted sample of the obtained nucleic acid extract, and a step specific to the microorganism or cell to be detected for each diluted sample. The process of performing PCR for amplifying the sequence and the amplification product composed of the double-stranded nucleic acid obtained by the PCR do not emit fluorescence when released, and
The fluorescence obtained by reacting a dye compound that emits fluorescence by reacting with the double-stranded nucleic acid is measured, and the number of microorganisms or cells to be detected, the number of specific genes, or the specific gene is determined from the dilution ratio of the diluted sample from which fluorescence was obtained. A measuring method characterized by obtaining the number of copies of. 21. The dye compound is represented by the following general formula [I]: (In the above general formula [I], Represents a heterocycle, X is O, S, Se or Te, represents a pyrylium ring or a pyrylium-like ring, and R ¹ and R ² are each independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, R represents a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group or a substituted or unsubstituted cycloalkyl group, R ³ is -A or -L-a, L ^{is, -L 1 -, - L 2} -L 3 - or -L ⁴ -L ⁵ -L ⁶ -
And L ^{1 to} L ⁶ are each independently-(CH = C
H) -, 2-valent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted lower alkylene group, or -CH = R ⁴ - (R ⁴ represents a ring structure having an oxo group)
Represents a substituted or unsubstituted aryl group, -CH = R
⁵ (R ⁵ represents a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group or a substituted or unsubstituted aromatic ring), and R ¹ , R ^{2 of} the pyrylium ring containing X or a ring similar thereto,
The hydrogen atom bonded to the carbon atom to which R ³ is not bonded is a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, It may be substituted with a substituted or unsubstituted aryl group or a substituted or unsubstituted lower aralkyl group, and Y ⁻ represents an anion. The measuring method according to claim 20, which is a compound represented by the formula (4). 22. A compound represented by the general formula [I]:
2,4-bis (N, N-dimethylaminophenyl) -6
-Methylpyrylium salt, or 2,4-bis (N, N-
The measuring method according to claim 21, which is a dimethylaminophenyl) -6-methylthiopyrylium salt. 23. The dye compound is insoluble in water, PCR is carried out in a reaction system containing the dye compound and an aqueous medium, and then a solvent is added to the reaction system to dissolve the dye compound for amplification. The measuring method according to any one of claims 20 to 22, which is caused to react with a product. 24. The measuring method according to claim 20, wherein the amplification product has a chain length of 100 base pairs or more. 25. The measuring method according to any one of claims 20 to 24, wherein the chain length of the primer is 30 bases or less. 26. A reaction vessel having a required amount of a dye compound which does not emit fluorescence when released and which emits fluorescence when reacted with a double-stranded nucleic acid, in each reaction area of a large number of PCR reaction areas, These reaction zones can be used to form serial dilutions of a nucleic acid extract extracted from a sample containing a microorganism or cell to be detected, and for amplifying a sequence unique to the microorganism or cell to be detected at each dilution. A kit for measuring the number of microorganisms or cells to be detected, the number of specific genes or the copy number of specific genes, which is characterized in that PCR can be performed. 27. The dye compound is represented by the following general formula [I]: (In the above general formula [I], Represents a heterocycle, X is O, S, Se or Te, represents a pyrylium ring or a pyrylium-like ring, and R ¹ and R ² are each independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, R represents a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group or a substituted or unsubstituted cycloalkyl group, R ³ is -A or -L-a, L ^{is, -L 1 -, - L 2} -L 3 - or -L ⁴ -L ⁵ -L ⁶ -
And L ^{1 to} L ⁶ are each independently-(CH = C
H) -, 2-valent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted lower alkylene group, or -CH = R ⁴ - (R ⁴ represents a ring structure having an oxo group)
Represents a substituted or unsubstituted aryl group, -CH = R
⁵ (R ⁵ represents a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group or a substituted or unsubstituted aromatic ring), and R ¹ , R ^{2 of} the pyrylium ring containing X or a ring similar thereto,
The hydrogen atom bonded to the carbon atom to which R ³ is not bonded is a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, It may be substituted with a substituted or unsubstituted aryl group or a substituted or unsubstituted lower aralkyl group, and Y ⁻ represents an anion. 27. The kit according to claim 26, which is a compound represented by the formula: 28. A compound represented by the general formula [I]:
2,4-bis (N, N-dimethylaminophenyl) -6
-Methylpyrylium salt, or 2,4-bis (N, N-
The kit according to claim 26 or 27, which is a dimethylaminophenyl) -6-methylthiopyrylium salt. 29. The dye compound according to claim 26, which is water-insoluble, and further comprises a solvent for dissolving the dye compound in a reaction solution after PCR in the reaction region to react with an amplification product. Kit described in Crab. 30. The kit according to claim 26, further comprising a primer for PCR amplification of a sequence unique to the microorganism or cell to be detected in the reaction region. 31. MPN-PC for a nucleic acid sample in the presence of a primer required for amplification of a specific sequence region of a target nucleic acid.
A dye that does not fluoresce when released and does not fluoresce in an amplified product consisting of a double-stranded nucleic acid that is amplified when the target nucleic acid is present in the sample and the process of performing R A process for measuring the intensity of fluorescence obtained when a compound is reacted and a method for quantifying a nucleic acid, which comprises determining the amount of target nucleic acid in the nucleic acid sample from the measured fluorescence intensity. 32. The dye compound is represented by the following general formula [I]: (In the above general formula [I], Represents a heterocycle, X is O, S, Se or Te, represents a pyrylium ring or a pyrylium-like ring, and R ¹ and R ² are each independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, R represents a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group or a substituted or unsubstituted cycloalkyl group, R ³ is -A or -L-a, L ^{is, -L 1 -, - L 2} -L 3 - or -L ⁴ -L ⁵ -L ⁶ -
And L ^{1 to} L ⁶ are each independently-(CH = C
H) -, 2-valent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted lower alkylene group, or -CH = R ⁴ - (R ⁴ represents a ring structure having an oxo group)
Represents a substituted or unsubstituted aryl group, -CH = R
⁵ (R ⁵ represents a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group or a substituted or unsubstituted aromatic ring), and R ¹ , R ^{2 of} the pyrylium ring containing X or a ring similar thereto,
The hydrogen atom bonded to the carbon atom to which R ³ is not bonded is a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, It may be substituted with a substituted or unsubstituted aryl group or a substituted or unsubstituted lower aralkyl group, and Y ⁻ represents an anion. 32. The method for quantifying a nucleic acid according to claim 31, which is a compound represented by the formula (1). 33. A compound represented by the general formula [I]:
2,4-bis (N, N-dimethylaminophenyl) -6
-Methylpyrylium salt, or 2,4-bis (N, N-
33. The method for quantifying a nucleic acid according to claim 32, which is a dimethylaminophenyl) -6-methylthiopyrylium salt. 34. MPN-PCR in a reaction system in which the dye compound is water-insoluble and contains the dye compound and an aqueous medium.
The reaction with the amplification product is performed by adding a solvent to the reaction system to dissolve the dye compound after carrying out
The method for quantifying a nucleic acid according to any one of 3 above. 35. The method for quantifying a nucleic acid according to claim 31, wherein the amplification product has a chain length of 100 base pairs or more. 36. The method for quantifying a nucleic acid according to claim 31, wherein the chain length of the primer is 30 bases or less. 37. A reaction vessel for MPN-PCR, comprising a reactor in which a necessary amount of a dye compound which does not emit fluorescence when released and which emits fluorescence by reacting with a double-stranded nucleic acid is arranged. A kit for quantifying nucleic acids. 38. The nucleic acid quantification kit according to claim 37, wherein a necessary amount of a primer necessary for amplification of MPN-PCR of a specific sequence region of a target nucleic acid is further arranged in the reaction region. 39. The dye compound is represented by the following general formula [I]: (In the above general formula [I], Represents a heterocycle, X is O, S, Se or Te, represents a pyrylium ring or a pyrylium-like ring, and R ¹ and R ² are each independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, R represents a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group or a substituted or unsubstituted cycloalkyl group, R ³ is -A or -L-a, L ^{is, -L 1 -, - L 2} -L 3 - or -L ⁴ -L ⁵ -L ⁶ -
And L ^{1 to} L ⁶ are each independently-(CH = C
H) -, 2-valent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted lower alkylene group, or -CH = R ⁴ - (R ⁴ represents a ring structure having an oxo group)
Represents a substituted or unsubstituted aryl group, -CH = R
⁵ (R ⁵ represents a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group or a substituted or unsubstituted aromatic ring), and R ¹ , R ^{2 of} the pyrylium ring containing X or a ring similar thereto,
The hydrogen atom bonded to the carbon atom to which R ³ is not bonded is a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, It may be substituted with a substituted or unsubstituted aryl group or a substituted or unsubstituted lower aralkyl group, and Y ⁻ represents an anion. 39. The nucleic acid quantification kit according to claim 37 or 38, which is a compound represented by the formula (4). 40. A compound represented by the general formula [I]:
2,4-bis (N, N-dimethylaminophenyl) -6
-Methylpyrylium salt, or 2,4-bis (N, N-
40. The nucleic acid quantification kit according to claim 39, which is a dimethylaminophenyl) -6-methylthiopyrylium salt. 41. The method according to claim 37, wherein the dye compound is insoluble in water, and further has a solvent for dissolving the dye compound in the reaction solution after MPN-PCR in the reaction region to react with the amplification product. The kit for quantifying nucleic acid according to any one of 1. 42. A reaction region for MPN-PCR, and a reagent region isolated from the reaction region, wherein the reagent region does not emit fluorescence when released, and emits fluorescence by reacting with a double-stranded nucleic acid. A kit for quantifying nucleic acid, wherein a required amount of a coloring compound to be emitted is arranged, and the coloring compound in the reagent area is provided so as to be added in the reaction area. 43. The nucleic acid quantification kit according to claim 42, wherein the reagent region also contains a reagent for MPN-PCR. 44. The nucleic acid quantification kit according to claim 43, wherein a reagent region is divided into a plurality of regions, and a reagent region containing the dye compound and a reagent region containing a reagent for MPN-PCR are provided. 45. The nucleic acid quantification kit according to claim 42, wherein a necessary amount of a primer necessary for amplification of MPN-PCR of a specific sequence region of a target nucleic acid is further arranged in the reaction region. 46. The dye compound is represented by the following general formula [I]: (In the above general formula [I], Represents a heterocycle, X is O, S, Se or Te, represents a pyrylium ring or a pyrylium-like ring, and R ¹ and R ² are each independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, R represents a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group or a substituted or unsubstituted cycloalkyl group, R ³ is -A or -L-a, L ^{is, -L 1 -, - L 2} -L 3 - or -L ⁴ -L ⁵ -L ⁶ -
And L ^{1 to} L ⁶ are each independently-(CH = C
H) -, 2-valent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted lower alkylene group, or -CH = R ⁴ - (R ⁴ represents a ring structure having an oxo group)
Represents a substituted or unsubstituted aryl group, -CH = R
⁵ (R ⁵ represents a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group or a substituted or unsubstituted aromatic ring), and R ¹ , R ^{2 of} the pyrylium ring containing X or a ring similar thereto,
The hydrogen atom bonded to the carbon atom to which R ³ is not bonded is a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, It may be substituted with a substituted or unsubstituted aryl group or a substituted or unsubstituted lower aralkyl group, and Y ⁻ represents an anion. The nucleic acid quantification kit according to any one of claims 42 to 45, which is a compound represented by the formula (4). 47. A compound represented by the general formula [I]:
2,4-bis (N, N-dimethylaminophenyl) -6
-Methylpyrylium salt, or 2,4-bis (N, N-
The nucleic acid quantification kit according to claim 46, which is a dimethylaminophenyl) -6-methylthiopyrylium salt. 48. The dye compound is water-insoluble, and further comprises a solvent for dissolving the dye compound in the reaction solution after MPN-PCR in the reaction region to react with the amplification product. The kit for quantifying nucleic acid according to any one of 1. 49. The M according to claim 42, wherein a solution of the dye compound is arranged in a reagent region.
A kit for quantifying a PN-PCR amplification product. 50. A process of extracting a nucleic acid from a sample containing a microorganism or a cell to be detected, a step of preparing a stepwise diluted sample of the obtained nucleic acid extract, and a step specific to the microorganism or cell to be detected for each diluted sample. The process of performing MPN-PCR for amplifying the sequence and the amplification product composed of the double-stranded nucleic acid obtained by the MPN-PCR do not emit fluorescence when released, and thus the fluorescence is obtained by reacting with the double-stranded nucleic acid. It is characterized in that the fluorescence obtained by reacting a coloring compound is measured, and the number of microorganisms or cells to be detected, the number of specific genes, or the copy number of specific genes is determined from the dilution ratio of the diluted sample from which fluorescence was obtained. Measuring method. 51. The dye compound is represented by the following general formula [I]: (In the above general formula [I], Represents a heterocycle, X is O, S, Se or Te, represents a pyrylium ring or a pyrylium-like ring, and R ¹ and R ² are each independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, R represents a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group or a substituted or unsubstituted cycloalkyl group, R ³ is -A or -L-a, L ^{is, -L 1 -, - L 2} -L 3 - or -L ⁴ -L ⁵ -L ⁶ -
And L ^{1 to} L ⁶ are each independently-(CH = C
H) -, 2-valent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted lower alkylene group, or -CH = R ⁴ - (R ⁴ represents a ring structure having an oxo group)
Represents a substituted or unsubstituted aryl group, -CH = R
⁵ (R ⁵ represents a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group or a substituted or unsubstituted aromatic ring), R ¹ or R ^{2 of} the pyrylium ring containing X or a ring similar thereto,
The hydrogen atom bonded to the carbon atom to which R ³ is not bonded is a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, It may be substituted with a substituted or unsubstituted aryl group or a substituted or unsubstituted lower aralkyl group, and Y ⁻ represents an anion. The measuring method according to claim 50, which is a compound represented by the formula (1). 52. A compound represented by the general formula [I]:
2,4-bis (N, N-dimethylaminophenyl) -6
-Methylpyrylium salt, or 2,4-bis (N, N-
The measuring method according to claim 51, which is a dimethylaminophenyl) -6-methylthiopyrylium salt. 53. MPN-PCR in a reaction system in which the dye compound is water-insoluble and contains the dye compound and an aqueous medium.
After carrying out, the reaction with the amplification product by adding a solvent to the reaction system to dissolve the dye compound.
2. The measuring method according to any one of 2. 54. The measuring method according to claim 50, wherein the amplification product has a chain length of 100 base pairs or more. 55. The measuring method according to claim 50, wherein the chain length of the primer is 30 bases or less. 56. A reactor provided with a necessary amount of a dye compound which does not emit fluorescence when released and which emits fluorescence when reacted with a double-stranded nucleic acid, in each of the reaction zones of a large number of MPN-PCR reaction zones. However, these reaction regions can be used to form a serial dilution of a nucleic acid extract extracted from a sample containing a microorganism or cell to be detected, and at each dilution, a sequence specific to the microorganism or cell to be detected is amplified. MP for
A kit for measuring the number of microorganisms or cells to be detected, the number of specific genes, or the copy number of specific genes, which is characterized by being able to perform N-PCR. 57. The dye compound is represented by the following general formula [I]: (In the above general formula [I], Represents a heterocycle, X is O, S, Se or Te, represents a pyrylium ring or a pyrylium-like ring, and R ¹ and R ² are each independently a hydrogen atom, a halogen atom, a sulfonate group, an amino group, R represents a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted lower aralkyl group or a substituted or unsubstituted cycloalkyl group, R ³ is -A or -L-a, L ^{is, -L 1 -, - L 2} -L 3 - or -L ⁴ -L ⁵ -L ⁶ -
And L ^{1 to} L ⁶ are each independently-(CH = C
H) -, 2-valent group derived from a substituted or unsubstituted aryl group, a substituted or unsubstituted lower alkylene group, or -CH = R ⁴ - (R ⁴ represents a ring structure having an oxo group)
Represents a substituted or unsubstituted aryl group, -CH = R
⁵ (R ⁵ represents a substituted or unsubstituted heterocycle, a substituted or unsubstituted cycloalkyl group or a substituted or unsubstituted aromatic ring), and R ¹ , R ^{2 of} the pyrylium ring containing X or a ring similar thereto,
The hydrogen atom bonded to the carbon atom to which R ³ is not bonded is a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a cyano group, a substituted or unsubstituted lower alkyl group, It may be substituted with a substituted or unsubstituted aryl group or a substituted or unsubstituted lower aralkyl group, and Y ⁻ represents an anion. 57. The kit according to claim 56, which is a compound represented by the formula: 58. A compound represented by the general formula [I]:
2,4-bis (N, N-dimethylaminophenyl) -6
-Methylpyrylium salt, or 2,4-bis (N, N-
The kit according to claim 56 or 57, which is a dimethylaminophenyl) -6-methylthiopyrylium salt. 59. The dye compound is water-insoluble, and further comprises a solvent for dissolving the dye compound in the reaction solution after MPN-PCR in the reaction region to react with the amplification product. The kit according to any one of 1. 60. The kit according to claim 56, further comprising a primer for MPN-PCR amplification of a sequence peculiar to the microorganism or cell to be detected in the reaction region.