JP2001269196A - Quantitative method for nucleic acid in test object and method for counting number of molecule of nucleic acid in test object - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for precisely and quantitatively determining a nucleic acid in a test object such as a small amount of a gene or a mRNA in a specimen. SOLUTION: The number of molecule of the nucleic acid in the test object initially existing in the specimen is counted and quantitatively determined by dividedly injecting a PCR solution containing a target DNA derived from the nucleic acid in the test object into a channel of a capillary plate, treating the capillary plate with temperature cycling of PCR without doing anything else, multiplying the target DNA in the channel by PCR and specifying and counting a channel exhibiting fluorescence caused by the multiplied target DNA through image analysis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for quantifying a nucleic acid such as a gene or mRNA present in a sample in a trace amount. More specifically, the present invention amplifies a nucleic acid such as a gene or mRNA present in a sample in a trace amount or a target DNA derived from the nucleic acid based on PCR, and counts the number of molecules of the nucleic acid originally present in the sample. The present invention relates to a method and a method for quantifying the concentration of a nucleic acid from the counting result.

[0002]

BACKGROUND OF THE INVENTION In vitro nucleic acid amplification technology has provided a powerful tool for the detection and analysis of small amounts of nucleic acids. Such techniques have provided significant advances in the diagnosis of infectious and genetic diseases, isolation of genes for biochemical analysis, and the detection of certain nucleic acids in forensic medicine. At present, the most versatile technology is the polymerase chain reaction (Polymerase Chain®) using a thermostable polymerase (Taq polymerase).
action (hereinafter referred to as PCR or PCR method)
Theoretically, DNA (fragment) can be amplified more than 1 billion times. However, the amplification efficiency and accuracy in PCR are affected by various factors such as the processing temperature, the base sequence of the nucleic acid, the concentration and type of the reaction solution components, and the number (concentration) of the nucleic acid molecules to be measured is determined by the final PCR. Accurate determination by product concentration measurements or real-time measurements during the PCR process has been difficult.

Under such circumstances, a technique called "digital PCR" has recently been proposed as an attempt to accurately quantify a trace amount of a gene. Bert Vogelstein et al.
Proc. Natl. Acad. Sci. USA, 96
Vol., Pages 9236-9241. Following this method,
The gene sample to be quantified is subjected to limiting dilution with a PCR solution and dispensed into wells of a titer plate. After performing PCR of the sample solution on a titer plate and amplifying the DNA derived from the gene, the fluorescent signal in each hole is measured using a fluorescent probe or the like, and the number of holes in which the amplified DNA emits fluorescence is measured. Is counted. Due to the limiting dilution, it may be assumed that one molecule of DNA is present in each well, so that the number of fluorescent wells corresponds to the number of DNA molecules in the sample, that is, the amount of mRNA in the sample, It can be quantified. This method unfortunately has a number of potential problems which are mentioned below.

[0004] PCR is performed on a titer plate, and D
60 cycles of PCR before NA amplification can be detected
You need to do it about once. However, when the number of PCR cycles exceeds 40, P
It is recognized by those skilled in the art that CR products are generated and contaminate the amplified product (hereinafter referred to as non-specific DNA amplification).
There are two possible causes for this. One is that a very small amount of DNA accidentally mixed into a PCR solution is amplified by PCR, and is generally called “carry over”. This phenomenon becomes more remarkable as the concentration of the target DNA in the PCR solution decreases. In the other, primers form a double strand, which serves as a template, and is amplified by PCR, resulting in a non-specific amplification product.
In order to avoid this, it is understood by those skilled in the art that the number of cycles is generally not more than 40. In any case, since the digital PCR method requires a cycle number of 60 or more times, it becomes difficult to distinguish the target PCR amplification product from the undesired mixed PCR amplification product, and an accurate DN
Quantification of A naturally becomes difficult. Such non-specific DN
Several countermeasures are already known to avoid A-amplification, but all require complicated operations or expensive reagents. For example, 1) one of the bases used as a substrate for DNA synthesis, dUTP is added in place of dTTP to synthesize DNA, and dU incorporated into DNA in the next PCR
A method of adding an enzyme that cleaves the TP portion, 2) a method of performing two-step PCR using two types of different primer pairs,
3) A method of removing a carry-over DNA by adding a restriction enzyme that specifically cleaves an appropriate base sequence in DNA to be amplified to a solution before PCR high-temperature treatment is known.

[0005] Furthermore, in the digital PCR method, conditions must be set so that DNA molecules are included in, for example, half of the total number of holes in a titer plate by diluting at various magnifications to obtain a limiting dilution.

[0006] In the digital PCR method, when limiting dilution is performed so that DNA molecules are contained in half of the total number of holes in the titer plate, DNs are included in one hole from Poisson distribution.
The probability that two molecules of A molecules enter is 12%, and the probability that three molecules enter A molecules is 3
%, And cannot be ignored. Therefore, the number of holes counted and DN
The number of A molecules does not match, the evaluation is fluctuated, and the reliability of quantification is reduced.

[0007]

In view of the above problems of the digital PCR method, a small amount of gene or mR
In order to accurately determine (ie, count) the number of molecules of nucleic acid such as NA and to quantify the concentration including the concentration of nucleic acid, a method alternative to digital PCR is desired. Therefore, the present invention
An object of the present invention is to solve the problems of the digital PCR method and to provide a method for simply and accurately quantifying an analyte nucleic acid in a sample or a method for counting the number of molecules of the analyte nucleic acid.

[0008]

That is, according to the present invention, there is provided a method for quantifying an analyte nucleic acid, which comprises a target DNA derived from the analyte nucleic acid from a sample, and a PC containing a fluorescent reagent.
A first step of preparing an R solution, a second step of placing the PCR solution on a capillary plate having a predetermined number of channels, and sealing both sides of the capillary plate with an elastic transparent plate; A third step of enclosing the PCR solution in each of the channels, and exposing the capillary plate whose both sides are sealed to a preset temperature cycle to perform PCR of the target DNA derived from the analyte nucleic acid.
Performing the fourth step, and subjecting the capillary plate whose both sides are sealed to fluorescence measurement to count the number of fluorescent channels that emit fluorescence, wherein the fluorescent channels are the analytes amplified by the PCR. A fifth step, which is a channel containing a target DNA derived from the nucleic acid, is provided.

Further, the present invention further comprises, following the fifth step, a step of obtaining the concentration of the analyte nucleic acid based on the counted number of the fluorescent channels. Provides a quantification method.

[0010] The present invention also provides the above method for quantifying an analyte nucleic acid, further comprising a step of pretreating a capillary plate with a protein adsorption inhibitor.

[0011] Preferably, in the above-described method for quantifying an analyte nucleic acid, the fluorescent reagent comprises a minor glove-bound fluorescent dye or a fluorescent probe oligonucleotide utilizing fluorescence energy transfer.

[0012] Preferably, in the above-described method for quantifying an analyte nucleic acid, the elastic transparent plate for sealing both sides of the capillary plate is made of silicone rubber.

Further, according to another aspect of the present invention, there is provided a method for counting the number of molecules of a nucleic acid to be analyzed, the method comprising a PC containing a target DNA derived from the nucleic acid to be analyzed from a sample and a fluorescent reagent.
A first step of preparing an R solution, a second step of placing the PCR solution on a capillary plate having a predetermined number of channels, and sealing both sides of the capillary plate with an elastic transparent plate; A third step of enclosing the PCR solution in each of the channels, and exposing the capillary plate whose both sides are sealed to a preset temperature cycle to perform PCR of the target DNA derived from the analyte nucleic acid.
Performing the fourth step, and subjecting the capillary plate whose both sides are sealed to fluorescence measurement to count the number of fluorescent channels that emit fluorescence, wherein the fluorescent channels are the analytes amplified by the PCR. A fifth step which is a channel containing a target DNA derived from the nucleic acid, and a method for counting the number of molecules of the analyte nucleic acid, the method comprising:

Hereinafter, the present invention will be described in detail according to preferred embodiments.

[0015]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A major aspect of the present invention is a method for quantifying the number or concentration of analyte nucleic acids, comprising the steps of: using a PCR solution containing a target DNA derived from an analyte nucleic acid from a sample and a fluorescent reagent; A first step of preparing, a second step of placing the PCR solution on a capillary plate having a predetermined number of channels, and sealing both sides of the capillary plate with an elastic transparent plate; A third step of enclosing the channel into the respective channels, and a fourth step of exposing the capillary plate having both sides sealed to a preset temperature cycle to perform PCR of the target DNA derived from the analyte nucleic acid, The two-sided sealed capillary plate is subjected to fluorescence measurement, and the number of fluorescent channels emitting fluorescence is counted. A method of quantifying analyte nucleic acids, characterized in that it comprises a fifth step is a channel containing the target DNA from the subject nucleic acid amplified by.

Therefore, the essence of the method for quantifying an analyte nucleic acid according to the present invention is that PCR comprising a target DNA derived from the analyte nucleic acid is performed.
Enclose the solution in the channel of the capillary plate,
The object of the present invention is to amplify the target DNA by PCR, detect and analyze the fluorescence emitted from the amplified target DNA, and thereby count the number of molecules of the analyte nucleic acid present in the sample first. Therefore, hereinafter, the “quantitative method of the present invention” includes the method of quantifying the concentration of the analyte nucleic acid and the method of counting the number of molecules of the analyte nucleic acid according to the present invention, and both or any of them are used.

As used herein, “analyte nucleic acid” refers to
It includes DNA or RNA (including mRNA) and may be single-stranded or double-stranded. However, in order to apply the quantification method of the present invention, the target nucleic acid itself or a target derived therefrom is required. It is necessary that the base sequence of DNA (described later) is known at least to the extent that PCR is possible. Also,
The analyte nucleic acid need not necessarily be in pure form, but may be only a fraction of a complex mixture (eg, a portion of a β-globulin gene). Furthermore, the analyte nucleic acid may be of any origin, such as natural or artificially synthesized, but in view of the field of application of the present invention, extracted from blood, cells, tissues, microorganisms or viruses. It is preferably DNA or RNA. As used herein, the term “sample” refers to a sample containing such an analyte nucleic acid, and is not particularly limited. However, for the same reasons as described above, biological samples (particularly, clinical samples and cytological samples) are preferably used in the quantification method of the present invention. Clinical samples include urine, feces, sputum,
Blood and the like. Examples of the cytological specimen include body cavity fluids such as liquid pleural effusion and ascites, and tumor cells obtained by a biopsy method such as needle aspiration. Usually, in order to obtain an analyte nucleic acid (DNA) from these specimens, the specimen is pre-treated to extract the nucleic acid. The preprocessing method differs depending on the type of the sample, and the established processing method may be applied to each sample. Known methods for extracting nucleic acids include the phenol-chloroform method and the guanidine thiocyanate method. On the other hand, when the test nucleic acid is RNA, the RNA itself cannot be a substrate for PCR. However, PCR can be applied to RNA analysis by synthesizing cDNA from RNA using reverse transcriptase. Therefore, by combining the reverse transcriptase reaction with PCR, the quantification method of the present invention can be applied even when the analyte nucleic acid is RNA. In particular, such a case is when the specimen contains an RNA virus (HIV, HCV, hunter virus, etc.). This is because RNA viruses have not chromosomes but DNA but RNA. Further, even when DNA can be analyzed as a test nucleic acid, if mRNA is used as the test nucleic acid instead of the DNA, the mRNA may have a higher copy number per cell than the DNA in some cases. It is more advantageous to analyze the mRNA than to analyze the DNA. Therefore,
As used herein, “target DN derived from analyte nucleic acid”
"A" is DNA amplified by PCR, and when the nucleic acid to be tested is DNA, refers to the DNA, and when the nucleic acid to be tested is RNA (mRNA), the DNA obtained therefrom
(CDNA).

The reverse transcriptase reaction can be easily carried out according to a conventional method. For example, for a sample, a commercially available standard cDNA synthesis kit (Amersham Pharm
Time Saver cDNA Syn manufactured by Acia
thesis Kit) to convert mRNA into cD
Synthesize NA. The optimum pH of the reverse transcriptase (around 8.3) in the reverse transcriptase reaction is determined by the pH of the PCR buffer.
(For example, 8.4 to 8.8), and Mg ²⁺ required for both reactions is also common. Therefore, after the reverse transcriptase reaction, by diluting the buffer with the buffer used for PCR without replacing the buffer, buffer conditions suitable for PCR can be obtained. Therefore, cD synthesized by reverse transcription
NA can be directly subjected to amplification by PCR. In this case, in a normal PCR, the double-stranded DN
Similarly to the case where A is denatured to form a single-stranded DNA and then PCR is performed, the mRNA-cDNA duplex is denatured to obtain a single-stranded cDNA. At this time, the reverse transcriptase used at the same time is inactivated.

The first step included in the quantification method of the present invention comprises preparing a PCR solution containing a target DNA (single strand) derived from a nucleic acid to be tested and a fluorescent reagent.
Specifically, a PCR solution is prepared by mixing the target DNA, a PCR buffer (comprising primers, dNTPs, Taq DNA polymerase, etc.) and a fluorescent reagent. The primers used herein are those obtained by synthesizing two types of primers on the 5 ′ side and 3 ′ side of the target DNA according to a conventional method. For primer design, please refer to GENET
Commercially available software such as YX (software development) can be used.

The fluorescent reagent is used for the purpose of detecting the amplified target DNA, and needs to have a property that its fluorescence intensity increases in proportion to the amount of the amplified DNA. In the present invention, a DNA-specific fluorescent dye of a minor groove binding type is preferably used as a fluorescent reagent.
This type of fluorescent dye binds to the minor groove of the DNA strand and is incorporated into the amplified DNA in proportion to its amount. In the absence of DNA, the fluorescent dye itself emits only weak fluorescence. Hoechst 3325 is a typical example of such a minor groove-binding fluorescent dye.
8 and Hoechst 33342.

A fluorescent probe utilizing fluorescence energy transfer can also be used as a fluorescent reagent in the quantification method of the present invention. Such a probe is an oligonucleotide in which a pair of two types of fluorescent dyes acting as a donor dye and an acceptor dye or a pair of a single type of fluorescent dye and a pair of dyes that quench the fluorescence thereof are used, and the PCR proceeds. As a result, the fluorescence energy transfer is eliminated by hybridization or decomposition of the probe, resulting in an increase in the fluorescence intensity of the donor dye. Thus, DNA amplification can be estimated from the fluorescence measurement.

The second step included in the quantification method of the present invention comprises placing the PCR solution on a capillary plate having a predetermined number of channels. The capillary plate used here is preferably composed of a glass or silicon material. 1a and 1b show an example of a capillary plate 1 used in the present invention. A typical one has an outer diameter of several to several tens mm, a diameter of several to about 200 μm, and a depth (thickness) of 0.5.
A predetermined number (thousands to several millions of which can be arbitrarily selected) of cylindrical columns (also referred to as channels 2) having a size of to several mm is provided. To place the PCR solution on the capillary plate 1, the PCR solution is brought into contact with one side (bottom surface) of the capillary plate 1. Then, a PCR solution is injected into each channel 2 by capillary action. This injection stage is schematically illustrated in FIG. 3A (described later). Alternatively, the PCR solution may be injected into the channel by pipetting from the top of the capillary plate.

The third step included in the quantification method of the present invention comprises sealing both sides of the capillary plate with an elastic transparent plate, and enclosing the PCR solution in each of the channels. Here, as shown in FIG. 2a, both sides of the capillary plate 1 are elastically transparent plates (for example, while the PCR solution is still being injected into each channel 2 of the capillary plate 1 in the second step). And sealing with a silicone rubber plate 3). When the capillary plate 1 is sealed, the PCR solution
And transfer of the PCR solution between channels does not occur. Thereafter, the outside of the silicon rubber plate 3 is covered with a cover glass 4.

In the fourth step included in the quantification method of the present invention, the above-mentioned capillary plate having both sides closed is exposed to a preset temperature cycle to perform PCR of the target DNA.
Is performed. This step is performed in the same manner as ordinary PCR. See Figure 2a and Figure 2b. First, in the third step, the capillary plate 1 whose both surfaces are sealed and covered with the cover glass 4 is housed in a chamber 5 designed for PCR. Chamber 5
Is made of a material having high thermal conductivity, for example, an oxygen-free copper or aluminum alloy plate, and the chamber 5 itself is directly heated / cooled as a heat block. After placing the capillary plate 1 in the chamber 5, tighten the fixing screw 6 to fix the capillary plate 1 (FIG. 2).
b). With the chamber 5 as it is, it is placed on an aluminum block 7 of a commercially available thermal cycler. The temperature, reaction time and cycle number of each cycle in the thermal cycler are set, and PCR is performed. The temperature cycle employed in the present invention is not particularly different from the temperature cycle of ordinary PCR. The amplification method itself of the quantification method of the present invention
Digital PCR is no different from normal PCR
It is preferable to minimize the "carryover" caused by the hybrid DNA as described in the method section and to suppress nonspecific DNA amplification (background). For this,
Various measures and ingenuity are as recognized by those skilled in the art. Thus, PCR is performed using the target DNA as a template, and the DNA is amplified.

However, the PCR reaction solution is directly enclosed in a channel of a capillary plate,
May not progress PCR. this is,
DNA polymerase is adsorbed on the inner wall of the channel,
This is because it has an adverse effect on PCR. Such a protein adsorption phenomenon can occur even when another reaction vessel is used, but is particularly remarkable in the case of a capillary plate because the total surface area of the reaction vessel becomes very large. Therefore, in a preferred embodiment of the present invention, the capillary plate is treated with a substance having a protein adsorption inhibitory action (referred to as a protein adsorption inhibitor) prior to PCR to prevent the adsorption phenomenon. The pretreatment step with the protein adsorption inhibitor is performed before the second step. Alternatively, PCR may be performed by mixing a protein adsorption inhibitor into the PCR solution. In this case, it is preferable that the protein adsorption inhibitor is in excess with respect to the DNA polymerase concentration. As an example of the protein adsorption inhibitor, bovine serum albumin is exemplified. Suitable concentrations are from about 0.01 to about 0.1%.

The fifth step included in the quantification method of the present invention comprises subjecting a capillary plate, whose both sides are sealed, to fluorescence measurement and counting the number of fluorescent channels that emit fluorescence. Here, the fluorescent channel is the P channel.
Target DN derived from subject nucleic acid amplified by CR
A channel including A. Specifically, after the fourth step, the capillary plate 1 is taken out of the chamber 5 and the surface of the capillary plate is observed under a fluorescence microscope with the cover glass 4 attached. The fluorescence microscope used in the present invention may be a commercially available product equipped with an excitation light source, an excitation light wavelength selection filter (in the case of Hoechst 33258, an ultraviolet transmission filter), a fluorescence wavelength selection filter, and the like. Further, a fluorescent image of the surface of the capillary plate is captured by a high-sensitivity camera (II-CCD camera), and the obtained image is analyzed by an image processing system. In this way, it is possible to measure the fluorescence signal from the fluorescent reagent for each channel of the capillary plate.
At the time of image processing, an appropriate threshold is set, and a background fluorescent signal below the threshold is removed by a filter. Then, only the channel whose fluorescence signal has changed (that is, the fluorescent channel that emits fluorescence) is specified. In order to count the number of such fluorescent channels, the number of channels having high luminance is visually counted on the image-processed fluorescent image. Alternatively, based on the fluorescence signal in the image processing system,
The processing may be performed by a microcomputer and counting may be performed automatically. In any case, the processing of these series of fluorescent signals is performed by a combination of known techniques.
Can be performed.

[0027] The fluorescent channel contains a target DNA amplified by PCR. This is because the PCR solution encapsulated in the third step in the channel contains a target DNA molecule derived from the analyte nucleic acid. The target DNA molecule is amplified in the fourth step, and the amplified DNA emits fluorescence with the fluorescent reagent, so that the fluorescence signal of the channel changes as compared to before the PCR. On the other hand, when the PCR solution in the channel does not contain the target DNA molecule,
No amplified DNA is produced by PCR. The fluorescence signal of the channel did not change from before the PCR, and this was also confirmed from experiments in Examples described later. Thus, by identifying the fluorescent channels and counting their total number on the capillary plate, the number of channels in which the target DNA molecule was present before PCR can be determined.

Under conditions where the number of target DNA molecules before PCR is sufficiently small relative to the total number of channels constituting the capillary plate, the number of target DNA molecules in most of the channels containing target DNA is 1 in the channels containing target DNA. If it is a numerator, it can be assumed from the Poisson distribution. Therefore, when the concentration of such target DNA is sufficiently low, the number of fluorescent channels will correspond to the number of molecules of target DNA.

This assumption is supported by the fact that the measured value substantially matches the expected value from the Poisson distribution in the experiments in the examples described later. As described above, according to the quantification method of the present invention, by counting the total number of fluorescent channels, the number of molecules of the target DNA in the PCR solution is counted, and further, the number of molecules of the analyte nucleic acid in the sample is determined. It will be possible to decide. If desired, the concentration of the target DNA (and thus the analyte nucleic acid) can be determined based on the number of molecules and the volume of the channel thus determined.

FIG. 3 schematically illustrates the quantification method of the present invention described above in detail for each of the steps corresponding to the first to fourth steps. According to the quantification method of the present invention,
A series of procedures can be easily understood by those skilled in the art with reference to the above description and this figure.

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

[0032]

EXAMPLES (Example 1) A PCR solution having the following composition was prepared. The target DNA (corresponding to the template DNA), the 5 ′ primer, and the 3 ′ primer used in this example have the following base sequences, respectively. These DNAs can be
It was obtained from an NA synthesizer (Vex Corporation). Target DNA: 5'-ATGGAAACCTGTTTGTTGGATATAAATACGTCGT
AGATGGGCACAGTGTG-3 '(described in SEQ ID NO: 1 in the sequence listing) 5' primer: 5'-CACACTGTGCCCATCTACGA-3 '(described in SEQ ID NO: 2 in the sequence listing) 3' primer: 5'-ATGGAAACCTGTTTGTTGGA-3 '(described in the sequence listing) PCR solution Target DNA 100 aM 5 ′ primer 1 μM 3 ′ primer 1 μM Mg ₂ Cl 6 mM BSA 0.1% each dNTP 0.5 mM Hoechst 33258 1 μM Taq antibody 0.056 μM Taq DNA polymerase 0.02 unit / μl Taq DNA polymerase 10-fold buffer solution 25 μl Distilled water Remaining volume 250 μl Using a capillary plate with an outer diameter of 25 mm provided with channels (about 100,000, volume of about 2 nl) having a diameter of 0.05 mm and a depth of 1 mm The PCR solution was dispensed into each channel by capillary action. Both sides of the capillary plate were sealed with a transparent silicon rubber plate, and the outside was further covered with a cover glass [FIG. 2 (a)]. This capillary plate is stored in the chamber, and the chamber is connected to a thermal cycler (PerkinElmer, DNA Thermal Cycler).
488) on the aluminum block [FIG. 2 (b)].

The temperature, reaction time and cycle number of each cycle in the thermal cycler were set as follows.
A CR was performed. That is, pretreatment at 95 ° C. for 3 minutes
The reaction was repeated twice, followed by a reaction at 95 ° C. for 2 minutes and then at 45 ° C. for 2 minutes, and this was repeated for 40 cycles. Further, the temperature was kept at 68 ° C. for 5 minutes, and the reaction was left at room temperature to terminate the PCR.

After completion of the PCR, the capillary plate was taken out of the chamber, and its surface was observed under an inverted fluorescence microscope (IX70, manufactured by Olympus, objective lens: 10 times) with the silicon rubber plate and the cover glass attached. A fluorescent image of the surface was taken with a high-sensitivity camera, II-CCD camera (ARGUS500 manufactured by Hamamatsu Photonics), and the obtained fluorescent image was recorded by an image processing system. This image is shown in FIG.
(A).

Further, a new PCR solution having the same composition as that of the above-mentioned PCR solution except that no target DNA was contained was prepared, and a control test was performed. FIG. 4 shows the obtained image.
(B).

Comparing the fluorescence image of FIG. 4A with the fluorescence image of FIG. 4B, the former image includes several tens of channels (fluorescence) which are clearly higher in brightness, and these channels have higher brightness. Are scattered between low channels. On the other hand, the latter image shows that there is no dark and high-luminance channel over all the channels. The fluorescent channel has increased brightness due to amplification of the target DNA molecule therein, indicating that the target DNA molecule was present in the channel before PCR. 4 used under the above PCR conditions
Even with a cycle number of 0 cycles, DNA amplification occurred sufficiently and was detectable. In addition, the results of the control test revealed that non-specific DNA amplification did not occur at all under these PCR conditions.

(Example 2) In the PCR solution used in Example 1, the concentration of the target DNA (100 aM) was changed variously, and a PCR solution having the same other composition was prepared. These PCR solutions were subjected to PCR in the same manner as in Example 1, fluorescence images were obtained for each concentration, and the number of fluorescent channels was counted. FIG. 5 shows the ratio of the obtained number of fluorescent channels to the total number of channels of the capillary plate, and plotted against the concentration of each target DNA. In FIG. 5, the curve indicates the ratio of the actually measured values thus obtained (the number of fluorescent channels: the total number of channels).
And the curve is a plot of expected values from a Poisson distribution. First, the curve is the target DN
This shows that the number of fluorescent channels increases in proportion to the concentration of A. The approximation of both curves suggests that the PCR solution used in this example follows the Poisson distribution. That is, in a region where the concentration of the target DNA is low (for example, the expected value is 0.1 or less), only one molecule of the target DNA is present in most of the fluorescent channels. One molecule is amplified by PCR, and the channel containing it emits fluorescence. Together with these facts and the results of Example 1, the number of fluorescent channels
It can be seen that the number of molecules of the target DNA in the R solution can be accurately estimated. Note that a part of the fluorescence image based on the data shown in FIG. 5 is directly shown in FIG. In the figure, the fluorescence images when the concentration of the target DNA is 100, 10, and 1 aM are (a), (b), and (c), respectively, and the left and right sides belong to a system obtained by diluting separate solutions. Since the counting results from the left and right fluorescent images almost match, it can be seen that the counting by the quantitative method of the present invention has reproducibility.

[0038]

According to the quantification method of the present invention, the amplified DNA can be detected with sufficient fluorescence even under the allowable number of cycles under ordinary PCR conditions. The decrease in counting accuracy is improved, and a reliable quantitative result is obtained.

For the same reason, the quantification method of the present invention does not require a special method or reagent for suppressing nonspecific DNA amplification, and is a simple quantification method.

Further, in the quantification method of the present invention, the number of channels corresponding to the wells of the titer plate employed in the digital PCR method is dramatically increased by using the capillary plate. Typically, tens of thousands or more of the hundreds of titer plates are obtained, and the DNA concentration range that can be analyzed (that is, the number of molecules of the test nucleic acid that can be quantified) is improved 100 times or more. Moreover, this range can be further increased by reducing the size of the capillary used and / or increasing the total area of the capillary plate. This involves the sample containing the analyte nucleic acid,
This means that quantification can be performed with a single measurement without repeating dilution at various concentrations.

The quantification method of the present invention is superior to the digital PCR method in terms of counting accuracy and simplicity of operation, and detects very small amounts of genes and other nucleic acids in a sample (particularly, a biological sample) and determines the number of molecules. Very useful for counting and quantifying. For example, by counting the number of expressed molecules of a specific gene using the quantification method of the present invention, it becomes possible to identify whether a cell is normal or abnormal.

[0042]

[Sequence List] SEQUENCE LISTING <110> Hamamatsu Photonics KK <120> Method for quantifying nucleic acid analyte and method for counting the molecular number of nucleic acid analyte <130> P99HP-257 <140> <141> <160> 3 <170 > PatentIn Ver. 2.1 <210> 1 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> PCR template <400> 1 atggaaacct gtttgttgga tataaatacg tcgtagatgg gcacagtgtg 50 <210> 2 <211> 20 <212 > DNA <213> Artificial Sequence <220> <223> PCR primer <400> 2 cacactgtgc ccatctacga 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 3 atggaaacct gtttgttgga 20 gga

[Brief description of the drawings]

FIG. 1A is a partially cutaway perspective view of a capillary plate according to the present invention. (B) is a schematic sectional view of the capillary plate according to the present invention.

FIG. 2A is a cross-sectional view schematically showing a state in which a capillary plate according to the present invention is accommodated in a PCR chamber. (B) is a cross-sectional view schematically showing a state in which the chamber shown in (a) is mounted on a thermal cycler and PCR is performed.

FIG. 3 is a schematic diagram for explaining the quantification method of the present invention step by step. In the figure, (a) shows a stage in which the PCR solution is being dispensed into the channel of the capillary plate according to the present invention, (b) shows a stage in which the PCR solution is sealed in the channel, and (c) shows a stage in which the PCR solution is sealed in the channel. PCR shows the stage where amplification of the target DNA occurs in some channels, and (d) shows the stage of observing the fluorescence from the channel of the capillary plate.

FIG. 4 is a fluorescence image (photograph) of a capillary plate obtained in Example 1. In the figure, (a) is a fluorescence image obtained from PCR of a PCR solution containing a target DNA, and (b) is a fluorescence image obtained from PCR of a PCR solution containing no target DNA.

FIG. 5 shows the test results of Example 2, where the target DN
5 is a graph in which the ratio of the number of fluorescent channels to the total number of channels is plotted against the concentration of A. In the figure,
Is a plot of the measured values, and is a plot of the expected values from the Poisson distribution.

FIG. 6 is a fluorescence image (photograph) of a capillary plate obtained in Example 2. In the figure, (a) is a fluorescence image obtained from PCR of a PCR solution containing a target DNA at a concentration of 100 aM, and (b) is a fluorescence image obtained from PCR of a PCR solution containing a target DNA at a concentration of 10 aM. (C) is a fluorescence image obtained from PCR of a PCR solution containing a target DNA at a concentration of 1 aM.

[Explanation of symbols]

1 ... capillary plate, 2 ... channel, 3 ...
Silicon rubber plate, 4 ... cover glass, 5 ... chamber, 6 ... fixing screw, 7 ... aluminum block of thermal cycler, 8 ... target DNA molecule, 9 ... amplified DN
A molecule, 10 ... fluorescent channel

 ────────────────────────────────────────────────── ─── Continued on the front page F term (reference) 2G045 AA40 BB60 DA12 DA13 DA14 FA11 FA16 FA19 FB12 GC15 JA01 JA07 4B063 QA01 QA18 QQ42 QQ53 QR32 QR62 QR66 QS25 QS32 QX02

Claims (7)

[Claims] 1. A method for quantifying an analyte nucleic acid, comprising: a first step of preparing a PCR solution containing a target DNA derived from an analyte nucleic acid from a sample and a fluorescent reagent; and a predetermined number of channels. PCR into a capillary plate
A second step of loading a solution, a third step of sealing both sides of the capillary plate with an elastic transparent plate, and sealing the PCR solution in each channel, and a capillary plate having both sides sealed. Is exposed to a preset temperature cycle, and the target DN derived from the analyte nucleic acid is exposed.
A fourth step of performing the PCR of A, and subjecting the capillary plate whose both sides are sealed to fluorescence measurement to count the number of fluorescent channels that emit fluorescence, wherein the fluorescent channels are amplified by the PCR. A fifth step which is a channel containing a target DNA derived from the test nucleic acid, and a method for quantifying the test nucleic acid. 2. The analyte according to claim 1, further comprising a step of calculating a concentration of the analyte nucleic acid based on the counted number of fluorescent channels, following the fifth step. A method for quantifying nucleic acids. 3. The method according to claim 1, further comprising a step of pre-treating the capillary plate with a protein adsorption inhibitor. 4. The method for quantifying an analyte nucleic acid according to claim 1, wherein the fluorescent reagent comprises a minor glove-bound fluorescent dye. 5. The method according to claim 1, wherein the fluorescent reagent comprises a fluorescent probe oligonucleotide utilizing fluorescence energy transfer. 6. The method according to claim 1, wherein the elastic transparent plate is made of silicone rubber. 7. A method for counting the number of molecules of an analyte nucleic acid, comprising: a first step of preparing a PCR solution containing a target DNA derived from the analyte nucleic acid from a sample and a fluorescent reagent;
A second step of placing the PCR solution on a capillary plate having a predetermined number of channels, and sealing both sides of the capillary plate with an elastic transparent plate, and enclosing the PCR solution in each channel. Step 3, a fourth step of exposing the capillary plate sealed on both sides to a preset temperature cycle to perform PCR of a target DNA derived from the subject nucleic acid, and a capillary plate closed on both sides. Is subjected to fluorescence measurement, the number of fluorescent channels that emit fluorescence is counted, wherein the fluorescent channel is a channel containing a target DNA derived from the analyte nucleic acid amplified by the PCR, a fifth step, A method for counting the number of molecules of a subject nucleic acid, comprising: