JP4427238B2 - Method for quantifying target nucleic acid, nucleic acid used therefor, probe-immobilized substrate and assay kit, and virus test method using the same - Google Patents

Description

【図面の簡単な説明】
【図１】Ｃ型肝炎ウイルスのウイルス量を測定するために用いたゲノム配列の一部を示す図。
【図２】競合的ＰＣＲの原理を示す図。
【図３】競合的ＰＣＲの原理を示す図。
【図４】本発明の態様であるプローブ固相化基体の例を示す図。
【図５】本発明の態様であるプローブ固相化基体の例を示す図。
【図６】本発明の態様であるプローブ固相化基体の例を示す図。
【図７】Ｃ型肝炎ウイルスのウイルス量測定に関するグラフを示す。
【図８】Ｃ型肝炎ウイルスのウイルス量測定に関するグラフを示す。
【図９】Ｃ型肝炎ウイルスのウイルス量測定に関するグラフを示す。
【図１０】Ｃ型肝炎ウイルスを測定する現行法の一つ、Ａｍｐｌｉｃｏｒｅ ｍｏｎｉｔｏｒ法での結果と本発明の方法での結果を比較して示す図。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for quantifying a target nucleic acid, a nucleic acid used therefor, a probe-immobilized substrate and an assay kit, and a virus test method using the same.
[0002]
For example, when the present invention starts antiviral treatment for a virus-infected person, the patient is infected for the purpose of knowing the degree of viremia, or for the purpose of tracing the treatment process and the therapeutic effect after treatment. The present invention relates to a method for measuring the concentration of a virus in blood. Furthermore, the present invention relates to a method for measuring the amount of mRNA expressed in a tissue for the purpose of examining drug responsiveness.
[0003]
[Prior art]
In general, blood virus concentration is said to be an important indicator of the degree of viremia. For example, in the case of hepatitis C virus (HCV), patients with low blood virus concentrations have numerical values AST and ALT indicating liver function and liver damage is minor, whereas patients with high blood virus concentrations are Liver damage is severe and liver function is often reduced. Furthermore, blood virus concentration is said to have an effect on the effects of antiviral treatment. In the case of the same HCV, it is said that interferon therapy is effective, but it is difficult to obtain a therapeutic effect in a case with a high viral viral load, and there is a tendency that a high therapeutic effect is high in a patient with a low viral load. is there.
[0004]
There are about 2 million people with HCV in Japan, about 70% of whom have chronic hepatitis. These patients are known to develop liver cancer at a high rate after the lapse of 10 to 20 years, and it is strongly desired to completely eliminate the virus in chronic hepatitis. Although the above-mentioned interferon therapy is considered to be an effective method for eliminating viruses, it has been found that it actually works only in about 30% of patients. In addition to such a situation, there are problems such as the fact that interferon therapy has serious side effects and is expensive to treat. Therefore, it is considered that it is important to reduce the burden on patients by predicting the effects of interferon therapy to some extent before starting treatment, and selecting patients who can be treated successfully.
[0005]
In addition, the effects of interferon therapy for chronic hepatitis C are often regulated mainly by viral factors. Factors that are considered to be particularly affected are virus type (genotype) and viral load. These two factors are related to each other, and there is a tendency that the amount of virus tends to increase in genotype 1b, and conversely, the amount of virus does not increase so much in types 2a and 2b. In addition, there is a report that the method of transition of the viral load after the start of treatment is reflected in the therapeutic effect (refer to Non-Patent Document 1), and measuring the viral load determines the treatment policy or advances the treatment. It will be an important decision material.
[0006]
In addition, measuring the amount of mRNA expressed in a tissue is considered to be an important indicator for observing an individual's reactivity to a certain drug. For example, when considering the effect of interferon therapy for chronic hepatitis C, hepatic tissues are collected before and after the start of treatment, and the amount of mRNA expressed in each is compared with several genes. It becomes possible to examine the reactivity of the individual to interferon. For this purpose, first, the expression patterns of several genes are compared before and after the start of treatment, and the pattern of meaningful gene expression change is specified in order to define the presence or absence of the effect of interferon. Attempts have been made to predict the therapeutic effect in advance by measuring the change in the expression level of a specific gene before and after the start of treatment based on the results.
[0007]
Conventional viral load measurements include Competitive PCR (see Non-Patent Document 2 and Non-Patent Document 3), Branched DNA probe assay (Bayer), and Amplore monitor method (Roche Diagnostics). It is used. For example, the Competitive PCR method calculates the viral load by competitive PCR using a concentration standard. In this method, electrophoresis is used for detection, and some experience is required to read the electrophoretic image. In addition, the procedure is complicated, such as applying several PCRs per sample. In order to perform Branched DNA probe assay, a special technique for synthesizing Branched DNA probe is required. The Amplcore method requires a special measuring device. Conventional methods for measuring the amount of mRNA in tissue include TaqMan method (Applied Biosystems Co., Ltd.) that requires a special device, methods using various microarrays, etc., except that it is roughly viewed by Northern blotting.
[0008]
[Non-Patent Document 1]
Chayama K et al. Author, Viral Hepatitis and Liver Disease. 1994, 617-620
[0009]
[Non-Patent Document 2]
Kaneko S et al. Written by J. Med. Virol. 37, 1992, 278-282
[0010]
[Non-Patent Document 3]
Chayama K et al. Written by J Gastroenterol. Hepatol. 8, 1993, S40-44
[0011]
[Problems to be solved by the invention]
In view of the above situation, an object of the present invention is to quantitate a target nucleic acid capable of easily quantifying a target nucleic acid in a sample, particularly a virus genome or mRNA, a nucleic acid used in the method, and a probe solid phase. And an assay kit, and a virus test method using the same.
[0012]
[Means for Solving the Problems]
The above problems are achieved by the present invention as follows. That is,
(1) A method for quantifying a target nucleic acid contained in a sample nucleic acid, comprising:
(A) a first target sequence comprising an av region and first and second primer binding regions at both ends across the av region; and
A bv region which is present at a position different from the first target sequence and is different from the av region, and third and fourth primer binding regions at both ends across the bv region A second target sequence comprising
A target nucleic acid comprising within one sequence;
An as region which is a sequence different from the sequence of the av region, the sequence of the bv region and their complementary sequences, and the first and second primers at both ends across the as region A first concentration standard having a first concentration including a binding region;
A first primer that binds to the first primer binding region;
A second primer that binds to the second primer binding region;
Reacting under conditions that provide adequate amplification;
Here, the amplification of the first target sequence using the first primer and the second primer competes with the amplification of the first concentration standard;
(B) the target nucleic acid;
A bs region different from the sequence of the bv region, the sequence of the av region, the sequence of the as region and their complementary sequences; and the third region at both ends across the as region. And a second concentration standard substance having a second concentration different from the first concentration including the fourth primer binding region, a third primer that binds to the third primer binding region,
A fourth primer that binds to the fourth primer binding region;
Reacting under conditions that provide adequate amplification;
Here, the amplification of the second target sequence in which the third primer and the fourth primer are used competes with the amplification of the second concentration standard;
(C) The amplification product obtained in (a) is appropriately combined with a first probe for detecting the sequence of the av region and a second probe for detecting the sequence of the as region. Reacting under conditions that allow hybridization;
(D) The amplification product obtained in (b) is appropriately combined with a third probe for detecting the sequence of the bv region and a fourth probe for detecting the sequence of the bs region. Reacting under conditions that allow hybridization;
(E) detecting the hybridization caused by (c);
(F) detecting the hybridization caused by (d); and
(G) determining the first target nucleic acid concentration and the second target nucleic acid concentration contained in the sample nucleic acid from the results obtained in (e) and (f);
Here, (c) and (d) are performed on one probe-immobilized substrate in which the first to fourth probes are immobilized on the probe-immobilized substrate.
(2) The method according to (1), wherein (a) and (b) are performed in one reaction solution.
(3) The method according to any one of (1) and (2) above, wherein the Tm value of the probe region of the amplification product obtained in (a) and the probe region of the amplification product obtained in (b) The Tm values of are equal.
(4) The method according to any one of (1) to (3), wherein the base length of the amplification product obtained in (a) is equal to the base length of the amplification product obtained in (b). A method characterized by.
(5) The method according to (1) above, wherein hybridization is detected electrochemically.
(6) The method according to any one of (1) to (5), wherein the first to fourth probes have a label.
(7) The method according to any one of (1) to (6), wherein the target nucleic acid is selected from the group consisting of a viral genome and mRNA.
(8) The method according to any one of (1) to (7), wherein the target nucleic acid contains at least a sequence represented by SEQ ID NO: 1 or a complementary sequence thereof, a nucleic acid derived from a hepatitis C virus genome And
The first concentration standard is a sequence shown in SEQ ID NO: 2 or a complementary sequence, and the second concentration standard is a sequence shown in SEQ ID NO: 3 or a complementary sequence;
The first to fourth primers are primers having different sequences, and the four types of primers are nucleic acid chains having the sequences represented by any one of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7. Or a complementary strand thereof, and at least four kinds of the probes are used, and the four kinds of probes are nucleic acid strands having a sequence represented by any one of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11. Or a complementary strand.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
I. Explanation of terms
As used herein, the term “nucleic acid” refers to any nucleic acid analog such as DNA and RNA, S-oligo, methylphosphonate oligo, PNA (ie, peptide nucleic acid) and LNA (ie, lock nucleic acid), etc. In addition, it is a term that collectively indicates substances whose partial structure can be represented by a base sequence. Such nucleic acids may be naturally occurring or artificially synthesized and may be a mixture thereof.
[0014]
II. Quantification method of target nucleic acid
Below, the 1st aspect of this invention is demonstrated using an example.
[0015]
1. Synthesis of a concentration standard used for measurement of blood concentration of target nucleic acid
In order to measure the blood concentration of the target nucleic acid, it is important to first select a region with high conservation of the target nucleic acid. Two or more regions with a length of about 80 bases or more are selected as much as possible in the region. The two selected locations are target sequences. In FIG. 1, the positional relationship of the target sequence of 2 area | region (a area | region and b area | region) set to 5'UTR of the example performed in the hepatitis C virus genome is shown.
[0016]
A probe region of 20 bases (that is, a v sequence) is set in each of the target regions of the two regions (or two or more regions) thus selected. The concentration standard substance a, which is the first concentration standard substance, consists of a sequence homologous to the a region of the target nucleic acid except for the region corresponding to the probe region. The concentration standard substance a has an s sequence in which mutation is intentionally incorporated in a region corresponding to the probe region. That is, the concentration standard substance a is a nucleic acid having the same sequence as the viral genome except for the s sequence. The concentration standard substance a may be synthesized as a nucleic acid chain such as a DNA chain of about 100 bases. Similarly, the concentration standard substance b which is the second concentration standard substance is also synthesized. Even when three or more target sequences are selected, the nth target sequence may be selected and the nth concentration standard substance n may be synthesized in the same manner as described above (where n is an integer of 3 or more). ).
[0017]
Here, the sequence shown in FIG. 1 describes a typical sequence of genotype 1b common to Japanese from the 5 ′ end. Two kinds of concentration standard substances a and b were designed in the part indicated by “−” along the sequence. The part indicated by “-” has the same sequence as the viral genome, and mutations were introduced concentrated on the part surrounded by a square. Then, two types of probe regions were designed, a virus genome-specific sequence v sequence and a sequence s-sequence specific to the concentration standard substance into which the mutation was introduced. The probe region can be hybridized with a probe for detecting a concentration standard and a target sequence. Such probe hybridization can be used to quantify the concentration standard and the target sequence for each sequence. Alternatively, the concentration standard substance and the target sequence may be recognized using the difference in the sequence of the probe region and quantified by a method known per se.
[0018]
The number of mutations arbitrarily incorporated into the concentration standard substance can be detected with a difference of one base in the probe region consisting of 20 bases, for example, but it is desirable to incorporate two or more bases. In that case, it is preferable to make the arrangement so that the probe region and the Tm value before the change do not change. Moreover, it is preferable that the position into which the mutation is incorporated is not near the end of the probe region but near the center.
[0019]
As the concentration standard substance, one artificially synthesized by the phosphoramidite method can be used. Alternatively, an artificially synthesized nucleotide chain having a mutation site can be amplified by PCR using the sequences at both ends, and the PCR product can be used as a concentration standard substance. It is also possible to use a PCR product amplified using a virus having a portion of a very rare sequence as a template. In that case, the region where accumulated mutations is used as a probe region, and a highly conserved region is used for amplification. It may be a primer region. The method for introducing mutation is not limited to the above method.
[0020]
The concentration standard substances a and b are prepared by changing the concentration for each region. In this example for quantification of the hepatitis C virus genome, two regions were selected as target sequences. Thus, for example, the first concentration standard is 10 ² copy / μl with 10% second concentration standard. ⁴ What is necessary is just to dilute to copy / microliter.
[0021]
Alternatively, when three regions are selected, for example, the first concentration standard substance is 10 ¹ copy / μl, 10% of the second concentration standard ³ copy / μl, 10 for the third concentration standard ⁵ copy / μl. When 5 areas are selected, for example, the first concentration standard substance is 10 ¹ copy / μl, 10% of the second concentration standard ² copy / μl, 10 for the third concentration standard ³ copy / μl, 10th concentration standard substance 10 ⁴ copy / μl, 10th concentration standard substance ⁵ What is necessary is just to carry out serial dilution according to the number of selected target sequences, such as copy / microliter. It is desirable to set an effective concentration in consideration of the normal abundance of the virus or considering the level of judgment necessary for treatment.
[0022]
In the above example, an example in which two regions having a length of about 80 bases or more in the region are selected as much as possible is selected. However, the present invention is not limited to this, and is intended for the purpose of the present invention. Based on this, the practitioner can arbitrarily select the length and the number of selection points.
[0023]
For example, as for the length of the target nucleic acid used in the embodiment of the present invention, it is preferable that at least two regions or more can be selected as the target sequence, and that there are about 100 bases or more between each region. An example of such a preferred target nucleic acid length is about 300 bp or more. However, the present invention is not limited to this, and it will be understood by those skilled in the art that the method for quantifying a target nucleic acid can be performed based on the above-described principle of the present invention, whether it is shorter or longer. Let's go.
[0024]
For example, the length of the target sequence used in the embodiment of the present invention may be a length that allows the complementary strand to hybridize to the target nucleic acid under conditions that allow appropriate hybridization. Similarly, the length of the concentration standard used in the embodiment of the present invention can be selected as desired according to the same criteria as the length of the target sequence.
[0025]
The length of the probe region may be a length that can maintain the Tm value necessary for the complementary strand to hybridize to the probe region under conditions where appropriate hybridization is obtained. In general, for example, about 15 mer to about 25 mer is preferable, but not limited thereto.
[0026]
2. Principle of competitive PCR for target nucleic acid concentration measurement
A DNA derived from a target nucleic acid purified from a test sample or a cDNA product after reverse transcription is used as a sample nucleic acid. A PCR reaction is carried out by bringing the sample containing the sample nucleic acid into the same volume of the concentration standard substance solution whose concentration has been adjusted in advance. Here, the “test sample” may be any cell, tissue, body fluid, or the like from an organism.
[0027]
For example, 1 μl of the virus-derived product as described above and 1 μl of a solution of concentration standard substances a and b diluted to the respective concentrations in the two regions are added and PCR reaction is performed in one tube. PCR may be carried out using a common commercially available enzyme in a total amount of 20 μl or more. The total amount of the reaction can be appropriately increased as the volume of the sample and the concentration standard substance solution brought in with respect to the total amount increases, but it is desirable that the upper limit is 50 μl.
[0028]
The schematic diagram shown in FIG. 2 illustrates the principle of competitive PCR. When the amount of virus is low and the concentration of virus amount and concentration standard substance a is approximately 1: 1, only the fragment having the s sequence is selectively amplified in the region of concentration standard substance b that is brought in a large amount. Concentration standard substance a having the same concentration amplifies s and v sequences to the same extent. If the amount of one virus is high and the concentration of the virus amount and the concentration standard substance b is almost 1: 1, the concentration standard substance b and the virus genome that are brought in a large amount will be amplified to the same extent, but the low concentration standard substance For a, only the v sequence is selectively amplified. The amount of virus is measured by comparing the amount produced. Hereinafter, a more specific example of density will be shown and described.
[0029]
Among the concentration standard substances, for example, the concentration standard substance a is 10 ² The concentration standard substance b is 10 in copy / μl. ⁴ Suppose that it is diluted to copy / μl and brought in.
[0030]
DNA or cDNA in virus-derived product 3 is 10 ² When the concentration is copy / μl, the virus-derived product and the concentration standard substance a-derived product 1 originally existed in the same amount in the region a, and thus increase equally. However, since there is a large amount of product 2 derived from the concentration standard substance b in the region b, the product 2 derived from the concentration standard substance b uses most of the primers for amplification, and the product 3 derived from the virus is amplified. (Figure 2).
[0031]
On the contrary, as shown in FIG. 3, DNA or cDNA which is the product 3 derived from the introduced virus is 10 ⁴ If it is copy / μl, the virus-derived product 3 and the concentration standard substance b-derived product 2 originally existed in the same amount in the b region, so that the number of virus-derived products 3 is large in the a region. Therefore, most of the primers are used and the product 1 of the concentration standard substance a cannot be amplified (FIG. 3).
[0032]
In addition, the virus-derived product 3 is 10 ³ In the case of copy / μl, since the virus-derived product 3 is 10 times more in the region a, most of the primers are used by the virus-derived product 3 and the product 1 of the concentration standard substance a can be amplified. Can not. In the region b, there are 10 times as many products 2 derived from the concentration standard substance b, so that most of the primers are used by the product 2 of the concentration standard substance b, and the virus-derived product 3 cannot be amplified. Alternatively, the viral DNA and cDNA in the virus-derived product 3 are 10 ¹ In the case of copy / μl, since there are many concentration standards in both regions, the products 1 and 2 of the concentration standards are preferentially amplified. In addition, virus-derived product 3 is 10 ⁵ When copy / μl is brought in, since there are many virus-derived products 3 in both regions, the virus-derived product 3 is amplified more dominantly than products 1 and 2 of the concentration standard substance. By comparing the above results for each sample by origin, the virus concentration can be measured.
[0033]
The primer set consisting of the forward primer and the reverse primer used here is the same primer set for each target sequence. For example, for the region a which is the first target sequence, the first primer set is used, whereby the sequence of any length including the probe region of the virus-derived product and the full length or part of the concentration standard a Can be amplified. Similarly, for the second target sequence b region, the second primer set is used, whereby the sequence of any length including the probe region of the virus-derived product and the full length or part of the concentration standard b Can be amplified. The same applies to three or more target sequences.
[0034]
Moreover, although the example which uses PCR method as an amplification means was shown above, according to the aspect of the present invention, the amplification means is not limited to PCR, and any amplification means known per se may be used. That is, any conventionally known primer extension method can be used in the embodiment of the present invention. For example, it is possible to use a method utilizing a polymerase chain reaction (generally referred to as PCR, hereinafter referred to as PCR) or reverse transcription PCR. Nucleic acid strand amplification (NASBA), Transcription mediated amplification (TMA), Ligase chain reaction (LCR), Strand displacement amplification (SDA), Isothermal and Chimeric primer-initiated Amplification of Nucleic acids (ICAN), Rolling circle amplification (RCA) Amplification methods such as method) can also be used.
[0035]
Moreover, since each density | concentration shown above was shown as an example, the practitioner can change as desired.
[0036]
3. Principle of quantification of hepatitis C virus by microtiter plate method
The viral genome extracted from the patient sample is subjected to a treatment such as converting RNA into cDNA by reverse transcription reaction as necessary, and the obtained sample nucleic acid is used as a template. Similarly, two or more kinds of concentration standard substances (for example, two kinds) as required are prepared as templates, and these are prepared by changing the concentration as described above. This is a method of performing competitive PCR by coexisting these. The primer used at that time is preferably provided with a label that can be detected later. The type of label is not particularly limited, and a chemical label such as biotin, a fluorescent label, and the like may be used per se. However, depending on the technology such as a DNA chip, primer labeling is not particularly necessary when a method is employed in which an intercalating agent is brought in at the time of hybridization and the signal from it is used for detection. Any of the means using a labeling substance and the means using an intercalating agent can be used in the embodiments of the present invention.
[0037]
Hybridization of competitive PCR products and probes on a probe-immobilized substrate obtained by immobilizing the probe on a microtiter plate, DNA chip, microarray or the like, which is a substrate used for detection, following the competitive PCR described above. I do. As the number of wells and spots on which the probe is solid-phased, the number corresponding to the type of region used may be used. For example, in the case of the quantification of hepatitis C virus performed in the Examples, since the two regions of the a region and the b region were used as target sequences, for example, the av sequence derived from the virus is attached to each of the two probe regions. 4 probes in total, probes for bv sequences, probes for a-s sequences to detect concentration standards a and b incorporating mutations and probes for bs sequences Wells may be used. FIG. 4 shows an example using four wells. In each well of the probe-immobilized substrate 10, a probe 4 'for the a-s sequence 4, a probe 6' for the b-s sequence 6, a probe 7 'for the av sequence 7, and Probe 8 'for av sequence 8 is immobilized. Such a probe-immobilized substrate can be used in the embodiments of the present invention, and such a probe-immobilized substrate is also within the scope of the present invention.
[0038]
In addition, when 3 regions are used, 6 wells may be required because the probe for v sequence and the probe for s sequence are solid-phased in each region.
[0039]
Competitive PCR products amplified by PCR are hybridized to respective probes of v and s sequences, depending on the number of concentration standards (ie, the number of target sequences). Depending on whether the amplified product obtained by competitive PCR has a v sequence or an s sequence, a probe that can be hybridized and a probe that cannot be hybridized are generated. Therefore, if a target substance such as biotin previously labeled on the PCR product is detected, the amount of hybridization between the two can be compared, so that the virus can be quantified from the concentration of the original concentration standard substance. .
[0040]
For example, the concentration standard substance a is 10 ² The concentration standard substance b is 10 in copy / μl. ⁴ Suppose that it is diluted to copy / μl and brought in. DNA or cDNA in the virus-derived product is 10 ² In the case of a concentration of copy / μl, the virus-derived product and the product derived from the concentration standard substance a were originally present in the same amount in the region a. Therefore, the product derived from the concentration standard substance a uses most of the primers for amplification, and the product derived from the virus cannot be amplified. In that case, the v and s probes show the same numerical value in the well or spot in the region a, but in the well or spot in the b region, the numerical value does not increase in the v probe and the numerical value increases only in the s probe.
[0041]
On the contrary, DNA or cDNA derived from the introduced virus is 10 ⁴ Assuming that copy / μl, the virus-derived product and the concentration standard substance b-derived product originally existed in the same amount in the b region, but the amount of virus-derived product is large in the a region. Since most of the primers are used, the product of the concentration standard substance a cannot be amplified. In that case, the v and s probes show the same numerical value in the well or spot in the b region, but in the well or spot in the a region, the numerical value does not increase in the s probe and the numerical value increases only in the v probe.
[0042]
In addition, the virus-derived products brought in are 10 ³ If it is copy / μl, since there are 10 times more virus-derived products in the region a, most of the primers are used by the virus-derived products, and the product of the concentration standard substance cannot be amplified. In the region b, there are 10 times as many products derived from the concentration standard, so that most of the primers are used by the concentration standard and the virus-derived product cannot be amplified. In that case, the v probe shows a lower value than the s probe in the well or spot in the a region, and the v probe shows a higher value than the s probe in the well or spot in the b region.
[0043]
10 viral DNAs and cDNAs in the virus-derived product ¹ In the case of copy / μl, there is a large concentration standard substance in either region, so that amplification is predominant. Therefore, the s probe shows a higher value in either well or spot.
[0044]
10 virus-derived products ⁵ When copy / μl is brought in, the amplification is predominant because there are many virus-derived products in both regions. Therefore it shows the v numerical value is higher of the probe in either well or spot.
[0045]
By comparing the values of the v probe and s probe in the used region by the above method, the amount of DNA or cDNA in the sample brought into the PCR can be calculated from the copy number of the concentration standard substance. . Thereafter, the concentration in the blood is calculated taking into account the concentration from the patient sample to the sample brought into the PCR. For example, it is assumed that 20 μl of a sample in the previous stage to be brought into PCR using 100 μl of serum is obtained. If 1 μl is used for competitive PCR, 1 μl brought into the PCR corresponds to the abundance in 5 μl of serum. Therefore, the obtained result needs to be multiplied by 100 when converted into 1 ml of blood.
[0046]
Thus, using a probe-immobilized substrate in which a single-stranded DNA typified by an oligonucleotide having a specific nucleotide sequence specific to a specific virus is immobilized on a substrate such as a microtiter plate, a DNA chip, or various microarrays, On the other hand, according to the embodiment of the present invention that measures the amount of purified amplification product by origin by hybridization of PCR products carried in several types of templates, it is possible to grasp the pathological condition of viral diseases and determine the treatment policy. It is possible to easily measure the amount of virus in the blood and the amount of mRNA (and the genotype of the virus at the same time as will be described later).
[0047]
Moreover, since each density | concentration shown above was shown as an example, the practitioner can change as desired.
[0048]
4). A method to detect multiple factors simultaneously.
[0049]
By using the present invention, it is possible to measure a plurality of factors with a single detection operation.
[0050]
For example, when examining the genotype of a virus at the same time, a characteristic region in which the genotype of the virus can be divided is selected, and a probe having a similar Tm value is designed as a probe for the amount of virus. In that case, if the genomic region used for measuring the viral load and the region for genotype are set apart (about 1000 bases or more), PCR and genotype for measuring viral load in the same single PCR tube PCR for detection may be performed simultaneously.
[0051]
By measuring the genotype and viral load of the virus thus infected at once, a method for easily measuring the degree of viremia for individual patients and a simple method for predicting in advance whether the effect of interferon therapy can be expected. And a simple method for measuring the amount of mRNA in a tissue reflecting drug responsiveness are provided.
[0052]
In the conventional method, only the amount of virus in the blood could be measured in each step, but in the present invention, after performing PCR, the amount of virus and genotype are detected on the same substrate (microtiter plate, DNA chip, microarray, etc.). It becomes possible, and two factors that are useful for understanding the disease state and determining the treatment method can be measured at a time. Furthermore, there is a possibility that drug response SNPs in the human genome that influence the therapeutic effect can be detected at the same time.
[0053]
5). Example of immobilization of nucleic acid on microtiter plate
The probe-immobilized substrate that can be used in the present invention may be any substrate in which the probe is immobilized on a substrate such as glass or plastic by any means known per se.
[0054]
For example, an example of a method capable of immobilizing DNA fragments on a microtiter plate at a high rate will be described below. However, the method for producing the probe-immobilized substrate of the present invention is not limited to this.
[0055]
As the material of the insoluble support for solid-phase the probe represented by the microtiter plate here, polystyrene, polyvinyl and the like are preferable. For example, ELISA-PLATE, MICROLON, 96W, FLAT-BOTT, MEDIUM BINDING (made by Greiner) can be mentioned. It is also possible to apply a solid phase directly to the PCR tube. Furthermore, other conventional methods for detecting nucleic acids based on hybridization of DNA chips, microarrays, etc. can be used.
[0056]
As a method for immobilizing the probe on the microtiter plate, several conventional methods can be used. In the present invention, the concentration of several salt solutions is adjusted in several steps, and the probe is diluted. The optimum conditions for the coating solution were sought, and the probe solid phase was more effectively performed. The method for solid-phase the probe on a DNA chip or microarray is not particularly specified.
[0057]
A probe consisting of about 20 bases is dissolved in this coating solution. The length of the probe is not particularly specified. For example, the probe is dissolved at a concentration of 0.4 μM, dispensed into a microtiter plate (appropriate amount so as not to cover the bottom of the well) and left overnight at 4 ° C. (or gently shaken at room temperature). 1 or 2 hours), the wells can be washed with a TBS (Tris Buffered Serine) buffer solution containing Tween 20, and the solid phase operation of the probe can be performed. The conditions of the solid phase and the washing operation are not limited to the described solutions, and other conventional washing solutions can be used.
[0058]
Subsequent blocking and hybridization conditions are not particularly limited, and can be appropriately selected from commonly used methods. For example, for blocking, a solution consisting of 6 × SSC, 0.01M EDTA (pH 8.0), 5 × Denhardt's solution, 100 μg / ml Sheared, denatured salmon super DNA is dispensed into each well, and lightly stirred at room temperature for about 1 hour. This can be done by incubating with shaking. Thereafter, the wells are washed with a TBS (Tris Buffered Serine) buffer containing Tween 20, and hybridization with the PCR product is performed. Hybridization is performed at 98 ° C. for 2 minutes in a solution consisting of, for example, 6 × SSC, 0.01M EDTA (pH 8.0), 5 × Denhardt's solution, 0.5% SDS, 100 μg / ml Sheared, densed salmon sperm DNA. The PCR product (with a detectable label) was added, dispensed to each well, and incubated by gently shaking at around 50 degrees for about 2 hours. At this time, it is necessary to adjust the hybridization temperature with reference to the Tm value calculated from the GC content of the probe. The amount of PCR product to be added is 10 per well for a 96-well plate. ⁹ Copy or more is desirable but not limited.
[0059]
After hybridization, the wells are washed with a conventional washing solution such as 2 × SSC buffer containing Tween 20 to remove excess DNA fragments that could not be hybridized.
[0060]
The method for detecting the label bound to the subsequent PCR product and confirming the state of hybridization can be appropriately selected from conventional methods. For example, when a biotin label is applied to the primer, the well is covered with 1 × TBS buffer containing 25% FCS dissolved in some labeled streptavidin to a final concentration of 7.5 mU and shaken. Then, the label is bound to biotin by incubating at room temperature for about 1 hour. Although the type of label is not specified, a commercially available reagent such as peroxidase-labeled streptavidin (manufactured by Roche Diagnostics) can be used as long as it is peroxidase-labeled. The wells were washed with 2 × SSC buffer containing Tween 20, and developed using a peroxidase chromogenic substrate, such as TMB (3,3 ′, 5,5′-tetramethylbenzidine) -ELISA solution (manufactured by Lifetech Oriental). Measure the absorbance.
[0061]
6). Probe immobilization substrate
The probe-immobilized substrate used in the embodiment of the present invention may be a plate-shaped substrate in addition to the one using the microtiter plate as described above. An example of such a probe-immobilized substrate will be described below.
[0062]
(A) First example
FIG. 5 schematically shows a first example of a probe-immobilized substrate that can be used in accordance with an embodiment of the present invention. The probe-immobilized substrate as a first example includes probes 11 to 14 that are immobilized by one or more in the substrate 16 and one or more immobilization regions 15 existing on the surface thereof (FIG. 5).
[0063]
Such a probe-immobilized substrate can be produced, for example, by immobilizing the probe to a substrate such as a silicon substrate by means known per se.
[0064]
According to the present invention, the number of immobilization regions 15 arranged on one substrate and the number of probes immobilized thereon are not limited to this, and may be changed as desired. A plurality of types of base sequences may be arranged as a probe on one substrate. A person skilled in the art can appropriately change the design of the solid phase pattern of a plurality of and / or a plurality of types of probes on the substrate. Such a probe-immobilized substrate is within the scope of the present invention.
[0065]
(B) Second example
A second example of a probe-immobilized substrate that can be used in the embodiment of the present invention will be described with reference to FIG. The probe-immobilized base body as the second example includes probes 17 to 20 fixed at one or more to one or more electrodes 23 provided on the base body 22 (FIG. 6). The electrode 23 is connected to a pad 24 for extracting electrical information.
[0066]
Such a probe-immobilized substrate can be manufactured, for example, by disposing an electrode on a substrate such as a silicon substrate by means known per se and immobilizing the probe on the electrode surface.
[0067]
In this embodiment, the number of electrodes is five, but the number of electrodes arranged on one substrate is not limited to this. Further, the arrangement pattern of the electrodes is not limited to that shown in FIG. 6, and those skilled in the art can appropriately change the design as necessary. You may provide a reference electrode and a counter electrode as needed. Such a probe-immobilized substrate is also within the scope of the present invention.
[0068]
In the case of a probe-immobilized substrate for performing fluorescence detection as described in the above example, the probe may be immobilized on any of the above-described substrates. In the case of a probe-immobilized substrate for performing electrochemical detection as described in the first example above, electrodes are arranged on any of the above substrates so that electrochemical detection is possible. And what is necessary is just to fix | immobilize a probe on the electrode.
[0069]
The electrode that can be used in the present invention is not particularly limited. For example, carbon electrodes such as graphite, glassy carbon, pyrolytic graphite, carbon paste, carbon fiber, platinum, platinum black, gold, palladium, Noble metal electrodes such as rhodium, oxide electrodes such as titanium oxide, tin oxide, manganese oxide, lead oxide, Si, Ge, ZnO, CdS, TiO ₂ And semiconductor electrodes such as GaAs, titanium and the like. These electrodes may be coated with a conductive polymer or a monomolecular film, and may be treated with other surface treatment agents as desired.
[0070]
In addition, probes having different base sequences may be immobilized on different electrodes, respectively, or may be immobilized on one electrode in a state where a plurality of types of probes having different base sequences are mixed. Good.
[0071]
(5) Detection
When using the probe-immobilized substrate according to the present invention, an electrochemical method as a means for detecting the presence of a double strand resulting from the hybridization reaction between the probe immobilized on the substrate and the target nucleic acid And fluorescence detection methods can be used.
[0072]
(A) Electrochemical detection
Electrochemical detection of double-stranded nucleic acid may be performed using, for example, a publicly known double-stranded recognition substance.
[0073]
The double-stranded recognizer used here is not particularly limited. For example, Hoechst 33258, acridine orange, quinacrine, dounomycin, metallointercalator, bisintercalator such as bisacridine, trisintercalator and polyintercalator Etc. can be used. Furthermore, these intercalators can be modified with an electrochemically active metal complex such as ferrocene or viologen. In addition, any other known double-stranded recognition substance is preferably used in the present invention.
[0074]
In the probe-immobilized substrate according to the present invention, the probe is fixed to the electrode. In the detection of double-stranded nucleic acid using such an electrode, a counter electrode or a reference electrode may be used in the same manner as in other general electrochemical detection methods. When arranging the reference electrode, for example, a general reference electrode such as a silver / silver chloride electrode or a mercury / mercury chloride electrode may be used.
[0075]
Subsequently, the reaction is performed under conditions that allow appropriate hybridization. Such appropriate conditions can be determined by those skilled in the art depending on various conditions such as the type of base contained in the target sequence, the type of probe provided on the probe-immobilized substrate, the type of sample nucleic acid, and the state thereof. It is possible to select appropriately. Although not limited to this, for example, the reaction may be performed under the following conditions.
[0076]
For example, the hybridization reaction solution is performed in a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10. In this solution, dextran sulfate, which is a hybridization accelerator, salmon sperm DNA, bovine thymus DNA, EDTA, and a surfactant may be added. The sample nucleic acid obtained here is added and heat denatured at 90 ° C. or higher. The probe-immobilized substrate may be inserted into the heat-denatured sample nucleic acid immediately after denaturation or after rapid cooling to 0 ° C. It is also possible to perform a hybridization reaction by dropping a solution on the substrate.
[0077]
During the reaction, the reaction rate may be increased by an operation such as stirring or shaking. The reaction temperature may be, for example, in the range of 10 ° C. to 90 ° C., and the reaction time may be about 1 minute to overnight. After the hybridization reaction, the electrode is washed. For washing, for example, a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10 may be used. When the target nucleic acid containing the target sequence is present in the sample nucleic acid, it hybridizes with the probe, thereby generating a double-stranded nucleic acid.
[0078]
Subsequently, the double-stranded nucleic acid generated by the following procedure is detected by electrochemical means. In general, after the hybridization reaction, the substrate is washed, and a double-stranded recognizer is allowed to act on the double-stranded portion formed on the electrode surface, and the resulting signal is electrochemically measured.
[0079]
The concentration of the double-stranded recognizer varies depending on the type, but is generally used in the range of 1 ng / mL to 1 mg / mL. At this time, a buffer solution having an ionic strength of 0.001 to 5 and a pH of 5 to 10 may be used.
[0080]
For example, the electrochemical measurement may be performed by applying a potential equal to or higher than the potential at which the double-stranded recognizer reacts electrochemically and measuring the reaction current value derived from the double-stranded recognizer. At this time, the potential may be swept at a constant speed, applied with a pulse, or a constant potential may be applied. In the measurement, for example, the current and voltage may be controlled by using a device such as a potentiostat, a digital multimeter, and a function generator. For example, the concentration of the target nucleic acid may be calculated from a calibration curve based on the obtained current value.
[0081]
In addition, electrochemical detection means known per se, for example, means disclosed in the following documents (Hashimoto et al. 1994, Wang et al. 1998) can be preferably used in the method of the present invention. In this document, Hashimoto et al. Reported sequence-specific gene detection using a gold electrode modified with a DNA probe and an electrochemically active dye. The anode current derived from the dye correlates with the concentration of the target DNA. Wang et al. Also reported indicator-free electrochemical DNA hybridization. This biosensor configuration includes immobilization of an inosine displacement probe (without guanine) to a carbon paste electrode and chronopotentiometric detection of duplex formation due to the presence of the guanine oxidation peak of the label. The detection means described in these documents may preferably be used in the present invention.
[0082]
(B) Fluorescence detection method
In the case of a method using a fluorescent labeling substance, the target nucleic acid may be labeled with a substance capable of obtaining fluorescent activity such as a fluorescent dye such as FITC, Cy3, Cy5 or rhodamine. Alternatively, a second probe labeled with the aforementioned substance may be used instead of such a label. A plurality of labeling substances may be used simultaneously.
[0083]
In some embodiments, a hybridization reaction between a nucleic acid component extracted from a sample and a probe immobilized on a probe-immobilized chip is performed, for example, as follows. For example, the hybridization reaction solution is performed in a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10. In this solution, dextran sulfate, which is a hybridization accelerator, salmon sperm DNA, bovine thymus DNA, EDTA, and a surfactant may be added. The nucleic acid component extracted here is added and heat-denatured at 90 ° C. or higher. The probe-immobilized chip may be inserted immediately after denaturation or after rapid cooling to 0 ° C. It is also possible to perform a hybridization reaction by dropping a solution on the substrate. During the reaction, the reaction rate may be increased by an operation such as stirring or shaking. The reaction temperature may be, for example, in the range of 10 ° C. to 90 ° C., and the reaction time may be about 1 minute to overnight. After the hybridization reaction, washing is performed. For the washing, for example, a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10 is used.
[0084]
In the case of fluorescence detection, the hybridization reaction is detected by detecting the labeled base sequence in the sample or the label in the secondary probe using an appropriate detection device corresponding to the type of label. When the label is a fluorescent substance, for example, the label may be detected using a fluorescence detector.
[0085]
7). Assay kit
The present invention also provides a probe, a probe-immobilized substrate, a primer, a nucleic acid chain constituting the primer, a concentration standard, and / or a hepatitis C virus genome as described below according to any of the embodiments described above. Nucleic acids can be selected according to the intended use, combined, and provided as an appropriate assay kit. Furthermore, in addition to them, buffer salts, other necessary reagents, labeling substances and / or reaction containers may be combined and provided as an assay kit.
[0086]
For example, the primer, nucleic acid and / or probe-immobilized substrate according to the present invention is provided as an assay kit in combination with a reaction vessel, buffer salts and / or other necessary reagents (eg, polymerase and substrate). Also good.
[0087]
8). Nucleic acids for quantification of hepatitis C virus genome
According to a further aspect of the invention, there are provided nucleic acids for quantification of the hepatitis C virus genome.
[0088]
For example, according to the above-described aspects of the present invention, an example of a sequence preferably used as a target nucleic acid for quantification of the hepatitis C virus genome is a nucleic acid represented by the sequence set forth in SEQ ID NO: 1 or a complementary sequence thereof, A nucleic acid containing the sequence, or a partial sequence of the sequence.
[0089]
For example, one example of a sequence preferably used as a concentration standard for quantification of hepatitis C virus genome according to the above-described aspects of the present invention is represented by the sequences set forth in SEQ ID NOs: 1 and 2 or their complementary sequences. Or a part of the sequence.
[0090]
For example, according to the above-described aspects of the present invention, the primer for quantification of the hepatitis C virus genome is represented by the sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 or their complementary sequences. A nucleic acid, a nucleic acid containing the sequence, or a partial sequence of the sequence. In particular, it is preferable to use SEQ ID NO: 4 and SEQ ID NO: 5 and SEQ ID NO: 6 and SEQ ID NO: 7 as a primer set having a forward primer and a reverse primer, respectively.
[0091]
For example, in accordance with the above-described aspects of the present invention, a probe for quantification of the hepatitis C virus genome is represented by the sequences shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 or their complementary sequences. A nucleic acid, a nucleic acid containing the sequence, or a partial sequence of the sequence.
[0092]
In particular, when quantifying the hepatitis C virus genome according to the embodiment of the present invention, the nucleic acid represented by the sequence shown in SEQ ID NO: 1 to SEQ ID NO: 11 or a complementary sequence thereof is used together. It is preferable to do. However, the present invention is naturally not limited to this.
[0093]
【Example】
1. HCV content measurement using synthetic oligonucleotides as concentration standards for two regions
1-1. Overview
One of the current methods for measuring the amount of HCV, 10 by the competitive PCR method ⁷ HCV RNA was extracted from serum collected from a patient with chronic hepatitis C measured as copy / ml, and the blood HCV level was measured using a concentration standard substance in two regions consisting of synthetic oligonucleotides. A microtiter plate was used as a substrate for detection.
[0094]
1-2. How to collect patient specimens
Peripheral blood was collected from the patient to be measured and collected in a blood collection tube such as a jelly tube. After sufficiently coagulating, centrifugation was performed at 3000 rpm for about 10 minutes to remove blood cell components and obtain serum. Serum was dispensed into several test tubes for storage and then stored in a freezer at -20 ° C or lower.
[0095]
1-3. Hepatitis C virus genome extraction method
The viral genome was extracted using 100 μl of serum per specimen. For the extraction, a kit such as Sepa-Gene-RV-R (manufactured by Sanko Junyaku Co., Ltd.) was used. This gave a fraction containing the required HCV RNA in pellets. The pellet was dissolved in 9 μl of a 1 mM DTT solution containing RNAse inhibitor (manufactured by TAKARA) at a concentration of 1 unit / μl to obtain an HCV RNA sample to be subjected to a reverse transcription reaction.
[0096]
1-4. Reverse transcription reaction of hepatitis C virus genomic RNA
The reverse transcription reaction was performed using Hexa-deoxyribonucleotide mixture (manufactured by TAKARA) in M-MLV Reverse Transscriptase (manufactured by LIFE TECHNOLOGIES) at a total volume of 20 μl, in which 9 μl of the HCV RNA sample was added for 20 minutes at 42 ° C. I went.
[0097]
1-5. Competitive PCR to measure viral load
PCR was performed using GeneTaq (manufactured by Wako Pure Chemical Industries, Ltd.) in a total amount of 50 μl. Among the primers used for PCR, the primer on the antisense side was labeled with biotin. As a template for amplification, 1 μl of the reverse transcription product of the above-mentioned viral genomic RNA, 10 of the concentration standard substance a (a region) ⁴ 1 μl of the diluted solution of copy / μl, 10 of the concentration standard substance b (b region) ² 1 μl of copy / μl dilution was added. Viral genomic RNA reverse transcripts were obtained as-is from a sample of 100 μl serum (10% in blood). ⁷ 10 out of cDNA by back calculation from copy / ml ⁵ In addition to (copy / μl), those diluted 100-fold with sterilized distilled water and those diluted 10000-fold were measured. Primers in the a and b regions were simultaneously introduced into the PCR reaction solution. The reaction was performed at 94 ° C. for 4 minutes, and then the reaction was repeated 35 times with 94 ° C. for 10 seconds and 62 ° C. for 1 second as one cycle.
[0098]
1-6. Probe solid phase and hybridization to microtiter plates
Dispensing the coating solution adjusted with 3M potassium carbonate solution so that the probe is 0.4 μM on ELISA-PLATE, MICROLON, 96W, FLAT-BOTT, MEDIUM BINDING (manufactured by Greiner), and incubate at 4 ° C. overnight. did. Four types of probes, av, as, bv, and bs, were solid-phased per specimen.
Next, the wells were washed with a TBS buffer containing Tween 20, and a solution consisting of 6 × SSC, 0.01M EDTA (pH 8.0), 5 × Denhardt's solution, 100 μg / ml Sheared, densed salmon sperm DNA was added to each well. 100 μl each was dispensed and blocked by light shaking at room temperature for about 1 hour. The wells were then washed with TBS buffer containing Tween 20, and consisted of 6 × SSC, 0.01M EDTA (pH 8.0), 5 × Denhardt's solution, 0.5% SDS, 100 μg / ml sheared, densed salmon sperm DNA. 50 μl of the hybridization solution was dispensed and heated to 50 ° C., which is the temperature of the current hybridization. The target PCR product was treated at 98 ° C. for 2 minutes, immediately transferred to ice to maintain the single-stranded state, and the hybridization solution was dispensed and added to the warmed wells. Hybridization was performed by incubating at 50 degrees for 2 hours with light shaking. After hybridization, the wells were washed with 2 × SSC buffer containing Tween 20.
[0099]
1-7. Detection method
Next, the wells were covered with 50 μl of peroxidase-labeled streptavidin (Roche Diagnostics) dissolved in TBS buffer containing 25% FCS to a final concentration of 7.5 mU, and shaken. The mixture was incubated at room temperature for about 1 hour. Further, the wells were washed with 2 × SSC buffer containing Tween 20, and 50 μl of TMB (3,3 ′, 5,5′-tetramethylbenzidine) -ELISA solution (manufactured by Lifetech Oriental Co.) was added. The mixture was allowed to stand in the dark until sufficiently colored (about 10 minutes), and then 50 μl of a reaction stop solution (2N sulfuric acid) was added, and the absorbance at 492 nm was measured. Absorbance was measured using a multi-scan bichromatic (Lab Systems Japan). The results are as shown in FIGS.
[0100]
1-8. Judgment result
The results were judged as follows. If the absorbance in a well in which a probe having a v sequence is solid-phased in both the a region and the b region is high, 10 ⁴ The virus genome was present at a concentration of copy / μl or more, and it was about 10 when converted into blood. ⁶ It is determined that copy / ml or more. If the absorbance in a well where an s-sequence probe is solid-phased is high in both the a region and the b region, ² The virus genome was present at a concentration of copy / μl or less. ⁴ It is determined that the copy / ml or less. If the absorbance of the s sequence is high in the a region and the absorbance of the v sequence is high in the b region, ⁴ copy / μl or less 10 ² The virus genome was present at a concentration of copy / μl or more, and converted to 10 in blood. ⁶ copy / μl or less 10 ⁴ It is determined that copy / μl or more. Furthermore, when the absorbances of the v and s sequences are almost the same in the a region or the b region, 10 ⁴ copy / μl, 10 ² The virus genome was present at a concentration of copy / μl. ⁶ copy / ml, 10 ⁴ Judge as copy / ml.
[0101]
The results are shown in FIGS. The concentration written in the upper part of each graph is the cDNA concentration of the test sample expected based on the measurement result by the current method and the dilution rate therefrom. No dilution 10 ⁵ When copy / μl cDNA was quantified, both the a and b regions showed a higher absorbance in the well in which the v probe was solid-phased. ⁴ It shows that the concentration is not less than copy / μl. 10 of the next 100-fold dilution ³ When copy / μl cDNA was quantified, the s probe in the a region and the v probe in the b region showed higher values. This is a measured sample of 10 ⁴ 10 at copy / μl or less ² It shows that the concentration is not less than copy / μl. 10 of the next 10,000-fold dilution ¹ When copy / μl cDNA was quantified, the s probe showed higher absorbance in both the a and b regions. ² It shows that the concentration is not more than copy / μl. Therefore, it was confirmed that the range of viral load can be accurately determined by using the present invention.
[0102]
2. A product obtained by amplifying a synthetic nucleotide having a mutation introduced by PCR is diluted and used as a concentration standard substance in two regions.
2-1. Overview
HCV RNA was extracted from serum collected from 24 patients with chronic hepatitis C whose blood HCV level was measured by the Amplicore monitor method (Roche Diagnostics), one of the current methods for measuring HCV levels. The results were compared with the quantitative methods in the present invention. In this quantification, the amount of HCV in blood was measured using a double-stranded DNA concentration standard substance prepared by amplifying synthetic nucleotides as a material by PCR.
[0103]
2-2. How to collect patient specimens
Peripheral blood was collected from the patient to be measured and collected in a blood collection tube such as a jelly tube. After sufficiently coagulating, centrifugation was performed at 3000 rpm for about 10 minutes to remove blood cell components and obtain serum. Serum was dispensed into several test tubes for storage and then stored in a freezer at -20 ° C or lower.
[0104]
2-3. Hepatitis C virus genome extraction method
The viral genome was extracted using 100 μl of serum per specimen. For the extraction, a kit such as Sepa-Gene-RV-R (manufactured by Sanko Junyaku Co., Ltd.) was used. This gave a fraction containing the required HCV RNA in pellets. The pellet was dissolved in 9 μl of a 1 mM DTT solution containing RNAse inhibitor (manufactured by TAKARA) at a concentration of 1 unit / μl to obtain an HCV RNA sample to be subjected to a reverse transcription reaction.
[0105]
2-4. Reverse transcription reaction of hepatitis C virus genomic RNA
The reverse transcription reaction was performed using Hexa-deoxyribonucleotide mixture (manufactured by TAKARA) in M-MLV Reverse Transscriptase (manufactured by LIFE TECHNOLOGIES) at a total volume of 20 μl, in which 9 μl of the HCV RNA sample was added for 20 minutes at 42 ° C. I went.
[0106]
2-5. PCR for measuring viral load
2-5-1. Synthesis method of concentration standard using PCR
Concentration standards can be quantified even with synthetic single-stranded nucleotides, but the concentration may become unstable with repeated freezing and thawing. Thus, synthetic nucleotides were amplified using PCR, and the resulting double-stranded PCR product was used as a concentration standard substance.
[0107]
For both the a and b regions, a primer of about 20 bases having the terminal sequence as it was was synthesized with respect to the synthesized 100 bases (a-forward: 5′-gattggggggcactactcc-3 ′, a-reverse: 5′-tgcacgactactactataat -3 ′, b-forward: 5′gactgctagccgagtaggtg-3 ′, b-reverse: 5′-atgggtgacacgtctacgag-3 ′). Amplification was performed using GeneTaq (manufactured by Wako Pure Chemical Industries, Ltd.) in a total amount of 50 μl. The reaction was carried out at 94 ° C. for 4 minutes, followed by 35 reactions of 94 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 10 seconds, and finally a treatment at 72 ° C. for 7 minutes. After the reaction, the entire amount of the reaction solution was electrophoresed on an agarose gel, and it was cut out after confirming that there was one band. Extract the product from the agarose gel using QIAquick Gel Extraction Kit (manufactured by QIAGEN), measure the OD and calculate the concentration of the product. ⁴ copy / μl, b region product is 10 ² It was diluted to copy / μl to obtain a concentration standard.
[0108]
2-5-2. Competitive PCR for viral load measurement
Competitive PCR was performed using GeneTaq (manufactured by Wako Pure Chemical Industries, Ltd.) in a total volume of 50 μl. The primer used for PCR was labeled with biotin. As a template for amplification, 1 μl of the reverse transcription product of the above-mentioned viral genomic RNA, 10 of the concentration standard substance a (a region) ⁴ 1 μl of the diluted solution of copy / μl, 10 of the concentration standard substance b (b region) ² 1 μl of copy / μl dilution was added. The a region primer and b region primer were brought into the PCR reaction solution at the same time. The reaction was performed at 94 ° C. for 4 minutes, and then the reaction was repeated 35 times with 94 ° C. for 10 seconds and 62 ° C. for 1 second as one cycle.
[0109]
2-6. Probe solid phase and hybridization to microtiter plates
Dispense the coating solution prepared with 3M potassium carbonate solution to ELISA-PLATE, MICROLON, 96W, FLAT-BOTT, MEDIUM BINDING (made by Greiner) so that the probe becomes 0.4 μM, and incubate at 4 ° C. overnight. did. Four types of probes, av, as, bv, and bs, were used per specimen.
[0110]
Next, the wells were washed with a TBS buffer containing Tween 20, and a solution consisting of 6 × SSC, 0.01M EDTA (pH 8.0), 5 × Denhardt's solution, 100 μg / ml Sheared, densed salmon sperm DNA was added to each well. 100 μl each was dispensed and blocked by light shaking at room temperature for about 1 hour. The wells were then washed with TBS buffer containing Tween 20, and consisted of 6 × SSC, 0.01M EDTA (pH 8.0), 5 × Denhardt's solution, 0.5% SDS, 100 μg / ml sheared, densed salmon sperm DNA. 50 μl of the hybridization solution was dispensed and heated to 50 ° C., which is the temperature of the current hybridization. The target PCR product was treated at 98 ° C. for 2 minutes, immediately transferred to ice to maintain the single-stranded state, and the hybridization solution was dispensed and added to the warmed wells. Hybridization was performed by incubating at 50 degrees for 2 hours with light shaking. After hybridization, the wells were washed with 2 × SSC buffer containing Tween 20.
[0111]
2-7. Detection method
Next, the wells were covered with 50 μl of peroxidase-labeled streptavidin (Roche Diagnostics) dissolved in TBS buffer containing 25% FCS to a final concentration of 7.5 mU, and shaken. The mixture was incubated at room temperature for about 1 hour. Further, the wells were washed with 2 × SSC buffer containing Tween 20, and 50 μl of TMB (3,3 ′, 5,5′-tetramethylbenzidine) -ELISA solution (manufactured by Lifetech Oriental Co.) was added. The mixture was allowed to stand in the dark until sufficiently colored (about 10 minutes), and then 50 μl of a reaction stop solution (2N sulfuric acid) was added, and the absorbance at 492 nm was measured. Absorbance was measured using a multi-scan bichromatic (Lab Systems Japan).
[0112]
The results were judged as follows. If the absorbance in a well in which a probe having a v sequence is solid-phased in both the a region and the b region is high, 10 ⁴ The virus genome was present at a concentration of copy / μl or more, and it was about 10 when converted into blood. ⁶ It is determined that copy / ml or more. If the absorbance in a well where an s-sequence probe is solid-phased is high in both the a region and the b region, ² The virus genome was present at a concentration of copy / μl or less. ⁴ It is determined that the copy / ml or less. If the absorbance of the s sequence is high in the a region and the absorbance of the v sequence is high in the b region, ⁴ copy / μl or less 10 ² The virus genome was present at a concentration of copy / μl or more, and converted to 10 in blood. ⁶ copy / μl or less 10 ⁴ It is determined that copy / μl or more. Furthermore, when the absorbances of the v and s sequences are almost the same in the a region or the b region, 10 ⁴ copy / μl, 10 ² The virus genome was present at a concentration of copy / μl. ⁶ copy / ml, 10 ⁴ Judge as copy / ml.
[0113]
2-8. result
The measurement results are shown in FIG. FIG. 10 is a graph relating to viral load measurement of hepatitis C virus. The copy number in 1 μl of cDNA of the sample used for the measurement is shown at the top of each graph. According to the method of the present invention, a specimen showing a numerical value of 200 IU / ml or less in the conventional Amplicon monitor method (referred to as a comparative example in FIG. 10) is 10 ⁴ Quantified to copy / ml or less. A sample showing a numerical value between 200 IU / ml and 1000 IU / ml by the Amplicore monitor method is 10 according to the method of the present invention. ⁴ copy / ml or more 10 ⁶ Quantified to a value less than copy / ml. A sample showing a numerical value of 1000 IU / ml or more by the Amplicore monitor method is 10 according to the method of the present invention. ⁶ It was found that each value was determined to be copy / ml or more. As a result of some results, divergence examples were confirmed by using another quantitative method, Competitive PCR. Despite the relatively low value of 170 IU / ml in the Amplicon monitor method, ⁴ copy / ml or more 10 ⁶ Two cases (cases Nos. 6 and 18) determined to be less than copy / ml were each 10 when measured by the Competitive PCR method. ⁵ copy / ml, 10 ^5.5 The result was a quantification result of copy / ml, which was similar to the quantification result of the present invention. On the contrary, in the present invention, although the high value of 1500 IU / ml is shown in the Amplicon monitor method, ⁴ copy / ml or more 10 ⁶ A case (Case No. 23), which is moderately discriminated as less than copy / ml, is 10 when measured by the Competitive PCR method. ⁵ The result was a quantification result of copy / ml, which was the same as the quantification result of the present invention. As described above, it was found that the present invention is not only simple but also provides more accurate quantitative results than the conventional method.
[0114]
In addition, clinically, it has been found that interferon treatment is effective in cases with low viral load. Even if the viral load is not displayed as an absolute value, it is sufficient to determine whether or not it is included or not included in the viral load range in which interferon is said to be effective. In that sense, the setting of this concentration standard is lowered and the effect of interferon treatment is influenced (approximately 10 points). ¹ Even a measurement system that shows a very simple tendency set to about copy / μl in cDNA seems to be useful for treatment.
[0115]
【The invention's effect】
According to the present invention as described above, a target nucleic acid, a nucleic acid used in the method, a probe-immobilized substrate and an assay, which can easily quantify a target nucleic acid in a sample, particularly a virus genome or mRNA, etc. A kit and a virus inspection method using the kit were provided.
[0116]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a diagram showing a part of a genome sequence used for measuring the viral load of hepatitis C virus.
FIG. 2 is a diagram showing the principle of competitive PCR.
FIG. 3 is a diagram showing the principle of competitive PCR.
FIG. 4 is a view showing an example of a probe-immobilized substrate which is an embodiment of the present invention.
FIG. 5 is a diagram showing an example of a probe-immobilized substrate that is an embodiment of the present invention.
FIG. 6 is a diagram showing an example of a probe-immobilized substrate that is an embodiment of the present invention.
FIG. 7 shows a graph relating to viral load measurement of hepatitis C virus.
FIG. 8 shows a graph relating to viral load measurement of hepatitis C virus.
FIG. 9 shows a graph relating to viral load measurement of hepatitis C virus.
FIG. 10 is a graph showing a comparison between the results of the current method for measuring hepatitis C virus, the Amplicore monitor method, and the results of the method of the present invention.

Claims (8)

A method for quantifying a target nucleic acid contained in a sample nucleic acid, comprising:
(A) the av region, a first target sequence including first and second primer binding regions at both ends across the av region, and a position different from the first target sequence In addition, a bv region having a sequence different from the av region and a second target sequence including third and fourth primer binding regions at both ends across the bv region are included in one sequence. A target nucleic acid comprising,
An as region which is a sequence different from the sequence of the av region, the sequence of the bv region and their complementary sequences, and the first and second primers at both ends across the as region A first concentration standard having a first concentration including a binding region;
A first primer that binds to the first primer binding region;
A second primer that binds to the second primer binding region;
Wherein the first target sequence and the first concentration standard are amplified using the first primer and the second primer, respectively. Competing;
(B) the target nucleic acid;
A bs region different from the sequence of the bv region, the sequence of the av region, the sequence of the as region and their complementary sequences; and the third region at both ends across the as region. And a second concentration standard substance having a second concentration different from the first concentration including the fourth primer binding region, a third primer that binds to the third primer binding region,
A fourth primer that binds to the fourth primer binding region;
Wherein the amplification of the second target sequence and the amplification of the second concentration standard using the third primer and the fourth primer are as follows: Competing;
(C) The amplification product obtained in (a) is appropriately combined with a first probe for detecting the sequence of the av region and a second probe for detecting the sequence of the as region. Reacting under conditions that allow hybridization;
(D) The amplification product obtained in (b) is appropriately combined with a third probe for detecting the sequence of the bv region and a fourth probe for detecting the sequence of the bs region. Reacting under conditions that allow hybridization;
(E) detecting the hybridization caused by (c);
(F) detecting the hybridization caused by (d); and (g) a first target nucleic acid concentration and a second target contained in the sample nucleic acid from the results obtained by (e) and (f) above. Determining the nucleic acid concentration;
Here, the (c) and (d) is carried out Te one probe-immobilized substrate on odor the first to fourth probes are immobilized on the probe-immobilized substrate. 2. The method according to claim 1, wherein the steps (a) and (b) are performed in one reaction solution . 3. The method according to claim 1, wherein the Tm value of the probe region of the amplification product obtained in (a) is equal to the Tm value of the probe region of the amplification product obtained in (b). A method characterized by.   The method according to any one of claims 1 to 3, wherein the base length of the amplification product obtained in (a) is equal to the base length of the amplification product obtained in (b). 2. The method according to claim 1 , wherein the detection of hybridization is performed electrochemically. The method according to claim 1 , wherein the first to fourth probes have a label. The method according to any one of claims 1 to 6 , wherein the target nucleic acid is selected from the group consisting of a viral genome and mRNA. The method according to any one of claims 1 to 7 , wherein the target nucleic acid is a nucleic acid derived from a hepatitis C virus genome including at least the sequence represented by SEQ ID NO: 1 or a complementary sequence thereof,
The first concentration standard is a sequence shown in SEQ ID NO: 2 or a complementary sequence, and the second concentration standard is a sequence shown in SEQ ID NO: 3 or a complementary sequence;
The first to fourth primers are primers having different sequences, and the four types of primers are nucleic acid chains having the sequences represented by any one of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7. Or a complementary strand thereof, and at least four kinds of the probes are used, and the four kinds of probes are nucleic acid strands having a sequence represented by any one of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11. Or a complementary strand.

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