JP2002153274A - MDR1 gene expression level quantitative measurement kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleic acid amplification primer, a nucleic acid capture probe, a nucleic acid detection probe, a quantitative measurement kit and a determination method which are used for rapidly, simply, specifically, highly sensitively and highly accurately determining the expression level of a MDR1 gene of medicine-resistant causative gene in a wide measurement range, and to detect the medicine-resistance of a subject and the changes of the medicine- resistance with the passage of time. SOLUTION: The method for detecting the medicine-resistant degree of the subject, comprises amplifying a nucleic acid extracted from a specimen collected from the subject by a nucleic acid amplification method using a kit comprising a probe and a primer using a part of the base sequence of MDR1 which is a medicine-resistant gene, capturing, detecting the nucleic acid corresponding to one part of the base sequence of the amplified MDR1, and thus determines the expression level of the MDR1 as the number of copies. The medicine- resistance subject is shown from the quantitatively detected expression level of the MDR1.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a gene diagnosis kit for quantitatively measuring the expression level of the MDR1 gene quickly, simply, specifically, with high sensitivity, high precision, and with a wide measurement range, and a gene using the kit. Diagnosis method.

[0002]

2. Description of the Related Art Drug resistance is widely recognized clinically, and in particular, multidrug resistance, which becomes resistant to a plurality of drugs having different action mechanisms and structures, is a major obstacle to drug treatment. Because the degree of drug resistance differs in each case even for the same disease type,
The same treatment does not necessarily give the same treatment result. Therefore, judging the effect of the test subject on the drug before the drug administration and tracking the change in the drug resistance of the test subject after the drug administration and verifying the effect of the drug over time can improve the therapeutic effect of the drug and increase the resistance of the cells. It is extremely useful for preventing useless side effects of a subject due to administration of a drug that is ineffective.

[0003] The MDR1 protein localized on the cell membrane has a function of actively excreting a drug outside the cell, and is a causative factor of drug resistance such as vincristine, doxorubicin, etoposide and an independent poor prognostic factor. It is. Up to now, the expression level of MDR1 in clinical samples has been determined by flow cytometry, immunohistochemistry, Western blot, in situ hybridization, Northern hybridizatio
It has been investigated by the n method. However, these experimental methods have drawbacks such as low detection sensitivity and quantitativeness, complexity of the experimental procedure, and a large amount of sample required for detection. Is not appropriate.

[0004] When the expression level of a gene is small, PCR or NAS
Nucleic acid amplification methods such as the BA method are extremely useful. Kobayashi et al. Co
MDR1 gene expression level was quantified using the mpetitive RT-PCR method, indicating that the MDR1 gene expression level correlates very well with the MDR1 protein amount on the cell membrane, the MDR1 protein reagent excretion ability, and the drug resistance level (Kobayashi H et al., (1997)
J. Clin. Lab. Anal. 11: 258-66 and Kawabata M et al., (199
7) Rinsho Byori 45: 891-8).

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to measure the expression level of MDR1, which is a gene responsible for drug resistance, quickly, simply, specifically, with high sensitivity, with high accuracy and broadly by using a nucleic acid amplification method. Provides a nucleic acid amplification primer, a nucleic acid capture probe, a nucleic acid detection probe, a quantitative measurement kit, and a quantitative method used for quantitative measurement with a range, and provides a rapid, simple, specific, and The object is to perform detection with high sensitivity, high accuracy, and a wide measurement range.

[0006]

Means for Solving the Problems In order to achieve the above object, the present inventors have intensively studied primers and probes that are a part of the nucleotide sequence of MDR1 mRNA, and completed the present invention. done.

That is, the present invention relates to an oligonucleotide comprising 15 to 30 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or one to several nucleotides in such an oligonucleotide as long as its function is not lost. Is deleted,
PC consisting of a substituted or added oligonucleotide
R (polymerase chain reaction) method, NASBA (nucleicaci
d sequence-based amplification) method, LAMP (Loop-media
ted isothemal amplification of DNA), TMA (Transcr
iption mediated amplification) method, ICAN (Isothermal
and chimeric primer-initiated amplification of nuc
A primer for nucleic acid amplification used for gene diagnosis of drug resistance by a nucleic acid amplification method such as a leic acids method.

Further, the present invention relates to an oligonucleotide having a promoter sequence of RNA polymerase at the 5 'end and consisting of 15 to 30 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or One or several bases are deleted as long as the function is not lost,
It is a primer for nucleic acid amplification used for gene diagnosis of drug resistance by a nucleic acid amplification method comprising a substituted or added oligonucleotide.

Further, the present invention relates to an oligonucleotide comprising 15 to 30 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or 1 to several nucleotides in such an oligonucleotide as long as its function is not lost. Is deleted,
A nucleic acid capture probe and a nucleic acid detection probe used for gene diagnosis of drug resistance by a nucleic acid amplification method comprising a substituted or added oligonucleotide.

[0010] The present invention further includes the above-mentioned primer, and optionally the above-mentioned capture probe and detection probe, and further comprises, if necessary, reverse transcriptase, ribonuclease H, RNA polymerase, DNA polymerase, dNTPs, NTPs and a buffering agent. A nucleic acid amplification reagent comprising:

Further, the present invention uses a sample collected from a subject as a sample to convert a part of the sequence corresponding to exons 6 to 9 of the nucleotide sequence of the drug resistance gene MDR1 into a forward primer and a reverse primer.
The drug resistance gene MDR1 amplified by the nucleic acid amplification method using seprimer and amplified in the case of a patient exhibiting drug resistance
This is a method for detecting the degree of drug resistance by capturing and detecting a nucleic acid corresponding to a part of the base sequence.

Further, the present invention is a method for quantifying MDR1 in the presence of a competitor as an internal standard when amplifying by nucleic acid amplification.

[0013]

DESCRIPTION OF THE PREFERRED EMBODIMENTS As a primer used in the present invention, a part of the nucleotide sequence of a nucleic acid corresponding to exons 6 to 9 of the drug resistance gene MDR1 is arbitrarily selected and used. MD
R1 is described, for example, in Chang-jie Chen et al., (1990) J. Biol.
m. 265: 506-14 "discloses its nucleotide sequence. The following primers are preferred.

As the second primer, an oligonucleotide consisting of consecutive 15 to 30 bases in the nucleotide sequence of SEQ ID NO: 1 is preferable, and an oligonucleotide consisting of the 81st to 102nd is particularly preferable. Further, as the second primer, the above oligonucleotide having an RNA polymerase promoter sequence at the 5 ′ end may be used.

As the first primer, an oligonucleotide consisting of 15 to 30 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 2 is preferable, and an oligonucleotide consisting of nucleotides 274 to 294 is particularly preferable. Further, as the first primer, the above-mentioned oligonucleotide having an RNA polymerase promoter sequence at the 5 ′ end may be used.

Examples of the promoter of the RNA polymerase include the T7 promoter of SEQ ID NO: 5. The promoter sequence may include a space up to the replication origin, and for example, a sequence such as 5'-AGGA-3 'may be bound to the 3' end of the promoter sequence as necessary.

Similarly, as a probe for capture or detection, a part of the nucleotide sequence of the nucleic acid corresponding to exons 6 to 9 of MDR1 is selected and used arbitrarily.
It is preferably a probe having a length of 0 nucleotides, and more preferably an oligonucleotide consisting of 15 to 30 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In particular, SEQ ID NO: 3 28-54 or 62-83
No. 67 and SEQ ID NO: 4
The oligonucleotide consisting of No. 93 or Nos. 38 to 59 is
Preferred as a probe for capture or detection.

[0018] Oligonucleotides in which one to several bases have been deleted, substituted or added within a range that does not lose their function in all oligonucleotides used in the present invention are also within the scope of the present invention.

The specimen used in the present invention can be any of serum, urine, saliva, sputum, ascites, pleural effusion, cerebrospinal fluid, bone marrow fluid, organ sections or peripheral blood, but the nucleic acid substance used for amplification is unstable. Therefore, it is better to start the extraction process as soon as possible. When not used immediately, it is necessary to store at −20 ° C. or lower or −80 ° C. to suppress nucleic acid degradation.

Next, it is preferable that the nucleic acid is extracted by basically performing phenol treatment as a denaturation treatment of enzymes and then performing alcohol precipitation. According to this method, the concentration of nucleic acid, the removal of phenol, salts and nucleotides present in the nucleic acid solution, and the replacement of the buffer can be simultaneously and quickly performed. When extracting RNA, RNA is susceptible to alkalis and temperature.
Degraded more easily than DNA. Since animal-derived materials contain a relatively large amount of a degrading enzyme, it is preferable to freeze instantaneously if not immediately extracted. In addition, it is preferable to immerse immediately in a solution containing a ribonuclease inhibitor after thawing. Many extraction methods have been developed and a simple method (EDTA-SDS-
Phenol-Ethanol), lithium chloride-urea method, protease
K-deoxyribonuclease method, phenol SDS method, guanidine thiocyanate cesium chloride method, guanidine thiocyanate cesium trifluoroacetate method, acid guanidine thiocyanate phenol chloroform method (AGP
C)) and the vanadyl ribonucleoside complex method, etc., but in the present invention, the AGPC method is preferred.

The primers and probes used in the present invention are as described above. The synthesis of these nucleotides uses a commercially available DNA / RNA synthesizer, and there is almost no problem if they are synthesized according to the synthesis conditions of the synthesizer. In order to improve the amplification efficiency, it is important to improve the purity of the primer. Preferably, the synthesized primers are fractionated by liquid chromatography and used.
The detection probe varies depending on the detection method,
A good result is obtained when the length is about 20 to 40 mer.

The present invention relates to the conventional methods for amplifying nucleic acids, including, for example, PCR, RT-PCR, NASBA, ligase chain reaction (LCR), self-sequence replication (3SD), TM
It can be applied to A method, LAMP method, ICAN method, etc.

The present inventors have concluded that the present invention provides an
It has been confirmed that it is particularly effective and useful when the SBA method is used. In the NASBA method, the target nucleic acid sequence (RN
The first primer having a sequence complementary to A) and a promoter sequence of RNA polymerase at the 5 ′ end thereof is hybridized to a target nucleic acid (RNA) in a sample in the presence of an appropriate buffer, and reverse transcriptase is used. DNA from dNTPs
(Step 1), only RNA of the RNA / DNA hybrid is degraded by ribonuclease H to obtain a single-stranded DNA (second template) (Step 2), and then the single-stranded DNA (second template)
Is hybridized with a second primer having a sequence complementary to the target nucleic acid sequence (DNA).
An extension reaction is performed with a polymerase to obtain a double-stranded DNA (Step 3). Next, a large number of RNA copies (third templates) are synthesized using NTPs as a material by using an RNA polymerase that recognizes a promoter sequence (step 4). Using the obtained RNA (third template) as a template, synthesis of an RNA / DNA hybrid with a second primer using reverse transcriptase (step 5), single-stranded DNA that degrades only RNA with ribonuclease H
Synthesis of (fourth template) (step 6), resulting single-stranded DNA
Using the (fourth template) as a template, synthesis of double-stranded DNA with the first primer using DNA polymerase (Step 7), R
Synthesis of multiple copies (third template) of RNA from the double-stranded DNA using NA polymerase (step 8). By repeating these steps (steps 5 to 8), the nucleic acid in the sample can be amplified.

The NASBA method is based on another measurement method (PCR method (JP-A-60-2
81) and the RT-PCR method), the gene can be quantitatively measured using a competitor as an internal standard (competitor). As a competitor, it is important to replace the sequence part of the capture probe in the amplification part with another sequence and use it in order to obtain good results. Preferably, the replacement sequences are approximately the same length and have no homology. Also,
It is desirable that the Tm value with the supplementary probe does not have a large difference and does not participate in the formation of the secondary structure. Competitor is about 10
Those having a size of about 00mer are preferable.

In the LAMP method, a gene can be qualitatively measured. Gene, 2 types of forward primer, 2 types of reve
When rse primer and DNA synthase are reacted at 65 ° C. for about 1 hour, the gene is amplified with high efficiency. When the target gene is RNA, reverse transcriptase is added to the above reaction solution.

In the ICAN method, a chimeric primer of DNA and RNA having RNA bonded to the 3 ′ side is used as a primer, and the gene is amplified at 50-60 ° C. for a certain time together with a gene, DNA synthase, RNaseH, and dNTPs.

[0027]

Next, the present invention will be described in more detail with reference to examples, but it goes without saying that the present invention is not limited to only the examples.

Example 1 [Measurement of MDR1 RNA Using Competitive NASBA Method] Synthesis of MDR1 standard RNA (WT) MDR1 cD containing NASBA amplified region corresponding to exons 6 to 9
Plasmid (pM65) with part of NA incorporated into pSP65 (Promega)
DR94) was used to synthesize synthetic RNA in vitro. That is, pMD
R94 is treated with a restriction enzyme and cut at the 3 'end of the cDNA.
After fragmentation, RNA was synthesized using MEGAscript kits. The copy number of the synthetic RNA was calculated from the absorbance at 260 nm.

2. MDR1 competitor RNA (QA) synthesis An RNA synthesis plasmid (pMDR94QA) was prepared in which only the binding sequence of the capture probe was changed to a sequence having no homology to standard RNA. Competitor RNA was synthesized according to a standard RNA synthesis method. The copy number of the synthetic RNA was calculated from the absorbance at 260 nm.

3. Samples Total RNA was extracted from eight clinical samples.

4. Dilution of RNA WT RNA was serially diluted to 10 ² copies / 5 μL. QA R
NA was diluted in 10 steps to 10 ⁴ copies / 5 μL. The total RNA derived from the sample was prepared at 0.1 μg / 5 μL.

5. Primer and probe A primer consisting of nucleotides 81 to 102 of SEQ ID NO: 1 as a forward primer, a primer consisting of oligonucleotides 274 to 294 of SEQ ID NO: 2 as a reverse primer and having a T7 promoter sequence at its 5 ′ end, and 28 to 54 of SEQ ID NO: 3 as capture probes
No. 62-83 of SEQ ID NO: 3 was used as a detection probe.

6. Competitive NASBA 40 μL NASBA reaction system with 10 ² , 10 ⁴ , 10 ⁷ copies of standard
Total RNA (0.1 [mu] g / 5 [mu] L) and 10 ⁴ copies of the competitor RNA from RNA or specimens was amplified reaction added. MDR1 / Q measurement of amplification products using an automatic analyzer (Toyobo Co., Ltd.)
A Using each detection plate, perform each signal ratio (log (W
T / QA)), and the expression level (copy / μg) was calculated from the regression equation.

7. Results FIG. 1 shows the performance of the MDR1 quantitative measurement kit. From FIG.
It was found that the logarithm of WT / QA and WT RNA expression had a good correlation with a correlation function of 0.99 over a wide range. Therefore,
By measuring WT / QA by competitive NASBA method, WT
MDR1 can be quantitatively measured with high sensitivity, high precision and a wide measurement range as the expression level of RNA, and it can be said that it is excellent also in terms of operability and quickness. FIG. 2 shows a high correlation with the RT-PCR method. RT-PCR is described in MiyachiH et al., (199
3) Performed according to a method according to Int. J. Hematol. 57: 31-7.

[0035]

As described above, according to the present invention, the expression level of MDR1 can be quantified rapidly, simply, specifically, with high sensitivity, with high precision, and in a wide measurement range. The degree of drug resistance of the subject is indicated by the expression level of MDR1 quantitatively detected.

[0036]

[Sequence List] <110> Kainos Laboratories, Inc. <120> A kit for quantitatively detecting the level of expression of MDR1 gene <130> 00PKN194 <160> 5 <210> 1 <211> 120 <212> DNA <213> Artificial Sequence <400> 1 ctgcttacat tcaggtttca ttttggtgcc tggcagctgg aagacaaata cacaaaatta 60 gaaaacagtt ttttcatgct ataatgcgac aggagatagg ctggtttgat gtgcacgatg 120 <210> 2 <211> 300 <212> DNA <213> Artificial Sequence <400> 2 tagaatattc ccctgagagg accaaggtgg tcccatacca gaaggccaga gcataagatg 60 catagatcag caggaaagca gcacctatag aaatattggc tgtaatagct ttctttatcc 120 caattctttt agcttcttct aaatttttgt tgtacctttc aagttctttc ttttgtcctc 180 caaatgcaat cacagttcta attgctgcca agacctcttc agctactgct ccagcttttg 240 catacgctaa gagttcttta tcagtaaatg aagatagtat ctttgcccag acagcagctg 300 <210> 3 <211> 120 <212> DNA <213> Artificial Sequence <400> 3 acaaaattgg aatgttcttt cagtcaatgg caacattttt cactgggttt atagtaggat 60 ttacacgtgg ttggaagcta acccttgtga ttttggccat cagtcctgtt cttggactgt 120 <210> 4 <211> 120 <212> DNA <213> Arti ficial Sequence <400> 4 acagtccaag aacaggactg atggccaaaa tcacaagggt tagcttccaa ccacgtgtaa 60 atcctactat aaacccagtg aaaaatgttg ccattgactg aaagaacatt ccaattttgt 120 <210> 5 <211> 27 <212> DNA <213> gg agtagcta catattac 400

[Brief description of the drawings]

FIG. 1 is a diagram showing the performance of an MDR1 quantitative measurement kit.

FIG. 2 is a diagram showing the correlation between the expression levels of MDR1 by the NASBA method and the RT-PCR method.

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Masako Kawase 1274-7 Asahi, Oka-shi, Ito-shi, Shizuoka Pref. F term (reference) 4B024 AA11 BA80 CA01 CA09 CA12 HA12 4B063 QA01 QA19 QQ42 QQ53 QR08 QR55 QR62 QS25 QS34 QS36 QX02

Claims (25)

[Claims] (1) a sequence consisting of consecutive 1s in the base sequence of SEQ ID NO: 1;
An oligonucleotide consisting of 5 to 30 bases, or 1 as long as the function of the oligonucleotide is not lost;
A primer for nucleic acid amplification, comprising an oligonucleotide in which one to several bases have been deleted, substituted or added, and used for quantification of MDR1 expression level by a nucleic acid amplification method. 2. An oligonucleotide having a promoter sequence of RNA polymerase at its 5 'end and comprising 15 to 30 consecutive nucleotides in the base sequence of SEQ ID NO: 1, or a range in which the function of the oligonucleotide is not lost. A primer for nucleic acid amplification used for quantifying the expression level of MDR1 by a nucleic acid amplification method, comprising an oligonucleotide in which one to several bases have been deleted, substituted or added. 3. The oligonucleotide of SEQ ID NO: 1
The primer according to claim 1 or 2, which is an oligonucleotide of # 102 or an oligonucleotide in which one to several bases have been deleted, substituted or added within a range not losing its function. 4. A continuous 1 in the nucleotide sequence of SEQ ID NO: 2.
An oligonucleotide consisting of 5 to 30 bases, or 1 as long as the function of the oligonucleotide is not lost;
A primer for nucleic acid amplification, comprising an oligonucleotide in which one to several bases have been deleted, substituted or added, and used for quantification of MDR1 expression level by a nucleic acid amplification method. 5. An oligonucleotide having an RNA polymerase promoter sequence at its 5 ′ end and comprising 15 to 30 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 2, or a range in which the function of the oligonucleotide is not lost A primer for nucleic acid amplification used for quantifying the expression level of MDR1 by a nucleic acid amplification method, comprising an oligonucleotide in which one to several bases have been deleted, substituted or added. 6. The oligonucleotide according to claim 2, wherein the oligonucleotide comprises 27 of SEQ ID NO: 2.
The primer according to claim 4 or 5, which is an oligonucleotide of Nos. 4 to 294, or an oligonucleotide in which one to several bases have been deleted, substituted or added within a range not losing its function. 7. A continuous 1 in the nucleotide sequence of SEQ ID NO: 3.
An oligonucleotide consisting of 5 to 30 bases, or 1 as long as the function of the oligonucleotide is not lost;
A nucleic acid capture probe comprising an oligonucleotide in which 1 to several bases have been deleted, substituted or added, and which is used for quantification of the MDR1 expression level by a nucleic acid amplification method. 8. The oligonucleotide according to claim 8, wherein the oligonucleotide is SEQ ID NO: 28.
The probe according to claim 7, which is an oligonucleotide of Nos. 54 to 54 or an oligonucleotide in which one to several bases have been deleted, substituted or added within a range not to lose its function in said oligonucleotide. 9. The continuous 1 in the nucleotide sequence of SEQ ID NO: 4.
An oligonucleotide consisting of 5 to 30 bases, or 1 as long as the function of the oligonucleotide is not lost;
A nucleic acid capture probe comprising an oligonucleotide in which 1 to several bases have been deleted, substituted or added, and which is used for quantification of the MDR1 expression level by a nucleic acid amplification method. 10. The oligonucleotide according to claim 6, wherein the oligonucleotide is SEQ ID NO: 4.
The probe according to claim 9, which is an oligonucleotide of Nos. 7 to 93, or an oligonucleotide in which one to several bases are deleted, substituted or added within a range not losing its function. 11. An oligonucleotide consisting of 15 to 30 contiguous bases in the base sequence of SEQ ID NO: 3, or one to several bases deleted, substituted or added within a range not to lose its function in the oligonucleotide. A nucleic acid detection probe comprising the oligonucleotide thus obtained and used for quantification of the MDR1 expression level by a nucleic acid amplification method. 12. The oligonucleotide of SEQ ID NO: 3-6
The probe according to claim 11, which is an oligonucleotide of Nos. 2 to 83, or an oligonucleotide in which one to several bases have been deleted, substituted or added within a range not losing its function. 13. An oligonucleotide consisting of 15 to 30 contiguous bases in the base sequence of SEQ ID NO: 4, or one to several bases deleted, substituted or added to the oligonucleotide within a range not to lose its function. A nucleic acid detection probe comprising the oligonucleotide thus obtained and used for quantification of the MDR1 expression level by a nucleic acid amplification method. 14. The oligonucleotide of SEQ ID NO: 4
14. The probe according to claim 13, which is an oligonucleotide of Nos. 8 to 59, or an oligonucleotide in which one to several bases have been deleted, substituted, or added within a range not losing its function. 15. A reagent for a nucleic acid amplification method, comprising the primer according to claim 1, 2 or 3, and the primer according to claim 4, 5 or 6. 16. The method of claim 7, 8, 9 or 10.
The reagent according to claim 15, comprising the capture probe of claim 11, and the detection probe of claim 11, 12, 13, or 14. 17. The reagent according to claim 15, further comprising the supplementary probe according to claim 11, 12, 13, or 14 and the detection probe according to claim 7, 8, 9, or 10. 18. The method according to claim 18, further comprising: reverse transcriptase, ribonuclease H, RNA polymerase, DNA polymerase, dNTPs, NT
18. The reagent according to claim 15, 16 or 17, comprising Ps and a buffer. 19. Using a sample collected from a subject as a sample, a part of the sequence corresponding to exons 6 to 9 of the nucleotide sequence of the drug resistance gene MDR1 is forward primer and reverse primate.
ER to detect the degree of drug resistance by capturing and detecting the nucleic acid corresponding to a part of the base sequence of the amplified drug resistance gene MDR1 in the case of a patient sample that exhibits drug resistance when amplified by the nucleic acid amplification method how to. 20. The diagnostic method according to claim 19, wherein the capture / detection is performed using a probe set between the forward primer and the reverse primer in the base sequence of MDR1. 21. The diagnostic method according to claim 19, wherein the forward primer is the primer according to claim 1, 2 or 3. 22. The method according to claim 19, wherein the reverse primer is the primer according to claim 4, 5 or 6.
Term diagnosis method. 23. The capture probe according to claim 7, 8, 9 or 10, and the detection probe according to claim 1.
21. The probe of claim 1, 12, 13, or 14.
Diagnostic method. 24. The capture probe according to claim 11, 12, 1, or
21. The diagnostic method according to claim 20, wherein the probe is 3 or 14, and the detection probe is the probe according to claim 7, 8, 9 or 10. 25. The method according to any one of claims 19 to 24, wherein MDR1 is quantified in the presence of a competitor as an internal standard during amplification by a nucleic acid amplification method.