CN112501265A - Reagent, detection method and kit for detecting CYP2D6 gene - Google Patents

Abstract

The invention relates to a CYP2D6 genotyping detection kit, which comprises a detection primer and a probe of CYP2D6 x 10, and a detection primer and a probe of an internal reference gene ALB. The invention can distinguish CYP2D6 x 10 and CYP2D6 x 5 by amplifying CYP2D6 and internal reference (ALB) genes. Provides a new way for simple judgment for clinical medication guidance.

Description

Reagent, detection method and kit for detecting CYP2D6 gene

Technical Field

The invention relates to the fields of molecular biology and medicine, in particular to a polymorphism detection reagent, a kit and a detection method for drug metabolism related genes CYP2D 6.

Background

According to the publication of the World Health Organization (WHO), the proportion of the patients WHO are hospitalized in all countries of the world to suffer from the adverse drug reactions is 10% -20%, wherein 5% of the patients die of the serious adverse drug reactions. Therefore, pharmacogenomics research has become a global focus, and personalized medicine and personalized treatment are the key points of discussion of international academic conference of various medical biology.

Cytochrome (CYP)2D6 is one of important members of CYP450 family, although the total amount only accounts for 2% -9% of liver enzymes, the Cytochrome (CYP)2D6 participates in 20% -30% of clinical drug metabolism, and comprises more than 80 drugs such as antihypertensive drugs, antidepressant drugs, antiarrhythmic drugs, antipsychotic drugs, antitumor drugs and the like.

There are many polymorphisms in the CYP2D6 gene, and more than 100 mutation sites have been found so far. Among them, the normal allele CYP2D6 x 1, the intermediate metabolic allele CYP2D6 x 10 and the slow metabolic allele CYP2D6 x 5 are the most common. CYP2D6 × 10 is 188C mutation on the 1 st exon of CYP2D6 is T, the allele frequency is 51.6%, CYP2D6 × 5 is deletion of the whole CYP2D6 gene, and the occurrence frequency is 3% -7%.

The Taqman probe fluorescence PCR method is a common SNP typing method, the method is mature in technology and can be developed into a commercialized kit. However, if the common Taqman probe method is used to type CYP2D6, there is a first problem that only CYP2D6 × 10 can be typed, and CYP2D6 × 5 cannot be typed.

The interpretation of the results of the polymorphism detection of the gene CYP2D6 is troubled by the CYP2D6 x 10 typing, which considers only the 188C mutation T on the 1 st exon of CYP2D6, and the CYP2D6 x 5 typing, which does not consider the deletion of the entire CYP2D6 gene. Since human beings have deletion mutations of CYP2D6 x 5 type, when the Taqman probe method is used to classify CYP2D6 x 10, there is no fluorescence signal when the deletion mutation of CYP2D6 x 5 type is encountered. In this case, the operator cannot judge whether the absence of the fluorescence signal is caused by various incidental factors such as too low a DNA concentration or degradation of the sample, an erroneous sample due to an operation error, and an unstable PCR system, or whether the sample itself is a deletion mutation of CYP2D6 × 5 type. Therefore, it is urgently needed to develop a new detection reagent and a new detection method which can simultaneously judge whether the gene CYP2D6 in a sample is wild-type CYP2D6 x 1, mutant CYP2D6 x 10 or deletion CYP2D6 x 5.

Disclosure of Invention

The invention provides a detection kit for judging mutant type and deletion type of CYP2D6 gene aiming at the defect of the existing Taqman probe fluorescence PCR method for detecting CYP2D6, and the detection kit comprises a primer and a probe for detecting CYP2D6 x 10, and a primer and a probe for detecting internal reference gene ALB.

The sequences of the primer and the probe for detecting CYP2D6 × 10 are respectively as follows:

CYP2D6 × 10 upstream primer: 5'-CCATTTGGTAGTGAGGCAGGT-3'

CYP2D6 × 10 downstream primer: 5'-ATGCAGCAGGTTGCCCA-3'

CYP2D6 × 10 wild-type probe: FAM-CACGCTACCCACCAGG-MGBNFQ

CYP2D6 × 10 mutant probe: VIC-ACGCTACTCACCAGG-MGBNBNFQ

The sequences of primers and probes for detecting the internal reference gene ALB are respectively as follows:

ALB upstream primer: 5'-TTGTGGGCTGTAATCATCGTCTA-3'

ALB downstream primer: 5'-GTTCTCTTTCACTGACATCTGCAAA-3'

ALB probe: CY5-CCCACACAAATCTCTCCCTGGCATTG-BHQ 3.

The invention also provides application of the primer and the probe in a drug-directed analysis kit.

The invention also provides a method for detecting the polymorphism of the CYP2D6 gene. The method comprises the following steps:

(1) extracting genomic DNA in a sample;

(2) and (3) performing fluorescent quantitative PCR on the DNA obtained in the step (1), wherein PCR reaction reagents comprise primers and probes for detecting CYP2D6 x 10 and primers and probes for detecting an internal reference gene ALB.

(3) And (5) judging the result. If CY5 and FAM have signals, the signal is CYP2D6 x 1 wild type; if CY5 and VIC have signals, the mutant is CYP2D6 x 10 homozygous mutant; if CY5, FAM and VIC have signals, the mutant is CYP2D6 x 1/x 10 hybrid mutant; if only CY5 has a signal, it is CYP2D6 × 5 type.

In the present invention, CYP2D6 × 1 indicates that the first exon of the gene CYP2D6 is wild type; CYP2D6 × 10 indicates a mutant form of 188C mutation to T on exon 1 of the gene CYP2D 6; CYP2D6 x 1/' 10 represents a hybrid mutant; CYP2D6 × 5 indicates a deletion type in which the entire CYP2D6 gene was deleted.

Compared with the prior art, the method can accurately detect the CYP2D6 x 10(188C > T) gene polymorphism in human peripheral blood samples, and the detectable polymorphism types comprise CYP2D6 x 1 wild type (C/C), CYP2D6 x 1/x 10 heterozygous mutant type (C/T), CYP2D6 x 10 homozygous mutant type (T/T) and CYP2D6 x 5 gene deletion type. The specific analysis is as follows:

1. the CYP2D6 x 1 homozygous wild type, CYP2D6 x 1/10 heterozygous mutant and CYP2D6 x 10 homozygous mutant can be distinguished in one reaction by specifically designing the CYP2D6 x 1 wild type probe and the CYP2D6 x 10 mutant probe.

2. The first function of the internal reference gene ALB in the invention is to judge whether the fluorescent quantitative PCR reaction is normal or not, and the second function is to judge whether a gene deletion type exists or not. The CYP2D6 x 5 gene deletion type is judged by a method of adding internal reference gene detection, and when the wild type and the mutant type of CYP2D6 x 10 have no signals, and the internal reference has signals, the gene deletion can be judged.

3. By using the optimized reaction system, the amplification of CYP2D6 and the internal reference gene ALB are simultaneously carried out in one PCR reaction hole, so that the cost is saved, the error between the holes can be completely avoided, and the reliability of the result is ensured.

4. The reaction system used in the invention has strong anti-inhibitor capability, and can amplify DNA samples and also directly amplify blood or blood pretreatment substances.

5. The whole method only needs one fluorescence PCR reaction, and the manual operation time, so that the whole detection only needs less than 2 hours, and the method has the advantages of time saving and labor saving.

Drawings

Fig. 1 amplification curve of CYP2D6 x 1 wild type (C/C).

FIG. 2 amplification curves for CYP2D6 x 10 homozygous mutant (T/T).

FIG. 3 amplification curves for CYP2D6 x 1/' 10 hybrid mutant (C/T).

Figure 4 amplification curve of CYP2D6 x 5 type deletion.

FIG. 5 amplification curves for the blank control.

Figure 6CYP2D6 × 10 wild type (C/C) generation sequencing map.

Figure 7CYP2D6 × 10 mutant (T/T) generation sequencing map.

FIG. 8CYP2D6 x 10 hybrid (C/T) generation sequencing profile.

FIG. 9 electrophoretogram of example 3.

Detailed Description

The invention provides a kit and a method capable of simultaneously typing CYP2D6 x 10 and CYP2D6 x 5. The specific fluorescent probe is combined with the real-time fluorescent PCR technology to detect the gene polymorphism of human CYP2D6 x 10(188C > T). According to the site sequence of human CYP2D6 x 10(188C > T) gene, a wild type probe and a mutant type probe with high specificity and an ALB reference gene probe are designed respectively and are marked by different fluorescent signal groups respectively. When the specific probe is completely complementary and matched with the corresponding allele type template, after the probe is combined with the template, the probe is hydrolyzed to generate a fluorescent signal and generate a corresponding Ct value. Human CYP2D6 (188C > T) genotype or CYP2D6 x 5 was judged on a fluorescence quantitative PCR instrument from information of fluorescence signals (Ct values) forming an amplification curve.

Example 1 detection kit Using fluorescent quantitative PCR detection System

The detection kit comprises an amplification primer, a probe and other matched reagents for carrying out fluorescent quantitative PCR detection. Amplification primers and probes were designed against both CYP2D6 gene CYP2D6 x 10 and CYP2D6 x 5 types.

First, design and synthesis of amplification primer

CYP2D6 × 10 was based on the 188C → T mutation on the 1 st exon of CYP2D6, and the primers and probes for detecting CYP2D6 × 10 were:

CYP2D6 × 10 upstream primer: 5'-CCATTTGGTAGTGAGGCAGGT-3'

CYP2D6 × 10 downstream primer: 5'-ATGCAGCAGGTTGCCCA-3'

CYP2D6 × 10 wild-type probe: FAM-CACGCTACCCACCAGG-MGBNFQ

CYP2D6 × 10 mutant probe: VIC-ACGCTACTCACCAGG-MGBNBNFQ

CYP2D6 × 5 is the deletion of the whole CYP2D6 gene, and the primers and probes for detecting CYP2D6 × 5 comprise the primers and probes for CYP2D6 × 10 and an internal reference gene ALB, which are respectively as follows:

CYP2D6 × 10 upstream primer: 5'-CCATTTGGTAGTGAGGCAGGT-3' (SEQ 1)

CYP2D6 × 10 downstream primer: 5'-ATGCAGCAGGTTGCCCA-3' (SEQ 2)

CYP2D6 × 10 wild-type probe: FAM-CACGCTACCCACCAGG-MGBNFQ (SEQ 3)

CYP2D6 × 10 mutant probe: VIC-ACGCTACTCACCAGG-MGBNFQ (SEQ 4)

ALB upstream primer: 5'-TTGTGGGCTGTAATCATCGTCTA-3' (SEQ 5)

ALB downstream primer: 5'-GTTCTCTTTCACTGACATCTGCAAA-3' (SEQ 6)

ALB probe: CY5-CCCACACAAATCTCTCCCTGGCATTG-BHQ3(SEQ 7)

The fluorescent signal group marked on the probe in this embodiment is only a specific embodiment, and one skilled in the art can also select different fluorescent signal groups to mark according to needs.

The composition of the kit is shown in table 1.

TABLE 1

Example 2 fluorescent quantitative PCR detection method

A method of simultaneously typing CYP2D6 x 10 and CYP2D6 x 5, comprising the steps of:

(1) blood pretreatment: the blood pretreatment reagent includes TE Buffer. The blood pretreatment method comprises taking 80 μ L of whole blood sample, placing in a 1.5mL centrifuge tube, adding 1mL purified water, mixing by inversion, and centrifuging at 12000 × g for 1 min. Discard the supernatant, add 50. mu.L of 1 XTE Buffer, vortex and mix well, resuspend the pellet to obtain the blood pretreatment.

(2) Fluorescent PCR system

Reaction components	Test sample	Positive control	Negative control
				CYP2D6 × 10PCR reaction solution	17.6μL	17.6μL	17.6μL
Reaction enzyme	0.4μL	0.4μL	0.4μL
				Blood pretreatment	2μL	/	/
CYP2D6 x 10 positive control	/	2μL	/
				CYP2D6 x 10 negative control	/	/	2μL

(3) Fluorescent PCR procedure

"+" indicates fluorescence signal collection site

(4) Interpretation of results

Ct value interpretation method:

if the Ct value of the internal reference signal CY5 is greater than 30, the sample is abnormal, and the sample needs to be reprocessed or collected again. The amplification curve interpretation method is shown in FIGS. 1 to 5.

The optimized PCR reaction solution adopted by the invention has strong anti-inhibitor capability, can amplify blood pretreatment substances, is not limited to the blood pretreatment substances, and can amplify extracted DNA samples or directly amplify blood as a template.

Example 3 analysis of clinical samples Using fluorescent quantitative PCR

32 clinical specimens were collected, genomic DNA was extracted using a commercial column extraction kit, and clinical specimens for CYP2D6 gene polymorphism were developed in examples 1 and 2. Meanwhile, the first-generation sequencing method is used for carrying out genotyping detection on the extracted DNA, and the genotyping result of the method is compared with the genotyping result of the first-generation sequencing. The results are shown in Table 2, with 100% accuracy. The sequencing results for the three genotypes C/C, C/T and T/T are shown in FIGS. 6 to 8.

Table 2:

sequencing validation method for CYP2D6 × 5 type:

this method is derived from the literature: hersberger M, Marti-Jaun J, Rentsch K, et al Rapid detection of the CYP2D6 x 3, CYP2D6 x 4, and CYP2D6 x 6 alloys by four primer PCR and of the CYP2D6 x 5 alloys by multiplex Long PCR [ J ]. Clinical Chemistry,2000, 46(8I): 1072-. The specific method comprises the following steps:

an amplification primer:

PCR reaction system

The template is 1 sample of C/C, C/T and T/T type, 2 samples of CYP2D6 x 5 deletion type.

PCR procedure:

sequencing analysis: the amplification product is sent to sequencing. The result shows that the typing result of the fluorescence PCR is consistent with the genotype of the sequencing result. The 5.1kb (CYP2D6 non-deleted) sequence is shown in SEQ 8, and the 3.5kb (CYP2D6 deleted) sequence is shown in SEQ 9.

5.1kb (CYP2D6 non-deleted) sequence (SEQ 8), wherein Y represents C or T. The wild type sequence Y is C, and the mutant type sequence Y is T.

GTTATCCCAGAAGGCTTTGCAGGCTTCAGGAGCTTGGAGTGGGGAGAGGGGGTGACT TCTCCGACCAGGCCCCTCCACCGGCCTACCCTGGGTAAGGGCCTGGAGCAGGAAGCA GGGGCAAGAACCTCTGGAGCAGCCCATACCCGCCCTGGCCTGACTCTGCCACTGGCA GCACAGTCAACACAGCAGGTTCACTCACAGCAGAGGGCAAAGGCCATCATCAGCTCC CTTTATAAGGGAAGGGTCACGCGCTCGGTGTGCTGAGAGTGTCCTGCCTGGTCCTCTG TGCCTGGTGGGGTGGGGGTGCCAGGTGTGTCCAGAGGAGCCCATTTGGTAGTGAGGC AGGTATGGGGCTAGAAGCACTGGTGCCCCTGGCCGTGATAGTGGCCATCTTCCTGCTCC TGGTGGACCTGATGCACCGGCGCCAACGCTGGGCTGCACGCTACY(C>T)

CACCAGGCCCCCTGCCACTGCCCGGGCTGGGCAACCTGCTGCATGTGGACTTCCAGAA CACACCATACTGCTTCGACCAGGTGAGGGAGGAGGTCCTGGAGGGCGGCAGAGGTGC TGAGGCTCCCCTACCAGAAGCAAACATGGATGGTGGGTGAAACCACAGGCTGGACCA GAAGCCAGGCTGAGAAGGGGAAGCAGGTTTGGGGGACKTCCTGGAGAAGGGCATTTA TACATGGCATGAAGGACTGGATTTTCCAAAGGCCAAGGAAGAGTAGGGCAAGGGCCT GGAGGTGGAGCTGGACTTGGCAGTGGGCATGCAAGCCCATTGGGCAACATATGTTATG GAGTACAAAGTCCCTTCTGCTGACACCAGAAGGAAAGGCCTTGGGAATGGAAGATGA GTTAGTCCTGAGTGCCGTTTAAATCACGAAATCGAGGATGAAGGGGGTGCAGTGACCC GGTTCAAACCTTTTGCACTGTGGGTCCTCGGGCCTCACTGCTCACCGGCATGGACCAT CATCTGGGAATGGGATGCTAACTGGGGCCTCTCGGCAATTTTGGTGACTCTTGCAAGGT CATACCTGGGTGACGCATCCAAACTGAGTTCCTCCATCACAGAAGGTGTGACCCCCAC CCCCGCCCCACGATCAGGAGGCTGGGTCTCCTCCTTCCACCTGCTCACTCCTGGTAGC CCCGGGGGTCGTCCAAGGTTCAAATAGGACTAGGACCTGTAGTCTGGGGDGATCCTGG CTTGACAAGAGGCCCTGACCCTCCCTCTGCAGTTGCGGCGCCGCTTCGGGGACGTGTT CAGCCTGCAGCTGGCCTGGACGCCGGTGGTCGTGCTCAATGGGCTGGCGGCCGTGCG CGAGGCGCTGGTGACCCACGGCGAGGACACCGCCGACCGCCCGCCTGTGCCCATCAC CCAGATCCTGGGTTTCGGGCCGCGTTCCCAAGGCAAGCAGCGGTGGGGACAGAGACA GATTTCCGTGGGACCCGGGTGGGTGATGACCGTAGTCCGAGCTGGGCAGAGAGGGCG CGGGGTCGTGGACATGAAACAGGCCAGCGAGTGGGGACAGCGGGCCAAGAAACCAC CTGCACTAGGGAGGTGTGAGCATGGGGACGAGGGCGGGGCTTGTGACGAGTGGGCGG GGCCACTGCCGAGACCTGGCAGGAGCCCAATGGGTGAGGCTGGCGCATTTCCCAGCT GGAATCCGGTGTCGAAGTGGGGGGCGGGGACCGCACCTGTGCTGTAAGCTCAGTGTG GGTGGCGCGGGGCCCGCGGGGTCTTCCCTGAGTGCAAAGGCGGTCAGGGTGGGCAGA GACGAGGTGGGGCAAAGCCCTGCCCCAGCCAAGGGAGCAAGGTGGATGCACAAAGA GTGGGCCCTGTGACCAGCTGGACAGAGCCAGGGACTGCGGGAGACCAGGGGGAGCAT AGGGTTGGAGTGGGTGGTGGATGGTGGGGCTAATGCCTTCATGGCCACGCGCACGTGC CCGTCCCACCCCCAGGGGTGTTCCTGGCGCGCTATGGGCCCGCGTGGCGCGAGCAGA GGCGCTTCTCCGTGTCCACCTTGCGCAACTTGGGCCTGGGCAAGAAGTCGCTGGAGC AGTGGGTGACCGAGGAGGCCGCCTGCCTTTGTGCCGCCTTCGCCAACCACTCCGGTG GGTGATGGGCAGAAGGGCACAAAGCGGGAACTGGGAAGGCGGGGGACGGGGAAGG CGACCCCTTACCCGCATCTCCCACCCCCAGGACGCCCCTTTCGCCCCAACGGTCTCTTG GACAAAGCCGTGAGCAACGTGATCGCCTCCCTCACCTGCGGGCGCCGCTTCGAGTACG ACGACCCTCGCTTCCTCAGGCTGCTGGACCTAGCTCAGGAGGGACTGAAGGAGGAGT CGGGCTTTCTGCGCGAGGTGCGGAGCGAGAGACCGAGGAGTCTCTGCAGGGCGAGCT CCCGAGAGGTGCCGGGGCTGGACTGGGGCCTCGGAAGAGCAGGATTTGCATAGATGG GTTTGGGAAAGGACATTCCAGGAGACCCCACTGTAAGAAGGGCCTGGAGGAGGAGGG GACATCTCAGACATGGTCGTGGGAGAGGTGTGCCCGGGTCAGGGGGCACCAGGAGAG GCCAAGGACTCTGTACCTCCTATCCACGTCAGAGATTTCGATTTTAGGTTTCTCCTCTG GGCAAGGAGAGAGGGTGGAGGCTGGCACTTGGGGAGGGACTTGGTGAGGTCAGTGG TAAGGACAGGCAGGCCCTGGGTCTACCTGGAGATGGCTGGGGCCTGAGACTTGTCCA GGTGAACGCAGAGCACAGGAGGGATTGAGACCCCGTTCTGTCTGGTGTAGGTGCTGA ATGCTGTCCCCGTCCTCCTGCATATCCCAGCGCTGGCTGGCAAGGTCCTACGCTTCCAA AAGGCTTTCCTGACCCAGCTGGATGAGCTGCTAACTGAGCACAGGATGACCTGGGACC CAGCCCAGCCCCCCCGAGACCTGACTGAGGCCTTCCTGGCAGAGATGGAGAAGGTGA GAGTGGCTGCCACGGTGGGGGGCAAGGGTGGTGGGTTGAGCGTCCCAGGAGGAATG AGGGGAGGCTGGGCAAAAGGTTGGACCAGTGCATCACCCGGCGAGCCGCATCTGGGC TGACAGGTGCAGAATTGGAGGTCATTTGGGGGCTACCCCGTTCTGTCCCGAGTATGCT CTCGGCCCTGCTCAGGCCAAGGGGAACCCTGAGAGCAGCTTCAATGATGAGAACCTG CGCATAGTGGTGGCTGACCTGTTCTCTGCCGGGATGGTGACCACCTCGACCACGCTGG CCTGGGGCCTCCTGCTCATGATCCTACATCCGGATGTGCAGCGTGAGCCCATCTGGGAA ACAGTGCAGGGGCCGAGGGAGGAAGGGTACAGGCGGGGGCCCATGAACTTTGCTGG GACACCCGGGGCTCCAAGCACAGGCTTGACCAGGATCCTGTAAGCCTGACCTCCTCCA ACATAGGAGGCAAGAAGGAGTGTCAGGGCCGGACCCCCTGGGTGCTGACCCATTGTG GGGACGCATGTCTGTCCAGGCCGTGTCCAACAGGAGATCGACGACGTGATAGGGCAG GTGCGGCGACCAGAGATGGGTGACCAGGCTCACATGCCCTACACCACTGCCGTGATTC ATGAGGTGCAGCGCTTTGGGGACATCGTCCCCCTGGGTGTGACCCATATGACATCCCGT GACATCGAAGTACAGGGCTTCCGCATCCCTAAGGTAGGCCTGGCGCCCTCCTCACCCC AGCTCAGCACCAGCACCTGGTGATAGCCCCAGCATGGCTACTGCCAGGTGGGCCCACT CTAGGAACCCTGGCCACCTAGTCCTCAATGCCACCACACTGACTGTCCCCACTTGGGT GGGGGGTCCAGAGTATAGGCAGGGCTGGCCTGTCCATCCAGAGCCCCCGTCTAGTGGG GAGACAAACCAGGACCTGCCAGAATGTTGGAGGACCCAACGCCTGCAGGGAGAGGG GGCAGTGTGGGTGCCTCTGAGAGGTGTGACTGCGCCCTGCTGTGGGGTCGGAGAGGG TACTGTGGAGCTTCTCGGGCGCAGGACTAGTTGACAGAGTCCAGCTGTGTGCCAGGCA GTGTGTGTCCCCCGTGTGTTTGGTGGCAGGGGTCCCAGCATCCTAGAGTCCAGTCCCC ACTCTCACCCTGCATCTCCTGCCCAGGGAACGACACTCATCACCAACCTGTCATCGGT GCTGAAGGATGAGGCCGTCTGGGAGAAGCCCTTCCGCTTCCACCCCGAACACTTCCTG GATGCCCAGGGCCACTTTGTGAAGCCGGAGGCCTTCCTGCCTTTCTCAGCAGGTGCCT GTGGGGAGCCCGGCTCCCTGTCCCCTTCCGTGGAGTCTTGCAGGGGTATCACCCAGGA GCCAGGCTCACTGACGCCCCTCCCCTCCCCACAGGCCGCCGTGCATGCCTCGGGGAGC CCCTGGCCCGCATGGAGCTCTTCCTCTTCTTCACCTCCCTGCTGCAGCACTTCAGCTTC TCGGTGCCCACTGGACAGCCCCGGCCCAGCCACCATGGTGTCTTTGCTTTCCTGGTGA GCCCATCCCCCTATGAGCTTTGTGCTGTGCCCCGCTAGAATGGGGTACCTAGTCCCCAG CCTGCTCCCTAGCCAGAGGCTCTAATGTACAATAAAGCAATGTGGTAGTTCCAACTCGG GTCCCCTGCTCACGCCCTCGTTGGGATCATCCTCCTCAGGGCAACCCCACCCCTGCCTC ATTCCTGCTTACCCCACCGCCTGGCCGCATTTGAGACAGGGGTACGTTGAGGCTGAGC AGATGTCAGTTACCCTTGCCCATAATCCCATGTCCCCCACTGACCCAACTCTGACTGCC CAGATTGGTGACAAGGACTACATTGTCCTGGCATGTGGGGAAGGGGCCAGAATGGGCT GACTAGAGGTGTCAGTCAGCCCTGGATGTGGTGGAGAGGGCAGGACTCAGCCTGGAG GCCCATATTTCAGGCCTAACTCAGCCCACCCCACATCAGGGACAGCAGTCCTGCCAGC ACCATCACAACAGTCACCTCCCTTCATATATGACACCCCAAAACGGAAGACAAATCAT GGCGTCAGGGAGCTATATGCCAGGGCTACCTACCTCCCAGGGCTCAGTCGGC

3.5kb (CYP2D6 deleted) sequence (SEQ 9):

CACACCGGGCACCTGTACTCCTCAGCCCTTGGGCAGTGGACGGGACCAGG TGCCGTGGAGCAGTGGGAGGCACCCATCCGGGAGGCTCGGGCCTCGCAGG GAGCCCACCGTAGGGAGGCTTGGGCATGGCAGGCTGCAAGTCCTGAGCCC TGCCCCGCGGGGAGGTGACTGAGGCCTGGCGACAATTCAAGTGTGGTGAG CGCCGGCAGGCCAGCAGTACTGGGGGACCCGGTGCCCCCTCTGCAGCTGC TGGCCCAGGTGCTAAGCCCCTCACTGCCTGGGGCCAGAGGCACCAGCCGG CCGCTCCGAGTGCAGGGCCCGCTGAGCCCCTGCCCACCCAGAACTGGTGC TGGCCCGCGAGCAACCCAGGTTCCCGCACACGCCTCTCCCTCCATACCTCC CCGCAAGCAGACGGAGCCGGCTCCAGCCTCCACCAGTCCAGAGAGGGGC TCCCACAGTGCAGCGCTGGGCTGAAGGGCTCCTCAAGTGTGGTCAGAGCA GAAGCTGAGGCCGAGGAGGCGCTGAGAGCGAGCGAGGACCACCAGCACG TTGACACCTCTCAACCTCACCACAGGACTGGCCACCTCTCTGGGCCCTCAG GGATGCTGCTGTCTGGACCCCTGACCAGTGACGAGTTCGCACTCAGGGCC AGGCTGGCGCTGGAGGAGGACACTTGTTTGGCTCCAACCCTAGGTACCATC CTCCCAGTAGGGATCAGGCAGGGCCCACAGGCCTGCCCTAGGGACAGGAG TCAACCTTGGACCCATAAGGCACTGGGGCGGGCAGAGAAGGAGGAGGTG GCATGGGCAGCTGAGAGCCAGAGACCCTGACCCTAGTCCTTGCTCTGCCAT TACCCCGTGTGACCCCGGGCCCACCCTTCCCCACCCTTCCCCACCCTTCCC CACCCCGGGCTTCTGTTTCCCTTCTGCCAACGAGAAGGCTGCTTCACCTGC CCCGAGTCCTGTCTTCCTGCTCTGCCTTCTGGGGCTGTGGCCCTTGCTGGC CTGGAGCCCCAACCAAGGGCAGGGACTGCTGTCCTCCACGTCTGTCCTCA CCGACATAATGGGCTGGGCTGGGCACACAGGCAGTGCCCAAGAGTTTCTA ATGAGCATATGATTACCTGAGTCCTGGGCAGACCTTCTTAGGGAACAGCCT GGGACAGAGAACCACAGACACTCTGAGGAGCCACCTGAGGCCTCTTTTGC CAGAGGACCCTACAGCCTCCCTGGCAGCAGTTCCGCCAGCATTTCTGTAAA TGCCCTCATGCCAGGGTGCGGCCCGGCTGTCAGCACGAGAGGGACGTTGG TCTGTCCCCTGGCACCGAGTCAGTCAGAAGGGTGGCCAGGGCCCCCTTGG GCCCCTCCAGAGACAATCCACTGTGGTCACACGGCTCGGTGGCAGGAAGT GCTGTTCCTGCAGCTGTGGGGACAGGGAGTGTGGATGAAGCCAGGCTGGG TTTGTCTGAAGACGGAGGCCCCGAAAGGTGGCAGCCTGGCCTATAGCAGC AGCAACTCTTGGATTTATTGGAAAGATTTTCTTCACGGTTCTGAGTCTTGGG GGTGTTAGAGGCTCAGAACCAGTCCAGCCAGAGCTCTGTCATGGGCACGT AGACCCGGTCCCAGGGCCTTTGCTCTTTGCTGTCCTCAGAGGCCTCTGCAA AGTAGAAACAGGCAGCCTTGTGAGTCCCCTCCTGGGAGCAACCAACCCTC CCTCTGAGATGCCCCGGGGCCAGGTCAGCTGTGGTGAAAGGTAGGGATGC AGCCAGCTCAGGGGAGTGGCCCAGAGTTCCTGCCCACCCAAGGAGGCTCC CAGGAAGGTCAAGGCACCTGACTCCTGGGCTGCTTCCCTCCCCTCCCCTCC CCAGGTCAGGAAGGTGGGAAAGGGCTGGGGTGTCTGTGACCCTGGCAGTC ACTGAGAAGCAGGGTGGAAGCAGCCCCCTGCAGCACGCTGGGTCAGTGG TCTTACCAGATGGATACGCAGCAACTTCCTTTTGAACCTTTTTATTTTCCTG GCAGGAAGAAGAGGGATCCAGCAGTGAGATCAGGCAGGTTCTGTGTTGCA CAGACAGGGAAACAGGCTCTGTCCACACAAAGTCGGTGGGGCCAGGATG AGGCCCAGTCTGTTCACACATGGCTGCTGCCTCTCAGCTCTGCACAGACGT CCTCGCTCCCCTGGGATGGCAGCTTGGCCTGCTGGTCTTGGGGTTGAGCCA GCCTCCAGCACTGCCTCCCTGCCCTGCTGCCTCCCACTCTGCAGTGCTCCA TGGCTGCTCAGTTGGACCCACGCTGGAGACGTTCAGTCGAAGCCCCGGGC TGTCCTTACCTCCCAGTCTGGGGTACCTGCCACCTCCTGCTCAGCAGGAAT GGGGCTAGGTGCTTCCTCCCCTGGGGACTTCACCTGCTCTCCCTCCTGGGA TAAGACGGCAGCCTCCTCCTTGGGGGCAGCAGCATTCAGTCCTCCAGGTCT CCTGGGGGTCGTGACCTGCAGGAGGAATAAGAGGGCAGACTGGGCAGAA AGGCCTTCAGAGCACCTCATCCTCCTGTTCTCACACTGGGGTGTCACAGTC CTGGGAAGTTCTTCCTTTTCAGTTGAGCTGTGGTAACCTTGTGAGTTTCCTG GAGGGGGCCTGCCACTACCCTTGGGACTCCCTGCCGTGTGTCTGGGTCTAA CTGAGCTCTGAAAGGAGAGAGCCCCAGCCCTGGGCCTTCCAGGGGAAGCC TTACCTCAGAGGTTGGCTTCTTCCTACTCTTGACTTTGCGTCTCTGCAGAGG GAGGTGGGAGGGGTGACACAACCCTGACACCCACACTATGAGTGATGAGT AGTCCTGCCCCGACTGGCCCATCCTTTCCAGGTGCAGTCCCCCTTACTGTG TCTGCCAAGGGTGCCAGCACAGCCGCCCCACTCCAGGGGAAGAGGAGTG CCAGCCCTTACCCACCTGAGTGGGCACAGTGTAGCATTTATTCATTAGCCCC CACACTGGCCTGACCATCTCCCCTGTGGGCTGCATGACAAGGAGAGAGAA CAGGCTGAGGTGAGAGCTACTGTCAACACCTAAACCTAAAAAATCTATAAT TGGGCTGGGCAGGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCC GAGATGGGTGGATCACCTGAGGTCAGATGTTCGAGACCAGCCTGGCCAAC ATGGTGAAACCCCGTCTCTACTAAAAATACAAAAAATTAGCTGGGCGTGGT GGTGGGTGCCTGTAATCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATTG CTTGAACCTGGGAGGCAGAGGCTGCAGTGAGCCGAGATCGCATCATTGCA CTCCAGCCTGGTCAACAAGAGTGAAACTGTCTTAAAAAAAAAATCTATAAT TGATATCTTTAGAAAGATAAAACTTTGCATTCATGAAATAAGAATAGGAGGG TCTAAAATAAAAATGTTCAAACACCCACCACCACTAATTCTTGACAAAAAT ATAGTCTGGGTGCCTTAGCTCATGCCTG

electrophoresis: and carrying out electrophoretic analysis on the amplification product. The results of the analysis are shown in FIG. 9.

Sequence listing

<110> Hangzhou Baimai biological shares Ltd

<120> reagent, detection method and kit for detecting CYP2D6 gene

<130> 202002

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ccatttggta gtgaggcagg t 21

<210> 2

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgcagcagg ttgccca 17

<210> 3

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cacgctaccc accagg 16

<210> 4

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

acgctactca ccagg 15

<210> 5

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ttgtgggctg taatcatcgt cta 23

<210> 6

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gttctctttc actgacatct gcaaa 25

<210> 7

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cccacacaaa tctctccctg gcattg 26

<210> 8

<211> 5104

<212> DNA

<213> Human Gene Sequence (Human Gene Sequence)

<400> 8

gttatcccag aaggctttgc aggcttcagg agcttggagt ggggagaggg ggtgacttct 60

ccgaccaggc ccctccaccg gcctaccctg ggtaagggcc tggagcagga agcaggggca 120

agaacctctg gagcagccca tacccgccct ggcctgactc tgccactggc agcacagtca 180

acacagcagg ttcactcaca gcagagggca aaggccatca tcagctccct ttataaggga 240

agggtcacgc gctcggtgtg ctgagagtgt cctgcctggt cctctgtgcc tggtggggtg 300

ggggtgccag gtgtgtccag aggagcccat ttggtagtga ggcaggtatg gggctagaag 360

cactggtgcc cctggccgtg atagtggcca tcttcctgct cctggtggac ctgatgcacc 420

ggcgccaacg ctgggctgca cgctacycac caggccccct gccactgccc gggctgggca 480

acctgctgca tgtggacttc cagaacacac catactgctt cgaccaggtg agggaggagg 540

tcctggaggg cggcagaggt gctgaggctc ccctaccaga agcaaacatg gatggtgggt 600

gaaaccacag gctggaccag aagccaggct gagaagggga agcaggtttg ggggacktcc 660

tggagaaggg catttataca tggcatgaag gactggattt tccaaaggcc aaggaagagt 720

agggcaaggg cctggaggtg gagctggact tggcagtggg catgcaagcc cattgggcaa 780

catatgttat ggagtacaaa gtcccttctg ctgacaccag aaggaaaggc cttgggaatg 840

gaagatgagt tagtcctgag tgccgtttaa atcacgaaat cgaggatgaa gggggtgcag 900

tgacccggtt caaacctttt gcactgtggg tcctcgggcc tcactgctca ccggcatgga 960

ccatcatctg ggaatgggat gctaactggg gcctctcggc aattttggtg actcttgcaa 1020

ggtcatacct gggtgacgca tccaaactga gttcctccat cacagaaggt gtgaccccca 1080

cccccgcccc acgatcagga ggctgggtct cctccttcca cctgctcact cctggtagcc 1140

ccgggggtcg tccaaggttc aaataggact aggacctgta gtctggggdg atcctggctt 1200

gacaagaggc cctgaccctc cctctgcagt tgcggcgccg cttcggggac gtgttcagcc 1260

tgcagctggc ctggacgccg gtggtcgtgc tcaatgggct ggcggccgtg cgcgaggcgc 1320

tggtgaccca cggcgaggac accgccgacc gcccgcctgt gcccatcacc cagatcctgg 1380

gtttcgggcc gcgttcccaa ggcaagcagc ggtggggaca gagacagatt tccgtgggac 1440

ccgggtgggt gatgaccgta gtccgagctg ggcagagagg gcgcggggtc gtggacatga 1500

aacaggccag cgagtgggga cagcgggcca agaaaccacc tgcactaggg aggtgtgagc 1560

atggggacga gggcggggct tgtgacgagt gggcggggcc actgccgaga cctggcagga 1620

gcccaatggg tgaggctggc gcatttccca gctggaatcc ggtgtcgaag tggggggcgg 1680

ggaccgcacc tgtgctgtaa gctcagtgtg ggtggcgcgg ggcccgcggg gtcttccctg 1740

agtgcaaagg cggtcagggt gggcagagac gaggtggggc aaagccctgc cccagccaag 1800

ggagcaaggt ggatgcacaa agagtgggcc ctgtgaccag ctggacagag ccagggactg 1860

cgggagacca gggggagcat agggttggag tgggtggtgg atggtggggc taatgccttc 1920

atggccacgc gcacgtgccc gtcccacccc caggggtgtt cctggcgcgc tatgggcccg 1980

cgtggcgcga gcagaggcgc ttctccgtgt ccaccttgcg caacttgggc ctgggcaaga 2040

agtcgctgga gcagtgggtg accgaggagg ccgcctgcct ttgtgccgcc ttcgccaacc 2100

actccggtgg gtgatgggca gaagggcaca aagcgggaac tgggaaggcg ggggacgggg 2160

aaggcgaccc cttacccgca tctcccaccc ccaggacgcc cctttcgccc caacggtctc 2220

ttggacaaag ccgtgagcaa cgtgatcgcc tccctcacct gcgggcgccg cttcgagtac 2280

gacgaccctc gcttcctcag gctgctggac ctagctcagg agggactgaa ggaggagtcg 2340

ggctttctgc gcgaggtgcg gagcgagaga ccgaggagtc tctgcagggc gagctcccga 2400

gaggtgccgg ggctggactg gggcctcgga agagcaggat ttgcatagat gggtttggga 2460

aaggacattc caggagaccc cactgtaaga agggcctgga ggaggagggg acatctcaga 2520

catggtcgtg ggagaggtgt gcccgggtca gggggcacca ggagaggcca aggactctgt 2580

acctcctatc cacgtcagag atttcgattt taggtttctc ctctgggcaa ggagagaggg 2640

tggaggctgg cacttgggga gggacttggt gaggtcagtg gtaaggacag gcaggccctg 2700

ggtctacctg gagatggctg gggcctgaga cttgtccagg tgaacgcaga gcacaggagg 2760

gattgagacc ccgttctgtc tggtgtaggt gctgaatgct gtccccgtcc tcctgcatat 2820

cccagcgctg gctggcaagg tcctacgctt ccaaaaggct ttcctgaccc agctggatga 2880

gctgctaact gagcacagga tgacctggga cccagcccag cccccccgag acctgactga 2940

ggccttcctg gcagagatgg agaaggtgag agtggctgcc acggtggggg gcaagggtgg 3000

tgggttgagc gtcccaggag gaatgagggg aggctgggca aaaggttgga ccagtgcatc 3060

acccggcgag ccgcatctgg gctgacaggt gcagaattgg aggtcatttg ggggctaccc 3120

cgttctgtcc cgagtatgct ctcggccctg ctcaggccaa ggggaaccct gagagcagct 3180

tcaatgatga gaacctgcgc atagtggtgg ctgacctgtt ctctgccggg atggtgacca 3240

cctcgaccac gctggcctgg ggcctcctgc tcatgatcct acatccggat gtgcagcgtg 3300

agcccatctg ggaaacagtg caggggccga gggaggaagg gtacaggcgg gggcccatga 3360

actttgctgg gacacccggg gctccaagca caggcttgac caggatcctg taagcctgac 3420

ctcctccaac ataggaggca agaaggagtg tcagggccgg accccctggg tgctgaccca 3480

ttgtggggac gcatgtctgt ccaggccgtg tccaacagga gatcgacgac gtgatagggc 3540

aggtgcggcg accagagatg ggtgaccagg ctcacatgcc ctacaccact gccgtgattc 3600

atgaggtgca gcgctttggg gacatcgtcc ccctgggtgt gacccatatg acatcccgtg 3660

acatcgaagt acagggcttc cgcatcccta aggtaggcct ggcgccctcc tcaccccagc 3720

tcagcaccag cacctggtga tagccccagc atggctactg ccaggtgggc ccactctagg 3780

aaccctggcc acctagtcct caatgccacc acactgactg tccccacttg ggtggggggt 3840

ccagagtata ggcagggctg gcctgtccat ccagagcccc cgtctagtgg ggagacaaac 3900

caggacctgc cagaatgttg gaggacccaa cgcctgcagg gagagggggc agtgtgggtg 3960

cctctgagag gtgtgactgc gccctgctgt ggggtcggag agggtactgt ggagcttctc 4020

gggcgcagga ctagttgaca gagtccagct gtgtgccagg cagtgtgtgt cccccgtgtg 4080

tttggtggca ggggtcccag catcctagag tccagtcccc actctcaccc tgcatctcct 4140

gcccagggaa cgacactcat caccaacctg tcatcggtgc tgaaggatga ggccgtctgg 4200

gagaagccct tccgcttcca ccccgaacac ttcctggatg cccagggcca ctttgtgaag 4260

ccggaggcct tcctgccttt ctcagcaggt gcctgtgggg agcccggctc cctgtcccct 4320

tccgtggagt cttgcagggg tatcacccag gagccaggct cactgacgcc cctcccctcc 4380

ccacaggccg ccgtgcatgc ctcggggagc ccctggcccg catggagctc ttcctcttct 4440

tcacctccct gctgcagcac ttcagcttct cggtgcccac tggacagccc cggcccagcc 4500

accatggtgt ctttgctttc ctggtgagcc catcccccta tgagctttgt gctgtgcccc 4560

gctagaatgg ggtacctagt ccccagcctg ctccctagcc agaggctcta atgtacaata 4620

aagcaatgtg gtagttccaa ctcgggtccc ctgctcacgc cctcgttggg atcatcctcc 4680

tcagggcaac cccacccctg cctcattcct gcttacccca ccgcctggcc gcatttgaga 4740

caggggtacg ttgaggctga gcagatgtca gttacccttg cccataatcc catgtccccc 4800

actgacccaa ctctgactgc ccagattggt gacaaggact acattgtcct ggcatgtggg 4860

gaaggggcca gaatgggctg actagaggtg tcagtcagcc ctggatgtgg tggagagggc 4920

aggactcagc ctggaggccc atatttcagg cctaactcag cccaccccac atcagggaca 4980

gcagtcctgc cagcaccatc acaacagtca cctcccttca tatatgacac cccaaaacgg 5040

aagacaaatc atggcgtcag ggagctatat gccagggcta cctacctccc agggctcagt 5100

cggc 5104

<210> 9

<211> 3507

<212> DNA

<213> Human Gene (Human Gene Sequence)

<400> 9

cacaccgggc acctgtactc ctcagccctt gggcagtgga cgggaccagg tgccgtggag 60

cagtgggagg cacccatccg ggaggctcgg gcctcgcagg gagcccaccg tagggaggct 120

tgggcatggc aggctgcaag tcctgagccc tgccccgcgg ggaggtgact gaggcctggc 180

gacaattcaa gtgtggtgag cgccggcagg ccagcagtac tgggggaccc ggtgccccct 240

ctgcagctgc tggcccaggt gctaagcccc tcactgcctg gggccagagg caccagccgg 300

ccgctccgag tgcagggccc gctgagcccc tgcccaccca gaactggtgc tggcccgcga 360

gcaacccagg ttcccgcaca cgcctctccc tccatacctc cccgcaagca gacggagccg 420

gctccagcct ccaccagtcc agagaggggc tcccacagtg cagcgctggg ctgaagggct 480

cctcaagtgt ggtcagagca gaagctgagg ccgaggaggc gctgagagcg agcgaggacc 540

accagcacgt tgacacctct caacctcacc acaggactgg ccacctctct gggccctcag 600

ggatgctgct gtctggaccc ctgaccagtg acgagttcgc actcagggcc aggctggcgc 660

tggaggagga cacttgtttg gctccaaccc taggtaccat cctcccagta gggatcaggc 720

agggcccaca ggcctgccct agggacagga gtcaaccttg gacccataag gcactggggc 780

gggcagagaa ggaggaggtg gcatgggcag ctgagagcca gagaccctga ccctagtcct 840

tgctctgcca ttaccccgtg tgaccccggg cccacccttc cccacccttc cccacccttc 900

cccaccccgg gcttctgttt cccttctgcc aacgagaagg ctgcttcacc tgccccgagt 960

cctgtcttcc tgctctgcct tctggggctg tggcccttgc tggcctggag ccccaaccaa 1020

gggcagggac tgctgtcctc cacgtctgtc ctcaccgaca taatgggctg ggctgggcac 1080

acaggcagtg cccaagagtt tctaatgagc atatgattac ctgagtcctg ggcagacctt 1140

cttagggaac agcctgggac agagaaccac agacactctg aggagccacc tgaggcctct 1200

tttgccagag gaccctacag cctccctggc agcagttccg ccagcatttc tgtaaatgcc 1260

ctcatgccag ggtgcggccc ggctgtcagc acgagaggga cgttggtctg tcccctggca 1320

ccgagtcagt cagaagggtg gccagggccc ccttgggccc ctccagagac aatccactgt 1380

ggtcacacgg ctcggtggca ggaagtgctg ttcctgcagc tgtggggaca gggagtgtgg 1440

atgaagccag gctgggtttg tctgaagacg gaggccccga aaggtggcag cctggcctat 1500

agcagcagca actcttggat ttattggaaa gattttcttc acggttctga gtcttggggg 1560

tgttagaggc tcagaaccag tccagccaga gctctgtcat gggcacgtag acccggtccc 1620

agggcctttg ctctttgctg tcctcagagg cctctgcaaa gtagaaacag gcagccttgt 1680

gagtcccctc ctgggagcaa ccaaccctcc ctctgagatg ccccggggcc aggtcagctg 1740

tggtgaaagg tagggatgca gccagctcag gggagtggcc cagagttcct gcccacccaa 1800

ggaggctccc aggaaggtca aggcacctga ctcctgggct gcttccctcc cctcccctcc 1860

ccaggtcagg aaggtgggaa agggctgggg tgtctgtgac cctggcagtc actgagaagc 1920

agggtggaag cagccccctg cagcacgctg ggtcagtggt cttaccagat ggatacgcag 1980

caacttcctt ttgaaccttt ttattttcct ggcaggaaga agagggatcc agcagtgaga 2040

tcaggcaggt tctgtgttgc acagacaggg aaacaggctc tgtccacaca aagtcggtgg 2100

ggccaggatg aggcccagtc tgttcacaca tggctgctgc ctctcagctc tgcacagacg 2160

tcctcgctcc cctgggatgg cagcttggcc tgctggtctt ggggttgagc cagcctccag 2220

cactgcctcc ctgccctgct gcctcccact ctgcagtgct ccatggctgc tcagttggac 2280

ccacgctgga gacgttcagt cgaagccccg ggctgtcctt acctcccagt ctggggtacc 2340

tgccacctcc tgctcagcag gaatggggct aggtgcttcc tcccctgggg acttcacctg 2400

ctctccctcc tgggataaga cggcagcctc ctccttgggg gcagcagcat tcagtcctcc 2460

aggtctcctg ggggtcgtga cctgcaggag gaataagagg gcagactggg cagaaaggcc 2520

ttcagagcac ctcatcctcc tgttctcaca ctggggtgtc acagtcctgg gaagttcttc 2580

cttttcagtt gagctgtggt aaccttgtga gtttcctgga gggggcctgc cactaccctt 2640

gggactccct gccgtgtgtc tgggtctaac tgagctctga aaggagagag ccccagccct 2700

gggccttcca ggggaagcct tacctcagag gttggcttct tcctactctt gactttgcgt 2760

ctctgcagag ggaggtggga ggggtgacac aaccctgaca cccacactat gagtgatgag 2820

tagtcctgcc ccgactggcc catcctttcc aggtgcagtc ccccttactg tgtctgccaa 2880

gggtgccagc acagccgccc cactccaggg gaagaggagt gccagccctt acccacctga 2940

gtgggcacag tgtagcattt attcattagc ccccacactg gcctgaccat ctcccctgtg 3000

ggctgcatga caaggagaga gaacaggctg aggtgagagc tactgtcaac acctaaacct 3060

aaaaaatcta taattgggct gggcagggtg gctcacgcct gtaatcccag cactttggga 3120

ggccgagatg ggtggatcac ctgaggtcag atgttcgaga ccagcctggc caacatggtg 3180

aaaccccgtc tctactaaaa atacaaaaaa ttagctgggc gtggtggtgg gtgcctgtaa 3240

tcccagctac tcaggaggct gaggcaggag aattgcttga acctgggagg cagaggctgc 3300

agtgagccga gatcgcatca ttgcactcca gcctggtcaa caagagtgaa actgtcttaa 3360

aaaaaaaatc tataattgat atctttagaa agataaaact ttgcattcat gaaataagaa 3420

taggagggtc taaaataaaa atgttcaaac acccaccacc actaattctt gacaaaaata 3480

tagtctgggt gccttagctc atgcctg 3507

Claims (10)

1. A CYP2D6 genotyping detection kit comprises a primer and a probe for detecting CYP2D6 x 10.

2. The test kit according to claim 1, wherein the sequences of the CYP2D6 x 10 primer and the probe are:

CYP2D6 × 10 upstream primer: 5'-CCATTTGGTAGTGAGGCAGGT-3'

CYP2D6 × 10 downstream primer: 5'-ATGCAGCAGGTTGCCCA-3'

CYP2D6 × 10 wild-type probe: 5'-CACGCTACCCACCAGG-3'

CYP2D6 × 10 mutant probe: 5'-ACGCTACTCACCAGG-3' are provided.

3. The detection kit according to one of claims 1 to 2, further comprising primers and probe sequences for detecting the internal reference gene ALB, wherein the primers and probe sequences are respectively:

ALB upstream primer: 5'-TTGTGGGCTGTAATCATCGTCTA-3'

ALB downstream primer: 5'-GTTCTCTTTCACTGACATCTGCAAA-3'

ALB probe: 5'-CCCACACAAATCTCTCCCTGGCATTG-3' are provided.

4. The test kit according to any one of claims 1 to 3, wherein the CYP2D6 x 10 wild type probe, CYP2D6 x 10 mutant probe, ALB probe are labeled with different fluorescent signaling groups, respectively.

5. The detection kit according to claim 4,

CYP2D6 × 10 wild-type probe: FAM-CACGCTACCCACCAGG-MGBNFQ

CYP2D6 × 10 mutant probe: VIC-ACGCTACTCACCAGG-MGBNFQ.

ALB probe: CY5-CCCACACAAATCTCTCCCTGGCATTG-BHQ 3.

6. A method for detecting CYP2D6 gene polymorphism. The method comprises the following steps:

(1) extracting genomic DNA in a sample;

(2) performing fluorescent quantitative PCR on the DNA obtained in the step 1, wherein PCR reaction reagents comprise a primer and a probe for detecting CYP2D6 x 10 and a primer and a probe for detecting an internal reference gene ALB;

(3) and (4) interpretation of results: if CY5 and FAM have signals, the signal is CYP2D6 x 1 wild type; if CY5 and VIC have signals, the mutant is CYP2D6 x 10 homozygous mutant; if CY5, FAM and VIC have signals, the mutant is CYP2D6 x 1/x 10 hybrid mutant; if only CY5 has a signal, it is CYP2D6 × 5 type.

7. The method according to claim 6, wherein the sequences of the primer and probe for detecting CYP2D6 x 10 are:

CYP2D6 × 10 upstream primer: 5'-CCATTTGGTAGTGAGGCAGGT-3'

CYP2D6 × 10 downstream primer: 5'-ATGCAGCAGGTTGCCCA-3'

CYP2D6 × 10 wild-type probe: FAM-CACGCTACCCACCAGG-MGBNFQ

CYP2D6 × 10 mutant probe: VIC-ACGCTACTCACCAGG-MGBNBNFQ

The sequences of primers and probes for detecting the internal reference gene ALB are respectively as follows:

ALB upstream primer: 5'-TTGTGGGCTGTAATCATCGTCTA-3'

ALB downstream primer: 5'-GTTCTCTTTCACTGACATCTGCAAA-3'

ALB probe: CY5-CCCACACAAATCTCTCCCTGGCATTG-BHQ 3.

8. A reagent composition capable of judging mutant types and deletion types of CYP2D6 genes comprises a fluorescent quantitative PCR amplification reagent, and is characterized in that the fluorescent quantitative PCR amplification reagent comprises a primer and a probe for detecting CYP2D6 x 10 and a primer and a probe for detecting internal reference genes ALB, wherein,

sequences of the primer and the probe for detecting CYP2D6 × 10 are respectively as follows:

CYP2D6 × 10 upstream primer: 5'-CCATTTGGTAGTGAGGCAGGT-3'

CYP2D6 × 10 downstream primer: 5'-ATGCAGCAGGTTGCCCA-3'

CYP2D6 × 10 wild-type probe: FAM-CACGCTACCCACCAGG-MGBNFQ

CYP2D6 × 10 mutant probe: VIC-ACGCTACTCACCAGG-MGBNBNFQ

The sequences of primers and probes for detecting the internal reference gene ALB are respectively as follows:

ALB upstream primer: 5'-TTGTGGGCTGTAATCATCGTCTA-3'

ALB downstream primer: 5'-GTTCTCTTTCACTGACATCTGCAAA-3'

ALB probe: CY5-CCCACACAAATCTCTCCCTGGCATTG-BHQ 3.

9. The reagent composition of claim 8, wherein the fluorescent quantitative PCR amplification reagent further comprises PCR Buffer, MgCl2, dNTP, and ROX fluorescent calibration solution.

10. The reagent composition of claim 8, further comprising a blood pretreatment reagent.

Images