CN106755360A - Nucleic acid, kit and method for detecting mankind's CYP2D6 gene pleiomorphisms - Google Patents

Abstract

The invention discloses the nucleic acid for quick detection CYP2D6 gene pleiomorphisms, kit and method, detected specifically for the loci polymorphism to CYP2D6*10 gene Cs 100T and G4268C, CYP2D6*14 gene G1758A.The detection kit of offer real-time fluorescence quantitative PCR quick detection CYP2D6 gene pleiomorphisms of the present invention can be used in the detection of clinical polytype sample, and with high specificity, sensitivity is high, experimental period is short, simple to operate, safety non-toxic, cost is low significantly excellent, its detection method, easy to operate, high degree of automation enormously simplify operating process, and pollution in operating process is reduced, Detection results are accurate, reliable.

Description

Nucleic acid, kit and method for detecting mankind's CYP2D6 gene pleiomorphisms

Technical field

The invention belongs to biological technical field, and in particular to a kind of core for detecting mankind's CYP2D6 gene pleiomorphisms Acid, kit and method.

Background technology

CYP2D6 is one of important member in CYP enzyme families, and the gene is located at 22q13.1, contains 9 extrons and 8 Individual introne, overall length is 7kb or so, is a complete functioning gene.Research shows that CYP2D6 only accounts for 2-9% in liver, But the metabolism of 20%~30% medicine is participated in, including antidepressants, antiarrhymic, antipsychotic drug, antalgesic etc.. CYP2D6 has extensive gene pleiomorphism, at present it has been reported that have 100 several genes make a variation, such as single base mutation, The missing of missing or insertion and large fragment.These genetic mutations can cause the difference of enzymatic activity and quantity, so as to cause medicine generation Thank to the generation of individual difference.The metabolic phenotype of CYP2D6 can be divided into ultra-rapid metabolism type (UMs), fast metabolic pattern (EMs), medium metabolic Type (IMs), slow inactivation (PMs).

TAM is also tamoxifen citrate (tamoxifen), is a kind of SERM (SERM).This medicines structure is similar to estrogen, can simulate the effect of estrogen, and answering for stabilization is combined to form with ERs Compound, and transport in core, prevent chromogene from opening, so that the growth and development of cancer cell is suppressed, it is current Treat one of active drug of some breast cancer and oophoroma and the proliferation of mammary gland.The main generation in liver after TAM is oral Thank, major metabolite is N- demethyls TAM and 4-OHT, affinity and suppression cancer with ERs The ability of cell proliferation is higher than TAM at least 100 times.

In recent years, it is the security and validity of raising TAM clinical practice, domestic and foreign scholars are to TAM medicine The influence factor of effect has carried out numerous studies, and correlative study result shows that the polymorphism of drug metabolism related gene is to cause individuality One of major reason of drug effect difference.Tamoxifen is mainly formed with by CYP2D6 catalysis in vivo as inactive prodrug The chemical composition Endoxifen of activity, enzyme activity change will be straight caused by playing curative effect of medication, therefore CYP2D6 gene pleiomorphisms Connect influence tamoxifen medication effect.So the parting detection of CYP2D6 genes has important for clinical tamoxifen medication guide Meaning.

Distribution frequency of the CYP2D6 gene pleiomorphisms in different ethnic populations has larger difference, is dashed forward in the crowd of east Frequency it is higher be CYP2D6*10 and CYP2D6*14 gene pleiomorphisms.CYP2D6*10 distribution frequencies in the crowd of east are high It is the distinctive allele of Asians up to 40%~49.5%, CYP2D6*14, distribution frequency is about 1%-2%, both genes Type is to cause the main cause of Asians's CYP2D6 enzymes slow inactivation and weak metabolic pattern.Therefore, comprehensive detection CYP2D6*10 and CYP2D6*14 gene pleiomorphisms can screen those in the patient that can be more benefited in TAM treatments, and this is for clinical individual The formulation for the treatment of scheme is significant.

At present, the main method of CYP2D6 genetic polymorphism detections has：PCR sequencing PCR, chip method and solubility curve method.Sequencing Method is the most common method of genetic polymorphism detection, is also the goldstandard of gene polymorphism sites detection, although the method has High flux, the advantage in many sites, but the method exists simultaneously, and cumbersome, sensitivity is low, sample easily pollutes, and causes false positive knot Really, and the problems such as instrument cost is high.Chip method needs the amplification of advanced performing PCR, will contain afterwards the PCR primer of purposeful SNP site with Mutant probe, the hybridization of wild probe, by comparing the signal intensity judgement sample genotype that two probes hybridize.The method is operated Process is cumbersome, it is impossible to realize mass and automation, and is as a result difficult interpretation, and false positive probability is high.For solubility curve method, no Pipe is dyestuff solubility curve or probe solubility curve method, all cannot effectively distinguish the various mutations type in same site.Liquid phase Chip method operating process is complicated, and easily pollution, and false positive rate is high.

The content of the invention

To overcome the shortcomings of prior art detection technique, it is used to detect CYP2D6 genes it is an object of the invention to provide one group The nucleic acid of polymorphism.It is a further object to provide a kind of kit for detecting CYP2D6 gene pleiomorphisms and its Detection method.

To achieve the above object, the present invention uses following technical scheme：

One group of nucleic acid for being used to detect CYP2D6 gene pleiomorphisms, the nucleic acid includes CYP2D6*10 genes and CYP2D6*14 The detection primer and detection probe of gene pleiomorphism, wherein, the detection primer of the CYP2D6*10 gene pleiomorphisms and detection are visited Pin includes C100T wild type sense primer 1W-F of the nucleotide sequence as shown in SEQ ID No.1 for C100T sites, such as C100T saltant type sense primer 1M-F shown in SEQ ID No.2 and the public downstreams of C100T as shown in SEQ ID No.3 are drawn Thing 1C-R, and C100T detection probe 1P of the nucleotide sequence as shown in SEQ ID No.4；

And for the G4268C wild type sense primer of the nucleotide sequence as shown in SEQ ID No.5 in G4268C sites 2W-F, the G4268C saltant type sense primer 2M-F as shown in SEQ ID No.6 and the G4268C as shown in SEQ ID No.7 are public Use anti-sense primer 2C-R, and G4268C detection probe 2P of the nucleotide sequence as shown in SEQ ID No.8；

The detection primer and detection probe of the CYP2D6*14 gene pleiomorphisms include nucleotide sequence such as SEQ ID G1758A wild type sense primers 3W-F shown in No.9, the G1758A saltant type sense primers as shown in SEQ ID No.10 The 3M-F and public anti-sense primer 3C-R of the G1758A as shown in SEQ ID No.11, and nucleotide sequence such as SEQ ID No.12 institutes The G1758A detection probes 3P for showing.

Nucleic acid as described above, it is preferable that the nucleic acid also includes inner quality control, it includes that Quality Control primer pair and Quality Control are visited Pin, the nucleotide sequence of the Quality Control primer pair as shown in SEQ ID No.13 and SEQ ID No.14, the Quality Control probe Nucleotide sequence is as shown in SEQ ID No.15.

Nucleic acid as described above, it is preferable that the nucleic acid also includes CYP2D6*10 gene C 100T sites, G4268C sites Wild-type positive tester corresponding with CYP2D6*14 gene G1758A sites, saltant type positive control and the Quality Control positive are right According to thing, wherein, the wild-type positive tester in the C100T sites contains the nucleotide sequence as shown in SEQ ID No.16, The saltant type positive control in the C100T sites contains the nucleotide sequence as shown in SEQ ID No.17, the G4268C The wild-type positive tester in site contains the nucleotide sequence as shown in SEQ ID No.18, the mutation in the G4268C sites Type positive control contains the nucleotide sequence as shown in SEQ ID No.19, the wild-type positive control in the G1758A sites Thing contains the nucleotide sequence as shown in SEQ ID No.20, the saltant type positive control in the G1758A sites contain as Nucleotide sequence shown in SEQ ID No.21, the Quality Control positive control contains the nucleotides as shown in SEQ ID No.22 Sequence.

A kind of kit for quick detection CYP2D6 gene pleiomorphisms, the kit includes nucleic acid as described above, Wherein, the 5 ＇ ends connection fluorophor of the detection probe of each gene, 3 ＇ ends connection quenching group.

Kit as described above, it is preferable that kit also includes 10 × PCR buffer solutions, archaeal dna polymerase, dNTPs, ore deposit Thing oil, negative control thing：Nuclease free water.

Kit as described above, it is preferable that the fluorophor is any one in FAM, JOE, CY3, HEX, described Quenching group is any one in MGB, BHQ1, TAMRA, BHQ2.

The application method of kit as described above, the method is comprised the following steps：

(1) extraction of sample to be tested genomic DNA；

(2) PCR reaction solutions are prepared：Saltant type detection architecture and wild type detection architecture are prepared respectively per pleomorphism site； Respectively to same sample DNA is added in the saltant type detection architecture and the wild type detection architecture, of short duration centrifugation, mixing is equal It is even；

(3) fluorescence quantitative PCR detection：The PCR system that will be prepared is put into fluorescent PCR instrument, carries out quantitative fluorescent PCR Augmentation detection；

(4) result interpretation：The poor △ Ct couple of the Ct values of Ct values and wild type detection architecture according to saltant type detection architecture The genotype of sample carries out interpretation, if △ Ct<- 3, then the sample gene loci genotype is saltant type；If -3≤△ Ct≤ 3, then the sample gene loci genotype is heterozygous；If △ Ct>3, then the sample gene loci genotype is wild type.

Application method as described above, it is preferable that in step (2), the response procedures of the real-time fluorescent PCR amplification are：

First stage：95℃5min；

Second stage：95 DEG C of 5s, 58 DEG C of 30s, 10 circulations；

Phase III：95 DEG C of 5s, 58 DEG C of 30s, 72 DEG C of 30s, 35 circulations；

Signal collection：Fluorescence signal is collected during 72 DEG C of phase III.

Application method as described above, it is preferable that in step (2), the wild type detection architecture and the saltant type Also included in detection architecture as the Quality Control primer pair and Quality Control probe of inner quality control, 5 ＇ ends of the Quality Control probe are connected with Different from the fluorophor of any detection probe.

Application method as described above, it is preferable that the 5 ＇ ends connection FAM of the detection probe of each gene, the connection of 3 ＇ ends MGB, the 5 ＇ ends connection JOE of the Quality Control probe, 3 ＇ ends connection BHQ1.

Application method as described above, it is preferable that in the step (4), detects the JOE of the inner quality control of sample Signal should have typically " S " type amplification curve, and Ct values are between 12~30, then the genotype for determining the sample DNA；Otherwise Detection should be re-started.

Application method as described above, it is preferable that in step (2), be additionally provided with positive control and negative control, the sun Property control be with the corresponding wild-type positive tester of each gene and saltant type positive control and Quality Control positive control as mould Plate, carries out the real-time fluorescent PCR amplification of the wild type detection architecture and saltant type detection architecture；The negative control is with water It is template, carries out the real-time fluorescent PCR amplification of the wild type detection architecture and saltant type detection architecture；

In step (4), the wild type reaction system and saltant type reaction system of the positive control of each gene should have There are typical " S " type amplification curve, and Ct value≤30；The wild type detection architecture and saltant type of each gene of the negative control Detection architecture should meet the requirements without amplification curve and Ct values；Otherwise, detection should be re-started.

The invention provides the nucleic acid that one group is used for quick detection CYP2D6 gene loci polymorphisms, while establishing one kind Than more efficient, the detection kit of sensitive CYP2D6 gene polymorphism sites and method for quick.It is right to implement The loci polymorphism of CYP2D6*10 gene Cs 100T and G4268C, CYP2D6*14 gene G1758A detected, its detection reagent Box, easy to use, easy to operate, high degree of automation enormously simplify operating process, and reduce the dirt in operating process Dye, Detection results are good, the characteristics of with high sensitivity, high specific, high accuracy, pinpoint accuracy.The detection method of offer is adopted Be finished the operation of fully closed pipe, it is simple, convenient it is quick, by the acquisition detection of fluorescence signal value during direct detection PCR Result it is not necessary to PCR post processings or electrophoresis detection, overcome the easy pollution of Standard PCR technology, false positive occur, can effectively keep away Exempt from non-specific amplification problem, and be adapted to the detection of high-volume sample.

Detection kit and method that the present invention is provided, for detecting during CYP2D6 gene polymorphism sites, in order to avoid Whether whether just missing inspection and preliminary judgement sample amount of DNA are provided with inner quality control, in order to judge detection architecture in allowed band Often, it is additionally provided with the positive control of negative control and detection primer；In order to avoid false negative and missing inspection, wild type, saltant type are provided with With the positive control of inner quality control, its positive control tests positive result illustrates that detection architecture has no problem, and is not in vacation Negative findings and missing inspection, such as positive control detect the result that is negative, and illustrate that detection architecture is problematic, or reagent has failed, should The system of reconfiguring is detected；In order to avoid false positive and missing inspection, negative control is provided with, its negative control detects the knot that is negative Really, illustrate that detection architecture has no problem, be not in false positive results and missing inspection, such as negative control tests positive result, say Bright detection architecture is problematic, has been contaminated, and the system that should reconfigure is detected；The present invention provides detection kit and method sets Meter is rigorous, is prevented effectively from the possibility of missing inspection, false retrieval.

The primer of present invention design and probe sequence sensitivity are high, can accurately detect the as little as genomic DNA of 1ng, special It is different in nature strong, can only specific amplification target fragment, without non-specific amplification；Detection method provided by the present invention and PCR sequencing PCR 100% coincide；The inventive method can accurately distinguish various genotype, and visual result, easy interpretation, inspection provided by the present invention Survey method can complete detection in 90 minutes, and required time is far below PCR sequencing PCR, be particularly suitable for clinical application diagnosis；Present invention examination Agent box and detection method are applied to the quantitative real time PCR Instruments such as ABI7500, Roche LC480, Bio-Rad CFX96, and applicability is good.

Brief description of the drawings

Fig. 1, Fig. 2 and Fig. 3 are that the amplification of the genomic DNA sample (sample 1) of EDTA anticoagulated whole bloods extraction in embodiment 4 is bent Line.

Fig. 4, Fig. 5 and Fig. 6 are the amplification curve of the genomic DNA sample (sample 2) of buccal swab extraction in embodiment 4.

Fig. 7, Fig. 8 and Fig. 9 are the amplification curve of currently preferred kit detection sensitivity.

The currently preferred kit of Figure 10, Figure 11 and Figure 12 detects the amplification curve of precision.

The currently preferred kit of Figure 13, Figure 14 and Figure 15 detects specific amplification curve.

Specific embodiment

The present invention is described in further details below in conjunction with instantiation, not limitation of the invention, it is of the invention Implementation method is not limited to this, and the complementary series of the nucleotide sequence that the present invention is provided can also realize the present invention, such as without special theory Bright, agents useful for same is conventional reagent, thus all this areas according to done by present disclosure equivalent, belong to this hair Bright protection domain.

The primer of embodiment 1, probe, validation template design

According to NCBI dbSNP databases (https://www.ncbi.nlm.nih.gov/SNP/) announce CYP2D6* 10 and CYP2D6*14 sequence informations, respectively for 2 kinds of allele such as table 1 of this CYP2D6 gene, design pleomorphism site Detection primer and probe, by screening, optimization, lot of experiments checking obtains primer and probe sequence is specific as follows：

(1) for the primer and probe of the detection of CYP2D6*10SNP single nucleotide polymorphisms：

C100T wild-type alleles sense primer (1W-F)：

5'-CGCCAACGCTGGGCTGCACGCTAC-3'(SEQ ID NO.1),

C100T mutant alleles sense primer (1M-F)：

5'-CGCCAACGCTGGGCTGCACGCTAT-3'(SEQ ID NO.2),

The public anti-sense primer of C100T allele (1C-R):

5'-CCTCAGGACCTCTGCCGCCCTCC-3'(SEQ ID NO.3),

C100T allele-specific probes (1P)：

5'-TGTTCTGGAAGTCCACATGCAGCA-3'(SEQ ID NO.4)；

G4268C wild-type alleles sense primer (2W-F)：

5'-TGTCTTTGCT TTCCTGGTGAG-3'(SEQ ID NO.5)；

G4268C mutant alleles sense primer (2W-F)：

5'-TGTCTTTGCT TTCCTGGTGAC-3'(SEQ ID NO.6)；

The public anti-sense primer of G4268C allele (2C-R)：

5'-GTGAGCAGGGGACCCGAGTTGG-3'(SEQ ID NO.7)；

G4268C allele-specific probes (2P)：

5'-GGCTGGGGACTAGGTACCCCATT-3'(SEQ ID NO.8)；

(2) for the primer and probe of the detection of CYP2D6*10SNP single nucleotide polymorphisms：

G1758A wild-type alleles sense primer (3W-F)：

5'-TGCCCTTCTGCCCATCACCCACG-3'(SEQ ID NO.9)；

G1758A mutant alleles sense primer (3M-F)：

5'-TGCCCTTCTGCCCATCACCCACA-3'(SEQ ID NO.10)；

The public anti-sense primer of G1758A allele (3C-R)：

5'-CGCGAGCAGAGGCGCTTCTCCGT-3'(SEQ ID NO.11)；

G1758A allele-specific probes (3P)：

5'-CTCCTCGGTCACCCACTGCTCCAGCGACTTC-3'(SEQ ID NO.12)。

2 kinds of allele of table 1.CYP2D6 genes

Wild type sense primer can be used as wild-type positive template, saltant type upstream with the PCR primer of anti-sense primer amplification Primer can be corresponding as saltant type positive template, each gene polynorphisms site with the PCR primer of common downstream primer amplification The wild-type positive tester in the following CYP2D6*10 gene Cs 100T sites of wild-type positive tester, saltant type positive control Containing the nucleotide sequence as shown in SEQ ID No.16, the saltant type positive control in C100T sites contains such as SEQ ID Nucleotide sequence shown in No.17, the wild-type positive tester in the G4268C sites of CYP2D6*10 genes contains such as SEQ ID Nucleotide sequence shown in No.18, the saltant type positive control in G4268C sites contains the core as shown in SEQ ID No.19 Nucleotide sequence, the wild-type positive tester in the G1758A sites of CYP2D6*14 genes contains as shown in SEQ ID No.20 Nucleotide sequence, the saltant type positive control in G1758A sites contains the nucleotide sequence as shown in SEQ ID No.21.

SEQ ID No.16：

5’-CGCCAACGCTGGGCTGCACGCTACCCACCAGGCCCCCTGCCACTGCCCGGGCTGGGCAACCTGCTG CATGTGGACTTCCAGAACACACCATACTGCTTCGACCAGGTGAGGGAGGAGGTCCTGGAGGGCGGCAGAGGTCCTGA GG-3’

SEQ ID No.17：

5’-CGCCAACGCTGGGCTGCACGCTATCCACCAGGCCCCCTGCCACTGCCCGGGCTGGGCAACCTGCTG CATGTGGACTTCCAGAACACACCATACTGCTTCGACCAGGTGAGGGAGGAGGTCCTGGAGGGCGGCAGAGGTCCTGA GG-3’

SEQ ID No.18：

5’-TGTCTTTGCTTTCCTGGTGAGCCCATCCCCCTATGAGCTTTGTGCTGTGCCCCGCTAGAATGGGGT ACCTAGTCCCCAGCCTGCTCCCTAGCCAGAGGCTCTAATGTACAATAAAGCAATGTGGTAGTTCCAACTCGGGTCCC CTGCTCAC-3’

SEQ ID No.19：

5’-TGTCTTTGCTTTCCTGGTGACCCCATCCCCCTATGAGCTTTGTGCTGTGCCCCGCTAGAAT GGGGTACCTAGTCCCCAGCCTGCTCCCTAGCCAGAGGCTCTAATGTACAATAAAGCAATG TGGTAGTTCCAACTCGGGTCCCCTGCTCAC-3’

SEQ ID No.20：

5’-TGCCCTTCTGCCCATCACCCACGGGAGTGGTTGGCGAAGGCGGCACAAAGGCAGGCGGCCTCCTCG GTCACCCACTGCTCCAGCGACTTCTTG CCCAGGCCCAAGTTGCGCAAGGTGGAGACGGAGAAGCGCCTCTGCTCGC G-3’

SEQ ID No.21：

5’-TGCCCTTCTGCCCATCACCCACAGGAGTGGTTGGCGAAGGCGGCACAAAGGCAGGCGGCCTCCTCG GTCACCCACTGCTCCAGCGACTTCTTG CCCAGGCCCAAGTTGCGCAAGGTGGAGACGGAGAAGCGCCTCTGCTCGC G-3’

Further, in order to carry out inner quality control to real-time fluorescence PCR reaction system and experimental implementation process, while detecting sample This quality, it is to avoid leakage plus detection sample, according to the sequence of mankind's house-keeping gene GAPDH (NC_000012.12), devises work It is a pair of Quality Control primer pairs and Quality Control probe of inner quality control, it is desirable to which the inner quality control acts not only as the Quality Control of kit, It is also used as the Quality Control of sample DNA, it is ensured that the DNA for extracting includes gene, the non-specific friendship of other sequences can be excluded Fork reaction, while it also will not be to the wild type of each gene pleiomorphism and the genes of interest and primer pair and detection probe of mutability Produce cross reaction, it is ensured that the accuracy of kit.By the Quality Control primer pair to inner quality control, the screening of Quality Control probe and body System's optimization, establishes real-time fluorescence PCR inner quality control detection architecture, it is preferable that Quality Control primer pair (R-F, R-R), Quality Control probe (RP) it is as follows：

Internal control sense primer (R-F)：5'-CATCAAGAAGGTGGTGAAGCAG-3'(SEQ ID NO.13),

Internal control anti-sense primer (R-R)：5'-TGTCGCTGTTGAAGTCAGAGGA-3'(SEQ ID NO.14),

Internal control probe (RP)：5'-TGGTGCTCAGTGTAGCCCAGGATGC-3'(SEQ ID NO.15).

When entering performing PCR amplification as the Quality Control primer pair of inner quality control, the size of its extension increasing sequence is 98bp.Sequence will be expanded Row, that is, comprising the sequence as shown in SEQ ID No.22, sample are detected as verifying whether Lou to add as inner quality control positive control The Quality Control positive control of product,

SEQ ID No.22：

5’-CATCAAGAAGGTGGTGAAGCAGGCGTCGGAGGGCCCCCTCAAGGGCATCCTGGGCTACACTGAGCA CCAGGTGGTCTCCTCTGACTTCAACAGCGACA-3’

When entering performing PCR amplification as kit specificity verification, human genome ABCB1 genes are selected, positioned at No. 7 dyeing On body, its C3435T gene pleiomorphism is also closely related with opioid drug personalized medicine.For kit has no cross reaction Needed for checking, its plasmid sequence sequence as shown in SEQ ID No.23, length is 100bp.

SEQ ID No.23：

5’-TGGAGACAACAGCCGGGTGGTGTCACAGGAAGAGATCGTGAGGGCAGCAAAGGAGGCCAACATACA TGCCTTCATCGAGTCACTGCCTAATGTAAGTCTC-3’。

Embodiment 2 is used to detect the kit of CYP2D6 gene polymorphism sites

Real-time fluorescent PCR reagent case for quick detection CYP2D6 gene polymorphism sites includes following component：Strictly according to the facts Apply the primer of each gene described in example 1, detection probe, wild-type positive tester, saltant type positive control, each gene The 5 ＇ ends connection fluorophor of detection probe, 3 ＇ ends connection quenching group；Wherein, fluorophor is in FAM, JOE, CY3, HEX Any one, the quenching group is any one in MGB, BHQ1, TAMRA, BHQ2.

In order to avoid missing inspection, false retrieval also includes：It is positive as the Quality Control primer pair of inner quality control, Quality Control probe and Quality Control Tester, sequence as described in example 1 above, and Quality Control probe fluorophor be different from detection probe fluorophor.

In order to prevent reaction system from being volatilized in PCR amplification procedures, Detection results, kit are influenceed also to include mineral Oil.

For the ease of the configuration of reaction system, kit also includes 10 × PCR buffer solutions, archaeal dna polymerase, dNTPs, feminine gender Tester：Nuclease free water.Wherein archaeal dna polymerase can use Taqman Master Mix full name TaqMan Real-Time PCR Master Mixes, purchased from Thermo Fisher Scientific (Thermo Fischer Scient Inc.)；10×PCR buffer(Mg²⁺Plus) using TaKaRa treasured bioengineering (Dalian) Co., Ltd, also can in application market other companies it is slow Fliud flushing and archaeal dna polymerase.

The preparation of the detection kit of embodiment 3

The kit of the present embodiment is that on the basis of embodiment 2, being prepared into after being directly added into detection sample is carried out The kit of detection, reacts bar and CYP2D6 positive controls is constituted, the composition in kit in each pipe by the PCR of CYP2D6 6 It is shown in Table 2.

The kit forms of table 2

The full name of Taqman Master Mix is TaqMan Real-Time PCR Master Mixes, purchased from Thermo Fisher Scientific (Thermo Fischer Scient Inc.)；10×PCR buffer(Mg²⁺Plus it is) precious biological using TaKaRa Engineering (Dalian) Co., Ltd, also can in application market other companies buffer solution and archaeal dna polymerase.

The application method of the kit of the present invention of embodiment 4

Normal person's EDTA anticoagulated whole bloods sample (being provided by Wu great the People's Hospitals) and buccal swab sample are provided in the present embodiment This (being provided by hospital of Tongji University) is a case each, and therefrom extracts genomic DNA, is detected with CYP2D6 genetic polymorphism detections kit CYP2D6 gene mutations state in sample to be tested.Comprise the following steps that：

1st, the extraction of clinical sample DNA

Carried with reference to Tiangeng biochemical technology Co., Ltd poba gene group DNA extraction kit and buccal swab genomic DNA Kit explanation is taken, sample gene group DNA is obtained.DNA is extracted after finishing with the ultramicrospectrophotometers pair of Nanodrop 2000 Sample DNA carries out concentration mensuration, and sample DNA concentration should be advisable in 20ng/ μ L~200ng/ μ L, and OD260/OD280 is 1.65 Between~1.80.

2nd, PCR reaction systems are prepared

Using the kit of the preparation of the embodiment of the present invention 3 to the DNA profiling of detection sample, CYP2D6*10 genes are carried out C100T sites, the G4268C sites of CYP2D6*10 genes, the G1758A loci polymorphism real-time fluorescences of CYP2D6*14 genes PCR augmentation detections.

Wherein, each polymorphic detection site includes saltant type detection architecture and wild type detection architecture respectively, specifically, Each detection architecture configuration can will be shown in Table 3 and table 4, wherein the 5 ＇ ends connection FAM of the detection probe of each gene, 3 ＇ ends connection MGB, institute State the 5 ＇ ends connection JOE of Quality Control probe, 3 ＇ ends connection BHQ1.Above detection architecture is dispensed into PCR reactions respectively after the completion of preparing Guan Zhong, to the 2.0 same sample DNAs of μ L are separately added into above PCR reaction tubes, the of short duration centrifugation several seconds, mixes PCR reaction systems Uniformly.

The CYP2D6*10 quantitative fluorescent PCR reaction systems of table 3

The CYP2D6*14 quantitative fluorescent PCR reaction systems of table 4

3rd, fluorescent PCR detection

The PCR system that will have been configured is put into ABI7500 fluorescent PCR instruments, carries out fluorescent PCR augmentation detection.Reaction bar Part is：

First stage：95 DEG C of predegeneration 5min；

Second stage：95 DEG C of 20s, 58 DEG C of 30s, 10 circulations；

Phase III:95 DEG C of 20s, 58 DEG C of 30s (collecting FAM and JOE signals), 35 circulations.

It is automatic that baseline and threshold line are set after PCR EPs (end of program), obtain the Ct values of sample.

4th, result interpretation

NTC and positive control are analyzed：NTC reacting holes otherwise, should illustrate possibility without amplification curve and Ct values under normal circumstances There is operational pollution, now, experiment is restarted behind the source that should decontaminate；Positive control reacting hole should have typically " S " type Amplification curve, and Ct value≤30, otherwise, illustrate that PCR reaction systems are likely to occur exception, have impact on amplification efficiency.

Internal control is analyzed：Usually, internal control JOE signals should have typically " S " type amplification curve, and Ct values 12~30 it Between.If Ct value≤12, show added sample DNA excessive concentration, should take the circumstances into consideration to reduce DNA applied sample amounts；If Ct >=30, show to be loaded This DNA copy number is relatively low or sample DNA in there may be PCR inhibitor；If internal control JOE signals are without amplification curve, Ke Nengcun Add in sample DNA leakage, experiment should be restarted.

Sample genotyping：Under the premise of NTC (negative control), positive control and internal control JOE signals are normal, root Interpretation is carried out according to saltant type detection architecture Ct values to the genotype of sample with the difference (△ Ct) of wild type detection architecture Ct values (to be shown in Table 5).For any one polymorphic detection site, if △ Ct<- 3, then the sample gene loci genotype is saltant type；If -3 ≤ △ Ct >=3, then the sample gene loci genotype is heterozygous；If △ Ct>3, then sample gene loci genotype It is wild type.

5 CYP2D6 genes of table, three pleomorphism site genotype results judge

The amplification curve of the genomic DNA sample (sample 1 is labeled as rm1) that EDTA anticoagulated whole bloods are extracted in the present embodiment is shown in Fig. 1, Fig. 2 and Fig. 3, gene pleiomorphism are shown in Table 6.

The amplification curve of the genomic DNA sample (sample 2 be labeled as rm2) that buccal swab is extracted in the present embodiment see Fig. 4, Fig. 5 and Fig. 6, gene pleiomorphism is shown in Table 6.

The pattern detection result of table 6

Explanation：The genomic DNA reagent of the invention that the genomic DNA and buccal swab that EDTA anticoagulations are extracted are extracted The result of box detection and the result of sanger sequencings are completely the same, and the kit degree of accuracy is high, adapts to sample wide.Sanger PCR sequencing PCRs It is the goldstandard detection method commonly used in clinical examination.

Embodiment 5：The performance evaluation experiment of kit of the present invention

CYP2D6 recombinant plasmids are purchased from Nanjing Genscript Biotechnology Co., Ltd., are manually closed with existing technique for gene engineering Into CYP2D6*10 gene C 100T pleomorphism sites contain SEQ ID NO:Sequence shown in 16 is wild plasmid, contains SEQ ID NO:Sequence shown in 17 is mutant plasmids；CYP2D6*10 gene G4268C pleomorphism sites contain SEQ ID NO:18 institutes Show that sequence, for wild plasmid, contains SEQ ID NO:Sequence shown in 19 is mutant plasmids；The G1758A of CYP2D6*14 genes Pleomorphism site contains SEQ ID NO:Sequence shown in 20 is wild plasmid, contains SEQ ID NO:Sequence shown in 21 is mutation Type plasmid；It is artificial synthesized to contain SEQ ID NO:Sequence shown in 22 is internal control plasmid.Each plasmid dry powder with TE (10mmol/L, PH8.0) redissolve, diluted after quantifying.

(1) sensitivity

Three kinds the 2 × 10 of gene loci are taken respectively¹The wild plasmid of copies/ μ L and internal control plasmid 1:1 mixing, configuration Into the CYP2D6 reference wild-types product of final concentration of 10copies/ μ L, (numbering is：CY100W-1、CY4268W-1、CY1758W- 1)；Three kinds the 2 × 10 of gene loci are taken respectively¹The mutant plasmids of copies/ μ L and internal control plasmid 1:1 mixing, is configured to end For the CYP2D6 saltant types reference material of 10copies/ μ L, (numbering is concentration：CY100M-1、CY4268M-1、CY1758M-1)；Point Do not take three kinds of gene locis takes 2 × 10¹The wild plasmid of copies/ μ L, mutant plasmids, internal control plasmid 1:1:2 volumes Than mixing, (numbering is to be configured to the CYP2D6 heterozygous reference material of final concentration of 10copies/ μ L：CY100WM-1、 CY4268WM-1、CY1758WM-1).With these three reference materials as template, carried out using fluorescence quantifying PCR method in embodiment 4 Amplification, amplification figure result is shown in Fig. 7, Fig. 8, Fig. 9.Testing result shows, fluorescence PCR method high specificity of the invention, sensitivity Height, the DNA profiling of 10copies/ μ L concentration can be detected.

(2) precision

Three kinds the 6 × 10 of gene loci are taken respectively³The wild plasmid of copies/ μ L, mutant plasmids, internal control plasmid 1: 1:2 volume ratios mix, and are configured to final concentration of 3 × 10³(numbering is the CYP2D6 heterozygosis reference material of copies/ μ L：CY100WM- 2、CY4268WM-2、CY1758WM-2).With these three reference materials as template, entered using fluorescence quantifying PCR method in embodiment 4 Row amplification, amplification figure result is shown in Figure 10, Figure 11, Figure 12.Result shows that real-time fluorescent PCR amplification method of the invention is reproducible (20 repetition experimental result stabilizations, CV<5%).

(3) specificity

ABCB1 plasmids are artificial synthesized in Nanjing Genscript Biotechnology Co., Ltd..It is artificial with existing technique for gene engineering What is synthesized contains SEQ ID NO:The gene plasmid of sequence ABCB1 shown in 23, takes 6 × 10 respectively⁴The open country of the CYP2D6 of copies/ μ L Raw type plasmid and ABCB1 gene plasmids and internal control plasmid 1:1 mixing, is configured to final concentration of 3 × 10⁴Copies/ μ L's is wild (sample number into spectrum is type reference material：CY100W-3, CY4268W-3, CY1758W-3, ABW-3), detection architecture numbering is designated as CYP2D6W-3；Take the 6 × 10 of above-mentioned two gene⁴Copies/ μ L mutant plasmids and internal control plasmid 1:1 mixing, is configured to end Concentration is 3 × 10⁴(numbering is the saltant type reference material of copies/ μ L：CY100M-3、CY4268M-3、CY1758M-3、ABM- 3), detection architecture numbering is designated as CYP2D6M3；Three kinds of wild plasmids of gene loci, mutant plasmids, ABCB1 bases are taken respectively Because of plasmid and internal control plasmid, final concentration of 3 × 10 are configured to⁴(numbering is the CYP2D6 heterozygous reference material of copies/ μ L： CY100WM-3, CY4268WM-3, CY1758WM-3, ABWM-3), detection architecture numbering is designated as CYP2D6M3.With above-mentioned three seed ginseng Product are examined for template, the kit prepared with the embodiment of the present invention 3 to carrying out real-time fluorescent PCR amplification detection, amplification figure see Figure 13, Figure 14, Figure 15.Result shows, fluorescence PCR method high specificity of the invention, without friendship between ABCB1 genes and CYP2D6 genes Fork reaction, 3 × 10⁴Can not be detected in each reaction system of ABCB1 genes of copies/ μ L concentration, show present invention design The primer pair of each gene loci, probe it is specific good.

Embodiment described above is only the preferred embodiment lifted to absolutely prove the present invention, protection model of the invention Enclose not limited to this.Equivalent substitute or conversion that those skilled in the art are made on the basis of the present invention, in the present invention Protection domain within.

SEQUENCE LISTING

<110>Wuhan Hygiea Bioscience Co., Ltd.

<120>Nucleic acid, kit and method for detecting mankind's CYP2D6 gene pleiomorphisms

<130>

<160> 23

<170> PatentIn version 3.5

<210> 1

<211> 24

<212> DNA

<213>It is artificial synthesized

<400> 1

cgccaacgct gggctgcacg ctac 24

<210> 2

<211> 24

<212> DNA

<213>It is artificial synthesized

<400> 2

cgccaacgct gggctgcacg ctat 24

<210> 3

<211> 23

<212> DNA

<213>It is artificial synthesized

<400> 3

cctcaggacc tctgccgccc tcc 23

<210> 4

<211> 24

<212> DNA

<213>It is artificial synthesized

<400> 4

tgttctggaa gtccacatgc agca 24

<210> 5

<211> 21

<212> DNA

<213>It is artificial synthesized

<400> 5

tgtctttgct ttcctggtga g 21

<210> 6

<211> 21

<212> DNA

<213>It is artificial synthesized

<400> 6

tgtctttgct ttcctggtga c 21

<210> 7

<211> 22

<212> DNA

<213>It is artificial synthesized

<400> 7

gtgagcaggg gacccgagtt gg 22

<210> 8

<211> 23

<212> DNA

<213>It is artificial synthesized

<400> 8

ggctggggac taggtacccc att 23

<210> 9

<211> 23

<212> DNA

<213>It is artificial synthesized

<400> 9

tgcccttctg cccatcaccc acg 23

<210> 10

<211> 23

<212> DNA

<213>It is artificial synthesized

<400> 10

tgcccttctg cccatcaccc aca 23

<210> 11

<211> 23

<212> DNA

<213>It is artificial synthesized

<400> 11

cgcgagcaga ggcgcttctc cgt 23

<210> 12

<211> 31

<212> DNA

<213>It is artificial synthesized

<400> 12

ctcctcggtc acccactgct ccagcgactt c 31

<210> 13

<211> 22

<212> DNA

<213>It is artificial synthesized

<400> 13

catcaagaag gtggtgaagc ag 22

<210> 14

<211> 22

<212> DNA

<213>It is artificial synthesized

<400> 14

tgtcgctgtt gaagtcagag ga 22

<210> 15

<211> 25

<212> DNA

<213>It is artificial synthesized

<400> 15

tggtgctcag tgtagcccag gatgc 25

<210> 16

<211> 145

<212> DNA

<213>It is artificial synthesized

<400> 16

cgccaacgct gggctgcacg ctacccacca ggccccctgc cactgcccgg gctgggcaac 60

ctgctgcatg tggacttcca gaacacacca tactgcttcg accaggtgag ggaggaggtc 120

ctggagggcg gcagaggtcc tgagg 145

<210> 17

<211> 145

<212> DNA

<213>It is artificial synthesized

<400> 17

cgccaacgct gggctgcacg ctatccacca ggccccctgc cactgcccgg gctgggcaac 60

ctgctgcatg tggacttcca gaacacacca tactgcttcg accaggtgag ggaggaggtc 120

ctggagggcg gcagaggtcc tgagg 145

<210> 18

<211> 151

<212> DNA

<213>It is artificial synthesized

<400> 18

tgtctttgct ttcctggtga gcccatcccc ctatgagctt tgtgctgtgc cccgctagaa 60

tggggtacct agtccccagc ctgctcccta gccagaggct ctaatgtaca ataaagcaat 120

gtggtagttc caactcgggt cccctgctca c 151

<210> 19

<211> 151

<212> DNA

<213>It is artificial synthesized

<400> 19

tgtctttgct ttcctggtga ccccatcccc ctatgagctt tgtgctgtgc cccgctagaa 60

tggggtacct agtccccagc ctgctcccta gccagaggct ctaatgtaca ataaagcaat 120

gtggtagttc caactcgggt cccctgctca c 151

<210> 20

<211> 143

<212> DNA

<213>It is artificial synthesized

<400> 20

tgcccttctg cccatcaccc acgggagtgg ttggcgaagg cggcacaaag gcaggcggcc 60

tcctcggtca cccactgctc cagcgacttc ttgcccaggc ccaagttgcg caaggtggag 120

acggagaagc gcctctgctc gcg 143

<210> 21

<211> 143

<212> DNA

<213>It is artificial synthesized

<400> 21

tgcccttctg cccatcaccc acaggagtgg ttggcgaagg cggcacaaag gcaggcggcc 60

tcctcggtca cccactgctc cagcgacttc ttgcccaggc ccaagttgcg caaggtggag 120

acggagaagc gcctctgctc gcg 143

<210> 22

<211> 98

<212> DNA

<213>It is artificial synthesized

<400> 22

catcaagaag gtggtgaagc aggcgtcgga gggccccctc aagggcatcc tgggctacac 60

tgagcaccag gtggtctcct ctgacttcaa cagcgaca 98

<210> 23

<211> 100

<212> DNA

<213>It is artificial synthesized

<400> 23

tggagacaac agccgggtgg tgtcacagga agagatcgtg agggcagcaa aggaggccaa 60

catacatgcc ttcatcgagt cactgcctaa tgtaagtctc 100

Claims (10)

1. one group is used to detect the nucleic acid of CYP2D6 gene pleiomorphisms, it is characterised in that the nucleic acid include CYP2D6*10 genes and The detection primer and detection probe of CYP2D6*14 gene pleiomorphisms, wherein, the detection of the CYP2D6*10 gene pleiomorphisms is drawn Thing and detection probe include being directed to that C100T wild type upstream of the nucleotide sequence in C100T sites as shown in SEQ ID No.1 is drawn Thing 1W-F, the C100T saltant type sense primer 1M-F as shown in SEQ ID No.2 and the C100T as shown in SEQ ID No.3 are public Use anti-sense primer 1C-R, and C100T detection probe 1P of the nucleotide sequence as shown in SEQ ID No.4；

And G4268C wild type sense primer 2W-F of the nucleotide sequence as shown in SEQ ID No.5 for G4268C sites, Under G4268C saltant type sense primer 2M-F as shown in SEQ ID No.6 and the G4268C as shown in SEQ ID No.7 are public Trip primer 2 C-R, and G4268C detection probe 2P of the nucleotide sequence as shown in SEQ ID No.8；

The detection primer and detection probe of the CYP2D6*14 gene pleiomorphisms include nucleotide sequence such as SEQ ID No.9 institutes The G1758A wild type sense primers 3W-F, the G1758A saltant type sense primer 3M-F as shown in SEQ ID No.10 that show and such as The public anti-sense primer 3C-R of G1758A shown in SEQ ID No.11, and nucleotide sequence is as shown in SEQ ID No.12 G1758A detection probes 3P.

2. nucleic acid as claimed in claim 1, it is characterised in that the nucleic acid also includes inner quality control, and it includes Quality Control primer Pair and Quality Control probe, the nucleotide sequence of the Quality Control primer pair is described as shown in SEQ ID No.13 and SEQ ID No.14 The nucleotide sequence of Quality Control probe is as shown in SEQ ID No.15.

3. nucleic acid described as claimed in claim 2, it is characterised in that the nucleic acid also includes CYP2D6*10 gene Cs 100T Point, the corresponding wild-type positive tester in G4268C sites and CYP2D6*14 gene G1758A sites, saltant type positive control And Quality Control positive control, wherein, the wild-type positive tester in the C100T sites contains as shown in SEQ ID No.16 Nucleotide sequence, the saltant type positive control in the C100T sites contains the nucleotide sequence as shown in SEQ ID No.17, The wild-type positive tester in the G4268C sites contains the nucleotide sequence as shown in SEQ ID No.18, the G4268C The saltant type positive control in site contains the nucleotide sequence as shown in SEQ ID No.19, the G1758A sites it is wild Type positive control contains the nucleotide sequence as shown in SEQ ID No.20, the saltant type positive control in the G1758A sites Thing contains the nucleotide sequence as shown in SEQ ID No.21, and the Quality Control positive control contains as shown in SEQ ID No.22 Nucleotide sequence.

4. a kind of kit for quick detection CYP2D6 gene pleiomorphisms, it is characterised in that the kit includes such as right It is required that any described nucleic acid in 1-3, wherein, the 5 ＇ ends connection fluorophor of the detection probe of each gene, the connection of 3 ＇ ends Quenching group.

5. kit as claimed in claim 4, it is characterised in that the kit also includes that 10 × PCR buffer solutions, DNA are polymerized Enzyme, dNTPs, negative control thing：Nuclease free water；The fluorophor is any one in FAM, JOE, CY3, HEX, described to quench The group that goes out is any one in MGB, BHQ1, TAMRA, BHQ2.

6. as described in claim 4 or 5 kit application method, it is characterised in that the method is comprised the following steps：

(1) extraction of sample to be tested genomic DNA；

(2) PCR reaction solutions are prepared：Saltant type detection architecture and wild type detection architecture are prepared respectively per pleomorphism site；Respectively To same sample DNA is added in the saltant type detection architecture and the wild type detection architecture, of short duration centrifugation is well mixed；

(3) fluorescence quantitative PCR detection：The PCR system that will be prepared is put into fluorescent PCR instrument, carries out fluorescent quantitative PCR Detection；

(4) result interpretation：The poor △ Ct of the Ct values of Ct values and wild type detection architecture according to saltant type detection architecture are to sample Genotype carry out interpretation, if △ Ct<- 3, then the sample gene loci genotype is saltant type；If -3≤△ Ct≤3, The sample gene loci genotype is heterozygous；If △ Ct>3, then the sample gene loci genotype is wild type.

7. application method as claimed in claim 6, it is characterised in that in step (2), the real-time fluorescent PCR amplification it is anti- The program is answered to be：

First stage：95℃5min；

Second stage：95 DEG C of 5s, 58 DEG C of 30s, 10 circulations；

Phase III：95 DEG C of 5s, 58 DEG C of 30s, 72 DEG C of 30s, 35 circulations；

Signal collection：Fluorescence signal is collected during 72 DEG C of phase III.

8. application method as claimed in claim 7, it is characterised in that in step (2), the wild type detection architecture and institute State and also include as the Quality Control primer pair and Quality Control probe of inner quality control, 5 ＇ of the Quality Control probe in saltant type detection architecture End is connected with the fluorophor different from any detection probe.

9. application method as claimed in claim 8, it is characterised in that the 5 ＇ ends connection FAM of the detection probe of each gene, 3 ＇ ends connect MGB, the 5 ＇ ends connection JOE of the Quality Control probe, 3 ＇ ends connection BHQ1；

In the step (4), detecting the JOE signals of the inner quality control of sample should have typically " S " type amplification curve, and Ct values are between 12~30, then the genotype for determining the sample DNA；Detection should otherwise be re-started.

10. method as claimed in claim 8 or 9, it is characterised in that in step (2), be additionally provided with positive control and feminine gender is right According to the positive control is with the corresponding wild-type positive tester of each gene and saltant type positive control and the Quality Control positive Tester is template, carries out the real-time fluorescent PCR amplification of the wild type detection architecture and saltant type detection architecture；The feminine gender Control is the real-time fluorescent PCR amplification that the wild type detection architecture and saltant type detection architecture are carried out with water as template；

In step (4), the wild type reaction system and saltant type reaction system of the positive control of each gene should have allusion quotation " S " type amplification curve of type, and Ct value≤30；Wild type detection architecture and the saltant type detection of each gene of the negative control System should meet the requirements without amplification curve and Ct values；Otherwise, detection should be re-started.