CN107090499A - A kind of kit of detection CYP21 gene mutations - Google Patents
A kind of kit of detection CYP21 gene mutations Download PDFInfo
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Abstract
The present invention relates to biological technical field, more particularly to a kind of kit of detection CYP21 gene mutations, the kit and method can detect 8 kinds of mutation types of CYP21 genes simultaneously using special primer sets and probe groups.The present invention also provides a kind of kit of detection CYP21 gene mutations.Compared to existing detection method and kit, the kit sensitivity height of the detection CYP21 gene mutations that the present invention is provided, high specificity, simple and quick and testing cost is low, auxiliary diagnosis and prevention available for congenital adrenal cortical hyper plasia disease, with real-time monitoring, quantitatively detect the characteristics of.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of kit of detection CYP21 gene mutations.
Background technology
Congenital adrenal cortical hyper plasia (congenital adrenal hyperplasia, CAH) is a kind of often dye
Recessive hereditary disease on colour solid, is mesh mainly due to caused by the necessary enzyme defect in adrenal gland sebaceous glands building-up process
It is preceding the reason for clinically androgyny is most commonly seen.Wherein using 21-hydroxylase defect as main pathogenesis etiology, its incidence of disease is 1:
12000-1:23000, account for 90%-the 95% of CAH.CAH can cause low blood sodium and hyperkalemia, severe patient to cause shock, to new
Raw youngster makes diagnosis as early as possible when symptom occur, and it is heavy to closing to efficiently reduce the incidence of disease and case fatality rate of adrenal gland danger disease
Want.
21 hydroxylases are synthesized by 21-hydroxylase gene (CYP21 genes) coding, and 1980s mid-term just has confirmed
The positive patient of 21-hydroxylase defect is with the different degrees of defect of CYP21 genes.It is short that CYP21 genes are located at No. 6 chromosomes
HLAIII areas on arm, the pseudogene (CYP21P gene) inactive with it is arranged in series in 3 ' ends of C4A and C4B genes, very
Pseudogene has 10 extrons, 9 intrones, and the homology of its extron and intron sequences is respectively 98% and 96%.
With the development of molecular biology, the understanding to CYP21 gene defects is also more and more deep, up to now, human mutation
Database includes 62 kinds of CYP21 gene mutations altogether, including missense/nonsense mutation in 40,5 kinds of shearing mutation, 6 kinds small missing, 2 kinds
Small insertion/deletion, 3 kinds big missing and 4 kinds of promoter region mutation in small insertion, 2.
At present, the detection method of 21-hydroxylase gene mutation mainly have ELISA, time-resolved fluoroimmunoassay,
PCR-RFLP (PCR-RFLP) etc., but these detection methods lack specificity and sensitive
Property, it is impossible to efficiently and accurately the catastrophe of 21-hydroxylase gene loci is detected.Wherein, PCR-RFLP apply compared with
Extensively, but the mutational site of two and the above can not be detected simultaneously for this method, it is necessary to multiple detecting systems are detected respectively,
The cumbersome and time is long；And this method needs to use a variety of restriction enzymes, cost is higher, and digestion post-fragment is too small,
Difficult and differentiation, is also faced with the incomplete problem of digestion.
The content of the invention
In view of this, the technical problems to be solved by the invention are to provide a kind of reagent of detection CYP21 gene mutations
Box, the kit can effectively detect CYP21 genes wild type and saltant type result, high specificity, it is simple and quick,
And testing cost is low.
In order to realize the purpose of the present invention, the present invention is adopted the following technical scheme that：
The invention provides a kind of kit of detection CYP21 gene mutations, including primer sets and probe groups；
It is the primer shown in SEQ ID No.4~19 that the primer sets, which include sequence,；It is SEQ that the probe groups, which include sequence,
Probe shown in ID No.20~SEQ ID No.27.
Auxiliary diagnosis or prevention congenital adrenal cortical hyper plasia examination are being prepared present invention also offers mentioned reagent box
Application in agent.
In the kit that provides of the present invention, for the primer that mutational site P30L is designed have as SEQ ID No.4~
Sequence shown in SEQ ID No.5, the probe of design has the sequence as shown in SEQ ID NO 20；
There is the sequence as shown in SEQ ID No.6~SEQ ID No.7 for the primer that mutational site I172N is designed,
The probe of design has the sequence as shown in SEQ ID NO 21；
There is the sequence as shown in SEQ ID No.8~SEQ ID No.9 for the primer that mutational site I236N is designed,
The probe of design has the sequence as shown in SEQ ID NO 22；
There is the sequence as shown in SEQ ID No.10~SEQ ID No.11 for the primer that mutational site V281L is designed
Row, the probe of design has the sequence as shown in SEQ ID NO 23；
There is the sequence as shown in SEQ ID No.12~SEQ ID No.13 for the primer that mutational site Q318X is designed
Row, the probe of design has the sequence as shown in SEQ ID NO 24；
There is the sequence as shown in SEQ ID No.14~SEQ ID No.15 for the primer that mutational site C655G is designed
Row, the probe of design has the sequence as shown in SEQ ID NO 25；
There is the sequence as shown in SEQ ID No.16~SEQ ID No.17 for the primer that mutational site I2g is designed,
The probe of design has the sequence as shown in SEQ ID NO 26；
There is the sequence as shown in SEQ ID No.18~SEQ ID No.19 for the primer that mutational site R356W is designed
Row, the probe of design has the sequence as shown in SEQ ID NO 27.
In some embodiments that the present invention is provided, sequence is the probe shown in SEQ ID No.20~SEQ ID No.23
5 ' ends connect different fluorophors respectively, the quencher of 3 ' end connection non-luminescent.Preferably, fluorophor be selected from FAM,
One kind in ROX, HEX, CY5.
In some embodiments that the present invention is provided, sequence is the probe shown in SEQ ID No.24~SEQ ID No.27
5 ' ends connect different fluorophors respectively, the quencher of 3 ' end connection non-luminescent.Preferably, fluorophor be selected from FAM,
One kind in ROX, HEX, CY5.
Preferably, the kit that provides of the present invention also include dNTP, Tag archaeal dna polymerase, pH 8.8 Tris-HCl,
(NH4)2SO4, 0.01%Tween-20 and MgCl2。
The kit that the present invention is provided, amplification program is as follows：
The first step：95℃3min
Second step：94 DEG C of 15s → 65 DEG C~45 DEG C 15s → 75 DEG C 15s, 15 circulations；
3rd step：94 DEG C of 15s → 55 DEG C 15s → 75 DEG C 15s, 40 circulations, the fluorescence of annealing stage is gathered at 55 DEG C
Signal；
4th step：94 DEG C of 1min → 35 DEG C 3min → 40 DEG C~85 DEG C, are dissolved with 0.4 DEG C/5s gradient increased temperature
The analysis of curve, and collect the fluorescence signal in this stage.
It is the primer shown in SEQ ID No.4~19 that the primer sets, which include sequence,；It is SEQ that the probe groups, which include sequence,
Probe shown in ID No.20~SEQ ID No.27.
In the specific embodiment that the present invention is provided, using two detection bodies when the kit that the present invention is provided is detected
System：System A and system B.Wherein, system A is：67mM Tris-HCl(pH 8.8),16.6mM(NH4)2SO4, 0.01%
Tween-20,1.5-3mM MgCl2, 0.2m dNTPs, shown in 0.3 μM of SEQ ID No.20~SEQ ID No.23 sequences
Probe, the primer shown in the sequence of 0.3 μM of SEQ ID No.4~11,0.2 μM of 50-100ng CYP21 gene-specific regions
Fragment, 0.5U Tag archaeal dna polymerases.
Wherein, probe sequence is shown in Table 1：
System B is：67mM Tris-HCl (pH 8.8), 16.6mM (NH4)2SO4, 0.01%Tween-20,1.5-3mM
MgCl2, dNTPs, the fluorescence probe shown in 0.3 μM of SEQ ID No.24~SEQ ID No.27 sequences, 0.3 μM of SEQ ID
Primer shown in the sequence of No.12~19,0.2 μM of 50-100ng CYP21 gene-specific regions fragment, 0.5U Tag DNA
Polymerase.
Wherein probe sequence is as shown in table 1：
The probe sequence of table 1
Fusing point (Tm values) of the invention by comparing solubility curve between testing sample and CYP21 gene wild type control product
Difference △ Tm, the catastrophe of judgement sample, △ Tm≤1 DEG C be P30L P30L, I172N, I236N, V281L, C655G,
I2g, Q318X, R356W site wild type, when △ Tm >=4 DEG C, are determined as that the passage is mutated according to corresponding fluorescence detection channel
Type, and then can distinguish and detect the saltant type and wild type in specific site.
The invention provides providing a kind of method of detection CYP21 gene mutations, the present invention at least with following advantage it
One：
1st, reduction false positive (whether reduction false positive is equal to high specificity)：The present invention is using the spy for expanding allele
Specific fragment, reduces the interference of CYP21P pseudogenes, the false positive interference of reduction detection.
2nd, simplicity quick, simple to operate is detected：This patent uses specific alleles real-time fluorescence quantitative PCR, substitutes
Traditional PCR-AFLP (PCR-RFLP), without multiple digestion and polyacrylamide gel,
Detection process only needs to carry out 2 PCR, a regular-PCR, a specific alleles real-time fluorescence quantitative PCR, whole behaviour
Make to complete in 4-6 hour, operating procedure simple and fast.
3rd, detection mutational site is more：The detection method that the present invention is provided can be while detect 8 kinds of mutation classes of CYP21 genes
Type, the covering of upper frequency is reached to OAH mutation in crowd.
4th, testing cost is low：Traditional detection method often carries out the sequencing of two generations, and testing cost is expensive, the side that the present invention is provided
Method also reduces the cost during digestion with restriction enzyme while sequencing cost is reduced.
5th, detection accuracy height, result are easy to read：The present invention is to judge whether mutation by △ Tm averages, as a result
Accurately and reliably and be easy to read.
Brief description of the drawings
Fig. 1 shows the fluorescent PCR amplification curve diagram of the sample 001 of the embodiment of the present invention 3, wherein, abscissa is temperature, ordinate
For fluorescence intensity, curve 1 shows the fluorescence channel detection of 10mM TE buffer solution negative controls；Curve 2 shows that CYP21 genes are wild
The fluorescence channel detection of type reference substance；Curve 3 shows the fluorescence channel detection of 001 sample I2g site mutation types；
Fig. 2 shows the fluorescent PCR amplification curve diagram of the sample 002 of the embodiment of the present invention 3, wherein, abscissa is temperature, ordinate
For fluorescence intensity, curve 1 shows the fluorescence channel detection of 10mM TE buffer solutions negative controls (NTC)；Curve 2 shows CYP21 genes
The fluorescence channel detection of wild type control product；Curve 3 shows the fluorescence channel detection of 002 sample I236N site mutation types；
Fig. 3 shows the fluorescent PCR amplification curve diagram of the sample 003 of the embodiment of the present invention 3, wherein, abscissa is temperature, ordinate
For fluorescence intensity, curve 1 shows the fluorescence channel detection of 10mM TE buffer solutions negative controls (NTC)；Curve 2 shows CYP21 genes
The fluorescence channel detection of wild type control product；Curve 3 shows the fluorescence channel detection of 003 sample Q318X site mutation types；
Fig. 4 shows the fluorescent PCR amplification curve diagram of the sample 004 of the embodiment of the present invention 3, wherein, abscissa is temperature, ordinate
For fluorescence intensity, curve 1 shows the fluorescence channel detection of 10mM TE buffer solutions negative controls (NTC)；Curve 2 shows CYP21 genes
The fluorescence channel detection of wild type control product；Curve 3 shows the fluorescence channel detection of 004 sample C655G site mutation types；
Fig. 5 shows the fluorescent PCR amplification curve diagram of the sample 005 of the embodiment of the present invention 3, wherein, abscissa is temperature, ordinate
For fluorescence intensity, curve 1 shows the fluorescence channel detection of 10mM TE buffer solutions negative controls (NTC)；Curve 2 shows CYP21 genes
The fluorescence channel detection of wild type control product；Curve 3 shows the fluorescence channel detection of 005 sample R356W site mutation types；
Fig. 6 shows the fluorescent PCR amplification curve diagram of the sample 006 of the embodiment of the present invention 3, wherein, abscissa is temperature, ordinate
For fluorescence intensity, curve 1 shows the fluorescence channel detection of 10mM TE buffer solutions negative controls (NTC)；Curve 2 shows CYP21 genes
The fluorescence channel detection of wild type control product；Curve 3 shows the fluorescence channel detection of 006 sample V281L site mutation types；
Fig. 7 shows the fluorescent PCR amplification curve diagram of the sample 007 of the embodiment of the present invention 3, wherein, abscissa is temperature, ordinate
For fluorescence intensity, curve 1 shows the fluorescence channel detection of 10mM TE buffer solutions negative controls (NTC)；Curve 2 shows CYP21 genes
The fluorescence channel detection of wild type control product；Curve 3 shows the fluorescence channel detection of 007 sample P30L site mutation types；
Fig. 8 shows the fluorescent PCR amplification curve diagram of the sample 008 of the embodiment of the present invention 3, wherein, abscissa is temperature, ordinate
For fluorescence intensity, curve 1 shows the fluorescence channel detection of 10mM TE buffer solutions negative controls (NTC)；Curve 2 shows CYP21 genes
The fluorescence channel detection of wild type control product；Curve 3 shows the fluorescence channel detection of 008 sample I172N site mutation types；
Fig. 9 shows the fluorescent PCR amplification curve diagram of the sample 009 of the embodiment of the present invention 3, wherein, abscissa is temperature, ordinate
For fluorescence intensity, curve 1 shows the fluorescence channel detection of 10mM TE buffer solution negative controls；Curve 2 shows that CYP21 genes are wild
The fluorescence channel detection of type reference substance；Curve 3 shows the fluorescence channel detection of 009 sample I2g site mutation types；
Figure 10 shows the fluorescent PCR amplification curve diagram of the sample 010 of the embodiment of the present invention 3, wherein, abscissa is temperature, indulges and sits
Fluorescence intensity is designated as, curve 1 shows the fluorescence channel detection of 10mM TE buffer solutions negative controls (NTC)；Curve 2 shows CYP21 bases
Because the fluorescence channel of wild type control product is detected；Curve 3 shows the fluorescence channel detection of 010 sample I236N site mutation types；
Figure 11 shows the fluorescent PCR amplification curve diagram of the sample 011 of the embodiment of the present invention 3, wherein, abscissa is temperature, indulges and sits
Fluorescence intensity is designated as, curve 1 shows the fluorescence channel detection of 10mM TE buffer solutions negative controls (NTC)；Curve 2 shows CYP21 bases
Because the fluorescence channel of wild type control product is detected；Curve 3 shows the fluorescence channel detection of 011 sample Q318X site mutation types；
Figure 12 shows the fluorescent PCR amplification curve diagram of the sample 012 of the embodiment of the present invention 3, wherein, abscissa is temperature, indulges and sits
Fluorescence intensity is designated as, curve 1 shows the fluorescence channel detection of 10mM TE buffer solutions negative controls (NTC)；Curve 2 shows CYP21 bases
Because the fluorescence channel of wild type control product is detected；Curve 3 shows the fluorescence channel detection of 012 sample C655G site mutation types；
Figure 13 shows the fluorescent PCR amplification curve diagram of the sample 013 of the embodiment of the present invention 3, wherein, abscissa is temperature, indulges and sits
Fluorescence intensity is designated as, curve 1 shows the fluorescence channel detection of 10mM TE buffer solutions negative controls (NTC)；Curve 2 shows CYP21 bases
Because the fluorescence channel of wild type control product is detected；Curve 3 shows the fluorescence channel detection of 013 sample R356W site mutation types；
Figure 14 shows the fluorescent PCR amplification curve diagram of the sample 014 of the embodiment of the present invention 3, wherein, abscissa is temperature, indulges and sits
Fluorescence intensity is designated as, curve 1 shows the fluorescence channel detection of 10mM TE buffer solutions negative controls (NTC)；Curve 2 shows CYP21 bases
Because the fluorescence channel of wild type control product is detected；Curve 3 shows the fluorescence channel detection of 014 sample V281L site mutation types；
Figure 15 shows the fluorescent PCR amplification curve diagram of the sample 015 of the embodiment of the present invention 3, wherein, abscissa is temperature, indulges and sits
Fluorescence intensity is designated as, curve 1 shows the fluorescence channel detection of 10mM TE buffer solutions negative controls (NTC)；Curve 2 shows CYP21 bases
Because the fluorescence channel of wild type control product is detected；Curve 3 shows the fluorescence channel detection of 015 sample P30L sites wild type；
Figure 16 shows the fluorescent PCR amplification curve diagram of the sample 016 of the embodiment of the present invention 3, wherein, abscissa is temperature, indulges and sits
Fluorescence intensity is designated as, curve 1 shows the fluorescence channel detection of 10mMTE buffer solutions negative controls (NTC)；Curve 2 shows CYP21 bases
Because the fluorescence channel of wild type control product is detected；Curve 3 shows the fluorescence channel detection of 016 sample I172N sites wild type.
Embodiment
The invention discloses a kind of kit of detection CYP21 gene mutations, those skilled in the art can use for reference herein
Content, is suitably modified technological parameter realization.In particular, all similar replacements and change are to people in the art
It is it will be apparent that they are considered as being included in the present invention for member.The method of the present invention and application are by preferably real
Apply example to be described, related personnel substantially can be not departing from present invention, in spirit and scope to method described herein
It is modified with application or suitably change is with combining, realizes and apply the technology of the present invention.
To the explanation of the disclosed embodiments, professional and technical personnel in the field are enable to realize or using the present invention.To this
A variety of modifications of a little embodiments will be apparent for those skilled in the art, as defined herein general
Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, the present invention will not
It can be intended to be limited to the embodiments shown herein, and be to fit to consistent with principles disclosed herein and features of novelty
Most wide scope.
With reference to embodiment, the present invention is expanded on further：
The preparation of the reference substance of embodiment 1
(1) preparation of CYP21 genes wild type control product
Using normal person DNA as template, with SEQ ID NO:Sequence shown in 1-2 is primer, and amplification obtains sequence such as SEQ ID
NO:Product shown in 3.On the purified rear clone of gained PCR primer to pMD18-T carriers and through sequencing identification, by sequence verification
Plasmid be transferred to escherichia coli cloning and obtain substantial amounts of recombinant plasmid, dilute recombinant plasmid, as CYP21 genes wild type control
Product.
(2) preparation of CYP21 gene masculines reference substance
Using recombinant plasmid made from embodiment 1 as template, with Stratagene many site-directed mutagenesis kits, to CYP21
It is prominent that 8 mutational sites P30L, I172N, I236N, V281L, C655G, I2g, Q318X, R356W of gene carry out fixed point respectively
Become, obtain 8 mutant plasmids, as saltant type positive reference substance, be respectively：P30L site mutation type positive controls product,
I172N site mutation type positive controls product, I236N site mutation type positive controls product, the V281L site mutation type positives are right
According to a group product, C655G site mutation type positive controls product, I2g site mutation type positive controls product, Q318X, site mutation type
Positive controls product, R356W site mutation type positive controls product.
(3) negative controls：Using 10mM TE buffer solutions as negative control, addition is identical with sample to be tested amount, is 5
μL。
Detection of the kit that the present invention of embodiment 2 is provided to CYP21 gene mutations
Using congenital adrenal cortical hyper plasia patient DNA to be measured as template, with SEQ ID NO:Sequence shown in 1-2 is to draw
Thing, amplification obtains CYP21 gene-specific regions fragment.
Detection method：
(1) reaction system：
System A：Take 19 μ L PCRMix A, 1 μ LTag archaeal dna polymerases, by sample to be tested CYP21 gene-specific regions piece
Section, 10mM TE buffer solutions, CYP21 gene wild type control product and saltant type positive controls product (P30L, I172N, I236N
With the mixture of V281L site mutation type positive controls product) 5 μ L are taken respectively, it is separately added into each PCR pipe, covers lid, instead
It is 25 μ L to answer system.
PCRMix A solution system is：67mM Tris-HCl (pH 8.8), 16.6mM (NH4)2SO4, 0.01%
Tween-20,3mM MgCl2(a specific concentration please be determine), 0.2mM dNTPs, 0.3 μM of SEQ ID No.20~SEQ
Probe shown in ID No.23 sequences, the primer shown in the sequence of 0.3 μM of SEQ ID No.4~11.
System B：19 μ L PCRMixB is taken, PCRMix B solution system is 67mM Tris-HCl (pH 8.8),
16.6mM(NH4)2SO4, 0.01%Tween-20,3mM MgCl2, 0.2mM dNTPs, 0.3 μM of SEQ ID No.24~SEQ
Probe shown in ID No.27 sequences, the primer shown in the sequence of 0.3 μM of SEQ ID No.12~19 is poly- with 1 μ L Tag DNA
Synthase mixing obtains blended liquid, and sample to be tested CYP21 gene-specific regions fragment, 10mM TE buffer solutions, CYP21 genes is wild
Raw type reference substance and saltant type positive controls product (C655G, I2g, Q318X and R356W site mutation type positive controls product
Mixture) be separately added into each PCR pipe, cover lid, reaction system is 25 μ L.
(2) PCR is expanded：Amplification program is：The first step：95℃3min
Second step：94 DEG C of 15s → 65 DEG C~45 DEG C 15s → 75 DEG C 15s, 15 circulations；
3rd step：94 DEG C of 15s → 55 DEG C 15s → 75 DEG C 15s, 40 circulations, the fluorescence of annealing stage is gathered at 55 DEG C
Signal；
4th step：94 DEG C of 1min → 35 DEG C 3min → 40 DEG C~85 DEG C, are dissolved with 0.4 DEG C/5s gradient increased temperature
The analysis of curve, and collect the fluorescence signal in this stage.
(3) fluoroscopic examination：PCR baseline to exceed the fluorescence curve of negative control just during fluoroscopic examination, if negative right
Peak of breaking forth is more early, then manually adjusts baseline and enter logarithmic phase position to big portion's sample fluorescence curve, read Tm values.
(4) interpretation of result：Result of the 10mM TE buffer solutions in two detection architectures is feminine gender, reads CYP21 genes
Wild type control product, saltant type positive controls product calculate CYP21 genes wild respectively in the Tm values of different fluorescence detection channels
The poor △ Tm of the Tm values and the Tm values of saltant type positive controls product in each channel of raw type reference substance in each channel, institute
The relation in △ Tm and mutational site is obtained, as shown in table 2：
The △ Tm of table 2 and the relation in mutational site
CYP21 gene wild type controls product, sample to be tested CYP21 gene-specific region fragments is read to examine in different fluorescence
The Tm values of passage are surveyed, the Tm values of CYP21 gene wild type controls product in each channel are calculated respectively with sample to be tested at each
The difference △ Tm of Tm values in passage：
Wherein, for the detection in P30L sites：△ Tm value≤1 DEG C, is P30L sites wild type；4.5 DEG C≤△ Tm values≤
6.1 DEG C, be P30L site mutation types；
For the detection in I172N sites：△ Tm value≤1 DEG C, is I172N sites wild type；6.78 DEG C≤△ Tm values≤
8.42 DEG C, be I172N site mutation types；
For the detection in I236N sites：△ Tm value≤1 DEG C, is I236N sites wild type；5.7 DEG C≤△ Tm value≤7.5
DEG C, it is I236N site mutation types；
For the detection in V281L sites：△ Tm≤1 DEG C, is V281L sites wild type；7.42 DEG C≤△ Tm value≤8.58
DEG C, it is that V281L site mutation type results are compareed as wild-type positive, through fluoroscopic examination
For the detection in C655G sites：△ Tm value≤1 DEG C, is C655G sites wild type；4.02 DEG C≤△ Tm values≤
5.38 DEG C, be C655G site mutation types；
For the detection in I2g sites：△ Tm value≤1 DEG C, is I2g sites wild type；6.09 DEG C≤△ Tm value≤7.51 DEG C,
For I2g site mutation types；
For the detection in Q318X sites：△ Tm value≤1 DEG C, is Q318X sites wild type；5.3 DEG C≤△ Tm value≤6.9
DEG C, it is Q318X site mutation types；
For the detection in R356W sites：△ Tm value≤1 DEG C, is R356W sites wild type；3.29 DEG C≤△ Tm values≤
5.11 DEG C, be R356W site mutation types.
16 congenital adrenal cortical hyper plasia clinical samples are detected using above-mentioned detection method, sample it is glimmering
Light PCR figures are shown in Fig. 1~16, and experimental result is shown in Table 3, while carrying out gene sequencing to sample to be tested.
The testing result of table 3
| Catalogue number(Cat.No.) | Testing result of the present invention | Sequencing result |
| 001 | I2g site mutations | I2g site mutations |
| 002 | I236N site mutations | I236N site mutations |
| 003 | Q318X site mutations | Q318X site mutations |
| 004 | C655G site mutations | C655G site mutations |
| 005 | R356W site mutations | R356W site mutations |
| 006 | V281L site mutations | V281L site mutations |
| 007 | P30L site mutations | P30L site mutations |
| 008 | I172N site mutations | I172N site mutations |
| 009 | I2g sites are without mutation | I2g sites are without mutation |
| 010 | I236N sites are without mutation | I236N sites are without mutation |
| 011 | Q318X sites are without mutation | Q318X sites are without mutation |
| 012 | C655G sites are without mutation | C655G sites are without mutation |
| 013 | R356W sites are without mutation | R356W sites are without mutation |
| 014 | V281L sites are without mutation | V281L sites are without mutation |
| 015 | P30L sites are without mutation | P30L sites are without mutation |
| 016 | I172N sites are without mutation | I172N sites are without mutation |
As seen from the results in Table 3, the primer sets and the testing result of kit that the present invention is provided are consistent with sequencing result, accurately
Property is up to 100%.Therefore, the kit accuracy height of the invention provided, feasibility are good.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
SEQUENCE LISTING
<110>Yunnan Ren Qiao medical science and technologies Co., Ltd
<120>A kind of kit of detection CYP21 gene mutations
<130> MP1702498
<160> 27
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
gtcggtggga gggtacctga a 21
<210> 2
<211> 25
<212> DNA
<213>Artificial sequence
<400> 2
aattaagcct caatcctctg cagcg 25
<210> 3
<211> 3297
<212> DNA
<213>Artificial sequence
<400> 3
tcggtgggag ggtacctgaa ggtggggtca agggaggccc caaaacagtc tacacagcag 60
gagggatggc tggggctctt gagctataag tggcacctca gggccctgac gggcgtctcg 120
ccatgctgct cctgggcctg ctgctgctgc tgcccctgct ggctggcgcc cgcctgctgt 180
ggaactggtg gaagctccgg agcctccacc tcccgcccct tgccccgggc ttcttgcact 240
tgctgcagcc cgacctcccc atctatctgc ttggcctgac tcagaaattc gggcccatct 300
acaggctcca ccttgggctg caaggtgaga ggctgatctc gctctggccc tcaccatagg 360
agggggcgga ggtgacggag agggtcctct ctccgctgac gctgctttgg ctgtctccca 420
gatgtggtgg tgctgaactc caagaggacc attgaggaag ccatggtcaa aaagtgggca 480
gactttgctg gcagacctga gccacttacc tgtaagggct gggggcattt tttctttctt 540
aaaaaaattt ttttttaaga gatgggttct tgctatgttg cccaggctgg tcttaaattc 600
ctagtctcaa atgatcctcc cacctcagcc tcaagtgtga gccacctttg gggcatcccc 660
aatccaggtc cctggaagct cttggggggg catatctggt ggggagaaag caggggttgg 720
ggaggcggaa gaaggtcagg ccctcagctg ccttcatcag ttcccaccct ccagccccca 780
actcctcctg cagacaagct ggtgtctagg aactacccgg acctgtcctt gggagactac 840
tccctgctct ggaaagccca caagaagctc acccgctcag ccctgctgct gggcatccgt 900
gactccatgg agccagtggt ggagcagctg acccaggagt tctgtgaggt aaggctgggc 960
tcctgaggcc acctcgggtc agccttgcct ctcacagtag cccccgccct gcccgctgca 1020
cagcggcctg ctgaactcac actgtttctc cacagcgcat gagagcccag cccggcaccc 1080
ctgtggccat tgaggaggaa ttctctctcc tcacctgcag catcatctgt tacctcacct 1140
tcggagacaa gatcaaggtg cctcacagcc cctcaggccc acccccagcc cctccctgag 1200
cctctccttg tcctgaactg aaagtactcc ctccttttct ggcaggacga caacttaatg 1260
cctgcctatt acaaatgtat ccaggaggtg ttaaaaacct ggagccactg gtccaccaaa 1320
ttgtggacgt gattcccttt ctcagggtga ggacctggag cctagacacc cctgggttgt 1380
aggggagagg ctggggtgga gggagaggct ccttcccaca gctgcattct catgcttcct 1440
gccgcagttc ttccccaatc caggtctccg gaggctgaag caggccatag agaagaggga 1500
tcacatcgtg gagatgcagc tgaggcagca caaggtgggg actgtgtgtg gacggcctcc 1560
cctcggccca cagccagtga tgctaccggc ctcagcattg ctatgaggcg ggttcttttg 1620
cataccccag ttatgggcct gttgccactc tgtactcctc tccccaggcc agccgctcag 1680
cccgctcctt tcaccctctg caggagagcc tcgtggcagg ccagtggagg gacatgatgg 1740
actacatgct ccaaggggtg gcgcagccga ccatggaaga gggctctgga cagctcctgg 1800
aagggcacgt gcacatggct gcagtggacc tcctgatcgg tggcactgag accacagcaa 1860
acaccctctc ctgggccgtg gtttttttgc ttcaccaccc tgaggtgcgt cctggggaca 1920
agcaaaaggc tccttcccag caacctggcc agggcggtgg gcaccctcac tcagctctga 1980
gcactgtgcg gctggggctg tgcttgcctc accggcactc aggctcactg ggttgctgag 2040
ggagcggctg gaggctgggc agctgtgggc tgctggggca ggactccacc cgatcattcc 2100
ccagattcag cagcgactgt aggaggagct agaccacgaa ctgggccctg gtgcctccag 2160
ctcccgggtc ccctacaagg accgtgcacg gctgcccttg ctcaatgcca ccatcgccga 2220
ggtgctgcgc ctgcggcccg ttgtgccctt agccttgccc caccgcacca cacggcccag 2280
caggtgactc ccgagggttg gggatgagtg aggaaagccc gagcccaggg aggtcctggc 2340
cagcctctaa ctccagcccc cttcagcatc tccggctacg acatccctga gggcacagtc 2400
atcattccga acctccaagg cgcccacctg gatgagacgg tctgggagag gccacatgag 2460
ttctggcctg gtatgtgggg ggccgggggc ctgccgtgaa aatgtggtgg aggctggtcc 2520
ccgctgccgc tgaacgcctc cccacccacc tgtccacccg cccgcagatc gcttcctgga 2580
gccaggcaag aactccagag ctctggcctt cggctgcggt gcccgcgtgt gcctgggcga 2640
gccgctggcg cgcctggagc tcttcgtggt gctgacccga ctgctgcagg ccttcacgct 2700
gctgccctcc ggggacgccc tgccctccct gcagcccctg ccccactgca gtgtcatcct 2760
caagatgcag cctttccaag tgcggctgca gccccggggg atgggggccc acagcccggg 2820
ccagagccag tgatggggca ggaccgatgc cagccgggta cctcagtttc tcctttattg 2880
ctcccgtacg aacccctccc ctcccccctg taaacacagt gctgcgagat cgctggcaga 2940
gaaggcttcc tccagcggct gggtggtgaa ggaccctggc tcttctctcg gggcgacccc 3000
tcagtgctcg gcagtcatac tggggtgcga gagaggtggg cagcagctca gcctcccccc 3060
gctggggagc gaaagtttct tggtctcagc ttcatttccg tgaagggcac cgagaactcg 3120
aagcccttcc agtggtacca gctcactccc tgggaaaggg gttgtcaaga gagagtcaaa 3180
gccggatgtc ccatctgctc ttcccgttcc ccttaaggag gtagctccca gcactcaacc 3240
aacctccccg cagagctccc ttcctgaccc tccgctgcag aggattgagg cttaatt 3297
<210> 4
<211> 16
<212> DNA
<213>Artificial sequence
<400> 4
cggagcctcc acctgc 16
<210> 5
<211> 22
<212> DNA
<213>Artificial sequence
<400> 5
ccgaatttct gagtcaggcc aa 22
<210> 6
<211> 21
<212> DNA
<213>Artificial sequence
<400> 6
tcctcacctg cagcagcatg a 21
<210> 7
<211> 26
<212> DNA
<213>Artificial sequence
<400> 7
tgtactttca gttcaggaca aggaga 26
<210> 8
<211> 21
<212> DNA
<213>Artificial sequence
<400> 8
agctgcatct ccacgatgtg a 21
<210> 9
<211> 23
<212> DNA
<213>Artificial sequence
<400> 9
agctgcattc tcatgcttcc tgc 23
<210> 10
<211> 19
<212> DNA
<213>Artificial sequence
<400> 10
tccactgcag ccatgtgga 19
<210> 11
<211> 24
<212> DNA
<213>Artificial sequence
<400> 11
cagtggaggg acatgatgga ctac 24
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence
<400> 12
ttcgtggtct agctcctcgt 20
<210> 13
<211> 21
<212> DNA
<213>Artificial sequence
<400> 13
actcaggctc actgggttgc t 21
<210> 14
<211> 20
<212> DNA
<213>Artificial sequence
<400> 14
cagcttgtct gcaggaggtg 20
<210> 15
<211> 22
<212> DNA
<213>Artificial sequence
<400> 15
aatccaggtc cctggaagct ct 22
<210> 16
<211> 19
<212> DNA
<213>Artificial sequence
<400> 16
cttccagtgc aggggacca 19
<210> 17
<211> 22
<212> DNA
<213>Artificial sequence
<400> 17
aatccaggtc cctggaagct ct 22
<210> 18
<211> 16
<212> DNA
<213>Artificial sequence
<400> 18
taagggcaca acgggc 16
<210> 19
<211> 23
<212> DNA
<213>Artificial sequence
<400> 19
caggaggagc tagaccacga act 23
<210> 20
<211> 21
<212> DNA
<213>Artificial sequence
<400> 20
agattgggag gtcgggctgc a 21
<210> 21
<211> 24
<212> DNA
<213>Artificial sequence
<400> 21
tcaaggtgcc tcacagcccc tcag 24
<210> 22
<211> 20
<212> DNA
<213>Artificial sequence
<400> 22
ccggaggctg aagcaggcca 20
<210> 23
<211> 19
<212> DNA
<213>Artificial sequence
<400> 23
tccatgctcg gctgcgcca 19
<210> 24
<211> 20
<212> DNA
<213>Artificial sequence
<400> 24
tgatcgggtg gagtcctgcc 20
<210> 25
<211> 23
<212> DNA
<213>Artificial sequence
<400> 25
tcatcagttc ccaccctcca gcc 23
<210> 26
<211> 23
<212> DNA
<213>Artificial sequence
<400> 26
tcatcagttc ccaccctcca gcc 23
<210> 27
<211> 23
<212> DNA
<213>Artificial sequence
<400> 27
tgcacggtcc ttgtagggga ccc 23
Claims (8)
1. a kind of kit of detection CYP21 gene mutations, it is characterised in that including primer sets and probe groups；
It is the primer shown in SEQ ID No.4~19 that the primer sets, which include sequence,；It is SEQ ID that the probe groups, which include sequence,
Probe shown in No.20~SEQ ID No.27.
2. kit according to claim 1, it is characterised in that the sequence is SEQ ID No.20~SEQ ID
5 ' ends of the probe shown in No.23 connect different fluorophors, the quencher of 3 ' end connection non-luminescent respectively.
3. kit according to claim 2, it is characterised in that the fluorophor is in FAM, ROX, HEX, CY5
One kind.
4. kit according to claim 1, it is characterised in that the sequence is SEQ ID No.24~SEQ ID
5 ' ends of the probe shown in No.27 connect different fluorophors, the quencher of 3 ' end connection non-luminescent respectively.
5. kit according to claim 4, it is characterised in that the fluorophor is in FAM, ROX, HEX, CY5
One kind.
6. the kit according to any one of Claims 1 to 5, it is characterised in that also including dNTP, Tag archaeal dna polymerase,
PH8.8 Tris-HCl, (NH4)2SO4, 0.01%Tween-20 and MgCl2。
7. the kit according to any one of claim 1~6, it is characterised in that the amplification program of the kit is as follows：
The first step：95℃3min
Second step：94 DEG C of 15s → 65 DEG C~45 DEG C 15s → 75 DEG C 15s, 15 circulations；
3rd step：94 DEG C of 15s → 55 DEG C 15s → 75 DEG C 15s, 40 circulations, the fluorescence signal of annealing stage is gathered at 55 DEG C；
4th step：94 DEG C of 1min → 35 DEG C 3min → 40 DEG C~85 DEG C, solubility curve is carried out with 0.4 DEG C/5s gradient increased temperature
Analysis, and collect the fluorescence signal in this stage.
8. the kit described in any one of claim 1 to 7 is preparing auxiliary diagnosis or prevention congenital adrenal cortical hyper plasia
Application in disease reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710266079.6A CN107090499A (en) | 2017-04-21 | 2017-04-21 | A kind of kit of detection CYP21 gene mutations |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710266079.6A CN107090499A (en) | 2017-04-21 | 2017-04-21 | A kind of kit of detection CYP21 gene mutations |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107090499A true CN107090499A (en) | 2017-08-25 |
Family
ID=59637039
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710266079.6A Pending CN107090499A (en) | 2017-04-21 | 2017-04-21 | A kind of kit of detection CYP21 gene mutations |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107090499A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154862A (en) * | 2020-01-17 | 2020-05-15 | 深圳会众生物技术有限公司 | Primer, probe composition, kit and method for detecting ABCB1 and LDLR gene polymorphism |
| CN111471761A (en) * | 2020-05-27 | 2020-07-31 | 东莞博奥木华基因科技有限公司 | Primer and kit for detecting CYP21 gene mutation and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102747159A (en) * | 2012-07-17 | 2012-10-24 | 广州达健生物科技有限公司 | B-raf mutation genotyping fluorescent quantitative PCR (polymerase chain reaction) kit and detection method |
| CN105274190A (en) * | 2014-07-22 | 2016-01-27 | 复旦大学附属华山医院 | HRM method for detecting genetic polymorphism of CYP3A4*1G and MDR1C1236T |
| CN105316401A (en) * | 2015-01-23 | 2016-02-10 | 复旦大学附属华山医院 | Method for measuring ABCC2 gene polymorphism |
-
2017
- 2017-04-21 CN CN201710266079.6A patent/CN107090499A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102747159A (en) * | 2012-07-17 | 2012-10-24 | 广州达健生物科技有限公司 | B-raf mutation genotyping fluorescent quantitative PCR (polymerase chain reaction) kit and detection method |
| CN105274190A (en) * | 2014-07-22 | 2016-01-27 | 复旦大学附属华山医院 | HRM method for detecting genetic polymorphism of CYP3A4*1G and MDR1C1236T |
| CN105316401A (en) * | 2015-01-23 | 2016-02-10 | 复旦大学附属华山医院 | Method for measuring ABCC2 gene polymorphism |
Non-Patent Citations (1)
| Title |
|---|
| YI-CHING LIN等: "High-resolution melting curve (HRM) analysis to establish CYP21A2 mutations converted from the CYP21A1P in congenital adrenal hyperplasia", 《CLINICA CHIMICA ACTA》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154862A (en) * | 2020-01-17 | 2020-05-15 | 深圳会众生物技术有限公司 | Primer, probe composition, kit and method for detecting ABCB1 and LDLR gene polymorphism |
| CN111471761A (en) * | 2020-05-27 | 2020-07-31 | 东莞博奥木华基因科技有限公司 | Primer and kit for detecting CYP21 gene mutation and application thereof |
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Application publication date: 20170825 |