CN101597638A - A kind of method and dedicated kit thereof that detects gene multi-mutant site - Google Patents
A kind of method and dedicated kit thereof that detects gene multi-mutant site Download PDFInfo
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Abstract
The invention discloses a kind of method and dedicated kit thereof that detects gene multi-mutant site.The method of described detection gene multi-mutant site is the method for A → G of 1555 of detection line plastochondria 12S rRNA gene and/or 1494 C → T sudden change, and the primer is 3, and primer sequence is respectively sequence 1 in the sequence table, sequence 2 or sequence 3; Carry out pcr amplification with described 3 primers, detect the melting curve of pcr amplification product, as melting temperature (Tm) to occur be 79.4 fusion peak that then 1555 of human mitochondrion 12S rRNA gene exist point mutation, mutation type is A → G; As melting temperature (Tm) to occur be 76.6 fusion peak, and then 1494 of human mitochondrion 12S rRNA gene exist point mutation, and mutation type is C → T.The present invention also discloses a kind of dedicated kit that detects gene multi-mutant site simultaneously.
Description
Technical field
The present invention relates to a kind of method and dedicated kit thereof that detects gene multi-mutant site.
Background technology
Base mutation is one type very important in the human genome sudden change, a lot of base mutations all with the confidential relation that has of disease, therefore detect some site mutation information, the clinical diagnosis and the guiding clinical treatment of disease had great significance.
Aminoglycosides antibiotics comprises gentamicin, Streptomycin sulphate, kantlex, tobramycin etc., and is effective especially because of its infection to gram-negative bacterium, for some gram positive bacterium synergetic antibacterial effect arranged also simultaneously.Therefore clinical treatment is widely used.But such medicine has serious ototoxicity side effect, may cause the irreversible damage of hearing during use.
Plastosome 12SrRNA gene is a mutantional hotspot zone of the non-syndrome type hearing loss that causes of aminoglycosides antibiotics.In these mutational sites, the homogeneous A1555G that is positioned at the area decoder of 12SrRNA gene high conservative suddenlys change with deaf relevant with C1494T, and these two sudden changes cause a lot of patients' aminoglycosides antibiotics toxic deafness.A1555G sudden change and C1494T sudden change meeting form new 1494C-G1555 or 1494U-A1555 base pair in the A position of the high conservative of 12SrRNA gene.These changes make 12SrRNA more similar to the secondary structure of the respective regions of the 16srRNA of bacterium on secondary structure.Therefore, because C1494T and A1555G sudden change forms G-C and the U-A pairing makes the combination of aminoglycosides antibiotics be more prone at 12SrRNA, deaf reason can appear or increase the weight of in the people why Here it is carries these sudden changes when having contacted aminoglycosides antibiotics.Therefore to the detection of these two point mutation, the important clinical meaning is arranged for prevention aminoglycosides antibiotics toxic deafness.
The at present disclosed round pcr that has only the relevant single mutational site of the deafness of detecting, urgent need can detect the PCR detection method in the relevant mutational site of two deafnesses simultaneously.
Summary of the invention
The purpose of this invention is to provide a kind of method and dedicated kit thereof that detects gene multi-mutant site.
The method of detection gene multi-mutant site provided by the present invention: may further comprise the steps:
1) it is right to design corresponding mutant primer respectively at two or more mutational sites of gene to be detected, and the purpose fragment that pcr amplification is obtained satisfies following two conditions:
A) include only a mutational site;
B) the segmental Tm value of two or more purposes that obtains of pcr amplification all differs more than 2 ℃;
The mutant base in the forward primer that described mutant primer is right or 3 ' terminal bases of reverse primer and mutational site to be amplified is mated fully, mates fully with the wild-type base in mutational site to be detected;
2) with the primer of step 1) to carrying out pcr amplification, detect the melting curve of pcr amplification product, according to the generation that judges whether sudden change that has or not at fusion peak.
Wherein, described pcr amplification is that fluorescent quantitative PCR or regular-PCR amplification add fluorescence dye simultaneously or regular-PCR amplification back adds fluorescence dye; Also can introduce the artificial mutation base in the described primer; Also can comprise a pair of interior label primer in the described method.
When the method for described detection gene multi-mutant site is the method for A → G of 1555 of detection line plastochondria 12S rRNA gene and/or C → T sudden change of 1494, the primer of described step 1) is 3 primers, and primer sequence is respectively sequence 1 in the sequence table, sequence 2 or sequence 3; Described step 2) for carrying out pcr amplification with 3 primers of step 1), the melting curve of detection pcr amplification product, as melting temperature (Tm) to occur be 79.4 fusion peak, and then 1555 of human mitochondrion 12S rRNA gene exist point mutation, and mutation type is A → G; As melting temperature (Tm) to occur be 76.6 fusion peak, and then 1494 of human mitochondrion 12S rRNA gene exist point mutation, and mutation type is C → T.
Wherein, the sequence of described a pair of Quality Control primer is respectively sequence 4 and sequence 5 in the sequence table.
Another object of the present invention provides a kind of dedicated kit that detects gene multi-mutant site.
When described detection gene multi-mutant site was detection of drugs induced deafness related locus A1555G and C1494T sudden change, described dedicated kit can include as the lower section:
(1) reagent of extraction blood sample DNA;
(2) PCR reaction reagent comprises dNTP, 10 * PCR damping fluid, Mg2
+, tri-distilled water and Taq enzyme;
(3) primer;
(4) positive sample contrast and the contrast of negative sample;
(5) specification sheets.
Described primer comprises following 3 primers, and their sequence is respectively sequence 1 in the sequence table, sequence 2 and sequence 3.
Wherein, also can comprise a pair of Quality Control primer in the primer of described dedicated kit, the sequence of this Quality Control primer is respectively sequence 4 and the sequence 5 in the sequence table.
The method that the present invention adopts is to design corresponding primer at each mutational site, each mutational site primer 3 ' end terminal bases and mutant base are mated fully, and with the wild-type base mispairing, also introduce other mutating alkali yl in the primer, to increase the specificity of reaction.Whether design a pair of interior label primer in the system in addition, the Tm value in the reaction between the PCR product of every pair of primer all differs more than 2 ℃, behind the fluorescence dye pcr amplification, carries out the melting curve analysis, by having the appearance at fusion peak to judge whether that sudden change takes place.The present invention realizes the detection of multisite mutation by the difference of design PCR product Tm value, instrument requires simple, expense is low, simple to operate, need not that enzyme is cut or electrophoresis, the generation of polluting is effectively avoided in the whole process operation that need not to uncap except that adding template simultaneously, detects when can realize two or more point mutation simply, fast and accurately or detection separately.
Description of drawings
Fig. 1 is for 12s RNA-1555G genome and 12s RNA-1494T genome being the PCR detection of template
Fig. 2 is for 12s RNA-1555G genome being the PCR detection of template
Fig. 3 is for 12s RNA-1494T genome being the PCR detection of template
Fig. 4 is for wild type gene group or aseptic double-distilled water being the PCR detection of template
Embodiment
Be example with a kind of method that detects A → G of 1555 of human mitochondrion 12S rRNA gene and/or 1494 C → T sudden change below, illustrate technical scheme of the present invention.This detects 1555 the A → G of human mitochondrion 12S rRNA gene and/or the method for C → T sudden change of 1494, be that genomic dna with the people is a template, carry out pcr amplification with 3 primers, detect the melting curve of pcr amplification product, as melting temperature (Tm) to occur be 79.4 fusion peak, then 1555 of human mitochondrion 12S rRNA gene exist point mutation, and mutation type is A → G; As melting temperature (Tm) to occur be 76.6 fusion peak, and then 1494 of human mitochondrion 12S rRNA gene exist point mutation, and mutation type is C → T.
The detection of embodiment 1, human mitochondrion 12S rRNA gene multi-mutant site
1, design of primers:
Adopt primer 5.0 softwares at 1555 and 1494 mutational sites design primer, catastrophe point is positioned at 3 of primer ' end, both shared downstream primers.The amplified production Tm value of design 1555 is 77.1 ℃, and the Tm value of 1494 amplified production is 80.1 ℃.
Be the specificity of intensified response, introduce the artificial mutation base in 3 of 1555 and 1494 primers ' end the 3rd base reciprocal.Primer sequence is as follows:
1555 site primer: 5 ' CCCTACGCATTTATATAGAGGCGG 3 ' (sequence 1)
1494 site primer: 5 ' CACACCGCCCGTCTCT 3 ' (sequence 2)
Common downstream primer: 5 ' GCACTTTCCAGTACACTTACCA 3 ' (sequence 3)
At one section conservative nucleic acid segment on the plastosome, the design interior label primer, the Tm value that designs its amplified production is 88.9 ℃.
Interior label primer F:5 ' ACATTACAGTCAAATCCCTTCTC 3 ' (sequence 4)
Interior label primer R:5 ' AGCTACCCCCAAGTGTTATG 3 ' (sequence 5)
Pcr amplification is template used to provide 1555,1494 mutants and wild type gene group for 301 Hospital.
Reaction system is as follows: 2 * Real time EvaGreen buffer (Bo Ao company), 10 μ l, Taq enzyme 0.2 μ l, 1555 site primers (10uM), 0.6 μ l, 1494 site primers (10uM), 0.4 μ l, common downstream primer (10uM) 0.8 μ l, interior label primer F (10uM) 0.4 μ l, interior label primer R (10uM) 0.4 μ l, EvaGreen 0.6 μ l, template 2 μ l, H
2O 4.6 μ l.
Wherein, the template concentrations of each PCR reaction is as follows: in first PCR reaction, the template concentrations of 12s RNA-1555G, 12s RNA-1494T is respectively 0.1ng and 0.1ng; In second PCR reaction, the template concentrations of 12sRNA-1555G is 0.1ng; In the 3rd the PCR reaction, the template concentrations of 12s RNA-1494T is 0.1ng; In the 4th the PCR reaction, the concentration template of 12s RNA is 1ng.
Being reflected at ABI 7700 fluorescent PCR previous steps carries out.
Response procedures is as follows:
At first carry out amplification procedure, amplification procedure is as follows: 94 ℃ of 15min; 94 ℃ of 15sec then, 54 ℃ of 20sec, 72 ℃ of 20sec, 40 circulations; Carry out the melting curve analysis then, melting curve is analyzed as follows: 94 ℃ of 10sec, 72 ℃ of 10sec, heat-up rate are 0.1 ℃/s, 94 ℃ of 10sec.
Experimental result shows, though the theory T m value that software provides and actual Tm value difference to some extent, difference and the expection between the Tm value of each product of PCR matches (＞2 ℃), and each fuses the peak and can well distinguish to each other.Above-mentioned four PCR reaction all amplifies specific product.Occurred melting temperature (Tm) in first PCR reaction and be 79.4,76.6 and 85.1 fusion peak, be respectively 1555 and be the amplified production of G, 1494 amplified productions (Fig. 1) for the amplified production of T and Quality Control primer; Having occurred melting temperature (Tm) in second PCR reaction and be 79.4 and 85.1 fusion peak, is 1555 amplified productions (Fig. 2) for the amplified production of G and Quality Control primer; Having occurred melting temperature (Tm) in the 3rd PCR reaction and be 76.6 and 85.1 fusion peak, is 1494 amplified productions (Fig. 3) for the amplified production of T and Quality Control primer; Only occurred melting temperature (Tm) in the 4th PCR reaction and be 85.1 fusion peak, be the amplified production of Quality Control primer, NTC any fusion peak do not occur for being template with the aseptic double-distilled water.(Fig. 4).
Sequence table
＜110〉Boao Biotech Co., Ltd.
＜120〉a kind of method and dedicated kit thereof that detects gene multi-mutant site
<130>CGGNARW81372
<160>5
<210>1
<211>24
<212>DNA
＜213〉synthetic
<220>
<223>
<400>1
ccctacgcat?ttatatagag?gcgg 24
<210>2
<211>16
<212>DNA
＜213〉synthetic
<220>
<223>
<400>2
cacaccgccc?gtctct 16
<210>3
<211>22
<212>DNA
＜213〉synthetic
<220>
<223>
<400>3
gcactttcca?gtacacttac?ca 22
<210>4
<211>23
<212>DNA
＜213〉synthetic
<220>
<223>
<400>4
acattacagt?caaatccctt?ctc 23
<210>5
<211>20
<212>DNA
＜213〉synthetic
<220>
<223>
<400>5
agctaccccc?aagtgttatg 20
Claims (8)
1, a kind of method that detects gene multi-mutant site, it is characterized in that: may further comprise the steps: it is right 1) to design corresponding mutant primer respectively at two or more mutational sites of gene to be detected, the purpose fragment that pcr amplification is obtained satisfies following two conditions: a) include only a mutational site, b) the segmental Tm value of two or more purposes that obtains of pcr amplification all differs more than 2 ℃; The mutant base in the forward primer that described mutant primer is right or 3 ' terminal bases of reverse primer and mutational site to be amplified is mated fully, mates fully with the wild-type base in mutational site to be detected;
2) with the primer of step 1) to carrying out pcr amplification, detect the melting curve of pcr amplification product, according to the generation that judges whether sudden change that has or not at fusion peak.
2, method according to claim 1 is characterized in that: described pcr amplification is that fluorescent quantitative PCR or regular-PCR amplification add fluorescence dye simultaneously or regular-PCR amplification back adds fluorescence dye.
3, method according to claim 1 is characterized in that: also introduce the artificial mutation base in the described mutant primer.
4, according to claim 1,2 or 3 described methods, it is characterized in that: also comprise a pair of Quality Control primer in the described method.
5, method according to claim 4, it is characterized in that: the method for described detection gene multi-mutant site is the method for A → G of 1555 of detection line plastochondria 12S rRNA gene and/or 1494 C → T sudden change, the primer of described step 1) is 3 primers, and primer sequence is sequence 1 in the sequence table, sequence 2 and sequence 3; Described step 2) for carrying out pcr amplification with 3 primers of step 1), the melting curve of detection pcr amplification product, as occur 1555 and be the fusion peak of G, then 1555 of human mitochondrion 12S rRNA gene exist point mutation; As occur 1494 and be the fusion peak of T, then 1494 of human mitochondrion 12S rRNA gene exist point mutation.
6, method according to claim 5 is characterized in that: the sequence of described a pair of Quality Control primer is respectively sequence 4 and sequence 5 in the sequence table.
7, the test kit of the deaf related locus sudden change of a kind of detection of drugs, this test kit includes reagent, PCR reaction reagent, primer, positive sample contrast and contrast of negative sample and the specification sheets that extracts blood sample DNA, it is characterized in that: described primer comprises following 3 primers, and their sequence is respectively sequence 1 in the sequence table, sequence 2 and sequence 3.
8, test kit according to claim 7 is characterized in that: also can comprise a pair of interior label primer in the primer of described test kit; The sequence of described interior label primer is respectively sequence 4 and the sequence 5 in the sequence table.
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| CN102154481A (en) * | 2011-02-11 | 2011-08-17 | 智海生物工程（北京）有限公司 | Method for detecting mitochondrial mutations and kit thereof |
| CN102586433A (en) * | 2012-02-14 | 2012-07-18 | 北京科聆金仪生物技术有限公司 | Deafness predisposing gene 12S rRNA (ribosomal ribonucleic acid) 1494C>T fluorescence detection kit and application thereof |
| CN104962637A (en) * | 2015-07-08 | 2015-10-07 | 智海生物工程（北京）有限公司 | Kit and primer pair group for detecting drug-induced deafness susceptibility gene mutation site |
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| CN106957902A (en) * | 2016-11-01 | 2017-07-18 | 复旦大学 | A kind of non-marked probe for being used to detect drug-induced deafness related locus |
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| CN1245522C (en) * | 2003-09-10 | 2006-03-15 | 金政策 | Testing method for matrilinear hereditary deaf mitochondria gene 1555 place A-G catastrophe and reagent boxes |
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| CN102154481B (en) * | 2011-02-11 | 2013-01-02 | 智海生物工程（北京）有限公司 | Method for detecting mitochondrial mutations and kit thereof |
| CN102586433A (en) * | 2012-02-14 | 2012-07-18 | 北京科聆金仪生物技术有限公司 | Deafness predisposing gene 12S rRNA (ribosomal ribonucleic acid) 1494C>T fluorescence detection kit and application thereof |
| CN102586433B (en) * | 2012-02-14 | 2014-01-29 | 北京科聆金仪生物技术有限公司 | Deafness predisposing gene 12S rRNA (ribosomal ribonucleic acid) 1494C>T fluorescence detection kit and application thereof |
| CN104962637A (en) * | 2015-07-08 | 2015-10-07 | 智海生物工程（北京）有限公司 | Kit and primer pair group for detecting drug-induced deafness susceptibility gene mutation site |
| CN105586402A (en) * | 2015-12-16 | 2016-05-18 | 北京晋祺生物科技有限公司 | Detection primer set for drug-induced epicophosis, reaction system constituted by detection primer set and application of detection primer set |
| CN105950771A (en) * | 2016-07-08 | 2016-09-21 | 上海市内分泌代谢病研究所 | ARMC5 gene detection kit and mutation site |
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| CN106957902B (en) * | 2016-11-01 | 2020-12-04 | 复旦大学 | Non-labeled probe for detecting drug-induced deafness related sites |
| CN107022614A (en) * | 2017-04-21 | 2017-08-08 | 唐旌生物科技（上海）有限公司 | A kind of method and kit for detecting gene mutation |
| CN109652522A (en) * | 2019-01-11 | 2019-04-19 | 深圳市双科生物科技有限公司 | A kind of double Quality Control detection methods |
| CN113736915A (en) * | 2021-08-05 | 2021-12-03 | 青岛国际旅行卫生保健中心(青岛海关口岸门诊部) | Novel coronavirus 3-point mutation S gene identification kit based on high-resolution melting curve and identification method thereof |
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