CN102787169A - Mixed liquor, kit and detection system for detecting A1555G and C1494T mutation of mitochondria DNA (deoxyribonucleic acid) - Google Patents
Mixed liquor, kit and detection system for detecting A1555G and C1494T mutation of mitochondria DNA (deoxyribonucleic acid) Download PDFInfo
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Abstract
The invention discloses mixed liquor, a kit and a detection system for detecting A1555G and C1494T mutation of mitochondria DNA (deoxyribonucleic acid). The wild type and the mutant type of a locus 1555 and a locus 1494 of the mitochondria DNA are repeatedly detected in real time in a reaction system by a pair of specific primers and four LNA (linolenic acid) probes carrying different fluorophores. The kit comprises human genome DNA extraction reagents, the mixed liquor of the primers and the probes, PCR (polymerase chain reaction) reaction mixed liquor, negative control DNA and positive control DNA. A1555G and C1494T mutation of the mitochondria DNA related to maternal inheritance drug-induced deafness can be rapidly detected by the kit, and compared with a method for individually detecting A1555G and C1494T mutation of mitochondria DNA, the method is simple and rapid to operate, visual in result, accurate, reliable, high in specificity and sensitivity and beneficial to popularization and application.
Description
Technical field: the present invention relates to a kind of test kit and method of use thereof that detects matrilinear inheritance induced deafness Mitochondrial DNA A1555G and C1494T sudden change situation; Particularly a kind of mixed solution and test kit and detection system that is applied to detection line mitochondrial DNA A1555G, C1494T sudden change.
Background technology: Mitochondrial DNA (MitochondriaDNA; MtRNA) sudden change is one of major reason that causes phonosensitive nerve deafness; These sudden changes mainly are to be positioned on plastosome 12SrRNA, the tRNA gene, and the homogeneity A1555G that is positioned at the 12SrRNA gene is deaf relevant with the anti-non-syndrome type that causes of C1494T sudden change and aminoglycoside.
Since 1962 found the first in the world mitochondrial disease patient, the mankind had had been found that a variety of diseases that cause owing to mitochondria dysfunction.Clear and definite several and deaf relevant mitochondrial mutations over past ten years.Because unique function of human mitochondrial is to participate in the generation of human body chemical energy through oxidative phosphorylation; Therefore the generation of mitochondrial mutations interfering energy and then cause the whole body neuromuscular dysfunction just not at all surprising causes also one of its characteristic just of hearing impairment.Yet it also is one of reason of non-syndrome induced deafness that surprisingly hereditary mitochondrial mutations comes to light, and it is one of reason of presbycusis that acquired mitochondrial mutations has been suggested.
The aminoglycosides antibiotics ototoxicity is the modal reason of the acquired deafness day after tomorrow.Not only heavy dose of, long medication can cause cochlea-vestibular infringement, and low dose of sometimes medication also can cause deafness, and there is the possibility of genetic predisposition in prompting.The data of the family of the tool aminoglycosides ototoxic deafness that Konigsmark etc. have found great majority in 1976 is summed up, and reaches a conclusion: heredity possibly be the autosomal dominant inheritance of companion's incomplete penetrance to aminoglycosides antibiotics ototoxicity susceptibility.Simultaneously, they also notice the non-paternal inheritance characteristic of this hereditary tool, and this heredity of reaching a conclusion thus maybe multifactor effect and produced.Propose after Higashi had looked back relevant document in 1989, most probable explanation is the Mitochondrial DNA defective to viewed matrilinear inheritance.1993, Prezant etc. reported three Chinese familys, and several members after using aminoglycosides antibiotics ototoxic deafness take place, and all confirmed to have the mtDNA 12SrRNA gene 1555 bases A → G sudden change that is matrilinear inheritance in three familys.Subsequently, also there are the family report in Africa, Spain, Japan, Mongolia, South America, Israel.At China, Japan, the Mongolian report that also has some Sporadic cases, these Sporadic cases do not have family history, and ototoxic deafness and mtDNA 1555A → G sudden change is all arranged.Same Mitochondrial DNA Mutation is present in nuclear gene genetic background very inequality with deafness and has more affirmed the pathogenic of this sudden change.
Mitochondrial DNA (mtDNA) 12SrRNA is the main 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 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.These changes make 12SrRNA on secondary structure, be more prone to the secondary structure of the respective regions of the 16SrRNA of bacterium, and 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.The biochemical character that carries the cell of A1555G sudden change and C1494T sudden change is that the mitochondrial protein synthetic also causes that thereupon the respiratory function of cell is unusual unusually.And when cultivating the clone of carrying these two sudden changes, can observe these cells and control cells system and compare that proteic translation can to occur in growth defect and the plastosome unusual with the DMEM substratum of paromycin that contains high density or Xin Meisu.These are viewed as the evidence that direct heredity and biochemical aspect are provided to draw a conclusion, i.e. A1555G sudden change and C1494T sudden change are the pathogenic mtDNA sudden changes that causes aminoglycosides antibiotics inductive non-syndrome type deaf.
Since nineteen ninety-three, the domestic and international method of detection line mitochondrial DNA A1555G and C1494T sudden change has that PCR directly checks order, PCR-restriction fragment length polymorphism (PCR-RFLP) technological, unit point optical dye PCR measures; Order-checking is difficult to accomplish clinical expansion; Though the enzyme incision technology cost is low, clinical manipulation bothers and is difficult to accomplish prevent and pollutes, because its technology will be uncapped once more after pcr amplification finishes and carried out follow-up enzyme and cut and run glue; Also has the technology that detects this sudden change with biochip technology; Biochip kit be for quick, the known genetic mutation of high-throughput examination site is custom-designed; But the biochip kit detection relates to specific apparatus and the bulk data Treatment Analysis is had relatively high expectations to laboratory equipment, environment and operator; And expensive is difficult to promote the use of in general hospital or laboratory; Optical dye PCR method detects, and can only detect a mutational site; Also have high resolving power solubility curve method, the specificity that detects with solubility curve is difficult to accomplish assurance, and requires the software analysis program complicated, needs special messenger's operation; Therefore press for a kind of can on a lot of hospitals existing installation, can the detection; And easy and simple to handle, quick, accuracy is high, the matrilinear inheritance induced deafness Mitochondrial DNA Mutation detection method and the test kit of high specificity, to satisfy the needs that Mitochondrial DNA A1555G and C1494T are suddenlyd change and carry out extensive examination.
Summary of the invention: the present invention is method and the test kit that a kind of fluorescent probe PCR detection line mitochondrial DNA A1555G, C1494T sudden change will be provided; It has accomplished only to use two primers and four LNA probes, uses the multi-fluorescence probe PCR detects two mutational sites simultaneously in a reaction system sudden change situation, has avoided the fluorescent signal phase mutual interference of fluorescence radiation group; Detection time is short, directly with extract good masterplate detect 1 hour general time spent get final product one manage out two sites the result, do not need the later stage to uncap, can in amplification procedure, directly read the site mutation situation.
One of technical scheme of the present invention:
A kind of mixed solution that is applied to detection line mitochondrial DNA A1555G, C1494T sudden change comprises the mixed solution of primers F 1, R1 and four probe N1, N2, M1, M2;
Article one, primer sequence F1 is a sense strand, is upper reaches universal primer, and second primer sequence R1 is an antisense strand, is the downstream universal primer;
Article four, probe is respectively with one of the wild-type probe of the human body Mitochondrial DNA design in Mitochondrial DNA A1555 site that is template and one of mutant probe, in addition at one of the wild-type probe of Mitochondrial DNA C1494 site design and one of mutant probe.
Preferably: said a pair of primer is according to disclosed human mtdna sequences Design; Article one, primer sequence F1 is a sense strand, is upper reaches universal primer, and between the nucleotide sequence 1445-1465bp of Mitochondrial DNA, its sequence is the SEQ ID NO:1 in the sequence table; Second primer sequence R1 is an antisense strand, is the downstream universal primer, and between the nucleotide sequence 1589-1608bp of Mitochondrial DNA, its sequence is the SEQ ID NO:2 in the sequence table, also comprises the complementary strand of these two primers.
Article four, can not exist four kinds of fluorescence radiation quencher groups of four probes of phase mutual interference not suppress each other or to offset between the probe, so that send four kinds of performances that the fluorescent signal of four kinds of different wave lengths can be distinguished two sites.
Said four probes are to comprise using TaqMan-LNA, TaqMan-MGB, TaqMan-AllGlo technology mark designed probe.
Article one, the sequence of probe N1 designs near the mutant site of Mitochondrial DNA C1494T, is sense strand, also comprises its complementary strand, and between the nucleotide sequence 1484-1497bp of Mitochondrial DNA, its sequence is the SEQ ID NO:3 in the sequence table; The sequence of second probe N2 designs near the wild-type site of Mitochondrial DNA 1494, is sense strand, also comprises its complementary strand, and between the nucleotide sequence 1483-1498bp of Mitochondrial DNA, its sequence is the SEQ ID NO:4 in the sequence table; Article three, the sequence of probe M1 designs near the mutant site of Mitochondrial DNA A1555G, is antisense strand, also comprises its complementary strand, and between the nucleotide sequence 1548-1563bp of Mitochondrial DNA, its sequence is the SEQ ID NO:5 in the sequence table; Article four, the sequence of probe M2 designs near the wild-type site of Mitochondrial DNA 1555, is antisense strand, also comprises its complementary strand, and between the nucleotide sequence 1548-1562bp of Mitochondrial DNA, its sequence is the SEQ ID NO:6 in the sequence table.
Said four probes adopt TaqMan-LNA technology mark designed probe, adopt TaqMan-LNA technology mark designed probe to have following luminescence quenching fluorophor and LNA base respectively; Article one, probe N1 is the mutant LNA probe of Mitochondrial DNA C1494T, its 5 ' end-labelled be CY5 fluorescence report group, 3 ' end-labelled be BHQ1 quenching of fluorescence group, add the LNA base; Second probe N2 is the wild-type LNA probe of Mitochondrial DNA 1494, its 5 ' end-labelled be ROX fluorescence report group, 3 ' end-labelled be BHQ1 quenching of fluorescence group, add the LNA base; Article three, probe M1 is the mutant LNA probe of Mitochondrial DNA A1555G, its 5 ' end-labelled be HEX fluorescence report group, 3 ' end-labelled be BHQ1 quenching of fluorescence group, add the LNA base; Article four, probe M2 is the wild-type LNA probe of Mitochondrial DNA 1555, its 5 ' end-labelled be FAM fluorescence report group, 3 ' end-labelled be BHQ1 quenching of fluorescence group, add the LNA base.
Each primer is:
Primers F 1:5 ' GAGTGCTTAGTTGAACAGGGC 3 ';
Primer R1:5 ' CACTCTGGTTCGTCCAAGTG 3 ';
The concrete sequence of probe:
Probe N1:5 ' CY5-CGCC+C+GTCA+CTCTC – BHQ13 ' ,+be the LNA base;
Probe N2:5 ' ROX-CCGCCCGTCACCCTCC – BHQ13 ';
Probe M1:5 ' HEX-ACGACTTGCCT+C+CT+CT – BHQ13 ' ,+be the LNA base;
Probe M2:5 ' FAM-CGA+CTTGTCT+C+CT+CT – BHQ13 ' ,+be the LNA base.
Two of technical scheme of the present invention:
A kind of test kit comprises above-described a kind of mixed solution that is applied to detection line mitochondrial DNA A1555G, C1494T sudden change, also comprises extracting genomic dna reagent, a pipe negative control DNA, a pipe positive control dna and a PCR reaction mixture.
Preferably: it comprises:
(1) extracts human genome DNA reagent;
(2) mixed solution of primers F 1, R1 and probe N1, N2, M1, M2;
(3) one pipe negative control DNA; One pipe carries the positive control dna in plastosome A1555G and C1494T mutational site simultaneously;
(4) the PCR reaction mixture comprises: Tag archaeal dna polymerase, MgCl
2, 10 * PCR damping fluid, dNTP and ddH
2O.
Three of technical scheme of the present invention:
A kind of detection system of multiple real time fluorescence probe PCR detection line mitochondrial DNA A1555G, C1494T sudden change of using comprises the fluorescent probe PCR appearance and the PCR pipe that can detect four look fluorescence; In the said PCR pipe the described mixed solution of claim 1-6, PCR reaction mixture are housed.
Preferably: primers F 1, R1 and probe N1, N2, M1, M2 design a pair of primer at Mitochondrial DNA; One of design wild-type probe M2 and mutant probe M1 are one in Mitochondrial DNA 1555 sites; In addition at one of Mitochondrial DNA 1494 site design wild-type probe N2 and one of mutant probe N1; With the human body Mitochondrial DNA is template, in a reaction system, uses the sudden change of multiple real time fluorescence probe PCR detection line mitochondrial DNA A1555G, C1494T.
Four of technical scheme of the present invention:
The detection method of detection system is: the mixed solution, PCR reaction mixture etc. that will extract good human DNA template, primers F 1, R1 and probe N1, N2, M1, M2 add in the PCR pipe together; Set up positive controls and negative control group more respectively; Application can be to detect the multiple real time fluorescence probe PCR appearance of four look fluorescence; Set reaction conditions, promptly the fluorescent signal according to different wave length obtains Mitochondrial DNA 1555 and 1494 site mutation situation.
Preferably: one section target sequence of pcr amplification promptly detects the SNP SNP in the two or more sites in this target sequence with the real-time fluorescence probe PCR, promptly utilizes a pair of primer and four or many two or more mutational sites of the real-time simultaneously detection of fluorescent probes; Utilizing this a pair of upstream and downstream primer in amplification extension process; Utilize 5 ' 3 ' the 5 prime excision enzyme activities probe degraded to combining respectively of taq archaeal dna polymerase with its target sequence; The upstream primer that promptly once circulates can detect the fluorescent signal of a probe when extending; Downstream primer also can detect the fluorescent signal of a probe when extending, so that send four kinds of performances that the fluorescent signal of four kinds of different wave lengths can be distinguished two sites in reaction tubes.
The present invention has following characteristics:
(1) used primer and probe all designs through a large amount of experiments are careful in the test kit of the present invention; Two primers and four LNA probes have been accomplished only to use; Use the multi-fluorescence probe PCR detects two mutational sites simultaneously in a reaction system sudden change situation, avoided the fluorescent signal phase mutual interference of fluorescence radiation group through the severe test debugging.
(2) in theory; Sample genomic dna is through a PCR reaction; The probe NI, the MI that promptly in reaction system, have only added to mutant site (A1555G, C1494T) just can detect the mutational site; But we have also used probe N2, the M2 of wild-type site (1555,1494) simultaneously, and the result is verified in the site of not sudden change, have confirmed this site mutation situation simultaneously.That is to say, same sample genomic dna same site process mutant and two probe simultaneous verifications of wild-type, thus make the result more accurate, credible.
(3) the present invention detects the detection of mutational site application fluorescent probe has the additive method incomparable advantage; Detection time is short; Directly with extract good masterplate detect 1 hour general time spent get final product one manage out two sites the result; Do not need the later stage to uncap, can in amplification procedure, directly read the site mutation situation.
(4) use detection line mitochondrial DNA A1555G/C1494T of the present invention mutational site than PCR sequencing PCR, Restriction Enzyme cutting method, solubility curve method etc., highly sensitive, specificity good, accuracy is high, linearity is stable, controlled contaminative is low, operating process simply, the result judges intuitively; All there is the real-time fluorescence quantitative PCR appearance in now general hospital, need not to put in addition equipment again and can realize popularizing in an all-round way; So this test kit can be fit to general hospital, Molecular Biology Lab promotes; Be convenient to implement the extensive examination and the preventive inspection of matrilinear inheritance medicine deafness mitochondrial DNAA155G, C1494T sudden change in China, the drug induced deafness chance take place thereby can effectively reduce the responsive crowd of aminoglycosides antibiotics.
Description of drawings
Fig. 1 is the sample pcr amplification graphic representation that carries Mitochondrial DNA A1555G mutant.
Fig. 2 is the sample pcr amplification graphic representation of Mitochondrial DNA 1555 wild-types.
Fig. 3 is the sample pcr amplification graphic representation that carries Mitochondrial DNA C1494T mutant.
Fig. 4 is the sample pcr amplification graphic representation of Mitochondrial DNA 1494 wild-types.
Embodiment
Below be to combine instance explanation Mitochondrial DNA 1555,1494 site mutations four look real-time fluorescence PCR detection method and test kits of the present invention, so that the user better understands the characteristics of the present invention in practical application.
1, primer and probe design
The Using P rimer5.0 software assistance is designed primer and probe, and (ncbi database sequence NC 01920) is template with disclosed human mtdna (12SrRNA) reference sequences, carries out primer and probe design, and plan is following:
(1) be placed between a pair of primer to Mitochondrial DNA 1555 and 1,494 two sites and effectively increase, 3 ' end of upstream primer F1 will be before site 1494, and 3 ' end of downstream primer R1 will be after site 1555;
Upstream primer F1:5 ' GAGTGCTTAGTTGAACAGGGC 3 ' (SEQ ID NO:1), long 21bp is between Mitochondrial DNA nucleotide sequence 1445-1465bp.
Downstream primer R1:5 ' CACTCTGGTTCGTCCAAGTG 3 ' (SEQ ID NO:2), long 20bp is the antisense strand between Mitochondrial DNA nucleotide sequence 1589-1608bp.
(2) add the LNA base in different fluorescence radiation group, 3 ' terminal BHQ1 quencher group and the centres of mark respectively to Mitochondrial DNA C1494T site mutation type and two of wild-type designs and upstream primer probe in the same way, and at its 5 ' end;
Mitochondrial DNA C1494T mutant probe N1:5 ' CY5-CGCC+C+GTCA+CTCTC – BHQ13 ' (SEQ ID NO:3), long 14bp, between Mitochondrial DNA nucleotide sequence 1484-1497bp ,+be the LNA base.
Mitochondrial DNA 1494 wild-type probe N2:5 ' ROX-CCGCCCGTCACCCTCC – BHQ13 ' (SEQ ID NO:4), long 16bp is between Mitochondrial DNA nucleotide sequence 1483-1498bp.
(3) add the LNA base in different fluorescence radiation group, 3 ' terminal BHQ1 quencher group and the centres of mark respectively to Mitochondrial DNA A1555G site mutation type and two of wild-type designs and upstream primer probe in the same way, and at its 5 ' end;
Mitochondrial DNA A1555G mutant probe M1:5 ' HEX-ACGACTTGCCT+C+CT+CT – BHQ13 ' (SEQ IDNO:5), long 16bp, between Mitochondrial DNA nucleotide sequence 1548-1563bp ,+be the LNA base.
Mitochondrial DNA 1555 wild-type probe M2:5 ' FAM-CGA+CTTGTCT+C+CT+CT – BHQ13 ' (SEQ ID NO:6), long 15bp, between Mitochondrial DNA nucleotide sequence 1548-1562bp ,+be the LNA base.
2, the composition (50 person-portions/box) of Mitochondrial DNA A1555G, C1494T site mutation four look real-time fluorescence PCR assay kits:
(1) extract genomic dna reagent:
(2) mixed solution of primer (F1, R1) and probe (N1, N2, M1, M2):
(3) 1 pipe negative control DNA, concentration 20ng/ul, 100ul; 1 pipe positive control dna, concentration 20ng/ul, 100ul;
(4) the PCR reaction mixture comprises: TagDNA polysaccharase, MgCl
2, 10 * PCR damping fluid, dNTP and ddH
2O.
3, detect sample
3 of 5 of the matrilinear inheritance drug induced deafness patients that Mitochondrial DNA A1555G sudden change is carried in selection and the matrilinear inheritance drug induced deafness patients who carries Mitochondrial DNA C1494T sudden change are as the person under inspection; Select 5 normal peoples as the person under inspection again, receive inspection number 13 people altogether.
4, extracting genome DNA
5, extract good sample DNA with the Multi-channel Real-time fluorescent PCR appearance augmentation detection that can detect four look wavelength;
With the above-mentioned a pair of primer that designs and four probe sequences; Through synthetic and mark (entrusting biotech firm); With the good sample DNA of said extracted is template, adds mixed solution, the PCR reaction mixture of primer (F1, R1) and probe (N1, N2, M1, M2); Set up positive control dna group, negative control DNA group, no template control group more simultaneously.
Pcr amplification reaction system of table 1
| 10ng masterplate DNA |
| Every kind of primer of 350-500nM |
| Every kind of probe of 200-300nM |
| 200uM dNTP |
| 3mM MgCL 2 |
| 2.00-3.00U TaqDNA polysaccharase |
| 5ul 10 * PCR damping fluid |
| Add d dH 2O is to 20ul |
Equipment and instrument: use Luo Shi light cycler 480 fluorescent PCR appearance and detect;
Cycling condition: 95 ℃ of preparatory sex change 10min; Then 95 ℃ of sex change 10s anneal for 58 ℃ and extend 45s (acquired signal), 40 circulations.
The data processing that allelotrope is analyzed has shown 4 kinds of results like Fig. 1-4,
Shown in Figure 1: a Mitochondrial DNA A1555G mutant person under inspection shows that the mutant of a site A1555G rather than the probe signals of wild-type are detected;
Shown in Figure 2: one 1555 wild-type person under inspection shows the fluorescent signal of 1555 wild-type probe;
Shown in Figure 3: as to show that the mutant of a site C1494T rather than the probe signals of wild-type are detected.
Shown in Figure 4: one 1494 wild-type person under inspection shows the fluorescent signal of 1494 wild-type probe.
6, the result judges
13 persons under inspection, 5 Mitochondrial DNA A1555G mutant patients by name, 3 Mitochondrial DNA C1494T mutant patients by name, 5 Mitochondrial DNAs 1555 by name and 1494 wild-types are individual.
7, result verification
Selected person under inspection is all carried out direct sequencing analysis with PCR sequencing PCR to Mitochondrial DNA (12S rRNA); The detected result of sequencing result and Mitochondrial DNA A1555G, C1494T site mutation four look real-time fluorescence PCR assay kits fits like a glove, and explains that the method that this test kit uses can be used for Mitochondrial DNA A1555G and C1495T sudden change detection fully.
Detected result table 2
| Person under inspection's numbering | Deafness patient whether | This test kit method detects | PCR sequencing PCR detects |
| S001 | Be | A1555G is positive | A1555G is positive |
| S002 | Be | A1555G is positive | A1555G is positive |
[0085]
| S003 | Be | A1555G is positive | A1555G is positive |
| S004 | Be | A1555G is positive | A1555G is positive |
| S005 | Be | A1555G is positive | A1555G is positive |
| S006 | Be | C1494T is positive | C1494T is positive |
| S007 | Be | C1494T is positive | C1494T is positive |
| S008 | Be | C1494T is positive | C1494T is positive |
| S009 | Be not | 1555,1494 feminine genders | 1555,1494 feminine genders |
| S010 | Be not | 1555,1494 feminine genders | 1555,1494 feminine genders |
| S011 | Be not | 1555,1494 feminine genders | 1555,1494 feminine genders |
| S012 | Be not | 1555,1494 feminine genders | 1555,1494 feminine genders |
| S013 | Be not | 1555,1494 feminine genders | 1555,1494 feminine genders |
Sequence table
< 110>Guo Limin
< 120>a kind of mixed solution and test kit and detection system that is applied to detection line mitochondrial DNA A1555G, C1494T sudden change
〈160>
〈170>
〈210>1
〈211>21
〈212>DNA
< 213>artificial sequence
〈400>1
GAGTGCTTAGTTGAACAGGGC
〈210>2
〈211>20
〈212>DNA
< 213>artificial sequence
〈400>2
CACTCTGGTTCGTCCAAGTG
〈210>3
〈211>14
〈212>DNA
< 213>artificial sequence
〈400>3
CGCCCGTCACTCTC
〈210>4
〈211>16
〈212>DNA
< 213>artificial sequence
〈400>4
CCGCCCGTCACCCTCC
〈210>5
〈211>16
〈212>DNA
< 213>artificial sequence
〈400>5
ACGACTTGCCTCCTCT
〈210>6
〈211>15
〈212>DNA
< 213>artificial sequence
〈400>6
CGACTTGTCTCCTCT。
Claims (13)
1. mixed solution that is applied to detection line mitochondrial DNA A1555G, C1494T sudden change is characterized in that said mixed solution comprises the mixed solution of primers F 1, R1 and four probe N1, N2, M1, M2;
Article one, primer sequence F1 is a sense strand, is upper reaches universal primer, and second primer sequence R1 is an antisense strand, is the downstream universal primer;
Article four, probe is respectively with one of the wild-type probe of the human body Mitochondrial DNA design in Mitochondrial DNA A1555 site that is template and one of mutant probe, in addition at one of the wild-type probe of Mitochondrial DNA C1494 site design and one of mutant probe.
2. a kind of mixed solution that is applied to detection line mitochondrial DNA A1555G, C1494T sudden change as claimed in claim 1 is characterized in that said a pair of primer is according to disclosed human mtdna sequences Design; Article one, primer sequence F1 is a sense strand, is upper reaches universal primer, and between the nucleotide sequence 1445-1465bp of Mitochondrial DNA, its sequence is the SEQ ID NO:1 in the sequence table; Second primer sequence R1 is an antisense strand, is the downstream universal primer, and between the nucleotide sequence 1589-1608bp of Mitochondrial DNA, its sequence is the SEQ ID NO:2 in the sequence table, also comprises the complementary strand of these two primers.
3. according to claim 1 or claim 2 a kind of is applied to the mixed solution of detection line mitochondrial DNA A1555G, C1494T sudden change; It is characterized in that to exist between four probes four kinds of fluorescence radiation quencher groups of four probes of phase mutual interference not suppress each other or to offset, so that send four kinds of performances that the fluorescent signal of four kinds of different wave lengths can be distinguished two sites.
4. like each described a kind of mixed solution that is applied to detection line mitochondrial DNA A1555G, C1494T sudden change of claim 1-3, it is characterized in that said four probes are to comprise using TaqMan-LNA, TaqMan-MGB, TaqMan-AllGlo technology mark designed probe.
5. like each described a kind of mixed solution that is applied to detection line mitochondrial DNA A1555G, C1494T sudden change of claim 1-4; The sequence that it is characterized in that article one probe N1; Near the mutant site of Mitochondrial DNA C1494T, designing, is sense strand, also comprises its complementary strand; Between the nucleotide sequence 1484-1497bp of Mitochondrial DNA, its sequence is the SEQ ID NO:3 in the sequence table; The sequence of second probe N2 designs near the wild-type site of Mitochondrial DNA 1494, is sense strand, also comprises its complementary strand, and between the nucleotide sequence 1483-1498bp of Mitochondrial DNA, its sequence is the SEQ ID NO:4 in the sequence table; Article three, the sequence of probe M1 designs near the mutant site of Mitochondrial DNA A1555G, is antisense strand, also comprises its complementary strand, and between the nucleotide sequence 1548-1563bp of Mitochondrial DNA, its sequence is the SEQ ID NO:5 in the sequence table; Article four, the sequence of probe M2 designs near the wild-type site of Mitochondrial DNA 1555, is antisense strand, also comprises its complementary strand, and between the nucleotide sequence 1548-1562bp of Mitochondrial DNA, its sequence is the SEQ ID NO:6 in the sequence table.
6. like each described a kind of mixed solution that is applied to detection line mitochondrial DNA A1555G, C1494T sudden change of claim 1-5; It is characterized in that said four probes adopt TaqMan-LNA technology mark designed probe, adopt TaqMan-LNA technology mark designed probe to have following luminescence quenching fluorophor and LNA base respectively; Article one, probe N1 is the mutant LNA probe of Mitochondrial DNA C1494T, its 5 ' end-labelled be CY5 fluorescence report group, 3 ' end-labelled be BHQ1 quenching of fluorescence group, add the LNA base; Second probe N2 is the wild-type LNA probe of Mitochondrial DNA 1494, its 5 ' end-labelled be ROX fluorescence report group, 3 ' end-labelled be BHQ1 quenching of fluorescence group, add the LNA base; Article three, probe M1 is the mutant LNA probe of Mitochondrial DNA A1555G, its 5 ' end-labelled be HEX fluorescence report group, 3 ' end-labelled be BHQ1 quenching of fluorescence group, add the LNA base; Article four, probe M2 is the wild-type LNA probe of Mitochondrial DNA 1555, its 5 ' end-labelled be FAM fluorescence report group, 3 ' end-labelled be BHQ1 quenching of fluorescence group, add the LNA base.
7. like each described a kind of mixed solution that is applied to detection line mitochondrial DNA A1555G, C1494T sudden change of claim 1-6, it is characterized in that;
Each primer is:
Primers F 1:5 ' GAGTGCTTAGTTGAACAGGGC 3 ';
Primer R1:5 ' CACTCTGGTTCGTCCAAGTG 3 ';
The concrete sequence of probe:
Probe N1:5 ' CY5-CGCC+C+GTCA+CTCTC – BHQ13 ' ,+be the LNA base;
Probe N2:5 ' ROX-CCGCCCGTCACCCTCC – BHQ13 ';
Probe M1:5 ' HEX-ACGACTTGCCT+C+CT+CT – BHQ13 ' ,+be the LNA base;
Probe M2:5 ' FAM-CGA+CTTGTCT+C+CT+CT – BHQ13 ' ,+be the LNA base.
8. test kit; It is characterized in that comprising each described a kind of mixed solution that is applied to detection line mitochondrial DNA A1555G, C1494T sudden change, also comprise and extract genomic dna reagent, a pipe negative control DNA, a pipe positive control dna and a PCR reaction mixture like claim 1-7.
9. a kind of test kit as claimed in claim 8 is characterized in that it comprises:
(1) extracts human genome DNA reagent;
(2) mixed solution of primers F 1, R1 and probe N1, N2, M1, M2;
(3) one pipe negative control DNA; One pipe carries the positive control dna in plastosome A1555G and C1494T mutational site simultaneously;
(4) the PCR reaction mixture comprises: Tag archaeal dna polymerase, MgCl
2, 10 * PCR damping fluid, dNTP and ddH
2O.
10. a detection system of using multiple real time fluorescence probe PCR detection line mitochondrial DNA A1555G, C1494T sudden change is characterized in that comprising the fluorescent probe PCR appearance and the PCR pipe that can detect four look fluorescence; In the said PCR pipe the described mixed solution of claim 1-6, PCR reaction mixture are housed.
11. a kind of detection system of using multiple real time fluorescence probe PCR detection line mitochondrial DNA A1555G, C1494T sudden change according to claim 10; It is characterized in that: primers F 1, R1 and probe N1, N2, M1, M2 design a pair of primer at Mitochondrial DNA; One of design wild-type probe M2 and mutant probe M1 are one in Mitochondrial DNA 1555 sites; In addition at one of Mitochondrial DNA 1494 site design wild-type probe N2 and one of mutant probe N1; With the human body Mitochondrial DNA is template, in a reaction system, uses the sudden change of multiple real time fluorescence probe PCR detection line mitochondrial DNA A1555G, C1494T.
12. like claim 10 or 11 described a kind of detection systems of using multiple real time fluorescence probe PCR detection line mitochondrial DNA A1555G, C1494T sudden change; The detection method that it is characterized in that detection system is: the mixed solution, PCR reaction mixture etc. that will extract good human DNA template, primers F 1, R1 and probe N1, N2, M1, M2 add in the PCR pipe together; Set up positive controls and negative control group more respectively; Application can be to detect the multiple real time fluorescence probe PCR appearance of four look fluorescence; Set reaction conditions, promptly the fluorescent signal according to different wave length obtains Mitochondrial DNA 1555 and 1494 site mutation situation.
13. a kind of detection system of using multiple real time fluorescence probe PCR detection line mitochondrial DNA A1555G, C1494T sudden change as claimed in claim 12; The detection method that it is characterized in that detection system is: one section target sequence of pcr amplification promptly detects the SNP SNP in the two or more sites in this target sequence with the real-time fluorescence probe PCR, promptly utilizes a pair of primer and four or many two or more mutational sites of the real-time simultaneously detection of fluorescent probes; Utilizing this a pair of upstream and downstream primer in amplification extension process; Utilize 5 ' 3 ' the 5 prime excision enzyme activities probe degraded to combining respectively of taq archaeal dna polymerase with its target sequence; The upstream primer that promptly once circulates can detect the fluorescent signal of a probe when extending; Downstream primer also can detect the fluorescent signal of a probe when extending, so that send four kinds of performances that the fluorescent signal of four kinds of different wave lengths can be distinguished two sites in reaction tubes.
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