CN100458419C - Mitochondria DNA11778 point mutation detecting method and reagent case thereof - Google Patents

Abstract

The invention relates to a gene testing method and its agent box, which provides a based molecular fluorescence, probe PCR mitochondria DNA11778 point mutation measuring method and its agent box. The invention first quotes weather existing mitochondria DNA11778 point mutation, and then designs lead substance at mitochondria genome conservation zone and molecular probe which is complemented with target series at mitochondria 11778 point, and then dose PCR growth process.

Description

Kit with 11778 point mutation of molecular probe technology detection line mitochondrial DNA

Technical field

The present invention relates to the kit that a kind of genetic test is used, more particularly, the present invention relates to a kind of kit based on 11778 point mutation of molecular fluorescence probe technique PCR detection line mitochondrial DNA.

Background technology

Leber ' s disease, i.e. Leber ' s hereditary optic neuropathy (LHON), be a kind ofly mainly involve retina, the papillomacular bundle fiber is looked in the lamina cribrosa of sclera front portion, causes the genetic disease of optic nerve retrogression.This sick transfer mode does not meet Mendelian inheritance, and mode of inheritance and pathogenesis also have arguement always so far.Mostly be between twenty and fifty morbidity clinically, eyes while or priority show as acute or subacute visual function grievous injury, still do not have specific treatment at present, causedly thus untimelyly clarify a diagnosis or carry out incorrect treatment and will bring many adverse consequencess to society, family and patient.Carry out the clear and definite molecular genetics diagnostic process of LHON clinically and can not only avoid expensive, repeat, damaging inspection, saved resource, reduced unnecessary waste for society and individual, also can avoid the unnecessary treatment that may be harmful to the patient; And help Susceptible population and individual determining, play an important role on purpose preventing and early examining early to control, carry out prenatal gene diagnosis at molecular level, also carry out EAB objective basis is provided to female carrier's high risk pregnancy, thereby be prenatal and postnatal care, carry out genetic counselling, provide new method and guidance this sick pre-natal diagnosis.

Since Wallace etc. was at first finding that this disease was to be caused by mitochondrial DNA (mtDNA) sudden change in 1988, it had mainly found mtNd4*LHON11778A, mtND1*LHON3460A, these three former mutational sites of mtND6*LHON14484C.China and to belong among the crowd such as Mongoloid Japan mtND4*LHON11778A together especially occurred frequently has report to account for about 90%.Clinically in the optic neuropathy patient group of the unknown cause that distributes, be diagnosed as LHON patient's ratio through molecular genetics and be respectively 50～80%, 50%, 40% in the U.S., Europe, Japan, also there is quite a few (report nearly 40% is arranged) the actual LHON of being patient in China.MtDNA, as unique cytoplasmic inheritance material, current many scholars have carried out long-term exploration to its effect in the some diseases pathogenesis, now the detection of main former the point mutation of unknown cause optic neuropathy patient row mtDNA are reached common understanding to be diagnosed as on the sick this point of Leber ' s.But present general MSP (allele-specific PCR) detects 11778 mutational site stability and accuracy is not good enough, method costs consuming time such as enzyme is cut, SSCP reaches directly order-checking are high, be unwell to clinical expansion, how the mtDNA of accurate easy detection optic neuropathy sudden change remains domestic and international one of the problem that emphasis solves that needs in clinical.Simultaneously, to the mutual relationship between Mitochondrial DNA Mutation ratio and the morbidity, whether there is the sudden change threshold value and also is individual problem that can not fine solution on methodology always with the generation problem of (different) matter and disease.May have the difference of Mitochondrial DNA Mutation ratio on the one hand between each tissue of human body, drawing materials of individual eye optic nerve tissue is also impossible, so also just extremely importantly existing to the accurate detection of peripheral blood sample.At present because to be used for quantitative methods all be to realize sxemiquantitative by PCR product electrophoresis gray scale scanning, so poor accuracy relatively.

The fluorescent PCR technology that develops rapidly in recent years is that the detection principle of PCR product is being done very big improvement, and the fluorescent PCR technology is different from the variation that other PCR part is to utilize the fluorescence luminous energy that fluorescent dye discharged under the effect of exciting light variation comes dynamically directly to reflect the pcr amplification product amount.Because of the fluorescence signal variable is directly proportional with the amplified production amount, self-reacting device by enough sensitivities to the collection of fluorescence with analyze realize to original template quantitatively, but the formation of the nucleic acid product of dynamic real-time monitoring simultaneously, need not last handling process, avoided the pollution of amplified production so to a certain extent, both shorten detection time, saved specific apparatus and professional's outfit again.Applied Biosystems company releases up-to-date TaqMan MGB fluorescence probe synthetic technology, and SNP (single nucleotide polymorphism) screening is detected better method.TaqMan MGB fluorescence probe is compared with conventional TaqMan fluorescence probe, and two main differences are arranged: the one, probe 3 ' end mark self non-luminous cancellation fluorescence molecule, to replace the Tamra fluorescence labeling that routine can be luminous.This new technology reduces the fluorescence background, and the fluorescence spectrum explanation is improved greatly, and especially the resolution between the different report fluorescence spectrums is improved greatly during snp analysis.The 2nd, ' end combines Minor groove binder bond to probe 3 in addition, makes the Tm value of probe improve, and has increased the hybrid stability of probe greatly.Therefore detect SNP with TaqMan MGB fluorescence probe, its probe length is generally 13 to 18 base scopes, and conventional TaqMan probe the AT base contents more for a long time its probe length will reach 30-40 base, probe length is long more, then its SNP discrimination is low more.

Summary of the invention

The objective of the invention is to overcome deficiency of the prior art, a kind of detection method based on 11778 point mutation of molecular fluorescence probe PCR mitochondrial DNA is provided.

Another object of the present invention is to provide a kind of kit that is used for molecular fluorescence probe PCR mitochondrial DNA 11778 point mutation detecting methods.

In order to solve the problems of the technologies described above, the present invention is achieved by the following technical solutions:

The invention provides a kind of mitochondrial DNA 11778 point mutation detecting methods, comprise the steps:

(1) extraction of cell genomic dna comprises PERIPHERAL BLOOD MONONUCLEAR CELL separation and DNA extraction;

(2) in the presence of molecular probe, the cell genomic dna that extracts is carried out pcr amplification;

(3) fluorescence intensity of mensuration PCR reaction system judges in the sample whether have mitochondrial DNA 11778 point mutation;

Described pcr amplification has used the PCR primer, at mitochondrial genomes conserved regions design primer sequence is:

MT-1：5’-CATCCTCATTACTATTCTGCCTAGCA-3’；

MT-2：5’-GGAGTAGAGTTTGAAGTCCTTGAGAGA-3’；

Described molecular probe: at the molecular probe of mitochondria 11778 sites design with target complement sequence, 5 ' end is used the FAM mark, other end MGBNFQ mark, and its sequence is:

MT-3：5’-FAM-CACTCACAGTCACATCA-MGBNFQ；

MT-4：5’-VIC-AGGATTATGATGCGACTGT-MGBNFQ。

The condition of pcr amplification of the present invention is:

95 ℃ 120 seconds → 93 ℃ 10 seconds → 60 ℃ 40 seconds,

Wherein: 93 ℃ 10 seconds → 60 ℃ are carried out 40 circulations between 40 seconds.

During the fluorescence intensity of mensuration of the present invention PCR reaction system, the fluorescence acquisition channel is selected FAM and VIC binary channels.

The present invention also provides a kind of kit that is used for molecular lampmark (MGB) fluorescent probe PCR mitochondrial DNA 11778 point mutation detecting methods, and this kit comprises:

2 * Taqman PCR buffer of half of reaction cumulative volume:

DATP, dTTP, dGTP, the dCTP of each 200 μ Mol/L;

Two primer MT-1, the MT-2 of each 0.1 μ Mol/L～2 μ Mol/L;

Two probe MT-3, the MT-4 of each 0.1 μ Mol/L～5 μ Mol/L;

1.5mMol/L the magnesium chloride of～2.5mMol/L or magnesium sulphate;

Surplus is a distilled water.

Compared with prior art, the invention has the beneficial effects as follows:

1, quantitative function: in the pcr amplification process, probe is degraded by Taq enzyme (having 5 ' 5 prime excision enzyme activity), fluorophor is away from quenching group, the fluorescence intensity that the fluorescence report group is produced in the reaction system is along with the PCR reaction is carried out, strengthen gradually, detect fluorescence signal by quantitative real time PCR Instrument.The period threshold value (Cycle threshold, Ct value) that detects gained is linear negative correlation with reaction original template amount logarithm value.Introducing standard ginseng is controlled product in the course of reaction, can set up the typical curve of Ct value and sample concentration.Unknown sample can be converted into sample size according to the Ct value.

2, accuracy is good: with the determining nucleic acid sequence somatotype relatively, both results rate 100% of coincideing.

3, effectively anti-pollution: PCR reaction and analysis are effectively taken precautions against the PCR product pollution carrying out under the stopped pipe condition fully, improve the reliability of testing result.

4, weak point simple to operate, consuming time, result judge ocular and clear.On the fluorescent PCR detector, increase and analyze, need not carry out steps such as electrophoresis, nucleic acid purification, uviol lamp observation, sequencing reaction.Only need 0.5～1.2 hour can finish the PCR process, help improving report speed.Only need directly to judge variation type according to binary channels fluorescence growth curve or Ct value.

Description of drawings

Fig. 1 is LHONmtDNA wild type test collection of illustrative plates;

Fig. 2 is LHONmtDNA anomaly test collection of illustrative plates;

Fig. 3 is LHONmtDNA variant and wild strain mixed type test collection of illustrative plates;

Embodiment

Below in conjunction with specific embodiment, set forth the present invention in further detail.

Specific embodiment 1:

Preparing the raw material of kit of the present invention selects for use as follows:.

The PCR primer, synthetic by Biosearch.INC company, primer sequence is:

MT-1：5’-CATCCTCATTACTATTCTGCCTAGCA-3’；

MT-2：5’-GGAGTAGAGTTTGAAGTCCTTGAGAGA-3’；

Molecular probe: at molecular lampmark (MGB) probe of cellular genome conserved regions design and target complement sequence, 5 ' end FAM mark, other end MGBNFQ ((Minor groove binder/Non-fluorescent quencher)) mark, its sequence is:

MT-3：5’-FAM-CACTCACAGTCACATCA-MGBNFQ；

MT-4：5’-VIC-AGGATTATGATGCGACTGT-MGBNFQ。

DNTPs (dATP, dTTP, dnP, dGTP): available from APPlied Biosystems company, concentration: 200 μ Mol/L.

The kit composition is as follows in the specific embodiment 1:

2 * Taqman PCR buffer of half of reaction cumulative volume;

DATP, dTTP, dGTP, the dCTP of each 200 μ Mol/L;

Two primer MT-1, the MT-2 of each 0.1 μ Mol/L;

Two probe MT-3, the MT-4 of each 0.1 μ Mol/L;

1.5mMol/L magnesium chloride;

Surplus is a distilled water.

Specific embodiment 2:

The kit composition is as follows:

2 * Taqman PCR buffer of half of reaction cumulative volume;

DATP, dTTP, dGTP, the dCTP of each 200 μ Mol/L;

Two primer MT-1, the MT-2 of each 2 μ Mol/L;

Two probe MT-3, the MT-4 of each 5 μ Mol/L;

2.5mMol/L magnesium chloride;

Surplus is a distilled water.

Specific embodiment 3:

The kit composition is as follows:

2 * Taqman PCR buffer of half of reaction cumulative volume;

DATP, dTTP, dGTP, the dCTP of each 200 μ Mol/L;

Two primer MT-1, the MT-2 of each 1 μ Mol/L;

Two probe MT-3, the MT-4 of each 2.5 μ Mol/L;

The magnesium chloride of 2mMol/L;

Surplus is a distilled water.

Specific embodiment 4:

The kit composition is as follows:

2 * Taqman PCR buffer of half of reaction cumulative volume;

DATP, dTTP, dGTP, the dCTP of each 200 μ Mol/L;

Two primer MT-1, the MT-2 of each 0.5 μ Mol/L;

Two probe MT-3, the MT-4 of each 1 μ Mol/L;

1.5mMol/L magnesium sulphate;

Surplus is a distilled water.

Specific embodiment 5:

The kit composition is as follows:

2 * Taqman PCR buffer of half of reaction cumulative volume;

DATP, dTTP, dGTP, the dCTP of each 200 μ Mol/L;

Two primer MT-1, the MT-2 of each 1.5 μ Mol/L;

Two probe MT-3, the MT-4 of each 3 μ Mol/L;

2.5mMol/L magnesium sulphate;

Surplus is a distilled water.

Specific embodiment 6:

The kit composition is as follows:

2 * Taqman PCR buffer of half of reaction cumulative volume;

DATP, dTTP, dGTP, the dCTP of each 200 μ Mol/L;

0.1 the primer MT-2 of the primer MT-1 of μ Mol/L, 2 μ Mol/L;

0.1 the probe MT-4 of the probe MT-3 of μ Mol/L, 5 μ Mol/L;

The magnesium sulphate of 2mMol/L;

Surplus is a distilled water.

Specific embodiment 7:

Step based on molecular fluorescence probe PCR mitochondrial DNA 11778 point mutation detecting methods of the present invention is as follows:

(1) extraction of cell genomic dna:

1, PERIPHERAL BLOOD MONONUCLEAR CELL (PBMCs) is separated:

The 5ml anticoagulation is behind 0.9%NaCl solution two-fold dilution, slowly join in the centrifuge tube that equal-volume Ficoll100 solution is housed, centrifugal 10 minutes of 2500rpm rotating speed, mononuclearcell is placed in the 1.5ml eppendorf pipe in the middle of drawing, centrifugal 5 minutes of 5000rpm, abandon supernatant, be precipitated as PERIPHERAL BLOOD MONONUCLEAR CELL.

2, DNA extraction:

In above-mentioned cell precipitation, add 100 μ l DNA extraction liquid (Hangzhou Bo Kang Bioisystech Co., Ltd), 99 ℃ 10 minutes, 13, centrifugal 10 minutes of 000rpm, supernatant promptly can be the template of pcr amplification.

(2) cell genomic dna that extracts is carried out pcr amplification:

1, PCR reaction mixture

2 * Taqman PCR reaction buffer, 20 μ l, article two, primer MT-1, MT-2 concentration are 0.75mol/L, article two, probe MT-3, MT-4 concentration are 0.25 μ mol/L, 1.5U Taq archaeal dna polymerase (ROCHE company product), and dna profiling 5 μ l complement to 40 μ l with tri-distilled water.

2, pcr amplification program: 95 ℃ of pre-sex change 2 minutes, entered amplification cycles then 93 ℃ * 10 seconds, 60 ℃ * 40 seconds, totally 40 circulations.The real-time quantitative PCR instrument is selected ABI 7000 for use, and the fluorescence acquisition channel is selected FAM and VIC binary channels.

(3) fluorescence intensity of mensuration PCR reaction system judges in the sample whether have mitochondrial DNA 11778 point mutation.

(4) test findings:

1, normal person's blood examination result, fluorescent PCR method detection VIC passage detects all positive, and the FAM passage detects all negative, is judged as the LHONmtDNA wild type, and normal person's sample sequencing result is wild type, and the result fits like a glove.

2, clinical suspicious patient LHON and 20 people of family member, sequencing result shows as LHONmtDNA anomaly patient 12 examples, the fluorescent PCR method detects the FAM passage and is the LHONmtDNA variation positive, what wherein have the mixing of LHONmtDNA variant and wild strain is 5 examples, in the mixed type, variant/wild strain ratio is all greater than 4: 1;

3, sequencing result shows as LHON mtDNA wild type 8 examples, and the fluorescent PCR method detects, and 5 examples are the LHONmtDNA wild type, and 3 examples are LHONmtDNA variant and wild strain mixed type, and in the mixed type, variant/wild strain ratio is all less than 1: 4.

4, fluorescence quantitative PCR detection LHONmtDNA G11778A point mutation is consuming time only is 80 minutes, much smaller than the sequencing time.

Specific embodiment 8:

Detect optic neuropathy patient 10 people of unknown cause, the result is with directly checking order relatively.

1, dna sample derives from gene studies institute of Chinese medicine Academy of Military Sciences.

2, PCR reaction mixture:

2 * Taqman PCR reaction buffer, 20 μ l, article two, primer MT-1, MT-2 concentration are 0.75 μ mol/L, article two, probe MT-3, MT-4 concentration are 0.25mol/L, 1.5U Taq archaeal dna polymerase (ROCHE company product), and dna profiling 5 μ l complement to 40 μ l with tri-distilled water.

3, pcr amplification program: 95 ℃ of pre-sex change 2 minutes, entered amplification cycles then 93 ℃ * 10 seconds, 60 ℃ * 40 seconds, totally 40 circulations.The real-time quantitative PCR instrument is selected ABI 7000 for use, and the fluorescence acquisition channel is selected FAM and VIC binary channels.

4, measure the fluorescence intensity of PCR reaction system, judge in the sample whether have mitochondrial DNA 11778 point mutation.

5, result: directly sequencing result shows as LHON mtDNA wild type 7 examples, anomaly 3 examples; The fluorescent PCR method detects, and 6 examples be the LHONmtDNA wild type, 4 examples for the LHONmtDNA anomaly wherein 1 example be mixed type, in the mixed type, variant/wild strain ratio is less than 1: 4, so can not judge its variation situation in direct the order-checking.

At last, it is also to be noted that what more than enumerate only is specific embodiments of the invention.Obviously, the invention is not restricted to above examples of implementation, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.

Claims (1)

1, a kind of kit with 11778 point mutation of molecular probe technology detection line mitochondrial DNA is characterized in that this kit comprises:

2 * Taqman PCR buffer of half of reaction cumulative volume;

DATP, dTTP, dGTP, the dCTP of each 200 μ mol/L;

Two primer MT-1, the MT-2 of each 0.1 μ mol/L～2 μ mol/L;

Two probe MT-3, the MT-4 of each 0.1 μ mol/L～5 μ mol/L;

1.5mmol/L the magnesium chloride of～2.5mmol/L or magnesium sulphate;

Surplus is a distilled water;

Described two primer MT-1, MT-2 are the PCR primer that uses in the pcr amplification process, are respectively at mitochondrial genomes conserved regions design primer sequence:

MT-1：5’-CATCCTCATTACTATTCTGCCTAGCA-3’；

MT-2：5’-GGAGTAGAGTTTGAAGTCCTTGAGAGA-3’；

Described two probe MT-3, MT-4 are that 5 ' end is used FAM and VIC mark respectively at the molecular probe of mitochondria 11778 sites design with target complement sequence, other end MGBNFQ mark, and its sequence is:

MT-3：5’-FAM-CACTCACAGTCACATCA-MGBNFQ；

MT-4：5’-VIC-AGGATTATGATGCGACTGT-MGBNFQ。

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