CN100348732C - Quantitative detection of Leber's genetic optic nerve disease - Google Patents
Quantitative detection of Leber's genetic optic nerve disease Download PDFInfo
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Abstract
The present invention relates to quantitative detection method for Leber's heritability optic neuropathy (LHON). The quantitative detection method of the present invention has the steps that DNA sequences (which cause LHON) of target gene mutation site regions to be detected are designed and synthesized into a pair of primers and a fluorescent probe arranged between the two primers; a positive or negative probe is respectively added into mitochondrial DNA samples to be detected under the same reaction conditions for PCR amplification; through the addition of standard samples with the known concentration for drawing a standard curve, the concentration of an initial template is calculated according to the position of the samples to be detected in the standard curve; DNA initial reaction copy numbers displayed by different probes are calculated according to the standard curve for determining the mutation proportion. The present invention provides a clinical quantitative LHON detection method with the advantages of high accuracy, god specificity, short time, low cost, small artificial error and good repeatability, so the existing qualitative detection LHON technology is led into the quantitative and accurate level.
Description
Technical field
The present invention relates to Leber ' s hereditary optic neuropathy patient's Mitochondrial DNA Mutation quantitative detecting method.
Background technology
Leber ' s hereditary optic neuropathy (LHON) is the ophthalmology inherited disease that Leber doctor at first reported in 1871.Discover that this sick mode of inheritance does not meet Mendelian inheritance, and do not find as yet that so far the male patient passes to the offspring to this disease that be women's vertical transmission, pointing out this disease is the plasma inheritance disease relevant with matrilinear inheritance.Clinical manifestation descends final optic atrophy for successively acute or subacute the carrying out property central vision of young adult's eyes.It is the common cause of young adult's blinding.
Prove that in recent years this disease is that (mitochondrial DNA, mtDNA) sudden change causes owing to Mitochondrial DNA.The philtrum that this dna mutation is arranged, women's 10% morbidity, the male sex's 50% morbidity promptly has incomplete penetrance.People mtDNA is 16, the closed loop double chain DNA molecule of 569bp, and its complete nucleotide order is measured, and full length sequence is seen Genebank, and accession number is AY275533.That has reported so far has nearly 20 kinds with LHON related mtDNA sudden change, and wherein 90%～97% LHON is by due to one in G11778A (G in 11778 sites → A transgenation), the G3460A (G in 3460 sites → A transgenation) of mtDNA or three former pathogenic mutations of T14484C (T in 14484 sites → C transgenation).Not agnate various former pathogenic mutation site distribution situation difference.In Chinese population, what LHON patient had a G11778A accounts for 92.9%, and what G3460A was arranged accounts for 1.4%, has that T14484C's account for 5.7%.And in white people, above-mentioned three kinds of primary sudden change proportion is respectively 50%～70%, 8%～25%, 10%～15%.
Current diagnosis LHON is the gene diagnosis based on Mitochondrial DNA Mutation, and classical detection means is a restriction endonuclease SfaN I identification catastrophe point, heteroduplex-single chain conformation polymorphism (HA-SSCP method), and sudden change Auele Specific Primer PCR (MSP-PCR) detects and dna sequencing.
The SSCP method of coming out in 1989, its principle is: the space multistory structure of single stranded DNA fragment complexity mainly is because the interior interaction force of its inner base pairing equimolecular is kept, when a base changes, can influence its space conformation more or less, conformation is changed, and by native polyacrylamide gel electrophoresis (PAGE), can very observantly discrepant molecular separation on the conformation be opened.This method be single strand conformation polymorphism (Single-Strand Conformation Polymorphism, SSCP), the detection primary process that is used for the LHON11778 site is: 1. pcr amplification target DNA (11641-11980); 2. with special pcr amplification product sex change, then snapback makes it to become the single strand dna with certain space structure; 3. an amount of single stranded DNA is carried out native polyacrylamide gel electrophoresis; 4. dye by radioactive automatic developing, silver at last or ethidium bromide chromogenic assay result.Change with comparing of normal control if find single stranded DNA band mobility, just can judge that this chain conformation changes, and then infer that 11778 site base mutations are arranged in this dna fragmentation.But its weak point is: 1) can only will determine the position and the type of sudden change at last as a kind of sudden change detection method, also need further order-checking; 2) deposition condition requires strict; 3) because SSCP causes that according to point mutation the change of single strand dna three-dimensional conformation realizes electrophoretic separation, in the time of point mutation when some position so just may occurring inoperative or effect be very little to the change of single strand dna three-dimensional conformation, add other condition effect, polyacrylamide gel electrophoresis can't be differentiated cause omission, false negative rate is higher.
The dna sequencing technology is meant that with the dna single chain be template, under given conditions, with special primer under the effect of order-checking level archaeal dna polymerase, according to the base complementrity pair principle, constantly 4 kinds of deoxyribonucleotides (dNTP) are added to 3 of primer '-C-terminal and make primer strand obtain extending.The order-checking of a general base costs an arm and a leg, and makes the detection cost higher, is difficult to apply.
Because SfaN I method and sequencing are very expensive, there are false-negative problem in SSCP and MSP, and previously to analyze the idiovariation ratio be to adopt semiquantitative method such as gel imaging system, only detect the product band after the PCR, tolerance range is not high, therefore makes troubles to clinical application.
1996 by U.S. Applied Biosystems company release real-time fluorescence quantitative PCR technology, this technology is to add fluorophor in the PCR reaction system, utilize the fluorescent signal accumulation whole PCR process of monitoring in real time, the method for by typical curve unknown template being carried out quantitative analysis at last.The real-time fluorescence quantitative PCR technology has solved traditional limitation that quantitatively can only end point determination effectively, has realized that each takes turns the intensity that circulation all detects the first order fluorescence signal, obtains quantitative result according to typical curve.It is quantitatively the most accurate up to now utilizing the real-time fluorescence quantitative PCR of outer typical curve, the quantivative approach that circulation ratio is best, obtained globally generally acknowledging, be widely used in gene expression research, transgenic research, numerous areas such as curative effect of medication examination, pathogen detection.This technology has not only realized the leap of PCR from qualitative to quantitative, and compares with conventional PCR, it have specificity stronger, effectively solve characteristics such as PCR pollution problem, level of automation height.The appearance of fluorescent quantitative PCR technique for the detection of LHON, especially detection by quantitative provide new approach, but has not yet to see report because many conditions grope prematurity still.
Summary of the invention
The object of the present invention is to provide the quantitative detecting method of a kind of Leber ' s hereditary optic neuropathy (LHON) patient's Mitochondrial DNA Mutation, still the technology that is at present qualitative detection ophthalmology inherited disease is incorporated into quantitative accurate level, helps further studying the relation between mutant proportion and the morbidity.
The quantitative detecting method of Leber ' s hereditary optic neuropathy of the present invention (LHON) patient's Mitochondrial DNA Mutation may further comprise the steps:
A) according to causing that the dna sequence dna of the mutant target gene site areas to be checked of Leber ' s hereditary optic neuropathy designs and synthesizes a pair of primer and the fluorescent probe between two primers, described fluorescent probe comprises positive probe and negative probe, the different fluorescence of mark separately, the DNA chain hybridization of positive probe and sudden change, negative probe and the hybridization of normal DNA chain;
B) the Mitochondrial DNA testing sample is added the positive or negative probe respectively under same reaction conditions and carry out pcr amplification, the variation of fluorescent signal in the monitoring reaction system;
C) by adding the standard model drawing standard curve of concentration known, wherein X-coordinate is represented the logarithm of initial copy number, ordinate zou is represented the Ct value, the cycle number that the Ct value is experienced when arriving the thresholding of setting for the fluorescent signal in each reaction tubes is extrapolated the concentration of original template then according to the position of testing sample in typical curve;
D) ask for the DNA initial reaction copy number that different probe shows according to typical curve, ask for the mean value of at least three reaction gained copy numbers of each probe, determine mutant proportion, the ratio of the copy number sum of the copy number that the positive fluorescence probe of this mutant proportion shows and two kinds of fluorescence probe demonstrations; The copy number difference that the sample of different concns records, and the mutant proportion of same sample is constant, obtains the detection by quantitative result in mitochondrial mutations site thus.
Described fluorescent probe is the Taqman-MGB fluorescent probe, perhaps uses the Taqman fluorescent probe, SYBR Green fluorescence dye.
The mode of the best of the present invention is the point mutation that fluorescent quantitative PCR technique is come detection by quantitative LHON in conjunction with the Taqman-MGB fluorescent probe.The Taqman-MGB fluorescent probe is a kind of oligonucleotide, and two ends are mark report fluorophor R and a cancellation fluorophor Q respectively.When probe was complete, the reporter group fluorescent signal emitted was absorbed by quenching group; During pcr amplification, 5 '-3 ' 5 prime excision enzyme activity of gold medal Taq enzyme is cut degraded with the probe enzyme, the report fluorophor is separated with the cancellation fluorophor, reporter group after the separation can send fluorescence, thereby the fluorescence monitoring system can receive fluorescent signal, be DNA chain of every amplification, just have a fluorescence molecule to form, realized that the accumulation of fluorescent signal and PCR product form fully synchronously.Concrete steps as shown in Figure 1.
Positive probe mark VIC fluorescence of the present invention, negative probe mark FAM fluorescence; Perhaps negative probe mark VIC fluorescence, positive probe mark FAM fluorescence.
Quantitative detecting method of the present invention can adopt multiple reaction system, as 10 μ l, 20 μ l or 50 μ l reaction systems, is listed below:
(1) the pcr amplification system is 10 μ l reaction systems, comprises:
Suitable template 1.80～2.20 μ l,
Water 1.70～1.90 μ l,
Quantitative fluorescent PCR reacts basic reagent 4.80～5.30 μ l,
Fluorescent probe 0.20～0.30 μ l,
Primer is a pair of, each 0.40～0.50 μ l;
Reaction conditions is:
48～52 ℃, 2 minutes; 91～93 ℃, 10 minutes; 93～96 ℃, 30 seconds; 58～61 ℃, 30 seconds; Back two step totally 50 circulations.
(2) the pcr amplification system is 20 μ l reaction systems, comprises:
Suitable template 3.60～4.40 μ l,
Water 3.50～3.90 μ l,
Quantitative fluorescent PCR reacts basic reagent 9.55～10.45 μ l,
Fluorescent probe 0.45～0.55 μ l,
Primer is a pair of, each 0.80～1.00 μ l;
Reaction conditions is:
48～52 ℃, 2 minutes; 91～93 ℃, 10 minutes; 93～96 ℃, 30 seconds; 58～61 ℃, 30 seconds; Back two step totally 50 circulations.
(3) the pcr amplification system is 50 μ l reaction systems, comprises:
Suitable template 9.50～10.5 μ l,
Water 9.0～9.40 μ l,
Quantitative fluorescent PCR reacts basic reagent 24.00～25.80 μ l,
Fluorescent probe 1.10～1.50 μ l,
Primer is a pair of, each 2.10～2.50 μ l;
Reaction conditions is:
48～52 ℃, 2 minutes; 91～93 ℃, 10 minutes; 93～96 ℃, 30 seconds; 58～61 ℃, 30 seconds; Back two step totally 50 circulations.
It is 2 that described quantitative fluorescent PCR reacts basic reagent
*Master Mix can buy the AppliedBiosystems company from the U.S..
Requiring of the suitable template that is used to react and the reaction of other quantitative fluorescent PCRs is identical, and the order of magnitude of conventional copy number is 10
4-5, the available wide range, 0.10～25.0ng/ μ l all can.
According to quantitative detecting method of the present invention, the contriver has realized the detection by quantitative to three main former pathogenic mutation sites of known person Mitochondrial DNA, and is specific as follows:
(1) G11778A mutational site:
Mutant target gene site areas to be checked is a Mitochondrial DNA 11701-11820 site areas, and base is G on normal 11778 sites, and base is A on 11778 sites that suddenly change; This regional sequence is: 11701 taatc gccca cggac ttaca tcctc attac tattc tgcct agcaa actca11751 aacta cgaac gcact cacct cacag tcGca tcata atcct ctctc aagga11801 cttca aactc tactc ccact
Primer sequence is:
Forward primer: 5 ' TCC TCA TTA CTA TTC TGC CTA GCA AA 3 ',
Reverse primer: 5 ' GGA GTA GAG TTT GAA GTC CTT GAG AGA 3 '.
Positive probe can be hybridized with Mitochondrial DNA 11769-11786 site, and its sequence is:
5’CTC?ACA?GTC?ACA?TCA?TAA?3’；
Negative probe can be hybridized with Mitochondrial DNA 11771-11786 site, and its sequence is:
5’CAC?AGT?CGC?ATC?ATA?A?3’。
(2) G3460A mutational site
Mutant target gene site areas to be checked is a Mitochondrial DNA 3401-3550 site areas, and base is G on normal 3460 sites, and base is A on 3460 sites that suddenly change; This regional sequence is:
3401?aacta?cgcaa?aggcc?ccaac?gttgt?aggcc?cctac?gggct?actac?aaccc
3451?ttcgc?tgacG?ccata?aaact?cttca?ccaaa?gagcc?cctaa?aaccc?gccac
3501?atcta?ccatc?accct?ctaca?tcacc?gcccc?gacct?tagct?ctcac?catcg
Described primer sequence is:
Forward primer FP:5 ' CCC CTA CGG GCT ACT ACA ACC 3 ',
Reverse primer RP:5 ' TTA GGG GCT CTT TGG TGA AGA G 3 ';
Described positive probe can be hybridized with Mitochondrial DNA 3451-3466 site, and its sequence is:
5’TTC?GCT?GAC?ACC?ATA?AA?3’；
Described negative probe can be hybridized with Mitochondrial DNA 3451-3467 site, and its sequence is:
5’TTC?GCT?GAC?GCC?ATA?A?3’。
(3) T14484C mutational site
Mutant target gene site areas to be checked is a Mitochondrial DNA 14421-14570 site areas, and base is T on normal 14484 sites, and base is C on 14484 sites that suddenly change; This regional sequence is:
14421?ctgac?cccca?tgcct?cagga?tactc?ctcaa?tagcc?atcgc?tgtag?tatat
14471?ccaaa?gacaa?ccaTc?attcc?cccta?aataa?attaa?aaaaa?ctatt?aaacc
14521?catat?aacct?ccccc?aaaat?tcaga?ataat?aacac?acccg?accac?accgc
Described primer sequence is:
Forward primer FP:5 ' CAA TAG CCA TCG CTG TAG TAT ATC CA 3 ',
Reverse primer RP:5 ' GGG AGG TTA TAT GGG TTT AAT AGT TTT TT 3 ';
Described positive probe can be hybridized with Mitochondrial DNA 14475-14490 site, and its sequence is:
5’AGA?CAA?CCA?CCA?TTC?C?3’；
Described negative probe can be hybridized with Mitochondrial DNA 14475-14490 site, and its sequence is:
5’AGA?CAA?CCA?TCA?TTC?C?3’。
Detect at present LHON and be qualitative methods such as adopting aforesaid SSCP, credible rate is 70%, that is to say: can measure feminine gender, but wherein have 30% this disease may still be arranged.Quantitative detecting method of the present invention then contained at present should disease main former mutational site, and, can low site and future can carry out detection by quantitative equally in detectable new mutant site to other mutation rates according to method of design of the present invention.If detect the related locus the sudden change whether patient has Leber ' s hereditary optic neuropathy really, can prove conclusively clinically diagnostic result by quantitative detecting method of the present invention.
In some patients' kinsfolk, the normal people of some phenotype also has mutant mtDNA, is that the ratio that accounts for of unusual mtDNA is less, therefore has research to think, may there be a threshold value in the disease that mtDNA causes unusually, has only when unusual mtDNA surpasses this threshold value and just falls ill.The present invention except more reliable qualitative, prior meaning is the result of detection by quantitative, the i.e. mutant proportion that is drawn, the copy number difference that the different concns sample draws, but mutant proportion is constant, and this ratio may be relevant with the weight of morbidity, to the clinical judgment state of an illness, estimate this sick prognosis and have the certain significance, can be in order to auxiliary diagnosis, guidance treatment and prompting prognosis.
There is false-negative phenomenon hardly in quantitative detecting method of the present invention, and can draw mutant proportion, and this ratio may be relevant with the weight of morbidity.If there is the people of clinical manifestation to detect as yet male is not arranged, can predict morbidity and hereditary situation in the future through method of the present invention; After treatment, change if can work out mutant proportion, also just significant to judging curative effect.
The present invention adopts a sample with carrying out two tube reactions under the condition simultaneously since in different pipes application of sample, may have error, so the present invention is provided with parallel reactor, every kind of probe carries out 3 secondary responses at least, to reduce error as far as possible, makes quantitative result accurate.
The present invention is through groping repeatedly, overcome at present can only qualitative detection LHON11778 site technical deficiency, creatively fluorescent quantitative PCR technique and TaqMan-MGB probe technique are combined the detection that is applied to the LHON inherited disease, and realized to G11778A (G in 11778 sites → A transgenation), the detection by quantitative of G3460A (G in 3460 sites → A transgenation) or three former pathogenic mutations of T14484C (T in 14484 sites → C transgenation), for clinical detection by quantitative LHON provides a kind of tolerance range height, specificity is good, time-consuming weak point, cost is low, personal errors is little, the method of good reproducibility, thus the technology that will still be in qualitative detection LHON at present is incorporated into quantitative accurate level.The present invention is applied to clinical and during scientific research, but detection by quantitative goes out above-mentioned 3 site mutation ratios of white corpuscle Mitochondrial DNA in the peripheral blood of patients usually, in order to auxiliary diagnosis, instruct treatment and prompting prognosis.
Description of drawings
Fig. 1 is a principle schematic of the present invention;
Fig. 2 is the synoptic diagram of the saltation zone of detection by quantitative LHON of the present invention;
Fig. 3 is the synoptic diagram of another saltation zone of detection by quantitative LHON of the present invention;
Fig. 4 is the synoptic diagram of the 3rd saltation zone of detection by quantitative LHON of the present invention;
Fig. 5 is an Application Example synoptic diagram of the present invention.
Embodiment
Quantitative detecting method with the G11778A mutational site of Leber ' s hereditary optic neuropathy (LHON) is example, and is specific as follows:
Mutant target gene site areas to be checked is a Mitochondrial DNA 11701-11820 site areas, and base is G on normal 11778 sites, and base is A on 11778 sites that suddenly change; This regional sequence is:
11701 taatc?gccca?cggac?ttaca?tcctc?attac?tattc?tgcct?agcaa?actca
11751 aacta?cgaac?gcact?cacct?cacag?tcGca?tcata?atcct?ctctc?aagga
11801 cttca?aactc?tactc?ccact
Designing two probes (base is G on normal 11778 sites, and LHON sports A, and it is made as G probe, A probe respectively) on the LHON11778 site respectively
G probe:, then can hybridize, by PCR reaction and display fluorescence G as normal mtDNA copy
A probe:, then can hybridize, by PCR reaction and display fluorescence A as sudden change mtDNA copy
The principle of this PCR reaction as shown in Figure 1.
In the specific embodiment, the positive probe of described A probe, mark VIC fluorescence, promptly fluorescence A is VIC; The negative probe of described G probe, flag F AM fluorescence, promptly fluorescence G is FAM.
Perhaps change negative probe mark VIC fluorescence, positive probe mark FAM fluorescence.
Can also adopt other mark fluorescents, as long as guarantee positive probe and the negative probe different fluorescence of mark separately.
The definite principle and the method for probe:
(1) keep G-C content between 30-80%.
(2) avoid same base to repeat too much.G particularly, can not surpass 4 and more than.
(3) 5 ' end can not be G.
(4) make Cs in the probe more than Gs as far as possible.If can not satisfy, then use the probe on the complementary strand.
(5) for single probe reaction, should be between 68-70 ℃ with the Tm value that Primer Express computed in software is come out.
Be preferably the TaqMan-MGB fluorescent probe, also can adopt the Taqman fluorescent probe, the SYBRGreen fluorescence dye.
The positive probe of design can be hybridized with Mitochondrial DNA 11769-11786 site, and its sequence is:
5 ' CTC ACA GTC ACA TCA TAA 3 '; It in Fig. 2 the A probe.
The negative probe of design can be hybridized with Mitochondrial DNA 11771-11786 site, and its sequence is:
5 ' CAC AGT CGC ATC ATA A 3 '; It in Fig. 2 the G probe.
The definite principle and the method for primer:
(1) after determining, probe selects primer again.
(2 primers will be as much as possible near probe, but not overlapping.
(3 keep G-C content between 30-80%.
(4) avoid same base to repeat too much.G particularly, can not surpass 4 and more than.
(5) the Tm value of coming out with Primer Express computed in software should be between 58-60 ℃.
The sum of G and/or C base can not be above 2 in 5 bases of (6) 3 ' end.
The primer sequence of design is:
Forward primer: 5 ' TCC TCA TTA CTA TTC TGC CTA GCA AA 3 ',
Reverse primer: 5 ' GGA GTA GAG TTT GAA GTC CTT GAG AGA 3 '.
As shown in Figure 2, each suitable template is with carrying out two tube reactions simultaneously under the condition, and a pipe is placed the G probe, and another pipe is placed the A probe; The design reaction system, reaction conditions, interpretation of result: the copy number that mutant proportion=A fluorescence probe shows/(copy number that the copy number that the A fluorescence probe shows+G fluorescence probe shows); Because may there be error in application of sample in different pipes, therefore carry out parallel reactor simultaneously, each sample is done six reactions at least, and promptly each probe is done three reactions at least, asks for the mean value of copy number.
10 μ l reaction systems are specific as follows:
Embodiment one:
Suitable template 1.80 μ l,
Water 1.90 μ l,
Quantitative fluorescent PCR reacts basic reagent 5.30 μ l,
Fluorescent probe 0.20 μ l,
Primer is a pair of, each 0.40 μ l;
Reaction conditions is:
48 ℃, 2 minutes; 91 ℃, 10 minutes; 93 ℃, 30 seconds; 58 ℃, 30 seconds.
Back two step totally 50 circulations.
Embodiment two:
Suitable template 2.00 μ l,
Water 1.85 μ l,
Quantitative fluorescent PCR reacts basic reagent 5.00 μ l,
Fluorescent probe 0.25 μ l,
Primer is a pair of, each 0.45 μ l;
Reaction conditions is:
50 ℃, 2 minutes; 92 ℃, 10 minutes; 94 ℃, 30 seconds; 60 ℃, 30 seconds.
Back two step totally 50 circulations.
Embodiment three:
Suitable template 2.20 μ l,
Water 1.70 μ l,
Quantitative fluorescent PCR reacts basic reagent 4.80 μ l,
Fluorescent probe 0.30 μ l,
Primer is a pair of, each 0.50 μ l;
Reaction conditions is:
52 ℃, 2 minutes; 93 ℃, 10 minutes; 96 ℃, 30 seconds; 61 ℃, 30 seconds.
Back two step totally 50 circulations.
20 μ l reaction systems are specific as follows:
Embodiment four:
Suitable template 3.60 μ l,
Water 3.90 μ l,
Quantitative fluorescent PCR reacts basic reagent 10.45 μ l,
Fluorescent probe 0.45 μ l,
Primer is a pair of, each 0.80 μ l;
Reaction conditions is:
48 ℃, 2 minutes; 91 ℃, 10 minutes; 93 ℃, 30 seconds; 58 ℃, 30 seconds.
Back two step totally 50 circulations.
Embodiment five:
Suitable template 4.00 μ l,
Water 3.70 μ l,
Quantitative fluorescent PCR reacts basic reagent 10.00 μ l,
Fluorescent probe 0.50 μ l,
Primer is a pair of, each 0.90 μ l;
Reaction conditions is:
50 ℃, 2 minutes; 92 ℃, 10 minutes; 94 ℃, 30 seconds; 60 ℃, 30 seconds.
Back two step totally 50 circulations.
Embodiment six:
Suitable template 4.40 μ l,
Water 3.50 μ l,
Quantitative fluorescent PCR reacts basic reagent 9.55 μ l,
Fluorescent probe 0.55 μ l,
Primer is a pair of, each 1.00 μ l;
Reaction conditions is:
52 ℃, 2 minutes; 93 ℃, 10 minutes; 96 ℃, 30 seconds; 61 ℃, 30 seconds.
Back two step totally 50 circulations.
50 μ l reaction systems are specific as follows:
Embodiment seven:
Suitable template 9.50 μ l,
Water 9.40 μ l,
Quantitative fluorescent PCR reacts basic reagent 25.80 μ l,
Fluorescent probe 1.10 μ l,
Primer is a pair of, each 2.10 μ l;
Reaction conditions is:
48 ℃, 2 minutes; 91 ℃, 10 minutes; 93 ℃, 30 seconds; 58 ℃, 30 seconds;
Back two step totally 50 circulations.
Embodiment eight:
Suitable template 10.00 μ l,
Water 9.25 μ l,
Quantitative fluorescent PCR reacts basic reagent 25.00 μ l,
Fluorescent probe 1.25 μ l,
Primer is a pair of, each 2.25 μ l;
Reaction conditions is:
50 ℃, 2 minutes; 92 ℃, 10 minutes; 94 ℃, 30 seconds; 60 ℃, 30 seconds;
Back two step totally 50 circulations.
Embodiment nine:
Suitable template 10.5 μ l,
Water 9.00 μ l,
Quantitative fluorescent PCR reacts basic reagent 24.00 μ l,
Fluorescent probe 1.50 μ l,
Primer is a pair of, each 2.50 μ l;
Reaction conditions is:
52 ℃, 2 minutes; 93 ℃, 10 minutes; 96 ℃, 30 seconds; 61 ℃, 30 seconds;
Back two step totally 50 circulations.
Among the above embodiment, quantitative fluorescent PCR reacts basic reagent and selects 2 for use
*Master Mix can buy the Applied Biosystems company from the U.S..Perhaps select for use quantitative fluorescent PCR homemade or that other companies provide to react basic reagent.
Requiring of the suitable template that is used to react and the reaction of other quantitative fluorescent PCRs is identical, and the order of magnitude of conventional copy number is 10
4-5, the available wide range, 0.10～25.0ng/ μ l all can.
In like manner, as shown in Figure 3, realize the detection by quantitative to the G3460A mutational site, difference is:
Mutant target gene site areas to be checked is a Mitochondrial DNA 3401-3550 site areas, and base is G on normal 3460 sites, and base is A on 3460 sites that suddenly change; This regional sequence is:
3401?aacta?cgcaa?aggcc?ccaac?gttgt?aggcc?cctac?gggct?actac?aaccc
3451?ttcgc?tgacG?ccata?aaact?cttca?ccaaa?gagcc?cctaa?aaccc?gccac
3501?atcta?ccatc?accct?ctaca?tcacc?gcccc?gacct?tagct?ctcac?catcg
Described primer sequence is:
Forward primer FP:5 ' CCC CTA CGG GCT ACT ACA ACC 3 ',
Reverse primer RP:5 ' TTA GGG GCT CTT TGG TGA AGA G 3 ';
Described positive probe can be hybridized with Mitochondrial DNA 3451-3466 site, and its sequence is:
5 ' TTC GCT GAC ACC ATA AA 3 '; It among the figure A probe.
Described negative probe can be hybridized with Mitochondrial DNA 3451-3467 site, and its sequence is:
5 ' TTC GCT GAC GCC ATA A 3 '; It among the figure G probe.
In like manner, as shown in Figure 4, realize the detection by quantitative to the T14484C mutational site, difference is:
Mutant target gene site areas to be checked is a Mitochondrial DNA 14421-14570 site areas, and base is T on normal 14484 sites, and base is C on 14484 sites that suddenly change; This regional sequence is:
14421?ctgac?cccca?tgcct?cagga?tactc?ctcaa?tagcc?atcgc?tgtag?tatat
14471?ccaaa?gacaa?ccaTc?attcc?cccta?aataa?attaa?aaaaa?ctatt?aaacc
14521?catat?aacct?ccccc?aaaat?tcaga?ataat?aacac?acccg?accac?accgc
Described primer sequence is:
Forward primer FP:5 ' CAA TAG CCA TCG CTG TAG TAT ATC CA 3 ',
Reverse primer RP:5 ' GGG AGG TTA TAT GGG TTT AAT AGT TTT TT 3 ';
Described positive probe can be hybridized with Mitochondrial DNA 14475-14490 site, and its sequence is:
5 ' AGA CAA CCA CCA TTC C 3 '; It in Fig. 4 the C probe.
Described negative probe can be hybridized with Mitochondrial DNA 14475-14490 site, and its sequence is:
5 ' AGA CAA CCA TCA TTC C 3 '; It in Fig. 4 the T probe.
Application Example:
As shown in Figure 5, the present invention is applied to the pedigree analysis among the figure:
No. 1 is mother, not morbidity, the SSCP gel electrophoresis results suggest positive, possible heterozygosis.
No. 2 is the elder brother, 22 years old, and eyes myopia-3.0DS → 1.2, SSCP gel electrophoresis results suggest may be normal.
No. 3 is younger brother, the propositus, and 21 years old, 20 years old began to fall ill the decline of carrying out property of eyesight, the SSCP gel electrophoresis results suggest positive.
No. 4 is younger sister, and weak-eyed is specifically not quite clear from childhood.SSCP gel electrophoresis results suggest homozygous mutation.
Use the result of quantitative detecting method of the present invention:
The copy number 45233 that No. 1 G probe (negative probe) detects
The copy number 107133 that A probe (positive probe) detects
Mutant proportion=107133/ (107133+45233)=70.3%
The copy number 235666 that No. 2 G probes (negative probe) detect
The copy number 0 that A probe (positive probe) detects
Mutant proportion=0%
The copy number 0 that No. 3 G probes (negative probe) detect
The copy number 1176000 that A probe (positive probe) detects
Mutant proportion=100%
The copy number 302 that No. 4 G probes (negative probe) detect
The copy number 6946 that A probe (positive probe) detects
Mutant proportion=6946/ (6946+302)=91.2%
In this example, No. 2 samples SSCP band at the beginning is very suspicious, can't judge that he is the normal people, other sudden change is still arranged, or this sudden change is arranged.But just make of quantitative detecting method of the present invention and can draw definite conclusion.Secondly, the characteristic of this family is: mother's ratio is low, do not fall ill, but really pass to the offspring, and the offspring who has has, and what have does not have; People's ratio height of morbidity, people's ratio of not falling ill is low---and this is highly significant made a definite diagnosis in the family everyone.
Sequence table
SEQUENCE?LISTING
＜110〉Zhongshan Ophthalmic Center, Sun Yat-sen University
＜120〉quantitative detecting method of Leber ' s hereditary optic neuropathy
<160>15
<170>Patentln?version?3.2
<210>1
<211>120
<212>DNA
＜213〉human mitochondrion (mitochondrion Homo sapiens)
<220>
<221>gene
<222>(11701)...(11820)
<400>1
taatcgccca?cggacttaca?tcctcattac?tattctgcct?agcaaactca?aactacgaac 60
gcactcacct?cacagtcgca?tcataatcct?ctctcaagga?cttcaaactc?tactcccact 120
<210>2
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉design is as forward primer (forward primer)
<400>2
tcctcattac?tattctgcct?agcaaa 26
<210>3
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉design is as reverse primer (reverse primer)
<400>3
ggagtagagt?ttgaagtcct?tgagaga 27
<210>4
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉design is as positive probe
<400>4
ctcacagtca?catcataa 18
<210>5
<211>16
<212>DNA
＜213〉artificial sequence
<220>
＜223〉design is as negative probe
<400>5
cacagtcgca?tcataa 16
<210>6
<211>360
<212>DNA
＜213〉human mitochondrion (mitochondrion Homo sapiens)
<220>
<221>gene
<222>(3401)...(3550)
<400>6
aactacgcaa?aggccccaac?gttgtaggcc?cctacgggct?actacaaccc?ttcgctgacg 60
ccataaaact?cttcaccaaa?gagcccctaa?aacccgccac?atctaccatc?accctctaca 120
tcaccgcccc?gaccttagct?ctcaccatcg 150
<210>7
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉design is as forward primer (forward primer)
<400>7
cccctacggg?ctactacaac?c 21
<210>8
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉design is as reverse primer (reverse primer)
<400>8
ttaggggctc?tttggtgaag?ag 22
<210>9
<211>16
<212>DNA
＜213〉artificial sequence
<220>
＜223〉design is as positive probe
<400>9
ttcgctgaca?ccataaa 17
<210>10
<211>17
<212>DNA
＜213〉artificial sequence
<220>
＜223〉design is as negative probe
<400>10
ttcgctgacg?ccataa 16
<210>11
<211>540
<212>DNA
＜213〉human mitochondrion (mitochondrion Homo sapiens)
<220>
<221>gene
<222>(14421)...(14570)
<400>11
ctgaccccca?tgcctcagga?tactcctcaa?tagccatcgc?tgtagtatat?ccaaagacaa 60
ccatcattcc?ccctaaataa?attaaaaaaa?ctattaaacc?catataacct?cccccaaaat 120
tcagaataat?aacacacccg?accacaccgc 150
<210>12
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉design is as forward primer (forward primer)
<400>12
caatagccat?cgctgtagta?tatcca 26
<210>13
<211>29
<212>DNA
＜213〉artificial sequence
<220>
＜223〉design is as reverse primer (reverse primer)
<400>13
gggaggttat?atgggtttaa?tagtttttt 29
<210>14
<211>16
<212>DNA
＜213〉artificial sequence
<220>
＜223〉design is as positive probe
<400>14
agacaaccac?cattcc 16
<210>15
<211>16
<212>DNA
＜213〉artificial sequence
<220>
＜223〉design is as negative probe
<400>15
agacaaccat?cattcc 16
Claims (10)
1, Leber ' s hereditary optic neuropathy patient's Mitochondrial DNA Mutation quantitative detecting method is characterized in that, may further comprise the steps:
A) according to causing that the dna sequence dna of the mutant target gene site areas to be checked of Leber ' s hereditary optic neuropathy designs and synthesizes a pair of primer and the fluorescent probe between two primers, described fluorescent probe comprises positive probe and negative probe, the different fluorescence of mark separately, positive probe can be hybridized with the DNA chain of sudden change, and negative probe can be hybridized with the normal DNA chain;
B) the Mitochondrial DNA testing sample is added the positive or negative probe respectively under same reaction conditions and carry out pcr amplification, the variation of fluorescent signal in the monitoring reaction system;
C) by adding the standard model drawing standard curve of concentration known, wherein X-coordinate is represented the logarithm of initial copy number, ordinate zou is represented the Ct value, the cycle number that the Ct value is experienced when arriving the thresholding of setting for the fluorescent signal in each reaction tubes is extrapolated the concentration of original template then according to the position of testing sample in typical curve;
D) ask for the DNA initial reaction copy number that different probe shows according to typical curve, ask for the mean value of at least three reaction gained copy numbers of each probe, determine mutant proportion, the ratio of the copy number sum of the copy number that the positive fluorescence probe of this mutant proportion shows and two kinds of fluorescence probe demonstrations; The copy number difference that the sample of different concns records, and the mutant proportion of same sample is constant, obtains the detection by quantitative result in mitochondrial mutations site thus.
2, Leber ' s hereditary optic neuropathy patient's according to claim 1 Mitochondrial DNA Mutation quantitative detecting method, it is characterized in that, described fluorescent probe is the Taqman-MGB fluorescent probe, perhaps uses the Taqman fluorescent probe, SYBR Green fluorescence dye.
3, Leber ' s hereditary optic neuropathy patient's according to claim 2 Mitochondrial DNA Mutation quantitative detecting method is characterized in that, described positive probe mark VIC fluorescence, negative probe mark FAM fluorescence; Perhaps negative probe mark VIC fluorescence, positive probe mark FAM fluorescence.
4, Leber ' s hereditary optic neuropathy patient's according to claim 1 Mitochondrial DNA Mutation quantitative detecting method is characterized in that, described pcr amplification system is 10 μ l reaction systems, comprises:
Suitable template 1.80～2.20 μ l,
Water 1.70～1.90 μ l,
Quantitative fluorescent PCR reacts basic reagent 4.80～5.30 μ l,
Fluorescent probe 0.20～0.30 μ l,
Primer is a pair of, each 0.40～0.50 μ l;
Reaction conditions is:
48～52 ℃, 2 minutes; 91～93 ℃, 10 minutes; 93～96 ℃, 30 seconds; 58～61 ℃, 30 seconds; Back two step totally 50 circulations.
5, Leber ' s hereditary optic neuropathy patient's according to claim 1 Mitochondrial DNA Mutation quantitative detecting method is characterized in that, described pcr amplification system is 20 μ l reaction systems, comprises:
Suitable template 3.60～4.40 μ l,
Water 3.50～3.90 μ l,
Quantitative fluorescent PCR reacts basic reagent 9.55～1 0.45 μ l,
Fluorescent probe 0.45～0.55 μ l,
Primer is a pair of, each 0.80～1.00 μ l;
Reaction conditions is:
48～52 ℃, 2 minutes; 91～93 ℃, 10 minutes; 93～96 ℃, 30 seconds; 58～61 ℃, 30 seconds; Back two step totally 50 circulations.
6, quantitative Leber ' s hereditary optic neuropathy patient's according to claim 1 Mitochondrial DNA Mutation quantitative detecting method is characterized in that, described pcr amplification system is 50 μ l reaction systems, comprises:
Suitable template 9.50～10.5 μ l,
Water 9.00～9.40 μ l,
Quantitative fluorescent PCR reacts basic reagent 24.00～25.80 μ l,
Fluorescent probe 1.10～1.50 μ l,
Primer is a pair of, each 2.10～2.50 μ l;
Reaction conditions is:
48～52 ℃, 2 minutes; 91～93 ℃, 10 minutes; 93～96 ℃, 30 seconds; 58～61 ℃, 30 seconds; Back two step totally 50 circulations.
7, according to the described Leber ' of one of claim 4 to 6 s hereditary optic neuropathy patient's Mitochondrial DNA Mutation quantitative detecting method, it is characterized in that: it is 2 that described quantitative fluorescent PCR reacts basic reagent
*Master Mix.
8, according to the described Leber ' of one of claim 1 to 6 s hereditary optic neuropathy patient's Mitochondrial DNA Mutation quantitative detecting method, it is characterized in that, described mutant target gene site areas to be checked is a Mitochondrial DNA 11701-11820 site areas, base is G on normal 11778 sites, and base is A on 11778 sites that suddenly change; This regional sequence is:
11701?taatc?gccca?cggac?ttaca?tcctc?attac?tattc?tgcct?agcaa?actca
11751?aacta?cgaac?gcact?cacct?cacag?tcGca?tcata?atcct?ctctc?aagga
11801?cttca?aactc?tactc?ccact
Described primer sequence is:
Forward primer FP:5 ' TCC TCATTA CTATTC TGC CTA GCAAA 3 ',
Reverse primer RP:5 ' GGA GTA GAG TTT GAA GTC CTT GAG AGA 3 ';
Described positive probe can be hybridized with Mitochondrial DNA 11769-11786 site, and its sequence is: 5 ' CTC ACA GTC ACA TCA TAA 3 ';
Described negative probe can be hybridized with Mitochondrial DNA 11771-11786 site, and its sequence is: 5 ' CACAGT CGCATCATAA 3 '.
9, according to the described Leber ' of one of claim 1 to 6 s hereditary optic neuropathy patient's Mitochondrial DNA Mutation quantitative detecting method, it is characterized in that, described mutant target gene site areas to be checked is a Mitochondrial DNA 3401-3550 site areas, base is G on normal 3460 sites, and base is A on 3460 sites that suddenly change; This regional sequence is:
3401?aacta?cgcaa?aggcc?ccaac?gttgt?aggcc?cctac?gggct?actac?aaccc
3451?ttcgc?tgacG?ccata?aaact?cttca?ccaaa?gagcc?cctaa?aaccc?gccac
3501?atcta?ccatc?accct?ctaca?tcacc?gcccc?gacct?tagct?ctcac?catcg
Described primer sequence is:
Forward primer FP:5 ' CCC CTACGG GCTACTACAACC 3 ',
Reverse primer RP:5 ' TTAGGG GCT CTTTGG TGAAGAG 3 ';
Described positive probe can be hybridized with Mitochondrial DNA 3451-3466 site, and its sequence is:
5’TTC?GCT?GACACCATAAA?3’；
Described negative probe can be hybridized with Mitochondrial DNA 3451-3467 site, and its sequence is: 5 ' TTC GCT GAC GCC ATAA 3 '.
10, according to the described Leber ' of one of claim 1 to 6 s hereditary optic neuropathy patient's Mitochondrial DNA Mutation quantitative detecting method, it is characterized in that, described mutant target gene site areas to be checked is a Mitochondrial DNA 14421-14570 site areas, base is T on normal 14484 sites, and base is C on 14484 sites that suddenly change; This regional sequence is:
14421?ctgac?cccca?tgcct?cagga?tactc?ctcaa?tagcc?atcgc?tgtag?tatat
14471?ccaaa?gacaa?ccaTc?attcc?cccta?aataa?attaa?aaaaa?ctatt?aaacc
14521?catat?aacct?ccccc?aaaat?tcaga?ataat?aacac?acccg?accac?accgc
Described primer sequence is:
Forward primer FP:5 ' CAATAG CCATCG CTG TAG TATATC CA 3 ',
Reverse primer RP:5 ' GGG AGG TTA TAT GGG TTTAAT AGT TTT TT 3 ';
Described positive probe can be hybridized with Mitochondrial DNA 14475-14490 site, and its sequence is:
5’AGA?CAA?CCA?CCA?TTC?C?3’；
Described negative probe can be hybridized with Mitochondrial DNA 14475-14490 site, and its sequence is: 5 ' AGA CAA CCA TCA TTC C 3 '.
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| CNB2004100276234A CN100348732C (en) | 2004-06-15 | 2004-06-15 | Quantitative detection of Leber's genetic optic nerve disease |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102465180A (en) * | 2010-11-19 | 2012-05-23 | 上海基康生物技术有限公司 | Method for detecting gene mutation of gene K-ras related to colorectal and rectal cancers and related kit |
| CN102146443B (en) * | 2011-01-06 | 2013-01-16 | 中国科学院昆明动物研究所 | Specific primer for detecting Leber hereditary optic neuropathy mitochondrial DNA mutation G10680A |
| CN103468822B (en) * | 2013-09-30 | 2015-08-26 | 吴南 | A kind of detection kit of Lumbar Intervertebral Disc Degeneration related SNP |
| CN103981255B (en) * | 2014-04-01 | 2016-06-01 | 重庆医科大学附属儿童医院 | A kind of Leber hereditary optic neuropathy external diagnosis reagent case |
| CN105331719A (en) * | 2015-11-27 | 2016-02-17 | 首都医科大学宣武医院 | Method, primers and kit for detecting LEBER (Leber Hereditary Optic Neuroretinopathy) pathogenic gene mutation |
| CN106755335B (en) * | 2016-11-30 | 2020-06-05 | 中山大学中山眼科中心 | Detection primer, kit and detection method for gene mutation of Leber hereditary optic neuropathy mitochondria DNA |
| CN109207562A (en) * | 2018-11-06 | 2019-01-15 | 郑州大学第附属医院 | Unicellular mitochondria copy number detection method |
| CN110172506A (en) * | 2019-05-05 | 2019-08-27 | 重庆医科大学附属儿童医院 | A kind of kit and its application for quantitative detection Leber hereditary optic neuropathy mutant |
| CN110894524B (en) * | 2019-05-13 | 2023-05-05 | 嘉兴雅康博医学检验所有限公司 | Method for rapidly preparing gene mutation reference |
| CN116334215A (en) * | 2023-04-04 | 2023-06-27 | 温州谱希基因科技有限公司 | LHON detection kit |
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| US5185244A (en) * | 1989-12-08 | 1993-02-09 | Emory University | Genetic test for hereditary neuromuscular disease |
| CN1143117A (en) * | 1996-04-01 | 1997-02-19 | 中国人民解放军第二军医大学 | Detecting method for Leber hereditary optic nurve pathologic change and its kit |
| US5670320A (en) * | 1994-11-14 | 1997-09-23 | Emory University | Detection of mitochondrial DNA mutation 14459 associated with dystonia and/or Leber's hereditary optic neuropathy |
| US6441149B1 (en) * | 1998-06-15 | 2002-08-27 | Mitokor | Diagnostic method based on quantification of extramitochondrial DNA |
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2004
- 2004-06-15 CN CNB2004100276234A patent/CN100348732C/en not_active Expired - Fee Related
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|---|---|---|---|---|
| US5185244A (en) * | 1989-12-08 | 1993-02-09 | Emory University | Genetic test for hereditary neuromuscular disease |
| US5670320A (en) * | 1994-11-14 | 1997-09-23 | Emory University | Detection of mitochondrial DNA mutation 14459 associated with dystonia and/or Leber's hereditary optic neuropathy |
| CN1143117A (en) * | 1996-04-01 | 1997-02-19 | 中国人民解放军第二军医大学 | Detecting method for Leber hereditary optic nurve pathologic change and its kit |
| US6441149B1 (en) * | 1998-06-15 | 2002-08-27 | Mitokor | Diagnostic method based on quantification of extramitochondrial DNA |
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| Title |
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| Rapid quantification of the heteroplasmy of mutantmitochondrial DNAs in Leber's hereditary optic neuropathyusing the Invader technology. Yukihiko Mashima et al. CLINICAL BIOCHEMISTRY,Vol.37. 2004 * |
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