CN1288254C - Kit for diagnosing gene of Leber optic neuropathy in heredity and detecting method - Google Patents
Kit for diagnosing gene of Leber optic neuropathy in heredity and detecting method Download PDFInfo
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- CN1288254C CN1288254C CNB2005100060978A CN200510006097A CN1288254C CN 1288254 C CN1288254 C CN 1288254C CN B2005100060978 A CNB2005100060978 A CN B2005100060978A CN 200510006097 A CN200510006097 A CN 200510006097A CN 1288254 C CN1288254 C CN 1288254C
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Abstract
The present invention discloses a kit for diagnosing genes of Leber optic neuropathy in heredity and a detecting method thereof, which belongs to the method for diagnosing hereditary diseases by using genes. In the present invention, a site specificity PCR primer is designed and synthesized on the basis that more than 90% of mitochondrial DNA abrupt change of patients with Leber optic neuropathy in heredity belongs to the abrupt change of three constitutional pathopoiesia sites (G11778A, G3460A and T14484C); a full blood sample is directly used as a DNA template of PCR; the allele specificity multiplex PCR technology and the disposable PCR reaction of a single tube are used; three constitutional pathopoiesia mutational sites of mitochondrion DNA of an LHON patient are simultaneously detected. The present invention has the advantages of convenience, quickness, high efficiency, low cost, good specificity, trace consumption of blood sample, etc., and provides a simple and reliable method and a kit for quickly diagnosing genes for LHON patients in clinic. The present invention has great popularization and application value.
Description
Technical field
The present invention relates to utilize the method for gene diagnosis heredopathia, a kind of specifically gene diagnosis kit and detection method thereof of Leber hereditary optic neuropathy.
Background technology
German eye doctor Theodor Leber reported first one familial inheritance optic neuropathy in 1871, mainly show as acute or inferior anxious bilateral central light loss, only transmit by maternal, often involve young man, now be called Leber hereditary optic neuropathy (Leber ' s hereditary optic neuropathy, LHON).Wallace in 1988 etc. find that first LHON is relevant with Mitochondrial DNA (mtDNA) G11778A sudden change, so far the related mtDNA sudden change of having reported has kind more than 40, wherein the LHON patient of 90%-95% is relevant with three primary mtDNA sudden changes (G11778A, G3460A and T14484C).Not agnate various former pathogenic mutation site distribution situation difference, LHON patient in the white race ethnic group more than 90% has G11778A, a sudden change in G3460A and the T14484C site, 3 sudden changes of this of European patient account for 50%～70%, 15%～25% and 15% respectively; G11778A site mutation rate is very high in the asian population LHON family, accounts for more than 90%.Guo Xiangming etc. are to 3 former pathogenic mutation G11778A of 140 routine Chinese LHON patient mtDNA, and the spectral decomposition that suddenlys change of G3460A and T14484C site finds that its mutation rate is respectively 92.9%, 1.4% and 5.7%.Therefore, utilizing gene differential diagnosis means examination LHON patient's first-selection is exactly to analyze 3 former pathogenic mutation sites of mtDNA.
Difficult at the early clinical diagnosis of Leber hereditary optic neuropathy, Sporadic cases (promptly not having family's case history) particularly utilizes gene diagnosis to become to make a definite diagnosis the powerful measure of this disease.The mtDNA site mutation detection method of LHON is a lot, its principle all is based on round pcr, comprise traditional allele-specific PCR, SSCP-PCR, RFLP-PCR and Mitochondrial Genome Overview dna sequencing etc., the first step of these methods all needs to extract the mtDNA in the peripheral blood sample, have also from isolated lymphocytes, required time is long, expense is higher.Traditional allele-specific PCR has characteristics such as quick, simple than additive method, but a PCR can only differentiate a mutational site; The used restriction enzyme of RFLP-PCR method is subjected to all multifactor impacts usually to cause endonuclease reaction to be failed, and causes the result to judge by accident; SSCP-PCR method expense height not only consuming time, and the more high factor affecting of requirement for experiment condition carrying out the clinical gene diagnosis of LHON; Reason only is adapted to scientific research and the mtDNA genome sequencing costs an arm and a leg etc.A kind of complete blood cell lysate that directly utilizes of reports such as Yohsuke Minatogawa carries out the PCR reaction as template, and this method needs microLYSISTM buffer (COSMO BIO Co.Ltd) to come lysing cell.
Patent of invention related to the present invention is on February 19th, 1997 disclosed " Leber hereditary optic neuropathy detection method and a test kit thereof (application number: 96116296.1) ", this invention is only at the LHON patient of Mitochondrial DNA G11778A site mutation, need hemolysate to extract total DNA through sex change, trivial step such as pcr amplification goal gene segment and SfaNI digestion with restriction enzyme and electrophoresis, not only consuming time but also increased corresponding cost.
Therefore, set up that a cover is quick, simple and easy, LHON gene diagnosis technical system that specificity is well highly sensitive again and prepare its test kit and will help the clinical gene diagnosis that penetration and promotion should disease.
Show that from the pertinent data retrieval that comprises Chinese patent utilize whole blood allele-specific multiple PCR technique, the disposable PCR of single tube detects 3 primary mutational sites of LHON Mitochondrial DNA simultaneously and do not see its relevant report as yet.
Summary of the invention
The objective of the invention is to the detection method and the test kit thereof that provide a kind of quick, simple and easy gene diagnosis Leber hereditary optic neuropathy for clinical.
The present invention is according to both at home and abroad to the result of the molecule genetics research of Leber hereditary optic neuropathy, the Mitochondrial DNA Mutation of finding human Leber hereditary optic neuropathy patient more than 90% belongs to 3 primary pathogenic mutation site (G11778A, G3460A and T14484C), utilize the point mutation of allelotrope locus specificity PCR method detection line mitochondrial DNA, just can reach the purpose that clearly detects this heredopathia.
The gene diagnosis kit of Leber hereditary optic neuropathy provided by the invention, be to make dna profiling with direct adding whole blood, utilize allelotrope locus specificity multiple PCR method, detect 3 former disease cause mutation sites (G11778A, G3460A and T14484C) of LHONmtDNA simultaneously.The upstream primer 3 '-end of 3 pairs of PCR primers that this test kit comprises is a mutating alkali yl, and by the PCR reaction conditions of optimizing, PCR only can carry out amplified reaction in having patient's blood sample of point mutation, and the normal people does not have the product of pcr amplification.Therefore, the PCR product has or not and the segmental size of PCR product through agarose gel electrophoresis, the pyridine dyeing of bromination second and ultraviolet detection PCR product, just can make a definite diagnosis this disease, is 222bp if promptly the PCR product is long, then the LHON patient who suddenlys change for G11778A; The PCR product is 294bp, then is T14484C sudden change patient; The PCR product is 360bp, is G3460A site mutation patient; If 2 bands occur, be 222bp such as a PCR product is long, another be 294bp, illustrates then that this LHON patient is contained G11778A and T14484C is two to suddenly change, remaining and the like; If 3 bands occur, illustrate that then this LHON patient contains G11778A, G3460A and T14484C three sudden changes.In general occur the dibit point mutation among the LHON patient and be very very rare, rarely seen external report 1 example, and the LHON patient who has these 3 site mutations does not simultaneously appear in the newspapers at present yet.
The gene diagnosis kit of Leber hereditary optic neuropathy provided by the invention, it is characterized in that: this test kit is primarily aimed at the gene diagnosis of the Leber hereditary optic neuropathy patient with 3 primary pathogenic mutation sites (G11778A, G3460A and T14484C).
The gene diagnosis kit of Leber hereditary optic neuropathy provided by the invention, it is characterized in that: this gene diagnosis kit is directly to utilize whole blood to make the dna profiling of PCR, do not need the DNA in the purifying blood sample, and only need the micro-blood sample of 0.5-1.0 μ l.
The gene diagnosis kit of Leber hereditary optic neuropathy provided by the invention, it is characterized in that: this gene diagnosis kit is to utilize allelotrope locus specificity multiplex PCR (MAS-PCR) method, by optimizing the PCR reaction conditions, realize that the disposable PCR of single tube reacts 3 former disease cause mutation sites (G11778A, G3460A and T14484C) of detecting mtDNA simultaneously and diagnoses the LHON disease.
The gene diagnosis kit building block of Leber hereditary optic neuropathy of the present invention sees the following form-1:
The gene diagnosis kit of table-1Leber hereditary optic neuropathy is formed
| 1.TaKaRa Ex Taq(5U/μl) 2.10×Ex Taq Buffer(Mg 2+Plus) 5. 6 * Loading Buffer of 3.dNTP Mixture (each 2.5mM) 4.MAS PCR primers mixture (each 20pM) | 50μl 1ml 800μl 600μl 1ml |
The PCR primer is according to human mitochondrial genomic dna (mtDNA) sequence and mtDNA G11778A, G3460A and T14484C point mutation design in the mentioned reagent box, promptly 3 '-tip designs at 3 upstream PCR primers is a mutating alkali yl, and its dna sequence dna sees the following form-2:
Table-2MAS PCR primers forms and sequence
| The primer title | The nucleic acid position | Primer sequence |
| MS11778F MS14484F MS3460F 11778R 14484R 3460R | 11759～11778nt 14460～14484nt 3441～3460nt 11961～11980nt 14734～14753nt 3781～3800nt | 5’-TACGAACGCACTCACAGACA-3’ 5′-CTGTAGTATATCCAAAGACAACGAC-3′ 5’-ACTACAACCCTTCGCTGTCA-3’ 5’-GGAGTATAGGGCTGTGACTA-3’ 5’-GGGTCATTGGTGTTCTTGTA-3’ 5’-AGGGTGGAGAGGTTAAAGGA-3’ |
Method with mentioned reagent box gene diagnosis Leber hereditary optic neuropathy of the present invention follows these steps to carry out:
1) according to the form below preparation MAS-PCR reaction system (25 μ l), mixing places on the PCR instrument;
| 10×Ex Taq Buffer(Mg 2+Plus) dNTP Mixture (each 2.5mM) MAS PCR primers mixture (each 20pM) whole blood sample TaKaRa Ex Taq (5U/ μ l) dH2O | 2.5μl 1.0μl 1.5μl 0.5μl 0.13μl 19.37μl |
2) by carrying out pcr amplification reaction with following condition:
The 94 ℃ of pre-sex change of 4min → → 72 ℃ of 4min → 4 ℃ preservations of 30 circulations of 94 ℃ of 30sec
59.8℃ 30sec
72℃ 45sec
3) after reaction finishes, add 5 μ l, 6 * Loading Buffer and mix, get 10-15 μ l and behind agarose gel electrophoresis 15-20 minute of 1.5%-1.8%, on ultraviolet device, observe.If the PCR product of 222bp size occurs, then be the LHON patient of G11778A point mutation; If the PCR product of 294bp size occurs, then be the LHON patient of T14484C point mutation; If the PCR product of 360bp size occurs, then be the LHON patient of G3460A point mutation; If 2 bands occur, be 222bp such as a PCR product is long, another be 294bp, illustrates then that this LHON patient is contained G11778A and T14484C is two to suddenly change, remaining and the like; If 3 bands occur, illustrate that then this LHON patient contains G11778A, three site mutations of G3460A and T14484C; If there is not specific PCR product band, then can gets rid of it more than 90% and be LHON patient.
The invention has the beneficial effects as follows: because the present invention is a dna profiling of making PCR with direct adding whole blood, utilize the MAS-PCR method, by optimizing the PCR reaction conditions, realize that the disposable PCR reaction of single tube detects 3 primary pathogenic mutation sites (G11778A, G3460A and T14484C) of LHON mtDNA simultaneously.Therefore, the method that this method detects LHON mtDNA site mutation more at present has tangible three big advantages: 1) directly adopt the periphery whole blood to make dna profiling, do not need extra lysis and purifying mtDNA, therefore shorten the time of diagnosis greatly and reduce corresponding cost; 2) required blood sample amount is few, only needs tip can diagnose once the sample of bleeding, and this has also solved the difficult problem of the elderly and child's blood specimen collection, and blood sample can be stored on the filter paper and diagnose by mailing; 3) the disposable PCR of single tube detects 3 former pathogenic mutation sites of LHONmtDNA simultaneously, can cover the diagnosis of the LHON patient more than 90%, and have advantages such as quicker, simple and easy and efficient.This shows that this method has overcome the shortcoming of the clinical gene diagnosis of present LHON, be very suitable for the quick clinical gene diagnosis of LHON, have huge penetration and promotion using value.
Description of drawings
Fig. 1: utilize test kit provided by the invention to carry out the electrophoresis result that allele-specific multiplex PCR method detects 3 primary pathogenic mutations of LHONmtDNA site.Wherein, M:DNA molecular weight standard X174-Hinc IIdigest; LHON patient's blood sample of A:mtDNA G11778A point mutation; LHON patient's blood sample of B:mtDNAT14484C point mutation; LHON patient's blood sample of C:mtDNA G3460A point mutation; The LHON patient of D:mtDNA G11778A and T14484C point mutation mixes blood sample; The LHON patient of E:mtDNA G11778A and G3460A point mutation mixes blood sample; The LHON patient of F:mtDNA T14484C and G3460A point mutation mixes blood sample; The LHON patient of G:mtDNA G11778A, T14484C and G3460A point mutation mixes blood sample; H: normal people's blood sample (negative control).
Embodiment
Below the invention will be further described by specific embodiment
Embodiment 1: the gene diagnosis kit of Leber hereditary optic neuropathy of the present invention
This test kit is formed (400 secondary response amount):
| 1.TaKaRa Ex Taq(5U/μl) 2.10×Ex Taq Buffer(Mg 2+Plus) 5. 6 * Loading Buffer of 3.dNTP Mixture (each 2.5mM) 4.MAS PCR primers mixture (each 20pM) | 50μl 1ml 800μl 600μl 1ml |
MAS PCR primers forms and dna sequence dna in the last table
| The primer title | The nucleic acid position | Primer sequence |
| MS11778F MS14484F MS3460F 11778R 14484R 3460R | 11759～11778nt 14460～14484nt 3441～3460nt 11961～11980nt 14734～14753nt 3781～3800nt | 5’-TACGAACGCACTCACAGACA-3’ 5′-CTGTAGTATATCCAAAGACAACGAC-3′ 5’-ACTACAACCCTTCGCTGTCA-3’ 5’-GGAGTATAGGGCTGTGACTA-3’ 5’-GGGTCATTGGTGTTCTTGTA-3’ 5’-AGGGTGGAGAGGTTAAAGGA-3’ |
Embodiment 2: the detection method of gene diagnosis Leber hereditary optic neuropathy
Use the test kit of embodiment 1, follow these steps to carry out:
1) according to the form below preparation MAS PCR reaction system (25 μ l), mixing places on the PCR instrument;
| 10×Ex Taq Buffer(Mg 2+Plus) dNTP Mixture (each 2.5mM) MAS PCR primers mixture (each 20pM) whole blood sample TaKaRa Ex Taq (5U/ μ l) dH2O | 2.5μl 1.0μl 1.5μl 0.5μl 0.13μl 19.37μl |
2) by carry out the PCR reaction with following condition:
The 94 ℃ of pre-sex change of 4min → → 72 ℃ of 4min → 4 ℃ preservations of 30 circulations of 94 ℃ of 30sec
59.8℃ 30sec
72℃ 45sec
3) should finish after, add 5 μ l, 6 * Loading Buffer and mix, get 10-15 μ l and behind agarose gel electrophoresis 15-20 minute of 1.5%-1.8%, on ultraviolet device, observe.If the PCR product of 222bp size occurs, then be the LHON patient of G11778A point mutation; If the PCR product of 294bp size occurs, then be the LHON patient of T14484C point mutation; If the PCR product of 360bp size occurs, then be the LHON patient of G3460A point mutation; If 2 bands occur, be 222bp such as a PCR product is long, another be 294bp, illustrates then that this LHON patient is contained G11778A and T14484C is two to suddenly change, remaining and the like; If 3 bands occur, illustrate that then this LHON patient contains G11778A, three site mutations of G3460A and T14484C; If there is not specific PCR product band, then can gets rid of it more than 90% and be LHON patient's (see figure 1).
Claims (1)
1, a kind of gene diagnosis kit of Leber hereditary optic neuropathy, it is characterized in that this test kit by concentration be 5U/ μ l TaKaRa Ex Taq 50 μ l, contain Mg
2+10 * Ex Taq damping fluid 1ml, dNTP mixture 800 μ l that concentration respectively is 2.5mM, concentration respectively be that the MAS PCR primer mixture 600 μ l of 20pM and 6 * sample-loading buffer 1ml form; Wherein, MAS PCR primer mixture is made up of MS11778F/11778R, MS14484F/14484R and three pairs of PCR primers of MS3460F/3460R; Described MS11778F primer sequence is 5 '-TACGAACGCACTCACAGACA-3 ', is positioned at mtDNA11759～11778nt; Described 11778R primer sequence is 5 '-GGAGTATAGGGCTGTGACTA-3 ', is positioned at mtDNA 11961～11980nt; Described MS14484F primer sequence is 5 '-CTGTAGTATATCCAAAGACAACGAC-3 ', be positioned at mtDNA 14460～14484nt; Described 14484R primer sequence is 5 '-GGGTCATTGGTGTTCTTGTA-3 ', is positioned at mtDNA 14734～14753nt; Described MS3460F primer sequence is 5 '-ACTACAACCCTTCGCTGTCA-3 ', is positioned at mtDNA 3441～3460nt; Described 3460R primer sequence is 5 '-AGGGTGGAGAGGTTAAAGGA-3 ', is positioned at mtDNA3781～3800nt.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101135666B (en) * | 2007-10-01 | 2010-04-21 | 中国人民解放军第三军医大学 | Reagent kit for detecting elevated plain pneumochysis susceptibility based on mitochondria DNA C3970T mononucleotide polymorphism |
| CN101135667B (en) * | 2007-10-01 | 2010-05-26 | 中国人民解放军第三军医大学 | Reagent kit for detecting elevated plain pneumochysis susceptibility based on mitochondria DNA G3010A mononucleotide polymorphism |
| CN101135665B (en) * | 2007-10-01 | 2010-05-26 | 中国人民解放军第三军医大学 | Reagent kit detecting elevated plain pneumochysis susceptibility based on mitochondria DNA T6680C mononucleotide polymorphism |
| 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 |
| CN104487575B (en) * | 2012-07-28 | 2017-09-19 | 深圳华大基因股份有限公司 | NMNAT1 mutators, the primer for detecting it, kit and method with and application thereof |
| 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 |
| CN104046687B (en) * | 2014-05-29 | 2015-11-25 | 同昕生物技术（北京）有限公司 | A kind of Leber hereditary optic neuropathy gene diagnosis kit or reagent |
| CN104805210B (en) * | 2015-04-30 | 2017-02-22 | 浙江大学 | Gene detection method of Leber's hereditary optic neuropathy, (LHON) gene chip and kit |
| 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 |
| 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 |
| CN114250296B (en) * | 2022-02-09 | 2024-02-20 | 深圳市妇幼保健院 | Primer group for detecting Leber hereditary optic neuropathy based on nucleic acid mass spectrum |
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