CN1143117A

CN1143117A - Detecting method for Leber hereditary optic nurve pathologic change and its kit

Info

Publication number: CN1143117A
Application number: CN 96116296
Authority: CN
Inventors: 周海林; 邹祝英; 胡育新
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 1996-04-01
Filing date: 1996-04-01
Publication date: 1997-02-19

Abstract

Based on the feature that the patient has point mutation of chondriosome, which results in the loss of restriction enzyme SfaNI recognition site in nomal chondriosome DNA, the present invention detects the said disease according to that the PCR cloning product of specific anticoagulant, hemolysis liquid and one pair of primers can't be digected and cut by restriction enzyme SfaNI. The present invention provides diagnosis method and reagent kit for the clear detection of Leber's hereditary optic hurve pathologic change.

Description

Leber hereditary optic neuropathy detection method and test kit thereof

The present invention relates to the physianthropy health field, is a kind of method and test kit thereof of the Leber of detection hereditary optic neuropathy.

Leber hereditary optic neuropathy is a kind of much illness in eye of matrilinear inheritance.Be everlasting pubescence morbidity, the male sex sees that it is impaired mainly to show as optic nerve more, promptly eyes successively or occur the rapid decline of central vision simultaneously present optic atrophy after the several months.Clinical symptom that this is sick and eyeground performance and acute retrobulbar neuritis, papillitis are very alike, therefore normal clinically easily mistaken diagnosis.(see: 1. " the four example reports of LeberShi disease one family " Chinese ophthalmology disease magazine was rolled up for the 2nd phase in 1993 the 9th for the main dependence familial inheritance history of clarifying a diagnosis clinically; 2. " LeberShi leber's disease " practical ophthalmology magazine was rolled up o. 11th in 1991 the 9th), but some patient is difficult to get the familial inheritance history, China carries out Population control at present in addition, and great majority are the only-child, and the familial inheritance history is difficult for appearing.Up to the present, yet there are no the method for casting aside the familial inheritance history and detecting Leber hereditary optic neuropathy.

The objective of the invention is to provides a kind of method and test kit thereof that need not rely on the familial inheritance history just can detect Leber hereditary optic neuropathy for clinical.

The present inventor finds that according to domestic and international and own to the result of this disease from molecule genetics research in recent years there is mitochondrial point mutation in the patient of Leber hereditary optic neuropathy.Because the existence of plastosome point mutation, make the recognition site forfeiture of the restriction enzyme SfaNI of human high conservative in the normal mtdna.And then, make plastosome production capacity decrease in efficiency, cause cellular oxidation and respiratory dysfunction, especially make the anterior optic nerve unmyelinated nerve fiber that is subject to the influence of plastosome production capacity most, the aixs cylinder of optic ganglion cell, because of the cell energy supply is not enough to keep their complete structure and function, degeneration takes place at last change.Mitochondrial point mutation is regarded as pathogenic relevant with this disease, utilizes PCR method to detect mitochondrial point mutation, just can reach and clearly detect this sick purpose.

Method and this test kit of use of detection Leber hereditary optic neuropathy provided by the invention, be in taking 1 milliliter of venous blood of patient, add specific antithrombotics, extract total DNA (comprising Mitochondrial DNA) through degeneration methods, with the mtDNA fragment of PCR method amplifying specific, again with the cutting of SfaNI restriction enzyme.The normal people, product through pcr amplification can be cut by the SfaNI restriction enzyme, and Leber hereditary optic neuropathy patient, its PGR amplified production then can not be cut by the SfaNI restriction enzyme, can find mitochondrial point mutation from the recognition site forfeiture of restriction enzyme SfaNI, thereby can clearly detect this disease.

Below further set forth concrete preparation of the present invention and operation steps.

The reagent of Leber hereditary optic neuropathy detection kit of the present invention and hemolysate preparation or production unit:

1, specific antithrombotics:

EDTANa 2g

NaCl 0.85g

Add water to the dissolving of 100ml room temperature, 1 milliliter of venous blood of the specific antithrombotics anti-freezing of per 100 microlitres.

2, a pair of primer: by Shanghai plant physiology institute or Shanghai finished product that biochemical research provides.

5 ' AGCCCTCGTAGTAACAGCCA-3 ' light chain POS.11641～11660, every microlitre 500 nanograms.

5 ' GGAGTATAGGGCTGTGACTA-3 ' heavy chain POS.11980～11961, every microlitre 500 nanograms.

3, PCR reagent:

The dNTP200 nanogram(ng)

10 * Buffer, 5 microlitres

TaqDNA polymerase 1.2 units

4, restriction enzyme SfaNI (New England Biolabs product)

NEBuffer3 (New England Biolabs product)

5, hemolysate: Tris-Hcl 10mmol/L PH7.5 NaCl 10mmol/L, MgCl ₂5mmol/L.

The composition of Leber hereditary optic nerve detection kit of the present invention

1, specific antithrombotics one is managed

2, each pipe of a pair of primer

3, dNTP one pipe

4, TaqDNA polymerase one pipe

5,10 * Buffer, one pipe

6, restriction enzyme SfaNI one pipe

7, NEBuffer3 one pipe

8, hemolysate one pipe

Concrete operation method of the present invention is as follows;

Extract 1 milliliter of patient's peripheric venous blood, add in specific antithrombotics 100 microlitres, become anticoagulation behind the mixing gently, get anticoagulation 50 microlitres again and place 400 microlitre hemolysates mixing gently, left standstill 5 minutes, abandon supernatant through 4000rpm after centrifugal 5 minutes, add 100 microlitre physiological saline mixings, put 100 ℃ of boiling water 7 minutes, 4000rpm of short duration centrifugal (being no more than 1 minute), get supernatant 10 microlitres and make each 20pmol of a pair of primer of template (i.e. 0.2 micrograms of DNA) adding, the dNTP200 nanogram(ng), 10 * Buffer, 5 microlitres add distilled water to 50 microlitre again, putting into the PCR thermal cycler increases: 95 ℃ of sex change 4 minutes, treat to take out when the temperature follow procedure is reduced to 70 ℃ automatically, add TaqDNA polymerase 1.2 units, continue then with 94 ℃ of sex change 40 seconds, 55 ℃ of renaturation 40 seconds, 70 ℃ are extended 30 seconds is a circulation, totally 30 circulations, last 72 ℃ are continued to extend 7 minutes.Learn from else's experience DNA fragment specific 20 microlitres behind the pcr amplification, put in 37 ℃ of water baths digestion 6 hours with 1 unit of SfaNI restriction endonuclease, 2.3 microlitre NEBuffer3, get 10 microlitres and add analysis under the 2% ethidium bromide electrophoresis apparatus, normal person presents two fragments of 190bp, 150bp, and Leber hereditary optic neuropathy patient only presents fragment of 340bp because of its DNA can not be cut by the SfaNI restriction enzyme.

The invention has the beneficial effects as follows to have solved and to lean on the familial inheritance history to diagnose the problem of Leber hereditary optic neuropathy in the past, for the clinical Leber hereditary optic neuropathy that clearly detects provides easy, a reliable method and test kit.

Claims

1, a kind of test kit of clinical detection Leber hereditary optic neuropathy, it is characterized in that forming, be specially by antithrombotics, hemolysate, a pair of primer, dNTP, TaqDNA polymerase, 10 * Buffer, SfaNI restriction enzyme, each pipe of NEBuffer3:

(1.1) specific antithrombotics

EDTANa ₂ 2g

NaCl 0.85g

Add water to 100ml

(1.2) each pipe of a pair of primer

5GGAGTATAGGGCTGTGACTA-3 ' heavy chain POS.11980～11961, every microlitre 500 nanograms.

(1.3)dNTP

DNTP 200 nanogram(ng)s;

(1.4) TaqDNA polymerase;

TaqDNA polymerase 1.2 units:

(1.5)10×Buffer

10 * Buffer, 5 microlitres;

(1.6) SfaNI restriction enzyme

SfaNI (New England Biolabs product)

(1.7)NEBuffer3

NEBuffer3 (New England Biolabs product) '

(1.8) hemolysate:

Tris-Hcl?10mmol/L.pH7.5

NaCl 10mmol/L，

MgCl ₂ 5mmol/L。

2, a kind of method of clinical detection Leber hereditary optic neuropathy is characterized in that in turn including the following steps:

(2.1) extract 1 milliliter of patient's peripheric venous blood, add in specific antithrombotics 100 microlitres, shake up gently, become anticoagulation;

(2.2) get anticoagulation 50 microlitres and place 400 microlitre hemolysates to shake up gently, left standstill 5 minutes, abandon supernatant after centrifugal 5 minutes through 4000rpm, add 100 microlitre physiological saline mixings, put 100 ℃ of boiling water 7 minutes, 4000rpm is of short duration centrifugal, gets supernatant 10 microlitres and makes template;

(2.3) add each 20pmol of a pair of primer in template 10 microlitres, the dNTP200 nanogram(ng), 10xBuffer 5 microlitres, add distilled water to 50 microlitre again, put in the PCR thermal cycler and circulate: 95 ℃ of sex change 4 minutes, treat to take out when the temperature follow procedure is reduced to 70 ℃ automatically, add TaqDNA polymerase 1.2 units, continuing then to extend 30 seconds for 40 seconds, 70 ℃ with 94 ℃ of sex change 40 seconds, 55 ℃ of renaturation is a circulation, totally 30 circulations, last 72 ℃ are continued to extend 7 minutes, become the DNA fragment specific behind pcr amplification;

(2.4) learn from else's experience DNA fragment specific 20 microlitres behind the pcr amplification were put in 37 ℃ of water baths digestion 6 hours with 1 unit of SfaNI restriction endonuclease and 2.3 microlitre NEBuffer3;

(2.5) get last resultant 10 microlitres in the step (2.4), add 2% ethidium bromide, analyze under electrophoresis apparatus, normal person presents two fragments of 190dp, 150dp, and Leber hereditary optic neuropathy patient only presents fragment of 340dp.