CN205528786U

CN205528786U - Mitochondria brain myopathy, lactic acidosis and palsy appearance disease gene sequencing kit

Info

Publication number: CN205528786U
Application number: CN201620183292.1U
Authority: CN
Inventors: 卿志荣; 郑帮
Original assignee: Shanghai Repodx Biotechnology Co Ltd
Current assignee: Jiangsu Rui Rui Biotechnology Co., Ltd.; SHANGHAI REPODX BIOTECHNOLOGY CO., LTD.
Priority date: 2016-03-10
Filing date: 2016-03-10
Publication date: 2016-08-31
Anticipated expiration: 2026-03-10

Abstract

The utility model discloses a mitochondria brain myopathy, lactic acidosis and palsy appearance disease gene sequencing kit, a serial communication port, the kit include box body 1, paper liner 2, paper liner 2 arrange box body 1 in, be equipped with 4 vessel bore and be equipped with 4 reagent pipes that are equipped with detect reagent on the paper liner 2, be respectively being equipped with PCR amplify forward primer reagent pipe 3 with be equipped with PCR amplify reverse primer reagent pipe 4, be equipped with the taq enzyme reagent pipe 5, the reagent pipe 6 of PCR reaction liquid is equipped with, wherein the reverse primer of PCR forward primer and PCR increase all can use as sequencing primer alone. This kit can be used for mitochondria brain myopathy, lactic acidosis and palsy appearance disease dependency basis to detect because of the order -checking of focus mutational site A3243G, T3271C, C3093G, G3244A, A3252G, C3256T, T3258C, A3260G, T3291C mononucleotide polymorphism simultaneously, compares in current kit smallly, and it is easy to make, easy operation, be convenient for transportation and use.

Description

Mitochondrial encephalomyopathy, lactic acidosis and apoplexy sample disease gene sequencing kit

Technical field

This utility model belongs to biological technical field, relates to a kind of gene pleiomorphism order-checking detection kit, specifically Relate to mitochondrial encephalomyopathy, lactic acidosis and apoplexy sample disease gene sequencing kit.

Background technology

Mitochondrial encephalomyopathy, lactic acidosis and apoplexy sample disease (MELAS) are that mitochondrial disease is modal The form of expression, and sickness rate is of a relatively high.MELAS causes brain periodically apopiecticus insultus, often makes patient occur Migraine, vomitting and faint from fear, sometimes causing brain permanent damage, the principal pathogenetic phase is in childhood and grows up in early days. The progressively accumulative effect of these events often leads to patient and occurs that motor skill is lost, sensation is undermined spirit and hinders Hinder.Lactic acid generally accumulates in blood or cerebrospinal fluid, or both have both at the same time.Research finds, Most patients All have mtDNA A3243G heterogeneity sudden change, also have some be by T3271C, C3093G, G3244A, The point mutation of A3252G, C3256T, T3258C, A3260G, T3291C site causes.

Therefore, research and development one mitochondrial encephalomyopathy fast and effectively, lactic acidosis and apoplexy sample disease (MELAS) A3243G、T3271C、C3093G、G3244A、A3252G、C3256T、T3258C、A3260G、T3291C The order-checking detection kit of loci gene type is particularly important.

Utility model content

This utility model offer one employing Sanger method order-checking (generation order-checking) detection mitochondrial encephalomyopathy, Lactic acidosis and apoplexy sample disease (MELAS) related gene A3243G, T3271C, C3093G, G3244A, The test kit of A3252G, C3256T, T3258C, A3260G, T3291C loci gene type, this utility model Can preferably be applied to 9 hot mutant site single nucleotide polymorphism detections of MELAS, volume is little, operation Simply, it is simple to transport is carried and uses.

Mitochondrial encephalomyopathy, lactic acidosis and the apoplexy sample disease gene sequencing reagent that this utility model provides Box, it is characterised in that described test kit includes that box body 1, paper liner 2, described paper liner 2 are put In box body 1, paper liner 2 is provided with 4 container holes and equipped with 4 Reagent Tubes equipped with detectable, Be respectively equipped with PCR amplification forward primer Reagent Tube 3 and equipped with PCR amplification reverse primer Reagent Tube 4, Reagent Tube 5 equipped with Taq enzyme, the Reagent Tube 6 equipped with PCR reactant liquor.

Further, described container hole is preferably 4, and container hole divides 2 in described paper liner Row's arrangement, and it is 2ml Reagent Tube.

In above-mentioned Reagent Tube 6 equipped with PCR reactant liquor main component comprise dNTPs and MgCL₂。

Above-mentioned Taq archaeal dna polymerase is this area routine high-fidelity Taq archaeal dna polymerase, and production firm is preferable Include TAKARA or TOYOBO (Japan is spun), the production firm of described specific primer is preferably LIFE Or TAKARA, described dNTPs, MgCL₂It is conventional dNTPs, MgCL₂。

The amplicon that described PCR amplification forward primer and PCR amplification reverse primer are formed comprise A3243G, T3271C, C3093G, G3244A, A3252G, C3256T, T3258C, A3260G, T3291C site.

Described PCR amplification forward primer is: 5'-TTTGTTCAACGATTAAAGTCCTACG-3'.

Described PCR amplification reverse primer is: 5'-ATTTTTCGTTCGGTAAGCA-3'.

Application the beneficial effects of the utility model:

The test kit that this utility model provides is arranged by orderly container hole, each container hole be respectively equipped with PCR expands the Reagent Tube 3 of forward primer and equipped with the Reagent Tube 4 of PCR amplification reverse primer, equipped with Taq enzyme Reagent Tube 5, equipped with the Reagent Tube 6 of PCR reactant liquor, facilitate user to have after obtaining and having measured Sequence put back to relevant position, easy to use；

Taq enzyme needed for PCR is expanded by the test kit that this utility model provides is individually placed, it is ensured that Taq The stability of enzyme, PCR Buffer and MgCL needed for PCR is expanded₂Mix as PCR reactant liquor etc. component, Make the most easily and fast, reduce the preparation steps of PCR amplification system reactant liquor；

In the test kit that this utility model provides, PCR amplification forward primer and PCR amplification reverse primer are all with lyophilizing The form of powder is stored in Reagent Tube, it is simple to storage and transport；

In the test kit that this utility model provides, PCR amplification forward primer and PCR amplification reverse primer all can be simultaneously Use as PCR amplification, it is possible to use as sequencing primer.

Accompanying drawing explanation

Fig. 1 is mitochondrial encephalomyopathy of the present utility model, lactic acidosis and apoplexy sample disease gene sequencing reagent Box structural representation 1 is box body in figure, 2 for paper liner, 3 be the examination expanding forward primer equipped with PCR Agent pipe, 4 be equipped with PCR amplification the Reagent Tube of reverse primer, 5 be the Reagent Tube equipped with Taq enzyme, 6 for fill There is the Reagent Tube of PCR reactant liquor.

Detailed description of the invention

In order to more clearly describe technology contents of the present utility model, below by detailed description of the invention and tie Conjunction accompanying drawing is described in further detail.

Mitochondrial encephalomyopathy, lactic acidosis and the apoplexy sample disease gene sequencing reagent that this utility model provides Box, it is characterised in that described test kit includes that box body 1, paper liner 2, described paper liner 2 are put In box body 1, paper liner 2 is provided with 4 container holes and equipped with 4 Reagent Tubes equipped with detectable, Be respectively equipped with PCR amplification forward primer Reagent Tube 3 and equipped with PCR amplification reverse primer Reagent Tube 4, Reagent Tube 5 equipped with Taq enzyme, the Reagent Tube 6 equipped with PCR reactant liquor, wherein PCR amplification forward primer and PCR amplification reverse primer all can use separately as sequencing primer, and described primer all stores up with the form of lyophilized powder It is stored in Reagent Tube, it is simple to storage and transport.

Further, described container hole is 4, and container hole is point 2 row's arrangements in described paper liner, Often row 2, and it is 2ml Reagent Tube.

Test kit provided by the utility model is stored in-20 DEG C, it is to avoid multigelation.

Mitochondrial encephalomyopathy, lactic acidosis and the apoplexy sample specifically used method of disease gene sequencing kit:

(1), when using for the first time, open test kit and first PCR is expanded forward primer and PCR amplification reversely Primer is of short duration centrifugal, adds ultra-pure water and is diluted to 10 μm ol/L, it is proposed that preserve in subpackage to different centrifuge tubes Avoid multigelation.

(2) preparation PCR amplification system reactant liquor: Taq enzyme 1ul (1u/uL), PCR reactant liquor 34ul, PCR amplification forward primer and each 1.5ul of reverse primer, template 0.2 to 1ug, adds ultra-pure water polishing to 50ul, Of short duration being centrifuged expands.

(3) PCR amplification: 98 DEG C 2 minutes, 98 DEG C 10 seconds, 56 DEG C 30 seconds, 68 DEG C 22 seconds, step 98 DEG C 10 seconds, 56 DEG C 30 seconds, 68 DEG C carry out 35 circulations for 22 seconds altogether, last 68 DEG C 7 minutes.

(4) pcr amplification product is carried out the agarose gel electrophoresis detection of 1.5% by electrophoresis: PCR after terminating.

(5) order-checking: PCR amplification forward primer and the PCR amplification reverse primer of this test kit all can individually be made Using for sequencing primer, experimental implementation person can arrange the parameter in sequencing reaction also according to the sequenator model of oneself Check order.

Above example is only preferred embodiment of the present utility model, but it is clear that still may be made that various repairing Change and convert without departing from the spirit and scope of the present invention.Therefore, specification and drawings is considered as illustrative And nonrestrictive.

In sum, this utility model simple in construction, easy to manufacture, easy to use, reasonable in design, detected Journey is susceptible to pollute, and uses the design of paper liner to make Reagent Tube avoid colliding with, and this test kit is at mitochondrion brain Myopathy, lactic acidosis and apoplexy sample disease related gene hot mutant site A3243G, T3271C, C3093G, The single nucleotide polymorphism order-checking inspection of G3244A, A3252G, C3256T, T3258C, A3260G, T3291C Have during survey sensitivity by force, the shortest, high specificity, facilitate the advantages such as transport.

Claims

1. a mitochondrial encephalomyopathy, lactic acidosis and apoplexy sample disease gene sequencing kit, it is characterized in that, described test kit includes box body (1), paper liner (2), described paper liner (2) is placed in box body (1), paper liner (2) is provided with 4 container holes and equipped with 4 Reagent Tubes equipped with detectable, is respectively the Reagent Tube (3) equipped with PCR amplification forward primer and expands the Reagent Tube (4) of reverse primer, the Reagent Tube (5) equipped with Taq enzyme, the Reagent Tube (6) equipped with PCR reactant liquor equipped with PCR.

Mitochondrial encephalomyopathy the most according to claim 1, lactic acidosis and apoplexy sample disease gene sequencing kit, it is characterized in that, described paper liner (2) is placed in box body (1), paper liner (2) is provided with 4 container holes and equipped with 4 Reagent Tubes equipped with detectable, is respectively the Reagent Tube (3) equipped with PCR amplification forward primer and expands the Reagent Tube (4) of reverse primer, the Reagent Tube (5) equipped with Taq enzyme, the Reagent Tube (6) equipped with PCR reactant liquor equipped with PCR.

Mitochondrial encephalomyopathy the most according to claim 2, lactic acidosis and apoplexy sample disease gene sequencing kit, it is characterised in that described 4 container holes in paper liner (2) are divided into 2 row's arrangement, often rows 2.