CN105861687A - Detection primer composition, reagent kit and method for mitochondria drug-induced deafness - Google Patents
Detection primer composition, reagent kit and method for mitochondria drug-induced deafness Download PDFInfo
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Abstract
The invention relates to the technical field of biological detection, in particular to detection primer composition, reagent kit and method for mitochondria drug-induced deafness. With the PNA Clamp LAMP technology having PNA and LAMP combined, C-alleles of 1494 loci are closed by PNA-4C, T-alleles of 1494 loci are closed by PNA-4T, A-alleles of 1555 loci are closed by PNA-5A, and G-alleles of 1555 loci are closed by PNA-5G. In this way, corresponding LAMP reaction cannot go on for the purpose of detecting sudden change of gene loci of the mitochondria. The method is simple in steps, good in specificity, convenient to identify, low in cost and convenient in large-scale popularization of clinical detection.
Description
Technical field
The invention belongs to technical field of biological, especially relate to the detection primer combination of a kind of mitochondrion drug induced deafness
Thing, test kit and method.
Background technology
Drug intoxication is deaf (abbreviation drug induced deafness), refers to that patient makes because using or contact some ototoxic drug
The deafness become, these medicines (including amino glucosides antibiotics, non-amino glucosides antibiotics, salicylate etc.) are equal
Human auditory nerve is had toxic and side effects, cochlea can be destroyed, typically result in bilateral, permanent deafness, how irreversible.
Showing according to China in 2006 the Second China National Sample Survey on Disability result, hearing loss patient numbers about 2004
Ten thousand people, account for the 24.16% of the whole nation 82,960,000 people with disability's sum, the wherein number of drug-induced deafness up to 8,000,000.Wherein,
The incorrect use of aminoglycoside antibiotics is the most common factor causing drug induced deafness to occur, and this kind of antibiotic includes
Common streptomycin, gentamycin etc..Result of study is additionally had to show, the generation of homo mitochondrion gene sudden change and medicine
Property deafness is closely related.In Chinese population, sending out of A1555G, C1494T the two site mutation of mitochondrial gene
Raw frequency is up to 4%.A1555G sudden change and C1494T sudden change can be formed in the A position of the high conservative of 12S rRNA
New 1494C-G1555 or 1494U-A1555 base pair.These change make 12S rRNA in secondary structure with
The secondary structure of the respective regions of the 16SrRNA of antibacterial is even more like, causes sugar aminoglycoside that " not should " occurs therewith
Combination, cause the Na+ on cell membrane, K+-ATP pumping function obstacle, cause damage of hair cell, cause Drug audition
Impaired even deaf.The method being currently used for detecting the two site mutation has a variety of, such as Sanger sequencing, this
Method can the catastrophe that must obtain purpose site the most directly perceived, but operation complexity, need twice PCR and substantial amounts of pure
Chemical industry is made, and costly, the longest, is not suitable for promoting the use of on a large scale；ApoE gene method,
This method, by design saltant type and the special PCR primer of wild type, compares wild type and saltant type PCR by electrophoresis
The sudden change situation varied in size to judge detection sample of product, advantage is low cost, but sample during extensive detection
Amount is relatively big, carries out substantial amounts of electrophoretic procedures simultaneously and will become the most loaded down with trivial details, and easily causes cross-contamination；Fluorescence is fixed
Amount PCR method is highly sensitive, it is not necessary to manual detection, and therefore at present Application comparison is extensive, however it still can not break away from right
The dependence of expensive equipment, meanwhile, the probe for detection is also a no small expenditure.Therefore, it is highly desirable to out
Send out a kind of can the most again can be cheap detection method.Isothermal duplication (LAMP) technology of ring mediation is day
A kind of isothermal amplification technique that this scientist proposed in 2000, it is mainly characterized by 6 region designs for target gene
Article 4, special primer, carries out constant-temperature amplification under the effect of strand displacement archaeal dna polymerase (Bst DNA polymerase),
Only need within 15-90 minute, 10 can be produced9-1010The product of the order of magnitude.Have that sensitivity is high, specificity is good, easy and simple to handle,
With low cost and result is prone to the advantage judged.
LAMP method has simple to operate, high specificity and advantage with low cost.LAMP method is relatively used for cause of disease at present
In the detection of body and some other microorganism, although for using LAMP method to carry out the most accidental report of detection of gene mutation,
But the LAMP method of these allele-specific primerses is the most unstable in actual application, does not the most also have
Gene mutation detection kit based on LAMP method is sold.Peptide nucleic acid(PNA) (PNA) is the DNA with class polypeptide backbone
Analog, skeleton is formed by connecting by methylene carbonyl with nucleic acid base by N (2-amino-ethyl)-glycine.PNA
Specifically can hybridize with DNA or RNA complementary therewith, form stable complex.When PNA and target sequence
During complete complementary, the stability of this species complex to be significantly larger than common DNA-DNA, DNA-RNA and RNA-RNA
Duplex structure.But, when PNA and target sequence incomplete pairing even only one of which base mispairing when, this
PNA-DNA complex just becomes highly unstable, it is easy to open double-strand.
Summary of the invention
The technical problem to be solved in the present invention is that the detection of existing mitochondrial gene site mutation exists costly, detection week
The problems such as the phase is longer, complex operation, are unfavorable for that Clinical Laboratory is popularized on a large scale.
For solving the problems referred to above, PNA with LAMP method is combined by the present invention, utilizes PNA-4C to close 1494 site C
Allele, PNA-4T closes 1494 site T allele, and PNA-5A closes 1555 site A allele,
PNA-5G closes 1555 site G allele, so that corresponding LAMP reaction cannot continue, realizes with this
The detection of mitochondrial gene site mutation, the method not only rapid sensitive but also with low cost.
First purpose of the present invention is to provide the detection primer compositions of a kind of mitochondrion drug induced deafness, including:
Forward Outside primer F3:
5’-TGGGCTACATTTTCTACCC-3’(SEQ ID No.1)；
Reversely Outside primer B3:
5’-ACACTTACCATGTTACGACT-3’(SEQ ID No.2)；
Forward inner primer FIP:
5’-AGCACTCTACTCTTAGTTTACTGCTCAGAAAACTACGATAGCCCTT-3’
(SEQ ID No.3)；
Reversely inner primer BIP:
5’-CCGTCACCCTCCTCAAGTATTGTCTCCTCTATATAAATGCGTA-3’(SEQ
ID No.4)；And
For closing 1494 site C allelic peptide nucleic acid sequence PNA-4C:
5’-CCGTCACCCTCCTCA-3’(SEQ ID No.5)；
For closing 1494 site T allelic peptide nucleic acid sequence PNA-4T:
5’-CCCGTCACTCTCCTCA-3’(SEQ ID No.6)；
For closing 1555 site A allelic peptide nucleic acid sequence PNA-5A:
5’-TACGACTTGTCTCCTCTA-3’(SEQ ID No.7)；
For closing 1555 site G allelic peptide nucleic acid sequence PNA-5G:
5’-TACGACTTGCCTCCTCT-3’(SEQ ID No.8)。
Second object of the present invention is to provide the detection kit of a kind of mitochondrion drug induced deafness, described detectable
Box includes above-mentioned primer and peptide nucleic acid sequence.
Further, described detection kit also includes dNTP, buffer, Bst polymerase and ultra-pure water.
Further, described detection kit is divided into four reaction systems, is respectively designated as detecting 1494 site C equipotential bases
Reaction system C of cause, detects 1494 allelic reaction systems T of site T, detects 1555 site A allele
Reaction system A and detection 1555 allelic reaction systems G of site G:
Reaction system C includes F3, B3, FIP, BIP, PNA-4C, dNTP, buffer, Bst polymerase and surpasses
Pure water；Reaction system T include F3, B3, FIP, BIP, PNA-4T, dNTP, buffer, Bst polymerase and
Ultra-pure water；Reaction system A includes F3, B3, FIP, BIP, PNA-5A, dNTP, buffer, Bst polymerase
And ultra-pure water；Reaction system G includes that F3, B3, FIP, BIP, PNA-5G, dNTP, buffer, Bst are polymerized
Enzyme and ultra-pure water.
Further, described four reaction systems also include the indicator for showing system color.
Preferably, described indicator is hydroxynaphthol blue.
Preferably, described indicator concentration in four reaction systems is identical, and the concentration of hydroxynaphthol blue is 120 μMs.
Further, described four reaction systems also include additive, dense in four reaction systems of described additive
Degree is consistent.
Preferably, described additive is glycine betaine, and the concentration of glycine betaine is 0.8M.
Preferably, in described four reaction systems, the concentration of buffer is identical, comprising: Tris-HCl 20mM, KCl 50
MM, (NH4)2SO410mM, MgSO44mM, Tween-20 0.1% (volume fraction).
Preferably, in described four reaction systems: the concentration of F3 is identical, is 0.4 μM；The concentration of B3 is identical, all
It it is 0.4 μM；The concentration of FIP is identical, is 1.6 μMs；The concentration of BIP is identical, is 1.6 μMs；Described four anti-
Answer the concentration of corresponding peptides nucleotide sequence in system identical, be 1 μM.
Preferably, in four reaction systems, the concentration of dNTP is identical, is 1.4mM；In two reaction systems, Bst gathers
The concentration of synthase is identical, is 8U.
Third object of the present invention is to provide the detection method of a kind of mitochondrion drug induced deafness, and it uses above-mentioned inspection
Test agent box, comprises the steps:
(1) venous blood samples preserves with EDTA anticoagulant tube；
(2) with DNA extraction kit extraction complete genome DNA as template to be detected, simultaneously with known mutations matter
Grain is as positive control；
(3) use PNA Clamp LAMP technology, template to be detected and mutant plasmid are carried out LAMP reaction, will
Template and mutant plasmid are added separately in reaction system C, reaction system T, reaction system A and reaction system G, in
React at 63 DEG C, then heat to 80 DEG C and keep 20min to carry out enzyme-deactivating process, lower the temperature afterwards, observe system color and become
Change.
Further, the described response time is 70～90min.
In four reaction systems of the present invention, when reaction system exists indicator, select hydroxynaphthol blue as finger
When showing agent, the magnesium ion in reacting precursor system chelates with dNTP, and system presents pansy；When there being positive reaction,
Magnesium ion forms precipitation with by-product pyrophosphoric acid, and pH value of solution changes, and color is gradually become sky blue from pansy.
Therefore, the change of observing response system color just can directly determine whether amplified reaction.
The present invention has the advantage that with good effect:
The present invention uses PNA Clamp LAMP technology, PNA with LAMP method is combined, and utilizes PNA by corresponding
Allele " close " so that LAMP reaction cannot continue；And when detection sample in exist another one or
During the several allele of person, PNA is owing to effectively can not carry out " closing " to it so that LAMP reaction is entered smoothly
OK, the purpose of mitochondrial gene site mutation detection is reached with this.
Compared with prior art, PNA Clamp LAMP method has the advantages that
(1) step is simple: LAMP method itself is not strict to the requirement of template, and its reaction template can even is that DNA
Crude extract, be placed in thermostat water bath after only needing reaction system configuration and carry out reaction and expand, it is not necessary in advance
First carry out double-stranded DNA degeneration and be similar to the degeneration repeatedly in PCR cycle reaction, anneal, the alternating temperature process such as extension；
(2) high specific: typical LAMP reaction needs four primers, they totally six different template binding region
Two primers in territory, significantly larger than regular-PCR, therefore the specificity of LAMP amplification is the highest, can be according to whether expand
Just can judge the presence or absence of target gene；
(3) amplified reaction is quickly, efficiently: whole amplification procedure is less than 1.5h, and productivity is high；
(4) simplicity is identified: observing by the naked eye color change after having reacted and get final product result of determination, the observation of result is permissible
The operation requirement and the instrument that depart from gel imaging limit；
(5) with low cost: whole detection reaction only needs water-bath just can carry out, it is not necessary to any other detects equipment.
Accompanying drawing explanation
Fig. 1-Fig. 2 is 1494 sites and the sequencer map in 1555 sites of detection sample M0 respectively；
Fig. 3 is the sequencer map in 1494 sites of detection sample M4；
Fig. 4 is the sequencer map in 1555 sites of detection sample M5.
Detailed description of the invention
In order to be better understood from the present invention, below in conjunction with specific embodiments and the drawings, the invention will be further described, but not
Limit protection scope of the present invention.
A kind of detection primer compositions of mitochondrion drug induced deafness, including:
Forward Outside primer F3:
5’-TGGGCTACATTTTCTACCC-3’(SEQ ID No.1)；
Reversely Outside primer B3:
5’-ACACTTACCATGTTACGACT-3’(SEQ ID No.2)；
Forward inner primer FIP:
5’-AGCACTCTACTCTTAGTTTACTGCTCAGAAAACTACGATAGCCCTT-3’
(SEQ ID No.3)；
Reversely inner primer BIP:
5’-CCGTCACCCTCCTCAAGTATTGTCTCCTCTATATAAATGCGTA-3’(SEQ
ID No.4)；And
For closing 1494 site C allelic peptide nucleic acid sequence PNA-4C:
5’-CCGTCACCCTCCTCA-3’(SEQ ID No.5)；
For closing 1494 site T allelic peptide nucleic acid sequence PNA-4T:
5’-CCCGTCACTCTCCTCA-3’(SEQ ID No.6)；
For closing 1555 site A allelic peptide nucleic acid sequence PNA-5A:
5’-TACGACTTGTCTCCTCTA-3’(SEQ ID No.7)；
For closing 1555 site G allelic peptide nucleic acid sequence PNA-5G:
5’-TACGACTTGCCTCCTCT-3’(SEQ ID No.8)。
The detection kit of a kind of mitochondrion drug induced deafness, described detection kit includes above-mentioned primer and peptide nucleic acid(PNA) sequence
Row, also include dNTP, buffer, Bst polymerase, ultra-pure water, hydroxynaphthol blue and glycine betaine.
Described detection kit is divided into four reaction systems, is respectively designated as detecting the 1494 allelic reactants of site C
It is C, detects 1494 allelic reaction systems T of site T, detect the 1555 allelic reaction systems of site A
A and detection 1555 allelic reaction systems G of site G:
Reaction system C includes F3, B3, FIP, BIP, PNA-4C, dNTP, buffer, Bst polymerase, surpasses
Pure water, hydroxynaphthol blue and glycine betaine；Reaction system T include F3, B3, FIP, BIP, PNA-4T, dNTP,
Buffer, Bst polymerase, ultra-pure water, hydroxynaphthol blue and glycine betaine；Reaction system A include F3, B3, FIP,
BIP, PNA-5A, dNTP, buffer, Bst polymerase, ultra-pure water, hydroxynaphthol blue and glycine betaine；Reaction system
G includes F3, B3, FIP, BIP, PNA-5G, dNTP, buffer, Bst polymerase, ultra-pure water, hydroxyl naphthalene
Phenol indigo plant and glycine betaine.
In four reaction systems, the concentration of each component can be arranged by the concentration that normal LAMP reacts, as a kind of embodiment,
In four reaction systems: the concentration of F3 is identical, it is 0.4 μM；The concentration of B3 is identical, is 0.4 μM；FIP's
Concentration is identical, is 1.6 μMs；The concentration of BIP is identical, is 1.6 μMs；Corresponding peptides nucleic acid sequence in four reaction systems
The concentration of row is identical, is 1 μM；The concentration of dNTP is identical, is 1.4mM；The concentration of Bst polymerase is identical,
It is 8U；The concentration of buffer is identical, all includes: Tris-HCl 20mM, KCl 50mM, (NH4)2SO410mM,
MgSO44mM, Tween-20 0.1% (volume fraction)；The concentration of hydroxynaphthol blue is identical, is 120 μMs；Radix Betae
The concentration of alkali is identical, is 0.8M.
The detection method of a kind of mitochondrion drug induced deafness, uses above-mentioned detection kit, particularly as follows: venous blood samples
Preserve with EDTA anticoagulant tube；With DNA extraction kit extraction complete genome DNA as template to be detected, simultaneously
Using known mutations plasmid as positive control；Use PNA Clamp LAMP technology, to template to be detected and sudden change matter
Grain carries out LAMP reaction, and template and mutant plasmid are added separately to reaction system C, reaction system T, reaction system
In A and reaction system G, react at 63 DEG C, then heat to 80 DEG C and keep 20min to carry out enzyme-deactivating process, afterwards
Cooling, observes the change of system color.As preferably, the described response time is 70～90min.
When being embodied as, the 1ml venous blood extracting a normal people of audition preserves with EDTA anticoagulant tube, and often pipe takes 200ul
Blood, by full formula gold or the DNA extraction kit of other company, reference reagent box description extracts complete genome DNA
As template to be detected, it is designated as M0, separately has the plasmid of two known 1494C > T and 1555A > G as positive control,
It is designated as M4 and M5 respectively；Each template is used PNA Clamp LAMP technology, makes template to be detected carry out LAMP
Reaction, is divided into four reaction systems, first the C allele for 1494 sites, i.e. system C；Second for
The T allele in 1494 sites, i.e. system T；3rd the A allele for 1555 sites, i.e. system A；
4th the G allele for 1555 sites, i.e. system G.Indicator in four systems is hydroxynaphthol blue,
Additive is glycine betaine.By template Mn (n be group numbering, n is 0,4,5) be added separately to reaction system C,
In reaction system T, reaction system A and reaction system G, template Mn concentration in four reaction systems is consistent, all
For 10ng/ μ L (practical operation can be 0.01-50ng/ μ L), during four reaction systems reactions, at 63 DEG C
Reaction 70min, is warming up to 80 DEG C afterwards and keeps 20min to carry out inactivation treatment, lower the temperature afterwards, observe the change of system color.
Template Mn is detected by four reaction systems utilizing detection kit, as a kind of embodiment, detection examination
Four reaction systems of agent box be respectively 25 μ L, its interior component and content as shown in following table 1-table 4:
The component of table 1 reaction system C and content
The component of table 2 reaction system T and content
The component of table 3 reaction system A and content
The component of table 4 reaction system G and content
Reaction is carried out according to above-mentioned detection method, and before reaction, system color is pansy, after reaction, and each group
The color of reaction system and genotype judge as shown in table 5 below:
The color of table 5 each group reaction system and genotype judge
For the accuracy of further confirmatory reaction, template M0 of experimental group, M4 and M5 carried out point by PCR sequencing
Type, 1494 sites of M0 and the sequencer map in 1555 sites as depicted in figs. 1 and 2, the order-checking in 1494 sites of M4
Figure as it is shown on figure 3, M5 1555 sites sequencer map as shown in Figure 4, contrast gene sequencing collection of illustrative plates and the present invention provide
Detection kit testing result understand, the testing result of the two is completely the same, and the result demonstrating the present invention is accurate.
Above embodiments of the invention are described in detail, but described content has been only presently preferred embodiments of the present invention, no
The practical range for limiting the present invention can be considered.All impartial changes made according to the present patent application scope and improvement etc.,
Within all should still belonging to the patent covering scope of the present invention.
Claims (14)
1. the detection primer compositions of a mitochondrion drug induced deafness, it is characterised in that: including:
Forward Outside primer F3:
5’-TGGGCTACATTTTCTACCC-3’(SEQ ID No.1)；
Reversely Outside primer B3:
5’-ACACTTACCATGTTACGACT-3’(SEQ ID No.2)；
Forward inner primer FIP:
5’-AGCACTCTACTCTTAGTTTACTGCTCAGAAAACTACGATAGCCCTT-3’
(SEQ ID No.3)；
Reversely inner primer BIP:
5’-CCGTCACCCTCCTCAAGTATTGTCTCCTCTATATAAATGCGTA-3’(SEQ
ID No.4)；And
For closing 1494 site C allelic peptide nucleic acid sequence PNA-4C:
5’-CCGTCACCCTCCTCA-3’(SEQ ID No.5)；
For closing 1494 site T allelic peptide nucleic acid sequence PNA-4T:
5’-CCCGTCACTCTCCTCA-3’(SEQ ID No.6)；
For closing 1555 site A allelic peptide nucleic acid sequence PNA-5A:
5’-TACGACTTGTCTCCTCTA-3’(SEQ ID No.7)；
For closing 1555 site G allelic peptide nucleic acid sequence PNA-5G:
5’-TACGACTTGCCTCCTCT-3’(SEQ ID No.8)。
2. the detection kit of a mitochondrion drug induced deafness, it is characterised in that: include the primer described in claim 1
And peptide nucleic acid sequence.
The detection kit of mitochondrion drug induced deafness the most according to claim 2, it is characterised in that: also include
DNTP, buffer, Bst polymerase and ultra-pure water.
The detection kit of mitochondrion drug induced deafness the most according to claim 3, it is characterised in that: described detection
Test kit is divided into four reaction systems, is respectively designated as detecting 1494 allelic reaction systems C of site C, detection
1494 allelic reaction systems T of site T, detect 1555 allelic reaction systems A of site A and detection 1555
Allelic reaction system G of site G:
Reaction system C includes F3, B3, FIP, BIP, PNA-4C, dNTP, buffer, Bst polymerase and surpasses
Pure water；
Reaction system T includes F3, B3, FIP, BIP, PNA-4T, dNTP, buffer, Bst polymerase and surpasses
Pure water；
Reaction system A includes F3, B3, FIP, BIP, PNA-5A, dNTP, buffer, Bst polymerase and surpasses
Pure water；
Reaction system G includes F3, B3, FIP, BIP, PNA-5G, dNTP, buffer, Bst polymerase and surpasses
Pure water.
The detection kit of mitochondrion drug induced deafness the most according to claim 4, it is characterised in that: described four
Reaction system also includes the indicator for showing system color.
The detection kit of mitochondrion drug induced deafness the most according to claim 5, it is characterised in that: described instruction
Agent is hydroxynaphthol blue.
The detection kit of mitochondrion drug induced deafness the most according to claim 6, it is characterised in that: described instruction
Agent concentration in four reaction systems is identical, and the concentration of hydroxynaphthol blue is 120 μMs.
The detection kit of mitochondrion drug induced deafness the most according to claim 4, it is characterised in that: described four
Also including additive in reaction system, described additive concentration in four reaction systems is consistent.
The detection kit of mitochondrion drug induced deafness the most according to claim 8, it is characterised in that: described interpolation
Agent is glycine betaine, and the concentration of glycine betaine is 0.8M.
10., according to the detection kit of the mitochondrion drug induced deafness according to any one of claim 4-9, its feature exists
In: in described four reaction systems, the concentration of buffer is identical, comprising: Tris-HCl 20mM, KCl 50mM,
(NH4)2SO410mM, MgSO44mM, Tween-20 0.1% (volume fraction).
11. according to the detection kit of the mitochondrion drug induced deafness according to any one of claim 4-9, and its feature exists
In: in described four reaction systems: the concentration of F3 is identical, is 0.4 μM；The concentration of B3 is identical, is 0.4 μM；
The concentration of FIP is identical, is 1.6 μMs；The concentration of BIP is identical, is 1.6 μMs；Phase in described four reaction systems
The concentration answering peptide nucleic acid sequence is identical, is 1 μM.
12. according to the detection kit of the mitochondrion drug induced deafness according to any one of claim 4-9, and its feature exists
In: in four reaction systems, the concentration of dNTP is identical, is 1.4mM；In four reaction systems, Bst polymerase is dense
Spend identical, be 8U.
The detection method of 13. 1 kinds of mitochondrion drug induced deafnesss, uses the arbitrary described detection kit of claim 3-12,
It is characterized in that: comprise the steps:
(1) venous blood samples preserves with EDTA anticoagulant tube；
(2) with DNA extraction kit extraction complete genome DNA as template to be detected, simultaneously with known mutations matter
Grain is as positive control；
(3) use PNA Clamp LAMP technology, template to be detected and mutant plasmid are carried out LAMP reaction, will
Template and mutant plasmid are added separately in reaction system C, reaction system T, reaction system A and reaction system G, in
React at 63 DEG C, then heat to 80 DEG C and keep 20min to carry out enzyme-deactivating process, lower the temperature afterwards, observe system color and become
Change.
The detection method of 14. mitochondrion drug induced deafnesss according to claim 13, it is characterised in that: described reaction
Time is 70～90min.
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