CN103451268B - A kind of standard substance of detection line plastochondria A3243G heterozygous mutant rate, test kit and detection method thereof - Google Patents
A kind of standard substance of detection line plastochondria A3243G heterozygous mutant rate, test kit and detection method thereof Download PDFInfo
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Abstract
The invention discloses a kind of standard substance of detection line plastochondria A3243G heterozygous mutant rate, test kit and detection method thereof.The standard substance of detection line plastochondria A3243G heterozygous mutant rate of the present invention, comprising: (1) is the carrier of the mitochondria DNA fragment of wild-type A containing 3243 sites; (2) containing 3243 sites be the carrier of mitochondria DNA fragment of saltant type G.Plasmid molecule standard substance provided by the invention, detection method and the regression equation for detecting Data correction can A3243G heterozygous mutant rates easily, accurately in detection line mitochondrial DNA, there is efficient, sensitive advantage, and for mutation rate lower than 5% heterozygous mutant also can accurately, quantitatively detect, this is other particular advantages not available for abrupt climatic change means existing.The present invention is also for other heterozygous mutant detection by quantitative in research and production practice provides good reference.
Description
Technical field
The present invention relates to biomedical engineering field, be specifically related to a kind of standard substance of detection line plastochondria A3243G heterozygous mutant rate, test kit and detection method thereof.
Background technology
A3243G sudden change is Mitochondrial Genome Overview DNA(mito-chondrialDNA, mtDNA) upper modal pathogenic mutation, it often exists with hetero forms, in a cell, namely both there is the mtDNA of wild-type, also there is the mtDNA of saltant type.A3243G sudden change is one of paathogenic factor of the disease such as early-onset diabetes and neural heariing loss, and A3243G mutation rate causes the light and heavy degree of clinical symptom relevant to it.Therefore, the accurate quantitative analysis detection for A3243G sudden change has very important significance.MtDNA3243 site is in 16SrRNA and tRNA
leu(UUR) to the research that mtDNA3243 site A/G suddenlys change, gene intersection, finds that A base is positioned at two hydrogen urine of tRNALeu (UUR) secondary structure addicted on steep ring, guards at evolution camber.The sudden change in this site may make tRNA
leu(UUR) normal three-dimensional arrangement changes, thus has influence on leucic in polypeptide chain synthesis mixing.
Mutation detection methods conventional at present mainly contains PCR-RFLP, PCR-SSCP, PCR-order-checking etc., but the detection sensitivity of these methods is lower, and generally cannot detect for the heterozygous mutant of mutation rate <5%.Due to the demand in application, be badly in need of at present a kind of new method for detection by quantitative heterozygous mutant of exploitation and detection test kit.
Summary of the invention
Technical problem to be solved by this invention is for lacking the examination criteria product of detection by quantitative plastosome A3243G heterozygous mutant rate and the existing sensitive not present situation of detection method for A3243G sudden change at present, the A3243G mutation detection methods providing a kind of sensitivity higher and the standard substance used in detection by quantitative and test kit thereof.
One of technical scheme that the present invention solves the problems of the technologies described above is: a kind of standard substance of detection line plastochondria A3243G heterozygous mutant rate, comprising:
(1) containing 3243 sites be the carrier of mitochondria DNA fragment of wild-type A; With
(2) containing 3243 sites be the carrier of mitochondria DNA fragment of saltant type G.
In the present invention, 3243 described sites refer to the 3243rd Nucleotide of human mitochondria gene group DNA.In the Mitochondrial Genome Overview DNA of normal people, mt3243 site is A, usually can sport G in patient mtDNA.
In the present invention, the described mitochondria DNA fragment containing 3243 sites refers to the Mitochondrial Genome Overview DNA fragmentation including 3243 sites.Preferably, 3243 described sites are the mitochondria DNA fragment length of wild-type A is 50-1000bp, more preferably 53bp; 3243 sites are the mitochondria DNA fragment length of saltant type G is 50-1000bp, more preferably 53bp.Most preferred, the nucleotide sequence of the mitochondria DNA fragment that 3243 described sites are wild-type A is as shown in SEQIDNO.1, and the nucleotide sequence of the mitochondria DNA fragment that 3243 sites are saltant type G is as shown in SEQIDNO.2.
In the present invention, described carrier can be any conventional carrier, as plasmid, clay (pHZ132), phage or virus vector (retrovirus vector, adenovirus carrier) etc.Preferably, described carrier is plasmid.Preferred, described carrier is T-carrier.The cloning vector more preferably can bred in intestinal bacteria, as pUC19, pUC18, pUC118, pUC119, pUC19, pBlueScriptIISK or pGEM serial carrier.Most preferred, described carrier is pGEM-T carrier.
In the present invention, preferably, be describedly the carrier of the mitochondria DNA fragment of wild-type A containing 3243 sites and be the vehicle group resulting mixture of the mitochondria DNA fragment of saltant type G containing 3243 sites.Preferably, the content being the carrier of the mitochondria DNA fragment of saltant type G containing 3243 sites in described mixture accounts for the arbitrary concentration in 0 ~ 100% of carrier total amount, be the content of the carrier of the mitochondria DNA fragment of wild-type A containing 3243 sites be surplus, between described mixture, form a series of carrier concn ratio gradient.Preferred, the content being the carrier of the mitochondria DNA fragment of saltant type G containing 3243 sites in described mixture accounts for carrier total amount 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% respectively, is that the content of the carrier of the mitochondria DNA fragment of wild-type A is surplus containing 3243 sites.
Preferred further, also comprise the medium dissolving carrier in described mixture, described medium is any solvent that can dissolve carrier, preferably comprises water, TE, PBS or physiological saline.Preferred, in described mixture, described carrier concentration is in media as well various available concentration, can be 1ng/ μ L-100ng/ μ L, preferred 50ng/ μ L.
Two of the technical scheme that the present invention solves the problems of the technologies described above is: a kind of test kit of detection line plastochondria A3243G heterozygous mutant rate, comprises the standard substance of described detection line plastochondria A3243G heterozygous mutant rate.
Wherein, described standard substance are described above.
Preferably, amplification is also comprised in described test kit containing the amplimer of the mitochondria DNA fragment in 3243 sites with for detecting containing one or both in the Pyrosequencing primer of the mitochondria DNA fragment in 3243 sites.The oligonucleotide chain of described amplimer to be length be 25 ± 15nt, it is identical with Mitochondrial Genome Overview DNA fragmentation or complementary, as long as described amplimer can increase containing the mitochondria DNA fragment in 3243 sites.Preferably, the nucleotide sequence of described amplimer is respectively as shown in SEQ ID NO.3 and SEQIDNO.4.Described Pyrosequencing primer is identical with Mitochondrial Genome Overview sequence dna fragment, only need be positioned at 3243 site upstream, the primer shown in preferred SEQIDNO.5.
Preferably, DNA extraction reagent and/or TE damping fluid is also comprised in described test kit.
Preferably, also specification sheets is comprised in described test kit.This specification sheets describes the using method of this test kit, i.e. the method for detection by quantitative plastosome A3243G heterozygous mutant rate, and the method comprises the following steps:
1) with the standard substance of above-mentioned different weight percentage for template, adopt amplimer amplification containing the mitochondria DNA fragment in 3243 sites;
2) Manganic pyrophosphate complex initiation is carried out to the PCR primer of step 1) gained;
3) according to step 2) in the result of Manganic pyrophosphate complex initiation draft regression equation for Data correction;
4) from sample to be checked, DNA is extracted;
5) with the DNA of step 4) gained for template, adopt amplimer amplification containing the mitochondria DNA fragment in 3243 sites;
6) Manganic pyrophosphate complex initiation is carried out to the PCR primer of step 5) gained;
7) to the sequencing result applying step 3 of step 6) gained) regression equation of gained carries out Data correction, obtains the plastosome A3243G heterozygous mutant rate of sample to be checked.
In the present invention, described Manganic pyrophosphate complex initiation is Manganic pyrophosphate complex initiation (Pyrosequencing) technology of this area routine, and this technology need not carry out electrophoresis, and DNA fragmentation also need not carry out fluorescent mark, operates very easy.Pyrosequencing techniques is by the enzyme cascade chemiluminescence reaction in 4 kinds of enzymatic same reaction systems, take turns in sequencing reaction at each, only add a kind of dNTP, if this dNTP and template are matched, polysaccharase just can be incorporated in primer strand and the tetra-sodium group (PPi) of mole number such as to be discharged.PPi finally can be converted into visible light signal, and is converted into a peak value by PyrogramTM.The height of each peak value is directly proportional to the nucleotide number mixed in reaction.Then add lower a kind of dNTP, continue the synthesis of DNA chain.Namely peak value figure according to obtaining can read DNA sequence dna information accurately, and the peak height that peak value figure shows can detect the frequency of hybrid dna template allelic.But the present invention finds, the result of Manganic pyrophosphate complex initiation and actual value have certain deviation, this deviation is relevant with the optimum determining scope of sequence measurement, be about 50% at heterozygosity, detected value and actual value are substantially identical, but in the region that heterozygosity is lower or higher, the deviation of detected value and actual value is larger; The running environment of this deviation and sequenator is also relevant with the setting of operating parameter, and on different sequenators, the degree of these deviations has difference slightly.The existence of these deviations, for the sample that reality detects, interpretation of result and application all can produce detrimentally affect, be necessary to overcome these deviations, obtain the detected result close with actual value.Therefore, the detected value of the present inventor to Manganic pyrophosphate complex initiation adopts standard substance of the present invention to carry out Data correction, obtain the data analysis regression equation that correction adopts, thus make the detected value of sample after overcorrection closer to actual value, be conducive to further Accurate Analysis and the application of result.
Three of the technical scheme that the present invention solves the problems of the technologies described above is: a kind of preparation method of standard substance of described detection line plastochondria A3243G heterozygous mutant rate, comprises the steps:
1) from the sample confirming as A3243G sudden change and in nonmutationed normal sample, DNA is extracted respectively;
2) adopt amplimer to carry out pcr amplification respectively to the DNA of step 1) gained, in amplified production, include 3243 sites of Mitochondrial DNA;
3) by step 2) gained PCR primer to be respectively charged in carrier and to carry out sequence verification, thus obtaining wild-type carrier and mutant vector;
4) standard substance of each concentration series are prepared according to the different ratios of wild-type and mutant vector.
Wherein, step 1) extracts the method for DNA from sample is ordinary method.Such as from the peripheral blood sample of patient, extract DNA, adopt genome DNA extracting method can extract complete Mitochondrial DNA.
Step 2) described in the oligonucleotide chain of amplimer to be length be 25 ± 15nt, it is identical with Mitochondrial Genome Overview DNA fragmentation or complementary, as long as described amplimer can increase containing the mitochondria DNA fragment in 3243 sites.Preferably, the nucleotide sequence of described amplimer is respectively as shown in SEQ ID NO.3 and SEQIDNO.4.Step 2) program of described pcr amplification is conventional, as long as corresponding fragment can be obtained by specific amplification, be preferably: 1. 94 DEG C, 5min; 2. 94 DEG C, 20s; 3. 55 DEG C, 20s; 4. 72 DEG C, 20s; Step 2. to cycle number be 4. 40; 5. 72 DEG C, 10min.
Method PCR primer being loaded carrier described in step 3) is the ordinary method of this area, generally with restriction enzyme respectively enzyme cut PCR primer and carrier, then connect with ligase enzyme.Described carrier is the various carriers for loading exogenous nucleic acid fragment that this area can adopt, as plasmid, clay (pHZ132), phage or virus vector (retrovirus vector, adenovirus carrier) etc.Preferably, described carrier is plasmid.Preferred, described carrier is T-carrier.The cloning vector more preferably can bred in intestinal bacteria, as pUC19, pUC18, pUC118, pUC119, pUC19, pBlueScriptIISK or pGEM serial carrier.Most preferred, described carrier is pGEM-T carrier, and the method for order-checking is also the ordinary method of this area, can adopt arbitrary in pcr amplification primer and check order.
Two kinds of carriers are mixed into the standard substance of each concentration series by step 4), preferably comprise two kinds of carriers concentration quantitatively extremely certain respectively, are then mixed in proportion.Preferably as being then mixed in proportion quantitative respectively for two kinds of carriers to 10-100ng/ μ L, prepare each concentration standards.Described concentration refers to that 3243 sites are the percentage concentration that the mutant vector of G accounts for the whole carrier summation of the wild-type carrier of A (3243 sites to be the saltant type of G and 3243 sites be) total amount.
Four of the technical scheme that the present invention solves the problems of the technologies described above is: a kind of method of detection by quantitative plastosome A3243G heterozygous mutant rate, comprises the steps:
1) with the standard substance of above-mentioned different weight percentage for template, adopt amplimer amplification containing the mitochondria DNA fragment in 3243 sites;
2) Manganic pyrophosphate complex initiation is carried out to the PCR primer of step 1) gained;
3) according to step 2) in the result of Manganic pyrophosphate complex initiation draft regression equation for Data correction;
4) from sample to be checked, DNA is extracted;
5) with the DNA of step 4) gained for template, adopt primer PCR amplification containing the mitochondria DNA fragment in 3243 sites;
6) Manganic pyrophosphate complex initiation is carried out to the PCR primer of step 5) gained;
7) sequencing result applying step 3) regression equation of gained carries out Data correction, obtains the plastosome A3243G heterozygous mutant rate of sample to be checked.
Wherein, the method extracting DNA described in step 4) from sample to be checked is ordinary method.Such as from the peripheral blood sample of patient, extract DNA, adopt Extraction Methods of Genome can extract complete Mitochondrial DNA.
Step 1) or 5) method of described amplification is with conventional.The oligonucleotide chain of described amplimer to be length be 25 ± 15nt, it is identical with Mitochondrial Genome Overview DNA fragmentation or complementary, as long as described amplimer can increase containing the mitochondria DNA fragment in 3243 sites.Preferably, the nucleotide sequence of described amplimer is respectively as shown in SEQ ID NO.3 and SEQIDNO.4.The condition of described pcr amplification is conventional, as long as can obtain amplified fragments by specific amplification, is preferably: 1. 94 DEG C, 5min; 2. 94 DEG C, 20s; 3. 55 DEG C, 20s; 4. 72 DEG C, 20s; Step 2. to cycle number be 4. 40; 5. 72 DEG C, 10min.
Step 2) or 6) method of described Manganic pyrophosphate complex initiation is also the ordinary method of this area, and the Pyrosequencing primer as shown in SEQIDNO.5 can be adopted to check order.
Data correction described in step 7) carries out data calibration to the regression equation described in the Manganic pyrophosphate complex initiation result employing of sample to be tested, thus draw final calculation result; This correction equation is preferably Y=-0.0000737578382X
3+ 0.01054497865X
2+ 0.71548332X-0.63357639446.
Beneficial effect of the present invention: the detection method of plastosome A3243G heterozygous mutant rate provided by the invention is highly sensitive, quantitatively accurately, more can detect mutation rate that other common detection methods cannot detect lower than 5% heterozygous mutant, there is great using value and theory significance, also for other abrupt climatic change in research and production practice provides good enlightenment.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, characteristic sum beneficial effect of the present invention is described.
Fig. 1 is the Manganic pyrophosphate complex initiation result figure of 0% standard substance;
Fig. 2 is the Manganic pyrophosphate complex initiation result figure of 2% standard substance;
Fig. 3 is the Manganic pyrophosphate complex initiation result figure of 5% standard substance;
Fig. 4 is the Manganic pyrophosphate complex initiation result figure of 10% standard substance;
Fig. 5 is the Manganic pyrophosphate complex initiation result figure of 20% standard substance;
Fig. 6 is the Manganic pyrophosphate complex initiation result figure of 30% standard substance;
Fig. 7 is the Manganic pyrophosphate complex initiation result figure of 40% standard substance;
Fig. 8 is the Manganic pyrophosphate complex initiation result figure of 50% standard substance;
Fig. 9 is the Manganic pyrophosphate complex initiation result figure of 60% standard substance;
Figure 10 is the Manganic pyrophosphate complex initiation result figure of 70% standard substance;
Figure 11 is the Manganic pyrophosphate complex initiation result figure of 80% standard substance;
Figure 12 is the Manganic pyrophosphate complex initiation result figure of 90% standard substance;
Figure 13 is the Manganic pyrophosphate complex initiation result figure of 95% standard substance;
Figure 14 is the Manganic pyrophosphate complex initiation result figure of 100% standard substance;
Figure 15 is result after utilizing software correction and regression equation;
Figure 16 is the negative Manganic pyrophosphate complex initiation result figure detecting sample F932-4;
Figure 17 is the Manganic pyrophosphate complex initiation result figure of positive detection sample F1096-8;
Figure 18 is the Manganic pyrophosphate complex initiation result figure of weak positive detection sample F625-5.
Embodiment
Below in conjunction with embodiment, the invention will be further described.Should be understood that following examples only for illustration of the present invention but not for limiting scope of the present invention.The experimental technique of unreceipted actual conditions in following embodiment, the condition that conveniently conditioned disjunction is advised according to manufacturer is usually carried out.
The preparation of embodiment 1 standard substance
1, respectively from confirm as A3243G sudden change patient and nonmutationed normal human peripheral blood extract DNA, adopt QIAGEN company peripheral blood DNA extract test kit extract.
2, adopt amplimer to carry out pcr amplification, in amplified production, include the site at A3243G sudden change place;
Primer sequence is respectively:
MT3243-F(SEQ ID NO.3):
5’-AGAACAGGGTTTGTTAAGATGGC-3’；
MT3243-R(SEQ ID NO.4):
5’-GTTTTAAGTTTTATGCGATTACCG-3’
5 ' of primer MT3243-R have employed biotin labeling.
The amplification program of PCR is:
1. 94 DEG C, 5min; 2. 94 DEG C, 20s; 3. 55 DEG C, 20s; 4. 72 DEG C, 20s; Step 2. to cycle number be 4. 40; 5. 72 DEG C, 10min.
3, PCR primer be respectively charged in pGEM-T carrier, because PCR primer end is all with an outstanding A, and pGEM-T carrier is with an outstanding T, and therefore PCR primer directly can be carried out sticky end with pGEM-T carrier be connected without the need to cutting through enzyme.
4, adopt conventional sequence measurement to verify 3243 site sequences loaded in carrier, the carrier getting wild-type and saltant type respectively as 0% and 100% standard substance;
1) the PCR primer total order that the peripheral blood sample increasing normal people obtains is classified as shown in SEQ ID NO.1.Carrier called after MT3243-0 containing this sequence.
2) the PCR primer total order that the peripheral blood sample increasing mutated patient obtains is classified as shown in SEQ ID NO.2.Carrier called after MT3243-100 containing this sequence.
5, dilute after above-mentioned two kinds of vector purification with TE respectively, and quantitatively to 50ng/ μ L.
6, according to certain ratio mixing MT3243-0 and MT3243-100 two kinds of carriers, the standard substance of 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% are prepared respectively; Prepare MT3243-0 and the MT3243-100 carrier consumption that each concentration standards uses as shown in the table:
7, get the standard substance 1 μ L of above-mentioned each concentration respectively, adopt amplimer MT3243-F and MT3243-R pairing, carry out pcr amplification, amplification program is the same, and amplification system is 25 μ L.
8, use Pyrosequencing primer MT3243-S to carry out Manganic pyrophosphate complex initiation the pcr amplification product of each concentration standards, and calculate heterozygosity.Each standard substance all carry out 4 experiments.The sequence of Pyrosequencing primer is:
MT3243-S:5’-GGTTTGTTAAGATGGCAG-3’（SEQIDNO.5）。
Specific experiment process, with reference to the step of QIAGEN Company, Instrument PyroMarkQ24 operation, is summarized as follows:
1) pcr amplification product of 10 μ about L and binding buffer liquid, avidin etc. are added in reaction tubes jointly, PCR primer (using biotin labeling) and avidin are combined.
2) PCR primer after combining is carried out adsorbing respectively, the reactions steps such as cleaning.
3) extension damping fluid, Pyrosequencing primer are added in Sptting plate jointly, and the PCR primer of absorption is eluted in Sptting plate.80 DEG C of sex change 1.5 minutes.
4) add enzyme, substrate and dNTP respectively in the reaction clip configured in instrument, react according to pre-set programs subsequently.
5) the software analysis result adopting instrument to carry, instrument will calculate the heterozygous mutant rate in mutational site automatically.
The sequencing result of each standard substance is as follows:
1 Manganic pyrophosphate complex initiation result in 4 tests of each standard substance is see Fig. 1-14.In figure, blue shading part is the sequencing result in this standard substance mutational site, has marked the ratio shared by A and G of mt3243 site in standard substance respectively.As in Fig. 1 0% standard substance practical measurement result be that A accounts for 99%, G and accounts for 1%.
Then adopt software to analyze the measured value abscissa value of each check point (namely in Figure 15) and theoretical value (per-cent namely in actual accepted standard product shared by 3243 site G is also the ordinate value of each check point in Figure 15), draft regression equation subsequently.
Correction result and regression equation are shown in Figure 15.Wherein, regression equation is: Y=-0.0000737578382X
3+ 0.01054497865X
2+ 0.71548332X-0.63357639446, this regression equation and standard substance will be further used for the detection by quantitative of each detection sample Mitochondria A3243G heterozygous mutant rate.
Embodiment 2 examination criteria product and regression equation application in the detection
After standard substance and regression equation make, we are applied to the detection by quantitative of early-onset diabetes patient peripheral blood sample Mitochondria A3243G heterozygous mutant rate.
One, method
1, from patient's peripheral blood, extract DNA, method is with embodiment 1;
2, the method for gel electrophoresis is adopted to detect the quality of extracting DNA, and by the concentration of spectrophotometric determination DNA;
3, the DNA of detection is all diluted to 50ng/ μ L with TE;
4, adopt amplimer MT3243-F and MT3243-R pairing, the DNA sample of amplification patient, amplification program is with embodiment 1;
5, pcr amplification product is after electroresis appraisal, and adopt Pyrosequencing primer MT3243-S to carry out Manganic pyrophosphate complex initiation, method is with embodiment 1;
6, each sample standard deviation carries out repeating experiment for 3 times, gets the measured value of mean value as sample of 3 numerical value;
7, the regression equation in the measured value Application Example 1 obtained by Manganic pyrophosphate complex initiation carries out Data correction, draw final calculation result, by measured value as X, substitute in aforesaid cubic regression equation and calculate, the Y value of acquisition is the net result of the A3243G heterozygous mutant rate after correction.
Two, result
Adopt aforesaid method altogether 80 routine early-onset diabetes family samples to be carried out to the quantitative analysis of A3243G heterozygous mutant rate, Manganic pyrophosphate complex initiation result is as follows with correction result:
1, F932-4 detects sample (see Figure 16) for negative.What show in this figure is a Manganic pyrophosphate complex initiation result of F932-4 sample, and mutation rate is 0%, and the mean value of this sample 3 order-checkings is 0.5%, and the value after regression equation corrects is-0.27%, and this shows the heterozygous mutant that there are not 3243 sites in this sample.Clinical information is also confirmed that it is the spouse of diabetic, is normal people.Completely the same with detected result.
2, F932-1 is positive detection sample (see Figure 17).This sample has carried out 3 times equally and has detected, and Figure 17 is the result of wherein 1 time, and mutation rate is 19%, and the mean value that this sample measures for 3 times is 18%, and the value after regression equation corrects is 15.2%, and this shows that the heterozygous mutant rate in 3243 sites of this sample is higher.Clinical information also confirms that this patient is typical early-onset diabetes patient.
3, F625-5 is weak positive detection sample (see Figure 18).This sample has carried out 3 times equally and has detected, and Fig. 8 is the result of wherein 1 time, and mutation rate is 2%, and the mean value that this sample measures for 3 times is 1.5%, and the value after regression equation corrects is 0.5%, and this shows that 3243 sites of this sample exist low-down heterozygous mutant.Clinical information also confirms that this patient shows as early-onset diabetes, and symptom is not clearly.
4, sample determination data
| Sample number | Measured value (%) | Correction value (%) |
| F1096-1 | 23 | 20.50342 |
| F1096-2 | 39 | 38.93394 |
| F1096-3 | 49.5 | 51.67479 |
| F1096-4 | 55 | 58.34511 |
| F1096-5 | 1.5 | 0.463126 |
| F1096-6 | 49 | 51.06606 |
| F1096-7 | 45.5 | 46.80394 |
| F1096-8 | 1.5 | 0.463126 |
| F1096-9 | 35 | 34.16357 |
| F932-1 | 18 | 15.23154 |
| F932-2 | 48 | 49.84823 |
| F932-3 | 1 | 0.092378 |
| F932-4 | 0.5 | -0.27321 |
| F637-1 | 29.5 | 27.75641 |
| F637-2 | 47.5 | 49.23922 |
| F637-3 | 40 | 40.13722 |
| F637-4 | 25.5 | 23.24511 |
| F637-5 | 48 | 49.84823 |
| F637-6 | 41.5 | 41.94835 |
| F637-7 | 66.5 | 71.88792 |
| F637-8 | 57 | 60.75017 |
| F637-10 | 4.5 | 2.792913 |
| F625-1 | 34 | 32.98387 |
| F625-2 | 23 | 20.50342 |
| F625-3 | 43.5 | 44.37246 |
| F625-4 | 1 | 0.092378 |
| F625-5 | 1 | 0.092378 |
| F625-6 | 1.5 | 0.463126 |
| F625-7 | 2 | 0.83898 |
| F625-8 | 1.5 | 0.463126 |
| F1256-1 | 30 | 28.32994 |
| F1256-2 | 32.5 | 31.2258 |
| F1256-3 | 2 | 0.83898 |
| F1256-4 | 1 | 0.092378 |
| F1256-5 | 1 | 0.092378 |
| F1256-6 | 1.5 | 0.463126 |
| F674-1 | 16 | 13.21156 |
| F674-2 | 2 | 0.83898 |
| F674-3 | 1.5 | 0.463126 |
| F656-1 | 19.5 | 16.78117 |
| F656-3 | 46.5 | 48.02132 |
| F656-4 | 39.5 | 39.53513 |
| F656-5 | 14.5 | 11.73315 |
| F656-6 | 2.5 | 1.219886 |
| F656-7 | 12 | 9.343247 |
| F924-1 | 14.5 | 11.73315 |
| F924-2 | 10 | 7.501997 |
| F924-3 | 39 | 38.93394 |
| F924-4 | 44.5 | 45.5875 |
| F924-5 | 35.5 | 34.75555 |
| F924-6 | 31 | 29.48281 |
| F924-7 | 30.5 | 28.90543 |
| F924-8 | 43.5 | 44.37246 |
| F924-9 | 34.5 | 33.57299 |
| F924-10 | 0.5 | -0.27321 |
| F924-11 | 1 | 0.092378 |
| F924-12 | 0.5 | -0.27321 |
| F924-13 | 0.5 | -0.27321 |
| F924-14 | 0.5 | -0.27321 |
| F924-15 | 1 | 0.092378 |
| F924-16 | 51.5 | 54.10709 |
| F924-17 | 22 | 19.42545 |
| F333-1 | 12.5 | 9.81356 |
| F333-2 | 13 | 10.28776 |
| F333-3 | 1 | 0.092378 |
| F333-4 | 0.5 | -0.27321 |
| F333-5 | 28.5 | 26.61543 |
| F333-6 | 0 | -0.63358 |
| F333-7 | 1 | 0.092378 |
| F333-8 | 0 | -0.63358 |
| F333-9 | 1 | 0.092378 |
| F333-10 | 0 | -0.63358 |
| F333-11 | 0.5 | -0.27321 |
| F333-12 | 28 | 26.04809 |
| F333-13 | 0.5 | -0.27321 |
| F333-14 | 0.5 | -0.27321 |
| F333-15 | 0.5 | -0.27321 |
| F333-16 | 0.5 | -0.27321 |
| F333-17 | 0.5 | -0.27321 |
| F333-18 | 0 | -0.63358 |
Claims (7)
1. one kind is applicable to the test kit of the detection line plastochondria A3243G heterozygous mutant rate of Manganic pyrophosphate complex initiation, it is characterized in that, it comprises the standard substance of detection line plastochondria A3243G heterozygous mutant rate, the amplimer containing the mitochondria DNA fragment in 3243 sites that increases, the Pyrosequencing primer containing the mitochondria DNA fragment in 3243 sites and specification sheets
Wherein, the standard substance of described detection line plastochondria A3243G heterozygous mutant rate comprise:
(1) containing 3243 sites be the carrier of mitochondria DNA fragment of wild-type A; (2) containing 3243 sites be the carrier of mitochondria DNA fragment of saltant type G;
The nucleotide sequence of the mitochondria DNA fragment that 3243 described sites are wild-type A is as shown in SEQIDNO.1, and the nucleotide sequence of the mitochondria DNA fragment that 3243 described sites are saltant type G is as shown in SEQIDNO.2; Described be the carrier of the mitochondria DNA fragment of wild-type A containing 3243 sites and be the vehicle group resulting mixture of the mitochondria DNA fragment of saltant type G containing 3243 sites; The content being the carrier of the mitochondria DNA fragment of saltant type G containing 3243 sites in described mixture accounts for the arbitrary concentration in 0 ~ 100% of carrier total amount, be the content of the carrier of the mitochondria DNA fragment of wild-type A containing 3243 sites be surplus, between described mixture, form a series of carrier concn ratio gradient;
The nucleotide sequence of described amplimer is respectively as shown in SEQ ID NO.3 and SEQIDNO.4; Described Pyrosequencing primer is as shown in SEQ ID NO.5;
Described specification sheets describes the using method of described test kit, and the method comprises the following steps:
1) with the standard substance of the described detection line plastochondria A3243G heterozygous mutant rate of different weight percentage for template, adopt amplimer amplification containing the mitochondria DNA fragment in 3243 sites;
2) to step 1) PCR primer of gained carries out Manganic pyrophosphate complex initiation;
3) according to step 2) in the result of Manganic pyrophosphate complex initiation draft regression equation for Data correction;
4) from sample to be checked, DNA is extracted;
5) with step 4) DNA of gained is template, adopts amplimer amplification containing the mitochondria DNA fragment in 3243 sites;
6) to step 5) PCR primer of gained carries out Manganic pyrophosphate complex initiation;
7) to step 6) the sequencing result applying step 3 of gained) regression equation of gained carries out Data correction, obtains the plastosome A3243G heterozygous mutant rate of sample to be checked;
Step 3) described in regression equation be Y=-0.0000737578382X
3+ 0.01054497865X
2+ 0.71548332X-0.63357639446.
2. test kit as claimed in claim 1, it is characterized in that, described carrier is plasmid.
3. test kit as claimed in claim 1, is characterized in that, also comprises the medium dissolving carrier in described mixture.
4. test kit as claimed in claim 1, it is characterized in that, described test kit also comprises DNA extraction reagent and/or TE damping fluid.
5. test kit as claimed in claim 1, it is characterized in that, the standard substance of described detection line plastochondria A3243G heterozygous mutant rate are obtained by the preparation method comprised the steps:
1) from the sample confirming as A3243G sudden change and in nonmutationed normal sample, DNA is extracted respectively;
2) amplimer is adopted to step 1) DNA of gained carries out pcr amplification respectively, includes 3243 sites of Mitochondrial DNA in amplified production;
3) by step 2) PCR primer of gained to be respectively charged in carrier and to carry out sequence verification, thus obtaining wild-type carrier and mutant vector;
4) standard substance of each concentration series are prepared according to the different ratios of wild-type and mutant vector.
6. a method for the diagnosis of non-diseases or the detection by quantitative plastosome A3243G heterozygous mutant rate of methods for the treatment of object, is characterized in that, comprise the steps:
1) with the standard substance of detection line plastochondria A3243G heterozygous mutant rate for template, adopt amplimer amplification containing the mitochondria DNA fragment in 3243 sites;
2) to step 1) PCR primer of gained carries out Manganic pyrophosphate complex initiation;
3) according to step 2) in the result of Manganic pyrophosphate complex initiation draft regression equation for Data correction;
4) from sample to be checked, DNA is extracted;
5) with step 4) DNA of gained is template, adopts amplimer amplification containing the mitochondria DNA fragment in 3243 sites;
6) to step 5) PCR primer of gained carries out Manganic pyrophosphate complex initiation;
7) sequencing result applying step 3) regression equation of gained carries out Data correction, obtains the plastosome A3243G heterozygous mutant rate of sample to be checked;
Step 1) standard substance of described detection line plastochondria A3243G heterozygous mutant rate comprise:
(1) containing 3243 sites be the carrier of mitochondria DNA fragment of wild-type A; (2) containing 3243 sites be the carrier of mitochondria DNA fragment of saltant type G;
The nucleotide sequence of the mitochondria DNA fragment that 3243 described sites are wild-type A is as shown in SEQIDNO.1, and the nucleotide sequence of the mitochondria DNA fragment that 3243 described sites are saltant type G is as shown in SEQIDNO.2; Described be the carrier of the mitochondria DNA fragment of wild-type A containing 3243 sites and be the vehicle group resulting mixture of the mitochondria DNA fragment of saltant type G containing 3243 sites; The content being the carrier of the mitochondria DNA fragment of saltant type G containing 3243 sites in described mixture accounts for the arbitrary concentration in 0 ~ 100% of carrier total amount, be the content of the carrier of the mitochondria DNA fragment of wild-type A containing 3243 sites be surplus, between described mixture, form a series of carrier concn ratio gradient;
Step 1) and step 5) described in the nucleotide sequence of amplimer respectively as shown in SEQ ID NO.3 and SEQIDNO.4;
Step 2) and step 6) described in the sequence of Manganic pyrophosphate complex initiation primer used as shown in SEQ ID NO.5;
Step 3) described in regression equation be Y=-0.0000737578382X
3+ 0.01054497865X
2+ 0.71548332X-0.63357639446.
7. method as claimed in claim 6, is characterized in that, step 1) or 5) program of described amplification is: 1. 94 DEG C, 5min; 2. 94 DEG C, 20s; 3. 55 DEG C, 20s; 4. 72 DEG C, 20s; Step 2. to cycle number be 4. 40; 5. 72 DEG C, 10min.
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| CN107955833B (en) * | 2017-11-20 | 2021-08-24 | 广州市达瑞生物技术股份有限公司 | Heterozygous mutant dry blood spot positive quality control product suitable for deafness mutant gene detection |
| CN108192965B (en) * | 2017-12-30 | 2021-09-10 | 北京中科唯新生物医学研究所有限公司 | Method for detecting heterogeneity of mitochondrial genome A3243G locus |
| CN109234380B (en) * | 2018-10-18 | 2019-08-27 | 东莞博奥木华基因科技有限公司 | Hereditary hearing impairment related gene inspecting reagent kit and specific primer group |
| 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 |
| CN110878333A (en) * | 2019-05-15 | 2020-03-13 | 嘉兴雅康博医学检验所有限公司 | Method for preparing gene mutation reference substance |
| CN114085903B (en) * | 2022-01-19 | 2022-04-26 | 广州嘉检医学检测有限公司 | Primer pair probe combination product for detecting mitochondria 3243A & gtG mutation, kit and detection method thereof |
| CN115404266B (en) * | 2022-09-29 | 2023-08-11 | 广州源井生物科技有限公司 | Preparation method of standard substances with different mutation rates based on humanized cells |
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Non-Patent Citations (3)
| Title |
|---|
| Detection and quantification of heteroplasmic mutant mitochondrial DNA by real-time amplification refractory mutation system quantitative PCR analysis：a single-step approach;Ren-kui Bai et al;《clinical chemistry》;20040630;第50卷(第6期);第996-1001页 * |
| 建立线粒体DNA中A3243G突变检测方法;张维延;《中国血液流变学杂志》;20061230;第16卷(第4期);第532-533页 * |
| 线粒体DNA A3243G点突变的临床异质性表现;张英等;《中国医学科学院学报》;20050228;第27卷(第1期);第77-80页 * |
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