CN104450925A - Primers, probes and kit for detecting gene mutation caused by G6PD deficiency disease - Google Patents
Primers, probes and kit for detecting gene mutation caused by G6PD deficiency disease Download PDFInfo
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Abstract
The invention relates to primers for detecting gene mutation caused by G6PD deficiency disease. The primers are as shown in SEQ ID NO: 1-32. The invention further relates to probes for detecting gene mutation caused by G6PD deficiency disease. The probes are as shown in the sequences SEQ ID NO: 33-67. The invention further relates to a kit for detecting gene mutation caused by G6PD deficiency disease. The kit comprises a PCR reaction liquid I, a PCR reaction liquid II and a PCR reaction liquid III. The kit for detecting gene mutation caused by G6PD deficiency disease provided by the invention has the advantages of high coverage rate, high detection rate of female heterozygote, low cost and high accuracy.
Description
Technical field
The present invention relates to technique of gene detection, particularly detect the primer of G6PD deficiency disease transgenation, probe and test kit.
Background technology
Glucose 6 phosphate dehydrogenase deficiency (Glucose-6-phosphate dehydrogenase deficiency, G6PD deficiency) is one of modal mankind's enzyme defect disease.The essence of G6PD deficiency disease is G6PD transgenation, is the chain incomplete dominant lnheritance of X.Patients with clinical manifestations's change differs.G6PD deficient patients, if be detected and give early intervention, usually can play good preventive effect.Because only having an X chromosome, therefore in colony, only there is the normal hemizygote two class crowd with significantly lacking in the male sex.Women has two X chromosomes, is divided into normally, heterozygote and homozygote crowd according to its different amts carrying G6PD mutator gene.Heterozygote accounts for the overwhelming majority of female patient.Confirm that G6PD lacks heterozygote and can fall ill both at home and abroad for a long time, and external perspective study shows that the danger of G6PD heterozygote generation hyperbilirubinemia of newborn will be significantly higher than the normal homozygote of G6PD, it is one of independent hazard factor of hyperbilirubinemia of newborn generation that G6PD lacks heterozygote.
The active Screening tests of the domestic G6PD adopted clinically has methemoglobin reduction test, nitro nitroblue tetrazolium (NBT) paper disk method and fluorescent stains at present.Part Experiment room adopts the examination of automatic clinical chemistry analyzer enzyme process detection by quantitative Cord Blood of Neonates G6PD deficiency disease.Many laboratories adopt fluorescent stains to make qualitative primary dcreening operation, then quantitatively make a definite diagnosis with ratioing technigue.Although two kinds of methods are had complementary advantages, examination susceptibility and diagnostic accuracy can be improved, all can accurately detect male sex's hemizygote and female homozygote, but it is still undesirable to the actual recall rate of women's heterozygote, reason may be many-sided, and the difference of operator itself may have certain influence to result.But from the pathogenesis of heterozygote, zymetology detection itself cannot detect most heterozygote and be only most the underlying cause.Molecular biology method detects the gold standard that transgenation is Heterozygote detation, domesticly at present some achievements are achieved in this respect, ARMS (cut model and flashlight model detector), restriction enzyme enzyme process, high resolving power melting curve analysis (HRM), denaturing high-performance liquid chromatography (DHPLC) method and gene chips have been widely used in the detection of G6PD deficiency disease known mutations gene, and achieve good effect.In general, though above-mentioned molecular biological method to the recall rate of heterozygote up to 90% ~ 99%, but technical sophistication, somewhat expensive, length consuming time, only for scientific research, not yet promote the use of at clinical hospitals at present, therefore develop a kind of simple, quick, cheap gene test screening method, improving the recall rate of heterozygote, is clinical primary demand.
More than 160 kind of G6PD genic mutation type has been reported in the whole world so far, at least 34 kinds are found in population of China, G1376T, G1388A, A95G are the modal 3 kinds of sudden changes of Chinese, present market there is no maturation and is applied to clinical G6PD deficiency disease gene diagnosis product, the shortcoming that the technological method such as ARMS, restriction enzyme enzyme process, HRM and the DHPLC method etc. that are applied to laboratory scientific research all have clinical application difficulty to promote.
Although existing molecular detecting method can solve the problem of G6PD deficiency disease women Heterozygote detation preferably, diverse ways has self limitation: (1) DHPLC is high to instrument requirements equipment, and applicability is not strong; (2) flux of ARMS detection is very limited, and needs PCR aftertreatment, easily occurs polluting the generation causing false positive results; (3) multiplex allele-specific PCR (multiplex allele specific PCR, MAS-PCR) method is comparatively easy, economical, reliable, but due in a reaction system, interference mutually is easily there is between multipair primer, affect PCR result, so a PCR reaction tubes only should detect limited several sudden changes, concerning the diagnosis of the so many sudden change of G6PD deficiency disease, recall rate is limited; (4) although fluorescent PCR melting curve method susceptibility is high, and accuracy is low, flux is low, the result interpretation that multiple site is detected with pipe is more difficult, and especially heterozygote melts cutting edge of a knife or a sword figure more complicated difficult interpretation, and clinical application is limited; (5) reverse dot blot hybridization method is sensitive, reliable, and cost is low, but complex operation, need to do aftertreatment to PCR primer, easily cause pollution, result interpretation is subject to subjective impact.(6) sequencing operation steps is various, and need special expensive instrument, testing cost is expensive.
The defect of aforesaid method directly limit their clinical applications in the gene test of G6PD deficiency disease, namely there is clinical application and promotes more difficult problem; In addition, also there is the not high defect of fraction of coverage in the gene detecting kit developed based on aforesaid method simultaneously.
Summary of the invention
Technical problem to be solved by this invention is: provide a kind of recall rate high and the test kit of the fluorescent PCR method of applicable clinical application detection G6PD deficiency disease transgenation.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is:
For detecting a primer for G6PD deficiency disease transgenation, comprise the primer of following sequence:
The sequence of the upstream primer F1 in specific amplification 1376T site, 1388A site and downstream primer R1, described F1 is if: the sequence of SEQ ID NO:1, described R1 is as SEQ ID NO:2; The sequence of the upstream primer F2 in specific amplification 871A site and downstream primer R2, described F2 is if: the sequence of SEQ ID NO:3, described R2 is as SEQ ID NO:4; The sequence of the upstream primer F3 in specific amplification 95G site and downstream primer R3, described F3 is if: the sequence of SEQ ID NO:5, described R3 is as SEQ ID NO:6; The sequence of the upstream primer F4 in specific amplification 392T site and downstream primer R4, described F4 is if: the sequence of SEQ ID NO:7, described R4 is as SEQ ID NO:8; The sequence of the upstream primer F5 in specific amplification 1024T site and downstream primer R5, described F5 is if: the sequence of SEQ ID NO:9, described R5 is as SEQ ID NO:10; The sequence of the upstream primer F6 in specific amplification 196A site, 202A site and downstream primer R6, described F6 is if: the sequence of SEQ ID NO:11, described R6 is as SEQ ID NO:12; The sequence of the upstream primer F7 in specific amplification 274T site and downstream primer R7, described F7 is if: the sequence of SEQ ID NO:13, described R7 is as SEQ ID NO:14; The sequence of the upstream primer F8 in specific amplification 383C site, 406T site, 442A site, 473A site and downstream primer R8, described F8 is if: the sequence of SEQ ID NO:15, described R8 is as SEQ ID NO:16; The sequence of the upstream primer F9 in specific amplification 487A site, 493G site, 517C site, 519G site, 563T site, 592T site and downstream primer R9, described F9 is if: the sequence of SEQ ID NO:17, described R9 is as SEQ ID NO:18; The sequence of the upstream primer F10 in specific amplification 691C site, 703T site and downstream primer R10, described F10 is if: the sequence of SEQ IDNO:19, described R10 is as SEQ ID NO:20; The sequence of the upstream primer F11 in specific amplification 835A site, 835C site and downstream primer R11, described F11 is if: the sequence of SEQ ID NO:21, described R11 is as SEQ ID NO:22; The sequence of the upstream primer F12 in specific amplification 1004T site and downstream primer R12, described F12 is if: the sequence of SEQ ID NO:23, described R12 is as SEQ ID NO:24; The sequence of the upstream primer F13 in specific amplification 1159T site, 1160A site, 1225T site and downstream primer R13, described F13 is if: the sequence of SEQ ID NO:25, described R13 is as SEQ ID NO:26; The upstream primer F14 in specific amplification 1311T site, 1339A site, 1340T site, 1360T site, 1381A site, 1387T site, 1414C site and downstream primer R14, the sequence of described F14 is if: the sequence of SEQ ID NO:27, described R14 is as SEQ ID NO:28; The sequence of the upstream primer F15 in specific amplification ACTBP site and downstream primer R15, described F15 is if: the sequence of SEQ ID NO:29, described R15 is as SEQ ID NO:30; And the sequence of general upstream primer F16 and downstream primer R16, described F16 is if: the sequence of SEQ ID NO:31, described R16 is as SEQ ID NO:32.
A kind of probe for detecting the transgenation of G6PD deficiency disease of the present invention, comprises the probe of following sequence: the sequence of the probe P1 in specific recognition 1376T site, described P1 is as SEQ ID NO:33; The sequence of the probe P2 in specific recognition 1388A site, described P2 is as SEQ ID NO:34; The sequence of the probe P3 in specific recognition 871A site, described P3 is as SEQ ID NO:35; The sequence of the probe P4 in specific recognition 95G site, described P4 is as SEQ ID NO:36; The sequence of the probe P5 in specific recognition 392T site, described P5 is as SEQ ID NO:37; The sequence of the probe P6 in specific recognition 1024T site, described P6 is as SEQ ID NO:38; The sequence of the probe P7 in specific recognition 196A site, described P7 is as SEQ ID NO:39; The sequence of the probe P8 in specific recognition 202A site, described P8 is as SEQ ID NO:40; The sequence of the probe P9 in specific recognition 274T site, described P9 is as SEQ IDNO:41; The sequence of the probe P10 in specific recognition 383C site, described P10 is as SEQ ID NO:42; The sequence of the probe P11 in specific recognition 406T site, described P11 is as SEQ ID NO:43; The sequence of the probe P12 in specific recognition 442A site, described P12 is as SEQ ID NO:44; The sequence of the probe P13 in specific recognition 473A site, described P13 is as SEQ ID NO:45; The sequence of the probe P14 in specific recognition 487A site, described P14 is as SEQ ID NO:46; The sequence of the probe P15 in specific recognition 493G site, described P15 is as SEQ ID NO:47; The sequence of the probe P16 in specific recognition 517C site, described P16 is as SEQ ID NO:48; The sequence of the probe P17 in specific recognition 519G site, described P17 is as SEQ ID NO:49; The sequence of the probe P18 in specific recognition 563T site, described P18 is as SEQ ID NO:50; The sequence of the probe P19 in specific recognition 592T site, described P19 is as SEQ ID NO:51; The sequence of the probe P20 in specific recognition 691C site, described P20 is as SEQ ID NO:52; The sequence of the probe P21 in specific recognition 703T site, described P21 is as SEQ IDNO:53; The sequence of the probe P22 in specific recognition 835A site, described P22 is as SEQ ID NO:54; The sequence of the probe P23 in specific recognition 835C site, described P23 is as SEQ ID NO:55; The sequence of the probe P24 in specific recognition 1004T site, described P24 is as SEQ ID NO:56; The sequence of the probe P25 in specific recognition 1159T site, described P25 is as SEQ ID NO:57; The sequence of the probe P26 in specific recognition 1160A site, described P26 is as SEQ ID NO:58; The sequence of the probe P27 in specific recognition 1225T site, described P27 is as SEQ ID NO:59; The sequence of the probe P28 in specific recognition 1311T site, described P28 is as SEQ ID NO:60; The sequence of the probe P29 in specific recognition 1339A site, described P29 is as SEQ ID NO:61; The sequence of the probe P30 in specific recognition 1340T site, described P30 is as SEQ ID NO:62; The sequence of the probe P31 in specific recognition 1360T site, described P31 is as SEQ ID NO:63; The sequence of the probe P32 in specific recognition 1381A site, described P32 is as SEQ ID NO:64; The sequence of the probe P33 in specific recognition 1387T site, described P33 is as SEQ ID NO:65; The sequence of the probe P34 in specific recognition 1414C site, described P34 is as SEQ IDNO:66; The sequence of the probe P35 in specific recognition ACTBP site, described P35 is as SEQ ID NO:67.
The present invention also discloses a kind of test kit for detecting the transgenation of G6PD deficiency disease, comprise PCR reaction solution I, PCR reaction solution II and PCR reaction solution III, described PCR reaction solution I comprises specific amplification 1376T site, the upstream primer F1 in 1388A site and downstream primer R1, the upstream primer F2 in specific amplification 871A site and downstream primer R2, the upstream primer F15 in specific amplification ACTBP site and downstream primer R15, general upstream primer F16 and downstream primer R16, the probe P1 in specific recognition 1376T site, the probe P2 in specific recognition 1388A site, and the probe P3 in specific recognition 871A site, described PCR reaction solution II comprises upstream primer F3 and the downstream primer R3 in specific amplification 95G site, the upstream primer F4 in specific amplification 392T site and downstream primer R4, the upstream primer F15 in specific amplification ACTBP site and downstream primer R15, general upstream primer F16 and downstream primer R16, the upstream primer F5 in specific amplification 1024T site and downstream primer R5, the probe P4 in specific recognition 95G site, the probe P5 in specific recognition 392T site, and the probe P6 in specific recognition 1024T site, described PCR reaction solution III comprises specific amplification 196A site, the upstream primer F6 in 202A site and downstream primer R6, the upstream primer F7 in specific amplification 274T site and downstream primer R7, specific amplification 383C site, 406T site, 442A site, the upstream primer F8 in 473A site and downstream primer R8, specific amplification 487A site, 493G site, 517C site, 519G site, 563T site, the upstream primer F9 in 592T site and downstream primer R9, specific amplification 691C site, the upstream primer F10 in 703T site and downstream primer R10, specific amplification 835A site, the upstream primer F11 in 835C site and downstream primer R11, the upstream primer F12 in specific amplification 1004T site and downstream primer R12, specific amplification 1159T site, 1160A site, the upstream primer F13 in 1225T site and downstream primer R13, specific amplification 1311T site, 1339A site, 1340T site, 1360T site, 1381A site, 1387T site, the upstream primer F14 in 1414C site and downstream primer R14, the upstream primer F15 in specific amplification ACTBP site and downstream primer R15, general upstream primer F16 and downstream primer R16, the probe P7 in specific recognition 196A site, the probe P8 in specific recognition 202A site, the probe P9 in specific recognition 274T site, the probe P10 in specific recognition 383C site, the probe P11 in specific recognition 406T site, the probe P12 in specific recognition 442A site, the probe P13 in specific recognition 473A site, the probe P14 in specific recognition 487A site, the probe P15 in specific recognition 493G site, the probe P16 in specific recognition 517C site, the probe P17 in specific recognition 519G site, the probe P18 in specific recognition 563T site, the probe P19 in specific recognition 592T site, the probe P20 in specific recognition 691C site, the probe P21 in specific recognition 703T site, the probe P22 in specific recognition 835A site, the probe P23 in specific recognition 835C site, the probe P24 in specific recognition 1004T site, the probe P25 in specific recognition 1159T site, the probe P26 in specific recognition 1160A site, the probe P27 in specific recognition 1225T site, the probe P28 in specific recognition 1311T site, the probe P29 in specific recognition 1339A site, the probe P30 in specific recognition 1340T site, the probe P31 in specific recognition 1360T site, the probe P32 in specific recognition 1381A site, the probe P33 in specific recognition 1387T site, and the probe P34 in specific recognition 1414C site.
Beneficial effect of the present invention is:
(1) apply and specially stick up the Taqman probe and specific site amplification method with universal amplification primer that tail modifies, independent somatotype is done to covering most of G6PD 6 kinds of crowd most common mutations points that suddenly change, by the adjacent sites fluorescent mark of the same race in 28 kinds of non-common site, both detection accuracy, specific requirement had been met, reduce again the rare genotypic scope of investigation further, being in charge of detection without the need to doing each rare site, reducing testing cost; (2) test kit for detecting the transgenation of G6PD deficiency disease of the present invention can complete the detection of 34 kinds of sudden changes in 34 sites in three pipe PCR reaction solutions, by machine on simple application of sample, whole operating in 1 ~ 1.5 hour can complete, and operation steps is few, consuming time short; (3) PCR reaction solution I, PCR reaction solution II and PCR reaction solution III first can get PCR reaction solution I and PCR reaction solution II detects 6 common site, after eliminating 6 common site, get PCR reaction solution III pair of extraneous sample of common site again detect, thus clinical detection cost can be reduced further.(4) the present invention is homogeneous phase detection system, and after adding template, PCR stopped pipe increases, and without the need to PCR aftertreatment, decreases PCR primer and pollutes probability; (5) can detect 34 kinds of mutation types in G6PD gene 34 sites, detection site is many simultaneously, high to the sudden change fraction of coverage of Chinese population, can meet the clinical examination demand to G6PD deficiency disease patient or carrier; (6) judge by amplification curve " having " or "None" " positive " or " feminine gender " in each mutational site, the easy interpretation of result, not easily make mistakes, specificity is high.
Accompanying drawing explanation
Amplification curve diagram when Fig. 1 is 1376G>T sudden change;
Amplification curve diagram when Fig. 2 is 1388G>A sudden change;
Amplification curve diagram when Fig. 3 is 871G>A sudden change;
Amplification curve diagram when Fig. 4 is 95A>G sudden change;
Amplification curve diagram when Fig. 5 is 1024C>T sudden change;
Amplification curve diagram when Fig. 6 is 392G>T sudden change;
Amplification curve diagram when Fig. 7 is 28 site mutation of not somatotype.
Embodiment
By describing technology contents of the present invention in detail, realized object and effect, accompanying drawing is coordinated to be explained below in conjunction with embodiment.
The design of most critical of the present invention is: specially stick up the Taqman probe and specific site amplification method with universal amplification primer that tail modifies, 34 types covering most of G6PD sudden change crowd are detected, there is the advantage that detection accuracy is high, specificity is high, testing cost is low.
Primer for detecting the transgenation of G6PD deficiency disease of the present invention, comprises the primer of following sequence: SEQ ID NO:1-32.Probe for detecting the transgenation of G6PD deficiency disease of the present invention, comprises the probe of following sequence: SEQ ID NO:33-67.The present invention also discloses a kind of test kit for detecting the transgenation of G6PD deficiency disease, comprises PCR reaction solution I, PCR reaction solution II and PCR reaction solution III.
Further, described PCR reaction solution I, PCR reaction solution II and PCR reaction solution III comprise upstream primer F15 and the downstream primer R15 in specific amplification ACTBP site respectively, and the probe P35 in specific recognition ACTBP site.Further, described PCR reaction solution I, PCR reaction solution II and PCR reaction solution III comprise 10 × PCR buffer, 5 × Probe qPCR buffer, dNTPs and Hotstart taq enzyme respectively.Further, 5 ' end of described probe carries out fluorescein-labelled, and described fluorescein is VIC, ROX, Cy5 or FAM.Further, also comprise positive quality control product and negative quality control product, described positive quality control product comprises 871G>A sudden change, 1004C>A sudden change and 1376G>T sudden change standard substance, and described negative quality control product is sterilized water.
The present invention sticks up the Taqman probe of tail modification and the specific site amplification method with universal amplification primer by special, detects each mutational site.Test kit for detecting the transgenation of G6PD deficiency disease of the present invention comprises A, B, C tri-pipe PCR reaction solution (PCR reaction solution I, PCR reaction solution II and PCR reaction solution III) and positive quality control product corresponding to each pipe, A pipe (PCR reaction solution I) comprises 3 sites, B pipe (PCR reaction solution II) comprises 3 sites and C pipe (PCR reaction solution III) comprises 28 sites, design three pipe multiple fluorescence PCR reaction solutions altogether, the upper machine after only needing to add DNA profiling that detects runs 1h, gather the fluorescent signal in PCR process, by Ct value, simple qualitative interpretation is carried out to amplification curve, easy and simple to handle, without the need to carrying out PCR primer electrophoresis or purifying, avoid the pollution that open pipe causes, be applicable to sample examination in enormous quantities to detect.
(1) probe, design of primers: the test kit for detecting the transgenation of G6PD deficiency disease of the present invention is high, the cheap test kit of a stability technically developed at multiple fluorescence PCR.Multiple fluorescence PCR system is the amplification system of a more complicated, should ensure the accuracy in each site, specificity, and the amplification efficiency taking into account each site is again close.Only design a large amount of primer for each site, probe screens, the clinical detection requirement of expection cannot be reached.
We are according to the G6PD gene order of GeneBank database and each site mutation base sequence, consume design optimization, sequence alignment that great effort carries out every point probe sequence, the mutational site design close to position merges amplimer, and amplimer contains specific primer sequences and universal amplification Tag fragment.Devise not isolabeling, part 5 ' end respectively for each catastrophe point and stick up the Taqman probe that tail is modified, avoid the primer dimer between each probe and non-specific binding.6 sites of PCR reaction solution I, PCR reaction solution II, devise 2 ~ 3 pairs of amplimers and Taqman probe respectively, the probe in each site is distinguished with different fluorescent marks respectively, 28 sites of PCR reaction solution III devise Taqman probe respectively, adjacent sites shares amplimer, and three tube reaction liquid share the amplimer of a pair reference gene and a probe does interior controlling/monitoring.
The primer in each site of this test kit, probe sequence are all through the distinguished sequence of optimal screening, after optimum probe, primer are selected in each site, be respectively a large amount of Orthogonal contrasts test and determine respective consumption with the primer in other sites, probe.The amplimer that table 1 is each site of the present invention and probe sequence table.
Table 1
(2) reaction solution configuration: table 2 is A tube reaction liquid formula.Table 3 is B tube reaction liquid formula.Table 4 is C tube reaction liquid formula.
Table 2
Table 3
Table 4
(3) PCR program: the present invention devises the PCR program in two stages simultaneously, after first making the specific amplification primer in each site carry out a small amount of enrichment to special masterplate by lower annealing temperature, recycling universal amplification primer carries out annealing, the amplification of higher anneal temperature, the specific fragment amplification efficiency in each site is controlled at similar level, make the detection efficiency in each site close, reach stable Detection results.
Concrete program is as follows: 94 DEG C of 5min, (94 DEG C of 10s, 60 DEG C of 20s) * 3cycles, and (94 DEG C of 10s, 69 DEG C of 32s) * 40cycles, at the collection step fluorescent signal of " 69 DEG C of 32s ".
(4) positive criteria product preparation: obtain the PCR primer containing mutant nucleotide sequence with the sudden change sample in 14 pairs of primer amplifications, 34 sites of A, B, C tri-in pipe, obtain the PCR primer containing reference gene sequence with F15, R15 amplification β actin reference gene simultaneously, by internal reference PCR primer with carry out Fusion after the PCR primer purifying of various sudden change and be connected and clone, the corresponding sudden change positive criteria product containing internal reference sequence of preparation, with these sudden change standard substance for template carries out simulation amplification to human genome DNA.
(5) probe specificity inspection: during with above-mentioned positive criteria product for template amplification, internal reference Cy5 signal is normal amplification (Ct value 22 ~ 38, Δ Rn>1.5*10
6) then experiment effectively, otherwise test invalid.
In the effective situation of experiment, there is normal amplification curve (Ct value 22 ~ 38, Δ Rn>1.5*10 in the probe in each site
6) be Positive mutants result, on the contrary then for sudden change is negative, each sudden change standard substance all have good specificity, and amplification curve is respectively as shown in Fig. 1 ~ Fig. 7 of subsequent drawings.
The process that test kit for detecting the transgenation of G6PD deficiency disease of the present invention detects for G6PD missing gene in turn includes the following steps:
A, extract sample DNA to be detected: extract genomic dna from peripheral blood, concentration is at 2 ~ 100ng/uL;
B, template are added: each experiment need arrange feminine gender (sterilized water does template), positive control (with the built-in positive quality control product of test kit for template), respectively get 2uL sample to be tested DNA single to be solely added into divide separately and to be filled to A, B, C in eight connecting legs tri-in tube reaction liquid, of short duration centrifugal after mixing;
C, fluorescent PCR increase: be placed on ABI 7500 fluorescent PCR instrument by the reaction solution that b step has added template, increase, collect real-time fluorescent signals after editting PCR program and sample information;
D, interpretation of result and interpretation: requirement listed by according to the form below 5 manually arranges threshold line, the baseline of each fluorescence channel, carries out interpretation to result.Table 5 is the threshold line of each fluorescence channel, baseline and result interpretation table.
Table 5
Embodiments of the invention one are:
1, main moiety: the main moiety for the test kit detecting the transgenation of G6PD deficiency disease of the present embodiment is as shown in table 6, and table 6 is the main moiety table of the test kit for detecting the transgenation of G6PD deficiency disease of the present embodiment.
Table 6
2, instrument is suitable for: fluorescent PCR amplification instrument (ABI 7500)
3, condition of storage and validity period: test kit lucifuge is stored in less than-18 DEG C, avoids multigelation.Validity period: 6 months.
4, sample requires: this test kit samples sources is anticoagulated whole blood, and antithrombotics used is Sodium Citrate or EDTA, can not use anticoagulant heparin.
5, the method for inspection: the extraction of a, DNA: the extracting method of this test kit to human gene group DNA does not specify requirement, general Available experimental room ordinary method (phenol-chloroform extraction process) or test kit extract human gene group DNA, and the whole blood DNA of recommendation QIAGEN company extracts test kit.Whole blood DNA according to QIAGEN extracts test kit, direct by specification application of sample; If extract DNA by phenol-chloroform or other method, then to measure DNA concentration, carry out if desired concentrating or diluting, after DNA concentration being adjusted to 2 ~ 100ng/ μ L, just can carry out test experience.B, pcr amplification: PCR reaction solution A, B, C of taking out test kit, get ABI7500 special PCR eight connecting leg ready, list stoichiometric number needed for single experiment, be dispensed in eight connecting legs by 23uL/ person-portion respectively, add the sample to be tested DNA 2 μ L extracted in order respectively, reaction is totally 25 μ L, positive and negative contrast is set, carry out mark, of short duration centrifugal in 5000rpm, be placed in fluorescent PCR detector and carry out increasing and signal collection.C, amplification program are arranged: 94 DEG C of 5min, (94 DEG C of 10s, 60 DEG C of 20s) * 3cycles, (94 DEG C of 10s, 69 DEG C of 32s) * 40cycles, at the collection step fluorescent signal of " 69 DEG C of 32s ".D, interpretation of result: by the threshold line, the baseline that manually arrange each fluorescence channel shown in table 5, interpretation is carried out to result.
Amplification curve diagram when Fig. 1 is 1376G>T sudden change.As shown in the figure, in figure, there are two bars curves, from being VIC fluorescence curve away from X-axis, represent 1376 jump signals, from being CY5 fluorescence curve close to X-axis, representing internal reference signal; Amplification curve diagram when Fig. 2 is 1388G>A sudden change.As shown in the figure, in figure, there are two bars curves, from being ROX fluorescence curve away from X-axis, represent 1388 jump signals, from being CY5 fluorescence curve close to X-axis, representing internal reference signal; Amplification curve diagram when Fig. 3 is 871G>A sudden change.As shown in the figure, in figure, there are two bars curves, from being FAM fluorescence curve away from X-axis, represent 871 jump signals, from being CY5 fluorescence curve close to X-axis, representing internal reference signal; Amplification curve diagram when Fig. 4 is 95A>G sudden change.As shown in the figure, in figure, there are two bars curves, from being FAM fluorescence curve away from X-axis, represent 95 jump signals, from being CY5 fluorescence curve close to X-axis, representing internal reference signal; Amplification curve diagram when Fig. 5 is 1024C>T sudden change.As shown in the figure, in figure, there are two bars curves, from being VIC fluorescence curve away from X-axis, represent 1024 jump signals, from being CY5 fluorescence curve close to X-axis, representing internal reference signal; Amplification curve diagram when Fig. 6 is 392G>T sudden change.As shown in the figure, in figure, there are two bars curves, from being ROX fluorescence curve away from X-axis, represent 392 jump signals, from being CY5 fluorescence curve close to X-axis, representing internal reference signal; Amplification curve diagram when Fig. 7 is 28 site mutation of not somatotype.As shown in the figure, in figure, there are two bars curves, from being ROX fluorescence curve away from X-axis, represent any one catastrophe point signal of 28 kinds of sudden changes, from being CY5 fluorescence curve close to X-axis, representing internal reference signal.
In sum, test kit for detecting the transgenation of G6PD deficiency disease provided by the invention contains 34 kinds of mutational sites that domestic literature has been reported, fraction of coverage is high, improves the recall rate of women's heterozygote, not yet has so comprehensively G6PD gene mutation detection kit at present clinically.Test kit of the present invention can realize 34 sites and detect, and realize stopped pipe operation, avoid polluting, with low cost, accuracy is high, within 1 hour, can complete detection, and result interpretation is easy, is applicable to Clinical screening and applies.The primer sets that the present invention comprises, probe groups are all that the distinguished sequence designed for each site is optimized screening.For the multiple system of complexity of many probes, many amplimer compositions, the present invention sticks up tail modification by adding at 5 ' end of part probe, avoid the primer dimer between each probe and non-specific binding, after the specific amplification primer in each site carries out a small amount of enrichment to special masterplate simultaneously, recycling universal amplification primer increases, the specific fragment amplification efficiency in each site is controlled at similar level, makes the detection efficiency in each site close, reach stable Detection results.
The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every equivalents utilizing specification sheets of the present invention and accompanying drawing content to do, or be directly or indirectly used in relevant technical field, be all in like manner included in scope of patent protection of the present invention.
Claims (6)
1. for detecting a primer for G6PD deficiency disease transgenation, it is characterized in that, comprising the primer of following sequence:
The sequence of the upstream primer F1 in specific amplification 1376T site, 1388A site and downstream primer R1, described F1 is if: the sequence of SEQ ID NO:1, described R1 is as SEQ ID NO:2;
The sequence of the upstream primer F2 in specific amplification 871A site and downstream primer R2, described F2 is if: the sequence of SEQ ID NO:3, described R2 is as SEQ ID NO:4;
The sequence of the upstream primer F3 in specific amplification 95G site and downstream primer R3, described F3 is if: the sequence of SEQID NO:5, described R3 is as SEQ ID NO:6;
The sequence of the upstream primer F4 in specific amplification 392T site and downstream primer R4, described F4 is if: the sequence of SEQ ID NO:7, described R4 is as SEQ ID NO:8;
The sequence of the upstream primer F5 in specific amplification 1024T site and downstream primer R5, described F5 is if: the sequence of SEQ ID NO:9, described R5 is as SEQ ID NO:10;
The sequence of the upstream primer F6 in specific amplification 196A site, 202A site and downstream primer R6, described F6 is if: the sequence of SEQ ID NO:11, described R6 is as SEQ ID NO:12;
The sequence of the upstream primer F7 in specific amplification 274T site and downstream primer R7, described F7 is if: the sequence of SEQ ID NO:13, described R7 is as SEQ ID NO:14;
The sequence of the upstream primer F8 in specific amplification 383C site, 406T site, 442A site, 473A site and downstream primer R8, described F8 is if: the sequence of SEQ ID NO:15, described R8 is as SEQ IDNO:16;
The sequence of the upstream primer F9 in specific amplification 487A site, 493G site, 517C site, 519G site, 563T site, 592T site and downstream primer R9, described F9 is if: the sequence of SEQ ID NO:17, described R9 is as SEQ ID NO:18;
The sequence of the upstream primer F10 in specific amplification 691C site, 703T site and downstream primer R10, described F10 is if: the sequence of SEQ ID NO:19, described R10 is as SEQ ID NO:20;
The sequence of the upstream primer F11 in specific amplification 835A site, 835C site and downstream primer R11, described F11 is if: the sequence of SEQ ID NO:21, described R11 is as SEQ ID NO:22;
The sequence of the upstream primer F12 in specific amplification 1004T site and downstream primer R12, described F12 is if: the sequence of SEQ ID NO:23, described R12 is as SEQ ID NO:24;
The sequence of the upstream primer F13 in specific amplification 1159T site, 1160A site, 1225T site and downstream primer R13, described F13 is if: the sequence of SEQ ID NO:25, described R13 is as SEQ ID NO:26;
The upstream primer F14 in specific amplification 1311T site, 1339A site, 1340T site, 1360T site, 1381A site, 1387T site, 1414C site and downstream primer R14, the sequence of described F14 is if: the sequence of SEQ ID NO:27, described R14 is as SEQ ID NO:28;
The sequence of the upstream primer F15 in specific amplification ACTBP site and downstream primer R15, described F15 is if: the sequence of SEQ ID NO:29, described R15 is as SEQ ID NO:30;
And the sequence of general upstream primer F16 and downstream primer R16, described F16 is if: the sequence of SEQ ID NO:31, described R16 is as SEQ ID NO:32.
2. for detecting a probe for G6PD deficiency disease transgenation, it is characterized in that, comprising the probe of following sequence:
The sequence of the probe P1 in specific recognition 1376T site, described P1 is as SEQ ID NO:33;
The sequence of the probe P2 in specific recognition 1388A site, described P2 is as SEQ ID NO:34;
The sequence of the probe P3 in specific recognition 871A site, described P3 is as SEQ ID NO:35;
The sequence of the probe P4 in specific recognition 95G site, described P4 is as SEQ ID NO:36;
The sequence of the probe P5 in specific recognition 392T site, described P5 is as SEQ ID NO:37;
The sequence of the probe P6 in specific recognition 1024T site, described P6 is as SEQ ID NO:38;
The sequence of the probe P7 in specific recognition 196A site, described P7 is as SEQ ID NO:39;
The sequence of the probe P8 in specific recognition 202A site, described P8 is as SEQ ID NO:40;
The sequence of the probe P9 in specific recognition 274T site, described P9 is as SEQ ID NO:41;
The sequence of the probe P10 in specific recognition 383C site, described P10 is as SEQ ID NO:42;
The sequence of the probe P11 in specific recognition 406T site, described P11 is as SEQ ID NO:43;
The sequence of the probe P12 in specific recognition 442A site, described P12 is as SEQ ID NO:44;
The sequence of the probe P13 in specific recognition 473A site, described P13 is as SEQ ID NO:45;
The sequence of the probe P14 in specific recognition 487A site, described P14 is as SEQ ID NO:46;
The sequence of the probe P15 in specific recognition 493G site, described P15 is as SEQ ID NO:47;
The sequence of the probe P16 in specific recognition 517C site, described P16 is as SEQ ID NO:48;
The sequence of the probe P17 in specific recognition 519G site, described P17 is as SEQ ID NO:49;
The sequence of the probe P18 in specific recognition 563T site, described P18 is as SEQ ID NO:50;
The sequence of the probe P19 in specific recognition 592T site, described P19 is as SEQ ID NO:51;
The sequence of the probe P20 in specific recognition 691C site, described P20 is as SEQ ID NO:52;
The sequence of the probe P21 in specific recognition 703T site, described P21 is as SEQ ID NO:53;
The sequence of the probe P22 in specific recognition 835A site, described P22 is as SEQ ID NO:54;
The sequence of the probe P23 in specific recognition 835C site, described P23 is as SEQ ID NO:55;
The sequence of the probe P24 in specific recognition 1004T site, described P24 is as SEQ ID NO:56;
The sequence of the probe P25 in specific recognition 1159T site, described P25 is as SEQ ID NO:57;
The sequence of the probe P26 in specific recognition 1160A site, described P26 is as SEQ ID NO:58;
The sequence of the probe P27 in specific recognition 1225T site, described P27 is as SEQ ID NO:59;
The sequence of the probe P28 in specific recognition 1311T site, described P28 is as SEQ ID NO:60;
The sequence of the probe P29 in specific recognition 1339A site, described P29 is as SEQ ID NO:61;
The sequence of the probe P30 in specific recognition 1340T site, described P30 is as SEQ ID NO:62;
The sequence of the probe P31 in specific recognition 1360T site, described P31 is as SEQ ID NO:63;
The sequence of the probe P32 in specific recognition 1381A site, described P32 is as SEQ ID NO:64;
The sequence of the probe P33 in specific recognition 1387T site, described P33 is as SEQ ID NO:65;
The sequence of the probe P34 in specific recognition 1414C site, described P34 is as SEQ ID NO:66;
The sequence of the probe P35 in specific recognition ACTBP site, described P35 is as SEQ ID NO:67.
3. for detecting a test kit for G6PD deficiency disease transgenation, it is characterized in that, comprising PCR reaction solution I, PCR reaction solution II and PCR reaction solution III,
Described PCR reaction solution I comprises specific amplification 1376T site, the upstream primer F1 in 1388A site and downstream primer R1, the upstream primer F2 in specific amplification 871A site and downstream primer R2, the upstream primer F15 in specific amplification ACTBP site and downstream primer R15, general upstream primer F16 and downstream primer R16, the probe P1 in specific recognition 1376T site, the probe P2 in specific recognition 1388A site, and the probe P3 in specific recognition 871A site;
Described PCR reaction solution II comprises upstream primer F3 and the downstream primer R3 in specific amplification 95G site, the upstream primer F4 in specific amplification 392T site and downstream primer R4, the upstream primer F5 in specific amplification 1024T site and downstream primer R5, the upstream primer F15 in specific amplification ACTBP site and downstream primer R15, general upstream primer F16 and downstream primer R16, the probe P4 in specific recognition 95G site, the probe P5 in specific recognition 392T site, and the probe P6 in specific recognition 1024T site;
Described PCR reaction solution III comprises specific amplification 196A site, the upstream primer F6 in 202A site and downstream primer R6, the upstream primer F7 in specific amplification 274T site and downstream primer R7, specific amplification 383C site, 406T site, 442A site, the upstream primer F8 in 473A site and downstream primer R8, specific amplification 487A site, 493G site, 517C site, 519G site, 563T site, the upstream primer F9 in 592T site and downstream primer R9, specific amplification 691C site, the upstream primer F10 in 703T site and downstream primer R10, specific amplification 835A site, the upstream primer F11 in 835C site and downstream primer R11, the upstream primer F12 in specific amplification 1004T site and downstream primer R12, specific amplification 1159T site, 1160A site, the upstream primer F13 in 1225T site and downstream primer R13, specific amplification 1311T site, 1339A site, 1340T site, 1360T site, 1381A site, 1387T site, the upstream primer F14 in 1414C site and downstream primer R14, the upstream primer F15 in specific amplification ACTBP site and downstream primer R15, general upstream primer F16 and downstream primer R16, the probe P7 in specific recognition 196A site, the probe P8 in specific recognition 202A site, the probe P9 in specific recognition 274T site, the probe P10 in specific recognition 383C site, the probe P11 in specific recognition 406T site, the probe P12 in specific recognition 442A site, the probe P13 in specific recognition 473A site, the probe P14 in specific recognition 487A site, the probe P15 in specific recognition 493G site, the probe P16 in specific recognition 517C site, the probe P17 in specific recognition 519G site, the probe P18 in specific recognition 563T site, the probe P19 in specific recognition 592T site, the probe P20 in specific recognition 691C site, the probe P21 in specific recognition 703T site, the probe P22 in specific recognition 835A site, the probe P23 in specific recognition 835C site, the probe P24 in specific recognition 1004T site, the probe P25 in specific recognition 1159T site, the probe P26 in specific recognition 1160A site, the probe P27 in specific recognition 1225T site, the probe P28 in specific recognition 1311T site, the probe P29 in specific recognition 1339A site, the probe P30 in specific recognition 1340T site, the probe P31 in specific recognition 1360T site, the probe P32 in specific recognition 1381A site, the probe P33 in specific recognition 1387T site, and the probe P34 in specific recognition 1414C site.
4. the test kit for detecting the transgenation of G6PD deficiency disease according to claim 3, it is characterized in that, described PCR reaction solution I, PCR reaction solution II and PCR reaction solution III comprise upstream primer F15 and the downstream primer R15 in specific amplification ACTBP site respectively, and the probe P35 in specific recognition ACTBP site.
5. the test kit for detecting the transgenation of G6PD deficiency disease according to claim 3, it is characterized in that, described PCR reaction solution I, PCR reaction solution II and PCR reaction solution III comprise 10 × PCRbuffer, 5 × Probe qPCR buffer, dNTPs and Hotstart taq enzyme respectively.
6. the test kit for detecting the transgenation of G6PD deficiency disease according to claim 3, it is characterized in that, also comprise positive quality control product and negative quality control product, described positive quality control product comprises 871G>A sudden change, 1004C>A sudden change and 1376G>T sudden change standard substance, and described negative quality control product is sterilized water.
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