CN101078025A - PCR kit for detecting SNP of COPD - Google Patents
PCR kit for detecting SNP of COPD Download PDFInfo
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- CN101078025A CN101078025A CNA2006100225373A CN200610022537A CN101078025A CN 101078025 A CN101078025 A CN 101078025A CN A2006100225373 A CNA2006100225373 A CN A2006100225373A CN 200610022537 A CN200610022537 A CN 200610022537A CN 101078025 A CN101078025 A CN 101078025A
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Abstract
A PCR kit for detecting SNP of COPD comprises a primer smad3PA, a primer smad3PB, a probe YBW 60315-FAM , a probe YBW 60315-VIC (g) and PCR reaction liquid which are separately packaged, wherein the sequences of the primers smad3PA and smad3PB are as follows: smad3PA 5-TGCACTGATCACTGTTCCTGTACTT-3; smad3PB 5-CATCGGTGT TTCCAGAGTCAGA-3; the sequences of the probe YBW 60315-FAM and the probe YBW 60315-VIC (g) are as follows: YBW 60315-FAM FAM-CCGCCCCATta CCCATCCATAGA-TAMRA; YBW 60315-VIC (g) VIC-CGCCCCATTgCCCATCCAT-TAMRA.
Description
Technical field
The present invention relates to molecular biology and medical field.Relate more specifically to single nucleotide polymorphism (the Single nucleotide polymorphism of SMAD3 gene, be abbreviated as SNP) and with chronic obstructive disease of lung (Chronic obstructive pulmonary disease, be abbreviated as COPD) dependency, relate to the test kit that detects these SNP.
Background technology
Chronic obstructive pulmonary disease (COPD) is the disease with flow limitation feature, and flow limitation is not exclusively reversible, can be with airway hyperreactivity, and be and carry out sexual development, relevant with lung to the abnormal inflammatory reaction of obnoxious flavour or deleterious particle.The high paid more and more attention of COPD morbidity and case fatality rate, COPD occupies the 4th of the current cause of death in the world, and by inference, to the year two thousand twenty, in the global range society being worked the mischief, COPD will drain into the 5th in the maximum disease.The U.S. has 2,880,000 people to die from COPD calendar year 2001, and sickness rate is constantly in rising trend.The sickness rate of China COPD is also in rising trend, find when in recent years China the north and rural area, middle area 102230 crowds that grow up being investigated, China more than 15 years old crowd's COPD sickness rate be about 3.17%.There are every year 1000000 people to die from COPD approximately.
The diagnosis of COPD should be determined according to medical history, symptom, sign, pulmonary function test and image check analysis-by-synthesis, is the essential condition of diagnosis COPD but there is incomplete reversibility flow limitation.Diagnostic method commonly used at present comprises:
(1) when the patient cough is arranged, cough up phlegm, have difficulty in breathing symptom and (or) during dangerous factor contact history, should consider COPD, but must clarify a diagnosis through pulmonary function test.Chronic cough, expectoration often exist many year prior to flow limitation, but not allly have the patient of cough, expectoration symptom all can develop into COPD, and small number of patients only has the non-reversibility flow limitation to change and do not have chronic cough, expectoration symptom.
(2) the early stage sign of sign can be not obvious, along with barrel chest can appear in disease progression, and lung's percussion hyperresonant note, the two pulmonary respiration sounds of auscultation weaken, and expiratory duration prolongs, audible and moist rales.
(3) pulmonary function test.Pulmonary function test all has the important clinical meaning for the diagnosis of COPD, the degree that is in a bad way assessment, disease progression, therapeutic response, prognosis judgement, is the gold standard of diagnosis COPD.After sucking bronchodilator, FEV is less than 80 predicated values, and FEV/FVC shows there is flow limitation, and can not reverse fully that less than 70 o'clock diagnosable is COPD simultaneously.
(4) diagnostic imaging of COPD.Image check is to determining excessive inflation of lung and complication and differentiating significant with other pulmonary disorder.Conventional rabat sign mainly shows as two enhanced lung markings, and when pulmonary emphysema occurring, the lung transparence increases, and diaphragm is low flat, and the hilus pulumonis vascular lake is the undesirable root shape, and the lung field external perihaemal canal is very thin, reduced number etc.But it is limited that conventional rabat is worth emophysematous early diagnosis, its mainly act on be used for determining lung excessively inflation degree and with the differential diagnosis of other disease.CT examination especially high-resolution ct (HRCT) is diagnosis pulmonary emphysema the most responsive imaging examination methods, and type, distribution and the severity of clear and definite lung qi kind all is better than conventional rabat.Emophysematous CT shows as no wall and the very thin lung tissue lucent area of wall, and wind-puff chamber and normal surrounding tissue boundary are clear.But be subjected to the influence of bed thickness and irradiation dose, CT (HRCT) can not be used for the assessment to full lung, and is limited to the diagnostic value of chronic bronchitis.
As everyone knows, COPD is the coefficient result of inherited genetic factors and environmental factors.Inherited genetic factors, existing certified is that alpha1-antitrypsin (α 1-AT) lacks; The environmental risk factor mainly is the smoking factor.Have 85% to be long-term smoker among the COPD patient, yet only have 10%～20% people to develop into Symptomatic COPD patient, individual susceptibility difference to COPD is described, and this susceptibility is the phenotype of gene pleiomorphism.Chinese scholars has been done a large amount of research for the susceptibility of COPD, has inquired into gene pleiomorphism and at COPD developing effect has taken place.
But, also do not report the research of the dependency report between SMAD gene and the chronic obstructive pulmonary disease (COPD), more do not confirm the report of dependency between the single nucleotide polymorphism (SNP) of SMAD gene and the chronic obstructive pulmonary disease (COPD).
There are some researches show: TGF-β 1 (transforming growth factor-beta 1) plays important effect in the developing of COPD disease.And the TGF signal is mainly by Smad family mediation, and TGF-β forms receptor complex as part, activates Smad and enters nuclear, activates or suppress transcribing of target gene that their regulate jointly.
Found to have 8 kinds of different Smad albumen in mammals altogether, these albumen can be divided into 3 different subtribes: R-Smad (receptor-regulated Smads); Co-Smad (common-partner Smads) and I-Smad (inhibitory Smads).The R-Smad subtribe can be further divided into two groups, i.e. BMP-Smad and TGF-β/activin-Smad.Smad1, Smad5, Smad8 are by BMP receptor (ALK22, ALK23 and ALK26) and ALK21 phosphorylation; Smad2 and Smad3 are activated by TGF-β/activin receptor (ALK25 and ALK24) and orphan receptor ALK27.Co-Smad can form different poly-complex body with the R-Smad of all phosphorylations, so it is the shared composition on TGF-β/activin and the BMP signal transduction pathway.Up to now, a kind of Co-Smad, i.e. Smad4 in mammals, have only been found.I-Smad, i.e. Smad6, Smad7 suppress the signal transduction of TGF-B superfamily by the activation of blocking-up R-Smad and Co-Smad.Smad albumen is by the Human genome group coding, and the gene of coding Smad2, Smad4 and Smad7 is positioned at 18q21-22; The gene of coding Smad3 and Smad6 is positioned at 15q21-22; The gene of coding Smad5, Smad1 and Smad8 lays respectively at 15q31.4 and 13.
In sum, for the final treatment COPD disease that realizes, this area presses for the tumor susceptibility gene of seeking the COPD disease, and develops test kit that detects the COPD disease and relevant medicine thus.
For this reason, the applicant is that to have submitted application number to China national Department of Intellectual Property be 200610021293.7 application for a patent for invention June 29 in 2006, in this application for a patent for invention, the applicant provides a kind of PCR test kit that detects the SNP relevant with chronic obstructive disease of lung, it is characterized in that by primer smad7PA, the primer smad7PB, the no MgCl that separate packing
2Damping fluid, archaeal dna polymerase, MgCl
2, the DNTP of deoxynucleotide unit and water forms.Wherein, the sequence of primer smad7PA, smad7PB is:
smad7PA 5-gtgtcttcattctaggagtc-3
smad7PB 5-cacccacacaccatccacct-3
Experimental result shows, owing to utilize detected SNP of this invention test kit and COPD to have very high cognation, therefore not only can diagnose and treat the COPD disease comparatively exactly, and can make susceptible COPD disease patient just not take reasonable precautions (as appropriate change living environment, living habit etc.) before the morbidity, thereby improve COPD susceptible person's lifetime and quality of life, therefore have earth shaking using value and social benefit.
Certainly, well-known, no matter be simplification from detecting operation, still from the cost of detecting operation and the multiple angles such as accuracy of detected result, the test kit that only has a kind of SNP of detection is far from being enough.Therefore, be necessary on the basis of above-mentioned prior art, the PCR test kit of the SNP that new being used to detect COPD is provided.
Look in the invention
Purpose of the present invention promptly provides a kind of new test kit that is used for diagnosing (especially early diagnosis) COPD disease.
The applicant is through deeply and extensive studies, and the SNP of a large amount of candidate genes is carried out deep research.Find first and proved that the part SNP and the COPD of SMAD3 gene are closely related, and find its new function.The difference of SMAD3 gene is an important factor of COPD disease incidence, wherein SNP (+239A/G) gene distribution in case and control group has significant difference (p＜0.05), therefore can be used as the specific SNP that detects the COPD disease, and we have finished the present invention on this basis.
According to test kit of the present invention, a kind of method that the COPD disease susceptibility of individuality is diagnosed can be provided, this method is:
Detect individual SMAD 3 genes, and compare with normal people's SMAD 3 genes, the possibility that there are differences with regard to showing this individuality trouble COPD is higher than normal population.
In the present invention, the single nucleotide polymorphism of described SMAD3 gene (SNP) is positioned on the 3rd intron of SMAD3 gene:
+239A→G
In the present invention, provide a kind of method that detects the single nucleotide polymorphism (SNP) that whether has the SMAD3 gene in the sample, this method comprises the steps:
(1) utilizes our self-designed test kit amplification SMAD3 gene, obtain pcr amplification product.
(2) utilize whether there is single nucleotide polymorphism in the TAQMAN technology for detection amplified production:
+239A→G
Among the present invention, the amplified production size is 78bp, wherein contains+239A → G
For realizing purpose of the present invention, basic fundamental route of the present invention is:
SNP molecular marker method in a kind of the 3rd intron identifying the SMAD3 gene, by extracting the genomic dna of host cell, adopt test kit provided by the invention to carry out pcr amplification, amplifying target genes, utilize the TAQMAN technology to detect, find that SNP exists, and utilizes the dna sequencing method to confirm the position of existence and the existence of this SNP.
SMAD3 gene intron sequence can obtain from the Genbank gene pool, and original gene group complete sequence is numbered AC012568 in the Genbank gene pool.
Those skilled in the art is clear, has a lot of analytical procedures can be used for detecting the gene intron sequence and has the single nucleotide polymorphism distribution frequency.These technology comprise: dna sequencing, PCR-SSCP, PCR-HPLC, PCR-TAQMAN etc.
The present invention provides the detection kit of a kind of easy to use, highly sensitive above-mentioned SNP of detection especially, it contains the PCR specific amplification primer and is used for PCR reaction solution (as conventional assembly such as reagent, damping fluid) that pcr amplification detects etc., and those skilled in the art know these conventional assembly and detection methods.When adopting TAQMAN technology for detection amplified production whether to have the comparison of sudden change, also need more needed conventional reagent of TAQMAN etc. with normal SMAD7 gene order.
Specifically, the invention provides the PCR test kit of the SNP of a kind of COPD of detection, it is characterized in that constituting by the primer smad3PA, the primer smad3PB that separate packing, probe YBW60315-FAM (a), probe YBW60315-VIC (g) and PCR reaction solution, wherein,
The sequence of primer smad3PA, smad3PB is:
Smad3PA 5-TGCACTGATCACTGTTCCTGTACTT-3
Smad3PB 5-CATCGGTGTTTCCAGAGTCAGA-3
The sequence of probe YBW60315-FAM (a), probe YBW60315-VIC (g) is:
YBW60315-FAM(a) FAM-CCGCCCCATTaCCCATCCATAGA-TAMRA
YBW60315-VIC(g) VIC-CGCCCCATTgCCCATCCAT-TAMRA
In the present invention, lay respectively at two kinds of fluorescent markers that FAM on probe YBW60315-FAM (a) and the probe YBW60315-VIC (g) and VIC are respectively different colours.
With regard to the present invention, the PCR reaction solution in the test kit can directly be served as by commercially available currently available products, the TaqMan universal master mix PCR reaction solution of producing as ABI company etc.; But it number is mentioned by the no MgCl that separates packing in 200610021293.7 the application for a patent for invention also can still adopting aforementioned application
2Damping fluid, archaeal dna polymerase, MgCl
2, the PCR reaction solution formed of the DNTP of deoxynucleotide unit and water.In the present invention, lay respectively at two kinds of fluorescent markers that FAM on probe YBW60315-FAM (a) and the probe YBW60315-VIC (g) and VIC are respectively different colours, this fluorescent marker also is existing commercially available prod, as the applicant at Chinese invention patent ZL03135755.5 number (patent name: the fluorescent marker that provides the Y chromosome multiple colour fluorescent composite amplification reagent kit) etc.
Major technique item of the present invention is offered and is:
1., study and confirmed that the difference of SMAD3 gene is an important factor of COPD disease incidence, wherein SNP (+therefore 239A/G) gene distribution in case and control group has significant difference (p＜0.05), can be used as the specific SNP that detects the COPD disease;
2., designed amplimer smad3PA and the smad3PB that is used for pcr amplification at the said mutation point.
3., utilize whether there is single nucleotide polymorphism in the TAQMAN technology for detection pcr amplification product:
+239A→G
And for this reason specialized designs probe YBW60315-FAM (a) and probe YBW60315-VIC (g).
Therefore, of particular note, with regard to test kit of the present invention, choosing of usage ratio between primer smad3PA, primer smad3PB, probe YBW60315-FAM (a), probe YBW60315-VIC (g) and the PCR reaction solution is accessory, and promptly this usage ratio chooses the prerequisite that does not constitute technical solution of the present invention.For example, narration with reference to the front can be found, when the PCR reaction solution in the test kit directly adopts commercially available currently available products (as the TaqMan universal master mix PCR reaction solution of ABI company production), amount ratio regular meeting between PCR reaction solution and primer smad3PA, primer smad3PB, probe YBW60315-FAM (a), the probe YBW60315-VIC (g) is different and different because of the commercially available prod, simultaneously, when still adopting by no MgCl
2Damping fluid, archaeal dna polymerase, MgCl
2, the DNTP of deoxynucleotide unit and water form the PCR reaction solution time, the usage ratio between PCR reaction solution and primer smad3PA, primer smad3PB, probe YBW60315-FAM (a), the probe YBW60315-VIC (g) also can change.Simultaneously, in the test kit product, the ratio between primer smad3PA, primer smad3PB, probe YBW60315-FAM (a) and the probe YBW60315-VIC (g) also can adjust at different customer requirements.
Certainly, in order to make the making of this test kit more easy, the present invention recommends to adopt following usage ratio scheme: when being benchmark with 25-μ l reaction system, comprise 12.5 μ l TaqMan universalmaster mix PCR reaction solutions (production of ABI company) in the test kit, 300nM primer smad3PA, 300nM primer smad3PB, 250nM probe YBW60315-FAM (a) and 250nM probe YBW60315-VIC (g).When detecting, take 5 μ l dna samples and add above-mentioned reaction system.By regulating the water yield that adds, allow the concentration of primer smad3PA and primer smad3PB be 300nM, the concentration of probe YBW60315-FAM (a) and probe YBW60315-VIC (g) is 250nM.
Owing to utilize detected SNP of test kit of the present invention and COPD to have very high cognation, therefore not only can diagnose and treat the COPD disease comparatively exactly, and can make susceptible COPD disease patient just not take reasonable precautions (as appropriate change living environment, living habit etc.) before the morbidity, thereby improve COPD susceptible person's lifetime and quality of life, therefore have earth shaking using value and social benefit.In addition, compare with aforementioned the applicant's No. 200610021293.7 disclosed technical schemes of application for a patent for invention, the key distinction of the present invention is as follows:
1, the goal gene of Shi Yonging is variant.Primer smad3PA, smad3PB in the test kit is at the SMAD3 gene design of individuality, and the part SNP and the COPD of individual SMAD3 gene are closely related.Experimental result shows, the difference of SMAD3 gene is an important factor of COPD disease incidence, wherein SNP (+therefore 239A/G) gene distribution in case and control group has significant difference (p＜0.05), can be used as the specific SNP that detects the COPD disease.
2, the detection method of Shi Yonging is variant.Design at the PCR-TAQMAN detection method at test kit of the present invention, and the test kit of aforementioned application for a patent for invention designs at the PCR-HPLC detection method, therefore, the design of the two is with different with the using method of the corresponding test kit of this design.
The primer difference that is adopted when 3, PCR reacts.Owing to need the gene locus of amplification that change has taken place, therefore redesigned amplimer smad3PA, smad3PB at novel site.
4, probe YBW60315-FAM (a), probe YBW60315-VIC (g) have been designed at catastrophe point.Those skilled in the art knows that the probe design in the TAQMAN technology is crucial, but also is very difficult, and the applicant has just designed probe YBW60315-FAM (a), probe YBW60315-VIC (g) by arduous creative work.
It should be noted that also the TAQMAN technology is a kind of technology well known to those skilled in the art, but according to the knowledge of the applicant, up to now, still nobody is applied to detect the SNP of COPD.For the ease of understanding, now the TAQMAN technology is summarized as follows:
Point mutation snp analysis: can detect with the Taqman probe of different fluorescence report group marks.Probe can mark on the luminophore of different colours, the difference between two probes mainly is that two base constitutes difference, depends on the type of SNP sudden change.The meaning is exactly that a kind of probe can only combine with a kind of SNP type, thereby sends a kind of fluorescence of color.Probe is hybridized with wild-type and mutator gene respectively.The efficient of probe hybridization is relevant with hybridization with excision thereafter.Illustrating that if a kind of fluorescent signal of dyestuff obviously is better than another kind it is a homozygote, is a heterozygote if two kinds of fluorescent signals all increase then show.
The data of real-time quantitative: PCR are marked on and distinguish special genotype on the xy coordinate axis.Utilize this method, can carry out gene type very soon in less than two hours 96 samples.Mention in the article that this method is delivered in " Methods " of calendar year 2001 by Sevall at first " Rapid allelic discriminationfrom real-time DNA amplification ".
In order to detect mutant, can design the probe of two kinds of different colours: a probe design detects wild-type, can flag F AM, and another probe design detects mutant, can mark VIC.The difference of common two probes has only 1 bp, because two probes differ minimum, and the therefore general strict reaction conditions of use of recommending.
Pass through analytical data, the result of two passages can be presented in the screen, the curve that does not have marker is CH1 result, that cycle number is arranged is CH2, can there be four probes (two mutant) can be analyzed at most, editting the genotype title, adjust to thresholding more than 0 after, the result will show in the form below figure.
Content of the present invention further illustrates with the following Examples, but content of the present invention is not limited only to content related among the embodiment.
Description of drawings
Fig. 1 is the gene type figure as a result among the embodiment.
Embodiment
Embodiment:
Test kit in the present embodiment is made at a pcr amplification.
Test kit in the present embodiment is to be made of the primer smad3PA, the primer smad3PB that separate packing, probe YBW60315-FAM (a), probe YBW60315-VIC (g) and PCR reaction solution, wherein,
The sequence of primer smad3PA, smad3PB is:
Smad3PA 5-TGCACTGATCACTGTTCCTGTACTT-3
Smad3PB 5-CATCGGTGTTTCCAGAGTCAGA-3
The sequence of probe YBW60315-FAM (a), probe YBW60315-VIC (g) is:
YBW60315-FAM(a) FAM-CCGCCCCATTaCCCATCCATAGA-TAMRA
YBW60315-VIC(g) VIC-CGCCCCATTgCCCATCCAT-TAMRA
On described probe YBW60315-FAM (a) and probe YBW60315-VIC (g), FAM and VIC are respectively two kinds of fluorescent markers of different colours.
Test kit in the present embodiment is that benchmark is made with 25-μ l reaction system, comprise 12.5 μ lTaqMan universal master mix PCR reaction solutions (production of ABI company) in the test kit, 300nM primer smad3PA, 300nM primer smad3PB, 250nM probe YBW60315-FAM (a) and 250nM probe YBW60315-VIC (g).When detecting, take 5 μ l dna samples and add reaction system.
For using method and the result of use that further specifies made test kit in the present embodiment, go on to say detailed process and result of use when the mentioned reagent box is used to detect the SNP relevant with chronic obstructive disease of lung below.
Experimental procedure:
1.DNA extract: the DNA extraction method according to routine is carried out DNA extraction.
2. the pcr amplification of goal gene (amplification system is a sample):
Adopt the mentioned reagent box that goal gene SMAD3 is carried out pcr amplification.The machine that uses during amplification is the Prism 7900HT of American AB I company.Condition is: 50 ℃, 2 minutes, 95 ℃ 10 minutes, activate AmpliTaqGold enzyme (archaeal dna polymerase).PCR condition: 40 circulations: 95 ℃, 15s; 60 ℃, 1min.
3. point mutation snp analysis.
Taqman probe with different fluorescence report group marks detects.In order to detect mutant, as previously mentioned, designed the probe of two kinds of different colours in the test kit of present embodiment, i.e. probe YBW60315-FAM (a) and probe YBW60315-VIC (g), wherein:
YBW60315-FAM (a) is
FAM-CCGCCCCATTaCCCATCCATAGA-TAMRA
YBW60315-VIC (g) is
VIC-CGCCCCATTgCCCATCCAT-TAMRA
Herein, a probe design detects wild-type, is labeled as FAM, and another probe is used for detecting mutant, is labeled as VIC, and the difference of two probes has only 1 bp.On the probe mark luminophore of different colours, i.e. FAM group and VIC group.Difference between these two probes mainly is that two base constitutes difference, depends on the type of SNP sudden change.The meaning is exactly that a kind of probe can only combine with a kind of SNP type, thereby sends a kind of fluorescence of color.In experimentation, probe YBW60315-FAM (a) and probe YBW60315-VIC (g) are hybridized with wild-type and mutator gene respectively.The efficient of probe hybridization is relevant with hybridization with excision thereafter.Illustrating that if a kind of fluorescent signal of dyestuff obviously is better than another kind it is a homozygote, is a heterozygote if two kinds of fluorescent signals all increase then show.
In experimentation, the data of PCR are marked on x, the y coordinate axis and distinguish special genotype.Utilize this method, can carry out gene type very soon in less than two hours 96 samples.
Pass through analytical data, the result of two passages can be presented in the screen, the curve that does not have marker is CH1 result, that cycle number is arranged is CH2, can have four probes (two mutant) can be analyzed at most, edit the genotype title, adjust to thresholding more than 0 after, the result will show in the form below screen, as shown in Figure 1.
In Fig. 1, blue a plurality of points are arranged in the zone shown in the label 1, that their reflect is homozygote AA, red a plurality of points are arranged in the zone shown in the label 2, that their reflect is homozygote GG, and viridescent a plurality of points in the zone shown in the label 3, that their reflect is heterozygote AG.
As previously mentioned, the experiment condition of present embodiment is as follows:
Reacted constituent: 25-μ l reaction system.Comprise 12.5 μ l TaqMan universal master mixPCR reaction solutions (production of ABI company), 300nM primer smad3PA, 300nM primer smad3PB, 250nM probe YBW60315-FAM (a), 250nM YBW60315-VIC (g), 5 μ l dna samples.
Need to prove: we adopt the mentioned reagent box, first by the PCR-TAQMAN technology, detect among the contrast crowd normally in 200 COPD patients of Chinese population and 200 ages, gender matched and to have A/G polymorphism SNP marker site in SMAD3 the 3rd intron+239 zones.By dna sequencing, confirmed the existence of catastrophe point.
Attached: genome sequence tabulation involved in the present invention:
<210>1
<211>78
<212>DNA
＜213〉people (Homo sapiens)
<220>
<221>PCR?Production_region
<222>44
<400>1
TGCACTGATC?ACTGTTCCTG?TACTTCTGTC?TTACCGCCCC?ATTACCCATC?CATAGATCTG?60
ACTCTGGAAA?CACCGATG 78
Claims (2)
1, a kind of PCR test kit that detects the SNP of COPD is characterized in that being made of the primer smad3PA, the primer smad3PB that separate packing, probe YBW60315-FAM (a), probe YBW60315-VIC (g) and PCR reaction solution, wherein,
The sequence of primer smad3PA, smad3PB is:
Smad3PA 5-TGCACTGATCACTGTTCCTGTACTT-3
Smad3PB 5-CATCGGTGTTTCCAGAGTCAGA-3
The sequence of probe YBW60315-FAM (a), probe YBW60315-VIC (g) is:
YBW60315-FAM(a) FAM-CCGCCCCATTaCCCATCCATAGA-TAMRA
YBW60315-VIC(g) VIC-CGCCCCATTgCCCATCCAT-TAMRA
On described probe YBW60315-FAM (a) and probe YBW60315-VIC (g), FAM and VIC are respectively two kinds of fluorescent markers of different colours.
2, test kit as claimed in claim 1, it is characterized in that: when being benchmark with 25-μ l reaction system, comprise 12.5 μ l TaqMan universal master mix PCR reaction solutions in the test kit, 300nM primer smad3PA, 300nM primer smad3PB, 250nM probe YBW60315-FAM (a) and 250nM probe YBW60315-VIC (g).
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| CNA2006100225373A CN101078025A (en) | 2006-12-19 | 2006-12-19 | PCR kit for detecting SNP of COPD |
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| CNA2006100225373A CN101078025A (en) | 2006-12-19 | 2006-12-19 | PCR kit for detecting SNP of COPD |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102388147A (en) * | 2009-02-24 | 2012-03-21 | 飞纳生物技术有限责任公司 | Genetic markers of the risk of suffering from restenosis |
| CN103320432A (en) * | 2013-05-27 | 2013-09-25 | 中国人民解放军第二军医大学 | Probe for recovering and identifying Smad3 target gene-correlative miRNA, kit and method |
| CN105018570A (en) * | 2014-04-16 | 2015-11-04 | 首都医科大学附属北京佑安医院 | Taqman fluorescent quantitative RT-PCR primers and probes for detecting IFITM3-rs12252 gene polymorphism |
| CN107385097A (en) * | 2017-09-13 | 2017-11-24 | 山东大学第二医院 | Detect the kit of SMAD4 gene V354L site mutations |
| CN113358866A (en) * | 2021-04-22 | 2021-09-07 | 四川大学华西医院 | Homogeneous phase ultrasensitive two-dimensional visualization and fluorescence analysis method for tetanus antigen based on triple parallel hybrid chain reaction and application |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102388147A (en) * | 2009-02-24 | 2012-03-21 | 飞纳生物技术有限责任公司 | Genetic markers of the risk of suffering from restenosis |
| CN103320432A (en) * | 2013-05-27 | 2013-09-25 | 中国人民解放军第二军医大学 | Probe for recovering and identifying Smad3 target gene-correlative miRNA, kit and method |
| CN103320432B (en) * | 2013-05-27 | 2015-11-25 | 中国人民解放军第二军医大学 | To be correlated with the probe of the recovery of miRNA and qualification, test kit and method for Smad3 target gene |
| CN105018570A (en) * | 2014-04-16 | 2015-11-04 | 首都医科大学附属北京佑安医院 | Taqman fluorescent quantitative RT-PCR primers and probes for detecting IFITM3-rs12252 gene polymorphism |
| CN107385097A (en) * | 2017-09-13 | 2017-11-24 | 山东大学第二医院 | Detect the kit of SMAD4 gene V354L site mutations |
| CN107385097B (en) * | 2017-09-13 | 2018-05-15 | 山东大学第二医院 | Detect the kit of SMAD4 gene V354L site mutations |
| CN113358866A (en) * | 2021-04-22 | 2021-09-07 | 四川大学华西医院 | Homogeneous phase ultrasensitive two-dimensional visualization and fluorescence analysis method for tetanus antigen based on triple parallel hybrid chain reaction and application |
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