CN104774947A - Fluorescent quantization PCR (polymerase chain reaction) method for detecting gene in heparin crude product - Google Patents
Fluorescent quantization PCR (polymerase chain reaction) method for detecting gene in heparin crude product Download PDFInfo
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Abstract
The invention relates to a fluorescent quantization PCR (polymerase chain reaction) method for detecting a gene in a heparin crude product. The fluorescent quantization PCR method comprises extraction of total DNA (deoxyribonucleic acid) in a heparin crude product, fluorescent PCR amplification reaction and data analysis. The fluorescent quantization PCR method is concise and clear, easy to operate, and high in accuracy; according to the adopted fluorescent quantization PCR detection method, the DNA efficient amplification of the PCR technology is ingeniously utilized; and the gene in the heparin crude product is efficiently and sensitively detected.
Description
Technical field
The present invention relates to a kind of method detecting heparin crude product gene in biological technical field, be specifically related to a kind of fluorescence quantifying PCR method detecting heparin crude product gene.
Background technology
Heparin is a kind of antithrombotics, is the polymer be alternately formed by connecting by two kinds of polysaccharide, has blood coagulation resisting function in vivo and in vitro.Be mainly used in thrombotic disease, myocardial infarction, operation on vessels of heart, cardia catheterization, extracorporeal circulation, hemodialysis etc. clinically.Along with pharmacology and clinical medical progress, the application of heparin constantly expands.First heparin finds from liver and gains the name, and it is also present in the tissues such as lung, vessel wall, intestinal mucosa, is a kind of natural anticoagulative substance in animal body.Naturally be present in mastocyte, now mainly extract from ox lung or intestinal mucosa.China is the maximum country of heparin output, and the heparin 70% of world market comes from China.Usual elder generation extracts heparin crude product from pig intestinal mucosa, then purifying, regenerative ratio heparin bulk drug.Thus, heparin crude product is as downstream raw material, very important to the detection of heparin crude product gene, directly affects the quality of the large class medicine of heparin one.
At present for the detection of gene mainly through the method for Standard PCR, and Standard PCR technology is carried out quantitatively and qualitative analysis the terminal product of pcr amplification reaction.To starting template accurate quantitative analysis, cannot cannot detect in real time amplified reaction.Quantitative fluorescent PCR refers to and add fluorophor in PCR reaction system, utilizes the whole PCR process of fluorescent signal accumulation Real-Time Monitoring, finally by typical curve, unknown template is carried out to the method for quantitative analysis.Fluorescent quantitative PCR technique is the change utilizing the change of fluorescent signal to detect each cyclic amplification product amount in pcr amplification reaction in real time, carries out quantitative analysis by the analysis of Ct value and typical curve to starting template.
Quantitative fluorescent PCR, as the detection technique of a kind of highly sensitive, high-speed, high-throughput and high specificity, also has broad application prospects at field of bioanalysis.The method is for gene amplification, specific amplification analysis, gene quantification analysis, gene test, gene type, snp analysis, RFLP polymorphism analysis, list/multi-gene expression research, the research of high-throughput gene expression profile.
Therefore, we devise the method for kind of quantitative fluorescent PCR to detect the gene in heparin crude product.Comprise the extraction of STb gene in heparin crude product, fluorescent PCR amplified reaction, data analysis.
Summary of the invention
The object of this invention is to provide a kind of fluorescence quantifying PCR method detecting heparin crude product gene.The object of the invention is to be achieved through the following technical solutions:
A kind of fluorescence quantifying PCR method detecting heparin crude product gene, described method comprises extraction, the design primer and probe, fluorescent PCR amplified reaction and data analysis of STb gene in heparin crude product, described primer is upstream primer and downstream primer, and described probe is fluorescent probe.
Further, in heparin crude product, the extraction of STb gene comprises: 1. prepare heparin crude product solution: take heparin crude product 30mg and be dissolved in 1ml purified water, then with heparinase effect damping fluid by above-mentioned solution dilution 100 times, obtain heparin crude product solution; 2. the deactivation of nuclease: by heparin crude product solution 85 DEG C of constant temperature 5min, then ice bath 10min on thermostat, and by nucleic acid quantification instrument mensuration DNA concentration wherein, be designated as C total; 3. heparinase degraded: the heparinase adding about 0.01IU at 200 μ l in the heparin crude product solution of nuclease deactivation, concussion mixing on constant temperature blending instrument, 25 DEG C of constant temperature 18h, centrifuging and taking supernatant liquor, 4 DEG C of preservations are stand-by, as the need testing solution after enzymolysis.
Further, described fluorescent PCR amplified reaction comprises preparation PCR system and PCR system amplified reaction.
Further, described preparation PCR system comprises need testing solution, negative control solution, upstream primer solution, downstream primer solution, probe solution, Master mix (PCR reagent premixed liquid) and water after DNA standard solution, enzymolysis; Described negative control solution is pure water.
Further, described fluorescent probe obtains fluorescent probe for adding fluorophor within the probe.
Further, described PCR system amplified reaction is: 1. denaturation 10min at 95 DEG C; 2. sex change 15s at 95 DEG C; 3. anneal 20s at 63 DEG C; 4. 72 DEG C of downward-extension 40s (fluorescent collecting); Times of thermal cycle is 40 times.
Further, described data analysis comprises the drafting of typical curve, system suitability judges and the calculating of each gene relative content of trial-product.
Further, being plotted as by each concentration DNA standard solution amplification curve Ct (threshold cycle) value corresponding with it of described typical curve, draws typical curve.
Further, in described data analysis, system suitability is judged as: drawing standard curve, the determination coefficient (R of typical curve
2)>=0.98, slope>=-4.0, Ct (threshold cycle) value of negative control should be greater than the Ct value of typical curve minimum concentration sample, or not amplification.
Further, being calculated as of each gene relative content of trial-product in described data analysis: after system suitability meets the requirements, draw each mrna concentration C in trial-product by typical curve
inspectionaccording to each kind gene content of formulae discovery:
The invention provides a kind of fluorescence quantifying PCR method detecting heparin crude product gene, its main beneficial effect is: the fluorescence quantifying PCR method for detecting heparin crude product gene provided by the invention, simple to operate, repeatable high, quantitative effect is served to heparin crude product gene, can be used for the quality identifying heparin crude product.
Accompanying drawing explanation
With reference to the accompanying drawings the present invention is described in further detail below.
Fig. 1 is the quantitative fluorescent PCR typical curve of the heparin crude product pig gene described in the embodiment of the present invention;
Fig. 2 is the quantitative fluorescent PCR typical curve of the heparin crude product cow genome described in the embodiment of the present invention.
Embodiment
A kind of fluorescence quantifying PCR method detecting heparin crude product gene described in the embodiment of the present invention.For specific experiment case, embodiment is described below, should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment 1
Fluorescence quantifying PCR method detects the pig gene in heparin crude product
1, solution preparation
(1) heparinase effect damping fluid: lime acetate 0.321mg/ml, bovine serum albumin 0.1mg/ml, the aqueous solution of 0.58% (v/v%) Glacial acetic acid, is finally adjusted to 7.0 by 2M NaOH solution by pH.
(2) heparin crude product solution: take heparin crude product 30mg and be dissolved in 1ml purified water, then with heparinase effect damping fluid by above-mentioned solution dilution 100 times, obtain heparin crude product solution.
(3) pig DNA standard solution: pig DNA standard substance are diluted to the standard solution that concentration is 10000pg/ μ l, 1000pg/ μ l, 100pg/ μ l, 10pg/ μ l by purified water.
(4) upstream and downstream primer and probe solution: carry out centrifugal before the primer of synthesis and probe open pipe, slowly open pipe lid, add purified water respectively and be dissolved to 100 μMs, continuation purified water dilutes upstream and downstream primer and the probe solution (lucifuge) of 20 times to 5 μMs respectively.
2, heparin enzymolysis
(1) deactivation of nuclease: by the heparin crude product solution of pig 85 DEG C of constant temperature 5min, then ice bath 10min on thermostat, and by nucleic acid quantification instrument mensuration DNA concentration wherein, be designated as C
always.
(2) heparinase degraded: the heparinase adding about 0.01IU at 200 μ l in the heparin crude product solution of nuclease deactivation, concussion mixing on constant temperature blending instrument, 25 DEG C of constant temperature 18h.Centrifuging and taking supernatant liquor, 4 DEG C of preservations are stand-by.
3, Q-PCR amplification (Q-PCR, i.e. Real-time Quantitative PCR Detecting System, real-time quantitative gene amplification fluorescence detecting system)
(1) configure PCR system, 20 μ l reaction systems are as follows:
1. pig DNA standard solution: 3 μ L
Upstream primer solution: 1 μ L
Downstream primer solution: 1 μ L
Probe solution: 1 μ L
Master mix (PCR reagent premixed liquid): 10 μ L
Water: polishing to 20 μ L
2. the need testing solution after enzymolysis: 3 μ L
Upstream primer solution: 1 μ L
Downstream primer solution: 1 μ L
Probe solution: 1 μ L
Master mix (PCR reagent premixed liquid): 10 μ L
Water: polishing to 20 μ L
3. negative control solution (purified water): 3 μ L
Upstream primer solution: 1 μ L
Downstream primer solution: 1 μ L
Probe solution: 1 μ L
Master mix (PCR reagent premixed liquid): 10 μ L
Water: polishing to 20 μ L
Upstream primer in above-mentioned, downstream primer and probe are all corresponding to upstream and downstream primer and the probe of pig gene.Wherein, pig upstream primer is (5 '-3 '): CTTgCA AAT CCT AAC Agg CCTgCA; Pig downstream primer is (5 '-3 '): CgT TTg CAT gTA gAT AgC gAA TAA CgT T; Pig probe: FAM-ACAGCTTTCTCATCAGTTAC-MGB.
(2) the PCR response procedures for above-mentioned three PCR system is: 95 DEG C, 10min denaturation; 95 DEG C, 15s sex change; 63 DEG C, 20s anneals; 72 DEG C, 40s extends (fluorescent collecting); Times of thermal cycle is 40 times.
4, data analysis
(1) system suitability is judged as: the amplification curve Ct value corresponding with it by each concentration pig DNA standard solution, draws typical curve, the determination coefficient (R of typical curve
2) should>=0.98, slope should>=-4.0, Ct (threshold cycle) value of negative control should be greater than the Ct value of typical curve minimum concentration sample, or does not increase.
(2) being calculated as of pig gene relative content: after system suitability meets the requirements, software draws pig mrna concentration C in heparin crude product by typical curve
inspection(this concentration C
inspectionnumerical value is consistent with the amount of X-coordinate in Fig. 2), according to formulae discovery pig gene content:
We take the heparin crude product of four batches of different manufacturers, and drawn the DNA amount in this four batch sample by pig DNA typical curve, as shown in Figure 1, the relative content that we measure pig DNA in these four batches of heparin crude products is all greater than 10000ppm.Because in qualified heparin crude product, pig DNA amount should be greater than 10000ppm, thus can judge that this four batch sample is as qualified samples.
Embodiment 2
Fluorescence quantifying PCR method detects the cow genome in heparin crude product
1, solution preparation
(1) heparinase effect damping fluid: lime acetate 0.321mg/ml, bovine serum albumin 0.1mg/ml, the aqueous solution of 0.58% (v/v%) Glacial acetic acid, is finally adjusted to 7.0 by 2M NaOH solution by pH.
(2) heparin crude product solution: take heparin crude product 30mg and be dissolved in 1ml purified water, then with heparinase effect damping fluid by above-mentioned solution dilution 100 times, obtain heparin crude product solution.
(3) ox DNA standard solution: ox DNA standard substance are diluted to the standard solution that concentration is 10000pg/ μ l, 1000pg/ μ l, 100pg/ μ l, 10pg/ μ l by purified water.
(4) upstream primer, downstream primer and probe solution: carry out centrifugal before the primer of synthesis and probe open pipe, slowly open pipe lid, add purified water respectively and be dissolved to 100 μMs, continuation purified water dilutes upstream and downstream primer and the probe solution (lucifuge) of 20 times to 5 μMs respectively.
2, heparin enzymolysis
(1) deactivation of nuclease: by heparin crude product solution 85 DEG C of constant temperature 5min, then ice bath 10min on thermostat, and by nucleic acid quantification instrument mensuration DNA concentration wherein, be designated as C
always.
(2) heparinase degraded: the heparinase adding about 0.01IU at 200 μ l in the heparin crude product solution of nuclease deactivation, concussion mixing on constant temperature blending instrument, 25 DEG C of constant temperature 18h.Centrifuging and taking supernatant liquor, obtains the need testing solution after enzymolysis, and 4 DEG C of preservations are stand-by.
3, Q-PCR amplification (Q-PCR, i.e. Real-time Quantitative PCR Detecting System, real-time quantitative gene amplification fluorescence detecting system)
(1) configure PCR system, 20 μ l reaction systems are as follows:
1. ox DNA standard solution: 3 μ L
Upstream primer solution: 1 μ L
Downstream primer solution: 1 μ L
Probe solution: 1 μ L
Master mix (PCR reagent premixed liquid): 10 μ L
Water: polishing to 20 μ L
2. the need testing solution after enzymolysis: 3 μ L
Upstream primer solution: 1 μ L
Downstream primer solution: 1 μ L
Probe solution: 1 μ L
Master mix (PCR reagent premixed liquid): 10 μ L
Water: polishing to 20 μ L
3. negative control solution (purified water): 3 μ L
Upstream primer solution: 1 μ L
Downstream primer solution: 1 μ L
Probe solution: 1 μ L
Master mix (PCR reagent premixed liquid): 10 μ L
Water: polishing to 20 μ L
Upstream primer in above-mentioned, downstream primer and probe are all corresponding to upstream and downstream primer and the probe of cow genome.Ox upstream primer is (5 '-3 '): AAC CAA ATA TTA CAA ACA CCA CTA ACA gct; Ox downstream primer is (5 '-3 '): CCT TgC gTA ggT AAT TCA TTC ATA Tg; Ox probe: 6-FAM-ACATAACACgCCCATACACAgACCAC-BHQ.
(2) the PCR response procedures for above-mentioned three PCR system is: 95 DEG C, 10min denaturation; 95 DEG C, 15s sex change; 63 DEG C, 20s anneals; 72 DEG C, 40s extends (fluorescent collecting); Times of thermal cycle is 40 times.
4, data analysis
(1) system suitability is judged as: the amplification curve Ct value corresponding with it by each concentration ox DNA standard solution, draws typical curve, the determination coefficient (R of typical curve
2)>=0.98, slope>=-4.0, the Ct value of negative control should be greater than the Ct value of typical curve minimum concentration sample, or not amplification.
(2) being calculated as of cow genome relative content: after system suitability meets the requirements, software draws pig mrna concentration C in heparin crude product by typical curve
inspection(this concentration C
inspectionnumerical value is consistent with the amount of X-coordinate in Fig. 2), according to following formulae discovery cow genome relative content:
We take four batches of heparin crude products, have been drawn the relative content of the ox DNA in this batch sample by ox DNA typical curve, and as shown in Figure 2, we show that the ox DNA amount of a batch sample is less than 0.01ppm, and its excess-three is criticized and do not detected.Because ox DNA amount should be less than 20ppm in qualified heparin crude product, thus can judge that this four batch sample is as qualified samples.
The present invention is not limited to above-mentioned preferred forms, anyone for the present invention any modification done under enlightenment of the present invention or change, and every have identical with the application or akin technical scheme, all drops within protection scope of the present invention.
Claims (10)
1. one kind is detected the fluorescence quantifying PCR method of heparin crude product gene, it is characterized in that: described method comprises extraction, the design primer and probe, fluorescent PCR amplified reaction and data analysis of STb gene in heparin crude product, described primer is upstream primer and downstream primer, and described probe is fluorescent probe.
2. the fluorescence quantifying PCR method of detection heparin crude product gene according to claim 1, is characterized in that: in heparin crude product, the extraction of STb gene comprises:
1. heparin crude product solution is prepared: take heparin crude product and be dissolved in purified water, then obtain heparin crude product solution with heparinase effect damping fluid by after above-mentioned solution dilution;
2. the deactivation of nuclease: by heparin crude product solution constant temperature 5min at 85 DEG C, then ice bath 10min, and by nucleic acid quantification instrument mensuration DNA concentration wherein, be designated as C
always;
3. heparinase degraded: add heparinase in the heparin crude product solution through nuclease deactivation, concussion mixing under constant temperature, 25 DEG C of constant temperature 18h, centrifuging and taking supernatant liquor, as the need testing solution after enzymolysis, 4 DEG C of preservations are stand-by.
3. the fluorescence quantifying PCR method of detection heparin crude product gene according to claim 1, is characterized in that: described fluorescent PCR amplified reaction comprises preparation PCR system and PCR system amplified reaction.
4. the fluorescence quantifying PCR method of detection heparin crude product gene according to claim 3, is characterized in that: described preparation PCR system comprises need testing solution/negative control solution, upstream primer solution, downstream primer solution, fluorescent probe solution, Master mix and water after DNA standard solution/enzymolysis; Described negative control solution is pure water.
5. the fluorescence quantifying PCR method of the detection heparin crude product gene according to claim 1 or 4, is characterized in that: described fluorescent probe obtains fluorescent probe for adding fluorophor within the probe.
6. the fluorescence quantifying PCR method of detection heparin crude product gene according to claim 3, is characterized in that: described PCR system amplified reaction is: 1. denaturation 10min at 95 DEG C; 2. sex change 15s at 95 DEG C; 3. anneal 20s at 63 DEG C; 4. fluorescent collecting is carried out at 72 DEG C of downward-extension 40s; Times of thermal cycle is 40 times.
7. the fluorescence quantifying PCR method of detection heparin crude product gene according to claim 1, is characterized in that: described data analysis comprises the drafting of typical curve, system suitability judges and the calculating of each gene relative content of trial-product.
8. the fluorescence quantifying PCR method of detection heparin crude product gene according to claim 7, is characterized in that: being plotted as by each concentration DNA standard solution amplification curve Ct value corresponding with it of described typical curve, draws typical curve.
9. the fluorescence quantifying PCR method of detection heparin crude product gene according to claim 7, it is characterized in that: in described data analysis, system suitability is judged as: drawing standard curve, determination coefficient >=0.98 of typical curve, slope >=-4.0, the Ct value of negative control is greater than the Ct value of typical curve minimum concentration sample, or not amplification.
10. the fluorescence quantifying PCR method of detection heparin crude product gene according to claim 7, it is characterized in that: being calculated as of each gene relative content of trial-product in described data analysis: after system suitability meets the requirements, draw each mrna concentration C in trial-product by typical curve
inspectionaccording to each kind gene content of formulae discovery:
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112080553A (en) * | 2019-06-13 | 2020-12-15 | 苏州融析生物科技有限公司 | qPCR detection method for sheep and pig gene content in crude sheep heparin sodium |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103937881A (en) * | 2014-03-26 | 2014-07-23 | 北京电子科技职业学院 | Fluorescent PCR detection reagent for identifying cattle gene source in heparin, preparation method and application |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103937881A (en) * | 2014-03-26 | 2014-07-23 | 北京电子科技职业学院 | Fluorescent PCR detection reagent for identifying cattle gene source in heparin, preparation method and application |
Non-Patent Citations (3)
| Title |
|---|
| CÉCILE AUGUSTE等: "New developments in quantitative polymerase chain reaction applied to control the quality of heparins", 《ANAL BIOANAL CHEM》 * |
| NICOLETTE PEGELS等: "Applicability assessment of a real-time PCR assay for the specific detection of bovine, ovine and caprine material in feedstuffs", 《FOOD CONTROL》 * |
| SOICHI TANABE等: "A Real-Time Quantitative PCR Detection Method for Pork, Chicken, Beef, Mutton, and Horseflesh in Foods", 《BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112080553A (en) * | 2019-06-13 | 2020-12-15 | 苏州融析生物科技有限公司 | qPCR detection method for sheep and pig gene content in crude sheep heparin sodium |
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