CN101434991A - Real time fluorescent PCR method for detecting aquatic product food allergen gene - Google Patents
Real time fluorescent PCR method for detecting aquatic product food allergen gene Download PDFInfo
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Abstract
The invention discloses a real-time fluorescent PCR method for testing the allergen genes of aquatic food, comprising the following steps: total RNA of samples is extracted; the allergen genes Par and TM of aquatic food, and internal standard gene Beta-actin are respectively tested by SYBR Green or TaqMan probe real-time fluorescent PCR technology; and result analysis is carried out to the tested samples according to the amplification kinetic curve and the Ct value. As the real-time fluorescent PCR technology is used for testing the allergen genes Par and TM of aquatic food, compared with other methods, the method has the advantages of high sensitivity, short testing time, available quantitative analysis, and the like, and can be used in the qualitative screening, quantitative analysis and confirmation identification of allergen genes in unknown species or mixed samples of aquatic food.
Description
Technical field
The present invention relates to the detection of food allergen gene, particularly relate to a kind of real-time fluorescent polyase chain reaction (PCR) method that detects aquatic product food allergen gene of inspection, the real-time fluorescence PCR that this method is mainly used in fish parvalbumin (Par) and crustaceans tropomyosin (TM) gene detects.
Background technology
Food anaphylaxis medically also claims transformation reactions.The material that causes the food anaphylaxis reaction is called food allergen, as fish, shrimp, crab, milk, eggs etc.Its reaction mechanism is that anaphylactogen enters after the body, can cause body that normal or excessive immunne response, i.e. allergy take place.A plurality of IgE molecules and antibodies when contacting identical anaphylactogen once more, the Fc end structure that causes IgE changes, cause cell degranulation, discharge biological respiratory activity material such as histamine, serotonin, leukotriene and act on effector, show as that smooth muscle contraction, telangiectasis, permeability increase, allergic, respiratory tract anaphylaxis, hemopoietic system allergy even anaphylactic shock (Zhao Junfang, Deng. food allergen detects and application prospect. Chinese laboratory medicine magazine, 2007,8:948-950).
The food anaphylaxis problem has caused consumers in general, food producer and scientific research person's common concern in recent years.According to epidemiology survey, the whole world has the adult of 2%-2.5% to be subjected to the puzzlement of food anaphylaxis disease approximately, and infant's sickness rate is higher, reaches 4%-6%, and children are about 2%-3%.In the irritated food of the whole world eight big classes that Food and Argriculture OrganizationFAO announces, fish and crustaceans fishery products are two wherein important big classes, its main anaphylactogen be respectively fish parvalbumin (Par) and crustaceans tropomyosin (TM) (Food and Agri cultureOrganization.Report of the FAO technical consultation on foodallergies.Rome, Italy.1995.).
For the present no special methods of treatment of food anaphylaxis, it is effective means that contact allergy food is avoided in strictness.Detection anaphylactogen method commonly used has anaphylactogen pricking method test in the body, vitro detection specific IgE (RAST suppresses experiment and EAST suppresses experiment), the rechallenge of double blinding food and enzyme linked immunosorbent assay etc.In these methods, anaphylactogen pricking method test is prone to false positive in the body; The deficiency that RAST inhibition experiment and EAST suppress to test is the dependency to human serum, and serum is that very difficult assurance is consistent, so this method is difficult to stdn; The rechallenge of double blinding food is subject to the other factors influence, and also not easy to operate in the practice, links such as test dose, placebo and double blinding control are still waiting perfect; Enzyme linked immunosorbent assay for trace of albumin be difficult to detect (Zhao Junfang, etc. food allergen detects and application prospect. Chinese laboratory medicine magazine, 2007,8:948-950).
In recent years gene tester (as PCR, nucleic acid hybridization) with its susceptibility height, specificity is good, easy and simple to handle and extremely inspection man author's favor.But PCR detection technique commonly used still need experience a process of amplified production being carried out electrophoretic analysis, time consumption is unavoidable, and employed bromination second pyridine dyestuff all has in various degree harm (Lee C Y to human and environment in the electrophoresis process, et al.Detection of pathogenic bacteria inshellfish using multiplex PCR followed by CovaLink NH microwellplate sandwich hybridization.Journal of Microbiology Methods, 2003,53:199-209).The amplification of real-time fluorescence PCR and product analysis whole process are all carried out under the single tube sealing condition, and pass through microcomputer control, realized PCR is detected the target of monitoring in real time and analyzing automatically, also fundamentally solved the contingent false positive pollution problem of conventional PCR.Do not detect aquatic product food allergen gene but still there is the real time fluorescent PCR method of employing at present, therefore, the public presses for the aquatic product food allergen gene detection method is applied to practice.
Summary of the invention
The object of the present invention is to provide a kind of can be quantitatively, fast, sensitivity detects the former real time fluorescent PCR method of food allergy to aquatic products.
For achieving the above object, technical solution of the present invention is: extract the total RNA of sample, detect aquatic product food allergen gene Par and TM respectively by SYBR Green or TaqMan probe method real-time fluorescence PCR technology, and internal standard gene β-actin, according to amplification kinetic curve and Ct value sample is carried out interpretation of result.
The present invention specifically may further comprise the steps:
(1) design is synthetic is used to detect 3 groups of former primers of food allergy to aquatic products and probe.
The upstream primer of Par gene: 5 '-ATGGCATTCGCTGGAATTCTG-3 ',
The downstream primer of Par gene: 5 '-TGCCATTTATGCCTTGACCAG-3 ',
The probe of Par gene: 5 '-FAM-AGGGCTGCCAAGCTGCTGACTCC-TAMRA-3 ';
The upstream primer of TM gene, TM-1:5 '-CGCATGGACGCCATCAAGAAGAAGATG-3 ',
The upstream primer of TM gene: 5 '-AGGTTAATAGCCAGACAGTTCGCT-3 ',
The probe of TM gene: 5 '-FAM-AGCAGCTGTCCGCCGCTAACACTAAG-TAMRA-3 ':
The upstream primer of actin gene: 5 '-AGAGCAAGAGAGGTATCTTGA-3 ',
The upstream primer of actin gene: 5 '-GGCGGTCTCGTGGATACCAG-3 ',
The probe of actin gene: 5 '-FAM-CACGGCATCATCACTAACTGGGACGA-TAMRA-3 '.
(2) real-time fluorescence PCR detects.Preparation fluorescent PCR amplification reaction system, system contains PCR damping fluid, MgCl
2, dNTP, reversed transcriptive enzyme, RNase inhibitor, archaeal dna polymerase, upstream primer and downstream primer, 0ligo dT
15, RNA template, sterilization ultrapure water (annotate: probe method need be added the TaqMan probe); The fluorescent PCR amplification reaction condition is 45-55 ℃ of reverse transcription 20-40min; 93-95 ℃ of pre-sex change 3-10min; 93-95 ℃/30-60S, 50-60 ℃/30-60S, 72 ℃/40-90S, 35-45 circulation (annotating: adopt SYBR Green method that 60 ℃ to 95 ℃ melting curve analysis need be set); Positive control (the total RNA of fishery products muscle), negative control (the total RNA of pork) and blank (sterilization ultrapure water) are set up in detection.Reaction according to the analysis software of fluorescent PCR instrument, is obtained the Ct value of each sample after finishing, and according to Ct value and amplification kinetic curve sample is analyzed.
The present invention can make the real-time fluorescent PCR reagent case that is used to detect aquatic product food allergen gene.
Because the present invention adopts SYBR Green or TaqMan probe method real-time fluorescence PCR to detect aquatic product food allergen gene.SYBR Green is a kind of and the fluorescence dye dna double chain combination, sends fluorescence when with the dna double chain combination; When DNA unwind, SYBR Green was released, and fluorescent signal sharply weakens, and fluorescence signal intensity has been represented the quantity of double chain DNA molecule in the reaction system; But SYBR Green dyestuff can with all dna double chain combinations, the specificity that the analysing amplified product of melting curve therefore must be set (is the melting temperature (Tm) of target nucleic acid sequence, Tm).The TaqMan probe be a kind of can with the oligonucleotide probe of PCR product hybridization, the probe two ends are mark fluorescent reporter group and quenching group respectively; When probe was complete, the reporter group fluorescent signal emitted was absorbed by quenching group; During pcr amplification, the 5 prime excision enzyme activity of archaeal dna polymerase is degraded probe, has destroyed the transmission ofenergy between two fluorescence molecules, thereby has sent fluorescence; The fluorescence molecule number that cuts down is directly proportional with the quantity of PCR product, the accumulation and the PCR product that are fluorescent signal form (Ke L D fully synchronously, et al.A reliabilitytest of standard-based quantitative PCR:exogenous vsendogenous standards.Molecular and Cellular Probes, 2000,14:127-135).The present invention also is provided with internal standard gene β-actin as detecting Quality Control in reaction, also can be used for the quantitative result error of goal gene is proofreaied and correct, β-actin is main component Actin muscle a kind of who constitutes cytoskeleton, it has the gene order high conservative, mRNA expresses the high and stable characteristics of quantity, the Chang Zuowei internal standard gene is widely used (Neuvians T P, et al.Standardization strategy forquantitative PCR in human seminoma and normal testis.Journalof Biotechnology, 2005,117:163-171).
The present invention utilizes the real-time fluorescence PCR technology to detect aquatic product food allergen gene Par and TM, with respect to other method, that this technology has is highly sensitive, detection time short and can quantitative analysis etc. advantage, the qualitative screening, quantitative analysis and the affirmation that can be used for anaphylactogen gene in the unknown kind of fishery products or the biased sample are identified.
The present invention is further illustrated below in conjunction with the drawings and specific embodiments.
Description of drawings
The kinetic curve of 10 times of gradient dilution carp RNA of Fig. 1 TaqMan probe method fluorescent PCR (Par gene) amplification;
The kinetic curve of 10 times of gradient dilution Young Crab RNA of Fig. 2 A SYBR Green fluorescent PCR (TM gene) amplification;
The melting curve of the SYBR Green fluorescence PCR products of Fig. 2 B gradient dilution Young Crab RNA;
Fig. 3 TaqMan fluorescence probe PCR detects the kinetic curve of unknown sample Mesichthyes Par gene;
Fig. 4 A SYBR Green fluorescent PCR detects the kinetic curve of crab class TM gene in the unknown sample;
Fig. 4 B SYBR Green fluorescent PCR detects the product melting curve of crab class TM gene in the unknown sample.
Embodiment
Embodiment 1: the real-time fluorescence PCR that is used to detect carp Par gene
With TaqMan fluorescence probe PCR method the total RNA of carp muscle of 10 times of gradient dilutions has been carried out the detection of Par gene, reaction system is PCR Master Mix 10 μ L, sterilization ultrapure water 6.4 μ L, MultiScribe Reverse Transcriptase 0.5 μ L, RNaseInhibiter 0.4 μ L, Par upstream region of gene primer 0.5 μ L, the downstream primer 0.5 μ L of Par gene, probe 0.3 μ L, the Oligo dT of Par gene
151 μ L and RNA template 0.4 μ L.Reaction parameter is 48 ℃ of reverse transcription 30min; 94 ℃ of pre-sex change 10min; 94 ℃/40S, 55 ℃/45S, 72 ℃/90S, totally 35 circulations.The result shows that the amplification kinetic curve is typical S type, and the translation backward gradually along with the reduction of sample concentration; The Ct value increases (as shown in Figure 1) gradually along with the reduction of sample concentration.
In Fig. 1, the kinetic curve of 10 times of gradient dilution carp RNA of TaqMan probe method fluorescent PCR (Par gene) amplification; A.1:10 dilution, Ct=15.3089; B.1:100 dilution, Ct=16.5373; C.1: 1000 dilutions, Ct=17.4470; D.1:10000 dilution, Ct=19.0486;
Embodiment 2: the real-time fluorescence PCR that is used to detect Young Crab TM gene
With SYBR Green fluorescent PCR method the total RNA of Young Crab muscle of 10 times of gradient dilutions has been carried out the detection of TM gene, reaction system is downstream primer 0.5 μ L, the Oligo dT of PCR Master Mix 10 μ L, sterilization ultrapure water 6.7 μ L, MultiScribe Reverse Transcriptase 0.5 μ L, RNase Inhibiter 0.4 μ L, TM upstream region of gene primer 0.5 μ L, TM gene
151 μ L and RNA template 0.4 μ L.Reaction parameter is 48 ℃ of reverse transcription 30min; 94 ℃ of pre-sex change 10min; 94 ℃/40S, 55 ℃/45S, 72 ℃/90S, totally 35 circulations; 60 ℃ to 95 ℃ melting curve analysis is set.The result shows that the amplification kinetic curve is typical S type, and the translation backward gradually along with the reduction of sample concentration; The Ct value increases (shown in Fig. 2 A) gradually along with the reduction of concentration; The melting curve collection of illustrative plates of amplified production shows that its Tm is 87 ℃, and has only a specific peak, shows that (shown in Fig. 2 B) appears in no primer dimer and non-specific amplification.
In Fig. 2 A, the kinetic curve of 10 times of gradient dilution Young Crab RNA of SYBR Green fluorescent PCR (TM gene) amplification; A.1:10 dilution, Ct=15.2523; B.1:100 dilution, Ct=16.5799; C.1:1000 dilution, Ct=18.0974; D.1:10000, Ct=20.7475.
Embodiment 3: unknown sample Mesichthyes Par gene test
With TaqMan fluorescence probe PCR method the fish Par gene in the unknown sample has been carried out the detection of Par gene, actual conditions is with reference to embodiment 1.The result shows that the amplification kinetic curve of unknown sample is typical S type, and negative control does not detect fluorescent signal to be changed, and shows and contains fish Par gene (as shown in Figure 3) in the unknown sample.
In Fig. 3, TaqMan fluorescence probe PCR detects the kinetic curve of unknown sample Mesichthyes Par gene; A. sample S-I, Ct=15.2695; B. sample S-II, Ct=16.0500; C. negative control, Ct=Undet.
Embodiment 4: crab class TM gene test in the unknown sample
With SYBR Green fluorescent PCR method the crab class TM gene in the unknown sample has been carried out the detection of TM gene, actual conditions is with reference to embodiment 2.The result shows that the amplification kinetic curve of unknown sample is typical S type, and negative control does not then detect fluorescent signal to be changed, and shows and contains crab class TM gene (shown in Fig. 4 A) in the unknown sample; The melting curve collection of illustrative plates of positive amplified production shows that its Tm is 87 ℃, and has only a specific peak, does not occur the fusion specific peak in the negative control, shows that positive products is specific amplification (shown in Fig. 4 B).
In Fig. 4 A, SYBR Green fluorescent PCR detects the kinetic curve of crab class TM gene in the unknown sample; A. sample S-I, Ct=15.2204; B. sample S-II, Ct=16.2523; C. negative control, Ct=Undet.
Claims (4)
1, a kind of real time fluorescent PCR method that detects aquatic product food allergen gene, it is characterized in that: its step comprises extracts the total RNA of sample, detect former fish parvalbumin of food allergy to aquatic products (Par) and crustaceans tropomyosin (TM) gene respectively by SYBR Green or TaqMan probe method real-time fluorescence PCR technology, and internal standard gene β-actin, according to amplification kinetic curve and Ct value sample is carried out interpretation of result.
2, the real time fluorescent PCR method of detection aquatic product food allergen gene according to claim 1 is characterized in that: specifically may further comprise the steps:
(1) design is synthetic is used to detect 3 groups of former primers of food allergy to aquatic products and probe;
The upstream primer of Par gene: 5 '-ATGGCATTCGCTGGAATTCTG-3 ',
The downstream primer of Par gene: 5 '-TGCCATTTATGCCTTGACCAG-3 ',
The probe of Par gene: 5 '-FAM-AGGGCTGCCAAGCTGCTGACTCC-TAMRA-3 ';
The upstream primer of TM gene, TM-1:5 '-CGCATGGACGCCATCAAGAAGAAGATG-3 ',
The upstream primer of TM gene: 5 '-AGGTTAATAGCCAGACAGTTCGCT-3 ',
The probe of TM gene: 5 '-FAM-AGCAGCTGTCCGCCGCTAACACTAAG-TAMRA-3 ';
The upstream primer of actin gene: 5 '-AGAGCAAGAGAGGTATCTTGA-3 ',
The upstream primer of actin gene: 5 '-GGCGGTCTCGTGGATACCAG-3 ',
The probe of actin gene: 5 '-FAM-CACGGCATCATCACTAACTGGGACGA-TAMRA-3 '.
(2) real-time fluorescence PCR detects.Preparation fluorescent PCR amplification reaction system, system contains PCR damping fluid, MgCl
2, dNTP, reversed transcriptive enzyme, RNase inhibitor, archaeal dna polymerase, upstream primer and downstream primer, Oligo dT
15, the RNA template, the sterilization ultrapure water; The fluorescent PCR amplification reaction condition is 45-55 ℃ of reverse transcription 20-40min; 93-95 ℃ of pre-sex change 3-10min; 93-95 ℃/30-60S, 50-60 ℃/30-60S, 72 ℃/40-90S, the 35-45 circulation; Positive control, negative control and blank are set up in detection; Reaction according to the analysis software of fluorescent PCR instrument, is obtained the Ct value of each sample after finishing, and according to Ct value and amplification kinetic curve sample is analyzed.
3, according to the real time fluorescent PCR method of claim 1 and 2 described detection aquatic product food allergen genes, it is characterized in that: detect aquatic product food allergen gene Par and TM respectively by SYBR Green or TaqMan probe method real-time fluorescence PCR technology.
4, according to the real time fluorescent PCR method of claim 1 and 2 described detection aquatic product food allergen genes, it is characterized in that: can make the real-time fluorescent PCR reagent case that is used to detect aquatic product food allergen gene.
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| CN101736086B (en) * | 2010-01-05 | 2013-07-10 | 长沙学院 | Rapid detection kit of myosin freshwater fish adjusting light chain mRNA expression and detection method thereof |
| CN101899501A (en) * | 2010-02-10 | 2010-12-01 | 江苏出入境检验检疫局动植物与食品检测中心 | Constant temperature amplification detection kit and method for detecting food allergen crustacean gene |
| CN101899501B (en) * | 2010-02-10 | 2012-06-20 | 江苏出入境检验检疫局动植物与食品检测中心 | Constant temperature amplification detection kit and method for detecting food allergen crustacean gene |
| CN103555730A (en) * | 2013-11-01 | 2014-02-05 | 集美大学 | Large yellow croaker tropomyosin gene as well as recombinant protein and application thereof |
| CN103555730B (en) * | 2013-11-01 | 2016-06-08 | 集美大学 | A kind of Carnis Pseudosciaenae tropomyosin gene and recombiant protein thereof and application |
| CN103642933A (en) * | 2013-12-24 | 2014-03-19 | 山东农业大学 | Method for detecting sensitization protein gene expression in transgenic soybean under adversity |
| CN106148506A (en) * | 2015-04-27 | 2016-11-23 | 重庆贝羿生物科技有限公司 | The antenatal detection kit of a kind of B race streptococcus quantitative fluorescent PCR and application thereof |
| CN105018627A (en) * | 2015-08-10 | 2015-11-04 | 江南大学 | PCR detecting technology of testa type allergenic substances in glucosamine products |
| CN105018627B (en) * | 2015-08-10 | 2018-01-16 | 江南大学 | The PCR detection techniques of crust source allergenic substance in ammonia sugar products |
| CN105154573A (en) * | 2015-10-21 | 2015-12-16 | 河北科技师范学院 | Primer set and probe for apple Actin gene real-time fluorescent quantitative PCR (polymerase chain reaction) detection and detection method by using same |
| CN105349540A (en) * | 2015-12-03 | 2016-02-24 | 浙江省检验检疫科学技术研究院 | Fluorescent quantitative PCR method for detecting fish parvalbumin and primer pair |
| CN105349540B (en) * | 2015-12-03 | 2018-08-28 | 浙江省检验检疫科学技术研究院 | Fluorescence quantifying PCR method for detecting fish parvalbumin and primer pair |
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