CN103060435B - Primers and probes for detecting fox component in food and feed - Google Patents
Primers and probes for detecting fox component in food and feed Download PDFInfo
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Abstract
The invention belongs to technology of qualitative detection of animal-derived components in food and feed, and particularly relates to a primer/probe group for detecting fox component in food and feed. The invention selects mitochondrion cytochrome C redox enzyme I subunit gene sequence of fox, and designs a group of specific primers and probes. After using the primers and probes, the real-time fluorescent PCR (polymerase chain reaction) technology can be adopted to quickly, sensitively and specifically detect the fox component in food and feed. The primers and probes can also be provided together with other reagents in the form of a kit, and is used for nucleic acid amplification reaction. The method is simple to operate and has favorable repetitiveness.
Description
Technical field
The present invention relates to a kind of method of utilizing nucleic acid amplification technologies to carry out animal derived materials rapid detection, specifically a kind of real-time fluorescence PCR of fox composition detects primer and the probe sequence of use.
Background technology
The continuous expansion that global economic integration and trade are international, for developing China, is that opportunity is also challenge.In recent years, China's Foreign Trade is the situation of sustained, stable growth.Data presentation, is sure to occupy the position of global foreign trade the second big country for continuous 3 years from 2009 Nian Qi China, export volume occupies first place in the world, and import volume occupies second place of the world.How supervision is supervised, and helps to instruct enterprise to improve the quality of products, and overcomes the technology barriers in the developed countries such as the U.S., European Union, Japan and area, further expands China's products export; Strict immigration supervision simultaneously, prevents that external substandard product from flowing into China, and protection China consumer's interests, are the important process tasks that the state administration competent authorities such as inspection and quarantine face.
With regard to the food and feed of importing and exporting, qualified product, not only nontoxic, meet safety and health request; And will adulterate without mixing up, meet quality requirements.Wherein quality is qualified, mainly comprises again the implication of two aspects: 1. raw material sources are consistent with label.2. component concentration is consistent with label.European Union came into effect new food hygiene law from 1 day January in 2006, new legislation given prominence in food production can tracing management and the trackability of food, emphasize the identity authentication mark and healthy mark of food." the import and export food tag control way " that China standard GB/T 7718-94 " the food labelling universal standard " and The State Administration for Entry-Exit Inspection and Quarantine issue, also clearly proposing food labelling marked content should conform to food.In addition, for preventing the propagation of mad cow disease, itch, a lot of countries such as European Union, the U.S. including China successively promulgate multiple rules, provide against use in ruminant feed, add the animal feedstuff product take mammal as raw material, effectively to prevent that mammiferous pathogenic agent is from animal infection to other animals and humans.The event that the means such as in the production and sales of food and feed, product is lack of standardization without label or label, and utilization doping is adulterated, adulterate deception human consumer reaps staggering profits, domestic, all generations repeatedly abroad.Various raw materials are processed to after food and feed finished product, cannot identify its composition from outward appearance.The producer is driven by interests, changes without authorization product composition, or mixes inexpensive substitute, or directly reduce raw material consumption, causes the real property of product and label not to be inconsistent.This imitation behavior not only relates to economy, nutritive value and religious belief problem, more may directly affect consumer health.
Fox, Mammals, belongs to Mammalia, Carnivora, Canidae (Canidae), fox subfamily (Vulpini).Except comprising that Vulpes (Vulpes) animal, fox also comprises the animal of other genus of fox subfamily: Japan Vulpes (Fennecus), otocyon megalotis belong to (Otocyon), South America Vulpes (Dusicyon), Urocyon (Urocyon), arctic Vulpes (Alopex) and and Cerdocyon genus (Cerdo-cyon).Fox distributes very wide, and all there are a large amount of distributions in North America, Europe, Asia.Its fur is more precious, beautiful in colour, and plate matter is pliable and tough, is the high-grade raw material of fur coat industry processed, is known as " soft gold ", is described as one of the world's three large fur coat pillars, at home and abroad on market, is in great demand very much.As a kind of important economic furbearer, fox aquaculture is very flourishing, and main breed variety comprises: rde fox, blue fox and Yin Hei fox.Its fur is mainly got in fox cultivation, and fox meat has peculiar smell, therefore low price, few people are edible.In recent years, fox meat is pretended to be dog meats, mutton through processing, has report while even making the news that skewer, sausage come into the market.Given this, set up as early as possible reliable and effective fox component detection method, for guaranteeing food and feed label true and accurate, strengthen food and feed identity management, improve food safety, Protection of consumer interests are significant.
In food and feed, animal derived materials detects, and often adopts the method for DNA detection.Method take DNA as tested object is compared with method take albumen as tested object, and the in the situation that of complicated component, target dna can effectively extract, and is subject to the impact of matrix less; In addition, DNA is more stable, does not resemble the easy impact that is subject to external environment factor and complete processing protein composition.The advantages such as current, in the detection method based on DNA, real-time fluorescence PCR method is easy and simple to handle, sensitive, special, quick, reproducible, quantitatively accurate with it, totally-enclosed reaction, obtain investigator's generally approval, become the important tool of detection.
Summary of the invention
The object of the invention is for fox mitochondrial cytochrome C oxydo-reductase I subunit gene sequence; design a group-specific primers and probe; and then set up a kind of rapid detection fox DNA method; to overcome detection method based on the albumen limitation in some food and feed detects; for fox composition detection provides effective tool; thereby guarantee the consistence of food and feed label, Protection of consumer interests.
Cardinal principle of the present invention is: design one group of primer (upstream primer: 5 '-TGGAGCATCAGTAGACCTTACAATTT-3 ' and downstream primer: 5 '-GGCGGGAGGTTTTATATTGATAATAG-3 ') and a Taqman probe (sequence is: 5 '-FAM-CCCTGCACCTGGCCGGAGTC-TAMRA-3 ') for conserved sequence.While carrying out detection of nucleic acids, template DNA is after being heated to 94-95 ℃ of certain hour, and DNA double chain dissociates, and primer and Taqman probe are combined with template specificity.5 ends of probe are marked with report fluorescence group, and 3 ends have cancellation fluorescence group.In the time that probe is complete, the fluorescent energy that report group is launched is quenched group and absorbs, and instrument can't detect signal.Along with the carrying out of PCR, Taq enzyme is in chain extension process, and while running into the probe of being combined with template, its 3 ' → 5 ' exonuclease activity will cut off probe, and report group is away from cancellation group, and its energy can not be absorbed, and produces fluorescent signal.Along with the increase of PCR circulation, object fragment exponentially increases, and fluorescent signal also synchronously strengthens, and the power of fluorescent signal directly reflects template number.
The fox composition real-time fluorescence PCR the present invention relates to detects primer and the probe of use, and its sequence is as follows:
(1) upstream primer: 5 '-TGGAGCATCAGTAGACCTTACAATTT-3 ';
(2) downstream primer: 5 '-GGCGGGAGGTTTTATATTGATAATAG-3 ';
(3) TaqMan probe: 5 '-CCCTGCACCTGGCCGGAGTC-3 ', its 5 ' end flag F AM report fluorescence group, 3 ' end mark TAMRA cancellation fluorescence group;
In application, the volume ratio of upstream primer, downstream primer, TaqMan probe is: 1: 1: 1;
Use mentioned reagent box to detect the method for garlic composition in food, comprise the following steps successively (1)-(3):
(1) extraction of sample DNA to be checked
DNA extraction can adopt common phenol-chloroform extraction process or use DNA extraction test kit.
(2) real-time fluorescence PCR of fox composition amplification
A. in reaction tubes, add 2 × PCR Master Mix12.5 μ L, the each 0.5-1 μ of upstream primer and downstream primer L, Taqman probe 0.5-1 μ L, the DNA1-2 μ L of 100ng/ μ L testing sample, supplements distilled water to 25 μ L, mixes;
B. PCR reaction tubes is put into quantitative real time PCR Instrument, completes pcr amplification by following reaction conditions:
94-95 ℃ of 5min, 1 circulation, denaturation;
94-95 ℃ of 10-20sec; 60 ℃ of 20-40sec, 45 circulations, pcr amplification.
When amplification, should set up three contrasts: positive control (get fox meat extract genomic dna), negative control (non-fox genomic dna), blank (containing DNA profiling, can water replace).
(3) application quantitative real time PCR Instrument accompanying software, analysing amplified result.
As there is amplification curve in testing sample, and Ct value is less than or equal to 41, negative control, positive control and blank result are all set up (be that typical positive amplification curve appears in positive, negative sample and blank are without amplification), can judge that this sample detects fox composition.
As amplification curve does not appear in testing sample, without Ct value, negative control, positive control and blank are all set up, and can judge that this sample does not detect fox composition.
If testing sample Ct value is between 41-45, answer the amplification of recast real-time fluorescence PCR.Result Ct value after amplification is still less than 45 again, and negative control, positive control and blank result all set up, and can judge that this sample detects fox composition; Again the result after amplification for without amplification curve without Ct value, and negative control, positive control and blank result all set up, and can judge that this sample does not detect fox composition.
Mitochondrial DNA is unique extranuclear inheritance information carrier that exists in animal body, is covalence closed double-stranded circular molecule, has the features such as matrilinear inheritance, rate of evolution are fast, species specificity.The research of the different tissues such as kidney to same individuality, heart, liver, skin, relatively show, the structure of Mitochondrial DNA does not have tissue specificity.Priorly be that its copy number is many, can reach thousands ofly to up to ten thousand, far above core DNA, even if testing sample is deep processed product, when the serious or DNA content of core DNA degradation is few, still can detect by pcr amplification Mitochondrial DNA.Mitochondrial DNA gene order has been widely used at present species and has identified.The complicated enzyme molecule that cytochrome C oxidase is formed, played an important role in organism metabolic process by multiple subunits, in its many subunit, the most conservative maximum subunit is cytochrome C oxydo-reductase I subunit, is encoded by Mitochondrial DNA.The present invention is according to fox mitochondrial cytochrome C oxydo-reductase I subunit gene sequence, uses Biodet software and Primer Express3.0 software to carry out sequential analysis and primer, probe design.This gene order obtains by the comparison of other animal nucleic acid sequences of Canidae to Genebank login, and the primer of designing and probe have been carried out to online Blast comparison inquiry, can guarantee detecting of fox composition.The present invention adopts real-time fluorescence PCR technology, and this technology high specificity, highly sensitive, easy and simple to handle is specially adapted to the detection of fox composition in the processed food of some complicated components and feed.
Accompanying drawing explanation
Fig. 1 is for using real-time fluorescence PCR technology for detection meat gruel sample DNA to be measured, the amplification collection of illustrative plates of sample; The amplification collection of illustrative plates of the negative contrast of Fig. 2 (capital bar dog DNA); Fig. 3 is the amplification collection of illustrative plates of blank (water); The positive contrast of Fig. 4 (fox DNA) amplification collection of illustrative plates.
Fig. 5 is for using real-time fluorescence PCR technology for detection Feed Sample DNA to be measured, the amplification collection of illustrative plates of sample; The amplification collection of illustrative plates of the negative contrast of Fig. 6 (capital bar dog DNA); Fig. 7 is the amplification collection of illustrative plates of blank (water); The positive contrast of Fig. 8 (fox DNA) amplification collection of illustrative plates.
Embodiment
Below in conjunction with embodiment, the present invention will be further described.
The real-time fluorescence PCR assay kit of making fox composition by following formula, reagent wherein comprises as follows:
(1)2×PCR?Master?Mix
Comprise 0.05u/ μ L Taq archaeal dna polymerase, reaction buffer, 4mmol/L magnesium chloride, 0.4mmol/L dNTP (dATP, dCTP, dGTP, dTTP);
Trihydroxy methyl aminomethane-hydrochloric acid, 100mmol/L Repone K and 2% triton x-100 that wherein reaction buffer contains 20mmol/LpH8.8;
(2) upstream primer: 10 μ mol/L, sequence is: 5 '-TGGAGCATCAGTAGACCTTACAATTT-3 ';
(3) downstream primer: 10 μ mol/L, sequence is: 5 '-GGCGGGAGGTTTTATATTGATAATAG-3 ';
(4) Taqman probe: 10 μ mol/L, sequence is: 5 '-CCCTGCACCTGGCCGGAGTC-3 ', its 5 ' end flag F AM report fluorescence group, 3 ' end mark TAMRA cancellation fluorescence group.
Detect according to following program:
(1) extraction of meat gruel sample DNA to be measured
A. take 0.3g meat gruel sample, add in 2mL centrifuge tube.Add CTAB lysis buffer and the 40 μ L Proteinase Ks of 600 μ L preheatings, after mixing gently, 65 ℃ of water bath heat preservation 90min;
B.2000g centrifugal 5min, gets supernatant in another clean 2mL centrifuge tube, adds the mixed solution (25: 24: 1) of isopyknic phenol, trichloromethane and primary isoamyl alcohol, and concussion mixes;
C.12000g centrifugal 10min, gets supernatant to clean 2mL centrifuge tube, adds equal-volume Virahol, puts upside down and mixes;
D.12000g centrifugal 10min, abandoning supernatant, with the TE damping fluid dissolution precipitation that is preheated to 65 ℃;
E. add 5 μ LRNA enzyme solution, 37 ℃ of 30min;
F. add 200 μ L trichloromethanes: primary isoamyl alcohol (24: 1), strongly vibration;
G.12000g centrifugal 10min, gets supernatant to clean 2mL centrifuge tube, adds equal-volume Virahol, puts upside down and mixes;
H.12000g centrifugal 10min, abandoning supernatant, by 70% ethanol 500 μ L of 4 ℃ of precoolings, vortex washing and precipitating;
I.12000g centrifugal 10min, abandoning supernatant, inversion adds 100 μ L TE damping fluid dissolution precipitations after drying, and-20 ℃ save backup.
(2) real-time fluorescence PCR of meat gruel sample DNA to be measured amplification
A. in PCR reaction tubes, add 2 × PCR Master Mix12.5 μ L as claimed in claim 1, the each 0.5 μ L of upstream primer and downstream primer, Taqman probe 0.5 μ L, the DNA1 μ L of 100ng/ μ L testing sample, supplements distilled water to 25 μ L, mixes;
B. PCR reaction tubes is put into quantitative real time PCR Instrument, completes pcr amplification by following reaction conditions:
95 ℃ of 2min, 1 circulation, denaturation;
95 ℃ of 15sec; 60 ℃ of 40sec, 45 circulations, pcr amplification.
(3) application quantitative real time PCR Instrument accompanying software, analysing amplified result.
There is positive amplification curve in sample, Ct value 17.8, negative control (capital bar dog DNA) and blank (water) are without amplification, positive control (fox DNA) produces typical positive amplification curve, see Fig. 1, Fig. 2, Fig. 3, Fig. 4, can judge that accordingly this sample detects fox composition.
The real-time fluorescence PCR assay kit of making fox composition by following formula, reagent wherein comprises as follows:
(1)2×PCR?Master?Mix
Comprise 0.05u/ μ L Taq archaeal dna polymerase Polymerase, reaction buffer, 4mmol/L magnesium chloride, 0.4mmol/L dNTP (dATP, dCTP, dGTP, dTTP);
Trihydroxy methyl aminomethane-hydrochloric acid, 100mmol/L Repone K and 2% triton x-100 that wherein reaction buffer contains 20mmol/L pH8.8;
(2) upstream primer: 10 μ mol/L, sequence is: 5 '-TGGAGCATCAGTAGACCTTACAATTT-3 ';
(3) downstream primer: 10 μ mol/L, sequence is: 5 '-GGCGGGAGGTTTTATATTGATAATAG-3 ';
(4) Taqman probe: 10 μ mol/L, sequence is: 5 '-CCCTGCACCTGGCCGGAGTC-3 ', its 5 ' end flag F AM report fluorescence group, 3 ' end mark TAMRA cancellation fluorescence group.
Detect according to following program:
(1) extraction of Feed Sample DNA to be measured
A. take 0.3g Feed Sample, add in 2mL centrifuge tube.Add CTAB lysis buffer and the 40 μ L Proteinase Ks of 600 μ L preheatings, after mixing gently, 65 ℃ of water bath heat preservation 90min;
B.2000g centrifugal 5min, gets supernatant in another clean 2mL centrifuge tube, adds the mixed solution (25: 24: 1) of isopyknic phenol, trichloromethane and primary isoamyl alcohol, and concussion mixes;
C.12000g centrifugal 10min, gets supernatant to clean 2mL centrifuge tube, adds equal-volume Virahol, puts upside down and mixes;
D.12000g centrifugal 10min, abandoning supernatant, with the TE damping fluid dissolution precipitation that is preheated to 65 ℃;
E. add 5 μ LRNA enzyme solution, 37 ℃ of 30min;
F. add 200 μ L trichloromethanes: primary isoamyl alcohol (24: 1), strongly vibration;
G.12000g centrifugal 10min, gets supernatant to clean 2mL centrifuge tube, adds equal-volume Virahol, puts upside down and mixes;
H.12000g centrifugal 10min, abandoning supernatant, by 70% ethanol 500 μ L of 4 ℃ of precoolings, vortex washing and precipitating;
The centrifugal 10min of 1.12000g, abandoning supernatant, inversion adds 100 μ L TE damping fluid dissolution precipitations after drying, and-20 ℃ save backup.
(2) real-time fluorescence PCR of Feed Sample DNA to be measured amplification:
A. in PCR reaction tubes, add 2 × PCR Master Mix12.5 μ L as claimed in claim 1, the each 0.5 μ L of upstream primer and downstream primer, Taqman probe 0.5 μ L, the DNA1 μ L of 100ng/ μ L testing sample, supplements distilled water to 25 μ L, mixes;
B. PCR reaction tubes is put into quantitative real time PCR Instrument, completes pcr amplification by following reaction conditions:
95 ℃ of 2min, 1 circulation, denaturation;
95 ℃ of 15sec; 60 ℃ of 40sec, 45 circulations, pcr amplification.
(3) application quantitative real time PCR Instrument accompanying software, analysing amplified result.
There is not positive amplification curve in sample, negative control (capital bar dog DNA) and blank (water) are without amplification, positive control (fox DNA) produces typical positive amplification curve, and Fig. 5, Fig. 6, Fig. 7, Fig. 8 can judge that this sample does not detect fox composition accordingly.
Claims (2)
1. fox composition rapid detection is used upstream primer and a downstream primer, is characterized in that:
Upstream primer sequence is: 5 '-TGGAGCATCAGTAGACCTTACAATTT-3 ';
Downstream primer sequence is: 5 '-GGCGGGAGGTTTTATATTGATAATAG-3 '.
Fox composition rapid detection use a Taqman probe, it is characterized in that:
Sequence is: 5 '-CCCTGCACCTGGCCGGAGTC-3 ', its 5 ' end flag F AM report fluorophor, 3 ' end mark TAMRA cancellation fluorophor.
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| CN105296648B (en) * | 2015-11-20 | 2018-08-03 | 华中农业大学 | Fox derived component identify and animal product in fox, rabbit, dog ingredient multiple PCR detection kit |
| CN108300771B (en) * | 2018-02-13 | 2021-07-20 | 广州海关技术中心 | Dual-digital PCR method for quantitatively detecting fox-derived components |
| CN108728556A (en) * | 2018-06-26 | 2018-11-02 | 东北林业大学 | The detection method of blue fox-fur robe leathercraft based on MGB probes |
| CN108753993A (en) * | 2018-06-27 | 2018-11-06 | 东北林业大学 | A kind of MGB probe differential methods of silver fox/rde fox fur products |
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| CN102634584A (en) * | 2012-04-18 | 2012-08-15 | 上海出入境检验检疫局工业品与原材料检测技术中心 | Fluorescence quantitative PCR (polymerase chain reaction) qualitative detection method for rabbit-derived fiber composition |
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| :HBL008452 cytochrome oxidase subunit 1 (COI) gene, partial cds * |
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| Eger JL et al..Accession No:JF443560.1,Vulpes vulpes voucher BIOUG<CAN>:HBL008452 cytochrome oxidase subunit 1 (COI) gene, partial cds |
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Effective date of registration: 20181127 Address after: 266000 West 001, 4th Floor, No. 177 Zhuzhou Road, Laoshan District, Qingdao City, Shandong Province Patentee after: Shandong Zhonghe Tiancheng Inspection Co., Ltd. Address before: 266002 Qutang Road, Qingdao City, Shandong Province, No. 70 Patentee before: Inspection and Quarantine Technology Center, Shandong Inspection and Quarantine |