CN102766685A - Primer and probe for detecting garlic component in food and beverage - Google Patents
Primer and probe for detecting garlic component in food and beverage Download PDFInfo
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Abstract
The invention belongs to the field of qualitative detection technology for plant derived components in food and beverage, and specifically provides a primer / probe group for detecting garlic component in food and beverage. According to the invention, a group of specific primers and a probe are designed based on a conservative sequence of an allinase gene in garlic. The primer group and the probe can be employed in real-time fluorescent PCR technology to detect garlic component in food and beverage rapidly, sensitively and specifically. The primer group and the probe can be provided together in the form of a kit or other reagents, and applied to nucleic acid amplification reaction. The method has the advantages of simple operation and good repeatability.
Description
Technical field
The present invention relates to a kind of method of utilizing nucleic acid amplification technologies to carry out plant-derived composition rapid detection, specifically is a kind of primer and probe sequence of real-time fluorescence PCR detection usefulness of garlic composition.
Background technology
The continuous expansion that global economic integration and trade are international for developing China, is that opportunity also is challenge.At present, China has become the fifth-largest in the world agricultural-exporting country, the sustainable growth of agricultural-food foreign trade.Developed countries and regional such as the U.S., European Union, Japan in order to safeguard the economic interests of this country; The contention world market; Do one's utmost through the Interventions Requested and strict even harsh technological standard of a multitude of names; Farm imports are provided with barrier to trade, cause the agricultural products in China export growth to suffer big pressure.With regard to the agricultural-food of importing and exporting, qualified product, not only nontoxic, satisfy safety and health request; And will not have to mix up and adulterate, satisfy quality requirements.Wherein quality is qualified, and mainly comprise the implication of two aspects again: 1. raw material sources are consistent with label.2. component concentration is consistent with label.European Union comes into effect new food hygiene law from January 1st, 2006, but new legislation has been given prominence to tracing management and the trackability of food in the food production, stresses the identity authentication sign and healthy sign of food." the import and export food tag control way " of China standard GB 7718-94 " the food labelling universal standard " and The State Administration for Entry-Exit Inspection and Quarantine's issue, also clearly proposing the food labelling marked content should conform to food.For this reason, State General Administration for Quality Supervision sends the documents specially, requires the severe bad illegal activities of false making system of hitting, and will confiscate and supervise destruction by law to counterfeit and shoddy goods, traces beginning of production and whereabouts, is strictly on guard against to flow into the consumption link.
In the production and sales of food and drink, product does not have label or label is lack of standardization, the incident of utilizing means deception human consumer such as mingle, adulterate to reap staggering profits, domestic, all generations repeatedly abroad.After various raw materials are processed to the food and drink finished product, can't identify its composition from outward appearance.The producer is driven by interests, changes product without authorization and forms, or mix inexpensive substitute, or directly reduce the raw material consumption, causes the true attribute and the label of product not to be inconsistent.This imitation behavior not only relates to economy, nutritive value, more directly affects human consumer's health, possibly cause the property anaphylaxis of food source.
Garlic has another name called the head of garlic, only garlic, giant garlic, is the bulb of monocotyledons Liliaceae allium garlic.Garlic nutrient is worth very abundant, contains protein 4.4 grams according to surveying and determination among the every 100g of fresh bulb of garlic, fat 0.2 gram, carbohydrate 23.6 balls, 5 milligrams of calcium, 0.4 milligram of iron, 0.24 milligram of VitB1,0.9 milligram of nicotinic acid, 3 milligrams in xitix.Contain 17 seed amino acids in the garlic, wherein 6 kinds must amino acid for human body, the highest with arginine content especially, account for 20.4% of total amino acid content, secondly be L-glutamic acid, account for 19.75% of total amino acid content.Also contain elements germanium (73.4mg/100g) and selenium (0.3-0.8mg/100g) in the garlic.It can seasoning, again can be aid digestion with promote appetite, still magical good medicine having multiple efficacies such as the cold dampness elimination of loosing, desinsection are detoxified, give protection against cancer, protected the liver, reducing blood-fat, step-down.Along with people's improves constantly garlic medical care effect understanding, is that novel food product and the beverage that raw material or additive are developed increases on market day by day with the garlic.Though garlic is nutritious, also be a kind of common food allergen simultaneously.Report is arranged during the anaphylaxis that causes because of edible garlic.Given this, set up reliable and effective garlic component detection method as early as possible, guarantee the food label true and accurate, take place for reducing the property anaphylaxis of food source, strengthen the food allergens identity management, improve food safety, the protection consumer's interests are significant.
Plant-derived composition detection in the food often adopts the method for DNA detection.With DNA is that the method for tested object is compared with the method that is tested object with albumen, and under the food composition complicated situation, target dna can effectively extract, and receives the influence of food matrix less; In addition, DNA is more stable, does not resemble the influence that protein receives geographical conditions, seasonal variation and complete processing forming.Advantages such as current, in the detection method based on DNA, the real-time fluorescence PCR method is easy and simple to handle, sensitive, special, quick with it, good reproducibility, quantitatively accurate, totally-enclosed reaction obtain investigator's generally approval, become the important tool of detection.
Summary of the invention
The objective of the invention is to garlic allinase gene; Design a group-specific primers and probe; And then set up a kind of rapid detection garlic DNA method; To overcome,, thereby guarantee the consistence of food labelling and human consumer's safe diet for the garlic composition detection provides effective tool based on the limitation of proteic detection method in some food inspection.
Cardinal principle of the present invention is: to conserved sequence design one group of primer (upstream primer: 5 '-GGAAATGCTGCGAACTATGTGA-3 ' and downstream primer: 5 '-TTGATTGGGCTGTAATGAGGC-3 ') and Taqman probe (sequence is: 5 '-FAM-TGGCAAGGAGACCTTCTGGGTTGTTAGG-TAMRA-3 ').When carrying out detection of nucleic acids, template DNA is after being heated to 94-95 ℃ of certain hour, and the dna double chain dissociates, and primer and Taqman probe combine with template specificity.5 ends of probe are marked with report fluorescence group, and 3 ends have cancellation fluorescence group.When probe was complete, report group institute emitted fluorescence energy was absorbed by cancellation group, and instrument detecting is less than signal.Along with the carrying out of PCR, the Taq enzyme is in the chain extension process, and when running into template bonded probe, 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 promptly produces fluorescent signal.Along with the PCR round-robin increases, purpose fragment exponentially increases, and fluorescent signal also strengthens synchronously, the strong and weak directly reflection template number of fluorescent signal.
The garlic composition real-time fluorescence PCR that the present invention relates to detects the primer and the probe of usefulness, and its sequence is following:
(1) upstream primer: 5 '-GGAAATGCTGCGAACTATGTGA-3 ';
(2) downstream primer: 5 '-TTGATTGGGCTGTAATGAGGC-3 ';
(3) TaqMan probe: 5 '-TGGCAAGGAGACCTTCTGGGTTGTTAGG-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 the mentioned reagent box to detect the method for garlic composition in the 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 the DNA extraction test kit.
(2) real-time fluorescence PCR of garlic composition amplification
A. in reaction tubes, add 2 * PCR Master Mix, 12.5 μ L, each 1-2 μ L of upstream primer and downstream primer, Taqman probe 0.5-1 μ L, the DNA 1-2 μ L of 100ng/ μ L testing sample replenishes distilled water to 25 μ L, mixing;
B. the PCR reaction tubes is put into quantitative real time PCR Instrument, accomplishes pcr amplification by following reaction conditions:
94-95 ℃ of 5min, 1 circulation, sex change in advance;
94-95 ℃ of 10-20sec; 60 ℃ of 20-40sec, 45 circulations, pcr amplification.
During amplification, should set up three contrasts: positive control (get fresh garlic extract genomic dna), negative control (non-garlic genomic dna), blank (do not contain dna profiling, can water replace).
(3) use quantitative real time PCR Instrument accompanying software, analysing amplified result.
Amplification curve appears like testing sample; And the Ct value is less than or equal to 41; Negative control, positive control and blank result set up (be that typical positive amplification curve appears in positive, negative sample and blank do not have amplification), and then this sample of decidable detects the garlic composition.
More than or equal to 45, negative control, positive control and blank are all set up like testing sample Ct value, and then this sample of decidable does not detect the garlic composition.
Between 41-45, answer the amplification of recast real-time fluorescence PCR like testing sample Ct value.The value of Ct as a result after the amplification is still less than 45 once more, and negative control, positive control and blank result set up, and then this sample of decidable detects the garlic composition; The value of Ct as a result after the amplification is greater than 45 once more, and negative control, positive control and blank result set up, and then this sample of decidable does not detect the garlic composition.
Garlicin (Allicin) is the main functional component of Liliaceae allium garlic, and its sterilizing power is strong, has a broad antifungal spectrum, and have antitumor, decreasing cholesterol, platelet aggregation-against, preventing cardiovascular disease, physiologic function such as hypotensive.But garlicin is not a naturally occurring activeconstituents in the garlic, and it is to be formed by the allinase in the garlic (Alliinase) catalytic substrate alliin.Allinase is a kind of endogenous enzyme that is present in the garlic; Its chemical name is S-alkyl-L-halfcystine sulfoxide enzyme; Account for the 10%-12% of soluble protein in the garlic, its molecular mass is about 10.2-10.4 ten thousand D, contains the subunit that 2 molecular masses are about 5.15 ten thousand D.Alliin is present in the vacuole of garlic cell under the normal circumstances, and allinase is present in the tenuigenin.When the garlic intact cell was impaired, the two met enzyme digestion reaction generation garlicin takes place.In view of the vital role of allinase in the garlicin generative process, domesticly allinase in the garlic separation and purification and gene clone have been carried out for the scholar.The present invention is according to garlic allinase gene conservative sequence, uses Biodet software and Primer Express 3.0 softwares to carry out sequential analysis and primer, probe design.This conservative gene sequence is passed through the comparison of the nucleotide sequence of the allinase of alliums such as the garlic of Genebank login, onion, leek, green onion is obtained, and primer and the probe of designing carried out online Blast comparison inquiry, can guarantee detecting of garlic.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 garlic composition in processed food and the beverage of some complicated components.
Description of drawings
Fig. 1 is with real-time fluorescence PCR technology for detection SUANRONG LAJIANG 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 (soy bean DNA); Fig. 3 is the amplification collection of illustrative plates of blank (water); The positive contrast of Fig. 4 (garlic DNA) amplification collection of illustrative plates.
Fig. 5 is with real-time fluorescence PCR technology for detection broad bean paste 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 (soy bean DNA); Fig. 7 is the amplification collection of illustrative plates of blank (water); The positive contrast of Fig. 8 (garlic DNA) amplification collection of illustrative plates.
Embodiment
Below in conjunction with embodiment the present invention is further specified.
By the real-time fluorescence PCR assay kit of following prescription making garlic composition, reagent wherein comprises as follows:
(1)2×PCR?Master?Mix
Comprise 0.05u/ μ L Taq archaeal dna polymerase, reaction buffer, the 4mmol/L magnesium chloride, 0.4mmol/L dNTP (dATP, dCTP, dGTP, dTTP);
Wherein reaction buffer contains trihydroxy methyl aminomethane-hydrochloric acid, 100mmol/L Repone K and 2% triton x-100 of 20mmol/L pH8.8;
(2) upstream primer: 10 μ mol/L, sequence is: 5 '-GGAAATGCTGCGAACTATGTGA-3 ';
(3) downstream primer: 10 μ mol/L, sequence is: 5 '-TTGATTGGGCTGTAATGAGGC-3 ';
(4) Taqman probe: 10 μ mol/L, sequence is: 5 '-TGGCAAGGAGACCTTCTGGGTTGTTAGG-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 SUANRONG LAJIANG sample DNA to be measured
A. take by weighing the 0.1g SUANRONG LAJIANG, add in the 1.5mL centrifuge tube.The lysis buffer that adds 600 μ L preheatings, gently behind the mixing, 65 ℃ of water bath heat preservation 10min;
B. in pipe, add equal-volume phenol/chloroform 600 μ L, the abundant mixing that turns upside down, extracting 2min;
C.12000g centrifugal 5min draws in the new centrifuge tube of supernatant to, adds and the isopyknic precipitated liquid of supernatant, and mixing, after normal temperature was placed 10min, the centrifugal 5min of 12000g removed supernatant, kept deposition;
D. in deposition, add 60 μ L RNA enzymes, 37 ℃ place 2min after, with the rifle head with its abundant mixing, in 37 ℃ of dissolution precipitations, adding 300 μ L damping fluids behind the 5min, mixing 10 times turns upside down;
E. take out centrifugal post, centrifugal post is placed on the sleeve pipe of 1 2mL, solution is joined in the centrifugal post, place 2min;
F. centrifugal post and 2mL sleeve pipe one are reinstated the centrifugal 30sec of 8000g, discard solution in the sleeve pipe, in centrifugal post, add 200 μ L washing lotions, the centrifugal 30sec of 8000g discards solution; Repeat this step once;
G. in centrifugal post, add 200 μ L70% ethanol, the centrifugal 30sec of 8000g discards solution; Repeat this step once;
H.12000g centrifugal 30sec removes trace residue solution in the centrifugal post;
I. centrifugal post is placed in the new 1.5mL centrifuge tube, the central 50 μ L elution buffers that add in centrifugal column bottom, behind 37 ℃ of placement 2min, the centrifugal 30sec of 12000g.Solution in the centrifuge tube promptly can be used as the template of PCR reaction.
(2) real-time fluorescence PCR of SUANRONG LAJIANG DNA to be measured amplification
A. in the PCR reaction tubes, add 2 * PCR Master Mix, 12.5 μ L as claimed in claim 1, each 1 μ L of upstream primer and downstream primer, Taqman probe 0.5 μ L, the DNA 1 μ L of 100ng/ μ L testing sample replenishes distilled water to 25 μ L, mixing;
B. the PCR reaction tubes is put into quantitative real time PCR Instrument, accomplishes pcr amplification by following reaction conditions:
94 ℃ of 5min, 1 circulation, sex change in advance;
94 ℃ of 30sec; 60 ℃ of 30sec, 45 circulations, pcr amplification.
(3) use quantitative real time PCR Instrument accompanying software, analysing amplified result.
Positive amplification curve appears in sample, and the Ct value is 28.4, and negative control (soy bean DNA) and blank (water) do not have amplification, and positive control (garlic DNA) produces typical positive amplification curve, sees Fig. 1, Fig. 2, Fig. 3, Fig. 4, and this sample of decidable detects the garlic composition in view of the above.
By the real-time fluorescence PCR assay kit of following prescription making garlic composition, reagent wherein comprises as follows:
(1)2×PCR?Master?Mix
Comprise 0.05u/ μ L Taq archaeal dna polymerase Polymerase, reaction buffer, the 4mmol/L magnesium chloride, 0.4mmol/L dNTP (dATP, dCTP, dGTP, dTTP);
Wherein reaction buffer contains trihydroxy methyl aminomethane-hydrochloric acid, 100mmol/L Repone K and 2% triton x-100 of 20mmol/L pH8.8;
(2) upstream primer: 10 μ mol/L, sequence is: 5 '-GGAAATGCTGCGAACTATGTGA-3 ';
(3) downstream primer: 10 μ mol/L, sequence is: 5 '-TTGATTGGGCTGTAATGAGGC-3 ';
(4) Taqman probe: 10 μ mol/L, sequence is: 5 '-TGGCAAGGAGACCTTCTGGGTTGTTAGG-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 broad bean paste sample DNA to be measured
A. take by weighing 0.1g broad bean paste, add in the 1.5mL centrifuge tube.The lysis buffer that adds 600 μ L preheatings, gently behind the mixing, 65 ℃ of water bath heat preservation 10min;
B. in pipe, add equal-volume phenol/chloroform 600 μ L, the abundant mixing that turns upside down, extracting 2min;
C.12000g centrifugal 5min draws in the new centrifuge tube of supernatant to, adds and the isopyknic precipitated liquid of supernatant, and mixing, after normal temperature was placed 10min, the centrifugal 5min of 12000g removed supernatant, kept deposition;
D. in deposition, add 60 μ L RNA enzymes, 37 ℃ place 2min after, with the rifle head with its abundant mixing, in 37 ℃ of dissolution precipitations, adding 300 μ L damping fluids behind the 5min, mixing 10 times turns upside down;
E. take out centrifugal post, centrifugal post is placed on the sleeve pipe of 1 2mL, solution is joined in the centrifugal post, place 2min;
F. centrifugal post and 2mL sleeve pipe one are reinstated the centrifugal 30sec of 8000g, discard solution in the sleeve pipe, in centrifugal post, add 200 μ L washing lotions, the centrifugal 30sec of 8000g discards solution; Repeat this step once;
G. in centrifugal post, add 200 μ L70% ethanol, the centrifugal 30sec of 8000g discards solution; Repeat this step once;
H.12000g centrifugal 30sec removes trace residue solution in the centrifugal post;
I. centrifugal post is placed in the new 1.5mL centrifuge tube, the central 50 μ L elution buffers that add in centrifugal column bottom, behind 37 ℃ of placement 2min, the centrifugal 30sec of 12000g.Solution in the centrifuge tube promptly can be used as the template of PCR reaction.
(2) real-time fluorescence PCR of broad bean paste DNA to be measured amplification:
A. in the PCR reaction tubes, add 2 * PCR Master Mix, 12.5 μ L as claimed in claim 1, each 1 μ L of upstream primer and downstream primer, Taqman probe 0.5 μ L, the DNA 1 μ L of 100ng/ μ L testing sample replenishes distilled water to 25 μ L, mixing;
B. the PCR reaction tubes is put into quantitative real time PCR Instrument, accomplishes pcr amplification by following reaction conditions:
94 ℃ of 5min, 1 circulation, sex change in advance;
94 ℃ of 30sec; 60 ℃ of 30sec, 45 circulations, pcr amplification.
(3) use quantitative real time PCR Instrument accompanying software, analysing amplified result.
Positive amplification curve does not appear in sample, and negative control (maize dna) and blank (water) do not have amplification, and positive control (garlic DNA) produces typical positive amplification curve, sees Fig. 5, Fig. 6, Fig. 7, Fig. 8,, this sample of decidable does not detect the garlic composition in view of the above.
Claims (2)
1. a upstream primer and downstream primer that garlic composition rapid detection is used is characterized in that:
The upstream primer sequence is: 5 '-GGAAATGCTGCGAACTATGTGA-3 ';
The downstream primer sequence is: 5 '-TTGATTGGGCTGTAATGAGGC-3 '.
A garlic composition rapid detection use the Taqman probe, it is characterized in that:
Sequence is: 5 '-TGGCAAGGAGACCTTCTGGGTTGTTAGG-3 ', its 5 ' end flag F AM report fluorescence group, 3 ' end mark TAMRA cancellation fluorescence group.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105510171A (en) * | 2014-09-27 | 2016-04-20 | 张瑞节 | Method for detecting garlicin by mercury nitrate precipitation method |
| CN111133116A (en) * | 2017-09-25 | 2020-05-08 | 好侍食品集团本社株式会社 | Onion discrimination method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN101899498A (en) * | 2009-05-15 | 2010-12-01 | 天津出入境检验检疫局动植物与食品检测中心 | Standard substance for detecting allergen almond components in foods |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899498A (en) * | 2009-05-15 | 2010-12-01 | 天津出入境检验检疫局动植物与食品检测中心 | Standard substance for detecting allergen almond components in foods |
| 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 |
Non-Patent Citations (2)
| Title |
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| 徐玲等: "蒜氨酸酶基因水平研究进展", 《时珍国医国药》 * |
| 麻丽丹等: "实时荧光PCR法检测食品中芝麻过敏原成分", 《食品科学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105510171A (en) * | 2014-09-27 | 2016-04-20 | 张瑞节 | Method for detecting garlicin by mercury nitrate precipitation method |
| CN111133116A (en) * | 2017-09-25 | 2020-05-08 | 好侍食品集团本社株式会社 | Onion discrimination method |
| CN111133116B (en) * | 2017-09-25 | 2021-03-30 | 好侍食品集团本社株式会社 | Onion discrimination method |
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