CN103614469B - Plant source protein composition discrimination method in a kind of raw dairy and milk - Google Patents
Plant source protein composition discrimination method in a kind of raw dairy and milk Download PDFInfo
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Abstract
The invention discloses plant source protein composition discrimination method in a kind of raw dairy and milk, it is characterized in that: comprise the following steps: one, DNA extraction, design identifies specific primer and the fluorescent probe of plant derived component, extract sample gene group DNA, utilize the method for real-time fluorescence quantitative PCR to carry out discriminating to judge, the sequence of described specific primer is: upstream primer 5 ｀-GGATTGAGCCTTGGTATGGA-3 ｀; Downstream primer 5 ｀-TTTCGGAAAACAGGATTTGG-3 ｀; Described probe sequence is: fluorescent probe 5 ｀-CAAATTCAGAGAAACCCTGGA-3 ｀; Two, fluorescence quantitative PCR detection.The invention has the beneficial effects as follows: with plant chloroplast gene conserved regions fragment for detecting target, designing special primer and fluorescent probe, the quantitative fluorescent PCR discrimination method set up, this kind discrimination method sensitivity is strong, specificity is high, simple fast.
Description
Technical field
The present invention relates to a kind of protein ingredient discrimination method, particularly relate to plant source protein composition discrimination method in a kind of raw dairy and milk.
Background technology
Milk-product are requisite integral parts in public diet.Under the ordering about of interests, the adulterated behaviors of milk-product such as raw dairy, milk remain incessant after repeated prohibition.Along with the continuous exposure of industry underlying rule and the raising gradually of detection level, the adulterated means of milk-product also improve constantly, from initial water mixing, urea, starch, volatile salt, trimeric cyanamide behavior, add the method for the vegetable-proteins such as some cheap soybean protein, wheat proteins or proteolysate relative " brilliant " up till now in the product, can say, adopt traditional protein detection method to be more and more difficult to the quality effectively determining raw dairy and milk products.
Therefore, for solving the problem, spy provides a kind of new technical scheme to satisfy the demands.
Summary of the invention
The invention provides a kind of quantitative fluorescent PCR discrimination method differentiating to mix in raw dairy and milk plant-derived albumen fast and accurately.
The technical solution used in the present invention is:
Plant source protein composition discrimination method in a kind of raw dairy and milk, comprises the following steps:
One, DNA extraction
Design identifies specific primer and the fluorescent probe of plant derived component, extracts sample gene group DNA, utilizes the method for real-time fluorescence quantitative PCR to carry out discriminating and judges,
The sequence of described specific primer is:
Upstream primer 5 ｀-GGATTGAGCCTTGGTATGGA-3 ｀;
Downstream primer 5 ｀-TTTCGGAAAACAGGATTTGG-3 ｀;
Described probe sequence is:
Fluorescent probe 5 ｀-CAAATTCAGAGAAACCCTGGA-3 ｀;
Two, fluorescence quantitative PCR detection:
Quantitative fluorescent PCR reaction system total amount is adopted to be 19.3-29.8 μ L, system composition comprises for fluorescence quantitative PCR reaction solution 12-13 μ L, concentration is the upstream primer 0.5-1.5 μ L of 10 μm of ol, concentration is the downstream primer 0.5-1.5 μ L of 10 μm of ol, concentration is the probe 0.3-0.8 μ L of 10 μm of ol, template DNA 1-3 μ L, ddH
2o5-10 μ L, amplification program is: each reagent in quantitative fluorescent PCR reaction system is joined fluorescent quantitation eight union, set up negative control pipe and positive control simultaneously, be placed in real-time fluorescence quantitative PCR instrument, control temperature is carried out denaturing treatment 1-2min at 94-96 DEG C to quantitative fluorescent PCR reaction system, maintain the temperature at 94-96 DEG C of denaturing treatment 5-10s, then reduce temperature and carry out anneal 25-35s to 55-60 DEG C, after annealing, raised temperature to 72 DEG C carries out prolonged treatment 25-50s, above-mentioned denaturing treatment-anneal-prolonged treatment three steps are repeated 35-45 circulation by above-mentioned order and processing requirement, be marked at 55-60 DEG C of end according to different fluorescent probe to start to collect fluorescent signal.
Described fluorescent probe 5 ｀ holds as FAM, HEX, ROX, JOE, FAM, SYBRGreenI, TET, Cy3, Cy5 or VIC fluorescent substance mark, and described fluorescent probe 3 ｀ end can connect TAMRA or BHQ fluorescence quenching.
Described vegetable-derived components is soybean, wheat, paddy rice or corn.
The invention has the beneficial effects as follows: with plant chloroplast gene conserved regions fragment for detecting target, design special primer and fluorescent probe, the quantitative fluorescent PCR discrimination method set up, this kind of discrimination method is higher than the accuracy rate of common PCR discrimination method, differentiating method is simple fast, because many probes, so quantitative fluorescent PCR is stronger than common PCR sensitivity, specificity is high.
Accompanying drawing explanation
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
Fig. 1 fluorescent quantitative PCR result schematic diagram.
Fig. 2 Artificial sample 1(soybean protein-containing sample) result schematic diagram, wherein: Artificial sample I is the raw dairy containing soybean protein isolate, and positive control is Soybean genomic DNA extracting solution, and negative control is natural cow's milk extracting genome DNA liquid.
Fig. 3 Artificial sample 2(is containing wheat protein crude extract sample) result schematic diagram, wherein: Artificial sample II is the raw dairy containing wheat protein crude extract, and positive control is Wheat DNA extracting solution, and negative control is Niu Tianran cow's milk extracting genome DNA liquid.
Embodiment
In order to deepen the understanding of the present invention, below in conjunction with embodiment and accompanying drawing, the invention will be further described, and this embodiment only for explaining the present invention, does not form limiting the scope of the present invention.
Embodiment 1
Plant source protein composition discrimination method in a kind of raw dairy and milk, comprises the following steps:
One, DNA extraction
Design identifies specific primer and the fluorescent probe of soybean components, extracts Soybean genomic DNA, utilizes the method for real-time fluorescence quantitative PCR to carry out discriminating and judges,
The sequence of described specific primer is:
Upstream primer 5 ｀-GGATTGAGCCTTGGTATGGA-3 ｀;
Downstream primer 5 ｀-TTTCGGAAAACAGGATTTGG-3 ｀;
Described probe sequence is:
Fluorescent probe 5 ｀-CAAATTCAGAGAAACCCTGGA-3 ｀;
Two, fluorescence quantitative PCR detection:
Adopt quantitative fluorescent PCR reaction system total amount to be 19.3 μ L, system composition comprises for fluorescence quantitative PCR reaction solution 12 μ L, and concentration is the upstream primer 0.5 μ L of 10 μm of ol, concentration is the downstream primer 0.5 μ L of 10 μm of ol, concentration is the probe 0.3 μ L of 10 μm of ol, template DNA 1 μ L, ddH
2o5 μ L, amplification program is: each reagent in quantitative fluorescent PCR reaction system is joined fluorescent quantitation eight union, set up testing sample group (template is the DNA solution that soybean to be measured is extracted), set up negative control pipe (template replaces with cow genome group DNA) simultaneously, positive control (template replaces with Soybean genomic DNA), be placed in real-time fluorescence quantitative PCR instrument, control temperature is carried out denaturing treatment 1min at 96 DEG C to quantitative fluorescent PCR reaction system, maintain the temperature at 96 DEG C of denaturing treatment 10s, then reduce temperature to 55 DEG C and carry out anneal 25s, after annealing, raised temperature to 72 DEG C carries out prolonged treatment 25s, above-mentioned denaturing treatment-anneal-prolonged treatment three steps are repeated 35 circulations by above-mentioned order and processing requirement, according to the situation of the ROX that fluorescent probe 5 ｀ marks, 3 ｀ ends connect BHQ, start to collect ROX fluorescent signal 55 DEG C of end.
Embodiment 2
Plant source protein composition discrimination method in a kind of raw dairy and milk, comprises the following steps:
One, DNA extraction
Design identifies specific primer and the fluorescent probe of Wheat components, extracts Wheat volatiles DNA, utilizes the method for real-time fluorescence quantitative PCR to carry out discriminating and judges,
The sequence of described specific primer is:
Upstream primer 5 ｀-GGATTGAGCCTTGGTATGGA-3 ｀;
Downstream primer 5 ｀-TTTCGGAAAACAGGATTTGG-3 ｀;
Described probe sequence is:
Fluorescent probe 5 ｀-CAAATTCAGAGAAACCCTGGA-3 ｀;
Two, fluorescence quantitative PCR detection:
Adopt quantitative fluorescent PCR reaction system total amount to be 25 μ L, system composition comprises for fluorescence quantitative PCR reaction solution 12.5 μ L, and concentration is the upstream primer 1 μ L of 10 μm of ol, concentration is the downstream primer 1 μ L of 10 μm of ol, concentration is the probe 0.5 μ L of 10 μm of ol, template DNA 2 μ L, ddH
2o8 μ L, amplification program is: each reagent in quantitative fluorescent PCR reaction system is joined fluorescent quantitation eight union, set up testing sample group (template is the DNA solution that wheat to be measured extracts), set up negative control pipe (template replaces with cow genome group DNA) simultaneously, positive control (template replaces with Wheat volatiles DNA), be placed in real-time fluorescence quantitative PCR instrument, control temperature is carried out denaturing treatment 1.5min at 95 DEG C to quantitative fluorescent PCR reaction system, maintain the temperature at 95 DEG C of denaturing treatment 5s, then reduce temperature to 58 DEG C and carry out anneal 30s, after annealing, raised temperature to 72 DEG C carries out prolonged treatment 30s, above-mentioned denaturing treatment-anneal-prolonged treatment three steps are repeated 40 circulations by above-mentioned order and processing requirement, according to the situation of the HEX that fluorescent probe 5 ｀ marks, 3 ｀ ends connect TAMRA, start to collect HEX fluorescent signal 58 DEG C of end.
Embodiment 3
Plant source protein composition discrimination method in a kind of raw dairy and milk, comprises the following steps:
One, DNA extraction
Design identifies specific primer and the fluorescent probe of paddy rice composition, extracts oryza sativa genomic dna, utilizes the method for real-time fluorescence quantitative PCR to carry out discriminating and judges,
The sequence of described specific primer is:
Upstream primer 5 ｀-GGATTGAGCCTTGGTATGGA-3 ｀;
Downstream primer 5 ｀-TTTCGGAAAACAGGATTTGG-3 ｀;
Described probe sequence is:
Fluorescent probe 5 ｀-CAAATTCAGAGAAACCCTGGA-3 ｀;
Two, fluorescence quantitative PCR detection:
Adopt quantitative fluorescent PCR reaction system total amount to be 29.8 μ L, system composition comprises for fluorescence quantitative PCR reaction solution 13 μ L, and concentration is the upstream primer 1.5 μ L of 10 μm of ol, concentration is the downstream primer 1.5 μ L of 10 μm of ol, concentration is the probe 0.8 μ L of 10 μm of ol, template DNA 3 μ L, ddH
2o10 μ L, amplification program is: each reagent in quantitative fluorescent PCR reaction system is joined fluorescent quantitation eight union, set up testing sample group (template is the DNA solution that paddy rice to be measured is extracted), set up negative control pipe (template replaces with cow genome group DNA) simultaneously, positive control (template replaces with oryza sativa genomic dna), be placed in real-time fluorescence quantitative PCR instrument, control temperature is carried out denaturing treatment 2min at 94 DEG C to quantitative fluorescent PCR reaction system, maintain the temperature at 94 DEG C of denaturing treatment 7s, then reduce temperature to 60 DEG C and carry out anneal 35s, after annealing, raised temperature to 72 DEG C carries out prolonged treatment 50s, above-mentioned denaturing treatment-anneal-prolonged treatment three steps are repeated 45 circulations by above-mentioned order and processing requirement, according to the situation of the FAM that fluorescent probe 5 ｀ marks, 3 ｀ ends connect TAMRA, start to collect FAM fluorescent signal 60 DEG C of end.
Embodiment 4
Plant source protein composition discrimination method in a kind of raw dairy and milk, comprises the following steps:
One, DNA extraction
Design identifies specific primer and the fluorescent probe of corn composition, extracts corn gene group DNA, utilizes the method for real-time fluorescence quantitative PCR to carry out discriminating and judges,
The sequence of described specific primer is:
Upstream primer 5 ｀-GGATTGAGCCTTGGTATGGA-3 ｀;
Downstream primer 5 ｀-TTTCGGAAAACAGGATTTGG-3 ｀;
Described probe sequence is:
Fluorescent probe 5 ｀-CAAATTCAGAGAAACCCTGGA-3 ｀;
Two, fluorescence quantitative PCR detection:
Adopt quantitative fluorescent PCR reaction system total amount to be 28 μ L, system composition comprises for fluorescence quantitative PCR reaction solution 12.5 μ L, and concentration is the upstream primer 1.2 μ L of 10 μm of ol, concentration is the downstream primer 1.3 μ L of 10 μm of ol, concentration is the probe 0.7 μ L of 10 μm of ol, template DNA 3 μ L, ddH
2o9.3 μ L, amplification program is: each reagent in quantitative fluorescent PCR reaction system is joined fluorescent quantitation eight union, set up testing sample group (template is the DNA solution that corn to be measured extracts), set up negative control pipe (template replaces with cow genome group DNA) simultaneously, positive control (template replaces with corn gene group DNA), be placed in real-time fluorescence quantitative PCR instrument, control temperature is carried out denaturing treatment 2min at 94 DEG C to quantitative fluorescent PCR reaction system, maintain the temperature at 94 DEG C of denaturing treatment 6s, then reduce temperature to 58 DEG C and carry out anneal 33s, after annealing, raised temperature to 72 DEG C carries out prolonged treatment 32s, above-mentioned denaturing treatment-anneal-prolonged treatment three steps are repeated 42 circulations by above-mentioned order and processing requirement, according to the situation of the JOE that fluorescent probe 5 ｀ marks, 3 ｀ ends connect TAMRA, start to collect JOE fluorescent signal 59 DEG C of end.
Fluorescent probe 5 ｀ end can also be FAM, SYBRGreenI, TET, Cy3, Cy5 or VIC fluorescent substance mark.
The invention has the beneficial effects as follows: with plant chloroplast gene conserved regions fragment for detecting target, design special primer and fluorescent probe, the quantitative fluorescent PCR discrimination method set up, this kind of discrimination method is higher than the accuracy rate of common PCR discrimination method, differentiating method is simple fast, because many probes, so quantitative fluorescent PCR is stronger than common PCR sensitivity, specificity is high.
Test knot of the present invention differentiates: quantitative fluorescent PCR reaction terminates, interpretation of result is carried out according to amplification curve, as shown in Figure 1, the precondition of result validity should meet negative control should without Ct value (without amplification curve) and positive control Ct < 35(typical case amplification curve), otherwise it is invalid to be considered as, when testing sample is without Ct value (without amplification curve), show sample not containing plant derived component; When testing sample is typical amplification curve, and during Ct value Ct < 35, show that sample contains plant derived component; When amplification curve appears in testing sample, and during 35≤Ct < 40, be suspicious specimen, should retest, if result is still in this scope, show that sample contains a small amount of plant derived component.
Applicating example of the present invention:
Manually to add sample (adding soybean protein isolate, wheat protein crude extract in raw dairy respectively), introduce this patent discrimination method.
1. reagent and instrument
Animal and plant genome DNA extracting reagent kit is purchased from Tian Gen biochemical technology company limited, and real-time fluorescence quantitative PCR reaction solution is purchased from Takara;
Primer and probe entrust precious biotechnology (Dalian) company limited to synthesize, and fluorescent probe 5 ｀ holds with FAM mark, and 3 ｀ ends connect TAMRA mark;
Artificial sample I: artificially add soybean protein isolate at random in raw dairy, mixing;
Artificial sample II: artificially random interpolation wheat protein crude extract in raw dairy, mixing;
The real-time fluorescence quantitative PCR instrument adopted is StratageneMx3000P (Agilent company of the U.S.), CF15RX whizzer (Japanese Hitachi company), micropipet (Thermo);
2.DNA extracts
The method provided according to Plant Genome test kit extracts soybean, wheat, Artificial sample I, Artificial sample II genomic dna, and the method provided according to Animal genome test kit extracts natural cow's milk genomic dna;
3. reaction system: related reagent is joined fluorescent quantitation reaction tubes according to following table formula, set up testing sample group (template is the DNA solution of artificial sample extraction), negative control pipe is become, respectively using the genomic dna of soybean, wheat extraction as positive control using cow genome group DNA as template sets;
| Reagent | Volume |
| Premix Ex Taq? (Perfect Real Time) | 12.5μL |
| Upstream primer (10 μm of ol) | 1μL |
| Downstream primer (10 μm of ol) | 1μL |
| Probe (10 μm of ol) | 0.5μL |
| Template DNA | 2μL |
| ddH 2O | 8 μL |
| Amount to | 25 μL |
4. reaction parameter: the reaction tubes adding reagent is placed in real-time fluorescence quantitative PCR instrument, mark corresponding pore, reaction parameter arranges as follows: 95 DEG C of sex change 1min30s, with sex change 95 DEG C of 5s, anneal 58 DEG C of 30s, and extend 72 DEG C of 30s and increase 40 and circulate, 58 DEG C of end start to collect FAM fluorescent signal;
5. result differentiates: quantitative fluorescent PCR reaction terminates, and carry out interpretation of result according to amplification curve, as shown in Figure 2 and Figure 3, can find that positive control is typical amplification curve, negative control is without amplification curve; Simultaneously, also there is amplification curve in Artificial sample I and the artificial Artificial sample II adding wheat protein crude extract in raw dairy at random of artificial random interpolation soybean protein isolate in raw dairy, Ct value is respectively 27.6 and 23.9, show to be identified containing plant source protein composition in sample, and then prove the accuracy of present method.
The above is only preferred embodiment of the present invention, is not restriction the present invention being made to any other form, and any amendment done according to technical spirit of the present invention or equivalent variations, still belong to the present invention's scope required for protection.
Claims (2)
1. a plant source protein composition discrimination method in raw dairy and milk, is characterized in that: comprise the following steps:
One, DNA extraction
Design identifies specific primer and the fluorescent probe of plant derived component, extracts sample gene group DNA, utilizes the method for real-time fluorescence quantitative PCR to carry out discriminating and judges,
The sequence of described specific primer is:
Upstream primer 5 ｀-GGATTGAGCCTTGGTATGGA-3 ｀;
Downstream primer 5 ｀-TTTCGGAAAACAGGATTTGG-3 ｀;
Described probe sequence is:
Fluorescent probe 5 ｀-CAAATTCAGAGAAACCCTGGA-3 ｀;
Two, fluorescence quantitative PCR detection
Quantitative fluorescent PCR reaction system total amount is adopted to be 19.3-29.8 μ L, system composition comprises for fluorescence quantitative PCR reaction solution 12-13 μ L, concentration is the upstream primer 0.5-1.5 μ L of 10 μm of ol, concentration is the downstream primer 0.5-1.5 μ L of 10 μm of ol, concentration is the probe 0.3-0.8 μ L of 10 μm of ol, template DNA 1-3 μ L, ddH
2o5-10 μ L, amplification program is: each reagent in quantitative fluorescent PCR reaction system is joined fluorescent quantitation eight union, set up negative control pipe and positive control simultaneously, be placed in real-time fluorescence quantitative PCR instrument, control temperature is carried out denaturing treatment 1-2min at 94-96 DEG C to quantitative fluorescent PCR reaction system, maintain the temperature at 94-96 DEG C of denaturing treatment 5-10s, then reduce temperature and carry out anneal 25-35s to 55-60 DEG C, after annealing, raised temperature to 72 DEG C carries out prolonged treatment 25-50s, above-mentioned denaturing treatment-anneal-prolonged treatment three steps are repeated 35-45 circulation by above-mentioned order and processing requirement, be marked at 55-60 DEG C of end according to different fluorescent probe to start to collect fluorescent signal,
Described plant source protein composition is soybean, wheat, paddy rice or corn.
2. plant source protein composition discrimination method in a kind of raw dairy according to claim 1 and milk, it is characterized in that: described fluorescent probe 5 ｀ holds as FAM, HEX, ROX, JOE, FAM, SYBRGreenI, TET, Cy3, Cy5 or VIC fluorescent substance mark, and described fluorescent probe 3 ｀ holds and connects TAMRA or BHQ fluorescence quenching.
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| CN102367472A (en) * | 2011-07-07 | 2012-03-07 | 哈尔滨工业大学 | Single/duplex PCR (polymerase chain reaction) detection method for botanical adding material in milk or milk powder |
| CN103014160A (en) * | 2012-12-11 | 2013-04-03 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | Method and primer for detecting lactobacilli in food |
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| CN102367472A (en) * | 2011-07-07 | 2012-03-07 | 哈尔滨工业大学 | Single/duplex PCR (polymerase chain reaction) detection method for botanical adding material in milk or milk powder |
| CN103014160A (en) * | 2012-12-11 | 2013-04-03 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | Method and primer for detecting lactobacilli in food |
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