CN109917032B - Quantitative detection method for Bt protein in Bt protein-transgenic food - Google Patents
Quantitative detection method for Bt protein in Bt protein-transgenic food Download PDFInfo
- Publication number
- CN109917032B CN109917032B CN201910187579.XA CN201910187579A CN109917032B CN 109917032 B CN109917032 B CN 109917032B CN 201910187579 A CN201910187579 A CN 201910187579A CN 109917032 B CN109917032 B CN 109917032B
- Authority
- CN
- China
- Prior art keywords
- protein
- family
- specific polypeptide
- follows
- detecting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a quantitative detection method of Bt protein in a transgenic Bt protein food, which comprises the steps of carrying out enzymolysis on the transgenic Bt protein food by adopting trypsin, selecting specific polypeptide in polypeptide generated by the enzymolysis of the Bt protein as a marker, artificially synthesizing the specific polypeptide as a standard substance, designing and synthesizing isotope-labeled specific polypeptide as an internal standard, carrying out quantitative detection on the specific polypeptide by adopting an isotope dilution liquid chromatography tandem mass spectrometry, and then converting to obtain the content of the Bt protein. The invention has the advantages of good stability, accurate quantification, good repeatability, high specificity and wide applicability.
Description
Technical Field
The invention relates to the technical field of food detection, in particular to a quantitative detection method of Bt protein in a transgenic Bt protein food.
Background
With the increasing maturity of transgenic technology, the industrialization of transgenic agricultural products and foods has presented a better trend. The transgenic agricultural product food brings great economic benefit and social benefit to human beings, and has potential environmental and food safety risks. In view of the safety of transgenic agricultural products, countries and organizations such as China, European Union, Japan and Korea have developed corresponding laws and regulations to identify the transgenic agricultural products and processed foods to protect the health and ecological environment diversity of human and animals. Since transgenic agricultural food and its products may have potential adverse effects on human health and ecological environment, the detection of transgenic agricultural food and its products is very important before it is uncertain whether transgenic agricultural food and its products are harmful.
At present, the detection methods of transgenic agricultural products and foods mainly comprise two methods: protein-based detection methods and nucleic acid-based detection methods.
Protein-based detection methods are generally antigen-antibody based detection methods, including ELISA, dipstick, and the like. However, the above techniques have the problems of false positive, false negative and inaccurate quantitative result. Due to the complex matrix in the agricultural product food, the antibody is easy to mistakenly identify the matrix protein with similar structure as Bt protein, and false positive is caused. During food processing, especially during heat processing, protein folding can change, leading to incorrect recognition of the protein by antibodies, leading to false negatives. Different batches of antibodies, the same antibody, all differ in affinity for different Bt proteins, leading to inaccuracies in the quantification results.
The detection method based on nucleic acid mainly refers to PCR technology, and specific primers are adopted to amplify the nucleotide sequence of the transferred exogenous gene. The presence, absence, amount, and amount of the amplification product are determined. This method cannot perform accurate quantification. Meanwhile, the loss and damage of DNA can be caused in the food processing process, and the correctness of the result is influenced.
Disclosure of Invention
The invention aims to solve the defects of the existing detection method and provides a quantitative detection method for Bt protein in a transgenic Bt protein food.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a quantitative detection method for Bt protein in a transgenic Bt protein food is characterized in that trypsin is adopted to carry out enzymolysis on the transgenic Bt protein food, specific polypeptide in polypeptide generated by the enzymolysis of the Bt protein is selected as a marker, the specific polypeptide is artificially synthesized to serve as a standard substance, isotope-labeled specific polypeptide is designed and synthesized to serve as an internal standard, isotope dilution liquid chromatography tandem mass spectrometry is adopted to carry out quantitative detection on the specific polypeptide, and then the content of the Bt protein is obtained through conversion.
According to the invention, Bt protein is subjected to enzymolysis by using enzyme, a specific enzymolysis polypeptide is selected as a marker, the specific polypeptide is artificially synthesized to serve as a standard substance, and quantitative detection is carried out by adopting an isotope dilution liquid chromatography tandem mass spectrometry method.
The specific operation of enzymolysis of the rotary Bt protein food by adopting trypsin is as follows: accurately weighing 0.1g of sample in a 2ml EP tube, adding 900mg of ammonium bicarbonate solution, then adding 10mg of DTT, carrying out water bath at 50 ℃ for 30min, then adding 30mg of IAA to react in a dark place for 30min, finally adding 10mg of trypsin, carrying out enzymolysis in water bath at 37 ℃ for 4h, taking out after the enzymolysis is finished, adding 20mg of 10% formic acid to stop the reaction, and finally adding 10mg of artificially synthesized isotope labeled specific polypeptide and mixing uniformly.
The concentration of ammonium bicarbonate solution is 50mM, DTT concentration is 100mM, IAA concentration is 100mM, trypsin concentration is 500mM, and the concentration of the synthetic isotope labeling specific polypeptide is 1000 mug/kg.
The amino acid sequence of the specific polypeptide for detecting the Bt protein Cry1 family is as follows: EKLEWETNIVYK, respectively;
the amino acid sequence of the specific polypeptide for detecting the Bt protein Cry2 family is as follows: TYMFLNVFEYVSIWSLFK are provided.
The amino acid sequence of the specific polypeptide for simultaneously detecting the Bt protein Cry1 family and Cry2 family is as follows: TFEAEYDLERA are provided.
The amino acid sequence of the specific polypeptide for detecting the Bt protein Cry3 family is as follows: IRELFSQAESHFR, respectively;
the amino acid sequence of the specific polypeptide for detecting the Bt protein Cry4 family is as follows: IDESKLKPYTR, respectively;
the amino acid sequence of the specific polypeptide for detecting the Bt protein Cry5 family is as follows: NLEKGINAGTYSK, respectively;
the amino acid sequence of the specific polypeptide for detecting the Bt protein Cry9 family is as follows: KMLLEAVR.
The amino acid sequence of the specific polypeptide for simultaneously detecting the Bt protein Cry3 family, Cry4 family and Cry5 family is as follows: MLALSNRMQK are provided.
The chromatographic conditions of the isotope dilution liquid chromatography tandem mass spectrometry are as follows: using C18 protein analytical column, wherein phase A is 0.1% formic acid water solution, and phase B is 0.1% formic acid acetonitrile solution; the elution conditions are that the B phase is from 3% to 30% in 0-9min, from 30% to 40% in 9-10min, from 10.1min to 100% and kept for 10.6min, and from 3% to 13min in 10.7 min. The total elution time was 13min and the flow rate was 0.3 mL/min.
The mass spectrum conditions of the isotope dilution liquid chromatography tandem mass spectrometry are as follows: capillary voltage: 3.0kv, desolventizing temperature: 550 ℃, desolventizing gas flow: 400L/min, cone hole back-blowing gas flow: 30L/hr, collision chamber pressure: 3.0X 10-3 mbar; low-end resolution 1: 2.5V, high-end resolution 1: 15.0V, ion energy 1: 0.6 eV; low-end resolution 2: 2.0V, high-end resolution 2: 15.0V, ion energy 2: 2.0 eV; ion source temperature: 150 ℃, extractor voltage: 5.0V, entrance lens voltage: 10V, outlet voltage: 10V.
A quantitative detection kit for Bt protein in food with transferred Bt protein comprises trypsin, ammonium bicarbonate solution, DTT, IAA, 10% formic acid and specific polypeptide for detecting Bt protein,
specific polypeptides for detecting Bt proteins are as follows:
the amino acid sequence of the specific polypeptide for detecting the Bt protein Cry1 family is as follows: EKLEWETNIVYK, respectively;
the amino acid sequence of the specific polypeptide for detecting the Bt protein Cry2 family is as follows: TYMFLNVFEYVSIWSLFK, respectively;
the amino acid sequence of the specific polypeptide for simultaneously detecting the Bt protein Cry1 family and Cry2 family is as follows: TFEAEYDLERA, respectively;
the amino acid sequence of the specific polypeptide for detecting the Bt protein Cry3 family is as follows: IRELFSQAESHFR, respectively;
the amino acid sequence of the specific polypeptide for detecting the Bt protein Cry4 family is as follows: IDESKLKPYTR, respectively;
the amino acid sequence of the specific polypeptide for detecting the Bt protein Cry5 family is as follows: NLEKGINAGTYSK, respectively;
the amino acid sequence of the specific polypeptide for detecting the Bt protein Cry9 family is as follows: KMLLEAVR;
the amino acid sequence of the specific polypeptide for simultaneously detecting the Bt protein Cry3 family, Cry4 family and Cry5 family is as follows: MLALSNRMQK are provided.
The invention has the beneficial effects that:
1. the stability is good: although the spatial structure of proteins is easily destroyed by food processing, the primary structure stability is very good.
2. The quantification is accurate: the isotope dilution liquid chromatography tandem mass spectrometry is a gold standard for quantitative detection, and the result is accurate and reliable.
3. The repeatability is good: the polypeptide chemically synthesized is adopted as a standard substance, can be synthesized in batches, and has stable quality.
4. The specificity is high: polypeptides are selected as standard substances, which have the characteristics of the genetic code and are capable of distinguishing the target polypeptide well from the matrix polypeptide.
5. The applicability is wide: the Bt protein is not a single protein, but a general name of a large class of proteins (Bt Cry1Aa, Bt Cry1Ab, Bt Cry1Ac, Bt Cry1Ah, Bt Cry1B, Bt Cry1C, Bt Cry1F, Bt Cry2A, Bt Cry3B and Bt Cry9C proteins), and various Bt proteins can be simultaneously and quantitatively detected by selecting different polypeptides, and each Bt protein can be respectively and quantitatively detected, so that various detection requirements are met.
Drawings
FIG. 1 is a diagram of a thermal denaturation test of Bt protein;
FIG. 2 is Cry1Aa transgenic soybean detection map-1;
FIG. 3 is Cry1Aa transgenic soybean detection map-2;
FIG. 4 is Cry1Aa transgenic soybean detection map-3;
FIG. 5 is a transgene-free soybean detection map-1;
FIG. 6 is a transgene-free soybean detection profile-2;
FIG. 7 is the detection map-3 of non-transgenic soybean.
Detailed Description
The technical solution of the present invention will be further specifically described below by way of specific examples.
In the present invention, the raw materials and equipment used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.
More than 100 Bt proteins, namely the pesticide crystal proteins, are found, 87 Bt proteins are verified by uniprot, and the homology among the Bt proteins is high or low. According to the homology classification, the method can meet various detection requirements by adopting a few specific polypeptides, and the specific polypeptides (containing isotope labeling sequences) are synthesized from Shanghai.
In the following examples: the concentration of ammonium bicarbonate solution is 50mM, DTT concentration is 100mM, IAA concentration is 100mM, trypsin concentration is 500mM, and the concentration of the synthetic isotope labeling specific polypeptide is 1000 mug/kg.
Example 1: detection of Cry1 family proteins in soymilk
1. Pretreatment of
Accurately weighing 0.1g of sample (soybean milk) in a 2ml EP tube, adding 900mg of ammonium bicarbonate solution, then adding 10mg of DTT, carrying out water bath at 50 ℃ for 30min, then adding 30mg of IAA to react in dark for 30min, finally adding 10mg of trypsin, carrying out enzymolysis in water bath at 37 ℃ for 4h, taking out after the enzymolysis is finished, adding 20mg of 10% formic acid to stop the reaction, finally adding 10mg of artificially synthesized isotope labeled specific polypeptide, mixing uniformly, and filtering and injecting samples.
2. Conditions of liquid chromatography
The reference conditions were as follows: a C18 protein analytical column (1.7 μm particle size, 2.1 mm. times.100 mm) was used. The phase A is 0.1% formic acid water solution, and the phase B is 0.1% formic acid acetonitrile solution; the elution conditions are that the B phase is from 3% to 30% in 0-9min, from 30% to 40% in 9-10min, from 10.1min to 100% and kept for 10.6min, and from 3% to 13min in 10.7 min. The total elution time was 13min and the flow rate was 0.3 mL/min.
3. Conditions of Mass Spectrometry
The mass spectrometric detection conditions were as follows: capillary voltage: 3.0kv, desolventizing temperature: 550 ℃, desolventizing gas flow: 400L/min, cone hole back-blowing gas flow: 30L/hr, collision chamber pressure: 3.0X 10-3mbar; low-end resolution 1: 2.5V, high-end resolution 1: 15.0V, ion energy 1: 0.6 eV; low-end resolution 2: 2.0V, high-end resolution 2: 15.0V, ion energy 2: 2.0 eV; ion source temperature: 150 ℃, extractor voltage: 5.0V, entrance lens voltage: 10V, outlet voltage: 10V.
TABLE 1 parameter conditions for Mass Spectrometry multiple reaction monitoring
Note: l is isotopically labelled [13C6,15N ] -leucine.
According to the method of this example, 2 soymilk made from non-transgenic soybeans and 2 soymilk made from transgenic soybeans were spot-tested, and the results are shown in the following table:
TABLE 2
| Sample numbering | Type (B) | Cry1 content [ mg/kg] |
| 1 | Non-transgenic | 0 |
| 2 | Non-transgenic | 0 |
| 3 | Transgenosis | 0.28 |
| 4 | Transgenosis | 0.79 |
Example 2: detection of Cry2 family proteins in corn preparations
The parameters of the steps 1 to 3 of this example are the same as those of example 1, except that:
TABLE 3 parameter conditions for mass spectrometric multiple reaction monitoring
Note: l is isotopically labelled [13C6,15N ] -leucine.
According to the method of this example, 2 non-transgenic and 2 transgenic maize were spot tested and the results are shown in the following table:
TABLE 4
| Sample numbering | Type (B) | Cry2 content [ mg/kg] |
| 1 | Non-transgenic | 0 |
| 2 | Non-transgenic | 0 |
| 3 | Transgenosis | 13.4 |
| 4 | Transgenosis | 2.0 |
。
Example 3: simultaneous detection of Cry1 and Cry2 in soybeans
The parameters of the steps 1 to 3 of this example are the same as those of example 1, except that:
TABLE 5 parameter conditions for Mass Spectrometry multiple reaction monitoring
Note: l is isotopically labelled [13C6,15N ] -leucine.
According to the method of this example, 2 non-transgenic soybeans and 2 transgenic soybeans were spot-tested, and the results are shown in the following table
TABLE 6
| Sample numbering | Type (B) | Cry1+ Cry2 content [ mg/kg] |
| 1 | Non-transgenic | 0 |
| 2 | Non-transgenic | 0 |
| 3 | Transgenosis | 14.3 |
| 4 | Transgenosis | 6.8 |
Examples 4 to 8 referring to example 1, the specific polypeptides were detected by replacing them with Cry3, Cry4, Cry5, Cry9, and Cry3, Cry4, and Cry5 polypeptides.
Methodology validation (linearity, precision, accuracy, sensitivity)
1. Standard curve
Purpose of the experiment: verification of the Linear Range of the method
Experimental methods
Accurately pipetting different volumes of peptide fragments [ EKLEWETNIVYK ] standard intermediate solution: mu.L, 10. mu.L, 20. mu.L, 40. mu.L, 60. mu.L, 80. mu.L and 100. mu.L were put in sample vials, 10. mu.L of isotope-specific peptide standard solutions were added, respectively, and then diluted to 1mL with 0.1% formic acid aqueous solution so that the concentrations were 400. mu.g/kg, 1000. mu.g/kg, 2000. mu.g/kg, 4000. mu.g/kg, 6000. mu.g/kg, 8000. mu.g/kg and 10000. mu.g/kg of standard series working curves (isotope internal standard concentration 1000. mu.g/kg), sample analysis was performed by linear fitting with the concentration of the standard solution as abscissa and the ratio of the peak area of the standard chromatographic peak to the peak area of the internal standard chromatographic peak as ordinate, as shown in Table 7.
TABLE 7 Standard Curve Linear and regression equations
Conclusion of the experiment
Linear r of standard curve2Are all larger than 0.99, and meet the requirements of detection and relevant regulations.
2. Precision experiment
Purpose of the experiment: the stability of the experimental method was investigated.
The experimental method comprises the following steps: one Cry1 Ab-transferred soybean was taken and assayed in parallel five times a day, repeated for 4 days, and pretreated (same as in example 1).
Table 8: day precision experimental result statistical table
Conclusion of the experiment
The RSD of the experimental result is less than or equal to 15 percent, and the requirements of detection and related regulations are met.
3. Accuracy/recovery experiment
Purpose of the experiment: the accuracy of the experimental results was examined by the recovery rate test of the spiked samples.
The experimental method comprises the following steps: taking non-transgenic soybean milk, respectively adding a proper amount of Cry1Aa standard protein to make the standard concentration of the sample be 500, 2000 and 8000 mug/kg, respectively, and paralleling the samples with three concentration points for five times and repeating for 4 days. Pretreatment (same as example 1).
The experimental results are as follows: see the following Table
Table 9: recovery rate experimental result statistical table
Conclusion of the experiment
According to the experimental result, the recovery rates of the three standard adding concentrations are all 80-120%, and the relative standard deviation RSD is less than 15%, so that the requirements of detection and related regulations are met.
4. Detection limit
Weighing 8 samples of the non-transgenic soybean milk, adding Cry1Aa standard protein close to detection, pretreating the samples (same as in example 1), detecting, calculating the result and standard deviation, taking the product of the variation coefficient f (n) and the standard deviation s when the confidence coefficient is 95% as the detection Limit (LOD) of the method, and the result is shown in Table 10, wherein the detection limit of the standard protein in the method is 55.7 mu g/kg, and the measurement requirement of daily samples is met.
TABLE 10 detection limits
Note: LOD ═ s × f (n); (n) the coefficient of variation when the confidence coefficient is 95%, and the coefficient of variation is obtained by table look-up; when n is 8, f (8) is 3.18.
5. Stability verification [ thermal denaturation experiment ]
Thermal denaturation test
The production process of soybean protein products is often accompanied with the generation of thermal denaturation, Bt protein denatured by heating cannot be detected by most methods such as an enzyme-linked immunosorbent assay, and the method can be used for detecting non-denatured Bt protein and thermal denatured Bt protein. To illustrate the problem, a transgenic soybean milk sample is respectively placed at the constant temperature of 0, 10, 20, 30, 40, 50, 60, 70, 80 and 90 ℃ for 1h, then the sample is respectively processed and detected according to the method and the enzyme-linked immunosorbent assay, the result is shown in figure 1, the result obtained by the enzyme-linked immunosorbent assay is gradually reduced along with the rise of the temperature, and when the temperature is 80 ℃, the result is reduced by more than 50%; by using the method, the detection result is not reduced along with the rise of the temperature; in the unheated or low-temperature region, the results are comparable. Therefore, the method has more accurate result for measuring Bt protein in soybean.
6. Specificity of
Detecting a plurality of soybeans transferred with Cry1Aa, wherein all soybeans are detected (figure 2-4); a number of non-transgenic soybeans were not detected (FIGS. 5-7), indicating that the method of the present invention is highly specific.
The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.
SEQUENCE LISTING
<110> Hangzhou dad evaluation technology Co Ltd
<120> quantitative detection method for Bt protein in food with transferred Bt protein
<130> 2019.03
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 12
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 1
Glu Lys Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys
1 5 10
<210> 2
<211> 18
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 2
Thr Tyr Met Phe Leu Asn Val Phe Glu Tyr Val Ser Ile Trp Ser Leu
1 5 10 15
Phe Lys
<210> 3
<211> 11
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 3
Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala
1 5 10
<210> 4
<211> 13
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 4
Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His Phe Arg
1 5 10
<210> 5
<211> 11
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 5
Ile Asp Glu Ser Lys Leu Lys Pro Tyr Thr Arg
1 5 10
<210> 6
<211> 13
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 6
Asn Leu Glu Lys Gly Ile Asn Ala Gly Thr Tyr Ser Lys
1 5 10
<210> 7
<211> 8
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 7
Lys Met Leu Leu Glu Ala Val Arg
1 5
<210> 8
<211> 10
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 8
Met Leu Ala Leu Ser Asn Arg Met Gln Lys
1 5 10
Claims (4)
1. A quantitative detection method of Bt protein in a food with transferred Bt protein is characterized in that: carrying out enzymolysis on the Bt protein food by adopting trypsin, selecting specific polypeptide in the polypeptide generated by the enzymolysis of the Bt protein as a marker, artificially synthesizing the specific polypeptide as a standard substance, designing and synthesizing isotope-labeled specific polypeptide as an internal standard, carrying out quantitative detection on the specific polypeptide by adopting an isotope dilution liquid chromatography tandem mass spectrometry, and then converting to obtain the content of the Bt protein;
the amino acid sequence of the specific polypeptide for detecting the Bt protein Cry1 family is as follows: EKLEWETNIVYK, respectively;
the amino acid sequence of the specific polypeptide for detecting the Bt protein Cry2 family is as follows: TYMFLNVFEYVSIWSLFK, respectively;
the amino acid sequence of the specific polypeptide for simultaneously detecting the Bt protein Cry1 family and Cry2 family is as follows: TFEAEYDLERA, respectively;
the amino acid sequence of the specific polypeptide for detecting the Bt protein Cry3 family is as follows: IRELFSQAESHFR, respectively;
the amino acid sequence of the specific polypeptide for detecting the Bt protein Cry4 family is as follows: IDESKLKPYTR, respectively;
the amino acid sequence of the specific polypeptide for detecting the Bt protein Cry5 family is as follows: NLEKGINAGTYSK, respectively;
the amino acid sequence of the specific polypeptide for detecting the Bt protein Cry9 family is as follows: KMLLEAVR;
the amino acid sequence of the specific polypeptide for simultaneously detecting the Bt protein Cry3 family, Cry4 family and Cry5 family is as follows: MLALSNRMQK, respectively;
the chromatographic conditions of the isotope dilution liquid chromatography tandem mass spectrometry are as follows: using C18 protein analytical column, wherein phase A is 0.1% formic acid water solution, and phase B is 0.1% formic acid acetonitrile solution; the elution conditions are that the B phase is eluted from 3% to 30% in 0-9min, from 30% to 40% in 9-10min, from 10.1min to 100% and kept for 10.6min, and from 3% to 13min in 10.7 min; the total elution time is 13min, and the flow rate is 0.3 mL/min;
the mass spectrum conditions of the isotope dilution liquid chromatography tandem mass spectrometry are as follows: capillary voltage: 3.0kv, desolventizing temperature: 550 ℃, desolventizing gas flow: 400L/min, cone hole back-blowing gas flow: 30L/hr, collision chamber pressure: 3.0X 10-3mbar; low-end resolution 1: 2.5V, high-end resolution 1: 15.0V, ion energy 1: 0.6 eV; low-end resolution 2: 2.0V, high-end resolution 2: 15.0V, ion energy 2: 2.0 eV; ion source temperature: 150 ℃, extractor voltage: 5.0V, entrance lens voltage: 10V, outlet voltage: 10V.
2. The quantitative determination method according to claim 1, characterized in that: the specific operation of enzymolysis of the rotary Bt protein food by adopting trypsin is as follows: accurately weighing 0.1g of sample in a 2ml EP tube, adding 900mg of ammonium bicarbonate solution, then adding 10mg of DTT, carrying out water bath at 50 ℃ for 30min, then adding 30mg of IAA to react in a dark place for 30min, finally adding 10mg of trypsin, carrying out enzymolysis in water bath at 37 ℃ for 4h, taking out after the enzymolysis is finished, adding 20mg of 10% formic acid to stop the reaction, and finally adding 10mg of artificially synthesized isotope labeled specific polypeptide and mixing uniformly.
3. The quantitative determination method according to claim 2, characterized in that: the concentration of ammonium bicarbonate solution is 50mM, DTT concentration is 100mM, IAA concentration is 100mM, trypsin concentration is 500mM, and the concentration of the synthetic isotope labeling specific polypeptide is 1000 mug/kg.
4. A quantitative detection box for Bt protein in a transgenic Bt protein food is characterized in that: comprises trypsin, ammonium bicarbonate solution, DTT, IAA, 10% formic acid and specific polypeptide for detecting Bt protein,
specific polypeptides for detecting Bt proteins are as follows:
the amino acid sequence of the specific polypeptide for detecting the Bt protein Cry1 family is as follows: EKLEWETNIVYK, respectively;
the amino acid sequence of the specific polypeptide for detecting the Bt protein Cry2 family is as follows: TYMFLNVFEYVSIWSLFK, respectively;
the amino acid sequence of the specific polypeptide for simultaneously detecting the Bt protein Cry1 family and Cry2 family is as follows: TFEAEYDLERA, respectively;
the amino acid sequence of the specific polypeptide for detecting the Bt protein Cry3 family is as follows: IRELFSQAESHFR, respectively;
the amino acid sequence of the specific polypeptide for detecting the Bt protein Cry4 family is as follows: IDESKLKPYTR, respectively;
the amino acid sequence of the specific polypeptide for detecting the Bt protein Cry5 family is as follows: NLEKGINAGTYSK, respectively;
the amino acid sequence of the specific polypeptide for detecting the Bt protein Cry9 family is as follows: KMLLEAVR;
the amino acid sequence of the specific polypeptide for simultaneously detecting the Bt protein Cry3 family, Cry4 family and Cry5 family is as follows: MLALSNRMQK are provided.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910187579.XA CN109917032B (en) | 2019-03-13 | 2019-03-13 | Quantitative detection method for Bt protein in Bt protein-transgenic food |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910187579.XA CN109917032B (en) | 2019-03-13 | 2019-03-13 | Quantitative detection method for Bt protein in Bt protein-transgenic food |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109917032A CN109917032A (en) | 2019-06-21 |
| CN109917032B true CN109917032B (en) | 2021-08-24 |
Family
ID=66964570
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910187579.XA Active CN109917032B (en) | 2019-03-13 | 2019-03-13 | Quantitative detection method for Bt protein in Bt protein-transgenic food |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109917032B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111323512B (en) * | 2020-03-26 | 2022-04-29 | 浙江大学医学院附属第四医院(浙江省义乌医院、浙江大学医学院附属第四医院医共体) | Kit for detecting inactivated virus and detection method |
| CN111323511B (en) * | 2020-03-26 | 2022-04-29 | 浙江大学医学院附属第四医院(浙江省义乌医院、浙江大学医学院附属第四医院医共体) | Rapid detection kit and method for inactivating new coronavirus |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1114358A (en) * | 1990-03-14 | 1996-01-03 | 加拿大国家研究院 | Isolation, quantitation and purification of insecticidal proteins from endotoxins of bacillus thuringiensis |
| CN101206226A (en) * | 2007-10-25 | 2008-06-25 | 中国农业科学院蜜蜂研究所 | Preparation and application of test paper for testing royal jelly freshness |
| CN102094038A (en) * | 2010-12-08 | 2011-06-15 | 上海市农业科学院 | Prokaryotic expression and purification method of transgenic rice Bt protein crylc |
| CN102094039A (en) * | 2010-12-08 | 2011-06-15 | 上海市农业科学院 | Prokaryotic expression and purification method of transgenic rice Bt protein cry2a |
| CN103616454A (en) * | 2013-12-06 | 2014-03-05 | 浙江贝因美科工贸股份有限公司 | Method and kit for quantitatively detecting human beta-casein content |
| CN105296522A (en) * | 2015-12-01 | 2016-02-03 | 中国农业科学院植物保护研究所 | Bt protein expression system |
| CN106852147A (en) * | 2014-10-16 | 2017-06-13 | 孟山都技术有限公司 | Lepidopteran-active Cry1Da1 variant amino acid sequence albumen |
| CN109097348A (en) * | 2018-07-26 | 2018-12-28 | 天津科技大学 | The application of alpha-galactosidase and its complex enzyme in galactomannan degradation |
-
2019
- 2019-03-13 CN CN201910187579.XA patent/CN109917032B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1114358A (en) * | 1990-03-14 | 1996-01-03 | 加拿大国家研究院 | Isolation, quantitation and purification of insecticidal proteins from endotoxins of bacillus thuringiensis |
| CN101206226A (en) * | 2007-10-25 | 2008-06-25 | 中国农业科学院蜜蜂研究所 | Preparation and application of test paper for testing royal jelly freshness |
| CN102094038A (en) * | 2010-12-08 | 2011-06-15 | 上海市农业科学院 | Prokaryotic expression and purification method of transgenic rice Bt protein crylc |
| CN102094039A (en) * | 2010-12-08 | 2011-06-15 | 上海市农业科学院 | Prokaryotic expression and purification method of transgenic rice Bt protein cry2a |
| CN103616454A (en) * | 2013-12-06 | 2014-03-05 | 浙江贝因美科工贸股份有限公司 | Method and kit for quantitatively detecting human beta-casein content |
| CN106852147A (en) * | 2014-10-16 | 2017-06-13 | 孟山都技术有限公司 | Lepidopteran-active Cry1Da1 variant amino acid sequence albumen |
| CN105296522A (en) * | 2015-12-01 | 2016-02-03 | 中国农业科学院植物保护研究所 | Bt protein expression system |
| CN109097348A (en) * | 2018-07-26 | 2018-12-28 | 天津科技大学 | The application of alpha-galactosidase and its complex enzyme in galactomannan degradation |
Non-Patent Citations (4)
| Title |
|---|
| Comparative Toxicity of Bacillus thuringiensis var. israelensis Crystal Proteins in aiuo and in tritvo;CHRIS N. CHILCOTT et al;《Journal of General Microbiology》;19881231;第134卷;2551-2558 * |
| Petition for Determination of Nonregulated Status for Insect-Resistant and Glufosinate Ammonium-Tolerant cotton;Alejandra L. Scott;《Bayer CropScience》;20081204;1-205 * |
| 苏云金芽孢杆菌杀虫晶体蛋白基于反相液相色谱分离的蛋白质组学研究;陈靖;《中国优秀硕士论文全文数据库 工程科技1辑》;20150615(第6期);B014-291 * |
| 转基因植物蛋白量值溯源核心技术及标准物质研究;毕佳明;《中国优秀硕士学位论文全文数据库 基础科学辑》;20121015(第10期);A006-448 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109917032A (en) | 2019-06-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108519485B (en) | Mass spectrum detection method of A1/A2 beta-casein | |
| Kurian et al. | Proteomic characterization of acid stress response in Synechocystis sp. PCC 6803 | |
| CN112225782B (en) | Specific peptide segment and method for determining content of structural protein in COVID-19 vaccine | |
| CN101042403A (en) | ELISA kit for detecting EPSPS gene in herbicide-tolerance soybeans and method of use thereof | |
| CN109917032B (en) | Quantitative detection method for Bt protein in Bt protein-transgenic food | |
| CN108469495A (en) | A method of detecting fish parvalbumin using Liquid Chromatography-Tandem Mass Spectrometry | |
| US8545773B2 (en) | Versatile multichannel capillary biosensor system | |
| CN108841828B (en) | Single-stranded DNA aptamer for specifically recognizing tobramycin and application thereof | |
| CN110672732B (en) | Determination method of active protein in pertussis toxin product and pertussis vaccine | |
| CN110850002B (en) | Method for quantifying honey MRJP2 in liquid chromatography tandem mass spectrometry | |
| CN114324626B (en) | Characteristic peptide segment and method for detecting content of spike protein of novel coronavirus | |
| CN114539362B (en) | Botulinum toxin specific substrate peptide, detection kit and detection method | |
| CN104987361B (en) | Polypeptide specifically bound with benzothiostrobin antibody and application thereof | |
| CN116804047A (en) | Method for detecting S protein content in novel coronavirus vaccine | |
| JP7502271B2 (en) | Compositions and methods for protein detection | |
| Zhang et al. | Quantification of Cry1Ab in genetically modified maize leaves by liquid chromatography multiple reaction monitoring tandem mass spectrometry using 18 O stable isotope dilution | |
| CN109142757A (en) | A kind of brucella indirect ELISA testing kit | |
| CN107817311B (en) | A kind of method that LC-MS detects casein phosphopeptide content in formula milk | |
| KR20210117268A (en) | High-Speed Sample Workflow for LC-MS-Based HBA1C Instrumentation | |
| CN114524863B (en) | Polypeptide sequence for quantitatively detecting content of G protein in rabies vaccine based on mass spectrometry and application of polypeptide sequence | |
| Chernukha et al. | Qualimetric assessment of methods for the determination of allergens in meat products | |
| CN113461778B (en) | Buffalo milk characteristic peptide and buffalo milk identification method | |
| Tang et al. | Detection of pesticide residues in vegetables by smartphone. | |
| CN118243763A (en) | Optimized CE-SDS purity detection method | |
| CN116930391A (en) | Screening method of characteristic peptide fragments for quantitative detection of proteins |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address |
Address after: Room 1203-3, Building 4, Chuangzhi Green Valley Development Center, No. 788, Hongpu Road, Shangcheng District, Hangzhou City, Zhejiang Province, 310000 Patentee after: Hangzhou Dad Evaluation Technology Co.,Ltd. Address before: 310000 room c2-701, building C, No. 459, Qianmo Road, Xixing street, Binjiang District, Hangzhou City, Zhejiang Province Patentee before: HANGZHOU DADDYLAB TECHNOLOGY Co.,Ltd. |
|
| CP03 | Change of name, title or address |