CN109917032A - A kind of quantitative detecting method turning Bt albumen in Bt protein food - Google Patents
A kind of quantitative detecting method turning Bt albumen in Bt protein food Download PDFInfo
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Abstract
The invention discloses a kind of quantitative detecting methods for turning Bt albumen in Bt protein food, it is digested using trypsase to Bt protein food is turned, the specific polypeptide in polypeptide for selecting Bt proteolysis to generate is as marker, the artificial synthesized specific polypeptide is as standard substance, the specific polypeptide of isotope labelling is designed and synthesized as internal standard, quantitative detection is carried out to specific polypeptide using isotopic dilution liquid chromatography tandem mass spectrometry, then convert to obtain Bt protein content.Stability of the present invention is good, quantitatively accurate, reproducible, specific height, applicability are wide.
Description
Technical field
The present invention relates to technical field of food detection, in particular to a kind of quantitative detection for turning Bt albumen in Bt protein food
Method.
Background technique
As increasingly maturation, the transgene agricultural product food industriesization of transgenic technology have showed the preferable impetus.Turn
For gene agricultural product food while bringing great economic benefit and social benefit to the mankind, there is also potential environment and food
Security risk.Consider for transgene agricultural product foodsafety, the country such as China, European Union, Japan, South Korea and tissue are numerous and confused
It formulates corresponding laws and rules requirement to be identified transgene agricultural product food and processed food, to be good for people, animal
Health and ecological environment diversity are protected.Since transgene agricultural product food and products thereof can to human health and ecological environment
There can be potential adverse effect, before whether transgene agricultural product food and products thereof is harmful without final conclusion, transgenosis agriculture
The detection of product food and products thereof is very important.
At present about there are mainly two types of the detection methods of transgene agricultural product food: detection method and base based on protein
In the detection method of nucleic acid.
Detection method based on protein is normally based on the side such as detection method of antigen-antibody, including ELISA, test strips
Method.But there is false positive, false negative, quantitative result inaccuracy in above-mentioned technology.Due to agricultural product food mesostroma
Complexity is very easy to cause antibody that the similar stroma protein of structure is accidentally identified as Bt protein, leads to false positive.In food
In process, especially in hot procedure, the folding of protein can change, and cause antibody that can not correctly identify albumen
Matter leads to false negative.The antibody of different batches is different from different Bt protein affinities with sample antibody, leads to quantitative knot
The inaccuracy of fruit.
Detection method based on nucleic acid refers mainly to round pcr, the nucleosides using specific primer to the foreign gene being transferred to
Acid sequence is expanded.According to amplified production with and without, it is more, less judge.This method can not carry out accurate quantitative analysis.Meanwhile
The loss and destruction that will lead to DNA in food processing process, influence the correctness of result.
Summary of the invention
It is an object of the invention to solve defect existing for existing detection method, one kind is provided and turns Bt egg in Bt protein food
White quantitative detecting method.
The technical solution adopted by the present invention to solve the technical problems is:
A kind of quantitative detecting method turning Bt albumen in Bt protein food is carried out using trypsase to Bt protein food is turned
Enzymatic hydrolysis, the specific polypeptide in polypeptide for selecting Bt proteolysis to generate are made as marker, the artificial synthesized specific polypeptide
For standard substance, the specific polypeptide of isotope labelling is designed and synthesized as internal standard, using isotopic dilution liquid chromatogram string
Join mass spectrography and quantitative detection is carried out to specific polypeptide, then convert to obtain Bt protein content.
The present invention first uses enzyme to digest Bt albumen, selects specific enzymolysis polypeptide as marker, artificial synthesized
The specific polypeptide carries out quantitative detection as standard substance, using isotopic dilution liquid chromatography tandem mass spectrometry, is to be based on
The detection method of specific polypeptide, although the space structure of protein is easy to be destroyed by food processing, primary structure is (more
Peptide) stability is very good, therefore can influence to avoid food processing process to testing result.
Using trypsase to turn Bt protein food carry out enzymatic hydrolysis concrete operations it is as follows: accurately weigh 0.1g sample in 2ml
EP pipe, is added 900mg ammonium bicarbonate soln, adds 10mg DTT, it is anti-to add the dark place 30mg IAA by 50 DEG C of water-bath 30min
30min is answered, 10mg trypsase is eventually adding, is taken out after 37 DEG C of water enzyme digestion 4h, enzymatic hydrolysis, 10% first of 20mg is added
Acid terminates reaction, is eventually adding artificial synthesized isotope labelling specific polypeptide 10mg and mixes.
Ammonium bicarbonate soln concentration is 50mM, and DTT concentration is 100mM, and IAA concentration is 100mM, and trypsinase concentration is
500mM, artificial synthesized isotope labelling specific polypeptide concentration are 1000 μ g/kg.
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry1 family are as follows: EKLEWETNIVYK；
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry2 family are as follows: TYMFLNVFEYVSIWSLFK.
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry1 family and Cry2 family simultaneously are as follows:
TFEAEYDLERA。
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry3 family are as follows: IRELFSQAESHFR；
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry4 family are as follows: IDESKLKPYTR；
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry5 family are as follows: NLEKGINAGTYSK；
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry9 family are as follows: KMLLEAVR.
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry3 family, Cry4 family and Cry5 family simultaneously
Are as follows: MLALSNRMQK.
The chromatographic condition of isotopic dilution liquid chromatography tandem mass spectrometry are as follows: use C18 analysis of protein column, A phase is 0.1%
Aqueous formic acid, B phase are 0.1% formic acid acetonitrile solution；Elution requirement be B phase 0-9min from 3% to 30%, 9-10min from
30% to 40%, 10.1min are raised to 100% and remain to 10.6min, and 10.7min returns to 3% and remains to 13min.Total elution time
For 13min, flow velocity 0.3mL/min.
The Mass Spectrometry Conditions of isotopic dilution liquid chromatography tandem mass spectrometry are as follows: capillary voltage: 3.0kv, desolventizing temperature:
550 DEG C, desolventizing gas flow: 400L/min, cone hole backflow airflow amount: 30L/hr collides chamber pressure: 3.0 × 10-3mbar；It is low
Hold resolution ratio 1:2.5V, high-end resolution ratio 1:15.0V, ion energy 1:0.6eV；Low side resolution ratio 2:2.0V, high-end resolution ratio
2:15.0V, ion energy 2:2.0eV；Ion source temperature: 150 DEG C, extractor voltage: 5.0V, entrance lens voltage: 10V, out
Mouth voltage: 10V.
A kind of quantitative detection box turning Bt albumen in Bt protein food, including trypsase, ammonium bicarbonate soln, DTT,
IAA, 10% formic acid and the specific polypeptide for detecting Bt albumen,
Specific polypeptide for detecting Bt albumen is specific as follows:
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry1 family are as follows: EKLEWETNIVYK；
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry2 family are as follows: TYMFLNVFEYVSIWSLFK；
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry1 family and Cry2 family simultaneously are as follows:
TFEAEYDLERA；
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry3 family are as follows: IRELFSQAESHFR；
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry4 family are as follows: IDESKLKPYTR；
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry5 family are as follows: NLEKGINAGTYSK；
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry9 family are as follows: KMLLEAVR；
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry3 family, Cry4 family and Cry5 family simultaneously
Are as follows: MLALSNRMQK.
The beneficial effects of the present invention are:
1, stability is good: although the space structure of protein is easy to be destroyed by food processing, primary structure is stablized
Property is very good.
2, quantitative accurate: isotopic dilution liquid chromatography tandem mass spectrometry is the gold standard of quantitative detection, as a result accurately
Reliably.
3, reproducible: using chemically synthesized polypeptide as standard substance, can to synthesize in batches, quality is stablized.
4, specificity is high: selecting polypeptide as standard substance, polypeptide has genetic code feature, can be by target polypeptides very
It is distinguished with matrix polypeptide well.
5, applicability is wide: Bt albumen not instead of single protein, general name (Bt Cry1Aa, Bt of a big albuminoid
Cry1Ab, Bt Cry1Ac, Bt Cry1Ah, Bt Cry1B, Bt Cry1C, Bt Cry1F, Bt Cry2A, Bt Cry3B and Bt
Cry9C albumen), by selecting different polypeptides, a variety of Bt albumen can be detected with simultaneous quantitative, it can also be to each Bt albumen point
Not carry out quantitative detection, meet various testing requirements.
Detailed description of the invention
Fig. 1 is Bt protein thermal denaturation Test Drawing；
Fig. 2 is Cry1Aa genetically engineered soybean test map -1；
Fig. 3 is Cry1Aa genetically engineered soybean test map -2；
Fig. 4 is Cry1Aa genetically engineered soybean test map -3；
Fig. 5 is no genetically engineered soybean test map -1；
Fig. 6 is no genetically engineered soybean test map -2；
Fig. 7 is no genetically engineered soybean test map -3.
Specific embodiment
Below by specific embodiment, technical scheme of the present invention will be further explained in detail.
In the present invention, if not refering in particular to, used raw material and equipment etc. are commercially available or commonly used in the art.
Method in following embodiments is unless otherwise instructed the conventional method of this field.
It has had now been found that 100 or more Bt protein Pesticidal crystal protein, has passed through
Uniprot verifying just has 87, and homology has height to have low between them.Classified according to homology, this method is using several
A specific polypeptide is just able to satisfy various detection demands, and specific polypeptide (sequence containing isotope labelling) is synthesized by the raw work in Shanghai.
In following embodiment: ammonium bicarbonate soln concentration is 50mM, and DTT concentration is 100mM, and IAA concentration is 100mM, pancreas
Protease concentration is 500mM, and artificial synthesized isotope labelling specific polypeptide concentration is 1000 μ g/kg.
Embodiment 1: Cry1 family protein in detection soya-bean milk
1, it pre-processes
It accurately weighs 0.1g sample (soya-bean milk) to manage in 2ml EP, 900mg ammonium bicarbonate soln is added, adds 10mg
DTT, 50 DEG C of water-bath 30min add the dark place 30mg IAA reaction 30min, 10mg trypsase are eventually adding, in 37 DEG C of water-baths
4h is digested, is taken out after enzymatic hydrolysis, 10% formic acid of 20mg is added and terminates reaction, is eventually adding artificial synthesized isotope labelling
Specific polypeptide 10mg is mixed, and filters sample introduction.
2, liquid phase chromatogram condition
Reference conditions are as follows: using C18 analysis of protein column (1.7 μm of partial sizes, 2.1mm × 100mm).A phase is 0.1% formic acid
Aqueous solution, B phase are 0.1% formic acid acetonitrile solution；Elution requirement be B phase 0-9min from 3% to 30%, 9-10min from 30% to
40%, 10.1min are raised to 100% and remain to 10.6min, and 10.7min returns to 3% and remains to 13min.Always elution time is
13min, flow velocity 0.3mL/min.
3, Mass Spectrometry Conditions
Mass Spectrometer Method condition is as follows: capillary voltage: 3.0kv, desolventizing temperature: 550 DEG C, desolventizing gas flow: 400L/
Min, cone hole backflow airflow amount: 30L/hr collides chamber pressure: 3.0 × 10-3mbar；Low side resolution ratio 1:2.5V, high-end resolution
Rate 1:15.0V, ion energy 1:0.6eV；Low side resolution ratio 2:2.0V, high-end resolution ratio 2:15.0V, ion energy 2:2.0eV；
Ion source temperature: 150 DEG C, extractor voltage: 5.0V, entrance lens voltage: 10V, exit potential: 10V.
The Parameter Conditions of 1 mass spectrum multiple-reaction monitoring of table
Note: L* is isotope labelling [13C6,15N]-leucine.
According to the method for the present embodiment, the soya-bean milk and 2 genetically engineered soybeans for having inspected 2 Non-transgenic soybean production by random samples are made
Soya-bean milk, as a result see the table below:
Table 2
| Sample number | Type | Cry1 content [mg/kg] |
| 1 | Non-transgenic | 0 |
| 2 | Non-transgenic | 0 |
| 3 | Transgenosis | 0.28 |
| 4 | Transgenosis | 0.79 |
。
Embodiment 2: the Cry2 family protein in detection corn product
The parameter of this example 1-3 step is with embodiment 1, the difference is that the following contents:
The Parameter Conditions of 3 mass spectrum multiple-reaction monitoring of table
Note: L* is isotope labelling [13C6,15N]-leucine.
According to the method for the present embodiment, inspects 2 non-transgenic corns and 2 transgenic corns by random samples, as a result see the table below:
Table 4
| Sample number | Type | Cry2 content [mg/kg] |
| 1 | Non-transgenic | 0 |
| 2 | Non-transgenic | 0 |
| 3 | Transgenosis | 13.4 |
| 4 | Transgenosis | 2.0 |
。
Embodiment 3: while detecting the Cry1 in soybean and Cry2
The parameter of this example 1-3 step is with embodiment 1, the difference is that the following contents:
The Parameter Conditions of 5 mass spectrum multiple-reaction monitoring of table
Note: L* is isotope labelling [13C6,15N]-leucine.
According to the method for the present embodiment, 2 Non-transgenic soybeans and 2 genetically engineered soybeans is inspected by random samples, as a result see the table below
Table 6
| Sample number | Type | Cry1+Cry2 content [mg/kg] |
| 1 | Non-transgenic | 0 |
| 2 | Non-transgenic | 0 |
| 3 | Transgenosis | 14.3 |
| 4 | Transgenosis | 6.8 |
Specific polypeptide need to only be replaced with Cry3, Cry4, Cry5, Cry9 by the method reference implementation example 1 of embodiment 4-8
And the corresponding polypeptide of Cry3, Cry4 and Cry5 is detected.
Methodology validation (linear, precision, accuracy, sensitivity)
1, standard curve
Experiment purpose: the range of linearity of this method is verified
Experimental method
Accurate peptide fragment [EKLEWETNIVYK] standard intermediate solution for drawing different volumes: 4 μ L, 10 μ L, 20 μ L, 40 μ L,
60 μ L, 80 μ L, 100 μ L are separately added into the 10 special peptide standard solution of μ L isotope, then with 0.1% formic acid in sample introduction bottle
Aqueous solution is diluted to 1mL so that concentration be 400 μ g/kg, 1000 μ g/kg, 2000 μ g/kg, 4000 μ g/kg, 6000 μ g/kg,
The standard series working curve (1000 μ g/kg of Isotopic Internal Standard concentration) of 8000 μ g/kg, 10000 μ g/kg, sample introduction is analyzed, with mark
The concentration of quasi- solution is abscissa, using the ratio of the peak area of reference colour spectral peak and the peak area of internal standard chromatographic peak as ordinate into
Row linear fit, as shown in table 7.
7 standard curve of table is linear and regression equation
Experiment conclusion
The linear r of standard curve2It is all larger than 0.99, meets detection and relevant laws and regulations requirement.
2, Precision Experiment
Experiment purpose: the stability of experimental method is investigated.
Experimental method: taking a kind of soybean for being transferred to Cry1Ab, and parallel five measurements, repeat 4 days, pre-process (with reality daily
It is identical to apply example 1).
Table 8: day to day precision experimental result statistical form
Experiment conclusion
Experimental result RSD≤15% meets detection and relevant laws and regulations requirement.
3, accuracy/rate of recovery experiment
Experiment purpose: the accuracy of experimental result is investigated by the rate of recovery test to mark-on sample.
Experimental method: taking a kind of no transgenosis soya-bean milk, adds appropriate Cry1Aa standard protein respectively and makes sample mark-on
Concentration is respectively 500,2000,8000 μ g/kg, the sample of three concentration points each parallel five times, is repeated 4 days.Pretreatment is (with implementation
Example 1 is identical).
Experimental result: it see the table below
Table 9: rate of recovery experimental result statistical form
Experiment conclusion
The rate of recovery of three spiked levels is between 80-120% according to the experimental results, and relative standard deviation RSD <
15%, meet detection and relevant laws and regulations requirement.
4, detection limit
8 parts are weighed without transgenosis soya-bean milk sample, is added the Cry1Aa standard protein of proximity test, sample preprocessing (with
Embodiment 1 is identical) it detects afterwards, calculated result and standard deviation, with the coefficient of variation f (n) and standard when confidence level is 95%
The product of deviation s limits (LOD) as the detection of method, and the results are shown in Table 10, and the detection of this method standard protein is limited to 55.7 μ
G/kg meets the measurement demand of daily sample.
The detection limit of table 10
Note: LOD=s × f (n)；Coefficient of variation when f (n) is confidence level 95%, tables look-up；As n=8, f (8)=
3.18。
5, stability verifying [heat denaturation experiment]
Heat denaturation experiment
The generation of thermal denaturation, most methods such as enzyme linked immunological are usually associated in the production process of soy protein products
Method etc. can not detect the Bt albumen because of heat denatured, and our rule can measure non denatured and thermal denaturation Bt albumen.To say
Then this bright problem, a transgenosis soya-bean milk sample are respectively placed at 0,10,20,30,40,50,60,70,80,90 DEG C
After constant temperature places 1h, carry out processing detection by this method and enzyme-linked immunization respectively, as a result as shown in Figure 1, with temperature raising,
Enzyme-linked immunization acquired results gradually decrease, until as a result reducing more than 50% at 80 DEG C；And with this method, testing result not with
The raising of temperature and reduce；And it is not heating or low-temperature space, the result of the two are comparable.It can be seen that this law measurement is big
Bt albumen result is more accurate in beans.
6, specific
Multiple soybean for being transferred to Cry1Aa are detected, all detection (Fig. 2-4)；Multiple no genetically engineered soybeans, are all not detected (Fig. 5-
7), it is seen that method high specificity of the invention.
Above-mentioned embodiment is only a preferred solution of the present invention, not the present invention is made in any form
Limitation, there are also other variations and modifications on the premise of not exceeding the technical scheme recorded in the claims.
SEQUENCE LISTING
<110>Hangzhou daddy evaluates and tests Science and Technology Ltd.
<120>a kind of quantitative detecting method for turning Bt albumen in Bt protein food
<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
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Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His Phe Arg
1 5 10
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<213>artificial sequence (Artificial sequence)
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Ile Asp Glu Ser Lys Leu Lys Pro Tyr Thr Arg
1 5 10
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Asn Leu Glu Lys Gly Ile Asn Ala Gly Thr Tyr Ser Lys
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Lys Met Leu Leu Glu Ala Val Arg
1 5
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Met Leu Ala Leu Ser Asn Arg Met Gln Lys
1 5 10
Claims (10)
1. a kind of quantitative detecting method for turning Bt albumen in Bt protein food, it is characterised in that: using trypsase to turning Bt egg
Food and drinks one gets without pay product are digested, and the specific polypeptide in polypeptide for selecting Bt proteolysis to generate is as marker, the artificial synthesized spy
Specific polypeptide designs and synthesizes the specific polypeptide of isotope labelling as internal standard, using isotopic dilution as standard substance
Liquid chromatography tandem mass spectrometry carries out quantitative detection to specific polypeptide, and then convert to obtain Bt protein content.
2. quantitative detecting method according to claim 1, it is characterised in that: using trypsase to turn Bt protein food into
Row enzymatic hydrolysis concrete operations are as follows: accurately weighing 0.1g sample and manage in 2ml EP, 900mg ammonium bicarbonate soln is added, adds
10mg DTT, 50 DEG C of water-bath 30min add the dark place 30mg IAA reaction 30min, 10mg trypsase are eventually adding, 37
DEG C water enzyme digestion 4h takes out after enzymatic hydrolysis, 10% formic acid of 20mg is added and terminates reaction, is eventually adding artificial synthesized same position
Element label specific polypeptide 10mg is mixed.
3. quantitative detecting method according to claim 2, it is characterised in that: ammonium bicarbonate soln concentration is 50mM, and DTT is dense
Degree is 100mM, and IAA concentration is 100mM, trypsinase concentration 500mM, and artificial synthesized isotope labelling specific polypeptide is dense
Degree is 1000 μ g/kg.
4. quantitative detecting method according to claim 1, it is characterised in that:
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry1 family are as follows: EKLEWETNIVYK；For detecting Bt
The amino acid sequence of the specific polypeptide of PROTEIN C ry2 family are as follows: TYMFLNVFEYVSIWSLFK.
5. quantitative detecting method according to claim 1, it is characterised in that: for simultaneously detect Bt PROTEIN C ry1 family and
The amino acid sequence of the specific polypeptide of Cry2 family are as follows: TFEAEYDLERA.
6. quantitative detecting method according to claim 1, it is characterised in that:
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry3 family are as follows: IRELFSQAESHFR；
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry4 family are as follows: IDESKLKPYTR；
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry5 family are as follows: NLEKGINAGTYSK；
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry9 family are as follows: KMLLEAVR.
7. quantitative detecting method according to claim 1, it is characterised in that: for simultaneously detect Bt PROTEIN C ry3 family,
The amino acid sequence of the specific polypeptide of Cry4 family and Cry5 family are as follows: MLALSNRMQK.
8. quantitative detecting method according to claim 1, it is characterised in that: isotopic dilution liquid chromatography tandem mass spectrometry
Chromatographic condition are as follows: use C18 analysis of protein column, A phase is 0.1% aqueous formic acid, and B phase is 0.1% formic acid acetonitrile solution；It washes
De- condition is B phase 0-9min from 3% to 30%, and from 30% to 40%, 10.1min is raised to 100% and remains to 9-10min
10.6min, 10.7min return to 3% and remain to 13min.Total elution time is 13min, flow velocity 0.3mL/min.
9. quantitative detecting method according to claim 1, it is characterised in that: isotopic dilution liquid chromatography tandem mass spectrometry
Mass Spectrometry Conditions are as follows: capillary voltage: 3.0kv, desolventizing temperature: 550 DEG C, desolventizing gas flow: 400L/min, taper hole blowback
Throughput: 30L/hr, collision chamber pressure: 3.0 × 10-3mbar；Low side resolution ratio 1:2.5V, high-end resolution ratio 1:15.0V, ion
Energy 1:0.6eV；Low side resolution ratio 2:2.0V, high-end resolution ratio 2:15.0V, ion energy 2:2.0eV；Ion source temperature: 150
DEG C, extractor voltage: 5.0V, entrance lens voltage: 10V, exit potential: 10V.
10. a kind of quantitative detection box for turning Bt albumen in Bt protein food, it is characterised in that: including trypsase, ammonium hydrogen carbonate
Solution, DTT, IAA, 10% formic acid and the specific polypeptide for detecting Bt albumen,
Specific polypeptide for detecting Bt albumen is specific as follows:
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry1 family are as follows: EKLEWETNIVYK；
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry2 family are as follows: TYMFLNVFEYVSIWSLFK；
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry1 family and Cry2 family simultaneously are as follows:
TFEAEYDLERA；
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry3 family are as follows: IRELFSQAESHFR；
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry4 family are as follows: IDESKLKPYTR；
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry5 family are as follows: NLEKGINAGTYSK；
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry9 family are as follows: KMLLEAVR；
For detecting the amino acid sequence of the specific polypeptide of Bt PROTEIN C ry3 family, Cry4 family and Cry5 family simultaneously are as follows:
MLALSNRMQK。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111323511A (en) * | 2020-03-26 | 2020-06-23 | 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 |
| CN111323512A (en) * | 2020-03-26 | 2020-06-23 | 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 |
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| 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 |
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