CN107090500A - A kind of method of utilization rpsL gene mutation rates check and evaluation food contaminant genetoxic - Google Patents
A kind of method of utilization rpsL gene mutation rates check and evaluation food contaminant genetoxic Download PDFInfo
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Abstract
The present invention relates to a kind of method of utilization rpsL gene mutation rates check and evaluation food contaminant genetoxic, it is characterised in that：Including extracting plasmid pMOL21, plasmid is transferred to host cell, food contaminant processing, the detection of gene mutation rate, food contaminant culture and the several steps of the assessment of food contaminant genetoxic, detected simultaneously using the positive screening system of rpsL gene mutation rates, it is a kind of sensitivity height and the external short-term mutagenic test method of easy-to-use, a kind of fast and effectively analysis method can be provided for the potential carcinogen produced in detection food processing process, should the genetoxic data that be obtained in this way by be food noxious material data bank strong supplement, there is important theoretical direction value to formulating food security regulation and standard.
Description
Technical field
The invention belongs to field of detection of food safety, it is related to the assessment of food contaminant genetoxic, especially a kind of profit
With the method for rpsL gene mutation rate check and evaluation food contaminant genetoxics.
Background technology
Food contaminant is to bring the polluter in food, aspergillus flavus into during production, processing, transport and storage etc.
Toxin, acrylamide, decis, dioxin and dioxin Polychlorinated biphenyls etc., food contaminant is it has been investigated that be respectively provided with
Mutagenicity, in food safety evaluation on toxicology, genetic toxicity test is tested frequently with bacterial mutation, i.e., mouse typhus is copied
Door Salmonella/experiment of mammal microsomal enzyme, i.e. Salmonella reversion test
Salmonella reversion test is widely used in the primary dcreening operation of mutagenesis chemicals, but still suffers from following ask in actual applications
Topic：
1st, Ames tests, which can only be detected, causes the change of a few base to cause mutation, and such as base substitution mutation, frameshit are dashed forward
The mutagen of change, and trigger the mutagen being mutated for causing big section of missing of DNA fracture and chromosome, then it can not examine
Their mutagenicity is surveyed, with certain limitation；
2nd, Ames testing experiments program is comparatively laborious, and method is not easy enough, awaits creating new more rapid sensitive, letter
Single easy short-term biological test method of mutagens.
Do not find that food pollution is being assessed in the detection of rpsL gene mutation rates by the retrieval to existing patent document
The application of thing genetoxic, i.e., influence the novelty and creativeness of the present invention as prior art without disclosed patent document.
The content of the invention
It is an object of the invention to overcome the deficiencies in the prior art part to be detected there is provided one kind using rpsL gene mutation rates
The method for assessing food contaminant genetoxic, this method sensitivity is high and simple and easy to do, can determine residues of pesticides, chemistry dirty
The genetoxic of many food contaminants such as thing, microbial toxin is contaminated, the genetoxic data obtained using this technology will
It is the strong supplement of food noxious material data bank, there is most important theories guiding value to formulating food security regulation and standard.
The present invention solves its technical problem and takes following technical scheme to realize：
A kind of method of utilization rpsL gene mutation rates check and evaluation food contaminant genetoxic, this method includes as follows
Step：
(1) plasmid pMOL21 is extracted：It is 3993bp by molecular weight, the rpsL gene orders with 375bp, and with ammonia benzyl
The plasmid pMOL21 of ampicillin resistance gene extracts the DNA by alkaline lysis or plasmid extraction kit；
(2) plasmid is transferred to host cell：Using CaCl2Method prepares MF101 competent cells, and pMOL21 plasmids are converted
To competent cell, host cell is sensitive to streptomysin after conversion；
(3) food contaminant is handled：Food liquid pollutant is directly detected that solid-state food pollutant is configured to molten
Liquid, adds water according to the property of different food products pollutant and organic solvent is dissolved, and carry out gradient dilution；
(4) detection of gene mutation rate：Escherichia coli MF101 with plasmid pMOL21 is cultivated to exponential phase,
OD600 is 0.5-0.7, with 0.5-1.5：100 are diluted in the fluid nutrient medium containing food contaminant, in 36-38 DEG C of progress
Shaken cultivation, and with the growth curve of the absorbance value measure cell at 550-650nm；
(5) food contaminant culture：Food contaminant is added in culture medium, and each group is used for after cultivating 10 hours
The measure of the rpsL frequencies of mutation, inoculum is coated on the flat board containing ampicillin to determine the total of transformed cells
Number, coated plate on containing ampicillin and streptomysin flat board to determine the sum of rpsL gene mutations, mutation rate be mutation bacterium number/
Total bacteria count, wherein mutation bacterium number of the bacterium number containing ampicillin and streptomysin flat board, bacterium of the total bacteria count containing ampicillin plate
Number, every group of at least three times repetition, total clump count is not less than 103It is individual；
(6) assessment of food contaminant genetoxic：RpsL gene mutations clone is chosen, DNA is extracted, rpsL is determined
Mutant nucleotide sequence, sequence alignment is carried out using DNAman softwares, and base replacement, frameshift mutation and large fragment are carried out for mutant nucleotide sequence
Miss statistics are analyzed, and determine the main Types of the frequency of mutation, gene mutation focus and gene mutation caused by food contaminant.
Moreover, the Escherichia coli MF101 with plasmid pMOL21 is cultivated to exponential phase in step (4), OD600 is
0.6, with 1：100 are diluted in the fluid nutrient medium containing food contaminant, are carried out in 37 DEG C at shaken cultivation, and 600nm
Absorbance value determine cell growth curve.
Moreover, the flat board in step (5) containing ampicillin and the concentration containing ampicillin and streptomysin flat board are 50 μ
g/mL。
Moreover, the detection of step (4) gene mutation rate also has another external detection method, this method step is such as
Under：
A uses the plasmid extraction kit with rpsL genes to extract plasmid pMOL21, utilizes ScaI linearization for enzyme restriction matter
Grain, and enter in performing PCR reaction, amplification in vitro plasmid pMOL21, PCR reaction system to add food using the primer of biotin labeling
Pollutant；
DNA cloning product by purifying, digestion, from coupled reaction, is transformed into host cell MF101 by b, blue or green using ammonia benzyl
Mycin and streptomysin screening rpsL gene mutation bacterium colonies；
C carries out data statistics, and calculates rpsL gene mutation rates, and mutation rate is mutation bacterium number/total bacteria count, wherein mutant bacteria
Bacterium number of the number containing ampicillin and streptomysin flat board, bacterium number of the total bacteria count containing ampicillin plate, every group of at least three times weights
Multiple, total clump count is not less than 106It is individual；
Moreover, described food contaminant refers to bring the pollution in food into production, processing, transport and storage
Material, mainly including the one or more in residues of pesticides, chemical pollutant, microbial toxin.
Advantages of the present invention effect：
1st, the present invention is screened using rpsL gene mutations and streptomycin resistance, while being sieved with the forward direction of rpsL gene mutation rates
System is selected to be detected, positive screening system refers to that the bacterial strain of only plasmid rpsL gene mutations can be detected, and plasmid is not
The bacterial strain of mutation must not be detected, and because streptomycin resistance is recessive genetic phenotype, Host Strains MF101 rpsL genes are mutation
, only plasmid pMOL21 rpsL genes possess streptomycin resistance while being induced mutation and can just ultimately result in host cell,
So as to reach the purpose of rpsL mutators in positive screening plasmid, forward direction screening can greatly improve detection time, entirely
Analysis process can realize in 24 hours,
2nd, rpsL genetic analysis background of the present invention is relatively low, and the method for being related to microorganism high-temperature cultivation is simply easy
OK, the evaluation for carrying out mutagenicity using rpsL forward selection systems does not need complicated experiment condition, and the spontaneous of TK genes dashes forward
Variability is 10-5, and rpsL spontaneous mutation rate as little as 10-6, far below similar positive screening system, use manpower and material resources sparingly, detect
Rapidly and efficiently.
3rd, the present invention uses internal level gene mutation rate to detect, utilizes the MF101 bacterial strains of rpsL detection in Gene Mutation
Ames experiments are carried out instead of the histidine auxotroph bacterial strain of salmonella typhimurium, mammal microsome enzyme system is added,
The deficiency that experiment in vitro lacks metabolism activation system is made up, and using the mutagenicity of flat board infiltration method detection food contaminant.
4th, rpsL gene mutations involved in the present invention selection system is that a kind of sensitivity is high and easy-to-use is short in vitro
Phase mutagenic test method, can provide one kind for the potential carcinogen produced in detection food processing process and fast and effectively divide
Analysis method, rpsL detection in Gene Mutation technologies are the duplication fidelities based on rpsL genes quantitatively to detect the side of genetoxic
Method, the genetoxic data obtained using this technology by be food noxious material data bank strong supplement, to formulate eat
Product security legislation and standard have most important theories guiding value.
Embodiment
Below by specific embodiment, the invention will be further described, and following examples are descriptive, is not limit
Qualitatively, it is impossible to which protection scope of the present invention is limited with this.
The present invention provides a kind of method of utilization rpsL gene mutation rates check and evaluation food contaminant genetoxic, the party
Method comprises the following steps：
(1) plasmid pMOL21 is extracted：It is 3993bp by molecular weight, the rpsL gene orders with 375bp, and with ammonia benzyl
The plasmid pMOL21 of ampicillin resistance gene extracts the DNA by alkaline lysis or plasmid extraction kit；
(2) plasmid is transferred to host cell：Using CaCl2Method prepares MF101 competent cells, and pMOL21 plasmids are converted
To competent cell, host cell is sensitive to streptomysin after conversion；
(3) food contaminant is handled：Food liquid pollutant is directly detected that solid-state food pollutant is configured to molten
Liquid, adds water according to the property of different food products pollutant and organic solvent is dissolved, and carry out gradient dilution；
(4) detection of gene mutation rate：Escherichia coli MF101 with plasmid pMOL21 is cultivated to exponential phase,
OD600 is 0.6, with 1：100 are diluted in the fluid nutrient medium containing food contaminant, and shaken cultivation is carried out in 37 DEG C, and
Absorbance value at 600nm determines the growth curve of cell；
(5) food contaminant culture：Food contaminant is added in culture medium, and each group is used for after cultivating 10 hours
The measure of the rpsL frequencies of mutation, inoculum is coated on the flat board containing ampicillin to determine the total of transformed cells
Number, coated plate on containing ampicillin and streptomysin flat board to determine the sum of rpsL gene mutations, mutation rate be mutation bacterium number/
Total bacteria count, wherein mutation bacterium number of the bacterium number containing ampicillin and streptomysin flat board, bacterium of the total bacteria count containing ampicillin plate
Number, every group of at least three times repetition, total clump count is not less than 103Individual, the flat board of ampicillin is with containing ampicillin and strepto-
The concentration of plain flat board is 50 μ g/mL.
(6) assessment of food contaminant genetoxic：RpsL gene mutations clone is chosen, DNA is extracted, rpsL is determined
Mutant nucleotide sequence, sequence alignment is carried out using DNAman softwares, and base replacement, frameshift mutation and large fragment are carried out for mutant nucleotide sequence
Miss statistics are analyzed, and determine the main Types of the frequency of mutation, gene mutation focus and gene mutation caused by food contaminant.
The detection of gene mutation rate also has another external detection method, and this method step is as follows：
A uses the plasmid extraction kit with rpsL genes to extract plasmid pMOL21, utilizes ScaI linearization for enzyme restriction matter
Grain, and enter in performing PCR reaction, amplification in vitro plasmid pMOL21, PCR reaction system to add food using the primer of biotin labeling
Pollutant；
DNA cloning product by purifying, digestion, from coupled reaction, is transformed into host cell MF101 by b, blue or green using ammonia benzyl
Mycin and streptomysin screening rpsL gene mutation bacterium colonies；
C carries out data statistics, and calculates rpsL gene mutation rates, and mutation rate is mutation bacterium number/total bacteria count, wherein mutant bacteria
Bacterium number of the number containing ampicillin and streptomysin flat board, bacterium number of the total bacteria count containing ampicillin plate, every group of at least three times weights
Multiple, total clump count is not less than 106It is individual；
Food contaminant described above refers to bring the pollution in food into production, processing, transport and storage
Material, mainly including the one or more in residues of pesticides, chemical pollutant, microbial toxin.
The present invention relates to the principle of method：
The present invention is undergone mutation in the cell or in extracellular duplication by rpsL genes, causes microbial cell strepto-
The character mutation of plain resistance, determining food contaminant influences the frequency of mutation of rpsL genes, and determines to dash forward by the method for sequencing
Deformation type, so that the purpose of the assessment food contaminant genetoxic reached.It is of the invention mainly to utilize rpsL gene mutation rates just
To screening system, the rpsL gene mutation rates that food contaminant triggers in vivo and in vitro are determined, are that a kind of sensitivity is high and easy
Easy external short-term mutagenesis appraisal procedure, can determine many food such as residues of pesticides, chemical pollutant, microbial toxin
The genetoxic of pollutant.
Wherein, mainly can be with strepto- by the ribosomal protein S1 2 of rpsL gene codes using the resistance of rpsL genes
Plain (Sm, streptomycin) formation compound, and strengthen the ability that streptomysin is combined with bacterial 16 S rRNA, so as to prevent
The starting of transcription, here it is the basis of streptomysin pharmacological action.If rpsL genes are undergone mutation, by destruction 16S rRNA and strepto-
The interaction of element, so as to cause microbial cell to produce drug resistance to streptomysin.
Positive screening system refers to that the bacterial strain of only plasmid rpsL gene mutations can be detected, and the unmutated bacterium of plasmid
Strain must not be detected.Because streptomycin resistance is recessive genetic phenotype, Host Strains MF101 rpsL genes are mutation, only
Plasmid pMOL21 rpsL genes possess streptomycin resistance while being induced mutation and can just ultimately result in host cell, so as to reach
The purpose of rpsL mutators in forward direction screening plasmid.
The histidine auxotroph bacterium of salmonella typhimurium is replaced using the MF101 bacterial strains of rpsL detection in Gene Mutation
Strain carries out Ames experiments, adds mammal microsome enzyme system, makes up experiment in vitro and lacks the deficiency of metabolism activation system, and adopts
The mutagenicity of food contaminant is detected with flat board infiltration method.
Embodiment 1
Influence of the nano material to rpsL gene mutation rates under the conditions of being determined in vivo using rpsL, is assessed food contaminant and received
The genetoxic of meter level food fresh keeping material.
This experiment chooses two kinds of nanoscale food fresh keeping materials and carries out mutation rate detection, and nano material adds culture medium, and
Add mammal microsome enzyme system, the induced mutation frequency in detection Escherichia coli MF101/pMOL21 rpsL sites.With molten
Agent group as a control group, every group of at least three times repetition, and detect using flat board infiltration method the mutagenicity of food contaminant, rpsL
Gene mutation rate the results are shown in Table 1, the wherein mutation rate 3.56 × 10 of material 1-6Higher than control group 1.29 × 10-6。
Under the conditions of in the body of table 1., influence of two kinds of nanoscale food fresh keeping materials to rpsL gene mutation rates
Embodiment 2
Influence of the nano material to rpsL gene mutation rates under conditions in vitro is determined using rpsL, food contaminant is assessed and receives
The genetoxic of meter level food fresh keeping material.
This experiment chooses two kinds of nanoscale food fresh keeping materials and carries out mutation rate detection, carries out outer-gene amplification plasmid
PMOL21, with solvent group as a control group, every group of at least three times repetition.Amplified production is attached and converted host cell
MF101, counts rpsL gene mutation rates, and mutation rate the results are shown in Table 2, and the mutation rate of nano material 2 is higher than control group for 2.64%.
Under the conditions in vitro of table 2., influence of two kinds of nanoscale food fresh keeping materials to rpsL gene mutation rates
Embodiment 3
Under the conditions of being determined in vivo using rpsL, food contaminant is assessed in influence of the acrylamide to rpsL gene mutation rates
The genetoxic of acrylamide.
The acrylamide of various concentrations is added in culture medium by this experiment, and each group is cultivated to be used to carry out after 10 hours
Culture medium flat plate is coated with, and is determined induction of the acrylamide of various concentrations to Escherichia coli MF101/pMOL21 rpsL sites and is dashed forward
Frequency.With solvent distilled water as a control group, total bacteria count is not less than 10-6, acquired results as shown in table 3, the third of gradient concentration
The frequency of mutation under acrylamide has certain dose-response relationship, and 1mg/mL acrylamide mutation rate reaches 28.11 × 10-6, far above control group 1.85 × 10-6。
Under the conditions of in the body of table 3., influence of the acrylamide to rpsL gene mutation rates
Claims (5)
1. a kind of method of utilization rpsL gene mutation rates check and evaluation food contaminant genetoxic, it is characterised in that：The party
Method comprises the following steps：
(1) plasmid pMOL21 is extracted：It is 3993bp by molecular weight, the rpsL gene orders with 375bp, and with ammonia benzyl mould
The plasmid pMOL21 of plain resistant gene extracts the DNA by alkaline lysis or plasmid extraction kit；
(2) plasmid is transferred to host cell：Using CaCl2Method prepares MF101 competent cells, and pMOL21 plasmids are converted to sense
Intracellular by state, host cell is sensitive to streptomysin after conversion；
(3) food contaminant is handled：Food liquid pollutant is directly detected that solid-state food pollutant is configured to solution, root
Water is added according to the property of different food products pollutant and organic solvent is dissolved, and carries out gradient dilution；
(4) detection of gene mutation rate：Escherichia coli MF101 with plasmid pMOL21 is cultivated to exponential phase, OD600
For 0.5-0.7, with 0.5-1.5：100 are diluted in the fluid nutrient medium containing food contaminant, and vibration training is carried out in 36-38 DEG C
Support, and with the growth curve of the absorbance value measure cell at 550-650nm；
(5) food contaminant culture：Food contaminant is added in culture medium, and each group is cultivated prominent for rpsL after 10 hours
The measure of Frequency, inoculum is coated on the flat board containing ampicillin to determine the sum of transformed cells, coated plate
To determine the sum of rpsL gene mutations on containing ampicillin and streptomysin flat board, mutation rate is mutation bacterium number/total bacteria count,
Wherein it is mutated bacterium number of the bacterium number containing ampicillin and streptomysin flat board, bacterium number of the total bacteria count containing ampicillin plate, every group
At least three times repetitions, total clump count is not less than 103It is individual；
(6) assessment of food contaminant genetoxic：RpsL gene mutations clone is chosen, DNA is extracted, rpsL mutation are determined
Sequence, sequence alignment is carried out using DNAman softwares, and base replacement, frameshift mutation and large fragment deletion are carried out for mutant nucleotide sequence
Statistical analysis, determines the main Types of the frequency of mutation, gene mutation focus and gene mutation caused by food contaminant.
2. a kind of side of utilization rpsL gene mutation rates check and evaluation food contaminant genetoxic according to claim 1
Method, it is characterised in that：The Escherichia coli MF101 with plasmid pMOL21 is cultivated to exponential phase in step (4), and OD600 is
0.6, with 1：100 are diluted in the fluid nutrient medium containing food contaminant, are carried out in 37 DEG C at shaken cultivation, and 600nm
Absorbance value determine cell growth curve.
3. a kind of side of utilization rpsL gene mutation rates check and evaluation food contaminant genetoxic according to claim 1
Method, it is characterised in that：Flat board in step (5) containing ampicillin and the concentration containing ampicillin and streptomysin flat board are 50
μg/mL。
4. a kind of side of utilization rpsL gene mutation rates check and evaluation food contaminant genetoxic according to claim 1
Method, it is characterised in that：The detection of step (4) gene mutation rate also has another external detection method, and this method step is such as
Under：
A uses the plasmid extraction kit with rpsL genes to extract plasmid pMOL21, using ScaI linearization for enzyme restriction plasmids, and
Entered using the primer of biotin labeling in performing PCR reaction, amplification in vitro plasmid pMOL21, PCR reaction system and add food pollution
Thing；
DNA cloning product by purifying, digestion, from coupled reaction, is transformed into host cell MF101, utilizes ampicillin by b
RpsL gene mutation bacterium colonies are screened with streptomysin；
C carries out data statistics, and calculates rpsL gene mutation rates, and mutation rate is mutation bacterium number/total bacteria count, wherein mutation bacterium number contains
The bacterium number of ampicillin and streptomysin flat board, bacterium number of the total bacteria count containing ampicillin plate, every group of at least three times repetition, always
Clump count is not less than 106It is individual.
5. one kind according to claim 1 or 4 utilizes rpsL gene mutation rate check and evaluation food contaminant genetoxics
Method, it is characterised in that：Described food contaminant refers to bring into food in production, processing, transport and storage
Polluter, mainly including the one or more in residues of pesticides, chemical pollutant, microbial toxin.
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Citations (4)
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|---|---|---|---|---|
| JPH08205708A (en) * | 1995-01-31 | 1996-08-13 | Rikagaku Kenkyusho | Detection of mutagenic substance with transgenic fishes |
| CN101280341A (en) * | 2008-05-21 | 2008-10-08 | 天津科技大学 | Method for quantitative determination of biological safety of nano-material by molecular biology technology |
| CN102021219A (en) * | 2009-09-23 | 2011-04-20 | 中国科学院生态环境研究中心 | Microbial cell sensor for detecting toluene organic pollutants |
| CN102605036A (en) * | 2012-04-01 | 2012-07-25 | 清华大学 | In-situ detection method for genetic toxicity of contaminated soil |
-
2017
- 2017-04-24 CN CN201710269594.XA patent/CN107090500A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08205708A (en) * | 1995-01-31 | 1996-08-13 | Rikagaku Kenkyusho | Detection of mutagenic substance with transgenic fishes |
| CN101280341A (en) * | 2008-05-21 | 2008-10-08 | 天津科技大学 | Method for quantitative determination of biological safety of nano-material by molecular biology technology |
| CN102021219A (en) * | 2009-09-23 | 2011-04-20 | 中国科学院生态环境研究中心 | Microbial cell sensor for detecting toluene organic pollutants |
| CN102605036A (en) * | 2012-04-01 | 2012-07-25 | 清华大学 | In-situ detection method for genetic toxicity of contaminated soil |
Non-Patent Citations (4)
| Title |
|---|
| SATU OLKKOLA ET AL: "Mutations in the rpsL Gene Are Involved in Streptomycin Resistance in Campylobacter coli", 《MICROBIAL DRUG RESISTANCE》 * |
| WENJUAN YANG ET AL: "Food storage material silver nanoparticles interfere with DNA replication fidelity and bind with DNA", 《NANOTECHNOLOGY》 * |
| 丁芳林主编: "《食品化学》", 31 August 2010, 华中科技大学出版社 * |
| 刘清岱 等: "用rpsL基因突变实验评价丙烯酰胺致突变性的方法初探", 《食品与生物技术学报》 * |
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Application publication date: 20170825 |