CN106636164A - Genetic toxicant detection vector and detection method - Google Patents
Genetic toxicant detection vector and detection method Download PDFInfo
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Abstract
The invention discloses a genetic toxicant detection vector and a detection method. The vector is a prokaryotic expression vector connected with a genetic toxicity response promoter, a bacteriophage lytic gene and an escherichia coli terminator in sequence from the end 5' to the end 3'. The detection method comprises the following steps: introducing the genetic toxicant response vector into escherichia coli to obtain recombinant bacteria, and then incubating the recombinant bacteria and a genetic toxicant to realize escherichia coli cell lysis, wherein the escherichia coli recombinant bacteria carry the genetic toxicant response vector; when the recombinant bacteria are in contact with the chemical genetic toxicant, cell lysis occurs in the recombinant bacteria, so that the method for quantitatively detecting the genetic toxicant according to the lysis efficiency is realized. The method is low in time consumption, high in detection sensitivity, easy and convenient for detection, feasible, low in cost and easy to popularize.
Description
Technical field
The present invention relates to the technical field to the Genotoxic detection in environment, specially using reporter gene
Recombination bacillus coli detection environment in Genotoxic.
Background technology
The detection method of Genotoxic is divided into long-term test and short-term test.Long-term method of testing is taken due to time-consuming
Power, it is costly to animal used as test long term maintenance, gradually replaced by short-term screening technique quick, with low cost.Short-term
Test is that the mutagenesis factor is screened with cytogenetics index, generally biological thin using plant, mammal, microorganism etc.
Born of the same parents monitor the genetoxic of residue.Development at present has Comet Assay, sister chromatid exchange test, SOS colour tests, journey
The outer DNA synthetic tests of sequence, reverse mutation, Prophage induction test etc..These detection methods generally have trivial operations,
Strict sterile working of detection time length, needs etc. is unfavorable for the factor promoted.And due to the reasons such as patent protection, Ge Biejian
Survey method is simultaneously unfavorable for Spread in China, limits it and widely uses.
Genetic toxicity test refers to the tested material damaged by the direct or indirect induction of genetic of different mechanisms for detection
In vitro and in vivo test, these tests can detect DNA damage.This DNA damage be malignant progression process link it
One.Establish the quick external genotoxicity testing method of some short-terms in recent years to detect the damage of DNA.It is traditionally used for losing
Passing the detection method of toxicant has gas chromatography-mass spectrum, liquid chromatography-mass spectrography or high pressure liquid chromatography-mass spectrometry skill
Art.These technologies can exactly, quantitatively detect hundreds of kind of above-mentioned chemical substance, and the degree of accuracy can reach trace level.But,
These methods are required to large-scale precision instrument and are detected, and these methods exist detection cycle length, it is cumbersome, costly and
The problems such as analysis personnel there must be higher analysis ability is required, the demand of Site Detection cannot be met.Therefore, develop a kind of
Easy, quick and economic detection means could meet the demand of Site Detection.The genetoxic short term detection set up at present
Method is more than 200 kinds.Wherein study and have using more genotoxicity testing method：Comet Assay (Coment assay),
Also known as single cell gel electrophoresis test (Single cell gel electrophoresis, SCGE), Sister chromatid exohange
Test (Sister chromatid exchange, SCE), unscheduled DNA synthesis (repair synthesis, Unscheduled also known as DNA
DNA synthesis, UDS), ames test (The reverse mutationtest of
Salmonella typhi munine, also known as Salmonella reversion test), SOS colour tests (SOS chromotest), prophage lure
Lead test method (Prophage induction assay) etc..In said method, Salmonella reversion test be in Analysis on genetic toxicity most
Conventional method.McCann Salmonella reversion tests detect that the result of 300 kinds of chemicals shows, most of carcinogens are mutagens, phase
Closing property is more than 80%.The advantage of Salmonella reversion test is that method is sensitive, and recall rate is high；In addition method is easier, easy, is not required to spy
Different equipment, easily promotes.It has the disadvantage：(1) the DNA repair systems of microorganism are simpler than mammal, and gene is dynamic not as lactation
Thing is more, it is impossible to represent the actual conditions of mammal completely；(2) sample containing histidine, glycine or lactose can not be detected；
(3) workload is big, and detection time is long, and bacterial strain is not easy to maintain.Even so, due to there is above-mentioned advantage, therefore causing to dash forward at present
Become in test and account for critical positions, become first-selected test method.Many countries such as Canada, the U.S., Japan are all in mutagenesis testing
Salmonella reversion test is listed in into preferred location in system, is combined with cytogenetics in vitro test or in vivo studies, can be used as the first rank
The means of section primary dcreening operation.
SOS colour tests, are to induce biological SOS to repair based on Genotoxic to start, express umuC genes and set
(G.Reifferscheid, J.Heil, Y.Oda and R.K.Zahn, the et al.A microplate version of of meter
the SOS/umu-test for rapid detection of genotoxins and genotoxic potentials
of environmental samples.Mutation Research,253(1991)215-222).Nineteen eighty-two,
Quillardet etc. detects first hereditary poisonous substance using SOS reaction principles, and what they built is fusion containing sifA-LacA
Plasmid Quillardet, P., Huisman, O., D ' Ari, R.et al.SOS chromotest, a direct assay of
induction of an SOS function in Escherichia coli K-12to measure
genotoxicity.Proceedings of the National Academy of Sciences of the United
States of America 79,5971-5975,1982).1985, researcher was according to the test to 83 kinds of different compounds
As a result, it is believed that the material that most Salmonella reversion tests are positive, SOS tests are also positive.1985, Oda etc. was used
UmuC and LacA merges, and umu tests are formally suggested (Oda, Y.Induction of SOS responses in
Escherichia coli by 5-fluorouracil.Mutation research 183,103-108,1987)。1986
Year, the method is classified as No. 83 publication of IARC one of the series methods of the short screen system of carcinogenic substance (Long-term and
short-term assays for carcinogens:a critical appraisal.Reports of an ad-hoc
working group.Lyons,2-6December 1985.IARC scientific publications,1-564,
1986.).1991, Reifferscheid etc. did further improvement to umu tests, and using micro- culture plate in 96 holes examination is replaced
Pipe, and combines computer technology, the method is automated, the hereditary poisonous substance of high-throughout screening.In Germany, umu tests are the
One standardized test (DIN 38415-3) for detecting waste water genetoxic (Reifferscheid, G., Heil, J.,
Oda,Y.et al.A microplate version of the SOS/umu-test for rapid detection of
genotoxins and genotoxic potentials of environmental samples.Mutation
research 253,215-222,1991.).The existing genetoxic based on SOS/umu detects detecting system, detection time
It is long, complex steps.Even if on the basis of detection bacterium is activated well, whole detection process is still needed 6 hours or so, and
Cellular lysate is needed to discharge BgaA, by enzyme activity determination BgaA enzyme activity.
Genetoxic pollutant is distributed more widely in China's environment, and species and quantity are larger, there is one in environmental contaminants
Fixed representativeness, it is larger to harm, set up quick, it is effectively, convenient and particularly significant beneficial to the detection method promoted.
The content of the invention
The present invention be directed to the deficiencies in the prior art, there is provided one kind operation is convenient, sensitivity is high, biological safety is good, nothing
Added regent cell lysis are needed, pigment interference is not received, is taken short, low cost, it is easy to accomplish the environment heredity poison of high flux examination
Property material method for quick.
Microbial DNA sustain damage (as ultraviolet irradiation, Genotoxic stimulate) when, a series of revision points can be caused
Great expression.And the great expression of these genes depends on recA albumen to activate under damaging action, aporepressor is hydrolyzed
LexA, promotes the revision points great expression in aporepressor downstream.These are repaired reaction and are referred to as SOS reactions.SOS reactions are wide
It is general to be present in prokaryotes and eucaryote, it is the instinct of the biological self-protection in adverse environment.Wherein LexA and reparation
The calmodulin binding domain CaM of upstream region of gene is referred to as SOS box, and this section of encoding gene is located at the promoter -35 of revision points and arrives in -10 regions.
This research is exactly, using SOS box response regions, regulation and control downstream reporter protein expression, to realize to Genotoxic in environment
Qualitative and quantitative analysis.
Colibacillary bacteriophage lambda is to study one of most clear, most widely used bacteriophage at present.Cause in bacteriophage
The gene of cell lysis includes tri- genes of S, R and Rz, and wherein R gene codes is a kind of water-soluble transglycosylase
(transglycosylase) hydrolysis of peptide bond, can be caused, decomposes the peptide glycan of cell membrane.The product of Rz genes is probably one
Kind of endopeptidase (endopepidase), it can cut between peptide glycan and oligosaccharides and/or peptide glycan and cell membrane outside
Connection between film.The function of R and Rz gene outcomes is all degradation of cell wall, and the effect of S gene outcomes is to change cytoplasm
The permeability of film, forms the structure of porous on cytoplasma membrane, so that the product of R and Rz genes passes through cytoplasma membrane, and acts on
In cell membrane, cell membrane is crushed, discharge intracellular organic matter.
The present invention detects bacterium by the use of recombination bacillus coli as Genotoxic.The bacterial strain is by specific genetoxic
The promoter sequence of substance responds is connected with phage splitting base Protein S RRz gene order, imports after being connected with plasmid vector big
Enterobacteria, forms the Escherichia coli for carrying phage splitting Protein S RRz, recombination bacillus coli as used in the present invention.When
Recombinant bacterium runs into Genotoxic, causes DNA damage, then start the expression of lysis genes SRRz, ultimately results in Escherichia coli
Rupture.By detecting cellular lysate efficiency, within the specific limits can quantitative determination pollutant load.
It is an object of the invention to provide a kind of Genotoxic detects carrier, the carrier is that from 5 ' to 3 ' ends are sequentially connected with
There is genetoxic to respond the coli expression carrier of promoter, phage splitting gene and Escherichia coli terminator.
Described Genotoxic response element, can be any element of SOS responses, SEQ ID preferably in sequence table
No.1 nucleotide sequences.
Described phage splitting gene, can be any one phage splitting gene, preferably lambda bacteriophages
Lysis genes SRRz, the nucleotide sequence with SEQ ID No.2 in sequence table.
Described Escherichia coli terminator can be any one Escherichia coli terminator, preferably T7 terminators.
The carrier that sets out for building the carrier can be for any one escherichia coli vector, preferred pBluescript,
PUC18, pUC19, pET serial carrier.With pUC18 as the carrier that sets out, the Escherichia coli cracking carrier of structure is pUST.
Second object of the present invention is to provide a kind of detection method of Genotoxic.
Genotoxic detection method provided by the present invention, is that above-mentioned Genotoxic response vector introduction is big
In enterobacteria, recombinant bacterium is obtained, then recombinant bacterium and Genotoxic are incubated, Bacillus coli cells cracking.The large intestine bar
Bacterium recombinant bacterium carries genetoxic response carrier, and recombinant bacterium can cause own cells when genotoxic chemotherapy material is contacted
Cracking, by the lysis efficiency detection method quantitative to Genotoxic.
The Escherichia coli preferred E.coli BL21, E.coli DH5a, E.coli XL1-blue or E.coli
HB101。
Above-mentioned Genotoxic detection method, comprises the following steps：
(1) E. coli detection liquid is prepared；
(2) testing sample is mixed with E. coli detection liquid, while adding the E. coli detection of same volume neat solvent
Liquid is compared；Two groups of samples continue to cultivate 0.3-1h；
(3) the E. coli detection liquid mixed with testing sample is measured respectively with the E. coli detection liquid of control group
OD600；
(4) lysis efficiency is calculated, Genotoxic concentration in testing sample is calculated according to lysis efficiency calibration curve.
The preparation of the E. coli detection liquid：
A) recombination bacillus coli reserve is cultivated with LB solid mediums so as to recovery activation；
B) the activation Escherichia coli single bacterium colony for obtaining is linked in LB fluid nutrient mediums carries out concussion and cultivate to logarithm life
The long-term later stage；
C) by the saturation bacterium solution for obtaining with 1:100 volume ratios are inoculated in fresh LB culture mediums, are cultivated to bacterium solution OD600
For 0.15-0.25, E. coli detection liquid is obtained.
Described Genotoxic includes methyl mesylate (MMS), 4- nitroquinolines 1- oxides (4-NQO), mitogen
Mycin C (MMC), 2- amino anthracenes (2-AA) and BaP (BaP).
The lysis efficiency calibration curve is as follows：
4-NQO, Y=-1.78+10.95X, R2=0.99 (1<X<5)
MMS, Y=-16.98+0.57X, R2=0.98 (40<X<100)
MMC, Y=-19.04+4.11X, R2=0.98 (5<X<20)
2-AA, Y=-8.32+86.24X, R2=0.99 (0.2<X<0.8)
BaP, Y=4.03+89.10X, R2=0.98 (0.1<X<0.8)
Wherein, X represents genetoxic compound concentration (mg/L), and Y represents cleavage rate (%), R2Represent that matched curve is related
Coefficient.
The recombination bacillus coli for responding carrier containing the Escherichia coli Genotoxic falls within the protection of the present invention
Scope.
Compared with prior art, beneficial effects of the present invention are as follows：
1st, operation object is Escherichia coli, easy to operation without risk of causing a disease.
2nd, test period is short, in 3h can detect complete (2h detection bacterium solution prepare, bacterium solution is contacted with testing sample within 0.5 hour
Culture, 0.5h determines bacterium solution OD600Value), the time required to substantially reducing conventional Genotoxic detection.
3rd, detection process need not add additional agents (such as zymolyte), with low cost.
4th, detection sensitivity is high, and the sensitive concentration scope of 4-NQO, MMS, MMC, 2-AA and BaP is respectively in 1-5,40-
100th, it is sensitive under the conditions of 5-20,0.2-0.8,0.1-0.8mg/L.
5th, the water sample Genotoxic content of detection, can be converted into the equivalent concentration of 4-NQO, make result more straight
Sight, unification.
6th, the method can be that burst water pollution and water factory's water quality conventional detection provide technical support.
Description of the drawings
Fig. 1 is vector construction schematic diagram.
Fig. 2 is wild-type e. coli (E.coli BL21/pUC18) and recombinant bacterium (E.coli BL21/pUST) and not
The lysis efficiency figure of culture 0.5h is contacted with Genotoxic.
Specific embodiment
The present invention is more specifically described in detail with reference to specific embodiment, but embodiments of the present invention are not limited
In this, for especially not dated technological parameter, can refer to routine techniques is carried out.
Embodiment 1
Genetoxic responds the structure of carrier
With pUC18 as the carrier that sets out, genetoxic response carrier pUST is built.Concrete construction method is as follows：
A) genetic response promoter P is synthesizedumu(SEQ ID No.1) and T7 terminator sequences, respectively at promoter and T7 ends
Only EcoRI and XbaI sites, downstream addition SpeI and PstI restriction enzyme sites are added in sub- upstream.
B) with lambda phage genomes DNA as template, SRRz genes are expanded.Also distinguish in the upstream and downstream of SRRz genes
Addition EcoRI and XbaI, SpeI and PstI restriction enzyme sites.
C) using biological building blocks (Biobricks) method, using digestion, method of attachment by each element according to promoter-split
Solution gene-terminator is linked in sequence, and in being inserted into pUC18 carriers, obtains genetoxic response carrier pUST.
D) carrier pUST is transferred to into E.coli BL21 competent cells, obtains the restructuring of Genotoxic detection
E. coli BL21/pUST.
Embodiment 2
Lysis efficiency is calculated
0.5h after E. coli detection liquid contact measured sample, determines respectively light absorbs of each test bacterium solution at 600nm
Value (OD600)。
Cleavage rate (%)=(A-B)/A*100%
Wherein, bacterium solution OD of A-addition methyl alcohol600
Bacterium solution OD of B-addition test specimens600。
Embodiment 3
Genotoxic is detected
1. the recovery activation of recombination bacillus coli
(1) by wild-type e. coli (E.coli BL21/pUC18) and recombinant bacterium (E.coli BL21/pUST) respectively
During LB plating mediums are lined from -80 ° of refrigerators, the renewal cultivation 14h under the conditions of 37 DEG C.
(2) picking single bacterium colony, during LB culture mediums are seeded to respectively, under the conditions of 37 DEG C, 250rpm 12-16h is cultivated.
2. prepared by E. coli detection liquid
By the bacterium solution of recovery activation with 1:100 volume ratios are seeded to fresh LB, and with 190 μ l volumes 96 are added to
In well culture plate, at 35-37 DEG C, cultivate under the conditions of 800rpm to OD600To 0.15-0.25.
3. contact with given the test agent
Take each 10 μ l of 4-NQO, MMS, MMC, 2-AA and BaP of methyl alcohol and variable concentrations to be added separately in 96 orifice plates,
35-37 DEG C, continue to cultivate 0.5h under the conditions of 800rpm, 3 parallel groups are set.
4. test result indicate that (Fig. 2), in the range of test concentrations, original strain E.coli BL21/pUC18 thalline are given birth to
It is long normal, do not observe cellular lysate.And contain the recombinant bacterium E.coli BL21/pUST that genetoxic responds carrier, there is difference
The cracking of degree.
According to detection Fig. 2, the examination criteria curve of corresponding Genotoxic is respectively：
4-NQO, Y=-1.78+10.95X, R2=0.99 (1<X<5)
MMS, Y=-16.98+0.57X, R2=0.98 (40<X<100)
MMC, Y=-19.04+4.11X, R2=0.98 (5<X<20)
2-AA, Y=-8.32+86.24X, R2=0.99 (0.2<X<0.8)
BaP, Y=4.03+89.10X, R2=0.98 (0.1<X<0.8)
Wherein, X represents genetoxic compound concentration (mg/L), and Y represents cleavage rate (%), R2Represent that matched curve is related
Coefficient.
Embodiment 4
Genotoxic detection in water sample
The method for quick of the present embodiment Genotoxic, with 4-NQO as standard toxicant.According to document report
Road, 4-NQO concentration is not to be exceeded 80ng/L (drinking watermeter with 2L/ days) in the drinking water of health adult's day intake
(Martijn,B.J.；Van Rompay,A.R.et al.,Development of a 4-NQO toxic equivalency
factor(TEF)approach to enable a preliminary risk assessment of unknown
genotoxic compounds detected by the Ames II test in UV/H2O2water treatment
samples.Chemosphere 2016,144,338-345.)。
The present embodiment is comprised the following steps：
(1) E. coli detection liquid is prepared according to the method for embodiment 3.
(2) water sample to be measured is taken, with the resin adsorption that the speed of 40mL/min is activated, then ethyl acetate wash-out.Wash-out
Liquid removes ethyl acetate using centrifugation freeze drying process, then with the distilled water sample dissolution of certain volume to volume required.Will be to be measured
Water sample mixes with E. coli detection liquid, while the E. coli detection liquid for adding same volume neat solvent is compareed；Two groups of samples
Continue to cultivate 1h.The water sample to be measured take respectively daily urban water (releasing of laboratory tap), urban water plant water source, certain
Chemical plant wastewater A and certain chemical plant wastewater B.
(3) the E. coli detection liquid mixed with water sample to be measured is measured respectively with the E. coli detection liquid of control group
OD600。
(4) lysis efficiency is calculated, Genotoxic 4-NQO in testing sample is calculated according to lysis efficiency calibration curve and is worked as
Amount concentration.Acquired results are as shown in table 1.
The result of table 1 shows that the equivalent concentration of the Genotoxic 4-NQO at daily urban water and urban water plant water source is low
In 25ng/L, and chemical plant wastewater A water sample 4-NQO equivalent concentration is about 460-470ng/L, and chemical plant wastewater B water samples 4-NQO works as
Amount concentration is about 290-300ng/L.
Above-mentioned testing result shows that the recombination bacillus coli of the genetoxic response of the present invention can be in response concentration model
Enclose the interior compound with genetoxic to determinand to detect.The method take it is short, detect it is simple and easy to do, be easy to push away
Extensively.
SEQUENCE LISTING
<110>South China Science ＆ Engineering University
<120>A kind of Genotoxic detection carrier and detection method
<130> 1
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 82
<212> DNA
<213> Artificial Sequence
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gaattctacg tctagagctt attgacatgc tggcaagaac agactactgt atataaaaac 60
agtataacta gttacgctgc ag 82
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<211> 1549
<212> DNA
<213> Artificial Sequence
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gccactgtct gtcctgaatt cattagtaat agttacgctg cggcctttta cacatgacct 60
tcgtgaaagc gggtggcagg aggtcgcgct aacaacctcc tgccgttttg cccgtgcata 120
tcggtcacga acaaatctga ttactaaaca cagtagcctg gatttgttct atcagtaatc 180
gaccttattc ctaattaaat agagcaaatc cccttattgg gggtaagaca tgaagatgcc 240
agaaaaacat gacctgttgg ccgccattct cgcggcaaag gaacaaggca tcggggcaat 300
ccttgcgttt gcaatggcgt accttcgcgg cagatataat ggcggtgcgt ttacaaaaac 360
agtaatcgac gcaacgatgt gcgccattat cgcctggttc attcgtgacc ttctcgactt 420
cgccggacta agtagcaatc tcgcttatat aacgagcgtg tttatcggct acatcggtac 480
tgactcgatt ggttcgctta tcaaacgctt cgctgctaaa aaagccggag tagaagatgg 540
tagaaatcaa taatcaacgt aaggcgttcc tcgatatgct ggcgtggtcg gagggaactg 600
ataacggacg tcagaaaacc agaaatcatg gttatgacgt cattgtaggc ggagagctat 660
ttactgatta ctccgatcac cctcgcaaac ttgtcacgct aaacccaaaa ctcaaatcaa 720
caggcgccgg acgctaccag cttctttccc gttggtggga tgcctaccgc aagcagcttg 780
gcctgaaaga cttctctccg aaaagtcagg acgctgtggc attgcagcag attaaggagc 840
gtggcgcttt acctatgatt gatcgtggtg atatccgtca ggcaatcgac cgttgcagca 900
atatctgggc ttcactgccg ggcgctggtt atggtcagtt cgagcataag gctgacagcc 960
tgattgcaaa attcaaagaa gcgggcggaa cggtcagaga gattgatgta tgagcagagt 1020
caccgcgatt atctccgctc tggttatctg catcatcgtc tgcctgtcat gggctgttaa 1080
tcattaccgt gataacgcca ttacctacaa agcccagcgc gacaaaaatg ccagagaact 1140
gaagctggcg aacgcggcaa ttactgacat gcagatgcgt cagcgtgatg ttgctgcgct 1200
cgatgcaaaa tacacgaagg agttagctga tgctaaagct gaaaatgatg ctctgcgtga 1260
tgatgttgcc gctggtcgtc gtcggttgca catcaaagca gtctgtcagt cagtgcgtga 1320
agccaccacc gcctccggcg tggataatgc agcctccccc cgactggcag acaccgctga 1380
acgggattat ttcaccctca gagagaggct gatcactatg caaaaacaac tggaaggaac 1440
ccagaagtat attaatgagc agtgcagata gagttgccca tatcgatggg caactcatgc 1500
aattattgtg agcaatacac acgcgcttcc agcggagtat aaatgccta 1549
Claims (10)
1. a kind of Genotoxic detects carrier, it is characterised in that the carrier is that from 5 ' to 3 ' ends have been sequentially connected with hereditary poison
Property response promoter, the coli expression carrier of phage splitting gene and Escherichia coli terminator.
2. Genotoxic according to claim 1 detects carrier, it is characterised in that described genetoxic response is opened
The sequence of mover is as shown in SEQ ID No.1.
3. Genotoxic according to claim 2 detects carrier, it is characterised in that described phage splitting gene
Base sequence as shown in SEQ ID No.2.
4. Genotoxic according to claim 3 detects carrier, it is characterised in that described terminator is T7 terminations
Son.
5. the Genotoxic according to claim 1 or 2 or 3 or 4 detects carrier, it is characterised in that：For building
That states carrier sets out carrier for pUC18, pUC19, pBluscript or pET30a.
6. the carrier described in any one of Claims 1 to 5 is transferred to into Escherichia coli, the recombination bacillus coli for obtaining.
7. a kind of Genotoxic detection method, it is characterised in that comprise the following steps：
(1) E. coli detection liquid is prepared using the recombination bacillus coli described in claim 6；
(2) testing sample is mixed with E. coli detection liquid, while the E. coli detection liquid for adding same volume neat solvent is made
Control；Two groups of samples continue to cultivate 0.3-1h；
(3) the E. coli detection liquid of the E. coli detection liquid mixed with testing sample and control group is measured into respectively OD600；
(4) lysis efficiency is calculated, Genotoxic concentration in testing sample is calculated according to lysis efficiency calibration curve.
8. Genotoxic detection method according to claim 7, it is characterised in that the E. coli detection liquid
Prepare：
A) recombination bacillus coli reserve is cultivated with LB solid mediums so as to recovery activation；
B) the activation Escherichia coli single bacterium colony for obtaining is linked in LB fluid nutrient mediums carries out concussion and cultivate to exponential phase
Later stage；
C) by the saturation bacterium solution for obtaining with 1:100 volume ratios are inoculated in fresh LB culture mediums, are cultivated to bacterium solution OD600For
0.15-0.25, obtains E. coli detection liquid.
9. the detection method according to claim 6 or 7 or 8, it is characterised in that described Genotoxic includes first sulphur
Sour methyl esters (MMS), 4- nitroquinolines 1- oxides (4-NQO), mitomycin C (MMC), 2- amino anthracenes (2-AA) and BaP
(BaP)。
10. detection method according to claim 9, it is characterised in that the lysis efficiency calibration curve is as follows：
4-NQO, Y=-1.78+10.95X, R2=0.99 (1<X<5)
MMS, Y=-16.98+0.57X, R2=0.98 (40<X<100)
MMC, Y=-19.04+4.11X, R2=0.98 (5<X<20)
2-AA, Y=-8.32+86.24X, R2=0.99 (0.2<X<0.8)
BaP, Y=4.03+89.10X, R2=0.98 (0.1<X<0.8)
Wherein, X represents genetoxic compound concentration (mg/L), and Y represents cleavage rate (%), R2Represent matched curve coefficient correlation.
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| AU2017393714A AU2017393714B2 (en) | 2017-01-18 | 2017-11-14 | Genotoxic substance detection vector and detection method thereof |
| PCT/CN2017/110897 WO2018133513A1 (en) | 2017-01-18 | 2017-11-14 | Genotoxic substance detection vector and detection method thereof |
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Cited By (6)
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| CN107478795A (en) * | 2017-08-24 | 2017-12-15 | 山东省城市供排水水质监测中心 | A kind of detection method of urban drinking water water body genetoxic |
| WO2018133513A1 (en) * | 2017-01-18 | 2018-07-26 | 华南理工大学 | Genotoxic substance detection vector and detection method thereof |
| CN109402155A (en) * | 2018-11-12 | 2019-03-01 | 川北医学院 | A kind of dual control delay cracking performance plasmid and its construction method and application |
| CN110873790A (en) * | 2018-09-03 | 2020-03-10 | 华南理工大学 | Whole-cell biosensor for detecting heavy metal ions in water-soluble sample and construction and application thereof |
| WO2021030030A1 (en) * | 2019-08-09 | 2021-02-18 | Nantomics, Llc | Machine methods to determine neoepitope payload toxicity |
| CN112680498A (en) * | 2020-12-28 | 2021-04-20 | 华南理工大学 | High-throughput screening method for genotoxic substances |
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| CN112680498A (en) * | 2020-12-28 | 2021-04-20 | 华南理工大学 | High-throughput screening method for genotoxic substances |
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| Publication number | Publication date |
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| AU2017393714A1 (en) | 2019-07-18 |
| AU2017393714B2 (en) | 2021-08-05 |
| WO2018133513A1 (en) | 2018-07-26 |
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