AU2017393714B2 - Genotoxic substance detection vector and detection method thereof - Google Patents

Abstract

A genotoxic substance detection vector and a detection method thereof are provided. The vector is an

Description

GENOTOXIC SUBSTANCE DETECTION VECTOR AND DETECTION METHOD THEREOF

FIELD OF THE INVENTION The present invention relates to the technical field of detecting genotoxic substances in the environment, in particular to detecting genotoxic substances in the environment by using recombinant Escherichia coli carrying a reporter gene.

BACKGROUND OF THE INVENTION The methods for detecting genotoxic substances are divided into long-term detection methods and short-term detection methods. The long-term detection methods are time-consuming and laborious, and have high maintenance costs for experimental animals; therefore, they have been gradually replaced by short-term screening methods that are fast and cost-effective. Short-term detection methods use cytogenetic indicators to screen for chemical mutagens, and usually use biological cells from plants, mammals, microorganisms and the like to monitor the genotoxicity of residues. The currently developed detection methods include comet assay, sister chromatid exchange, SOS chromotest, unscheduled DNA synthesis, bacterial reverse mutationtest, and prophage induction assay, and so on. These detection methods are generally not popular due to factors such as complicated operation, long detection time, and strict aseptic operation. In addition, due to patent protection and other reasons, individual detection methods are not conducive to domestic promotion, which limits their widespread use. The genotoxicity test, which can detect DNA damage, refers to in vitro and in vivo tests for detecting a test substance that is directly or indirectly induced to have genetic damage through different mechanisms. This DNA damage is one of the links in the development of malignant tumors. In recent years, some rapid short-term in vitro genotoxicity detection methods have been established to detect DNA damage. Conventional methods for the detection of genotoxic substances include gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, and high-pressure liquid chromatography-mass spectrometry. These technologies are capable of accurately and quantitatively detecting hundreds of the above chemical substances with accuracy at trace levels. However, these methods require large-scale precision instruments for testing, and have problems such as long detection period, cumbersome operation, high cost, and requiring analysts with high analytical capabilities, which cannot meet the needs of on-site detection. Therefore, a simple, fast and economical detection means can be developed to meet the needs. At present, more than 200 short-term methods for detecting genotoxicity have been established. Among them, the genotoxicity detection methods that have been more studied and applied are listed as follows: comet assay, also known as single cell gel electrophoresis (SCGE); sister chromatid exchange (SCE); unscheduled DNA synthesis (UDS), also known as DNA repair synthesis; the reverse mutationtest of salmonella typhi munine, also known as Ames test; SOS chromotest; prophage induction assay; and the like. Among the above methods, the Ames test is the most conventional method in genotoxicity analysis. McCann's test of 300 chemicals using the Ames test showed that most carcinogens are mutagens with a correlation of more than 80%. The Ames test has the following advantages: It is sensitive and has a high detectable rate; and it is relatively easy and simple without requiring special equipment, easy to promote. It has the following disadvantages: (1) Compared with mammals, microorganisms have simpler DNA repair systems and fewer genes, unable to fully represent the actual situation of mammals; (2) a sample containing histidine, glycine or lactose cannot be detected; and 3) the workload is large, the detection time is long, and the strain is not easy to store. Despite these, due to the above advantages, the Ames test still plays an important role in a mutagenicity test and is the preferred detection method. Many countries, such as Canada, the United States, and Japan, have taken the Ames test as the first choice in the mutagenesis test system. It can be combined with in vitro or in vivo cytogenetic tests to act as a means of screening in the first stage.

The SOS chromotest was designed based on the phenomenon that genotoxic substances induce SOS repair initiation of organisms and express umuC genes (G. Reifferscheid, J. Heil, Y. Oda and R.K. Zahn, et al. A microplate version of the SOS/umu-test for rapid detection of genotoxins and genotoxic potentials of environmental samples. Mutation Research, 253 (1991) 215-222). In 1982, Quillardet et al. first used the SOS reaction principle to detect genotoxic substance, and constructed a plasmid containing the sifA-LacA fusion gene (Quillardet, P., Huisman, 0., D'Ari, R. et al. SOS chromotest, a direct assay of induction of an SOS function in Escherichia coli K-12 to measure genotoxicity. Proceedings of the National Academy of Sciences of the United States of America 79, 5971-5975, 1982). In 1985, based on the test results of 83 different compounds, the researchers considered that most of the substances that were positive for the Ames test were also positive for the SOS test. In 1985, Oda et al. fused umuC and LacA, thus officially proposing the umu test (Oda, Y. Induction of SOS responses in Escherichia coli by 5-fluorouracil. Mutation research 183, 103-108, 1987). In 1986, IARC Publication No. 83 listed this method as one of a series of methods for short-term assays for carcinogens (Long-term and short-term assays for carcinogens: a critical appraisal. Reports of an ad-hoc working group. Lyons, 2-6 December 1985. IARC scientific publications, 1-564, 1986). In 1991, Reifferscheid et al. further improved the umu test by using a 96-well microplate instead of a test tube, and the computer technology, to enable automated, high-throughput screening of genotoxic substances. In Germany, the umu test is the first standardized test (DIN 38415-3) for the detection of genotoxicity of wastewater (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 SOS/umu-based genotoxicity detection system has a long detection time and is cumbersome. Even after the detection bacteria have been activated, the entire detection process still takes about 6 h, and the bacteria are required to be lysed to release beta-galactosidase, with the beta-galactosidase enzyme activity determined by the enzyme activity.

Genotoxic pollutants are widely distributed in China's environment, having a wide variety and a large amount. They are representative of environmental pollutants and are more harmful to humans. Therefore, it is very important to establish a detection method that is rapid, effective, convenient, and easy to promote.

CONTENTS OF THE INVENTION In view of the deficiencies of the prior art, the present invention provides a rapid detection method for environmental genotoxic substances, which desirably has the advantages of convenient operation, high sensitivity, good biosafety, no need for external reagents to lyse cells, no pigment interference, short time, low cost, and easiness in achieving high throughput screening. When the microbial DNA is damaged (for example, by ultraviolet radiation or stimulation of genotoxic substances), mass expression of a series of repair genes will be caused. The mass expression of these genes is dependent on the activation and hydrolysis of the repressor protein LexA by the recA protein under the action of damage, which promotes the mass expression of the repair gene downstream of the repressor protein. These repair reactions are called SOS reactions. SOS reactions, widely found in prokaryotes and eukaryotes, are instinct for organisms to protect themselves in adverse environments. The binding region of LexA to the upstream of the repair gene is called an SOS box, and the coding gene is located in the region of the promoter from -35 to -10 of the repair gene. This study is to use the SOS box response region to regulate the expression of downstream reporter proteins, so as to achieve qualitative and quantitative detection of genotoxic substances in the environment. X bacteriophage of Escherichia coli is one of the most well-studied and widely used bacteriophages currently studied. The genes that cause cell lysis in the bacteriophage include S, R and Rz genes, wherein the R gene encodes a water-soluble transglycosylase, which can cause hydrolysis of peptide bonds and decomposition of the peptidoglycan of the cell wall. The product of the Rz gene may be an endopepidase that can cleave the linkage between the peptidoglycan and the oligosaccharide and/or between the peptidoglycan and the cell wall outer membrane. The function of the products of the R and Rz genes is to degrade the cell wall; the product of the S gene acts to alter the permeability of the cytoplasmic membrane and form a porous structure on the cytoplasmic membrane, so as to allow the products of the R and Rz genes to cross the cytoplasmic membrane and act on the cell wall to break the cell wall and release intracellular substances. The present invention utilizes recombinant Escherichia coli as a genotoxic substance detection bacterium. The strain connects a specific genotoxic substance response promoter sequence to the bacteriophage lysis-based protein SRRz gene sequence, and Escherichia coli is introduced after connection with a plasmid vector to form Escherichia coli carrying the bacteriophage lysis protein SRRz, which is the recombinant Escherichiacoli used in the present invention. If the recombinant strain encounters a genotoxic substance to cause DNA damage, the expression of the lysis gene SRRz is initiated, which eventually leads to rupture of Escherichia coli. By detecting the bacterial lysis efficiency, the content of pollutants can be quantitatively detected within a certain range. In a first aspect, the present invention provides a genotoxic substance detection vector, which is an Escherichia coli expression vector sequentially having, from a 5' end to a 3' end, a genotoxic response promoter, a bacteriophage lysis gene, and an Escherichiacoli terminator. The genotoxic substance response element may be any element of the SOS response, preferably the nucleotide sequence of SEQ ID No. 1 in the Sequence Listing. The bacteriophage lysis gene may be any bacteriophage lysis gene, preferably a lysis gene SRRz of lambda bacteriophage, having the nucleotide sequence of SEQ ID No. 2 in the Sequence Listing. The Escherichia coli terminator may be any Escherichia coli terminator, preferably a T7 terminator.

The starting vector for constructing the vector may be any one of Escherichia coli vectors, preferably one of a series of vectors of pBluescript, pUC18, pUC19, and pET. The Escherichia coli lysis vector constructed with pUC18 as the starting vector is pUST. In a second aspect of the present invention, there is provided a method for detecting genotoxic substances. The method for detecting genotoxic substances provided by the present invention includes introducing the above genotoxic substance response vector into Escherichia coli to obtain recombinant bacteria, incubating the recombinant bacteria and genotoxic substances, and lysing the Escherichia coli. The recombinant Escherichia coli has the genotoxic response vector, and a self-cell lysis is initiated when the recombinant bacteria contact the genotoxic substances. The detection method quantitates genotoxic substances by means of lysis efficiency. The Escherichia coli is preferably E. coli BL21, E. coli DH5a, E. coliXL1-blue or E. coli HB101. The above method for detecting genotoxic substances comprises the following steps: (1) preparing an Escherichia coli detection solution using a recombinant Escherichia coli comprising a genotoxic substance detection vector, wherein the vector is an Escherichiacoli expression vector sequentially having, from a 5' end to a 3' end, a genotoxic response promoter, a bacteriophage lysis gene, and an Escherichia coli terminator and wherein the sequence of the genotoxic response promoter is shown in SEQ ID No. 1; (2) preparing a sample group of a volume by mixing a sample to be tested with the Escherichia coli detection solution, and preparing a control group by adding an Escherichia coli detection solution of pure solvent of the same volume; continuing to culture both groups of samples for 0.3-1 h; (3) measuring OD 6 0 0 ofthe Escherichia coli detection solution mixed with the sample to be tested and the Escherichia coli detection solution of the control group, respectively; and (4) calculating the lysis efficiency, and then calculating the concentration of genotoxic substances in the sample to be tested according to the standard curve of the lysis efficiency. Preferably, the base sequence of the bacteriophage lysis gene is shown in SEQ ID No. 2. Preferably, the terminator is a T7 terminator. Preferably, a starting vector used to construct the vector is pUC18, pUC19, pBluscript or pET30a. In embodiments, the preparation of the Escherichia coli detection solution comprises the following steps: a) culturing a stored recombinant Escherichia coli with an LB solid medium to recover and activate the Escherichia coli to obtain an activated Escherichia co/i; b) placing a single colony of the activated Escherichia coli in an LB liquid medium for shaking culture to the late logarithmic growth phase to obtain a saturated bacterial solution; and c) inoculating a fresh LB medium with the saturated bacterial solution at a volume ratio of 1:100, and culturing until the OD6 0 0 of the bacterial solution is 0.15-0.25, such that the Escherichia coli detection solution is obtained. In embodiments, the genotoxic substances are selected from the group consisting of: methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4-NQO), mitomycin C (MMC), 2-aminoindole (2-AA), and benzopyrene (BaP). The standard curve of the lysis efficiency is as follows: 4-NQO, Y =-1.78 + 10.95X, R2 = 0.99 (1 < X < 5) MMSY= -16.98+ 0.57X, R2 = 0.98 (40 < X < 100) MMC, Y =-19.04 + 4.11X, R 2 =0.98 (5 < X < 20) 2-AA, Y= -8.32 + 86.24X, R2 = 0.99 (0.2 < X < 0.8) BaP, Y = 4.03 + 89.1OX, R2 = 0.98 (0.1 < X < 0.8) Wherein X represents the concentration of a genotoxic compound (mg/L), Y represents the lysis efficiency (%), and R 2 represents a fitting curve correlation coefficient. In embodiments, the recombinant Escherichia coli containing the Escherichia coli genotoxic substance response vector is also within the scope of the present invention. Compared with the prior art, the present invention desirably has the following beneficial effects: 1. The operation object is Escherichia coli, which has no pathogenic risk and is easy to operate. 2. Having a short test cycle, the detection can be completed within 3 h (2 h for the preparation of the detection bacterial solution, 0.5 h for the contact of the bacterial solution with the sample to be tested, and 0.5 h for the determination of OD 6 0 0value of the bacterial solution), greatly shortening the time required for the detection of genotoxic substances in the past. 3. There is no need to add additional reagents (such as a zymolyte) during the detection process, and the cost is low. 4. The present invention has high detection sensitivity, having the sensitive concentration ranges of 1-5 mg/L, 40-100 mg/L, 5-20 mg/L, 0.2-0.8 mg/L, and 0.1-0.8 mg/L respectively for 4-NQO, MMS, MMC, 2-AA, and BaP. 5. The content of the water-like genotoxic substance detected by the present invention can be converted into the equivalent concentration of 4-NQO, so that the result is more intuitive and uniform. 6. This method can provide technical support for sudden water pollution and routine test of water quality in water plants.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic diagram of the construction of the vector. Fig. 2 is a graph showing the lysis efficiency of the wild-type Escherichia coli (E. coli BL21/pUC18) and the recombinant bacteria (E. coli BL21/pUST) in contact with different genotoxic substances for 0.5 h.

DETAILED DESCRIPTION OF THE EMBODIMENTS The present invention will be further described in detail below with reference to specific examples; however, the embodiments of the present invention are not limited thereto, and the process parameters not specifically noted can be determined by reference to conventional techniques. Example 1 Construction of genotoxic response vector pUC18 was used as a starting vector to construct a genotoxic response vector pUST. The specific construction method was as follows: a) synthesizing the genetic response promoter Pum, (SEQ ID No. 1) and T7 terminator sequences, respectively adding EcoRI and XbaI sites upstream of the promoter and T7 terminator, and adding Spel and PstI restriction sites downstream; b) amplifying the SRRz gene by using lambda bacteriophage genomic DNA as a template; also adding EcoRI and XbaI, Spel and PstI restriction sites respectively upstream and downstream of the SRRz gene; c) with the Biobricks method, the elements were linked in the order of promoter-lysis gene-terminator by enzyme digestion and connection, and inserted into the pUC18 vector to obtain a genotoxic response vector pUST; and d) the vector pUST was transferred to E. coli BL21 competent cells to obtain recombinant Escherichia coli (E. coli BL21/pUST) for the detection of genotoxic substances. Example 2 Calculation of lysis efficiency The light absorption value of each detection bacterial solution at 600 nm (OD6 0 0 )

was measured 0.5 h after the Escherichia coli detection solution was exposed to the sample to be tested.

Lysis efficiency(%)= (A-B)/A*100%, wherein A-OD 6 0 0of the bacterial solution containing methanol, and B-OD 6 0 0of the bacterial solution containing the test sample. Example 3 Detection of genotoxic substances 1. Recovery and activation of recombinant Escherichiacoli (1) Wild-type Escherichia coli (E. coli BL21/pUC18) and recombinant bacteria (E. coli BL21/pUST) were streaked from a -80°C refrigerator into an LB plate medium, and recovered to culture at 37°C for 14 h. (2) Single colonies were picked and inoculated into the LB medium, and cultured at 37°C and 250 rpm for 12-16 h. 2. Preparation of Escherichiacoli detection solution The recovered and activated bacterial solution was inoculated to a fresh LB medium at a volume ratio of 1:100, and added to a 96-well culture plate in a volume of 190 pl, and then cultured at 35°C to 37°C and 800 rpm to OD6 0 0 of 0.15-0.25. 3. Contact with the test sample Respectively putting 10 pl of methanol and different concentrations of 4-NQO, MMS, MMC, 2-AA and BaP into the 96-well plate, and continuing to culture for 0.5 h at 35°C to 37°C and 800 rpm, with 3 parallel groups set. 4. The experimental results (Fig. 2) showed that the original strain E. coli BL21/pUC18 grew normally in the tested concentration range, and no bacterial lysis was observed. The recombinant strain E. coli BL21/pUST containing the genotoxic response vector had different degrees of lysis. According to Fig. 2, the detection standard curves of the corresponding genotoxic substances were as follows: 4-NQO, Y =-1.78 + 10.95X, R2 = 0.99 (1 < X < 5) MMSY= -16.98+ 0.57X, R2 = 0.98 (40 < X < 100) MMC, Y =-19.04 + 4.11X, R 2 = 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 the concentration of a genotoxic compound (mg/L), Y represents the lysis efficiency (%), and R 2 represents a fitting curve correlation coefficient. Example 4 Detection of genotoxic substances in water samples The rapid detection method of genotoxic substances in this example was based on 4-NQO as a standard toxic substance. According to the literature, the concentration of 4-NQO in drinking water per day for healthy adults should not exceed 80 ng/L (based on 2L/day of drinking water) (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/H202 water treatment samples. Chemosphere 2016, 144, 338-345). This example comprises the following steps: (1) An Escherichia coli detection solution was prepared in accordance with the method of Example 3. (2) The water sample to be tested was adsorbed by an activated resin at a rate of 40 mL/min, and then eluted with ethyl acetate. The eluent was subjected to centrifugal freeze-drying to remove ethyl acetate, and then the sample was dissolved in a certain volume of distilled water to a desired volume. The water sample to be tested was mixed with the Escherichia coli detection solution, and the Escherichia coli detection solution of pure solvent of the same volume was added as a control; both samples were further cultured for 1 h. The water samples to be tested were taken from daily urban water (water from laboratory taps), water source of urban waterworks, wastewater A of a chemical plant, and wastewater B of a chemical plant. (3) Measuring OD 6 0 0of the Escherichia coli detection solution mixed with the water sample to be tested and the Escherichia coli detection solution of the control group, respectively.

(4) Calculating the lysis efficiency, and then calculating the equivalent concentration of the genotoxic substance 4-NQO in the sample to be tested according to the standard curve of the lysis efficiency. The measurement results are shown in Table 1. Equivalent Equivalent amount of amount of Water Concentrating 4-NQO in the 4-NQO in sample ratio efficiency concentrated the original sample sample (mg/L) (ng/L) Daily urban 4000:1 3.22 <1 < 250 water 40000:1 2.48 <1 < 25 Water 4000:1 5.89 <1 < 250 source of 40000:1 3.13 <1 < 25 urban waterworks Wastewater 4000:1 18.95 1.89 472.5 A of a 10000:1 49.03 4.64 464.1 chemical plant Wastewater 4000:1 11.25 1.19 297.5 B of a 10000:1 29.76 2.88 288.5 chemical plant

The results in Table 1 show that the equivalent concentration of the genotoxic substance 4-NQO in the daily urban water and water source of urban waterworks was less than 25 ng/L, while the equivalent concentration of 4-NQO in the wastewater A of a chemical plant was about 460-470 ng/L, and the equivalent concentration of

4-NQO in the wastewater B of a chemical plant was about 290-300 ng/L. The above test results indicate that the genotoxic response recombinant Escherichia coli of the present invention can detect genotoxic compounds of the test substance within the response concentration range. This method consumes little time, and is easy to use and promote. The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that such prior art forms part of the common general knowledge. It will be understood that the terms "comprise" and "include" and any of their derivatives (e.g. comprises, comprising, includes, including) as used in this specification, and the claims that follow, is to be taken to be inclusive of features to which the term refers, and is not meant to exclude the presence of any additional features unless otherwise stated or implied. In some cases, a single embodiment may, for succinctness and/or to assist in understanding the scope of the disclosure, combine multiple features. It is to be understood that in such a case, these multiple features may be provided separately (in separate embodiments), or in any other suitable combination. Alternatively, where separate features are described in separate embodiments, these separate features may be combined into a single embodiment unless otherwise stated or implied. This also applies to the claims which can be recombined in any combination. That is a claim may be amended to include a feature defined in any other claim. Further a phrase referring to "at least one of' a list of items refers to any combination of those items, including single members. As an example, "at least one of: a, b, or c" is intended to cover: a, b, c, a-b, a-c, b-c, and a-b-c. It will be appreciated by those skilled in the art that the disclosure is not restricted in its use to the particular application or applications described. Neither is the present disclosure restricted in its preferred embodiment with regard to the particular elements and/or features described or depicted herein. It will be appreciated that the disclosure is not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope as set forth and defined by the following claims.

SEQUENCE LISTING

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<120> Genotoxic substance detection vector and detection method thereof

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Claims (8)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. A method for detecting genotoxic substances, comprising the following steps: (1) preparing an Escherichia coli detection solution using a recombinant Escherichia coli comprising a genotoxic substance detection vector, wherein the vector is an Escherichia coli expression vector sequentially having, from a 5' end to a 3' end, a genotoxic response promoter, a bacteriophage lysis gene, and an Escherichia coli terminator and wherein the sequence of the genotoxic response promoter is shown in SEQ ID No. 1; (2) preparing a sample group of a volume by mixing a sample to be tested with the Escherichia coli detection solution, and preparing a control group by adding an Escherichia coli detection solution of pure solvent of the same volume; continuing to culture both groups of samples for 0.3-1 h; (3) measuring OD 6 0 0 ofthe Escherichia coli detection solution mixed with the sample to be tested and the Escherichia coli detection solution of the control group, respectively; and (4) calculating lysis efficiency, and then calculating the concentration of genotoxic substances in the sample to be tested according to a standard curve of the lysis efficiency.
2. 2. The method for detecting genotoxic substances according to claim 1, wherein the base sequence of the bacteriophage lysis gene is shown in SEQ ID No. 2.
3. 3. The method for detecting genotoxic substances according to claim 2, wherein the terminator is a T7 terminator.
4. 4. The method for detecting genotoxic substances according to any one of claims 1 to 3, wherein a starting vector used to construct the vector is pUC18, pUC19, pBluscript or pET30a.
5. 5. The method for detecting genotoxic substances according to any one of claims 1 to 4, wherein the preparation of the Escherichiacoli detection solution comprises the following steps: a) culturing a stored recombinant Escherichia coli with an LB solid medium to recover and activate the Escherichia coli to obtain an activated Escherichia co/i; b) placing a single colony of the activated Escherichia coli in an LB liquid medium for shaking culture to the late logarithmic growth phase to obtain a saturated bacterial solution; and c) inoculating a fresh LB medium with the saturated bacterial solution at a volume ratio of 1:100, and culturing until the OD6 0 0 of the bacterial solution is 0.15-0.25, such that the Escherichiacoli detection solution is obtained.
6. 6. The detection method according to any one of claims 1 to 5, wherein the genotoxic substances are selected from the group consisting of: methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4-NQO), mitomycin C (MMC), 2-aminoindole (2-AA), and benzopyrene (BaP).
7. 7. The detection method according to claim 6, wherein the standard curve of the lysis efficiency is as follows: 4-NQO, Y =-1.78 + 10.95X, R 2 = 0.99 (1 < X < 5) MMSY= -16.98+ 0.57X, R2 = 0.98 (40 < X < 100) MMC, Y =-19.04 + 4.11X, R 2 = 0.98 (5 < X < 20) 2-AA, Y= -8.32 + 86.24X, R2 = 0.99 (0.2 < X < 0.
8. 8) BaP, Y = 4.03 + 89.10X, R2 = 0.98 (0.1 < X < 0.8) wherein X represents the concentration of a genotoxic compound (mg/L), Y represents the lysis efficiency (%), and R 2 represents a fitting curve correlation coefficient.