CN107201327B

CN107201327B - Luminescent bacterium and application thereof in chlorothalonil residue detection

Info

Publication number: CN107201327B
Application number: CN201710409780.9A
Authority: CN
Inventors: 段效辉; 李金庆; 赵钰玲; 王海涛; 王颖; 刘鹏; 尚迪; 贺丽娜; 颜显辉
Original assignee: Inspection & Quarantine Technology Center Of Yantai Entry-Exit Inspection And Quarantine Bureau
Current assignee: Inspection & Quarantine Technology Center Of Yantai Entry-Exit Inspection And Quarantine Bureau
Priority date: 2017-06-02
Filing date: 2017-06-02
Publication date: 2020-06-16
Anticipated expiration: 2037-06-02
Also published as: CN107201327A

Abstract

The invention discloses a luminous bacterium and application thereof in chlorothalonil residue detection, and belongs to the technical field of microorganisms. The bacterium is a luminous bacterium Fc-11-1, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has the preservation date of 2016, 12 and 05 months, has the preservation number of CGMCC No.13424, and is named in classification as: vibrio toranziae. The invention also discloses application of the luminescent bacteria in chlorothalonil residue detection. The luminous bacteria Fc-11-1 enriches the species resource of the luminous bacteria, and is a novel luminous strain different from 11 kinds of luminous bacteria reported in the prior art. The invention provides an application of luminous bacteria in chlorothalonil residue detection, and the detection method has the advantages of low cost, simplicity in operation, no need of expensive or large-scale instruments and equipment and the like.

Description

Luminescent bacterium and application thereof in chlorothalonil residue detection

Technical Field

The invention relates to a luminous bacterium and application thereof in chlorothalonil residue detection, belonging to the technical field of microorganisms.

Background

Chlorothalonil (CHT) with the chemical name of 2,4,5, 6-tetrachloro-1, 3-dicyanobenzene is a broad-spectrum protective bactericide and is mainly used for preventing and treating fungal diseases of fruits and vegetables in agricultural production. The chlorothalonil has good adhesion to plant bodies and wide application, so that the chlorothalonil and metabolites thereof have large residues in fruits, vegetables, soil and water. The united states national environmental protection agency (u.s.epa) has listed chlorothalonil as one of the substances that may be carcinogenic to humans, which has a potential threat to environmental and food safety. The chlorothalonil is converted into hydroxyl chlorothalonil after being acted by light, microorganisms or enzymes in soil, has increased toxicity and stability, can be dissolved in water, and poses more serious threats to environment and food safety. At present, the detection methods of chlorothalonil mainly comprise gas chromatography, liquid chromatography and enzyme-linked immunosorbent assay, but the methods have the defects of complex pretreatment, expensive instrument reagents and the like in practical application.

The method for detecting the luminous bacteria is a biological method for detecting harmful substances, can determine the concentration of the harmful substances or evaluate the toxicity of the harmful substances through the change of luminous intensity of the luminous bacteria, has the advantages of sensitivity, rapidness, low cost and the like, and is increasingly widely applied to the fields of food safety, environmental monitoring and the like.

Disclosure of Invention

The inventor of the application aims to separate and screen the luminous bacteria in the seawater environment and marine organisms, and preliminarily applies the luminous bacteria to the biological detection of the chlorothalonil residues of the fruit and vegetable pollutants so as to establish a quick and simple method for detecting the chlorothalonil.

One of the purposes of the invention is to provide a luminous bacterium. The luminous bacterium Fc-11-1 enriches the species resource of the luminous bacterium, and is a novel luminous strain different from 11 reported luminous bacteria.

The technical scheme for solving the technical problems is as follows: a luminescent bacterium Fc-11-1 which is preserved in the general microbiological culture collection center (CGMCC for short) of China Committee for culture Collection of microorganisms of the institute of Chinese academy of sciences in the Yangtze area, Beijing, at the Tungxun road of the sunward area, 12 months and 05 days in 2016, has the preservation number of CGMCC No.13424 and is classified and named as Vibriotornizianae, and has the following characteristics:

(1) the light-emitting material emits bright blue-green visible light in a dark room and has the light-emitting characteristic;

(2) gram-negative bacteria;

(3) the colony on the luminous solid culture medium is round and similar to a steamed bread, the surface is smooth and moist, the edge is neat, and the periphery of the colony is in a sawtooth shape after more than 24 hours;

(4) observing under an oil lens, and taking the shape of a rod or an arc;

the method for separating the photobacteria Fc-11-1 comprises the following steps:

(1) preparation of the Medium

Solid culture medium for luminescent bacteria (isolation medium): 3mL of glycerol, 5g of yeast powder, 5g of tryptone, 1g of calcium carbonate, 20g of agar and 1000mL of aged seawater, wherein the pH value is 7.8-8.0, and the raw materials are sterilized for 20 minutes at 121 ℃;

luminous bacteria liquid culture medium: 5g of yeast powder, 5g of tryptone, 30g of sodium chloride, 5g of disodium hydrogen phosphate, 1g of monopotassium phosphate and 3mL of glycerol, wherein the pH value is 7.6, and the yeast powder is sterilized for 20 minutes at 121 ℃;

seawater 2216E agar slant culture medium: 5g of peptone, 1g of yeast powder, 0.01g of iron phosphate, 20g of agar and 1000mL of aged seawater, wherein the pH value is 7.6, and the sterilization is carried out for 20 minutes at 121 ℃;

(2) washing impurities on the surface of a fish body of a freshly captured marine fish by using a sterile 3% NaCl solution in a sterile environment, respectively collecting 5-6 fish scales, gills and 80% intestines of the fish, respectively putting 45mL of the sterile 3% NaCl solution containing glass beads, placing the mixture on a vortex oscillator, oscillating for 2-3 minutes, fully and uniformly mixing, and placing the mixture on a sterile super clean bench;

(3) respectively sucking 200 μ L of the mixed fish scale, fish gill and fish intestine suspension, injecting into 2216E seawater agar culture medium plate, coating with coating rod, standing for 1min, placing into 25 deg.C incubator, and performing inverted culture;

(4) picking the growing bacterial colony with an inoculating needle, streaking and inoculating the bacterial colony in a luminous bacteria solid culture medium plate, putting the luminous bacteria solid culture medium plate into an incubator at 25 ℃, carrying out inverted culture until bacterial colony is generated, and taking out; observing in a dark room, marking the colonies which emit fluorescence, and numbering;

(5) and (4) picking the marked luminous colonies by using an inoculating needle, and carrying out continuous streak inoculation until pure colonies which can emit fluorescence in a dark room are obtained.

The stale seawater is seawater obtained by storing seawater which is not polluted by land dirt or is rarely mixed with fresh water in a glass bottle for several weeks.

The identification method of the luminous bacteria Fc-11-1 comprises the following steps:

(1) observing the morphological size of plate colonies, and observing the cell morphology by gram staining;

(2) biolog identification

According to an operation manual provided by a Biolog microorganism automatic identifier, the turbidity of the inoculated strain suspension is adjusted, the inoculated strain suspension is inoculated into a 96-hole GN2 micropore identification plate, the plate is cultured for 24 hours, 48 hours and 72 hours at the temperature of 25 ℃, and the plate reading modes of manual operation and automatic operation are combined to respectively measure for 3 times, so that the identification result is obtained.

(3) Preparation of PCR template

The overnight cultured suspension of the photobacteria was formulated to 10⁸CFU/mL (OD600 ═ 0.1), 3. mu.L of the suspension was transferred to a 1.5mL centrifuge tube containing 100. mu.L of sterile double distilled water, and the suspension was subjected to water bath at 99 ℃ for 10min, centrifugation at 10000g for 10min, and after cooling, the supernatant was used as a template for PCR reaction. The extracted DNA template can be stored in a refrigerator at-20 ℃.

(4)16S rDNA PCR amplification

The primers used for the PCR amplification of the 16S rDNA of the luminous bacteria are bacterial 16S rDNA universal primers, and the upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', respectively; a downstream primer 1492R: 5'-TACGGTTACCTTGTTACGACTT-3' are provided.

PCR amplified system (25. mu.L): GoTaq Green Master Mix 12.5. mu.L, upstream primer 27F 1mu.L, the downstream primer 1492R 1. mu.L, the template DNA 2.5. mu.L, ddH₂O8. mu.L. The procedure for PCR amplification was: pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, extension at 72 ℃ for 1min, 30 cycles, and extension at 72 ℃ for 10 min.

The PCR product was detected by electrophoresis on a 1.5% agarose gel.

(5)16S rDNA PCR product sequencing and phylogenetic tree construction

The 16S rDNA PCR product is purified and then sent to the Boehringer Bioengineer (Dalian) Co., Ltd for sequencing. The gene sequence obtained by sequencing is submitted to NCBI website (http:// Blast. NCBI. nlm. nih. gov) for Blast alignment. The construction of the phylogenetic tree was carried out 1000 times by self-development with a Neighbor-join statistical method using MEGA 5.05 software.

The second purpose of the present invention is to provide the application of the above mentioned luminescent bacteria in the detection of chlorothalonil residues. The luminous bacterium Fc-11-1 can be used for detecting chlorothalonil residues in fruits and vegetables, and has the advantages of low cost, simplicity in operation, no need of expensive or large-scale instruments and equipment and the like.

The technical scheme for solving the technical problems is as follows: the application of a luminous bacterium in chlorothalonil residue detection.

The invention also aims to provide a method for detecting chlorothalonil residues. The detection method has the advantages of sensitive reaction, low cost, simple operation, no need of large-scale expensive instruments and equipment and the like.

The technical scheme for solving the technical problems is as follows: a method for detecting chlorothalonil residues comprises the following steps:

(1) weighing 200g of fruit and vegetable samples to be detected, homogenizing or stirring, crushing and homogenizing to obtain samples to be detected;

(2) placing 25g of the sample to be detected obtained in the step (1) into a 250mL conical flask, adding 60mL of acetone and 2mL of phosphoric acid with the mass percentage of 50%, shaking for 2min, filtering, washing the conical flask with 20mL of acetone for 2 times, completely transferring the filtrate into a 250mL separating funnel, adding 100mL of 20g/L sodium sulfate solution, shaking uniformly, extracting with 60mL of cyclohexane for 3 times, standing for layering, drying the extract by an anhydrous sodium sulfate funnel, concentrating under reduced pressure to be nearly dry, blowing dry by nitrogen, fixing the volume by 25mL of sterile double distilled water, carrying out vortex mixing, transferring the mixture into a test tube, and obtaining a chlorothalonil extract;

(3) preparing chlorothalonil standard solution with the concentration of 0-100 mug/kg;

(4) inoculating the separated single colony of the luminous bacteria Fc-11-1 into 10mL of luminous bacteria liquid culture medium, and carrying out shake culture at 25 ℃ and 150r/min for 20h to obtain seed liquid; inoculating 1mL of the seed solution into 150mL of liquid luminous culture medium, and carrying out shaking culture at 25 ℃ at 150r/min to obtain a test bacterial solution;

(5) and (3) when the photon count per second of the luminous intensity of the test bacteria liquid obtained in the step (4) is 3900000-.

On the basis of the technical scheme, the invention can be further improved as follows.

Further, the luminous bacteria liquid culture medium in the step (4) is 5g of yeast powder, 5g of tryptone, 30g of sodium chloride, 5g of disodium hydrogen phosphate, 1g of monopotassium phosphate and 3mL of glycerol, the pH value is 7.6, and the luminous bacteria liquid culture medium is sterilized for 20 minutes at 121 ℃.

Further, the calculation formula of the luminescence inhibition efficiency in the step (5) is as follows:

in the formula:

x%: a luminescence suppression efficiency;

K_c: the luminous intensity of luminous bacteria in the chlorothalonil sample is not added;

K_t: and (3) the luminous intensity of the luminous bacteria in the solution to be detected.

The invention has the beneficial effects that:

1. the invention separates out a luminous bacterium. The luminous bacteria Fc-11-1 enriches the species resource of the luminous bacteria, and is a novel luminous strain different from 11 kinds of luminous bacteria reported in the prior art.

2. The invention provides an application of luminous bacteria in chlorothalonil residue detection.

3. The luminous bacteria of the invention is used for detecting chlorothalonil residues in fruits and vegetables, and has the advantages of low cost, simple operation, no need of expensive or large-scale instruments and equipment, and the like.

Drawings

FIG. 1 is a colony map of a luminescent bacterium Fc-11-1.

FIG. 2 is a cell morphology observation chart of a luminescent bacterium Fc-11-1.

FIG. 3 is a graph showing the growth and luminescence intensity of a luminescent bacterium Fc-11-1.

FIG. 4 is a phylogenetic diagram of a luminescent bacterium Fc-11-1.

FIG. 5 is a standard curve of chlorothalonil concentration versus Fc-11-1 luminescence inhibition.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

Example 1 isolation and identification of Photobacterium Fc-11-1

As shown in FIG. 1 and FIG. 2, 1 luminescent bacterium (FIG. 1) was obtained after isolation, purification and culture, and emitted bright blue-green visible light in a dark room, and had luminescent properties; the gram-stained thalli is red, so that the gram-negative bacteria can be judged; the colony on the luminous solid culture medium is round and similar to a steamed bread, the surface is smooth and moist, the edge is neat, and the periphery of the colony is in a sawtooth shape after more than 24 hours; observing under an oil lens, and taking the shape of a rod or an arc; emits blue-green fluorescence in the dark.

Example 2Biolog identification

The Biolog identification analysis result of the luminous bacteria Fc-11-1 has low similarity with the standard strains in the database, and the species cannot be identified. But the similarity (SIM value 0.128) and the distance (DIST value 8.955) obtained from the statistical evaluation results of the Biolog evaluation system (Table 1) are shown in the same manner asThe Vibrio has high homology, and the physiological and biochemical characteristics of the Vibrio are highly consistent with the characteristics of Vibrio toranziae described by Aide Lasa et al^[1]。

TABLE 1 Biolog identification of Photobacterium Fc-11-1

Note: "+" indicates positive reaction, and "-" indicates negative reaction.

EXAMPLE 3 Effect of incubation time on the growth and luminescence intensity of luminescent bacteria

Effect of the culture time on the growth and luminescence intensity (photon counts per second, measured by a weak luminometer) of the photogenic bacteria Fc-11-1 in a luminescence medium under shaking culture conditions of 150rpm/min at 25 ℃ (FIG. 3). As can be seen from fig. 3, the luminescence and growth of the luminescent bacterium Fc-11-1 were not synchronized, and the luminescence was started when the bacterial concentration reached OD600nm ═ 0.1. When the luminous bacteria grow into the logarithmic growth phase and the bacterial density reaches about OD600 nm-0.7, the luminous bacteria can continuously and stably emit light for 12 h. In the actual detection, the strain with short initial luminescence time and long continuous stable luminescence time can shorten the detection period and is easy to operate and control, and the Fc-11-1 is suitable for detecting toxic substances.

Example 416S rDNA sequencing and phylogenetic analysis

The 16S rDNA sequence of the photobacteria Fc-11-1, with a length of 1471bp, was sequenced and the results were submitted to the NCBI for alignment, as shown in Table 2. The morphological identification result and the highest similarity of the experimental strain 16S rDNA sequence are combined to obtain the separated luminescent bacteria, and the similarity of the separated luminescent bacteria and Vibrio torazoniae is the highest and reaches 100 percent.

TABLE 2 16S rDNA sequence similarity of Photobacterium Fc-11-1

The isolated photobacteria Fc-11-1 were aligned and phylogenetic tree analyzed as shown in FIG. 4.

The results of physiological and biochemical identification and molecular biological identification prove that the luminous bacteria Fc-11-1 obtained by separation is Vibrio torazoniae, and the luminous bacteria are not reported at home.

Example 5 Effect of the luminescent status of the luminescent bacterium Fc-11-1 on its use in chlorothalonil assays

Inoculating the separated single colony of the luminous bacteria Fc-11-1 into 10mL of liquid culture medium, and carrying out shake culture at 25 ℃ and 150r/min for 20h to obtain a seed solution. Inoculating 1mL of the seed solution into 150mL of liquid luminescence culture medium, and performing shaking culture at 25 ℃ at 150 r/min. Transferring the bacteria liquid into test tubes with chlorothalonil concentration of 0, 20, 40, 60, 80 and 100 mu g/kg every 4h under the condition of luminescence. The chlorothalonil solution and the bacterial solution in the reaction system are mixed uniformly in equal volume, then the mixture is subjected to shaking culture at 25 ℃ and 150r/min for 30min, the luminous intensity is measured, 3 groups are parallel respectively, and the results are averaged. Sampling is carried out for 24 h. A standard curve is established by taking the chlorothalonil concentration as an abscissa and the luminescence inhibition rate of the photobacteria Fc-11-1 as an ordinate, and is shown in Table 3. The results in Table 3 demonstrate that the initial luminescence intensity of the luminescent bacteria is 3900000-²The values are all larger than 0.98, and the detection requirement is met.

TABLE 3 Effect of different luminescent states of the luminescent bacterium Fc-11-1 on its use in chlorothalonil assays

Example 6 Effect of duration of action on the use of the luminescent bacterium Fc-11-1 for chlorothalonil detection

Inoculating the separated single colony of the luminous bacteria Fc-11-1 into 10mL of liquid culture medium, and carrying out shake culture at 25 ℃ and 150r/min for 20h to obtain a seed solution. Inoculating 1mL of the seed solution into 150mL of liquid luminescence culture medium, and performing shaking culture at 25 ℃ at 150 r/min. Measuring the luminous intensity to 3900000-60, 80, 100. mu.g/kg. 5mL of chlorothalonil solution and 5mL of bacterial liquid in the reaction system. After mixing evenly, shaking and culturing for 15min, 30min and 60min at 25 ℃ and 150r/min respectively, measuring the luminous intensity, and taking an average value of the results, wherein 3 of each group are parallel. A standard curve was established with chlorothalonil concentration as the abscissa and the luminescence inhibition ratio against the luminescent bacterium Fc-11-1 as the ordinate, as shown in Table 4. The results in Table 4 prove that when the action time of the luminous bacteria for detecting chlorothalonil is 30min, the linear equation is that y is 0.3975x +0.3682, and R is²The value is more than 0.99, which is obviously superior to the condition that the action time is 15min and 60 min.

TABLE 4 determination of the optimal action time of chlorothalonil with the luminescent bacterium Fc-11-1

Example 7 application of Photobacterium Fc-11-1 to the detection of chlorothalonil residues in strawberry

(1) Weighing 200g of fresh strawberries to be detected without internal standards, homogenizing, and then adjusting the pH value of the sample to 7.0-7.6 by using Tris buffer solution;

(2) placing 25mL of the Chinese strawberry homogenate sample in the step (1) into a 250mL conical flask, adding 60mL of acetone and 2mL of phosphoric acid with the mass percentage of 50%, fully shaking for 2min, filtering, washing the conical flask with 20mL of acetone for 2 times, completely transferring the filtrate into a 250mL separating funnel, adding 100mL of 20g/L sodium sulfate solution, shaking uniformly, co-extracting with 60mL of cyclohexane for 3 times, standing for layering, drying the extract by an anhydrous sodium sulfate funnel, concentrating under reduced pressure to be completely dry, blow-drying with nitrogen, fixing the volume by 25mL of sterile double distilled water, carrying out vortex mixing for 1min, transferring the mixture into a test tube, and obtaining a chlorothalonil extract;

(3) preparation of strawberry juice samples with different addition concentrations of chlorothalonil

Respectively taking 25mL of the strawberry juice obtained in the step (1), adding a chlorothalonil standard substance to enable the final concentration of the chlorothalonil to reach 10, 20, 50, 100 and 200 mu g/kg respectively, obtaining an exogenous chlorothalonil-added strawberry sample, and preparing standard solutions with the chlorothalonil concentration of 0, 20, 40, 60, 80 and 100 mu g/kg for drawing a standard curve;

(4) inoculating the separated single colony of the luminous bacteria Fc-11-1 into 10mL of liquid culture medium, and carrying out shake culture at 25 ℃ and 150r/min for 20h to obtain seed liquid; inoculating 1mL of the seed solution into 150mL of liquid luminous culture medium, and carrying out shaking culture at 25 ℃ at 150r/min to obtain a test bacterial solution;

(5) and (3) when the luminous intensity of the bacteria liquid for testing obtained in the step (4) reaches 3900000-4200000 in photon count per second, taking 5mL of the chlorothalonil extract constant volume liquid obtained in the step (2), 5mL of the bacteria liquid for testing obtained in the step (4) and uniformly mixing the solution with the solution, respectively taking 5mL of the bacteria liquid for testing obtained in the step (4) and 5mL of the standard solution with the chlorothalonil concentration of 0, 20, 40, 60, 80 and 100 mu g/kg and uniformly mixing the solution with the solution, culturing the mixture at 25 ℃ for 30min, detecting the luminous intensity of the luminous bacteria, calculating the luminous inhibition efficiency, calculating the concentration of the chlorothalonil according to a standard curve, calculating indexes such as the recovery rate and the like, and obtaining the results shown in a table 5 and a figure 5.

Table 5 recovery of different chlorothalonil concentrations in strawberries (n ═ 6)

In sample detection, when the adding concentration of the chlorothalonil in the strawberry is lower than 20 mug/kg, the recovery rate is lower and is lower than 90%, and along with the increase of the adding concentration, when the adding concentration is higher than 50 mug/kg, the recovery rate of the chlorothalonil is higher than 90%, the precision is 4.95% -10.73%, and the requirements of biological detection are met.

Example 8 application of Photobacterium Fc-11-1 to the detection of chlorothalonil residues in frozen spinach

(1) Weighing 200g of frozen spinach, and stirring and homogenizing;

(2) preparation of frozen spinach samples with different addition concentrations of chlorothalonil

Respectively taking 25g of the frozen spinach obtained in the step (1), adding chlorothalonil standard substances to enable the final concentration of chlorothalonil to reach 10, 20, 40, 50, 100 and 200 mu g/kg respectively, obtaining exogenous chlorothalonil added spinach samples, and preparing standard solutions with chlorothalonil concentration of 0, 20, 40, 60, 80 and 100 mu g/kg for drawing standard curves;

(3) taking 25g of spinach samples, carrying out sample pretreatment in the same manner as the step (3) in the example 7, and extracting chlorothalonil for later use;

(4) luminescent bacteria suspension prepared in the same steps (4) and (5) as in example 7 was reacted with the sample to be tested or the chlorothalonil standard solution, the luminescent intensity of the luminescent bacteria was measured, the inhibition of luminescence was calculated, the measured chlorothalonil concentration was calculated according to the standard curve, and the index such as recovery rate was calculated, and the results are shown in table 6.

TABLE 6 recovery of various chlorothalonil concentrations in frozen spinach (n ═ 6)

In the sample detection, when the concentration of chlorothalonil in spinach is 10, 20 and 40 mu g/kg, the recovery rate is below 90 percent, but when the concentration is 40 mu g/kg, the recovery rate reaches 89.98 percent. When the adding concentration is increased to 50 mug/kg, the recovery rate is over 90 percent, the precision is between 3.07 percent and 8.88 percent, and the requirement of biological detection is met.

The result shows that the detection lower limit of the luminescent bacterium method for detecting the residual chlorothalonil in fruits and vegetables can reach 50 mu g/kg.

Reference to the literature

[1].Aide Lasa,Ana L.Dieguez,Jesus L.Romalde，Vibrio toranzoniaesp.nov.,a new member of the Splendidus clade in the genus Vibrio[J].Systematic and Applied Microbiology,2013,(36):96-100.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

Inspection and quarantine technology center of < 110 > cigarette table entry and exit inspection and quarantine bureau

Luminous bacterium strain of < 120 > and application thereof in chlorothalonil residue detection

〈160〉1

〈170〉PatentIn version 3.5

〈210〉1

〈211〉1471

〈212〉DNA

〈213〉Vibrio toranzoniae

〈400〉1

CTAACACATGCAAGTCGAGCGGTAACGACACTAACAATCCTTCGGGTGCGTTAATGGGCGTCGAGCGGCGGACGGGTGAGTAATGCCTAGGAAATTGCCTTGATGTGGGGGATAACCATTGGAAACGATGGCTAATACCGCATGATGCCTACGGGCCAAAGAGGGGGACCTTCGGGCCTCTCGCGTCAAGATATGCCTAGGTGGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGTTGTGAGGAAGGGGGTAACGTTAATAGCGCTATCTCTTGACGTTAGCAACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCATGCAGGTGGTTCATTAAGTCAGATGTGAAAGCCCGGGGCTCAACCTCGGAACTGCATTTGAAACTGGTGAACTAGAGTACTGTAGAGGGGGGTAGAATTTCAGGTGTAGCGGTGAAATGCGTAGAGATCTGAAGGAATACCAGTGGCGAAGGCGGCCCCCTGGACAGATACTGACACTCAGATGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCTACTTGGAGGTTGTGGCCTTGAGCCGTGGCTTTCGGAGCTAACGCGTTAAGTAGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAATCCAGCGGAGACGCAGGAGTGCCTTCGGGAGCTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTGTTTGCCAGCGAGTCATGTCGGGAACTCCAGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAGAGGGCAGCGAGCCAGCGATGGTAAGCGAATCCCAAAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGCTGCAAAAGAAGTAGGTAGTCTAACCTTCGGGAGGACGCTTACCACTTTGTGGTTCATGACTGGGGTGAAGTCGTAACAAGGTA 1471

Claims

1. A luminescent bacterium is characterized in that the luminescent bacterium is Fc-11-1, is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.13424 and is named in classification as: vibriotorzoniae.

2. Use of the luminescent bacterium of claim 1 in the detection of chlorothalonil residues.

3. A method for detecting chlorothalonil residues is characterized by comprising the following steps:

(4) inoculating the isolated single colony of the photobacteria Fc-11-1 of the claim 1 into 10mL photobacteria liquid culture medium, and performing shaking culture at 25 ℃ and 150r/min for 20h to obtain seed liquid; inoculating 1mL of the seed solution into 150mL of liquid luminous culture medium, and carrying out shaking culture at 25 ℃ at 150r/min to obtain a test bacterial solution;

4. The method for detecting chlorothalonil residues according to claim 3, wherein the luminous bacterial liquid culture medium in the step (4) is composed of 5g of yeast powder, 5g of tryptone, 30g of sodium chloride, 5g of disodium hydrogen phosphate, 1g of potassium dihydrogen phosphate and 3mL of glycerol, has a pH value of 7.6, and is sterilized at 121 ℃ for 20 minutes.

5. The method for detecting chlorothalonil residues according to claim 3, wherein the calculation formula of the luminescence inhibition efficiency in the step (5) is as follows:

in the formula:

x%: a luminescence suppression efficiency;