CN114058672A - Caenorhabditis elegans infection model for high-throughput rapid detection of salmonella virulence and preparation method and application thereof - Google Patents
Caenorhabditis elegans infection model for high-throughput rapid detection of salmonella virulence and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a caenorhabditis elegans infection model for high-throughput rapid detection of salmonella virulence, a preparation method and application thereof, and belongs to the technical field of biology. The model comprises: the caenorhabditis elegans is sek-1, glp-1 gene defect type caenorhabditis elegans; the culture medium for salmonella to infect the caenorhabditis elegans is an S-medium buffer solution, and the pH value is 7.0 +/-0.2; the infection concentration of the salmonella is 107‑1011CFU/mL. Obvious difference appears in the survival rate of the nematodes when the nematodes are infected at the 2 nd to 4 th, and pathogenicity is preliminarily judged. Therefore, the invention can realize the rapid realization by establishing the model of salmonella infecting caenorhabditis elegansThe method is simple, convenient and efficient in detecting the salmonella virulence, and provides a theoretical basis for the research on the pathogenic mechanism of the salmonella and the screening of related drugs.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a caenorhabditis elegans infection model for quickly detecting salmonella virulence in high flux, a preparation method and application thereof.
Background
Salmonella is the causative agent of salmonellosis, belongs to the family enterobacteriaceae, gram-negative enterobacteriaceae, and there are currently nearly thousands of species. The size of the thallus is (0.6-0.9) × (1-3) μm, no spores are generated, no capsules are generated generally, and the whole body flagella are mostly generated except for the salmonella pullorum and the salmonella gallinarum. The nutrition requirement is not high, and the separation culture usually adopts an intestinal tract selection identification culture medium. The biochemical reaction has important reference significance for the identification of the bacteria. Salmonella is highly pathogenic and one of the important pathogens causing zoonosis, including bacteria causing food poisoning, causing gastroenteritis, typhoid and paratyphoid. They can infect many animals including mammals, birds, reptiles, fish, amphibians, and insects in addition to humans. After infection of human and livestock, the disease can be asymptomatic and infected with bacteria, and can also be manifested as clinical symptoms, which can aggravate the disease state or death rate, or reduce the reproductive productivity of animals and bring huge economic loss to the livestock breeding industry, so that a simple and convenient method for detecting the virulence of salmonella and researching the pathogenic mechanism thereof is very necessary.
Caenorhabditis elegans (Caenorhabditis elegans) is a small nematode that lives in soil, feeds on bacteria, is a typical model organism, and has several characteristics: the body is transparent, the structure is simple, and the observation is easy; the life cycle is short and is divided into 4 stages: embryonic development stage, growth development stage, reproductive stage and late reproductive stage. Under the environment of 20 ℃, a life cycle can be completed within 3.5 days, and the test time can be greatly shortened; the gene sequence is known, and the origin of each cell is completely clear; the genetic sequence has 40% homology with mammals and the like. These characteristics make caenorhabditis elegans have very big simplicity and practicality with experimental animals such as mouse, rat, rabbit, more save time, energy and cost. At present, caenorhabditis elegans is widely applied to the researches of toxicology, disease models, pathogenic bacteria pathogenic models and the like. Caenorhabditis elegans is frequently reported for aging research, drug toxicology, drug screening, etc., and researchers have established over 50 nematode models including bacteria, fungi, and viruses. The bacteria include Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and the like. However, the pathogenic model of Salmonella infection of caenorhabditis elegans has not been reported in China.
At present, the caenorhabditis elegans infection model adopts a classical solid plate nematicidal method, but the solid plate nematicidal method has a plurality of defects: the cost is high, and time and labor are wasted; the nematode can avoid pathogenic bacteria according to different odors of the pathogenic bacteria and refuses to eat, so that the purpose of infection cannot be achieved; the number of nematodes climbing on the plate wall to avoid pathogenic bacteria also does not account for the mortality of pathogenic bacteria, resulting in test errors. Therefore, a method for simply, efficiently and quickly detecting the toxicity of pathogenic bacteria is urgently needed to provide a scientific theoretical basis for researching pathogenic mechanisms of pathogenic bacteria and clinical anti-infection treatment.
Disclosure of Invention
The invention aims to provide a caenorhabditis elegans infection model for quickly detecting the toxicity of salmonella in a high-throughput manner, a preparation method and application, so as to solve the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a caenorhabditis elegans infection model for high-throughput rapid detection of salmonella virulence, wherein the caenorhabditis elegans is sek-1, glp-1 gene-defective caenorhabditis elegans; the culture medium for salmonella to infect the caenorhabditis elegans is an S-medium buffer solution, and the pH value is 7.0 +/-0.2; the infection concentration of the salmonella is 107-1011CFU/mL, the survival rate of the 2 nd to 4 th infected nematodes is obviously different, so that pathogenicity can be preliminarily judged, and the aim of quick screening can be achieved.
The invention also provides application of the model in screening of anti-salmonella medicaments.
The invention also provides a preparation method of the model, which comprises the following steps:
(1) culturing salmonella and preparing a bacterial liquid;
(2) performing synchronous culture on caenorhabditis elegans to obtain L4-stage nematode;
(3) and (3) co-culturing the L4 stage nematodes obtained in the step (2) in the salmonella bacteria liquid obtained in the step (1) for 2-4 days.
Preferably, the preparation of the bacterial liquid comprises the following steps:
inoculating salmonella into a Macconk solid culture medium for recovery and purification, and then selecting a single colony to be inoculated into an LB liquid culture medium for culture to obtain a culture solution;
centrifuging the obtained culture medium, discarding supernatant, washing with S-medium buffer solution, and adjusting the concentration of the bacterial liquid to 107-1011CFU/mL, obtaining bacterial liquid with different concentrations.
Preferably, the culture condition of the single colony in the LB culture medium is 100-135r/min shake culture for 16-18 h.
Preferably, the specific steps of step (2) include:
s1: after the freezing tube containing the caenorhabditis elegans is quickly melted, centrifuging, removing supernatant, and pouring the caenorhabditis elegans into an NGM flat plate to recover the caenorhabditis elegans;
s2: selecting NGM plate containing a large amount of nematodes and eggs, repeatedly washing with M9 buffer solution, collecting washing solution, splitting, centrifuging, precipitating, transferring to NGM plate containing Escherichia coli OP50, placing in biochemical incubator at 20 deg.C, and allowing nematodes to grow to L4 stage after 40 hr.
Preferably, the temperature of the medium melting in S1 is 37 ℃, and the centrifugation is carried out for 1min under the centrifugation condition of 3000 r/min.
Preferably, the cracking time in S2 is 4min, and after cracking and centrifugation, the pellet containing the worm eggs is resuspended by using S basic solution and then is placed under the conditions of 20 ℃ and 135r/min for shake culture for 12-18 h; after centrifugation at 3000rpm for 1min, the pellet was transferred to an NGM plate containing OP 50.
Preferably, the step (3) further comprises the following steps:
adding L4 stage nematode into Salmonella bacteria solution, adjusting pH to 7.0 + -0.2, and culturing at 25 deg.C.
The invention also provides application of the preparation method in screening drugs, which is used for screening drugs having inhibitory action on salmonella in defective caenorhabditis elegans.
The invention discloses the following technical effects:
the infection model and the method can be used for quickly detecting the toxicity of the salmonella with high flux, can be used for screening various medicines which have the inhibiting effect on the salmonella in the caenorhabditis elegans, and have the characteristics of low preparation cost, short period and easy operation compared with other animal models. Compared with a solid culture medium for preparing the caenorhabditis elegans infection model, the method is time-saving and labor-saving, reduces the cost, avoids the trouble of plate rotation every day, ensures that each caenorhabditis elegans in a liquid culture medium can be infected, reduces the test error of the caenorhabditis elegans evading pathogenic bacteria and refusing to eat, and is a method for detecting the virulence of pathogenic bacteria more simply, conveniently, efficiently and quickly by using the caenorhabditis elegans infection model. Can reduce test error, and is favorable for providing scientific theoretical basis and test data for drug screening and clinical use. Therefore, the invention provides a simple, efficient and rapid method for detecting the toxicity of salmonella, which can lay a foundation for researching pathogenic mechanisms of pathogenic bacteria and provide scientific theoretical basis for clinical anti-infection treatment.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 shows Salmonella carrying green fluorescence under a fluorescence microscope;
FIG. 2 is a fluorescent microscope infected with Salmonella carrying green fluorescence;
FIG. 3 is 109Lethal results for CFU/mL of Salmonella.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
The following examples refer to the main test materials:
mackanka agar, LB broth from Beijing Luqiao technology, Inc.; a bacterial DNA extraction kit, 2 × ES Taq MasterMix, DNA Marker purchased from Beijing kang, century Biotechnology Co., Ltd; agarose gel, available from spanish GENE; PCR instruments were purchased from Bio-Rad, USA, and gel imaging analyzers were purchased from Alpha Inotech, USA; peristaltic pumps were purchased from pump industry ltd, baoding. NGM culture medium, SK culture medium, M9 buffer solution, nematode lysate, S basic solution, S-medium buffer solution, S-medium solid culture medium, etc. by the method of Abalay et al.
The salmonella is a green fluorescence-carrying strain SM022 stored in a bacterial research laboratory of the Guangxi Zhuang autonomous region veterinary institute.
sek-1, glp-1 gene-deficient C.elegans was complimentary to the molecular genetics and microbiology laboratories of the university of Duke college of medicine.
EXAMPLE 1 preparation of Salmonella and nematodes
1. Resuscitation of Salmonella
Inoculating the preserved salmonella to a Macconk solid culture medium, culturing overnight at 37 ℃, selecting a single colony, streaking again, purifying and culturing, and observing under a fluorescence microscope.
The results show that: the recovered strain is observed under a fluorescence microscope, and the colony is green, which indicates that the recovered strain is salmonella SM022 (shown in figure 1) carrying green fluorescence and preserved by a bacterial research laboratory of the autonomous region veterinary research institute of Guangxi province.
2. Preparation of salmonella liquid with different concentrations
Selecting single colony from solid culture medium, culturing in LB liquid culture medium under shaking at 100r/min for 16h, centrifuging at 4000rpm for 10min, discarding supernatant, washing the colony with S-medium solution for 3 times, and adjusting the concentration of the bacteria solution to 10 with McLeeb tube7-1011CFU/mL, spare.
3. Recovery of nematodes
Taking out the freezing tube containing the nematodes from the liquid nitrogen tank, placing the tube in a prepared water bath kettle at 37 ℃, rapidly thawing the nematode liquid, centrifuging at 3000rpm, discarding the supernatant, repeatedly washing with M9 buffer solution for 3 times to remove the freezing liquid in the tube, and finally pouring the nematodes into the NGM culture medium to finish the recovery of the nematodes.
4. Passage of nematodes
Selecting an NGM flat plate which is free from mixed bacteria pollution and contains a large amount of nematodes and worm eggs, sterilizing a clean blade by using an alcohol lamp, cutting a culture medium and reversely buckling the cut culture medium in a new NGM culture medium, so that the nematodes can climb from the old flat plate to the new flat plate, and the passage of the nematodes is finished.
5. Synchronization treatment of nematodes
Selecting an NGM flat plate which is free from mixed bacteria pollution and contains a large amount of pregnant insects and insect eggs, repeatedly washing the NGM flat plate with M9 buffer solution to wash the insect eggs and the bacterial solution, collecting the washed NGM flat plate into a sterile 15mL centrifugal tube, adding M9 buffer solution to supplement to 10mL, centrifuging at 3000rpm for 1min, discarding 9mL supernatant, adding 1mL nematode lysate, performing vortex oscillation for 4min, centrifuging at 3000rpm for 1min, discarding supernatant, adding M9 buffer solution to supplement to 10mL, washing for three times to remove lysate, adding S basic solution to supplement to 10mL, and oscillating at 20 ℃ and 100r/min for 12 h. After 12h, centrifuging at 3000rpm for 1min, transferring the precipitate to NGM plate containing OP50, placing in biochemical incubator at 20 deg.C, and after 40h, the nematode will grow to L4 stage for use.
Example 2 establishment of a C.elegans infection model for high-throughput rapid detection of Salmonella virulence
Firstly, the ratio of salmonella bacteria liquid and S-medium added into a twenty-four pore plate is preliminarily searched, and the method comprises the following steps:
(1)5 mu L of salmonella liquid and 2mL of S-medium buffer solution;
(2)10 mu L of salmonella liquid and 2mL of S-medium buffer solution;
(3)50 mu L of salmonella liquid and 2mL of S-medium buffer solution;
(4)100 mu L of salmonella liquid and 2mL of S-medium buffer solution;
(5)200 mu L of salmonella liquid and 2mL of S-medium buffer solution;
(6)500 mu L of salmonella liquid and 2mL of S-medium buffer solution;
(7)1mL of salmonella liquid and 1mL of S-medium buffer solution;
the preliminary screening result shows that 100 mu L of salmonella liquid and 2mL of S-medium buffer solution are in the optimal proportion.
Next, the optimum infection concentration of the Salmonella bacteria liquid was investigated, and 100. mu.L of the Salmonella bacteria liquid prepared in example 1 and having different concentrations was added to 24-well plates containing 2mL of S-medium buffer solution, and 3 replicate wells were made for each concentration. The L4-phase nematodes on the NGM plates in example 1 were washed with M9 buffer solution repeatedly for 2 times, then washed with S-medium buffer solution for 2 times to remove mixed bacteria, and then cultured with the L4-phase nematodes in the 24-well plates at an average ratio of 15-20 nematodes per well, and the pH was adjusted to 6.8 at 25 ℃. Observing the nematodes under a body type microscope every 24h by taking Escherichia coli OP50 as a negative control, recording the survival rate, observing the infection of the nematodes under a fluorescence microscope and taking pictures, wherein the number of the negative controls is 109The lethality results for CFU/mL Salmonella are shown in FIG. 3 and Table 1.
Watch 1109Lethal results for CFU/mL Salmonella
As a result, it was found that: nematodes infected with salmonella carrying green fluorescence could be clearly seen and distinguished under the fluorescence microscope (see fig. 2). And at different infection concentrations, 109The infection effect of CFU/mL is the best. At 109At the infection concentration of CFU/mL, the lethality showed a significant difference from the negative control OP50, and the survival rate began to drop significantly at 2-4d of infection, with the nematodes showing slow movement, decreased feeding, movement with needle stimulation of the teaser or even immobility, and all the 22 nd beetles died (see fig. 3 and table 1).
Comparative example 1
The difference from the embodiment 2 lies in that the solid culture method is adopted, and specifically comprises the following steps: adding 100 mu L of salmonella bacterial liquid prepared in the example 1 into an SK solid culture medium, uniformly coating the bottom of a sterilization test tube to ensure that the bacterial liquid is completely paved on the culture medium, placing the culture medium in a constant-temperature incubator at 37 ℃ for overnight drying to form bacterial lawn, and picking the L4 phase gene-defective nematodes which are treated synchronously into the SK solid culture medium containing the Salmonella fungal lawn by using a pest picking needle which is burnt and recovered to room temperature, wherein each plate contains 20 worms, and each group is repeated for 3 times. And E.coli OP50 is used as a negative control, the nematodes are observed under a body type microscope, the survival rate is recorded, and the infection conditions of the nematodes are observed under a fluorescence microscope.
As a result, it was found that 10 out of various infection concentrations were observed when the culture was carried out in the solid medium10The infection effect of CFU/mL is the best. At 1010The lethality at the infection concentration of CFU/mL compared to the negative control OP50 showed a significant difference, beginning to drop significantly at 4-6d survival of the infection, but not all of the 30 th aeriform insects died.
Comparative example 2
The difference from example 2 is that: the pH was adjusted to 6.5, 7.0, 7.2, 7.8, 8.0, 8.5, the other steps being the same.
As a result, it was found that: the pH values of 6.5, 7.8 and 8.0 can influence the shape of the nematode polypide, are not beneficial to observation after salmonella infection, and are easy to cause test errors. And when the pH values are 7.0, 7.2 and 7.8, the normal development of the nematode can be observed as in example 2, and the nematode morphology cannot be influenced after the infection of bacteria, so that the observation is convenient, and the experimental error caused by the change of the nematode morphology cannot be caused.
Comparative example 3
The difference from example 2 is that: wild caenorhabditis elegans is adopted, and other steps are the same.
As a result, it was found that: to avoid interference with counting of the larvae produced, the nematodes were transferred from the old plate to the new plate every day; in order to keep the solid plate free from the pollution of fungi and mould, the plate is coated every day and then placed in an incubator to be dried for standby, which consumes manpower, financial resources and time.
Comparative example 4
The difference from the embodiment 2 is that: after the L4 stage nematodes were added to the 24-well plates, the pH was adjusted to 6.8 and the cells were cultured at 20 ℃. The other steps are the same.
As a result, it was found that: the number of nematodes per well increases and, due to the partial reproduction of caenorhabditis elegans under suitable conditions, the resulting larvae can affect the count, causing experimental errors.
Simple analysis from the above examples and comparative examples: the main reasons for selecting sek-1, glp-1 gene defective caenorhabditis elegans in the invention are: firstly, when liquid test is carried out in a 24-pore plate, the plate does not need to be changed every day on a solid flat plate, if wild caenorhabditis elegans is selected as a test animal, the produced larvae interfere with counting, so that the result is inaccurate, and the selective gene defective nematodes are sensitive to temperature due to lack of glp-1 genes and lose fertility at 25 ℃, so that the number of nematodes in the pores is not increased, thereby avoiding the experimental error; and secondly, because the gene-deficient nematode lacks sek-1 gene, the gene-deficient nematode is more sensitive to pathogenic bacteria compared with wild caenorhabditis elegans, and the immunity of the nematode for resisting pathogenic bacteria is lower, thereby achieving the purpose of more rapid infection of the nematode.
Different from a solid plate nematode killing method, when a liquid culture medium is used for infecting nematodes, the selection of the concentration of pathogenic bacteria is crucial, different infection concentrations have different degrees of difference on the killing effect of the nematodes and are important determinants of the infection degree of the nematodes, and the optimal infection effect cannot be achieved when the concentration is low or high. The invention comprehensively considers the infection concentration and other factors, and tests show that 109The infection effect of CFU/mL is the best, the lethality of the CFU/mL is obviously different from that of negative control OP50, the survival rate of the CFU/mL is obviously reduced at the 2 nd to 4d of infection, and the lethality rate of the CFU/mL is obviously improved compared with that of a solid plate nematicidal method, so that a liquid test infection model of Salmonella infected caenorhabditis elegans is successfully established. Compared with a solid plate method, the liquid test model disclosed by the invention has the advantages of saving time and labor, reducing cost, avoiding the trouble of plate rotation every day, ensuring that each nematode in a liquid culture medium can be infected, reducing the test error of eating refusal of the nematodes due to escape of pathogenic bacteria, and finally, because the solid plate is extremely easy to cause fungal and mold pollution and is difficult to perceive in the preparation and storage processes under the hot and humid environment of southern climate, the solid plate can generate interference effect when judging the pathogenicity test of the pathogenic bacteria of the test on the nematodes, the real pathogenicity of the pathogenic bacteria of the test can not be known exactly, and the pollution problem can be avoided by using the twenty-four pore plates to carry out the liquid testA method for efficiently and quickly detecting the virulence of pathogenic bacteria.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (10)
1. A caenorhabditis elegans infection model for high-throughput rapid detection of salmonella virulence is characterized in that the caenorhabditis elegans is sek-1, glp-1 gene-deficient caenorhabditis elegans; the culture medium for salmonella to infect the caenorhabditis elegans is an S-medium buffer solution, and the pH value is 7.0 +/-0.2; the infection concentration of the salmonella is 107-1011CFU/mL。
2. Use of the model of claim 1 for screening for a drug against salmonella.
3. A method of making a mold as defined in claim 1, comprising the steps of:
(1) culturing salmonella and preparing a bacterial liquid;
(2) performing synchronous culture on caenorhabditis elegans to obtain L4-stage nematode;
(3) and (3) co-culturing the L4 stage nematodes obtained in the step (2) in the salmonella bacteria liquid obtained in the step (1) for 2-4 days.
4. The method according to claim 3, wherein the preparation of the bacterial liquid comprises the following steps:
inoculating salmonella into a Macconk solid culture medium for recovery and purification, and then selecting a single colony to be inoculated into an LB liquid culture medium for culture to obtain a culture solution;
centrifuging the obtained culture medium, discarding supernatant, washing with S-medium buffer solution, and adjusting the concentration of the bacterial liquid to 107-1011CFU/mL，Obtaining bacterial liquids with different concentrations.
5. The method of claim 4, wherein the single colony is cultured in LB medium under shaking conditions of 100-.
6. The method according to claim 3, wherein the specific steps of step (2) include:
s1: after the freezing tube containing the caenorhabditis elegans is quickly melted, centrifuging, removing supernatant, and pouring the caenorhabditis elegans into an NGM flat plate to recover the caenorhabditis elegans;
s2: selecting NGM plate containing a large amount of nematodes and eggs, repeatedly washing with M9 buffer solution, collecting washing solution, splitting, centrifuging, precipitating, transferring to NGM plate containing Escherichia coli OP50, placing in biochemical incubator at 20 deg.C, and allowing nematodes to grow to L4 stage after 40 hr.
7. The method according to claim 6, wherein the thawing temperature in S1 is 37 ℃ and the centrifugation is performed at 3000r/min for 1 min.
8. The method as claimed in claim 6, wherein the lysis time in S2 is 4min, the pellet containing the ova after lysis and centrifugation is resuspended in S basic solution, and then shake-cultured at 20 ℃ and 135r/min for 12-18 h; after centrifugation at 3000rpm for 1min, the pellet was transferred to an NGM plate containing OP 50.
9. The method according to claim 3, wherein the step (3) further comprises the steps of:
adding L4 stage nematode into Salmonella bacteria solution, adjusting pH to 7.0 + -0.2, and culturing at 25 deg.C.
10. Use of the process according to any one of claims 3 to 9 for screening drugs for inhibition of salmonella in c.
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