CN112725474B - Listeria monocytogenes standard strain containing specific molecular target and detection and application thereof - Google Patents
Listeria monocytogenes standard strain containing specific molecular target and detection and application thereof Download PDFInfo
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Abstract
The invention provides a specific molecular target for detecting Listeria monocytogenes, and a strain containing the molecular target and a quantitative preservation method thereof, wherein the specific molecular target comprises a nucleotide sequence shown in SEQ ID NO 1-4, the 4 strains contain the Listeria monocytogenes with the molecular target specificity, the preservation numbers of the Listeria monocytogenes are GDMCC 60836, GDMCC60838, GDMCC60839 and GDMCC 60840 respectively, the strain has standard Listeria monocytogenes bacterial microscopical forms and physiological and biochemical characteristics, and can be used for detecting the accuracy of a Listeria monocytogenes chromogenic plate and a standard strain verified by related reagents. The invention also provides a freeze-drying protective agent which is easy to form, has good water solubility, can be completely dissolved within 1-2 seconds, can be stored for at least more than one year at the temperature of-20 ℃, and can be used for storing quality control strains for a long time.
Description
Technical Field
The invention belongs to the technical field of microbial detection, and particularly relates to a molecular target for detecting Listeria monocytogenes and a standard bacterium thereof.
Background
Listeria monocytogenes (Listeria monocytogenes) is a gram-positive facultative anaerobic bacterium, an important food-borne pathogenic bacterium in China, and widely exists in edible fungi, livestock and poultry meat, frozen food and various natural environments. The consumer eating the food of the pollution listeria monocytogenes can cause infection, the main symptoms comprise severe listeriosis such as bacteremia, septicemia, meningoencephalitis and abortion, sensitive people comprise newborns, pregnant women, old people and people with low immunity, the fatality rate is up to 20% -30%, and under normal conditions, the number of the edible pollution listeria monocytogenes exceeds 105The CFU food can cause human infection, and has lower infection amount for people with low immunity. Therefore, the listeria monocytogenes is one of the key monitoring objects of food-borne pathogenic microorganisms, the pollution conditions of the listeria monocytogenes in different kinds of foods are known by various detection methods, the propagation rule and the potential pathogenicity of the listeria monocytogenes in the foods are the basis for effectively preventing and controlling the food-borne diseases caused by the listeria monocytogenes, and the reliability of the research result is determined by whether a proper standard strain is used as a contrast. The standard strain is from the collection of international or domestic strain collection center, and has genetic characteristics and stabilityThe qualitative results are confirmed and guaranteed and traceable strains. At present, standard listeria monocytogenes strains are mainly derived from American Type Culture Collection (ATCC), chinese medical bacterial Collection management center (CMCC), chinese industrial microbial Collection management center (CICC), and the like.
In the aspect of research on a long-term preservation method of a listeria monocytogenes standard substance, the protective agent adopted in the current report is basically used for qualitatively storing the listeria monocytogenes, and living bacteria can be ensured to exist in the shelf life. Chinese patent CN102140423B adopts 0.1-10 parts by mass of water-soluble sugar, 0.1-5 parts by mass of defatted milk powder, 0.1-20 parts by mass of gelatin and 0-10 parts by mass of active carbon as a protective agent for quantitatively preserving single listeria monocytogenes, the protective agent does not mention the freeze-drying survival rate, and the solubility of the protective agent is influenced under the normal temperature condition or in winter due to the fact that the protective agent contains a certain concentration of gelatin, and the dissolution rate is slow; in addition, the protective agent contains active carbon which is insoluble in aqueous solution, is precipitated at the bottom of a bottle after freeze-drying and is scattered on the surface of a flat plate during recovery, so that the appearance is influenced and the automatic counting of bacterial colonies is influenced. Chinese patent CN201910388287.2 discloses 40-60% of skimmed milk, 0.2-0.8% of polyvinylpyrrolidone, 2-6% of sucrose, 0.2-0.8% of tyrosine and sterile steamed water as a solvent, which is used for freeze-drying protection and preservation of a standard substance of Listeria monocytogenes, but the viable bacteria rate of the standard substance is maintained at a stable level of only about 30% within 30 days. In the prior art, no special freeze-drying protective agent with excellent protective performance for quantifying the Listeria monocytogenes standard substance exists, so that a method for quantitatively preserving standard strains for a long time, which can accurately control the bacterial count, has good stability and is convenient to use, for the Listeria monocytogenes standard substance is very necessary to be developed.
At present, most standard strains for researching the evolution law of the strain and potential pathogenic use are strains from American model culture collection and storage, and cannot well reflect the propagation characteristics of the strain in food, according to early domestic research reports, the Listeria monocytogenes has pollution of different levels in food samples such as instant food, edible fungi, aquatic products, frozen food and the like (Chen et al, food control, 2014; Chen et al, Frontiers in microbiology, 2015; Chen et al, Frontiers in microbiology,2018), but lacks representative isolates to research the genetic structure, propagation law and potential hazards of the strain in China. The research team systematically finishes the food source pathogenic microorganism risk investigation of 4300 parts of food samples in 43 representative cities/regions in China for the first time, and at present, more than 45 Sequence Type (ST) 2500 Listeria monocytogenes strains are separated and preserved, and are the strain resource library with the largest amount of food source type Listeria monocytogenes preserved strains in China, wherein ST87 is one of the superior ST types of food source type Listeria monocytogenes in China and has high toxicity characteristics. At present, most of standard strains used for researching the evolution law and pathogenicity of the strain are strains from American microbial strain preservation center, the propagation characteristics of the strain in food cannot be well reflected, the detection rate of Listeria monocytogenes in food in China is always at a high level, and a representative isolate is lacked for researching the genetic structure and the propagation law of the strain in China.
In addition, the preparation of the freeze-drying protective agent for quantifying the listeria monocytogenes standard substance is the key for long-term stable and effective preservation of the standard substance. At present, only Chinese patent CN102140423B adopts 0.1-10 parts by mass of water-soluble sugar, 0.1-5 parts by mass of skimmed milk powder, 0.1-20 parts by mass of gelatin and 0-10 parts by mass of active carbon as a protective agent for quantitatively preserving Listeria monocytogenes, the protective agent does not mention the freeze-drying survival rate, and the solubility of the protective agent is influenced and the dissolution speed is slow under the normal temperature condition or in winter due to the gelatin with a certain concentration; in addition, the protective agent contains active carbon which is insoluble in aqueous solution, is precipitated at the bottom of a bottle after freeze-drying and is scattered on the surface of a flat plate during recovery, so that the appearance is influenced and the automatic counting of bacterial colonies is influenced. Chinese patent CN201910388287.2 discloses 40-60% of skimmed milk, 0.2-0.8% of polyvinylpyrrolidone, 2-6% of sucrose, 0.2-0.8% of tyrosine and sterile steamed water as a solvent, which is used for freeze-drying protection and preservation of Listeria monocytogenes standard substances, but the viable bacteria rate of the Listeria monocytogenes standard substances is maintained at a stable level of only about 30% within 30 days. Therefore, the freeze-drying protective agent which has high freeze-drying survival rate and can quantitatively store the listeria standard substance for a long time is of great significance.
Disclosure of Invention
In order to solve the problems, the genetic diversity of the listeria monocytogenes is analyzed by multi-site sequence typing, 4 listeria monocytogenes dominant isolates and high-toxicity isolates which pollute food samples in China are provided, and the strains have stable and typical physiological and biochemical characteristics of the listeria monocytogenes, can better reflect the genetic background of the listeria monocytogenes in food source isolates in China and have good representativeness. The strain 428-1LM is separated from the brine duck, and the preservation number is as follows: GDMCC 60836, classification name: listeria monocytoenes; strain 678-1LM is separated from the brine duck, and the preservation number is as follows: GDMCC60838, classification name: listeria monocytoenes; strain 833-1LM is separated from frozen mutton roll, and the preservation number is as follows: GDMCC60839, classification name: listeria monocytoenes; strain 1382-1LM was isolated from frozen drumsticks with the accession number: GDMCC 60840, classification name: listeria monocytoenes; the 4 separated listeria monocytogenes strains are all preserved in Guangdong province microorganism strain preservation center with the address: the preservation date of the five storied building of the experimental building of the microbiological institute, namely 100 province, of the first furious Zhonglu city in China is as follows: 27 days 10 and 2019.
The technical scheme adopted by the invention is as follows:
the invention provides a specific molecular target for detecting Listeria monocytogenes, which is:
(a) 1-4 of any one or more nucleotide sequences shown in SEQ ID NO; alternatively, the first and second electrodes may be,
(b) the nucleotide sequence in (a) is subjected to substitution, deletion or addition of one or more nucleotides, and has more than 90% homology with the nucleotides in (a). According to the invention, the specific molecular target of the invention is obtained by pan-genomic analysis and comparison of newly obtained Listeria monocytogenes, and the specific Listeria monocytogenes can be specifically detected, so that the specific Listeria monocytogenes has strong specificity.
The invention also provides a primer for detecting the specific molecular target, and the PCR primer for amplifying the nucleotide sequence shown as SEQ ID NO. 1 comprises: an upstream primer shown as SEQ ID NO. 5 and a downstream primer shown as SEQ ID NO. 6; the PCR primer for the nucleotide sequence amplification shown as SEQ ID NO. 2 comprises: an upstream primer shown as SEQ ID NO. 7 and a downstream primer shown as SEQ ID NO. 8; the PCR primer for the amplification of the nucleotide sequence shown as SEQ ID NO. 3 comprises an upstream primer shown as SEQ ID NO. 9 and a downstream primer shown as SEQ ID NO. 10; the PCR primers for the amplification of the nucleotide sequence shown in SEQ ID NO. 4 include an upstream primer shown in SEQ ID NO. 11 and a downstream primer shown in SEQ ID NO. 12.
The present invention also provides a Listeria monocytogenes (Listeria monocytogenes) which is (a), (b), (c) or (d):
(a) strain 428-1LM, comprising the nucleotide sequence shown in SEQ ID NO. 1;
(b) strain 678-1LM, containing the nucleotide sequence shown in SEQ ID NO. 2;
(c) strain 833-1LM, which contains the nucleotide sequence shown in SEQ ID NO. 3;
(d) strain 1382-1LM, contains the nucleotide sequence shown in SEQ ID NO. 4.
Preferably, said strain 428-1LM further comprises at least one of the following virulence genes: llsX, actA, mpl, prfA, hly, inlA, inlB, iap, plcA, and plcB.
Preferably, said strain 678-1LM further comprises at least one of the following virulence genes: actA, mpl, prfA, hly, inlA, inlB, iap, plcA, and plcB.
Preferably, said strain 833-1LM further comprises at least one of the following virulence genes: actA, mpl, prfA, hly, inlA, inlB, iap, plcA, and plcB; or at least one of the following antibiotic resistance genes: kanamycin, clindamycin, streptomycin, sulfamethoxazole, erythromycin, tetracycline or chloramphenicol.
Preferably, said strain 1382-1LM further comprises at least one of the following virulence genes: actA, mpl, prfA, hly, inlA, inlB, iap, plcA, plcB and ptsA.
Preferably, the strain 428-1LM has a deposit number of: GDMCC 60836; the preservation number of the strain 678-1LM is GDMCC 60838; the preservation number of the strain 833-1LM is GDMCC 60839; the strain 1382-1LM has a collection number of GDMCC 60840.
The invention also provides application of the listeria monocytogenes in researching the antibiotic resistance of the listeria monocytogenes. Preferably, the antibiotic resistance of the strain 833-1LM is resistance to kanamycin, clindamycin, streptomycin, compound sulfamethoxazole, erythromycin, tetracycline or chloramphenicol
The invention also provides application of the listeria monocytogenes in improving the accuracy of detecting the listeria monocytogenes color development plate.
The invention also provides a freeze-drying protective agent for listeria monocytogenes, which comprises the following components in parts by weight: 0.1-3 parts of potassium lactate, 7-15 parts of skimmed milk powder, 1-4 parts of sodium alginate, 0.2-5 parts of bovine serum albumin and 0.1-3 parts of glycine.
The components are selected because sodium alginate has strong hydrophilicity, during freezing or drying, polar groups of thallus protein form hydrogen bonds to protect structural and functional integrity of cell membranes and proteins, skim milk powder and bovine serum albumin can wrap the outer layer of the thallus to protect the thallus, and amino acid ions have acid-base amphipathy, so that the pH value in a bacterial suspension can be stabilized and the pH value can be adjusted to the most stable region of an active substance during freezing, drying and storage. Potassium lactate acts as an antioxidant, reducing cellular oxidase activity during the freeze-drying process and long-term storage, preventing oxidative deterioration of the lyophilized product.
Preferably, the lyoprotectant comprises the following components in parts by weight: 0.5 part of potassium lactate, 12 parts of skimmed milk powder, 3 parts of sodium alginate, 3 parts of bovine serum albumin and 0.5 part of glycine.
The invention has the beneficial effects that: the standard substance of the listeria monocytogenes strain has standard bacillus microscopic morphology and physiological and biochemical characteristics, and can be used for detecting the accuracy of a listeria chromogenic culture medium. The standard strains have clear sources of standard substances, clear sequence types, specific situations of carried virulence genes and clear drug resistance characteristics and genetic backgrounds, are the only dominant strains and high-virulence strains which can reflect the genetic backgrounds of food source listeria monocytogenes in China at present compared with other standard strains of the species, and can be used as reference strains for scientific research.
The quantitative listeria monocytogenes prepared by the freeze-drying protective agent has the following advantages:
1. good molding, beautiful appearance and good water solubility, and can be completely dissolved within 1-2 seconds.
2. The freeze-drying survival rate can reach more than 90 percent.
3. Can be stored for at least more than one year at the temperature of 2-8 ℃, has no change in quantity value, and can be used for long-term storage of quantitative quality control strains.
Drawings
FIG. 1 is a morphological diagram of Listeria monocytogenes strain by microscopic examination (wherein a: 428-1LM strain, b: 678-1LM strain, c: 833-1LM strain, d: 1382-1LM strain).
FIG. 2 is a colony morphology chart of the Listeria monocytogenes strain (wherein a: 428-1LM grows colony morphology on Listeria chromogenic plate, b: 428-1LM grows colony morphology on PALCAM plate, c: 678-1LM grows colony morphology on Listeria chromogenic plate, d: 678-1LM grows colony morphology on PALCAM plate, e: 833-1LM grows colony morphology on Listeria chromogenic plate, f: 833-1LM grows colony morphology on PALCAM plate, g: 1382-1LM grows colony morphology on Listeria chromogenic plate, h: 1382-1LM grows colony morphology on PALCAM plate).
FIG. 3 is a schematic representation of the biochemical identification of API Listeria of 4 strains of Listeria monocytogenes (wherein a: 428-1LM strain; b: 678-1LM strain; c: 833-1LM strain; d: 1382-1LM strain).
FIG. 4 is a PCR amplification chart of the unique gene fragment of the standard substance of the Listeria monocytogenes strain (wherein a: 428-1LM strain specific molecular target amplification result; b: 678-1LM strain specific molecular target amplification result; c: 833-1LM strain specific molecular target amplification result; d: 1382-1LM strain specific molecular target amplification result), it can be seen that the strain marked "+" in the figure has a target band amplified by a specific molecular target, namely the strain of the invention, and 1-74 are the strains of other sequence types (1,2,3,5,7,8,9,11,14,20,37,59,87,91,101,120,155,199, 204,224,288,296,297,307,310,323,325,330,378,391,429,504,506,515,619, 621,705,767,804,805,807,1166) of the listeria monocytogenes strain, and no band amplified wood is found except the listeria monocytogenes strain standard substance, which indicates that the specific gene fragment of the listeria monocytogenes strain standard substance has specificity.
FIG. 5 is a schematic diagram showing the changes of Listeria monocytogenes in the storage period of quantitative quality control bacteria.
Detailed Description
In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the technical solutions of the present invention is provided with reference to specific embodiments and accompanying drawings.
EXAMPLE 1 isolation and culture of Listeria monocytogenes strains
Completely shearing food samples under the aseptic condition, uniformly mixing, adding 25g (mL) of aseptic-operation samples into a homogenizing bag containing 225mL of LB1 enrichment broth, continuously homogenizing for 1-2min on a slapping homogenizer, culturing for 24 +/-1 h at 30 +/-1 ℃, transferring 0.1mL of the product into 10mL of LB2 enrichment broth, and culturing for 24 +/-1 h at 30 +/-1 ℃. Taking 10 mu L of LB2 secondary enrichment broth, streaking the 10 mu L of secondary enrichment broth on a Listeria monocytogenes chromogenic culture medium and a PALCAM selective plate, culturing the mixture for 25 +/-1 h at 37 +/-1 ℃, observing bacterial colonies growing on each plate, wherein the Listeria monocytogenes is in a circular blue-green color on the Listeria monocytogenes chromogenic culture medium, and the typical white halo bacterial colonies are arranged around the Listeria monocytogenes, namely the Listeria monocytogenes. And (3) streaking the target colony on a TSA-YE plate, culturing for 24 +/-1 h at 37 +/-1 ℃, and selecting single colonies with gram-positive staining, oxidase negative and hydrogen peroxide positive reactions for identification of morphological characteristics, physiological biochemistry, serotype, molecular biology and the like. At the same time, the target colonies were transferred from TSA to tryptone soy broth and allowed to recover overnight at 37 ℃. Adding the bacterial liquid into a glycerol tube with the final concentration of 30% under the aseptic condition, storing in a refrigerator at minus 80 ℃, and performing freeze-drying tube storage.
Example 2 physio-biochemical characteristics and drug sensitivity characteristics of Listeria monocytogenes
(1) And (3) dyeing microscopic examination: the colonies were smeared, gram stained, and the morphology was visualized by microscopic examination. The single gram positive bacterium is short rod-shaped, has no spore and has flagella (figure 1).
(2) Growth of Listeria monocytogenes in selective medium
The circular blue-green color is formed on the color development culture medium of the Listeria monocytogenes of the Kjeldahl, and the colony with a typical white halo is arranged around the circular blue-green color; colonies with black halos around the dark gray color on PALCAM selective plates, as shown in FIG. 2.
The result shows that the bacterial strain generates a round blue-green bacterial colony on a chromogenic culture medium of the Listeria cyclokamii, and a bacterial colony with a typical white halo is arranged around the bacterial colony; colonies with a black central depression were grown on PALCAM selection.
(3) API Listeria qualification: using API Listeria biochemical identification test strips, single colonies were scraped from Tryptic Soy Agar (TSA) plates, prepared as cell suspensions with appropriate turbidity using the kit, 100. mu.L of the bacterial suspension was added to each well, incubated at 37 ℃ for 24. + -.2 h, and the results of the biochemical identification test strips were recorded (FIG. 3).
As a result, the strain had standard listeria monocytogenes biochemical characteristics: in API Listeria biochemical assays, Listeria monocytogenes gram-positive brevibacterium oxidase: and (4) negativity. Identification of Strain 428-1LM code: 6510, identification of coincidence: 98.6%, T value: 1.0; identification code for Strain 678-1 LM: 6510, identification of coincidence: 98.6%, T value: 1.0; identification code of strain 833-1 LM: 6510, identification of coincidence: 98.6%, T value: 1.0; identification and coding of strain 1382-1 LM: 6510, identification of coincidence: 98.6%, T value: 1.0.
(4) serotype analysis
1) Serogroup analysis
The listeria monocytogenes serogroup was determined using multiplex PCR. Primers and amplification methods were reported in the previous literature. The 5 primers used were synthesized by Saimerfi (Shanghai) Co., Ltd. (see Table 1 for primer sequences). The reaction system (25. mu.L) contained: 2 XDS PCR mix, 12.5. mu.L; 0.5. mu.M of upstream and downstream primers; ddH2O, 9.5. mu.L and genomic DNA, 1. mu.L. mu.L of the PCR product was applied to a 1.5% agarose gel for electrophoretic separation (120V, 40min) using DL2000 DNA Marker.
TABLE 1 Listeria monocytogenes serotype primer sequences
2) Identification of O antigen
The serotype of Listeria monocytogenes was further confirmed by seroagglutination (Japanese Utility Co., Ltd.) based on the result of serogroup identification by multiplex PCR. The colonies of Listeria monocytogenes grown on the TSA-YE plate were collected, resuspended in 1mL of 0.2% NaCl solution, boiled in boiling water for 30min, centrifuged, and the supernatant discarded. Resuspend with 300. mu.L of 0.2% NaCl solution. mu.L of the bacterial liquid was added to 7. mu.L of polyvalent O serum (LI-XIII) to carry out a slide agglutination test on the slide glass, and a 0.2% NaCl solution was used as a control. The sample was not typed when self-coagulated in 0.2% NaCl solution. Observing agglutination to determine O antigen serum type.
3) Identification of H antigen
The colonies of Listeria monocytogenes grown on TSA-YE plates were picked, inoculated into a semisolid BHI medium (0.15% agar) and cultured overnight at 30 ℃ for flagella induction, after induction twice, inoculated into BHI broth for overnight culture, 1mL was aspirated, centrifuged, and resuspended in physiological saline and 1% formalin in equal volume. Dripping 180 μ L of the bacterial solution onto a glass slide, adding 20 μ L of diagnostic serum (AB, ABC, BD) respectively, and observing agglutination in a water bath at 51 deg.C for about 3.5 h.
According to the agglutination phenomenon of the O antigen and the H antigen, the O antigen of the Listeria monocytogenes strain is I, II and III, the H-antigen is ABC, and the serotype of the Listeria monocytogenes strain is obtained by looking up the table.
Results the strain listeria monocytogenes 428-1LM serotype is 4 b; 678-1LM serotype 4 b; 833-1LM serotype 1/2 a; serotype 1382-1LM 1/2 b.
Example 3 Listeria monocytogenes drug resistance characteristics
Activating Listeria monocytogenes strain by TSA plate, adding normal saline to dilute to final concentration of about 105cfu/mL was evenly spread on MH plates, antibiotic paper sheets were attached to the surface of the medium, and incubated at 37 ℃ for 24 hours. And measuring the size of the bacteriostatic zone by using a vernier caliper to be accurate to 0.01 mm. The antibiotics selected were as follows: ampicillin (AMP, 10. mu.g), cephalomycin (CEP, 30. mu.g), chloramphenicol (CHL, 30. mu.g), ciprofloxacin (CIP, 5. mu.g), erythromycin (ERY, 15. mu.g), gentamicin (GEN, 10. mu.g), kanamycin (KAN, 30. mu.g), rifampicin (RIF, 5. mu.g), doxycycline (DOX, 30. mu.g), levofloxacin (LEV, 5. mu.g), clindamycin (DA, 2. mu.g) streptomycin (S, 5. mu.g) penicillin (PEN, 10U), tetracycline (TET, 30. mu.g), vancomycin (VAN, 30. mu.g), sulfamethoxazole/trimethoprim (Compound sulfamethoxazole SXT, 23.75/1.25. mu.g), sulbactam/ampicillin (SAM; 10/10. mu.g). Escherichia coli ATCC25922 and Staphylococcus aureus ATCC25923 were used as the quality control strains. The listeria monocytogenes strains were sensitive to all of the above tested antibiotics.
As a result, the 428-1LM, 678-1LM and 1382-1LM strains have no drug resistance, and the 833-1LM drug resistance spectrum is K-DA-SXT-ERY-TET-S-CHL.
Example 4 virulence factor-carrying characteristics of Listeria monocytogenes
And (3) confirming the carrying condition of the virulence factor of the listeria monocytogenes strain by adopting a PCR method. Primers and amplification methods are described in the literature. The 10 pairs of primers used were synthesized by England Shafer-based Shanghai trade Limited (see Table 2 for primer sequences). Reaction system 25 μ L in total: 2 XHS Mix 12.5. mu.L (Dongsheng Innovation), 0.5. mu.L primer, 1-2. mu.L template, and ultrapure water.
The reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 deg.C for 1 min; annealing at 60 deg.C for 1 min; stretching at 72 deg.C for 1-2 min; 35 cycles are carried out; final extension at 72 ℃ for 10 min.
TABLE 2 Listeria monocytogenes virulence factor amplification primer sequences
Results Listeria monocytogenes 428-1LM strain carries llsX-actA-mpl-prfA-hly-inlA-inlB-iap-plcA-plcB, Listeria monocytogenes 678-1LM carries actA-mpl-prfA-hly-inlA-inlB-iap-plcA-plcB; the Listeria monocytogenes 833-1LM strain carries actA-mpl-prfA-hly-inlA-inlB-iap-plcA-plcB; listeria monocytogenes 1382-1LM strain carries actA-mpl-prfA-hly-inlA-inlB-iap-plcA-plcB-ptsA.
Example 5 Multi-site sequence (MLST) typing assay for Listeria monocytogenes
MLST analysis was performed by amplifying 7 housekeeping genes of Listeria monocytogenes (abcZ, bglA, cat, dapE, dat, ldh and lhkA), and primers and amplification methods for the 7 housekeeping genes were synthesized by Shanghai Biometrics, Inc. (see Table 3 for primer sequences) with reference to the previous reports. The PCR system was 50. mu.L, containing 5 XPrimeSTAR buffer (Mg)2+Plus) 10. mu.L, dNTP mix (10mmol/L) 4. mu.L, forward and reverse primers 0.3. mu. mol/L each, 2. mu.L of genomic DNA and 1.25U of DNA polymerase. PCR amplification conditions of pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s (bglA is 45 ℃), extension at 72 ℃ for 2min, and extension at 72 ℃ for 10min after 35 cycles. The amplified products were subjected to two-way sequencing, MLST analysis was performed using Listeria MLST database (http:/bigsdb. pateur. fr/Listeria. html), and seven housekeeping gene loci were obtained respectivelyAnd forming a corresponding allele spectrum, and judging the sequence type and the clone complex type of the allele.
TABLE 3 Listeria monocytogenes MLST typing primer sequences
Results the strain listeria monocytogenes 428-1LM multi-site sequence typing (MLST) results in Sequence Type (ST) 1, belonging to Clonal Complex (CC) 1; 678-1LM MLST typing result is ST2/CC 2; the 833-1LM MLST typing result is ST9/CC 9; the 1382-1LM MLST typing results are ST87/CC 87.
Example 6 signature sequence analysis of Listeria monocytogenes strains
The non-essential genes specific to the strain are obtained mainly from the results of pan-genomic analysis of Listeria monocytogenes. The genome sequences of 153 representative listeria monocytogenes were co-selected for genome-wide analysis. The Pan-genome is analyzed by using an MP method in prokaryotic Pan-genome automated Analysis software (PGAP), and the Analysis result is processed by using a local Perl script to obtain the information of the core genes and the non-core genes of all strains.
Extracting the specific non-core gene protein sequence of the strain, and respectively comparing the non-core gene protein sequence with the total protein library of Listeria monocytogenes and the NCBI non-redundant protein database (NR) by local Blast. The sequences of known Listeria monocytogenes proteins were removed, and the remaining genes were unique to the standard strain. The specificity of the unique genes was examined by PCR amplification in each of the standard strains and in other Listeria monocytogenes strains of the sequence types (1,2,3,5,7,8,9,11,14,20,37,59,87,91,101,120,155,199, 204,224,288,296,297,307,310,323,325,330,378,391,429,504,506,515,619, 621,705,767,804,805,807,1166) (FIG. 4).
The strains of the invention all carry a special gene, the sequence is shown as SEQ ID No. 1-4, the gene can be amplified and tested by the following primers:
the specific sequence in the Listeria monocytogenes 428-1LM strain (deposit number: GDMCC 60836) is shown as SEQ ID No. 1, and the amplification primers are as follows:
428-1F：5'-GCAGGATAAACCGGCGACAAC-3'(SEQ ID No.6)
428-1R：5'-TGTAAGCTAACGGGCCGCAAT-3'(SEQ ID No.7)
the product size was: 323 bp;
the specific sequence of the Listeria monocytogenes 678-1LM strain (deposit number: GDMCC60838) is shown as SEQ ID No. 2, and the amplification primers are as follows:
678-1F：5'-TGGAAGGTCCGATGAGTATTAGCG-3'(SEQ ID No.10)
678-1R：5'-AGGCCAATCTTGTTGTTGCAGGA-3'(SEQ ID No.11)
the product size was: 732 bp;
the specific sequence of the Listeria monocytogenes 833-1LM strain (deposit number: GDMCC60839) is shown as SEQ ID No. 3, and the amplification primers are as follows:
833-1F：5'-CTCCCCCTTGGTGGAAACAGATAG-3'(SEQ ID No.12)
833-1R：5'-TTGGGTGCCGAAATGTTGTAGTAGTC-3'(SEQ ID No.13)
the product size was: 296 bp;
the specific sequence of the Listeria monocytogenes 1382-1LM strain standard substance (preservation number: GDMCC 60840) is shown as SEQ ID No. 4, and the amplification primers are as follows:
1382-1F：5'-GGACTTTTGGTGGCGATGA-3'(SEQ ID No.14)
1382-1R：5'-TCTTCATTAGCCCATACCA-3'(SEQ ID No.15)
the product size was: 394 bp.
The invention also provides a freeze-drying protective agent for quantitatively storing the standard strain of the listeria monocytogenes, and the components of the freeze-drying protective agent in different embodiments and comparative examples are shown in the table 4:
TABLE 4 composition of the lyoprotectants of each group
The parts by weight of the skim milk powder were increased in the comparative example to make up the total amount due to its lack of components.
The freeze-drying survival rate of the freeze-dried strain (GDMCC 60840) of the protective agent in examples 7 to 9 and comparative examples 1 to 3 is as follows:
after the recovery of the strains, inoculating the strains into a culture medium, culturing until a proper amount of the strains are selected and added into the protective agents of the examples 7-9 and the comparative examples 1-3 at the early stage of the logarithmic phase to the stationary phase, uniformly mixing, subpackaging the mixture into penicillin bottles, taking samples, diluting and counting to obtain the bacterial content A0 before freeze-drying. Transferring the half-stoppered split penicillin bottles into a freeze dryer for pre-freezing at the temperature of minus 40 ℃ for 3 hours, starting main drying for 20-25 hours, then entering an analysis drying stage for 6-8 hours, finishing drying, pressing the stoppered split penicillin bottles in a vacuum state, moving the split penicillin bottles out of the freeze dryer, automatically capping the split penicillin bottles, ensuring the complete vacuum state of the samples, and storing the split penicillin bottles at the low temperature of minus 20 ℃. And (3) taking the freeze-dried sample for dilution counting, wherein the counting result is the bacteria content A after freeze-drying, and the freeze-drying survival rate is the percentage of A to A0, and the result is shown in the table 5:
TABLE 5 comparison of freeze-drying survival rates of lyoprotectants of different compositions
As can be seen from Table 5, the lyoprotectant in example 9 is the best example of the present invention, and therefore, comparative examples 1 to 3 are prepared by taking example 9 as a comparison object, and as a result, as shown in Table 5, the protective effects of comparative examples 1 to 3 are all worse than those of the examples due to the absence of one of the components of the lyoprotectant in the invention, respectively, thereby illustrating the synergistic effect among the components of potassium lactate, sodium alginate and glycine in the lyoprotectant of the present invention, and the absence of any one of the components cannot achieve the effect of the protectant of the present invention.
The freeze-drying stability of the freeze-dried strains of the protective agents in the examples 7 to 9 and the comparative examples 1 to 3 is compared, and the specific method is as follows:
preparing quantitative quality control bacteria by using different protective agents according to the preparation method, storing at-20 ℃, and extracting 3 bacteria every month to test the bacteria content according to the counting method. In order to better compare the effect of each protective agent in the long-term storage process, the number of viable bacteria before freeze-drying is calculated according to the freeze-drying survival rate of each protective agent when quantitative quality control bacteria are prepared, so that the bacteria content of each protective agent is about 3000 cfu/bottle after freeze-drying. The change of the bacterial content of the quantitative quality control bacteria prepared by each protective agent after 12 months of storage is shown in the attached figure 5.
As can be seen from the attached figure 5, the bacteria content protected by the cryoprotectants of examples 7 to 9 did not change significantly after 12 months of storage, while the bacteria content protected by the cryoprotectants of comparative examples 1 to 3 significantly decreased after 2 months, and was close to 0 after 12 months.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
SEQUENCE LISTING
<110> Guangdong province institute for microbiology (Guangdong province center for microbiological analysis and detection)
<120> Listeria monocytogenes standard strain containing specific molecular target, and detection and application thereof
<130> 12.3
<160> 15
<170> PatentIn version 3.3
<210> 1
<211> 897
<212> DNA
<213> bacterium
<400> 1
atgcagaggc aggataaacc ggcgacaaca taccaaattc aaattgaaaa gatgaaacaa 60
aaaaatataa taattgaaga tgaggcattt gcaattagtt ttttaaaaaa ggttcaatat 120
tatagactaa gtggatactg gttatcatat tttgaagata gagaaaaaga tattttgaaa 180
cctggaataa catttgaaaa aatttcttca gtttatttat ttgataaaga attaagaaat 240
attttattgt ctatgctaga tacgattgaa acggaattta aatctgttct tgcttatgat 300
ttttcgcata attgcggccc gttagcttac aaatatgcta ataattttaa taaaccagaa 360
tactacgcaa aatggcttaa taaattttat agtagcataa gttattctga tactaataga 420
gaactttata ttgaatggta taaaaaagaa tataacggga aatttccttt ttggattgta 480
gtagaagttt gcaatttcaa tgatatttct aagttttata gtaatctaca tattaaaatt 540
aaaaagaaaa tggttaaaat ttatggatat gatgcagaat atattcaaag ttggttacac 600
acagttgtgt taatacgaaa tatatgcgcc cataatggga ggctctataa tagaacgata 660
acagtatcgc caaaattacc taatgggacg gctaaactaa atataaagag aatatttatt 720
gttatattta tattcaaatt tttgtgcgtt gatcaaacag agtgggaaat atttgtaaat 780
aaaatagaag aattaataca aaaataccaa gaggttattg aactagaaat gattggtttt 840
cctgaaacgt ggaaagaaat gttaattgac agagtggtag ttaattcgaa taactag 897
<210> 2
<211> 2061
<212> DNA
<213> bacterium
<400> 2
atgttaggaa aagttattga tgattataag ctggtggaat ttatcggaaa agggtcgttt 60
ggtactgtct atagagcaga aaaaaatggt tcaaattatg cattgaaaat atttaattat 120
gattacgttt tccaggagtt taagaggaat ggtgaaaata ataggatttc aagagaaatt 180
gctgtattgg aaaaagtaga gcatataaat gttactaaat atatagataa aggaagctat 240
agtgataaca cgcagtcata tttgtattta gtaatggaat atattaatgg tagtgatttg 300
aaaacccatt taaactcgtt ggaaggtccg atgagtatta gcgaaacaaa taattatgtg 360
caacaaattc tttctggatt agatgcagtt cataaacata atatagttca tagagactta 420
aaacccgaaa atatttttgt tactaaaaat ggaaccatta aaatactaga ttttgggtta 480
tcaaagctaa ttgattatac ttcaataact tcaacaggaa gtactgtagg aagtccactt 540
tatatgtcgc ctgaacagat tagagatagt aaatcaattg actacagatc agattattat 600
tcattaggtg taatcattta tgagatgtta gcaaaatgta gtccttatgg ggatgttgat 660
tcaatacatc aattgtatta caaaattcta aatgaacccc cgaaatctat acttctatat 720
aattatgaaa ttcctaatta tatagataat ttgattaatt ctttgttgag caaaaataat 780
tatgaaagac caaattctat agaaaatata aaggatttgt ttagatcaga agtggtatcc 840
gttgaaatag aaaatagttc acaagaaaaa tcagagtttg tcctgcgagt ctggaatgaa 900
aaaagtatac tatctgattt tgctcaagat ggttttcgca taccaaattg catctttcca 960
atcaatcatc aagatcaaca aaaaggtctg ttaaaattaa ttcaatctaa ttctaatttt 1020
tttattgatc ctgcaacaac aagattggcc tatgattcat attctgatgt aaaaggattg 1080
gttgctttac cttatgctcc gaaagggttc gataggttag aaataccatc ttttgaagag 1140
tattcaaaaa tacaagaata tgtagaacta gtggtagaag agcaattaaa gtttaatccg 1200
aaccaaattg ttgcaccttt tcatgtcagt aacaacactt ctgataatgc ggctttaaag 1260
tattcagatg aatcttggtt ctctttagat gttaagtttt taaaagaagc taaagactat 1320
ttagttaaga ataatattaa aaaagaacta attatgggtg tatgtatcaa gagtgagcta 1380
ctgtctattg cctctgaacg cgaatatttt attaatgtgc taagttcgtt accagcagac 1440
gtgtattgga tatatgttga tagtataaac tacgactcag gtgtttctca aatttttaat 1500
tatataaaga ctttgttagc aattcagaat tcaacaggaa aaaaagtaat tgctggtaga 1560
gttggtagta tagggatgct attgaattca tttggaattt atggatttga atcgggtgct 1620
tctagatttg aaacattttc tgaagatttg tttaagtcat cagaagacag ctacaacatg 1680
tacattagtt attattttcc agacctatta cgatcagtcc caattttaag aaaagacccc 1740
tcgaagctag tttctatatt tgattcttct gtaggagatg gaataaaatg caactgtccg 1800
tactgtaaag ataaagaaat tagtgctgta ctgaaagaac caaatgtaaa gaaacatttt 1860
ctataccata gaaatatgga gatggaaaaa atgaattcct tcgagagtat ttctgagaaa 1920
attgattact tttatgatag aatgagtaag gcacttattt tatatcaaaa tttaggacct 1980
atttttaaac ccaatcaata tcagtttgta aagacttgga tgcaagtggt tgagaaactt 2040
agaatagaag taggggtgta a 2061
<210> 3
<211> 630
<212> DNA
<213> bacterium
<400> 3
atgagtgaaa tagtcgtgag aaaaattgct gtcgaattcg aagaaaatag gaaatatcgt 60
tattcttggt cgatatcgta cgaaagtgac agttcacttc cacatgatct gcttgtaatt 120
atgatgaatc ctagcaaagc agatgaaagt ggtccagaca gaacaataaa gaagataatg 180
gattggaaca aagataaacg tctttgtgaa aattcatcat ataagagtat cataattatg 240
aatatctccc ccttggtgga aacagatagt aagaaggcct taaatgtatt tataaaaaat 300
gaaattccat gcgatatttt aaaagataac ctttcaaagc tggcatgtga aatctccgaa 360
gtgaaaaata taattattgc ttgggggatc ataggggaca aaatcttccg tgaagtttta 420
gaaaagtatc caaacaatga acaagtaaaa tgtttagtat ctgcattaaa agaaaaagtg 480
tccgcaaggt atgtgaaatg cttagaaatg aatgagacta ctacaacatt tcggcaccca 540
agaagagctt gggaagatga taaaaaagaa aactttataa aaactgttga gcaagaagat 600
tttcctgcgc tatatgaaga tcaaaagtaa 630
<210> 4
<211> 759
<212> DNA
<213> bacterium
<400> 4
atgttgttga agtcacgtgt tttatttgat aagttcgttt tgaaacgtga gtatgctcgt 60
gattataaag aaacagggaa atggtcactt caaaagcttg agaaatatac ggataataaa 120
ggtaataagc caaaatacgt tgggactttt ggtggcgatg aagataatcg aaataaacaa 180
attcgcacat tacaatctgc acttcgtatt acctacacat cacctaaaac tatgcattgg 240
atttctctaa ttctgacaag ctgtttaaat aatgatgatt ctgatatttt acaaattctt 300
gaagcttatg gtcaaactaa agtagatgct tcgggataca caacggcgac tggattcagt 360
tttgaaagaa ttgtatttag ctatttggat tatcttcttt atcgaaacgg atattcatat 420
aatggaaaag aagtggtaag acctcttgct gatgactggc aattccagtt tagaagttca 480
atagaacatt tttatccaca gcatcctact gaattggtgg tatgggctaa tgaagatttg 540
aactgtttcg ggaacttagc actaattaca gtttcaggga attcaacgtt taacaatgcg 600
atgccggttg ggaaagctag caccaaccct ggaattattg aacaaagttt gaagttaaaa 660
ataatgactg agatgatgag acagaatgat aataactgga atcaagaatt agctcataaa 720
catcaaaggg aaatgtttaa cgttcttgag aaaggctga 759
<210> 5
<211> 21
<212> DNA
<213> Synthesis
<400> 5
gcaggataaa ccggcgacaa c 21
<210> 6
<211> 21
<212> DNA
<213> Synthesis
<400> 6
tgtaagctaa cgggccgcaa t 21
<210> 7
<211> 24
<212> DNA
<213> Synthesis
<400> 7
tggaaggtcc gatgagtatt agcg 24
<210> 8
<211> 23
<212> DNA
<213> Synthesis
<400> 8
aggccaatct tgttgttgca gga 23
<210> 9
<211> 24
<212> DNA
<213> Synthesis
<400> 9
ctcccccttg gtggaaacag atag 24
<210> 10
<211> 26
<212> DNA
<213> Synthesis
<400> 10
ttgggtgccg aaatgttgta gtagtc 26
<210> 11
<211> 19
<212> DNA
<213> Synthesis
<400> 11
<210> 12
<211> 19
<212> DNA
<213> Synthesis
<400> 12
Claims (2)
1. The application of the primer for detecting the specific molecular target in the preparation of the listeria monocytogenes detection reagent is characterized in that the specific molecular target is as follows:
a nucleotide sequence shown as SEQ ID NO. 2 or SEQ ID NO. 4;
wherein, the primer for detecting the specific molecular target shown in SEQ ID NO. 2 is used for preparing the Listeria monocytogenes (L.) (Listeria monocytogenes) 678-1LM, with accession number GDMCC 60838;
wherein, the primer for detecting the specific molecular target shown in SEQ ID NO. 4 is used for preparing the Listeria monocytogenes (L.) (Listeria monocytogenes) 1382-1LM, deposited as GDMCC 60840.
2. The use according to claim 1,
the primers for detecting the specific molecular target shown as SEQ ID NO. 2 comprise: an upstream primer shown as SEQ ID NO. 7 and a downstream primer shown as SEQ ID NO. 8;
the primers for detecting the specific molecular target shown as SEQ ID NO. 4 comprise: an upstream primer shown as SEQ ID NO. 11 and a downstream primer shown as SEQ ID NO. 12.
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| CN102586413A (en) * | 2011-09-09 | 2012-07-18 | 山东轻工业学院 | Fluorescent quantitative PCR (polymerase chain reaction) reagent kit for detecting Listeria monocytogenes |
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| CN111118182A (en) * | 2019-12-30 | 2020-05-08 | 广东省微生物研究所（广东省微生物分析检测中心） | Specific novel molecular target of serotype listeria monocytogenes and rapid detection method thereof |
| KR20200059761A (en) * | 2018-11-21 | 2020-05-29 | 경희대학교 산학협력단 | Development of a Singleplex Real-Time PCR Kit for Rapid Detection of Listeria monocytogenes ssrA target gene |
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| FR2814754B1 (en) * | 2000-10-04 | 2005-09-09 | Pasteur Institut | LISTERIA INNOCUA, GENOME AND APPLICATIONS |
| US7368547B2 (en) * | 2003-02-03 | 2008-05-06 | Mississippi State University | Use of novel virulence-specific genes as targets for diagnosis and potential control of virulent strains of Listeria monocytogenes |
| EP3169802B8 (en) * | 2014-07-18 | 2019-07-03 | Qualicon Diagnostics LLC | Sequences and their use for detection of listeria monocytogenes |
| CN112538544B (en) * | 2020-12-30 | 2022-06-14 | 广东省微生物研究所（广东省微生物分析检测中心） | Detection method and application of food-borne pathogenic bacteria standard strain viable bacteria with specific molecular targets |
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| CN102586413A (en) * | 2011-09-09 | 2012-07-18 | 山东轻工业学院 | Fluorescent quantitative PCR (polymerase chain reaction) reagent kit for detecting Listeria monocytogenes |
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| KR20200059761A (en) * | 2018-11-21 | 2020-05-29 | 경희대학교 산학협력단 | Development of a Singleplex Real-Time PCR Kit for Rapid Detection of Listeria monocytogenes ssrA target gene |
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