CN110643691A - Method for monitoring salmonella - Google Patents
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- CN110643691A CN110643691A CN201911076136.XA CN201911076136A CN110643691A CN 110643691 A CN110643691 A CN 110643691A CN 201911076136 A CN201911076136 A CN 201911076136A CN 110643691 A CN110643691 A CN 110643691A
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- 241000607142 Salmonella Species 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 24
- 238000012544 monitoring process Methods 0.000 title claims abstract description 15
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- 238000012360 testing method Methods 0.000 claims abstract description 5
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- 235000019319 peptone Nutrition 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 11
- 230000001580 bacterial effect Effects 0.000 claims description 9
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 8
- 239000008101 lactose Substances 0.000 claims description 8
- 235000015278 beef Nutrition 0.000 claims description 7
- 239000011259 mixed solution Substances 0.000 claims description 7
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 6
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- 229920001817 Agar Polymers 0.000 claims description 4
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 claims description 4
- 239000008272 agar Substances 0.000 claims description 4
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- HXCILVUBKWANLN-UHFFFAOYSA-N brilliant green cation Chemical compound C1=CC(N(CC)CC)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](CC)CC)C=C1 HXCILVUBKWANLN-UHFFFAOYSA-N 0.000 claims description 4
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 239000001509 sodium citrate Substances 0.000 claims description 4
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- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 3
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims description 3
- 229930195725 Mannitol Natural products 0.000 claims description 3
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- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 3
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 claims description 3
- 150000001413 amino acids Chemical class 0.000 claims description 3
- 238000000137 annealing Methods 0.000 claims description 3
- 239000003833 bile salt Substances 0.000 claims description 3
- 239000004202 carbamide Substances 0.000 claims description 3
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- NNFCIKHAZHQZJG-UHFFFAOYSA-N potassium cyanide Chemical compound [K+].N#[C-] NNFCIKHAZHQZJG-UHFFFAOYSA-N 0.000 claims description 3
- 238000012257 pre-denaturation Methods 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- 101150021607 rppH gene Proteins 0.000 claims description 3
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- 101150070603 yadA gene Proteins 0.000 claims description 3
- 239000012295 chemical reaction liquid Substances 0.000 claims description 2
- 239000004158 L-cystine Substances 0.000 claims 1
- 235000019393 L-cystine Nutrition 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 10
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- 238000009629 microbiological culture Methods 0.000 description 2
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 206010004022 Bacterial food poisoning Diseases 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
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- 241001465754 Metazoa Species 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 206010039438 Salmonella Infections Diseases 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010876 biochemical test Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
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- 206010039447 salmonellosis Diseases 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- WBWWGRHZICKQGZ-HZAMXZRMSA-N taurocholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS(O)(=O)=O)C)[C@@]2(C)[C@@H](O)C1 WBWWGRHZICKQGZ-HZAMXZRMSA-N 0.000 description 1
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Abstract
The invention discloses a salmonella monitoring method, which comprises the following steps of mixing a proper amount of a sample to be detected with BPW liquid, grinding/stirring to obtain a suspension/mixed liquid, centrifuging to remove insoluble substances, washing bacteria with physiological saline, boiling to crack cells, centrifuging to obtain a supernatant, placing the supernatant in a sterile test tube, culturing and enriching for 8-10 h at 36 ℃ in a constant temperature box, inoculating a BPW liquid culture into a selective enrichment liquid SC of salmonella, inoculating an enrichment culture liquid on an SS culture medium in a circulating manner, selecting a medium-sized circular colony with a black center on the SS culture medium for pure culture, and simultaneously carrying out double PCR and biochemical identification on the pure-cultured colony; the invention shortens the detection time of salmonella, accelerates the detection process, reduces the complexity of operation, reduces labor intensity, reduces detection cost, realizes quick, simple and sensitive detection of the target object, and improves the accuracy by simultaneously carrying out PCR and biochemical dual identification.
Description
Technical Field
The invention relates to the technical field of salmonella monitoring, in particular to a method for monitoring salmonella.
Background
Salmonella is a common food-borne pathogenic bacterium, and is a gram-negative intestinal bacterium parasitized in cells. The bacterium is widely existed in nature, not only can cause acute, chronic or recessive infection of livestock, poultry and other animals, but also can cause food poisoning of people by polluting food, thereby causing great threat to human beings. According to statistics, the food poisoning caused by salmonella is often listed as the top in various countries in the world of bacterial food poisoning. Salmonella causes about forty thousand americans to fall and about 600 people die each year. Salmonella is the most common pathogenic bacterium in food poisoning in China, and accounts for the first place of food poisoning. The clinical symptoms of salmonellosis mainly comprise headache, abdominal pain, fever and the like, the death rate is 1 percent, and the harm to people is great.
Accurate and timely detection means are the key to preventing and controlling the spread of pathogenic microorganisms. At present, the salmonella is mostly detected by adopting a traditional microbial culture method, the traditional microbial culture method mainly comprises the steps of pre-enrichment, selective enrichment, plate separation, biochemical test, serological identification and the like, the related experiments are more, the operation is more complicated, the detection period is longer (generally needs 4-7 d), the preparation and ending work is heavy, and the practical requirement of rapid detection of pathogenic bacteria in the food at the present stage cannot be met.
Based on this, the present invention has devised a salmonella monitoring method to solve the above-mentioned problems.
Disclosure of Invention
The present invention is directed to a method for monitoring salmonella, which solves the above problems of the prior art.
In order to achieve the purpose, the invention provides the following technical scheme: a method of monitoring salmonella comprising the steps of:
s1, mixing a proper amount of solid/liquid sample to be detected with BPW liquid, grinding/stirring to obtain suspension/mixed liquid, centrifuging to remove insoluble substances, washing the bacteria with physiological saline, boiling to crack cells, centrifuging to obtain supernatant;
s2, placing the supernatant obtained after centrifugation in the step 1 into a sterile test tube, culturing and enriching for 8-10 h at 36 ℃ in a constant temperature box, inoculating BPW liquid culture into selective enrichment liquid SC of salmonella, and selectively enriching for 16-22 h at 42 ℃ in the constant temperature box;
s3, taking the enrichment culture solution in the step 2, inoculating the enrichment culture solution on an SS culture medium in a loop, and culturing at 37 ℃ for 18-24 hours;
s4, selecting a medium-sized and central black circular colony on an SS culture medium for pure culture, and simultaneously carrying out double PCR and biochemical identification on the pure-cultured colony;
s5, PCR identification: extracting strain genome DNA by a boiling method, wherein a reaction system is a mixed solution of upstream and downstream primers of invA and tuf genes, TaqDNA polymerase, template DNA and sterilized deionized water, the strain genome DNA is placed in the mixed solution, pre-denaturation is carried out for 5min at 92 ℃, the cycle is 28-32 times, annealing is carried out for 32s at 53 ℃, extension is carried out for 42s at 70 ℃, a pre-denatured product is added into 1% agarose gel electrophoresis, electrophoresis is carried out for 28-30 min at 120V voltage, a DNA marker DL2000 is taken as a reference, and a gel imager is used for imaging and observing a result;
s6, biochemical identification: and (3) adding the bacterial colony selected in the step (4) into 0.85% sterilized normal saline to prepare 0.5 McLeeb bacterial suspension, inoculating the bacterial suspension into a biochemical identification reagent tube, adding a reaction solution into the reagent tube, culturing for 20-24 h at 37 ℃, and observing the result.
Preferably, the BPW culture solution is 10g/L of peptone, 5g/L of sodium chloride, 3.57g/L of anhydrous disodium hydrogen phosphate, 1.5g/L of potassium dihydrogen phosphate and has the pH value of 7-7.4.
Preferably, the selective enrichment fluid SC is 5g/L of peptone, 4g/L of lactose, 4g/L of sodium hydrogen selenite, 10g/L, L g/L of disodium hydrogen phosphate and 0.01g/L of cystine, and the pH value is 6.9-7.2.
Preferably, the SS culture medium is beef extract powder 5g/L, peptone 5g/L, beef bile salt 8.5g/L, agar 16g/L, lactose 10g/L, sodium citrate 8.5g/L, sodium thiosulfate 8.5g/L, ferric citrate 1g/L, neutral red 0.025g/L, brilliant green 0.00033g/L, and pH7.1-7.3.
Preferably, the reaction solution in the step 6 is peptone water, urea, lazy amino acid decarboxylase, potassium cyanide, mannitol, sorbitol and galactose glycol mixed solution.
Compared with the prior art, the invention has the beneficial effects that: the invention shortens the detection time of salmonella, accelerates the detection process, reduces the complexity of operation, reduces labor intensity, improves the detection efficiency, reduces the detection cost, realizes the quick, simple and sensitive detection of the target object, and improves the accuracy by simultaneously carrying out PCR and biochemical dual identification.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The invention provides a technical scheme of a salmonella monitoring method, which comprises the following steps: the method comprises the following steps:
s1, mixing a proper amount of solid/liquid sample to be detected with BPW liquid, grinding/stirring to obtain suspension/mixed liquid, centrifuging to remove insoluble substances, washing the bacteria with physiological saline, boiling to crack cells, centrifuging to obtain supernatant;
s2, placing the supernatant obtained after centrifugation in the step 1 into a sterile test tube, culturing and enriching for 8-10 h at 36 ℃ in a constant temperature box, inoculating BPW liquid culture into selective enrichment liquid SC of salmonella, and selectively enriching for 16-22 h at 42 ℃ in the constant temperature box;
s3, taking the enrichment culture solution in the step 2, inoculating the enrichment culture solution on an SS culture medium in a loop, and culturing at 37 ℃ for 18-24 hours;
s4, selecting a medium-sized circular colony with black center on an SS culture medium for pure culture, and performing PCR identification on the pure-cultured colony;
s5, PCR identification: extracting strain genome DNA by a boiling method, wherein a reaction system is a mixed solution of upstream and downstream primers of invA and tuf genes, TaqDNA polymerase, template DNA and sterilized deionized water, putting the strain genome DNA into the mixed solution, performing pre-denaturation at 92 ℃ for 5min, circulating 28-32 times in this way, annealing at 53 ℃ for 32s, extending at 70 ℃ for 42s, adding a pre-denatured product into 1% agarose gel electrophoresis, performing electrophoresis at 120V for 28-30 min, and imaging and observing a result by using a gel imager with a DNA marker DL2000 as a reference.
Wherein the BPW culture solution is peptone 10g/L, sodium chloride 5g/L, anhydrous disodium hydrogen phosphate 3.57g/L, potassium dihydrogen phosphate 1.5g/L, and pH 7-7.4; the selective enrichment fluid SC is 5g/L of peptone, 4g/L of lactose, 4g/L of sodium hydrogen selenite, 10g/L, L g/L of disodium hydrogen phosphate-0.01 g/L of cystine, and the pH value is 6.9-7.2; the SS culture medium comprises 5g/L of beef extract powder, 5g/L of peptone, 8.5g/L of taurocholate, 16g/L of agar, 10g/L of lactose, 8.5g/L of sodium citrate, 8.5g/L of sodium thiosulfate, 1g/L of ferric citrate, 0.025g/L of neutral red, 0.00033g/L of brilliant green and pH7.1-7.3.
Example 2
The invention provides a technical scheme of a salmonella monitoring method, which comprises the following steps: the method comprises the following steps:
s1, mixing a proper amount of solid/liquid sample to be detected with BPW liquid, grinding/stirring to obtain suspension/mixed liquid, centrifuging to remove insoluble substances, washing the bacteria with physiological saline, boiling to crack cells, centrifuging to obtain supernatant;
s2, placing the supernatant obtained after centrifugation in the step 1 into a sterile test tube, culturing and enriching for 8-10 h at 36 ℃ in a constant temperature box, inoculating BPW liquid culture into selective enrichment liquid SC of salmonella, and selectively enriching for 16-22 h at 42 ℃ in the constant temperature box;
s3, taking the enrichment culture solution in the step 2, inoculating the enrichment culture solution on an SS culture medium in a loop, and culturing at 37 ℃ for 18-24 hours;
s4, selecting a medium-sized and central black circular colony on an SS culture medium for pure culture, and carrying out biochemical identification on the pure-cultured colony;
s5, adding the bacterial colony selected in the step 4 into 0.85% sterilized normal saline to prepare 0.5 McLee' S bacterial suspension, inoculating the bacterial suspension into a biochemical identification reagent tube, adding the reaction liquid into the reagent tube, culturing for 20-24 h at 37 ℃, and observing the result.
Wherein the BPW culture solution is peptone 10g/L, sodium chloride 5g/L, anhydrous disodium hydrogen phosphate 3.57g/L, potassium dihydrogen phosphate 1.5g/L, and pH 7-7.4; the selective enrichment fluid SC is 5g/L of peptone, 4g/L of lactose, 4g/L of sodium hydrogen selenite, 10g/L, L g/L of disodium hydrogen phosphate-0.01 g/L of cystine, and the pH value is 6.9-7.2; the SS culture medium is beef extract powder 5g/L, peptone 5g/L, beef bile salt 8.5g/L, agar 16g/L, lactose 10g/L, sodium citrate 8.5g/L, sodium thiosulfate 8.5g/L, ferric citrate 1g/L, neutral red 0.025g/L, brilliant green 0.00033g/L, pH7.1-7.3, and the reaction solution in step 6 is a mixture of peptone water, urea, lazy amino acid decarboxylase, potassium cyanide, mannitol, sorbitol and galactose glycol.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
Claims (5)
1. A method for monitoring Salmonella, which is characterized by comprising the following steps: the method comprises the following steps:
s1, mixing a proper amount of solid/liquid sample to be detected with BPW liquid, grinding/stirring to obtain suspension/mixed liquid, centrifuging to remove insoluble substances, washing the bacteria with physiological saline, boiling to crack cells, centrifuging to obtain supernatant;
s2, placing the supernatant obtained after centrifugation in the step 1 into a sterile test tube, culturing and enriching for 8-10 h at 36 ℃ in a constant temperature box, inoculating BPW liquid culture into selective enrichment liquid SC of salmonella, and selectively enriching for 16-22 h at 42 ℃ in the constant temperature box;
s3, taking the enrichment culture solution in the step 2, inoculating the enrichment culture solution on an SS culture medium in a loop, and culturing at 37 ℃ for 18-24 hours;
s4, selecting a medium-sized and central black circular colony on an SS culture medium for pure culture, and simultaneously carrying out double PCR and biochemical identification on the pure-cultured colony;
s5, PCR identification: extracting strain genome DNA by a boiling method, wherein a reaction system is a mixed solution of upstream and downstream primers of invA and tuf genes, TaqDNA polymerase, template DNA and sterilized deionized water, the strain genome DNA is placed in the mixed solution, pre-denaturation is carried out for 5min at 92 ℃, the cycle is 28-32 times, annealing is carried out for 32s at 53 ℃, extension is carried out for 42s at 70 ℃, a pre-denatured product is added into 1% agarose gel electrophoresis, electrophoresis is carried out for 28-30 min at 120V voltage, a DNA marker DL2000 is taken as a reference, and a gel imager is used for imaging and observing a result;
s6, biochemical identification: and (3) adding the bacterial colony selected in the step (4) into 0.85% sterilized normal saline to prepare 0.5 McLeeb bacterial suspension, inoculating the bacterial suspension into a biochemical identification reagent tube, adding a reaction solution into the reagent tube, culturing for 20-24 h at 37 ℃, and observing the result.
2. The method for monitoring salmonella as claimed in claim 1, wherein: the BPW culture solution is 10g/L of peptone, 5g/L of sodium chloride, 3.57g/L of anhydrous disodium hydrogen phosphate, 1.5g/L of potassium dihydrogen phosphate and has the pH value of 7-7.4.
3. The method for monitoring salmonella as claimed in claim 1, wherein: the selective enrichment fluid SC is 5g/L of peptone, 4g/L of lactose, 4g/L of sodium hydrogen selenite, 0.01g/L of disodium hydrogen phosphate 10g/L, L-cystine and has the pH value of 6.9-7.2.
4. The method for monitoring salmonella as claimed in claim 1, wherein: the SS culture medium is beef extract powder 5g/L, peptone 5g/L, beef bile salt 8.5g/L, agar 16g/L, lactose 10g/L, sodium citrate 8.5g/L, sodium thiosulfate 8.5g/L, ferric citrate 1g/L, neutral red 0.025g/L, brilliant green 0.00033g/L and pH7.1-7.3.
5. The method for monitoring salmonella as claimed in claim 1, wherein: the reaction liquid in the step 6 is a mixed liquid of peptone water, urea, lazy amino acid decarboxylase, potassium cyanide, mannitol, sorbitol and galactose glycol.
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| CN113621718A (en) * | 2021-08-09 | 2021-11-09 | 浙江舟鲜生食品科技有限公司 | Method for rapidly detecting salmonella in food |
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