CN110724753A - Method for rapidly detecting multiple bacteria in food, liquid phase chip and kit - Google Patents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Abstract
The invention discloses a rapid detection method, a liquid chip and a kit for various bacteria in food, which mainly comprise 50-80 parts of deionized water, 10-20 parts of a salmonella monoclonal antibody, 10-15 parts of staphylococcus aureus protein A, 5-10 parts of serine acetyltransferase, 30-50 parts of a liquid culture medium, 20-50 parts of amino acid, 10-20 parts of oligonucleotide for detection, 5-10 parts of PCR primer and 5-10 parts of PCR amplification reaction reagent, wherein the liquid culture medium comprises glucose, peptone, yeast extract and sodium chloride, and the invention relates to the technical field of food detection. The kit comprises the liquid chip and the internal reference nucleotide according to claim 1, can perform rapid and convenient qualitative and quantitative detection on various known food bacteria, has the advantages of high flux, high precision, good stability and the like, improves the efficiency of food bacteria detection, shortens the time spent on detection, and is simple to operate.
Description
Technical Field
The invention relates to the technical field of food detection, in particular to a method for rapidly detecting various bacteria in food, a liquid-phase chip and a kit.
Background
The food refers to various finished products and raw materials for people to eat or drink and the products which are both food and traditional Chinese medicinal materials according to the tradition, but does not include the products aiming at treatment, and the definition of the food is as follows: substances which can be eaten or drunk by human beings, including processed foods, semi-finished products and unprocessed foods, do not include tobacco or substances which are only used as medicines, and generally can be divided into two major parts, namely an endogenous substance component and an exogenous substance component, wherein the endogenous substance component is a component of the foods, and the exogenous substance component is other components which are artificially added or mixed in the whole process from the processing to the ingestion of the foods.
The bacterial contamination of food refers to the phenomenon that food exposed to the environment is contaminated by bacteria through different ways, is decayed under the action of bacteria, loses the nutritional ingredients which the food is supposed to have, and thus affects the edibility and the safety of the food, after people eat the food contaminated by harmful bacteria, various poisoning phenomena can occur, the bacterial contamination is one of the most common food contamination, in all outbreak cases of food-borne diseases all over the world, more than 60 percent of the common bacterial contamination is caused by the contamination of food by bacterial pathogenic bacteria, and some common bacterial contamination are: salmonella contamination, staphylococcus aureus, yersinia enterocolitica, campylobacter jejuni, and the like.
At present, in the process of carrying out bacteria detection on food, once can not detect multiple bacteria, the time of detection cost is longer, the efficiency of food bacteria detection is reduced, and when detecting food bacteria at present, the precision of detection is not high enough, and stability is relatively poor.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a method, a liquid phase chip and a kit for rapidly detecting various bacteria in food, and solves the problems that various bacteria cannot be detected at one time, the detection time is long, the food bacteria detection efficiency is reduced, the detection precision is not high enough, and the stability is poor.
(II) technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme: a rapid detection method, a liquid phase chip and a kit for various bacteria in food mainly comprise 50-80 parts of deionized water, 10-20 parts of salmonella monoclonal antibody, 10-15 parts of staphylococcus aureus protein A, 5-10 parts of serine acetyltransferase, 30-50 parts of liquid culture medium, 20-50 parts of amino acid, 10-20 parts of oligonucleotide for detection, 5-10 parts of PCR primer and 5-10 parts of PCR amplification reaction reagent.
Preferably, the raw materials comprise the following components: 80 parts of deionized water, 20 parts of a salmonella monoclonal antibody, 15 parts of staphylococcus aureus protein A, 10 parts of serine acetyltransferase, 50 parts of a liquid culture medium, 50 parts of amino acid, 20 parts of oligonucleotide for detection, 10 parts of PCR primers and 10 parts of PCR amplification reaction reagent.
Preferably, the raw materials comprise the following components: 65 parts of deionized water, 15 parts of salmonella monoclonal antibody, 12.5 parts of staphylococcus aureus protein A, 7.5 parts of serine acetyltransferase, 40 parts of liquid culture medium, 35 parts of amino acid, 15 parts of oligonucleotide for detection, 7.5 parts of PCR primer and 7.5 parts of PCR amplification reaction reagent.
Preferably, the raw materials comprise the following components: 50 parts of deionized water, 1 part of salmonella monoclonal antibody, 10 parts of staphylococcus aureus protein A, 5 parts of serine acetyltransferase, 30 parts of liquid culture medium, 20 parts of amino acid, 10 parts of oligonucleotide for detection, 5 parts of PCR primer and 5 parts of PCR amplification reaction reagent.
Preferably, the liquid medium comprises glucose, peptone, yeast extract and sodium chloride, and the amino acid is one or more of glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tyrosine, cysteine, methionine, asparagine, glutamine, threonine and aspartic acid.
Preferably, the liquid phase chip further comprises an internal reference nucleotide capture probe.
The invention also discloses a kit for multiple bacteria in food, which comprises the liquid phase chip of claim 1 and internal reference nucleotide.
The invention also discloses a method for rapidly detecting various bacteria in food, which specifically comprises the following steps:
s1, firstly putting the liquid culture medium into a stirrer, wherein the temperature of the stirrer is 36-40 ℃, stirring for 20-30 minutes until the solute is completely dissolved, then cooling to room temperature, and transferring the liquid culture medium into a centrifuge;
s2, inoculating a sample to be detected into a culture medium, adding deionized water, centrifuging for 30-40 minutes, carrying out PCR reaction, carrying out denaturation at 94 ℃, carrying out denaturation at 72 ℃, entering circulation, and terminating extension at 72 ℃ after 35 cycles to obtain a sample PCR product;
and S3, detecting the prepared sample PCR product, and detecting through a PCR detector, namely a fluorescence detector, so as to obtain the content and the type of the bacteria in the food.
(III) advantageous effects
The invention provides a method for rapidly detecting various bacteria in food, a liquid chip and a kit. Compared with the prior art, the method has the following beneficial effects:
(1) the kit comprises the liquid phase chip and the internal reference nucleotide according to claim 1, can carry out rapid and convenient qualitative and quantitative detection on various known food bacteria, has the advantages of high flux, high precision, good stability and the like, improves the detection efficiency of the food bacteria, shortens the detection time, and is simple to operate.
(2) The invention utilizes artificially synthesized labeled molecules with a certain amount of detectable groups to realize accurate quantitative labeling of microsphere carriers, and obviously improves the stability and the discrimination of the labeling of the same type of microsphere carriers.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described, 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.
The embodiment of the invention provides three technical schemes: a method for rapidly detecting various bacteria in food specifically comprises the following embodiments:
example 1
S1, firstly putting 50 parts of liquid culture medium into a stirrer, wherein the temperature of the stirrer is 40 ℃, stirring for 30 minutes until solute is completely dissolved, then cooling to room temperature, and transferring 50 parts of liquid culture medium into a centrifuge;
s2, inoculating a sample to be detected into a culture medium, adding 80 parts of deionized water, centrifuging for 40 minutes, carrying out PCR reaction, carrying out denaturation at 94 ℃, carrying out denaturation at 72 ℃, entering circulation, and terminating extension at 72 ℃ after 35 cycles to obtain a sample PCR product;
and S3, detecting the prepared sample PCR product, and detecting through a PCR detector, namely a fluorescence detector, so as to obtain the content and the type of the bacteria in the food.
Example 2
S1, firstly, putting 40 parts of liquid culture medium into a stirrer, wherein the temperature of the stirrer is 38 ℃, stirring for 25 minutes until solute is completely dissolved, then cooling to room temperature, and transferring 40 parts of liquid culture medium into a centrifuge;
s2, inoculating a sample to be detected into a culture medium, adding 65 parts of deionized water, centrifuging for 35 minutes, carrying out PCR reaction, carrying out denaturation at 94 ℃, carrying out denaturation at 72 ℃, entering circulation, and terminating extension at 72 ℃ after 35 cycles to obtain a sample PCR product;
and S3, detecting the prepared sample PCR product, and detecting through a PCR detector, namely a fluorescence detector, so as to obtain the content and the type of the bacteria in the food.
Example 3
S1, firstly, putting 30 parts of liquid culture medium into a stirrer, wherein the temperature of the stirrer is 36 ℃, stirring for 20 minutes until solute is completely dissolved, then cooling to room temperature, and transferring 30 parts of liquid culture medium into a centrifuge;
s2, inoculating a sample to be detected into a culture medium, adding 50 parts of deionized water, centrifuging for 30 minutes, carrying out PCR reaction, carrying out denaturation at 94 ℃, carrying out denaturation at 72 ℃, entering circulation, and terminating extension at 72 ℃ after 35 cycles to obtain a sample PCR product;
and S3, detecting the prepared sample PCR product, and detecting through a PCR detector, namely a fluorescence detector, so as to obtain the content and the type of the bacteria in the food.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A liquid chip of various bacteria in food is characterized in that: the kit mainly comprises 50-80 parts of deionized water, 10-20 parts of salmonella monoclonal antibody, 10-15 parts of staphylococcus aureus protein A, 5-10 parts of serine acetyltransferase, 30-50 parts of liquid culture medium, 20-50 parts of amino acid, 10-20 parts of oligonucleotide for detection, 5-10 parts of PCR primer and 5-10 parts of PCR amplification reaction reagent.
2. The liquid phase chip for multiple bacteria in food according to claim 1, wherein: the raw materials comprise the following components: 80 parts of deionized water, 20 parts of a salmonella monoclonal antibody, 15 parts of staphylococcus aureus protein A, 10 parts of serine acetyltransferase, 50 parts of a liquid culture medium, 50 parts of amino acid, 20 parts of oligonucleotide for detection, 10 parts of PCR primers and 10 parts of PCR amplification reaction reagent.
3. The liquid phase chip for multiple bacteria in food according to claim 1, wherein: the raw materials comprise the following components: 65 parts of deionized water, 15 parts of salmonella monoclonal antibody, 12.5 parts of staphylococcus aureus protein A, 7.5 parts of serine acetyltransferase, 40 parts of liquid culture medium, 35 parts of amino acid, 15 parts of oligonucleotide for detection, 7.5 parts of PCR primer and 7.5 parts of PCR amplification reaction reagent.
4. The liquid phase chip for multiple bacteria in food according to claim 1, wherein: the raw materials comprise the following components: 50 parts of deionized water, 1 part of salmonella monoclonal antibody, 10 parts of staphylococcus aureus protein A, 5 parts of serine acetyltransferase, 30 parts of liquid culture medium, 20 parts of amino acid, 10 parts of oligonucleotide for detection, 5 parts of PCR primer and 5 parts of PCR amplification reaction reagent.
5. The liquid phase chip of a plurality of bacteria in a food according to any one of claims 1 to 4, wherein: the liquid culture medium comprises glucose, peptone, yeast extract and sodium chloride, and the amino acid is one or more of glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tyrosine, cysteine, methionine, asparagine, glutamine, threonine and aspartic acid.
6. The liquid phase chip of a plurality of bacteria in a food according to any one of claims 1 to 4, wherein: the liquid phase chip also comprises an internal reference nucleotide capture probe.
7. A kit for multiple bacteria in a food product, comprising: the kit comprises the liquid phase chip of claim 1 and an internal reference nucleotide.
8. A method for rapidly detecting various bacteria in food is characterized by comprising the following steps: the method specifically comprises the following steps:
s1, firstly putting the liquid culture medium into a stirrer, wherein the temperature of the stirrer is 36-40 ℃, stirring for 20-30 minutes until the solute is completely dissolved, then cooling to room temperature, and transferring the liquid culture medium into a centrifuge;
s2, inoculating a sample to be detected into a culture medium, adding deionized water, centrifuging for 30-40 minutes, carrying out PCR reaction, carrying out denaturation at 94 ℃, carrying out denaturation at 72 ℃, entering circulation, and terminating extension at 72 ℃ after 35 cycles to obtain a sample PCR product;
and S3, detecting the prepared sample PCR product, and detecting through a PCR detector, namely a fluorescence detector, so as to obtain the content and the type of the bacteria in the food.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201911132172.3A CN110724753A (en) | 2019-11-19 | 2019-11-19 | Method for rapidly detecting multiple bacteria in food, liquid phase chip and kit |
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| CN201911132172.3A CN110724753A (en) | 2019-11-19 | 2019-11-19 | Method for rapidly detecting multiple bacteria in food, liquid phase chip and kit |
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| CN101974641A (en) * | 2010-11-19 | 2011-02-16 | 南京市产品质量监督检验院 | Multiple fluorescence quantitative PCR method for simultaneous and fast detecting three types of pathogenic bacteria in food |
| CN105316398A (en) * | 2014-07-30 | 2016-02-10 | 益善生物技术股份有限公司 | Amplification primer for detecting food-borne pathogenic microorganisms and liquid chip kit |
| CN105316391A (en) * | 2014-06-23 | 2016-02-10 | 北京市理化分析测试中心 | Method of detecting salmonella, shigella and staphylococcus aureus |
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2019
- 2019-11-19 CN CN201911132172.3A patent/CN110724753A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974641A (en) * | 2010-11-19 | 2011-02-16 | 南京市产品质量监督检验院 | Multiple fluorescence quantitative PCR method for simultaneous and fast detecting three types of pathogenic bacteria in food |
| CN105316391A (en) * | 2014-06-23 | 2016-02-10 | 北京市理化分析测试中心 | Method of detecting salmonella, shigella and staphylococcus aureus |
| CN105316398A (en) * | 2014-07-30 | 2016-02-10 | 益善生物技术股份有限公司 | Amplification primer for detecting food-borne pathogenic microorganisms and liquid chip kit |
Non-Patent Citations (1)
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| 党亚丽;周亭屹;: "单增李斯特菌、副溶血弧菌和沙门氏菌液相芯片检测方法的建立" * |
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