CN116656849A - Primer group, kit and detection method for rapidly detecting listeria monocytogenes - Google Patents
Primer group, kit and detection method for rapidly detecting listeria monocytogenes Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 69
- 241000186779 Listeria monocytogenes Species 0.000 title claims abstract description 54
- 239000011324 bead Substances 0.000 claims abstract description 68
- 238000006243 chemical reaction Methods 0.000 claims abstract description 28
- 230000035945 sensitivity Effects 0.000 claims abstract description 5
- 239000000126 substance Substances 0.000 claims abstract description 5
- 108020004414 DNA Proteins 0.000 claims description 49
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- 239000003153 chemical reaction reagent Substances 0.000 claims description 16
- 238000012408 PCR amplification Methods 0.000 claims description 15
- 239000012634 fragment Substances 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 12
- 238000007400 DNA extraction Methods 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 10
- 210000002421 cell wall Anatomy 0.000 claims description 10
- 238000001502 gel electrophoresis Methods 0.000 claims description 10
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 9
- 239000012807 PCR reagent Substances 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 230000003321 amplification Effects 0.000 claims description 7
- 239000011536 extraction buffer Substances 0.000 claims description 7
- 238000012986 modification Methods 0.000 claims description 7
- 230000004048 modification Effects 0.000 claims description 7
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 7
- 108020004711 Nucleic Acid Probes Proteins 0.000 claims description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 6
- 230000009471 action Effects 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 6
- 210000004027 cell Anatomy 0.000 claims description 6
- 238000013461 design Methods 0.000 claims description 6
- 239000002853 nucleic acid probe Substances 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 4
- 238000012136 culture method Methods 0.000 claims description 4
- 230000004544 DNA amplification Effects 0.000 claims description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 239000000969 carrier Substances 0.000 claims description 3
- 238000012790 confirmation Methods 0.000 claims description 3
- 150000002500 ions Chemical class 0.000 claims description 3
- 238000005191 phase separation Methods 0.000 claims description 3
- 238000012257 pre-denaturation Methods 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 239000007790 solid phase Substances 0.000 claims description 3
- 238000003786 synthesis reaction Methods 0.000 claims description 3
- 235000013305 food Nutrition 0.000 abstract description 6
- 230000007613 environmental effect Effects 0.000 abstract description 4
- 108090000623 proteins and genes Proteins 0.000 abstract description 4
- 238000012544 monitoring process Methods 0.000 abstract description 2
- 239000000306 component Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 244000052616 bacterial pathogen Species 0.000 description 2
- 239000008358 core component Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 235000021067 refined food Nutrition 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
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- 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
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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Abstract
The invention discloses a primer group, a kit and a detection method for rapidly detecting listeria monocytogenes, wherein the primer sequence is as follows: forward, 5'-GCCACGTTCTTCAGGCTGGTT-3'; reverse, 5'-TTCCCCATTGCGTCACCTATCC-3'. The magnetic bead-based PCR detection method has the main advantages that the magnetic beads are used for enriching target DNA, interference substances can be removed, detection sensitivity and specificity are improved, meanwhile, influence on PCR reaction is avoided, meanwhile, the PCR gene detection method has a certain detection limit and detection speed, listeria monocytogenes in food samples, environmental samples and the like can be accurately screened, the PCR gene detection method and the magnetic bead-based PCR detection method are combined for detection, the Listeria monocytogenes can be detected from multiple dimensions, detection accuracy and reliability are improved, and the magnetic bead-based PCR detection method has great application value in the fields of food safety assurance, environmental monitoring and the like.
Description
Technical Field
The invention relates to the field of bacteria detection, in particular to a primer group, a kit and a detection method for rapidly detecting listeria monocytogenes.
Background
The listeria monocytogenes is a pathogenic bacterium, is usually present in food and seriously threatens the health of people, is a common food-borne pathogenic bacterium, has stronger viability and better adaptability to processed food, so that a primer group, a kit and a detection method for rapidly and accurately detecting the listeria monocytogenes are very important for guaranteeing the safety of food;
the detection methods for rapidly detecting the listeria monocytogenes are numerous and comprise a traditional culture method, an immunological detection method, a genetic detection method and the like, and a single detection method has certain defects and shortcomings, and meanwhile, the defects in the respective detection methods cannot be overcome by single detection, so that the reliability and the accuracy after detection are insufficient, and therefore, a primer group, a kit and a detection method for rapidly detecting the listeria monocytogenes need to be designed.
Disclosure of Invention
The invention aims to provide a primer group, a kit and a detection method for rapidly detecting listeria monocytogenes, so as to solve the problems in the background technology.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a primer set for rapidly detecting listeria monocytogenes comprises
A primer set, the primer sequence being as follows:
Forward:5’-GCCACGTTCTTCAGGCTGGTT-3’；
Reverse:5’-TTCCCCATTGCGTCACCTATCC-3’。
a kit for rapidly detecting Listeria monocytogenes comprises
The kit comprises the following components:
DNA extraction buffer: can rapidly decompose cell walls and release DNA in cells;
magnetic beads: the magnetic beads are carriers commonly used in the solid phase separation technology, in the listeria monocytogenes detection kit, the surfaces of the magnetic beads are provided with specific nucleic acid probes, the specific nucleic acid probes can be combined with target listeria monocytogenes DNA fragments, the magnetic beads can enable the target DNA to be combined with the probes in the reaction, and the magnetic beads can be adsorbed and separated under the action of a magnetic field;
specific primers: designing a specific primer is an important step in a detection kit, wherein the design of the primer should pay attention to a specific region of target DNA so as to increase the specificity and sensitivity of detection, and in the PCR reaction, the specific primer can regulate a region of interest of target DNA amplification and avoid influencing a detection result;
PCR reagent: PCR is a key step of primer amplification of PCR products, and the PCR enzyme in the detection kit is a thermostable DNA polymerase;
buffer solution: the buffer solution contains ions with a certain concentration, so that the pH value of the reaction can be adjusted and the stability of the reaction can be maintained;
PCR product detection reagent: for detecting specific PCR products, such as gel electrophoresis reagents, colloidal gold reagents, etc.;
magnetic bead separator: for separating out the magnetic beads.
Further, the preparation of the kit specifically comprises:
preparation of DNA extraction buffer: the buffer contains chemical substances, which can rapidly decompose cell walls and release DNA in cells, such as BufferATL, bufferAL;
design and synthesis of specific primers: according to the DNA sequence of Listeria monocytogenes, selecting specific primers, wherein the primers should be prevented from being matched with the DNA sequence of non-target bacteria as much as possible;
preparation of PCR reagents: preparing reagents required by PCR reaction, including PCR enzyme, buffer solution, dNTPs, primers and the like, for amplifying the specific DNA fragment of the listeria monocytogenes;
preparation and modification of magnetic beads: covalent modification is carried out on magnetic beads with proper sizes and specific probes, and the modified magnetic beads can selectively carry out specific recognition on certain molecules in the cell wall of the listeria monocytogenes;
magnetic bead-based DNA extraction assay: adding a sample and magnetic beads into the packed microplate or tube, combining the DNA with the magnetic beads, separating and purifying the DNA by using the magnetic beads, and recovering the purified DNA after washing.
A detection method for rapidly detecting listeria monocytogenes, comprising the steps of:
step S1, sample treatment: treating a sample to be detected by adopting a traditional enrichment culture method, and extracting DNA in the sample;
step S2, PCR amplification: performing PCR amplification on the extracted DNA to obtain a PCR product;
step S3, magnetic bead separation: adding the PCR product into a magnetic bead system, enriching target DNA by the magnetic beads, and washing to remove non-specific products;
step S4, product analysis: performing PCR amplification on the product with the non-specificity removed, and performing gel electrophoresis analysis or color reaction such as colloidal gold on the PCR product;
step S5, observation and confirmation: by observing the presence or absence and size of the gel strips, it is determined whether listeria monocytogenes is present.
Further, in the step S2, the purified DNA is added into a PCR reaction system for amplifying the DNA fragment of the Listeria monocytogenes by a specific primer.
Further, in the step S3, the magnetic bead purification of the PCR product is performed by absorbing the product by the magnetic bead, removing the non-specific PCR product, washing again, and recovering the purified PCR product.
Further, in the step S4, the PCR product detection is performed on the recovered PCR product by various detection methods, such as gel electrophoresis, colloidal gold, and the like.
Further, in the step S2, the reaction conditions for PCR amplification are as follows:
pre-denaturation procedure: 95 ℃ for 5min;
35 rounds of amplification: 95 ℃ for 30s;58 ℃ for 30s;72 ℃,30s;
and (3) terminating the program: 72℃for 10min.
Compared with the prior art, the invention has the beneficial effects that:
1. the magnetic bead-based PCR detection method has the main advantages that the magnetic beads are used for enriching target DNA, interference substances can be removed, detection sensitivity and specificity are improved, meanwhile, influence on PCR reaction is avoided, meanwhile, a PCR gene detection rule has a certain detection limit and detection speed, listeria monocytogenes in food samples, environmental samples and the like can be accurately screened, the Listeria monocytogenes can be detected from multiple dimensions by combining the PCR gene detection method and the magnetic bead-based PCR detection method, and the detection method has great application value in the fields of food safety assurance, environmental monitoring and the like;
2. the DNA extraction buffer solution can effectively decompose the cell wall of the listeria monocytogenes to release DNA in the listeria monocytogenes, so that a foundation is provided for subsequent PCR amplification and magnetic bead separation, a specific primer is a key component part of the PCR amplification reaction, a target DNA fragment of the listeria monocytogenes can be accurately amplified, the amplification of DNA of non-target bacteria is avoided, a PCR reagent is a core component part of the PCR amplification reaction, and the PCR reagent comprises PCR enzyme, buffer solution, dNTPs, primers and the like, so that the target DNA fragment of the listeria monocytogenes can be amplified;
3. the magnetic beads are one of the core components of the kit, are high-sensitivity and high-specificity target DNA separation tools, can rapidly and accurately separate specific DNA fragments of Listeria monocytogenes, the magnetic bead separators are used for separating magnetic beads from detection samples, and recovering PCR products, and the PCR product detection reagents are reagents for detecting specific PCR products, including gel electrophoresis reagents, colloidal gold reagents and the like, can be used for detecting PCR products successfully amplified, and can be used for verifying the accuracy of detection results.
Drawings
FIG. 1 is a schematic flow chart of a primer set, a kit and a detection method for rapidly detecting Listeria monocytogenes.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1, the present invention provides a technical solution:
a primer set for rapidly detecting listeria monocytogenes comprises
A primer set, the primer sequence being as follows:
Forward:5’-GCCACGTTCTTCAGGCTGGTT-3’；
Reverse:5’-TTCCCCATTGCGTCACCTATCC-3’。
a kit for rapidly detecting Listeria monocytogenes comprises
The kit comprises the following components:
DNA extraction buffer: can rapidly decompose cell walls and release DNA in cells;
magnetic beads: the magnetic beads are carriers commonly used in the solid phase separation technology, in the listeria monocytogenes detection kit, the surfaces of the magnetic beads are provided with specific nucleic acid probes, the specific nucleic acid probes can be combined with target listeria monocytogenes DNA fragments, the magnetic beads can enable the target DNA to be combined with the probes in the reaction, and the magnetic beads can be adsorbed and separated under the action of a magnetic field;
specific primers: designing a specific primer is an important step in a detection kit, wherein the design of the primer should pay attention to a specific region of target DNA so as to increase the specificity and sensitivity of detection, and in the PCR reaction, the specific primer can regulate a region of interest of target DNA amplification and avoid influencing a detection result;
PCR reagent: PCR is a key step of primer amplification of PCR products, and the PCR enzyme in the detection kit is a thermostable DNA polymerase;
buffer solution: the buffer solution contains ions with a certain concentration, so that the pH value of the reaction can be adjusted and the stability of the reaction can be maintained;
PCR product detection reagent: for detecting specific PCR products, such as gel electrophoresis reagents, colloidal gold reagents, etc.;
magnetic bead separator: for separating out the magnetic beads.
In the invention, the preparation of the kit specifically comprises the following steps:
preparation of DNA extraction buffer: the buffer contains chemical substances, which can rapidly decompose cell walls and release DNA in cells, such as BufferATL, bufferAL;
design and synthesis of specific primers: according to the DNA sequence of Listeria monocytogenes, selecting specific primers, wherein the primers should be prevented from being matched with the DNA sequence of non-target bacteria as much as possible;
preparation of PCR reagents: preparing reagents required by PCR reaction, including PCR enzyme, buffer solution, dNTPs, primers and the like, for amplifying the specific DNA fragment of the listeria monocytogenes;
preparation and modification of magnetic beads: covalent modification is carried out on magnetic beads with proper sizes and specific probes, and the modified magnetic beads can selectively carry out specific recognition on certain molecules in the cell wall of the listeria monocytogenes;
magnetic bead-based DNA extraction assay: adding a sample and magnetic beads into the packed microplate or tube, combining the DNA with the magnetic beads, separating and purifying the DNA by using the magnetic beads, and recovering the purified DNA after washing.
A detection method for rapidly detecting listeria monocytogenes, comprising the steps of:
step S1, sample treatment: treating a sample to be detected by adopting a traditional enrichment culture method, and extracting DNA in the sample;
step S2, PCR amplification: performing PCR amplification on the extracted DNA to obtain a PCR product;
step S3, magnetic bead separation: adding the PCR product into a magnetic bead system, enriching target DNA by the magnetic beads, and washing to remove non-specific products;
step S4, product analysis: performing PCR amplification on the product with the non-specificity removed, and performing gel electrophoresis analysis or color reaction such as colloidal gold on the PCR product;
step S5, observation and confirmation: by observing the presence or absence and size of the gel strips, it is determined whether listeria monocytogenes is present.
In the present invention, in the step S2, the purified DNA is added to a PCR reaction system, and the DNA fragment of listeria monocytogenes is amplified by a specific primer.
In the present invention, in the step S3, the magnetic bead purification of the PCR product is performed by absorbing the product by the magnetic bead, removing the non-specific PCR product, washing again, and recovering the purified PCR product.
In the present invention, in the step S4, the PCR product detection is performed on the recovered PCR product by various detection methods, such as gel electrophoresis and colloidal gold.
In the present invention, in the step S2, the reaction conditions for PCR amplification are as follows:
pre-denaturation procedure: 95 ℃ for 5min;
35 rounds of amplification: 95 ℃ for 30s;58 ℃ for 30s;72 ℃,30s;
and (3) terminating the program: 72℃for 10min.
It is noted that relational terms such as first and second, and the like are 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. Moreover, 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 understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein 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 primer group for rapidly detecting listeria monocytogenes is characterized by comprising
A primer set, the primer sequence being as follows:
Forward:5’-GCCACGTTCTTCAGGCTGGTT-3’；
Reverse:5’-TTCCCCATTGCGTCACCTATCC-3’。
2. a kit for rapidly detecting listeria monocytogenes is characterized in that: comprising
The kit comprises the following components:
DNA extraction buffer: can rapidly decompose cell walls and release DNA in cells;
magnetic beads: the magnetic beads are carriers commonly used in the solid phase separation technology, in the listeria monocytogenes detection kit, the surfaces of the magnetic beads are provided with specific nucleic acid probes, the specific nucleic acid probes can be combined with target listeria monocytogenes DNA fragments, the magnetic beads can enable the target DNA to be combined with the probes in the reaction, and the magnetic beads can be adsorbed and separated under the action of a magnetic field;
specific primers: designing a specific primer is an important step in a detection kit, wherein the design of the primer should pay attention to a specific region of target DNA so as to increase the specificity and sensitivity of detection, and in the PCR reaction, the specific primer can regulate a region of interest of target DNA amplification and avoid influencing a detection result;
PCR reagent: PCR is a key step of primer amplification of PCR products, and the PCR enzyme in the detection kit is a thermostable DNA polymerase;
buffer solution: the buffer solution contains ions with a certain concentration, so that the pH value of the reaction can be adjusted and the stability of the reaction can be maintained;
PCR product detection reagent: for detecting specific PCR products, such as gel electrophoresis reagents, colloidal gold reagents, etc.;
magnetic bead separator: for separating out the magnetic beads.
3. A detection method for rapidly detecting listeria monocytogenes is characterized by comprising the following steps: the method comprises the following steps:
step S1, sample treatment: treating a sample to be detected by adopting a traditional enrichment culture method, and extracting DNA in the sample;
step S2, PCR amplification: performing PCR amplification on the extracted DNA to obtain a PCR product;
step S3, magnetic bead separation: adding the PCR product into a magnetic bead system, enriching target DNA by the magnetic beads, and washing to remove non-specific products;
step S4, product analysis: performing PCR amplification on the product with the non-specificity removed, and performing gel electrophoresis analysis or color reaction such as colloidal gold on the PCR product;
step S5, observation and confirmation: by observing the presence or absence and size of the gel strips, it is determined whether listeria monocytogenes is present.
4. The kit for rapid detection of listeria monocytogenes of claim 2, wherein: the preparation of the kit specifically comprises the following steps:
preparation of DNA extraction buffer: the Buffer contains chemical substances, which can rapidly decompose cell walls and release DNA in cells, such as Buffer ATL, buffer AL, etc.;
design and synthesis of specific primers: according to the DNA sequence of Listeria monocytogenes, selecting specific primers, wherein the primers should be prevented from being matched with the DNA sequence of non-target bacteria as much as possible;
preparation of PCR reagents: preparing reagents required by PCR reaction, including PCR enzyme, buffer solution, dNTPs, primers and the like, for amplifying the specific DNA fragment of the listeria monocytogenes;
preparation and modification of magnetic beads: covalent modification is carried out on magnetic beads with proper sizes and specific probes, and the modified magnetic beads can selectively carry out specific recognition on certain molecules in the cell wall of the listeria monocytogenes;
magnetic bead-based DNA extraction assay: adding a sample and magnetic beads into the packed microplate or tube, combining the DNA with the magnetic beads, separating and purifying the DNA by using the magnetic beads, and recovering the purified DNA after washing.
5. The method for rapid detection of listeria monocytogenes as claimed in claim 3, wherein: in the step S2, the purified DNA is added into a PCR reaction system for amplifying the DNA fragment of the listeria monocytogenes by a specific primer.
6. The method for rapid detection of listeria monocytogenes as claimed in claim 3, wherein: in the step S3, the magnetic bead purification of the PCR product is to remove the nonspecific PCR product by absorbing the product with the magnetic bead, and then washing again to recover the purified PCR product.
7. The method for rapid detection of listeria monocytogenes as claimed in claim 3, wherein: in the step S4, the PCR product detection is performed on the recovered PCR product by various detection methods, such as gel electrophoresis, colloidal gold, and the like.
8. The method for rapid detection of listeria monocytogenes as claimed in claim 3, wherein: in the step S2, the reaction conditions for PCR amplification are as follows:
pre-denaturation procedure: 95 ℃ for 5min;
35 rounds of amplification: 95 ℃ for 30s;58 ℃ for 30s;72 ℃,30s;
and (3) terminating the program: 72℃for 10min.
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