CN110938704A - LAMP (loop-mediated isothermal amplification) synchronous detection method and kit for staphylococcus aureus and salmonella in liquid milk - Google Patents
LAMP (loop-mediated isothermal amplification) synchronous detection method and kit for staphylococcus aureus and salmonella in liquid milk Download PDFInfo
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Abstract
The invention relates to a LAMP (loop-mediated isothermal amplification) synchronous detection method and kit for staphylococcus aureus and salmonella in liquid milk, belonging to the technical field of food safety rapid detection. Extracting the DNA of the liquid milk genome on site, designing a primer group based on the specific molecular marker characteristics of staphylococcus aureus and salmonella genomes, and synchronously detecting the polluted staphylococcus aureus and salmonella in the liquid milk by adopting a loop-mediated isothermal amplification technology. The requirements of the whole detection process on the environment and equipment are low, and whether the sample to be detected contains staphylococcus aureus or salmonella is determined by adding the color developing agent to observe the color change in the tube. The method has the advantages of high sensitivity, good specificity, safety, harmlessness, no organic reagent, no nucleic acid binding dye, and capability of directly releasing all reagents into the environment, and has important significance for improving the quality safety of dairy food and improving the analysis and detection technology of food-borne pathogenic bacteria.
Description
Technical Field
The invention relates to the technical field of rapid detection of food safety, in particular to rapid detection of two important food-borne pathogenic bacteria, namely staphylococcus aureus and salmonella, and particularly relates to a preparation method of a detection sample, a detection primer set, a detection kit and a specific detection method of an LAMP detection method for detecting staphylococcus aureus and salmonella in dairy products.
Background
Staphylococcus aureus (Staphylococcus aureus) is a widely distributed gram-positive bacterium that can be widely distributed in soil, air, water, and human skin. Staphylococcus aureus, as a common pathogenic bacterium, can produce various virulence factors such as enterotoxin. The virulence factors have strong virulence, low virulence dose and good stability, are difficult to remove in the food processing process and have serious harmfulness. The contamination of various foods such as pork, chicken, milk powder, egg, bread, ice cream and the like by staphylococcus aureus can be reported. Staphylococcus aureus causes food poisoning and is characterized by rapid onset (2-8h), usually accompanied by symptoms of nausea, vomiting, abdominal pain and diarrhea. According to the literature report, the separation rate of staphylococcus aureus in fresh milk is 43% -75%, the pollution rate is very high, so that staphylococcus aureus can grow and enterotoxin can be produced if the fresh milk is stored for too long at room temperature, the temperature is too high, or the production line is stopped for too long in the process of processing dairy products, and serious food-borne diseases can be caused. Food poisoning events caused by staphylococcus aureus contamination of milk have become a major concern in the milk production process.
Salmonella (Salmonella) is a facultative anaerobic gram-negative bacterium, has a wide distribution range, is distributed in sewage, soil and animal bodies, has extremely strong viability, and can survive for months once polluting food, thereby being an important food-borne pathogenic bacterium. Over 2500 serotypes are found on a global scale, with a wide range of prevalence. As reported by the U.S. CDC, salmonella causes 100 million cases of illness, 19,000 hospitalizations, and 380 deaths in the united states each year. Meanwhile, the salmonella has higher susceptibility, and according to reports, the low content of salmonella (10) in the food can cause the disease of eaters. In the process of processing dairy products, the salmonella pollution rate is higher, and researches indicate that the separation rate in fresh milk is 2.6-11.8%. The separation rate is related to the production process and environment of dairy products, and the detection rate of some fresh milk is as high as 28.1%.
Notomi of Japan scholars in 2000 established a new Isothermal nucleic acid Amplification technology, Loop-mediated Isothermal Amplification (LAMP). The LAMP technology has the following advantages: (1) and (3) the speed is high. The whole reaction process is carried out under the isothermal condition and can be finished only in 30-60 min; (2) the result is easy to identify. The precipitate generated by combining specific dyes (calcein, HNB, SYBR Green) or pyrophosphate ions and magnesium ions can be directly judged, and an instrument can be used for result analysis; (3) the sensitivity is strong. The detection process only needs extremely low substrate template concentration; (4) the specificity is strong. The reaction involves a plurality of pairs of specific primers, and can accurately target a target sequence; (5) the reaction cost is low. The whole reaction is completed under the isothermal condition, so the amplification can be completed in a water bath kettle or a metal bath, and the requirement on equipment is extremely low. Currently, as a rapid detection method based on molecular biology, LAMP has been widely used in the field of food pathogenic bacteria.
The LAMP technology is high in sensitivity, strong in specificity, simple to operate, high in speed, suitable for on-site and easy to standardize, and the LAMP technology is recommended to be used for detecting transgenic components in food according to various import and export industry standards. Aiming at enterprise requirements, the LAMP synchronous detection kit for two kinds of poisoning bacteria in the visual liquid milk is developed by utilizing the LAMP technology, the result is directly obtained, the instrument is separated, the detection time is shortened, and the requirement for rapidly detecting the same-quality and same-standard prevention risk is met.
Staphylococcus aureus and salmonella are two food poisoning bacteria which are easily polluted by liquid milk, and cannot be detected in the liquid milk specified by the national standard, but the existing detection method for the two pathogenic bacteria by the national standard takes 3 days, so that the problem that an enterprise cannot put the fresest product on the market in time and send the fresest product to a consumer is caused. A fast and accurate technical scheme is urgently needed to solve the technical problem.
Disclosure of Invention
The LAMP synchronous detection method and kit for staphylococcus aureus and salmonella in liquid milk provided by the invention have the advantages of accurate qualitative determination, good repeatability, high sensitivity, rapidness, simplicity and lower application cost, and can effectively ensure the food safety of the liquid milk.
The technical problem solved by the invention is to provide a method for extracting the total genome of the liquid milk, which comprises the steps of taking a proper amount of the liquid milk, and centrifugally collecting and precipitating. Washing the precipitate with normal saline for 3 times; adding lysozyme into the precipitate, and adding lysate, 10min at 37 deg.C, protease K, 10min at 37 deg.C; (ii) a Centrifuging at 12000 Xg for 2min, collecting supernatant, extracting with phenol chloroform, washing the extract with anhydrous ethanol, and adding ddH2And dissolving the precipitate by using O to obtain a genome sample to be detected.
The invention aims to provide an LAMP detection primer group for simultaneously detecting staphylococcus aureus and salmonella, which respectively consists of a pair of outer primers and an inner primer, and the specific nucleotide sequence comprises the following components:
the detection primer group of the salmonella primer group has the following sequence:
stn-F3：5’-ACCAGATTCAGGGAGTGAG-3’
stn-B3：5’-CGCGCACGAAATTCGTAAC-3
stn-FIP：5’-ACCGGGTGGTAAGCGAATTGCGAGGTTAACCGTCTGGAGC-3’
stn-BIP：5’-GGCCTCTTTGGCCATCACCTGGCTGGCGAAATACTTTGC-3’
the staphylococcus aureus primer group has the following sequence:
rpoB-F3：5’-CCAATTAGTACGTGTATATATCGTT-3’
rpoB-B3：5’-ACTTGTCCGATGTTCATACG-3
rpoB-FIP：
5’-AGGAACAATCTTAGAAATGACACCTAATTCATGTTGGTGATAAGATGTG-3’
rpoB-BIP：5’-GATATGCCTTACTTACCAGATGGACAGATGGTACACCAAGAGGATT-3’
the invention also solves the technical problem of providing the LAMP detection kit for simultaneously detecting staphylococcus aureus and salmonella. Wherein, the main reagent components are as follows: LAMP buffer solution, betaine, Bst DNA polymerase, magnesium chloride solution, negative control solution, positive control solution and double distilled water.
Wherein the using amounts of the reagents are respectively as follows:
the dosage of the LAMP buffer solution is as follows: 2-3 mu L; bst DNA polymerase was used in the following amounts: 0.5-1.5 μ L; the dosage of betaine is as follows: 5-15M; the dosage of the magnesium chloride solution is as follows: 15-25 Mm; the working concentration of the forward outer primer (F3) and the reverse outer primer (B3) is 10-15 mu M, and the dosage is 0.3-0.7 mu L; the working concentration of the Forward Inner Primer (FIP) and the reverse inner primer (BIP) is 10-15 mu M, and the dosage is 1-3 mu L; the working concentration of dNTP is 5-15 Mm, and the dosage is 1.5-3.5 muL. In the detection process, 1-3 mu L of sample DNA template is added, and the molar ratio of the primers is 1:1:4:4:2: 2.
The invention also solves the technical problem of providing a method for simultaneously detecting staphylococcus aureus and salmonella by using the LAMP detection kit. Reaction conditions are as follows: keeping the temperature for 1h at 65 ℃. The detection method comprises the following steps: 10 μ L of the reaction product was mixed with the loading buffer and detected by 2% gel electrophoresis.
The invention also provides a visual result identification method. Hydroxyl Naphthol Blue (HNB) is used as a color developing agent of the LAMP visualization method, and the reaction progress is judged according to the change (positive reaction is changed from purple to sky blue) of the hydroxyl naphthol blue acting on the LAMP before and after reaction. In order to ensure the specificity of the detection result, negative and positive controls are simultaneously arranged in the detection process.
As an important index influencing the detection effect, the selection of the target fragment is important. In nucleic acid detection, considering the length of staphylococcus aureus and salmonella genomes, the adoption of a full-series detection is unrealistic, time-consuming, labor-consuming and possible to detect results, the invention searches for the specific nucleic acid sequences of staphylococcus aureus and salmonella by comparing the nucleic acid sequences with other target genes, verifies the specificity of the nucleic acid sequences by comparing the nucleic acid sequences with other target genes, finally determines the specific nucleic acid sequences of staphylococcus aureus and salmonella as gene sequences as amplified target fragments, and combines the analysis and the technical effects in the embodiment of the invention to know that the invention at least comprises the following beneficial effects:
(1) the operation is simple and convenient: the DNA extraction step is simple and effective, the template can be directly used for subsequent amplification reaction, and special equipment such as a PCR instrument can be selected or not required for isothermal amplification;
(2) the amplification is rapid: isothermal amplification only needs 60min, the result can be directly interpreted after the amplification is finished, and the time required by the whole detection process is within 2 h.
(3) The specificity is high: the detection primer group comprises six specific primers, only can effectively amplify the Mycobacterium kansasii, but cannot effectively amplify other pathogens such as Mycobacterium tuberculosis complex, Mycobacterium cheloniae, Mycobacterium abscessus, Mycobacterium avium, M.
(4) The sensitivity is high: the lowest value that can be detected is 100 copies.
(5) And (3) pollution avoidance: the fluorescent dye or the color-developing agent is added before the reaction, and the fluorescent dye or the color-developing agent is added without opening a cover after the reaction, so that the diffusion and the pollution of the amplification product can be effectively prevented.
Drawings
FIG. 1 is a drawing of the genome extraction of the present invention;
FIG. 2 is a LAMP detection graph of the present invention;
FIG. 3 is a visual inspection diagram of the present invention.
Detailed Description
For better understanding of the present invention, the following detailed description of the present invention is provided in connection with the following examples, which are provided for the purpose of illustration only and are not intended to limit the present invention. Any replacement or modification of the method, steps or conditions of the present invention by non-creative efforts based on the embodiments of the present invention falls within the protection scope of the present invention.
Example 1 liquid milk sample Collection
And a clean sampling tool and a clean sample bottle are adopted, and the sample bottle cannot leave disinfectant, water and detergent. Respectively sampling in a milk station milking hall, a milk station milk storage tank, a milk carrying vehicle milk tank, a liquid milk production line and a supermarket, recording in detail, and immediately detecting the collected fresh samples.
Example 2
A method for detecting staphylococcus aureus and salmonella in liquid milk by a loop-mediated isothermal amplification technology comprises the following steps:
s1, extracting genome
(1) Sample preparation: taking 1ml of liquid milk, putting the liquid milk into a 1.5ml centrifuge tube, centrifuging the liquid milk for 5min at 12000rpm, and removing supernatant; adding 1ml of normal saline, stirring uniformly, centrifuging at 12000rpm for 1 minute, discarding the supernatant, adding 1ml of normal saline into the precipitate, stirring uniformly.
(2) Bacterial lysis: adding lysozyme into the precipitate obtained in the step (1) at 37 ℃ for 10min, then adding lysis solution, uniformly mixing, carrying out lysis at 37 ℃ for 5min, then adding protease K, and carrying out lysis at 70 ℃ for 10min to obtain lysis solution.
(3) DNA extraction: and (3) extracting the lysate obtained in the step (2) by using phenol chloroform to obtain a white suspension, carefully collecting a supernatant, adding isopropanol and sodium acetate, uniformly mixing, centrifuging and collecting a precipitate. Washing the precipitate with 75% anhydrous ethanol, air drying at room temperature for 5min, and removing residual ethanol; ddH2O was added and incubated at room temperature for 3min to obtain the genome to be detected (FIG. 1).
S2, design and synthesis of loop-mediated isothermal amplification (LAMP) primers
Firstly, by a method of comparing genomics, searching for specific gene sequences of staphylococcus aureus and salmonella, applying special LAMP primer design software to design primers by taking the sequences as target genes, and finally analyzing and optimizing the designed primers on primer-blast. The final determined primer sequence.
Comparative experiments to verify the unique gene sequences of staphylococcus aureus and salmonella included the following amplification primers:
the detection primer group of the salmonella primer group has the following sequence:
F3：5’-ACCAGATTCAGGGAGTGAG-3’
B3：5’-CGCGCACGAAATTCGTAAC-3
FIP：5’-ACCGGGTGGTAAGCGAATTGCGAGGTTAACCGTCTGGAGC-3’
BIP：5’-GGCCTCTTTGGCCATCACCTGGCTGGCGAAATACTTTGC-3’
the staphylococcus aureus primer group has a sequence shown in SEQ NO.5-SEQ NO. 8:
F3：5’-CCAATTAGTACGTGTATATATCGTT-3’
B3：5’-ACTTGTCCGATGTTCATACG-3
FIP：
5’-AGGAACAATCTTAGAAATGACACCTAATTCATGTTGGTGATAAGATGTG-3’
BIP：5’-GATATGCCTTACTTACCAGATGGACAGATGGTACACCAAGAGGATT-3’
the 4 pairs of primers are used for respectively detecting two target genes (a staphylococcus aureus rpoB gene and a salmonella stn gene) of food-borne strains of staphylococcus aureus and salmonella, amplification positive results of the two target genes are compared, the accuracy of the amplification positive results is analyzed, the higher the accuracy is, the higher the specificity of the amplification detection of the target genes on the staphylococcus aureus and the salmonella is, and the two target genes are more suitable for being used for detecting the staphylococcus aureus and the salmonella in the process of processing dairy products.
S3.LAMP detection
Isothermal amplification temperature is 65 ℃ and constant temperature water bath is 1h, and then 80 ℃ is used for inactivating the used polymerase. In a 20-mu-L reaction system, the concentrations of an outer primer F3, an outer primer B3, an inner primer FIP, an inner primer BIP, a loop primer LF and a loop primer LB are all 10 mu M, the addition amounts are 0.5 mu L, 2 mu L, 1 mu L and 1 mu L respectively, and the molar ratio in the reaction system is 1:1:4:4:2: 2; the amount of 0.16U/. mu.LBst DNA polymerase added was 0.4. mu.L.
S4. visual analysis of LAMP detection results
The experiment adopts an agarose gel electrophoresis method: and respectively taking 5 mu L of amplification products, carrying out electrophoresis in 30g/L agarose gel at the voltage of 100V for 30min, and placing the amplification products in a gel imaging system for photographing, wherein if a gradient band appears, the result is positive, and if the gradient band does not appear, the result is negative. Detecting Staphylococcus aureus and Salmonella concentration by agarose gel electrophoresis at 10-108CFU/mL (i.e., dilution factor of 10 in order)1、102、103、104、105、106、107、108Fold) of the amplification product corresponding to the contaminated sample showed a clear ladder-like band, and was positive (FIG. 2).
Note: the selected skim milk powder samples were all proved to be free of staphylococcus aureus and salmonella by the traditional method recommended by the FDA.
In the experiment, hydroxynaphthol blue (HNB) is used as a color developing agent of the LAMP visualization method, and the reaction progress is judged according to the change (positive reaction is changed from purple to sky blue) of the hydroxynaphthol blue (100 mu mol/L) before and after the LAMP reaction. The experiment was set up for blank control and positive groups and observed for color change (figure 3).
Staphylococcus aureus in contaminated samplesAnd Salmonella concentration of 101、102、103、104、105、106、107、108CFU/mL (i.e., dilution factor of 10 in order)1、102、103、104、105、106、107、108Multiple), the corresponding detection tube obviously shows fluorescence blue, namely the detection is positive; when the concentration of staphylococcus aureus and salmonella in the polluted sample is 100CFU/mL (namely the dilution factor is 10)8Double), the corresponding detection tube is still light orange before reaction, which is negative detection.
Example 3
A method for detecting staphylococcus aureus and salmonella in liquid milk by using a loop-mediated isothermal amplification technology, which is different from the method in example 2:
s3.LAMP detection
Isothermal amplification temperature is 60 ℃ and constant temperature water bath is used for 1h, and then used polymerase is inactivated at 80 ℃. In a 20-mu-L reaction system, the concentrations of an outer primer F3, an outer primer B3, an inner primer FIP, an inner primer BIP, a loop primer LF and a loop primer LB are all 10 mu M, the addition amounts are 0.5 mu L, 2 mu L, 1 mu L and 1 mu L respectively, and the molar ratio in the reaction system is 1:1:4:4:2: 2; the amount of 0.16U/. mu.LBst DNA polymerase added was 0.4. mu.L.
S4. visual analysis of LAMP detection results
In the experiment, hydroxynaphthol blue (HNB) is used as a color developing agent of the LAMP visualization method, and the reaction progress is judged according to the change (positive reaction is changed from purple to sky blue) of the hydroxynaphthol blue (100 mu mol/L) before and after the LAMP reaction. Blank control group and positive group were set up for the experiment and color change was observed.
The concentration of Staphylococcus aureus and Salmonella in the contaminated sample was 101、102、103、104、105、106、107、108CFU/mL (i.e., dilution factor of 10 in order)1、102、103、104、105、106、107、108Multiple times) ofThe corresponding detection tube obviously shows fluorescence blue, namely the detection is positive; when the concentration of staphylococcus aureus and salmonella in the polluted sample is 100CFU/mL (namely the dilution factor is 10)8Double), the corresponding detection tube is still light orange before reaction, which is negative detection.
Agarose gel electrophoresis: and respectively taking 5 mu L of amplification products, carrying out electrophoresis in 30g/L agarose gel at the voltage of 100V for 30min, and placing the amplification products in a gel imaging system for photographing, wherein if a gradient band appears, the result is positive, and if the gradient band does not appear, the result is negative. Detecting Staphylococcus aureus and Salmonella concentration by agarose gel electrophoresis at 10-108CFU/mL (i.e., dilution factor of 10 in order)1、102、103、104、105、106、107、108Double) and the amplification product corresponding to the polluted sample shows obvious ladder-shaped bands, and the detection is positive.
Note: the selected skim milk powder samples were all proved to be free of staphylococcus aureus and salmonella by the traditional method recommended by the FDA.
Claims (10)
1. An LAMP synchronous detection method for staphylococcus aureus and salmonella in liquid milk is characterized in that: the method comprises a method for extracting genome in liquid milk, wherein the genome is directly extracted from the liquid milk by using a lysate, the lysate mainly comprises SDS (sodium dodecyl sulfate), and the method comprises the following steps of: SDS, EDTA, Triton, Tris-HCl, and NaCl.
2. The LAMP synchronous detection method for staphylococcus aureus and salmonella in liquid milk according to claim 1, characterized in that: the method for extracting the genome from the liquid milk comprises the following steps:
(1) sample preparation: taking 1ml of liquid milk, putting the liquid milk into a 1.5ml centrifuge tube, centrifuging the liquid milk for 5min at 12000rpm, and removing supernatant; adding 1ml of normal saline, blowing, stirring and mixing uniformly, centrifuging at 12000rpm for 1 minute, removing supernatant, adding 1ml of normal saline into the precipitate, blowing, stirring and mixing uniformly;
(2) bacterial lysis: adding lysozyme into the precipitate obtained in the step (1), adding lysis solution, uniformly mixing, carrying out lysis at 37 ℃ for 5min, then adding protease K, and carrying out lysis at 70 ℃ for 10min to obtain lysis solution;
(3) DNA extraction: and (3) extracting the lysate obtained in the step (2) by using phenol chloroform to obtain a white suspension, carefully collecting a supernatant, adding isopropanol and sodium acetate, uniformly mixing, centrifuging and collecting a precipitate. Washing the precipitate with 75% anhydrous ethanol, air drying at room temperature for 5min, and removing residual ethanol; ddH2O is added and incubated for 3min at room temperature to obtain the genome to be detected.
3. A loop-mediated isothermal amplification primer group for staphylococcus aureus and salmonella is characterized in that target genes of the primer group are specific rpoB genes of staphylococcus aureus and specific stn genes of salmonella, sequence design and optimization are carried out, the target genes respectively comprise a pair of outer primers and a pair of inner primers, a detection primer group of the salmonella primer group has a sequence shown as the following, and the molar ratio in a reaction system is 1:1:4:4:2: 2;
stn-F3：5’-ACCAGATTCAGGGAGTGAG-3’
stn-B3：5’-CGCGCACGAAATTCGTAAC-3
stn-FIP：5’-ACCGGGTGGTAAGCGAATTGCGAGGTTAACCGTCTGGAGC-3’
stn-BIP：5’-GGCCTCTTTGGCCATCACCTGGCTGGCGAAATACTTTGC-3’
the staphylococcus aureus primer group has a sequence shown as the following, wherein the molar ratio of the staphylococcus aureus primer group to the staphylococcus aureus primer group in a reaction system is 1:1:4:4:2: 2;
rpoB-F3：5’-CCAATTAGTACGTGTATATATCGTT-3’
rpoB-B3：5’-ACTTGTCCGATGTTCATACG-3
rpoB-FIP：
5’-AGGAACAATCTTAGAAATGACACCTAATTCATGTTGGTGATAAGATGTG-3’
rpoB-BIP：5’-
GATATGCCTTACTTACCAGATGGACAGATGGTACACCAAGAGGATT-3’。
4. a LAMP synchronous detection kit for staphylococcus aureus and salmonella is characterized by comprising all or part of the following components: (1) positive and negative controls; (2) a fluorescent dye or color-developing agent; (3) an LAMP reaction system; (4) and (3) a primer group.
5. The LAMP simultaneous detection kit for Staphylococcus aureus and Salmonella according to claim 5, characterized in that: the LAMP reaction system comprises dNTP, reaction buffer solution and MgSO4And betaine, the DNA polymerase is Bst DNA polymerase.
6. The LAMP simultaneous detection kit for Staphylococcus aureus and Salmonella according to claim 4, characterized in that: the positive control is vector clone containing staphylococcus aureus rpoB gene fragment and salmonella stn gene fragment, and the negative control is water without staphylococcus aureus rpoB gene fragment and salmonella stn gene fragment.
7. The LAMP simultaneous detection kit for Staphylococcus aureus and Salmonella according to claim 4, characterized in that: the hydroxynaphthol blue (HNB) is a main dye.
8. The LAMP simultaneous detection kit for Staphylococcus aureus and Salmonella according to claim 4, comprising the following components:
(1) a primer group: comprises the LAMP detection primer group of claim 1, wherein the concentrations of the outer primer, the inner primer and the loop primer are all 10 μ M;
(2) reaction system: including Bst 3.0DNA polymerase (0.16U/. mu.L), MgSO4Betaine, dNTP mix, sterilized non-nucleic acid water, and adjusting the working concentration in equal proportion;
(3) fluorescent dyes or color developers: the color developing agent is Hydroxy Naphthol Blue (HNB);
(4) sealing liquid: is mineral oil for sealing.
9. The method for detecting staphylococcus aureus and salmonella by using the LAMP simultaneous detection kit for staphylococcus aureus and salmonella as claimed in claim 8, comprising the steps of:
(1) pretreatment and genome extraction of dairy samples
Directly detecting liquid milk, putting 1ml of the liquid milk into a 1.5ml centrifuge tube, centrifuging at 12000rpm for 5min, and discarding the supernatant; adding 1ml of normal saline, mixing uniformly, centrifuging at 12000rpm for 1 minute, removing supernatant, and repeating for three times; adding 1ml of normal saline, mixing well, adding lysozyme for heavy suspension, suspending at 37 ℃ for 30min, adding lysate for 1min at 37 ℃, adding proteinase K, mixing well, centrifuging at 70 ℃ for 10min, and centrifuging at 12000g for 1min to obtain lysate. Extracting lysate with phenol chloroform, collecting supernatant, washing with 75% anhydrous ethanol, and adding ddH2Dissolving the precipitate by using O to obtain a genome to be detected;
(2) loop-mediated isothermal amplification reaction
Adding 3 mu L of the DNA template of the sample to be detected obtained in the step (1) or the negative and positive controls into a loop-mediated isothermal amplification reaction system, uniformly mixing, carrying out isothermal reaction at 65 ℃ for 50min for amplification, and then carrying out isothermal reaction at 80 ℃ for 10min to terminate the reaction; the LAMP reaction system comprises the LAMP detection primer group for simultaneously detecting staphylococcus aureus and salmonella as described in claim 3;
(3) result judgment
After the reaction is finished, the result can be judged according to the change of the color of the liquid in the PCR tube observed by adding the color developing agent.
10. The method for synchronously detecting staphylococcus aureus and salmonella by using the LAMP detection kit for detecting staphylococcus aureus and salmonella as claimed in claim 9, wherein: the LAMP reaction system is a 25-microliter reaction system.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113621723A (en) * | 2021-09-14 | 2021-11-09 | 贵州中烟工业有限责任公司 | LAMP (loop-mediated isothermal amplification) detection primer system, detection kit and detection method for salmonella and staphylococcus aureus in buccal tobacco |
| CN114657273A (en) * | 2022-05-06 | 2022-06-24 | 华中农业大学 | Primer pair and probe combination for detecting multiple bovine mastitis pathogens and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113621723A (en) * | 2021-09-14 | 2021-11-09 | 贵州中烟工业有限责任公司 | LAMP (loop-mediated isothermal amplification) detection primer system, detection kit and detection method for salmonella and staphylococcus aureus in buccal tobacco |
| CN113621723B (en) * | 2021-09-14 | 2023-06-23 | 贵州中烟工业有限责任公司 | LAMP (loop-mediated isothermal amplification) detection primer system, detection kit and detection method for salmonella and staphylococcus aureus in buccal cigarettes |
| CN114657273A (en) * | 2022-05-06 | 2022-06-24 | 华中农业大学 | Primer pair and probe combination for detecting multiple bovine mastitis pathogens and application thereof |
| CN114657273B (en) * | 2022-05-06 | 2023-11-14 | 华中农业大学 | Primer pair and probe combination for detecting multiple bovine mastitis pathogens and application of primer pair and probe combination |
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