LAMP synchronous detection method for staphylococcus aureus and salmonella in liquid milk and kit

A technology for Staphylococcus and Salmonella, applied in biochemical equipment and methods, microbial measurement/inspection, recombinant DNA technology, etc., can solve the problems that the freshest products cannot be put on the market and delivered to consumers, so as to prevent proliferation and Contamination, high sensitivity, and easy operation

Pending Publication Date: 2020-03-31
JILIN BUSINESS & TECH COLLEGE
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AI-Extracted Technical Summary

Problems solved by technology

[0006] Staphylococcus aureus and Salmonella are two food-poisoning bacteria that are easily contaminated by liquid milk. The national standard stipulates that they must not be detected in liquid milk, but the existing n...
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Abstract

The invention relates to a LAMP synchronous detection method for staphylococcus aureus and salmonella in liquid milk and a kit, which belong to the technical field of food safety rapid detection. Themethod comprises the following steps: extracting genomic DNA of the liquid milk on site, designing a primer group on the basis of specific molecular marker characteristics of genomes of staphylococcusaureus and salmonella, and synchronously detecting polluted staphylococcus aureus and salmonella in the liquid milk by adopting a loop-mediated isothermal amplification technology. The whole detection process has low requirements on environment and equipment, and whether a to-be-detected sample contains staphylococcus aureus or salmonella or not is determined by adding a color developing agent toobserve the color change in the tube. The method has the advantages of high sensitivity, good specificity, safety, harmlessness, no organic reagent and no nucleic acid binding dye, all reagents can be directly released into the environment, and the method has important significance for improving the quality safety of dairy foods and improving the food-borne pathogenic bacteria analysis and detection technology.

Application Domain

Microbiological testing/measurementAgainst vector-borne diseases +1

Technology Topic

genomic DNABiotechnology +14

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  • LAMP synchronous detection method for staphylococcus aureus and salmonella in liquid milk and kit
  • LAMP synchronous detection method for staphylococcus aureus and salmonella in liquid milk and kit
  • LAMP synchronous detection method for staphylococcus aureus and salmonella in liquid milk and kit

Examples

  • Experimental program(3)

Example Embodiment

[0036] Example 1 Liquid milk sample collection
[0037] Use clean sampling tools and sample bottles, and the sample bottles must be free of disinfectant, water and detergent. Samples were taken in the milking parlor of the milk station, the milk storage tank in the milk station, the milk tank of the milk truck, the liquid milk production line and the supermarket, and detailed records were taken. The fresh samples collected were immediately tested.

Example Embodiment

[0038] Example 2
[0039] A method for detecting Staphylococcus aureus and Salmonella in liquid milk by a loop-mediated isothermal amplification technology includes the following steps:
[0040] S1. Genome extraction
[0041] (1) Sample preparation: Take 1ml of liquid milk and put it in a 1.5ml centrifuge tube, centrifuge at 12000rpm for 5min, discard the supernatant; add 1ml of saline to mix well, centrifuge at 12000rpm for 1 minute, discard the supernatant, and add 1ml of saline to the pellet Mix by pipetting.
[0042] (2) Bacterial lysis: Add lysozyme to the precipitate obtained in step (1) at 37°C for 10 minutes, then add the lysing solution and mix well, 37°C for 5 minutes, lyse, and then add proteinase K, 70°C for 10 minutes, Get the lysate.
[0043] (3) DNA extraction: extract the lysate obtained in step (2) with phenol and chloroform to obtain a white suspension, carefully collect the supernatant, add isopropanol and sodium acetate, mix, and centrifuge to collect the precipitate. Wash the precipitate with 75% absolute ethanol, dry it at room temperature for 5 minutes to remove residual ethanol; add ddH2O and incubate at room temperature for 3 minutes to obtain the genome to be tested ( figure 1 ).
[0044] S2. Design and synthesis of primers for loop-mediated isothermal amplification (LAMP)
[0045] Firstly, through comparative genomics method, find the unique gene sequence of Staphylococcus aureus and Salmonella, use the special LAMP primer design software to design the primer with this sequence as the target gene, and finally analyze the designed primer on the primer-blast And optimization. The final primer sequence.
[0046] The comparative test to verify the unique gene sequences of Staphylococcus aureus and Salmonella included the following amplification primers:
[0047] The detection primer set of the Salmonella primer set has the sequence shown below:
[0048] F3: 5’-ACCAGATTCAGGGAGTGAG-3’
[0049] B3: 5’-CGCGCACGAAATTCGTAAC-3
[0050] FIP: 5’-ACCGGGTGGTAAGCGAATTGCGAGGTTAACCGTCTGGAGC-3’
[0051] BIP: 5’-GGCCTCTTTGGCCATCACCTGGCTGGCGAAATACTTTGC-3’
[0052] The Staphylococcus aureus primer set has the sequence shown in SEQ NO.5-SEQ NO.8:
[0053] F3: 5’-CCAATTAGTACGTGTATATATCGTT-3’
[0054] B3: 5’-ACTTGTCCGATGTTCATACG-3
[0055] FIP:
[0056] 5’-AGGAACAATCTTAGAAATGACACCTAATTCATGTTGGTGATAAGATGTG-3’
[0057] BIP: 5’-GATATGCCTTACTTACCAGATGGACAGATGGTACACCAAGAGGATT-3’
[0058] The above 4 pairs of primers were used to detect the two target genes (staphylococcus aureus rpoB gene and Salmonella stn gene) of food-borne strains of Staphylococcus aureus and Salmonella respectively, and compare the positive results of amplification of the two target genes and analyze their correct rates , The higher the accuracy rate, the higher the specificity of the target gene amplification for detecting Staphylococcus aureus and Salmonella, and it also indicates that these two target genes are more suitable for the detection of Staphylococcus aureus and Salmonella during dairy processing. .
[0059] S3.LAMP detection
[0060] The isothermal amplification temperature is 65°C constant temperature water bath for 1h, and then 80°C to inactivate the used polymerase. In the 20μL reaction system, the concentration of outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, loop primer LF and loop primer LB are all 10μM, and the added amounts are 0.5μL, 0.5μL, 2μL, 2μL, 1μL, 1μL, the molar ratio in the reaction system is 1:1:4:4:2:2; the added amount of 0.16U/μLBst DNA polymerase is 0.4μL.
[0061] S4. Visual analysis of LAMP detection results
[0062] This experiment uses agarose gel electrophoresis: take 5μL of the amplified products separately, electrophoresis in 30g/L agarose gel, voltage 100V, time 30min, put it on the gel imaging system to take pictures, if gradient bands appear, then A positive result is a negative result if no gradient band appears. The concentration of Staphylococcus aureus and Salmonella detected by agarose gel electrophoresis is 10-10 8 CFU/mL (that is, the dilution factor is 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 Times) the amplified product corresponding to the contaminated sample shows obvious ladder-like bands, it is a positive test ( figure 2 ).
[0063] Note: The selected skimmed milk powder samples have been proven to be free of Staphylococcus aureus and Salmonella using traditional methods recommended by the FDA.
[0064] In this experiment, hydroxynaphthol blue (HNB) was used as the developer of the LAMP visualization method, and the reaction was judged based on the changes before and after the LAMP reaction (the positive reaction changed from purple to sky blue) after hydroxynaphthol blue (100μmol/L) was applied. The progress. The experiment set up a blank control group and a positive group to observe the color changes ( image 3 ).
[0065] When the concentration of Staphylococcus aureus and Salmonella in the contaminated sample is 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 CFU/mL (that is, the dilution factor is 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 When the concentration of Staphylococcus aureus and Salmonella in the contaminated sample is 100CFU/mL (that is, the dilution factor is 10 8 Times), the corresponding detection tube is still light orange before the reaction, that is, the test is negative.

Example Embodiment

[0066] Example 3
[0067] A method for detecting Staphylococcus aureus and Salmonella in liquid milk by a loop-mediated isothermal amplification technique, which differs from Example 2 in:
[0068] S3.LAMP detection
[0069] The isothermal amplification temperature is a constant temperature water bath at 60°C for 1 hour, and then the polymerase used is inactivated at 80°C. In the 20μL reaction system, the concentration of outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, loop primer LF and loop primer LB are all 10μM, and the added amounts are 0.5μL, 0.5μL, 2μL, 2μL, 1μL, 1μL, the molar ratio in the reaction system is 1:1:4:4:2:2; the added amount of 0.16U/μLBst DNA polymerase is 0.4μL.
[0070] S4. Visual analysis of LAMP detection results
[0071] In this experiment, hydroxynaphthol blue (HNB) was used as the developer of the LAMP visualization method, and the reaction was judged based on the changes before and after the LAMP reaction (the positive reaction changed from purple to sky blue) after hydroxynaphthol blue (100μmol/L) was applied. The progress. The experiment set up a blank control group and a positive group to observe the color changes.
[0072] When the concentration of Staphylococcus aureus and Salmonella in the contaminated sample is 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 CFU/mL (that is, the dilution factor is 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 When the concentration of Staphylococcus aureus and Salmonella in the contaminated sample is 100CFU/mL (that is, the dilution factor is 10 8 Times), the corresponding detection tube is still light orange before the reaction, that is, the test is negative.
[0073] Agarose gel electrophoresis method: Take 5μL of the amplified products separately, electrophoresis in a 30g/L agarose gel, voltage 100V, time 30min, put it on the gel imaging system to take pictures, if gradient bands appear, it is a positive result. If there is no gradient band, the result is negative. The concentration of Staphylococcus aureus and Salmonella detected by agarose gel electrophoresis is 10-10 8 CFU/mL (that is, the dilution factor is 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 If the amplified product corresponding to the contaminated sample shows obvious ladder-like bands, the test is positive.
[0074] Note: The selected skimmed milk powder samples have been proven to be free of Staphylococcus aureus and Salmonella using traditional methods recommended by the FDA.

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