Search

CN-121975961-A - Method for semi-quantitatively chromogenic detection of staphylococcus aureus in bulk instant food

CN121975961ACN 121975961 ACN121975961 ACN 121975961ACN-121975961-A

Abstract

The invention belongs to the detection field and discloses a method for semi-quantitatively chromogenic detection of staphylococcus aureus in bulk instant food, which comprises the steps of designing a specific primer aiming at tuf genes of staphylococcus aureus, extracting DNA from a bulk instant food sample to be detected as a template, adding the template DNA into an isothermal amplification reaction system for carrying out isothermal nucleic acid amplification reaction mediated by recombinase, wherein the isothermal amplification reaction system comprises the specific primer and SYBR Green I fluorescent dye in the step S1, and observing a color change judgment result of the reaction system after the amplification reaction is finished, wherein the judgment result shows that yellow-Green fluorescence is positive and orange color is negative under an ultraviolet lamp.

Inventors

  • HU LIANXIA
  • SUN RUIJIA
  • LU HAN

Assignees

  • 石家庄学院

Dates

Publication Date
20260505
Application Date
20260209

Claims (10)

  1. 1. The method for semi-quantitatively chromogenic detection of staphylococcus aureus in bulk instant food is characterized by comprising the following steps of: S1, designing a specific primer aiming at tuf genes of staphylococcus aureus; s2, extracting DNA from a bulk instant food sample to be detected as a template; s3, adding template DNA into an isothermal amplification reaction system to perform isothermal nucleic acid amplification reaction mediated by recombinase, wherein the isothermal amplification reaction system comprises the specific primer and SYBR Green I fluorescent dye in the step S1; And S4, after the amplification reaction is finished, judging that the color change of the reaction system is positive by observing the yellow-green fluorescence under an ultraviolet lamp, and judging that the color change is negative by taking the orange color.
  2. 2. The method for semi-quantitatively chromogenic detection of staphylococcus aureus in bulk ready-to-eat food according to claim 1, wherein the sequence of the specific primer in the step S1 is as follows: an upstream primer Tuf F:5'-TAAATTATTAGACTACGCTGAAGCTGGTGA-3'; The downstream primer Tuf R:5'-GAAGAATGGAGTGTGACGTCCACCTTCGT-3'.
  3. 3. The method for detecting staphylococcus aureus in bulk instant food according to claim 2, wherein in the step S2, when the sample to be detected is the bulk instant food, the method for extracting DNA is a lysozyme combined with a thermal cracking method, and the method comprises the steps of preprocessing a sample homogeneous solution by using absolute ethyl alcohol, ammonia water and petroleum ether, and then carrying out lysozyme treatment and boiling cracking.
  4. 4. A method for semi-quantitatively chromogenic detection of Staphylococcus aureus in bulk ready-to-eat food according to claim 3, wherein the total volume of the isothermal amplification reaction system in step S3 is 25. Mu.L, comprising 10. Mu.L of system buffer, 6.25. Mu.L of A buffer, 1.25. Mu.L of protein premix of recombinase, recombinase-assisted protein, single-stranded binding protein and polymerase, 1.0. Mu.L of upstream and downstream primers with a concentration of 10. Mu.M, 0.3. Mu.L of 10000 XSYBR Green I fluorescent dye diluted 200 times, 1.3. Mu.L of dNTPs of 10 mM, 1. Mu.L of template DNA, 1.65. Mu.L of sterilized distilled water, and 1.25. Mu.L of B buffer are pre-added on the reaction tube cover.
  5. 5. The method of semi-quantitatively detecting Staphylococcus aureus in a bulk instant food product according to claim 4, wherein the bulk instant food product is at least one selected from the group consisting of freshly squeezed fruit and vegetable juice, ice cream, cooked meat product, cooked egg product, raw meat and raw fresh nut.
  6. 6. The method for semi-quantitatively chromogenic detection of staphylococcus aureus in bulk ready-to-eat food according to claim 5, wherein the method further comprises the step of estimating the initial concentration of staphylococcus aureus in the sample according to the time when the amplification positive result appears in semi-quantitative analysis by referring to a standard curve, wherein the standard curve is plotted with the logarithmic value of the concentration of pure culture of staphylococcus aureus as the abscissa and the time when the corresponding amplification positive result appears as the ordinate.
  7. 7. The method for semi-quantitatively detecting staphylococcus aureus in bulk instant foods according to claim 6, wherein in the step S2, the pretreatment of extracting staphylococcus aureus DNA from the bulk instant foods comprises degreasing and deproteinizing the food sample homogeneous solution.
  8. 8. The method for semi-quantitatively detecting staphylococcus aureus in bulk instant food according to claim 7, wherein the time used by the method for semi-quantitatively detecting staphylococcus aureus in bulk instant food is 15 min.
  9. 9. The method for semi-quantitatively detecting staphylococcus aureus in bulk instant foods according to claim 8, wherein when the number of S.aureus bacteria is less than or equal to 1000 CFU/g (mL) (1 CFU/mu L), the reaction system is orange red, weak fluorescence is emitted, and the result is negative.
  10. 10. The method for semi-quantitatively detecting staphylococcus aureus in bulk instant foods according to claim 9, wherein when the number of S.aureus bacteria is more than 1000 CFU/g (mL) (2 CFU/. Mu.L), the reaction system emits green fluorescence, and the result is positive.

Description

Method for semi-quantitatively chromogenic detection of staphylococcus aureus in bulk instant food Technical Field The invention belongs to the field of staphylococcus aureus detection, and particularly relates to a method for detecting staphylococcus aureus in bulk instant food by semi-quantitative color development. Background Staphylococcus aureus (Staphylococcus aureus, s.aureus) widely exists in nature, has stronger viability, can survive in extreme environments such as low temperature, drying and the like, and pollutes various kinds of bulk instant foods which are exposed to the naked world through various ways, thereby causing food poisoning. The second to food poisoning caused by s.aureus annually in the united states, food poisoning caused by s.aureus in our country accounts for 20% -25% of bacterial food poisoning events. The national publication and implementation of pathogenic bacteria limit in bulk instant food of national food safety standard (GB 31607-2021) prescribes the detection standard (GB 4789.10-2016) and limit requirement (less than or equal to 1000 CFU/g (mL), namely less than or equal to 1 CFU/mu g (mu L) of S.aureus in bulk instant food, the national food safety standard GB4789.10-2016 is a gold standard method for S.aureus detection, but the detection period is as long as 4 d, the immunological detection method is simple and convenient to operate and has faster detection time, but the specificity and sensitivity are not ideal, along with the rapid development of molecular biology, the non-isothermal nucleic acid amplification PCR technology is mature gradually, has the characteristics of high sensitivity and high speed, but has higher requirements on detection personnel and equipment, and is unfavorable for popularization on a basic layer and on site. Recombinase-mediated isothermal amplification detection (recombinase-aided amplification, RAA) technology is a novel domestic isothermal nucleic acid amplification technology developed in 2012, and RAA amplification relies on the synergistic interaction of multiple enzymes (recombinase, recombinase-assisted protein, single-stranded binding protein, DNA polymerase) during its reaction. In the initial stage of RAA reaction, a primer and recombinase are combined to form a complex, DNA double chains are specifically identified with the help of recombinase auxiliary proteins, the identified DNA double chains and the complex are bound, single-chain binding protein (SSB) is combined with another single-chain DNA to prevent the DNA from reforming into a double-chain structure, the recombinase on the 3 '-end of the primer drops off, DNA polymerase replaces the recombinase to be combined with the primer on the 3' -end of the primer, so that the primer extends forwards, the replication process is immediately started, and the procedure is repeated, and the target fragment is amplified in an exponential manner. The amplification result can be displayed by agarose gel electrophoresis, SYBR Green I fluorescent dye color development technology, lateral flow immunochromatography and the like. Fluorescent dye (such as SYBR Green I) is added into a reaction system of the RAA, the DNA quantity starts to increase along with the progress of the amplification reaction, the RAA amplification process is monitored in the whole process through a real-time fluorescent detector, the increase of the fluorescence intensity is in direct proportion to the quantity of double-stranded DNA generated by the RAA system, and the SYBR Green I dye method is combined with a recombinase-mediated isothermal amplification (SQ-RAA) technology. The tuf gene is the coding gene of bacterial extension factor protein, has the main biological function of being responsible for the extension of peptide chains in the protein synthesis process, is a housekeeping gene, is only one set of genes in gram positive bacteria, is a single copy gene, can be used for quantitative analysis, has higher sequence difference than 16SrDNA among different strains of certain bacteria, and is a novel target for researching the identification of kindred species and strains in particular to the identification and the identification of the kindred species. Disclosure of Invention In order to overcome the defects of the prior art and solve at least one technical problem in the background art, the invention develops the SQ-RAA technology for rapidly and quantitatively detecting S.aureus in bulk instant foods, and 49 min is only needed for realizing sample processing, DNA extraction, DNA amplification and detection. The amplification and detection can be completed in one step by only 15 min tubes, and the detection result can be accurately, simply, conveniently and quickly judged by the presence or absence of an S-shaped curve and the change of the color of a reaction system. The technical scheme adopted for solving the technical problems is that the method for detecting staphylococcus aureus in bulk instant food