Test kit and testing method for staphylococcus aureus toxin gene in food

A staphylococcus, golden yellow technology, applied in the directions of microorganism-based methods, biochemical equipment and methods, and microorganism determination/inspection, etc., to achieve the effects of short detection time, labor and financial saving, and simple operation.

Inactive Publication Date: 2009-11-18
曹际娟
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in winter, if the contaminated food is stored indoors at a higher temperature, Staphylococcus can also multiply and produce toxins

Method used

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  • Test kit and testing method for staphylococcus aureus toxin gene in food
  • Test kit and testing method for staphylococcus aureus toxin gene in food
  • Test kit and testing method for staphylococcus aureus toxin gene in food

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, establishment of detection kit and detection method of staphylococcus aureus toxin gene in food

[0052] (1) Design, synthesis and kit assembly of primers, the primers are as follows:

[0053]

[0054]

[0055] On this basis, a kit for mPCR-DHPLC detection is designed:

[0056] The kit includes detection solution A and detection solution B:

[0057] Detection solution A contains 10mM Tris Cl, 50mM KCl, 25mM MgCl 2 , 2.5mM each of dNTP, 5U / μL of Taq DNA polymerase, and 10μM each of the six toxin gene primer pairs mentioned above: ETA, SEB, SEC, SEE, SEI and SEA;

[0058] Detection solution B contains 10mM Tris Cl, 50mM KCl, 25mM MgCl 2 , 2.5 mM each of dNTP, 5 U / μL of Taq DNA polymerase, and 10 μM each of the six toxin gene primer pairs of SEJ, ETB, TSST, SED, SEH and SEG.

[0059] (2) Establishment of mPCR-DHPLC detection method

[0060] This detection method uses the mPCR-DHPLC detection kit that the present embodiment establishes, comprises th...

Embodiment 2

[0084] Embodiment 2, multiple reaction system detection comparison test

[0085] PCR-electrophoresis and the PCR-DHPLC method established in Example 1 were used for detection and comparison.

[0086] This embodiment establishes two groups of composite PCR systems, the detection objects of the first composite PCR system include ETA, SEB, SEC, SEE, SEI, SEA genes; the detection objects of the second composite PCR system include ETASEJ, ETB, TSST, SED, SEH, SEG Gene.

[0087] The expected PCR amplification fragments of the six toxin genes of the first complex PCR system: ETA, SEB, SEC, SEE, SEI, and SEA are: 119bp, 257bp, 317bp, 350bp, 375bp, and 478bp; attached figure 1 As shown, mPCR-electrophoresis detection results are shown in the attached figure 2 Shown; mPCR-DHPLC test results are shown in the attached image 3 shown.

[0088] from by figure 1 with figure 2 It can be seen that when these 6 kinds of toxin genes are subjected to single PCR amplification and electr...

Embodiment 3

[0091] Embodiment 3, detection method specificity test

[0092] The reference bacterial strains listed in Table 1 were taken and detected according to the method established in Example 1.

[0093] Table 1

[0094]

[0095]

[0096]

[0097]Take the test strains listed in Table 1, extract the genomic DNA after culture, and establish the template library. Then, using these genomic DNAs as templates, the six toxin genes of ETA, SEB, SEC, SEE, SEI, and SEA in the first composite PCR system and SEJ, ETB, TSST, SED, SEH, The six toxin genes of SEG were used as target genes for mPCR-DHPLC analysis and detection using the conditions established in Example 1. The results showed that in the first multiplex PCR reaction system, only the ETA-producing Staphylococcus aureus strain had an ETA absorption peak at 2 min, and the other strains were all negative results; the SEB-producing Staphylococcus aureus strain had an SEB absorption peak at 3.6 min , other strains were all nega...

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Abstract

The invention provides a test kit a testing method for staphylococcus aureus toxin gene in food. The kit comprises test solution A and test solution B, wherein the test solutions A and B respectively contain 10mM of Tris.Cl, 50mM of KCl, 25mM of MgCl2, 2.5mM of dNTP respectively, 5U / mu L Taq DNA polymerase, and 10 mu M of toxin gene primers of ETA, SEB, SEC, SEE, SEI and SEA respectively, or 10 mu M of toxin gene primers of SEJ, ETB, TSST, SED, SHE and SEG. The test kit and the testing method can high sensitively test 12 staphylococcus aureus toxin genes in food, have short testing time and simple operation, can save mass labor and financial power, and can meet the requirement of quick testing.

Description

technical field [0001] The invention relates to a detection kit and a detection method for Staphylococcus aureus toxin gene in food, in particular to a PCR-DHPLC-based rapid detection kit and detection method. Background technique [0002] Staphylococcus aureus produces different enterotoxins under different food, temperature and pH conditions. Staphylococcus aureus enterotoxin (SE) has 12 serotypes, namely exfoliation toxin ETA (exfoliative toxin A), SEB, SEC, SEE, SEI, SEA, SEJ, ETB, toxic shock toxin TSST (toxic shock syndrome toxin) , SED, SEH, SEG. [0003] Staphylococcus aureus seldom produces enterotoxin when the pH value is lower than 5, and when the pH value is higher than 9.8, no matter what type of staphylococcus enterotoxin can be produced. Staphylococcus produces toxins on milk and grain porridge at 20°C to 37°C for 4h to 8h, and at 5°C to 6°C for 18 days or does not produce toxins. Staphylococcus is more likely to multiply and produce toxins in foods contain...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N30/02C12R1/445
Inventor 曹际娟
Owner 曹际娟
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