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Detection kit for detecting three spore production bacteria in food and detection method thereof

A technology for detection kits and detection methods, applied in the direction of microorganism-based methods, biochemical equipment and methods, measurement/inspection of microorganisms, etc., can solve problems such as unsatisfactory, single target, and affect the detection and identification cycle, so as to save labor and financial resources, simple operation, and short inspection time

Inactive Publication Date: 2009-12-02
曹际娟 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In my country, the detection of spore-forming bacteria still remains at the level of low detection efficiency and a single target, and mainly relies on traditional methods of enrichment, separation, and biochemical identification. Generally speaking, it takes 10 to 20 days to identify a spore-forming bacterium , which affects the cycle of testing and identification, which obviously cannot meet the needs of the current rapid development of food safety
Even though PCR and real-time PCR technologies have been developed, they can only detect known conventional pathogens
However, there is a lack of stable and reliable identification methods for mixed microbial samples, uncommon, harsh, and mutated spore bacteria

Method used

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  • Detection kit for detecting three spore production bacteria in food and detection method thereof
  • Detection kit for detecting three spore production bacteria in food and detection method thereof
  • Detection kit for detecting three spore production bacteria in food and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, the establishment of the detection kit of three kinds of spore-forming bacteria in food and its detection method

[0043] (1) Design and synthesis of primers and assembly of kits:

[0044] The present embodiment determines that the primer sequences and amplified fragment lengths used for detection are shown in Table 2:

[0045] Table 2

[0046]

[0047] Among them, N 1 is A, T or C; N 2 is A or G; N 3 is C or T; N 4 is A or T; N 5 is A or T; N 6 is G or A; N 7 is C or T; N 8 is C or T; N 9 is C or T;

[0048] On this basis, a kit for mPCR-DHPLC detection was designed. The kit includes Taq DNA polymerase and PCR reaction solution at a concentration of 5U / μL; the PCR reaction solution contains 10mM Tris HCl, 50mM KCl, 25mM MgCl 2 2.5 mM each of dNTP (dATP, dGTP, dCTP and dTTP) and 10 μM each of the primer pairs of the three spore-forming bacteria in Table 2.

[0049] (2) The mPCR-DHPLC method using this kit to detect three kinds of spore-formin...

Embodiment 2

[0072] Embodiment 2, specificity test

[0073] Genomic DNA was extracted from all reference strains in Table 1 according to the method established in Example 1, and then using these genomic DNA as templates, mPCR-DHPLC was used to determine whether there were false positives and false negatives. The results show that the detection kit and the PCR-DHPLC detection method established in Example 1 have high specificity in detecting three kinds of spore-forming bacteria in food, and there are no false positive and false negative results. In order to confirm that the positive absorption peaks of the detected samples were fragments of the specific genes of Bacillus cereus, Clostridium botulinum and Clostridium perfringens, the positive absorption peak products of DHPLC were recovered and cloned and sequenced respectively. The results were compared with the DNA sequences of the three spore-forming bacteria, which proved that the positive absorption peaks of DHPLC were the specific gen...

Embodiment 3

[0074] Embodiment 3, the application in actual sample detection

[0075] (1) Keep positive sample test results

[0076] Using the mPCR-DHPLC detection kit and detection method for three kinds of spore-forming bacteria in food established in Example 1, the remaining positive samples were detected.

[0077] Take 2 positive strains of Clostridium botulinum isolated and retained from the actual test samples, and use the detection kit and detection method established in Example 1 to carry out the detection test. Results The positive amplification absorption peaks of the two Clostridium botulinum isolates were detected by DHPLC, and the peak time and peak shape reproducibility were good. The detection results of Salmonella as negative control were negative.

[0078] Take 3 remaining isolated strains of Clostridium perfringens isolated from the actual test samples, and use the detection kit and detection method established in Example 1 to carry out the detection test. Results The a...

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Abstract

The invention relates to a detection kit for sensitively and quickly detecting three spore production bacteria in food and a detection method thereof. The kit comprises a 5 U / mu L Taq DNA polymerase and PCR reaction solution, wherein the PCR reaction solution contains 10 mM of Tris.HCl, 50 mM of KCl, 25 mM of MgCl2, 2.5 mM of dNTP and 10 mu M of each primer of the three spore production bacteria. By using the detection kit and the detection method, the clostridium botulinum, the clostridium perfringens and the bacillus cereus in the food can be detected with high sensitivity; besides, the detection takes a short time and has simple operations, thus the detection kit and the detection method can save a large number of labor and financial capacities and are suitable for the requirement on quick detection.

Description

technical field [0001] The invention relates to a detection kit for three kinds of spore-forming bacteria in food and a detection method thereof. In particular, it relates to a reagent kit for rapid high-throughput detection of PCR-DHPLC and a corresponding detection method. Background technique [0002] It has always been believed that heat treatment during food processing will destroy all microbial communities, but actual research has found that heat-resistant bacteria and spores will still remain in ultra-high temperature processed foods; especially pasteurized foods can only kill most of them Bacteria, most thermophilic bacteria, heat-resistant bacteria and spores still exist. At the same time, even with ultra-high temperature treatment, the toxins produced by the spores will not be inactivated, and the remaining spores and toxins are potential biological risk factors in heat-processed foods. Under favorable conditions, the remaining bacteria reproduce and produce toxi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N30/02C12R1/085C12R1/145
Inventor 郑秋月
Owner 曹际娟
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