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Detection kit and detection method for 9 species of pathogenic organisms in marine products

A microbial detection and pathogenicity technology, applied in the field of kits, various pathogenic microorganisms, and the composition used for detection, can solve the problems of time-consuming, expensive reagents and consumables, and easy pollution, and achieve simple operation, The effect of short inspection time, saving labor and financial resources

Inactive Publication Date: 2009-11-04
曹际娟 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing detection technologies such as microbial culture and biochemical identification, immunological technology, PCR technology, etc. have some problems: conventional biochemical identification methods are complicated to operate and time-consuming, and often only one or a few types of bacteria can be identified in one detection experiment; Although immunological technology is highly sensitive, it is prone to contamination and often has false positives; PCR technology is generally used in combination with gel electrophoresis technology, and the results of electrophoresis cannot be used as the final conclusion, and other probe hybridization experiments are required
The former and the reagents and consumables used are extremely expensive and difficult to popularize and apply; the latter requires special laboratory conditions to complete, and the test results are affected by many factors, resulting in poor repeatability and stability

Method used

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  • Detection kit and detection method for 9 species of pathogenic organisms in marine products
  • Detection kit and detection method for 9 species of pathogenic organisms in marine products
  • Detection kit and detection method for 9 species of pathogenic organisms in marine products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1, nine kinds of pathogenic microorganism detection kits and detection methods thereof in aquatic products

[0046] (1) Design and synthesis of primers and assembly of kits:

[0047] The present embodiment determines that the primer sequences and amplified fragment lengths used for detection are shown in Table 2:

[0048] Table 2

[0049]

[0050]

[0051] 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 the primer pairs of the above nine pathogenic microorganisms (concentrations are shown in Table 2).

[0052] (2) Establishment of mPCR-DHPLC detection method:

[0053] This detection method uses the detection kit established in this embodiment, comprising the following steps:

[0054] ①Preparation of the sample to be tested: t...

Embodiment 2

[0079] Embodiment 2, specificity test

[0080] All reference strains in Table 1 were taken to extract genomic DNA according to the method described in Example 1, and a template library was established. Then, using these genomic DNAs as templates, mPCR amplification and DHPLC detection were performed according to the method described in Example 1. The results showed that the one-time mPCR-DHPLC detection of 9 kinds of pathogenic microorganisms in aquatic products using the detection kit and detection method of Example 1 had strong specificity and no false positive or false negative results.

Embodiment 3

[0081] Embodiment 3, sensitivity test

[0082] According to the method for enriching bacteria established in Example 1, use the turbidimetric method to quantify the concentration of the cultured compound enriched bacteria: first, use the McFarland colorimetric tube kit to make the OD550 value—the number of bacteria in each mL of bacteria solution Then measure the OD550 value of the cultured bacterial solution, and substitute the OD550 value into the standard curve to obtain the concentration of the cultured bacterial solution, in CFU / mL. The enrichment broth of pathogenic bacteria was serially diluted to obtain 10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 、10 -9 、10 -10 Bacteria solution, and then enriched according to the compound enrichment method. According to the method established in Example 1, extract the bacterial solution DNA of compound enriched bacteria with different gradients, take 2 μL each as a template, carry out mPCR-DHPLC detection according to the method ...

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Abstract

The invention discloses a detection kit and a detection method for 9 species of pathogenic organisms in marine products. The kit comprises Taq DNA polymerase with a concentration of 5U / muL and a PCR reaction solution, wherein the PCR reaction solution contains 10 millimols of Tris.HCl, 50 millimols of KCl, 25 millimols of MgCl2, 2.5 millimols of dNTP and primer pairs of the 9 species of pathogenic organisms. The kit and the method can synchronously detect the 9 species of pathogenic organisms and can detect salmonella with a concentration of 11 CFU / mL, staphylococcus aurei with a concentration of 2,000 CFU / mL, monocytogenes with a concentration of 120 CFU / mL, enterohemorrhagic Escherichia coli O157:H76 with a concentration of CFU / mL, comma bacillus with a concentration of 220 CFU / mL, Vibrio parahaemolyticus with a concentration of 60 CFU / mL, Vibrio vulnficus with a concentration of 9 CFU / mL and Vibrio alginolyicus with a concentration of 230 CFU / mL. The detection method short in detection time and simple and quick in operation, and can save a large amount of labor and financial resources and meet requirements for quick detection.

Description

technical field [0001] The invention relates to a method for detecting pathogenic microorganisms in food, in particular to a method for detecting multiple pathogenic microorganisms in aquatic products by using multiple PCR and denaturing high performance liquid chromatography (DHPLC) techniques. It also relates to the composition used for the detection, ie the kit. Background technique [0002] With the improvement of living standards and the gradual opening of the market after my country's entry into the WTO, people's demand for aquatic products continues to increase, and the import and export trade of aquatic products is also expanding. There may be a variety of bacteria that can cause human intestinal diseases in aquatic products. Vibrio, Salmonella, Staphylococcus aureus, enterohemorrhagic Escherichia coli O157:H7, Listeria monocytogenes, etc. are common pathogens in aquatic products Bacteria. The characteristics of food-borne pathogenic bacteria are many types, small ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N30/02C12R1/63C12R1/42C12R1/185C12R1/445C12R1/01
Inventor 郑秋月
Owner 曹际娟
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