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Molecular biological quantitative detecting method for pathogenicity paratypic hemolytic vibrio in aquatic products

A quantitative detection method, molecular biology technology, applied in biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc. question

Inactive Publication Date: 2006-09-13
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For such a large sample size, for the microbes to be tested with a very small content, after a series of processing procedures, the nucleic acid of the microbes to be tested with a very small content in the obtained nucleic acid sample is easily lost, resulting in false negative test results
Therefore, in the actual test, it is difficult to realize the real quantitative

Method used

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  • Molecular biological quantitative detecting method for pathogenicity paratypic hemolytic vibrio in aquatic products
  • Molecular biological quantitative detecting method for pathogenicity paratypic hemolytic vibrio in aquatic products
  • Molecular biological quantitative detecting method for pathogenicity paratypic hemolytic vibrio in aquatic products

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Basic method description

[0107] 1. Sample processing:

[0108] 1. Sterile and quantitatively take samples to be tested (such as farmed or wild aquatic animals, seafood, finished or semi-finished aquatic products, or various water samples).

[0109] The purpose is to test its freshness and quality: the test site is the bacterial content in the muscle of aquatic food, so as to judge its freshness and quality.

[0110] For the purpose of testing whether a certain pathogenic bacteria is contaminated: the test site should be the digestive tract (or visceral mass) and gills and other respiratory organs: the intestine and gills of fish are cut; the hepatopancreas and digestive caecum in the cephalothorax of shrimp , the intestinal tube at the extension of the abdominal segment; crabs cut off the viscera and gills; shellfish snails cut the part below the gastropod muscle, and shellfish bivalves cut off the outer layer of the axopod muscle Viscera and flap gills.

[0111] S...

Embodiment 2

[0135] Example 2 Rapid quantitative detection of pathogenic Vibrio parahaemolyticus in seafood

[0136] Using a rapid quantitative detection method, samples such as salmon meat in a restaurant in Qingdao City, live shrimp, and live crabs in an aquarium were enriched and cultured, and three pairs of specific primers for Vibrio parahaemolyticus were designed for PCR amplification and electrophoresis detection. Determine the most probable number of pathogenic Vibrio parahaemolyticus in the sample to be tested.

[0137] Material method:

[0138] 1. Test materials: live shrimp, live crab, clams, and salmon meat in the aquarium of a restaurant in Qingdao.

[0139] Sample processing: Take out 0.234g of shrimp hepatopancreas, 0.221g of crab viscera, 0.231g of crab gills, 0.216g of salmon meat, 0.286g of clam water pipe and skirt tissue, 0.232g of prawn muscle (tissue control), 0.228g of crab muscle ( Tissue control), tissue homogenate respectively, and dilute to 10mL.

[0140] 2. A...

example 1

[0188] Example 1: Tube group

[0189] Tube number 3 3 3

[0190] Sample amount in each tube 10g 1g 0.1g

[0191] Number of positive tubes 3 0 0

[0192] Therefore, N=10, A=10×3+1×3+0.1×3=33.3, B=1×3+0.1×3=3.3

[0193] Substitute into the formula (1) to calculate the results of the column "3", "0" and "0" in the table.

[0194] 2. When there are positive tubes in the two dilution tube groups (denoted as positive tube group 1 and positive tube group 2), use formula (2) to calculate:

[0195] N 1 λ = 2.303 × lg ( A - N 2 γ - A × 10 - 0.4343 N 2 λ ...

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Abstract

The invention relates to a method to test pathogenicity vibrio parahaemolyticus in aquatic product that the feature is including the following steps: designing, filtering, and manufacturing the specificity primer of the testing strain, expanding PCR and testing, distilling DNA, PCR expanding of the testing strain specificity gene section, agarose gel electrophoresis of the PCR product and testing the specificity section; MPN method testing and calculating the testing result. The invention discloses a method to rapidly testing bacilli in testing sample.

Description

technical field [0001] The invention belongs to a microorganism detection method, in particular to a molecular biology method for rapid and quantitative detection of pathogenic Vibrio parahaemolyticus in aquatic products. [0002] Scope of application: This method is suitable for rapid quantitative detection of any microorganisms with specific enrichment medium, specific gene fragments with strong specificity, and specific primers can be designed. Background technique [0003] Vibrio parahaemolyticus is a pathogenic halophilic bacteria widely distributed in oceans and salt lakes. It has been paid attention to because it can cause food poisoning, especially in coastal areas in summer and autumn, often due to eating seafood containing a large amount of Vibrio parahaemolyticus, causing explosive food poisoning; in non-coastal areas, eating food contaminated by this bacterium Salted food is also often poisoned; it is a mandatory inspection item in commodity inspection and quara...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCY02A50/30
Inventor 李筠陈吉祥栾晓燕杨慧魏鉴腾焦爽李洪鹏张晓华池政豪公衍军李采风
Owner OCEAN UNIV OF CHINA
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