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Vibrio parahemolyticus detection kit and detection method thereof

A hemolytic vibrio, detection kit technology, applied in biochemical equipment and methods, microbial determination/inspection and other directions, can solve the problem of pretreatment process affecting detection efficiency and other problems, achieve labor saving, short time-consuming, operation process easy effect

Inactive Publication Date: 2012-07-18
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the technology can complete the detection within 1 hour, when it is applied to the target gene diagnosis of Vibrio parahaemolyticus in actual samples, the complicated sample pretreatment process affects the detection efficiency of the technology
There is no report on the high-throughput detection of Vibrio parahaemolyticus using IMS-LAMP

Method used

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  • Vibrio parahemolyticus detection kit and detection method thereof
  • Vibrio parahemolyticus detection kit and detection method thereof
  • Vibrio parahemolyticus detection kit and detection method thereof

Examples

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Embodiment 1

[0056] The specific content of the present invention is described further below:

[0057] 1. Using the polyclonal antibody of Vibrio parahaemolyticus, using the EDC / NHS activation method to couple the polyclonal antibody of Vibrio parahaemolyticus to magnetic beads coated with carboxyl groups on the surface to obtain the preparation of Vibrio parahaemolyticus Bacterial immunomagnetic beads.

[0058] The specific operation of carboxyl magnetic beads coupling Vibrio parahaemolyticus polyclonal antibody is as follows:

[0059] Take 1 ml of magnetic beads in a 2 ml centrifuge tube, and wash the magnetic beads twice with 25 mM 2-(N-morpholino)ethanesulfonic acid MES (pH 5). Then add 500 μl EDC (carbodiimide, 50 mg / ml) and 500 μl NHS (N-hydroxysuccinimide, 50 mg / ml), mix well, and react at room temperature for 30 minutes. Add 200 μl of 6.39mg / ml polyclonal antibody to the activated magnetic beads, then add MES to make up 1ml, mix well, and incubate at room temperature for 3h. Add...

Embodiment 2

[0085] Embodiment 2 Sensitivity detection:

[0086] The Vibrio parahaemolyticus culture solution of 8.79log CFU / ml is carried out 10 times of gradient dilutions, gets each dilution (10 -1 -10 -6 ) each 1ml of the bacterial solution was inoculated on the surface of the boiled shrimp body. After drying, add 225ml of APW (alkaline peptone water), and beat at a low speed for 90s. Take 25ml of the homogeneous solution and add it to a 50ml centrifuge tube, centrifuge at 4000rpm for 5min, remove the supernatant, add 50ml of Tris-HCl (pH6.4), add 100μl of immunomagnetic beads, add 35ml of PBS (+0.05% Tween -20), install the reaction kit, and carry out immune enrichment. After the reaction, the result was detected by the LAMP method, the color change was observed and the result was detected by electrophoresis. The electrophoresis detection result shows that the sensitivity that LAMP can detect the artificial contamination shrimp sample is 3.79log CFU / 25g (see image 3 ). Using th...

Embodiment 3

[0089] Embodiment 3 specific detection:

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Abstract

The invention relates to a vibrio parahemolyticus detection kit. The vibrio parahemolyticus detection kit is characterized by comprising the following components: (1) an immune enrichment reaction system component, (2) a loop-mediated isothermal amplification (LAMP) system component and (3) a set of specific primers based on an LAMP technology of vibrio parahemolyticus tlh genes, wherein the specific primers comprise two outer primers F3 and B3, two inner primers FIP and BIP and two ring primers LF and LB. The detection method for the vibrio parahemolyticus detection kit comprises the following steps of: detecting the vibrio parahemolyticus by adopting a method of combining immune enrichment and the LAMP technology; preparing an immunomagnetic bead by adopting a vibrio parahemolyticus polyclonal antibody; preliminarily screening the vibrio parahemolyticus in an actual sample by adopting an immunology method; and designing an LAMP specific primer according to the species specificity gene of the vibrio parahemolyticus. According to the invention, molecular detection is carried out on a nucleic acid level, therefore, the condition of false positive or undetection of a simple immunology method or a molecular method is effectively avoided, and the invention is a new development direction of the quick detection of the vibrio parahemolyticus.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a detection kit and detection method for Vibrio parahaemolyticus, which can be widely used in food and drug supervision and management agencies at all levels, entry-exit inspection and quarantine agencies, disease prevention and control agencies, etc. Detection and monitoring of Vibrio parahaemolyticus in aquatic foods. Background technique [0002] Vibrio parahaemolyticus (Vibrio parahaemolyticus, Vp) is a halophilic bacterium belonging to the genus Vibrio in the Vibrio family. It was discovered and isolated in a food poisoning incident in Japan in 1950. It mainly exists in seawater, fish, shrimp, shellfish and marinated raw animal seafood. The food poisoning caused by it is clinically characterized by abdominal pain, vomiting, and diarrhea as the main symptoms. At present, food poisoning caused by Vibrio parahaemolyticus has surpassed Salmonella, Escherichia co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 孙晓红苏晨曦卢瑛赵勇潘迎捷
Owner SHANGHAI OCEAN UNIV
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