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Assistant hemolysis vibrion constant temperature gene amplification fast detecting reagent box and methods

A constant-temperature gene amplification and detection kit technology, which is applied in biochemical equipment and methods, microbial measurement/inspection, material analysis through observation of the influence of chemical indicators, etc., can solve the problem of limited development, lack of clarity and accuracy, Harsh problems, to achieve the effect of simple identification, high sensitivity and high yield

Inactive Publication Date: 2008-05-14
SOUTH CHINA UNIV OF TECH
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AI Technical Summary

Problems solved by technology

Although the disclosed LAMP technology has high sensitivity and can efficiently amplify nucleic acid under isothermal conditions, the existing technology also has shortcomings, mainly: 1. Its special primer requirements are extremely harsh and limit the wide application of this method; 2. .It is not clear and accurate enough to judge the result only by the milky white precipitate generated by the reaction, and it is not easy to realize the miniaturization of the reaction, which limits its further development

Method used

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Embodiment Construction

[0023] Prepare Vibrio parahaemolyticus isothermal gene amplification rapid detection kit according to the following formula:

[0024] ——Primer mixture: the concentration of the four primers is 10 pmol / μl, and the primer sequences are such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4;

[0025] ——Bst DNA polymerase: the concentration is 8 U / μl;

[0026] ——Reaction solution: 80μl 10mM dNTP, 50μl 10×ThermoPol reaction buffer, 20μl 150mM MgSO 4 ;

[0027] ——Sample pretreatment solution: 20mM Tris-HCl (pH8.0), 2mM EDTA, 1.2% TritonX-100;

[0028] —— Chromogenic agent: fluorescent dye 1×SYBR Green I;

[0029] ——The positive control is the genomic DNA of Vibrio parahaemolyticus, and the negative control is the reaction mixture without template.

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Abstract

The invention discloses a vibrio parahaemolyticus constant temperature gene amplification fast-detection kit and a method thereof, the kit includes primer mixed liquid with a sequence of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and so on, Bst DNA polymerase, reaction solution, sample pretreatment solution, chromogenic agent, positive control and negative control. The invention can make use of the kit to realize the high specific, rapid, high sensitive and simple detection of the vibrio parahaemolyticus by the extraction of the vibrio parahaemolyticus gene group DNA, constant temperature gene amplification reaction and the judgement of the results of the reaction products.

Description

technical field [0001] The invention relates to a microorganism detection reagent and a method, in particular to a rapid detection kit and a method for constant temperature gene amplification of Vibrio parahaemolyticus. Background technique [0002] Vibrio parahaemolyticus (VP ​​for short) is a halophilic bacterium widely distributed in oceans and salt lakes, and is often found in seafood. Eating raw or undercooked seafood contaminated with Vibrio parahaemolyticus can lead to severe acute gastroenteritis. In the composition of bacterial food poisoning, acute gastroenteritis caused by Vibrio parahaemolyticus occupies the first place. Therefore, rapid and sensitive detection methods are the key to prevent and control the spread of Vibrio parahaemolyticus. Usually the traditional identification methods are isolation culture, microscopic observation, biochemical identification, halophilic test, etc. These detection methods are not only time-consuming but also complicated to ope...

Claims

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Application Information

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IPC IPC(8): G01N21/78C12Q1/68C12Q1/24
CPCY02A50/30
Inventor 鲁曦李琳王丽刘芳石磊耿予欢
Owner SOUTH CHINA UNIV OF TECH
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