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Detection method for quantitatively detecting vibrio parahaemolyticus in aquaculture environment by probe method

A technology for aquaculture and Vibrio hemolyticus, which is applied in the directions of microorganism-based methods, microbial determination/inspection, biochemical equipment and methods, etc., can solve problems such as inability to quantify samples, and achieve high sensitivity, reliable results, and strong specificity. Effect

Pending Publication Date: 2021-03-16
福州市疾病预防控制中心
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although this method has the characteristics of short time-consuming, easy operation, high specificity and sensitivity, conventional qPCR detection is only used for qualitative detection of food and environmental samples, and cannot quantify the samples. Therefore, a probe method for quantitative detection is proposed. Detection method for detection of Vibrio parahaemolyticus in aquaculture environment

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  • Detection method for quantitatively detecting vibrio parahaemolyticus in aquaculture environment by probe method
  • Detection method for quantitatively detecting vibrio parahaemolyticus in aquaculture environment by probe method
  • Detection method for quantitatively detecting vibrio parahaemolyticus in aquaculture environment by probe method

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Embodiment 1

[0027] see Figure 1-6 , the present invention provides a technical solution: a method for quantitatively detecting Vibrio parahaemolyticus in an aquaculture environment by a probe method, comprising the following steps:

[0028] S1. Connect the Tlh ​​gene of Vibrio parahaemolyticus to the carrier, prepare positive quantitative reference substances of different concentrations, and use the quantitative reference substance to make a standard curve;

[0029] S2. Cultivate the standard strain of Vibrio parahaemolyticus, prepare bacterial suspension samples with different concentrations, extract DNA respectively, and perform Taqman probe fluorescent quantitative PCR detection at the same time as the positive quantitative reference product, and calculate the different samples through the regression equation of the standard curve Concentration of Vibrio parahaemolyticus, at the same time, the traditional culture method was used to count the colonies of bacteria suspension samples wit...

Embodiment 2

[0040] see Figure 1-6 , the present invention also provides a technical solution: a detection method for the quantitative detection of Vibrio parahaemolyticus in the aquaculture environment by a probe method, comprising the following steps:

[0041] S1. Connect the Tlh ​​gene of Vibrio parahaemolyticus to the carrier, prepare positive quantitative reference substances of different concentrations, and use the quantitative reference substance to make a standard curve;

[0042] S2. Cultivate the standard strain of Vibrio parahaemolyticus, prepare bacterial suspension samples with different concentrations, extract DNA respectively, and perform Taqman probe fluorescent quantitative PCR detection at the same time as the positive quantitative reference product, and calculate the different samples through the regression equation of the standard curve Concentration of Vibrio parahaemolyticus, and at the same time, culture and count the bacteria suspension samples with different concen...

Embodiment 3

[0053] see Figure 1-5 , the present invention also provides a technical solution: a detection method for the quantitative detection of Vibrio parahaemolyticus in the aquaculture environment by a probe method, comprising the following steps:

[0054] S1. Connect the Tlh ​​gene of Vibrio parahaemolyticus to the carrier, prepare positive quantitative reference substances of different concentrations, and use the quantitative reference substance to make a standard curve;

[0055] S2. Cultivate the standard strain of Vibrio parahaemolyticus, prepare bacterial suspension samples with different concentrations, extract DNA respectively, and perform Taqman probe fluorescent quantitative PCR detection at the same time as the positive quantitative reference product, and calculate the different samples through the regression equation of the standard curve Concentration of Vibrio parahaemolyticus, and at the same time, culture and count the bacteria suspension samples with different concen...

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Abstract

The invention discloses a method for quantitatively detecting vibrio parahaemolyticus in an aquaculture environment by a Taqman probe method. According to the method, the species specificity of a thermolabile hemolysin (Tlh) gene of the vibrio parahaemolyticus and the characteristic of a linear relation between the Tlh gene and the quantity of the vibrio parahaemolyticus are utilized, the Tlh geneis connected with a cloning vector by using a molecular cloning technology, and five positive quantitative reference substances with different concentrations are prepared; the Taqman probe fluorescent quantitative PCR method and a traditional bacterial colony counting method are used for quantitatively detecting samples in different culture environments at the same time, and results show that theprobe method and the traditional bacterial colony counting method have no significant difference for the quantitative results of the vibrio parahaemolyticus; and meanwhile, the Taqman probe method for quantitatively detecting the vibrio parahaemolyticus is high in specificity, reliable in result and suitable for quantitatively detecting the vibrio parahaemolyticus in the water environment of a livestock farm.

Description

technical field [0001] The invention belongs to the fields of environmental sample and food inspection and quarantine and molecular biology detection, and specifically relates to a detection method for the quantitative detection of Vibrio parahaemolyticus in aquaculture environment by probe method, in particular to a detection method for environments such as oceans and aquaculture farms. Fluorescent PCR quantitative detection method based on Taqman probe for Vibrio parahaemolyticus in samples. Background technique [0002] Vibrio parahaemolyticus (Vibrio parahaemolyticus) is a Gram-negative halophilic bacterium that is widely distributed in oceans, salt lakes, and seafood farms. If it pollutes marine fish, shrimp, shellfish, and shrimp, it will After eating, it will cause acute gastroenteritis. The clinical symptoms include diarrhea, vomiting, nausea, abdominal cramps, low fever, chills, etc. Nearly 50% of food poisoning in coastal areas is caused by Vibrio parahaemolyticus....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/06C12R1/63
CPCC12Q1/6851C12Q1/689C12Q2531/113C12Q2561/101C12Q2545/114
Inventor 潘洁茹江小斌叶海梅张善霹蔡雷鸣刘杰郭睿王伟
Owner 福州市疾病预防控制中心
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