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High-pathogenicity Vibrio parahaemolyticus rapid detection primer and kit

A technology of Vibrio hemolyticus and high pathogenicity is applied in the field of rapid detection primers and kits of Vibrio parahaemolyticus, which can solve the problems of inability to separate Vibrio parahaemolyticus, complicated operation, long time consumption, etc. Achieve programming and standardization, good specificity and accuracy, and small fish damage

Inactive Publication Date: 2016-06-01
天津市水产技术推广站
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection of highly pathogenic Vibrio parahaemolyticus in shrimp mainly adopts traditional bacterial isolation and identification, serological reaction and PCR detection technology. Vibrio is separated from traditional Vibrio parahaemolyticus, and the PCR method can accurately identify specific Vibrio parahaemolyticus, but the cost is high, professional equipment and technical personnel are needed, and there is an urgent need for a method that can be applied to the breeding site in aquaculture production. Highly pathogenic Vibrio parahaemolyticus detection technology and its products that are fast, accurate, and easy to operate
There is no report on the detection technology of highly pathogenic Vibrio parahaemolyticus LAMP

Method used

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Experimental program
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Effect test

Embodiment 1

[0033] The highly pathogenic Vibrio parahaemolyticus rapid detection primer is composed of an outer primer and an inner primer, and the outer primer is composed of an outer primer upstream primer shown in SEQ ID NO.1 and an outer primer downstream primer shown in SEQ ID NO.2; the inner primer is composed of The internal primer upstream primer shown in SEQ ID NO.3 and the internal primer downstream primer shown in SEQ ID NO.4 are composed.

Embodiment 2

[0035] Highly pathogenic Vibrio parahaemolyticus rapid detection kit, including:

[0036] (1) TE buffer solution, its composition is 20mmol / LTris-HCl, 2mmol / LEDTA, solvent is distilled water, pH=8.0;

[0037] (2) BstDNA polymerase: BstDNA polymerase with a concentration of 8U / μl;

[0038] (3) LAMP reaction solution, the composition of 24 μl LAMP reaction solution is: 2.5 μl 10× reaction buffer solution; 3 μl concentration is the dNTP of 2.5mmol / L; 1 μl concentration is the external primer upstream primer shown in SEQIDNO. The concentration of 1 μl is 5 μmol / L of the outer primer downstream primer shown in SEQIDNO.2, the concentration of 1 μl is 40 μmol / L of the inner primer upstream primer shown in SEQIDNO.3, and the concentration of 1 μl is 40 μmol / L of the primer shown in SEQIDNO.4. The downstream primer of the inner primer shown; 1 μl complex formed by calcein and manganese ion; 13.5 μl DEPC water;

[0039] (4) Positive control membrane, made by the following method: abso...

Embodiment 3

[0043] Establishment of detection method for rapid detection kit of highly pathogenic Vibrio parahaemolyticus

[0044](1) Template preparation: DNA extraction kit (Quick DNA Extraction and Detection Kit KG203 from Tiangen) was used to extract the purely cultured highly pathogenic Vibrio parahaemolyticus genomic DNA as a positive control solution, and the purely cultured highly pathogenic Vibrio parahaemolyticus liquid was used as a test sample to test the feasibility of the established detection method.

[0045] (2) Design and synthesis of primers

[0046] Using BLAST software to analyze the highly pathogenic Vibrio parahaemolyticus gene (KM035408.1) sequence, screen out the nucleic acid sequence of the highly pathogenic Vibrio parahaemolyticus gene Vibrioparahaemolyticus, and design LAMP primers for this fragment according to the primer design principle of LAMP technology And synthesized, the primers used are as follows:

[0047]

Primer sequence (5'-3')

...

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PUM

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Abstract

The invention discloses a high-pathogenicity Vibrio parahaemolyticus rapid detection primer and kit. The primer comprises an outer primer and an inner primer, and the outer primer is composed of an outer primer upstream primer shown in SEQ ID No. 1 and an outer primer downstream primer shown in SEQ ID No. 3; the inner primer is composed of an inner primer upstream primer shown in SEQ ID No. 3 and an inner primer downstream primer shown in SEQ ID No. 4. The problems that the prior art has long cycle length and high detection cost in detecting high-pathogenicity Vibrio parahaemolyticus and is unable to serve for field detection are solved, a detection process is made quick and ordered, all detective operations may be finished within 2 h, the detection process is programmed and standardized, operation is specified and rarely erred, and damage to a fish body is low. The primer of the invention is effective in detecting high-pathogenicity Vibrio parahaemolyticus and has good specificity and accuracy.

Description

technical field [0001] The invention relates to a primer and a kit for rapid detection of highly pathogenic Vibrio parahaemolyticus, belonging to the field of rapid detection of aquatic pathogens. Background technique [0002] my country is the largest country in the world in the production of prawn farming. In 2012, the output was 1.65 million tons, accounting for 40% of the global aquaculture production. In 2013, acute hepatopancreatic necrosis of prawns led to a 23% reduction in the global aquaculture production of prawns. Thailand reduced production by 54%, and my country reduced production by 17%. %, Guangdong reduced production by 30%, and the incidence of acute hepatopancreatic necrosis of prawns in ponds that have been cultured for more than 80 days exceeded 80%, and the annual direct economic loss exceeded 5 billion yuan. The disease has become more serious in the past two years. The main pathogenic bacterium of acute hepatopancreatic necrosis is a specific Vibrio p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6806C12Q1/6848C12Q1/689Y02A50/30
Inventor 姚学良徐晓丽张振奎包海岩蔡琰
Owner 天津市水产技术推广站