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Kit for fluorescence real-time quantitative PCR detection of Laribacter hongkongensis and detection method

A real-time quantitative, kit-based technology, applied in microorganism-based methods, fluorescence/phosphorescence, biochemical equipment and methods, etc., can solve problems such as troublesome and carcinogenic substances, and achieve the effect of simplifying experimental steps and improving detection limits.

Inactive Publication Date: 2010-01-06
SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Ordinary PCR technology requires gel electrophoresis, which is very troublesome, and most of the reagents used are carcinogenic substances

Method used

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  • Kit for fluorescence real-time quantitative PCR detection of Laribacter hongkongensis and detection method
  • Kit for fluorescence real-time quantitative PCR detection of Laribacter hongkongensis and detection method
  • Kit for fluorescence real-time quantitative PCR detection of Laribacter hongkongensis and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The kit for the detection of Hong Kong gull-type bacteria by fluorescent real-time quantitative PCR provided in this embodiment contains the following reagents in separate packages:

[0053] (1) 1 bottle of lysate, containing CTAB solution with a mass concentration of 10%, NaCl solution with a mass concentration of 4.1%, and SDS with a mass concentration of 0.1-0.5%;

[0054] (2) 1 bottle of proteinase K, containing 1mg / mL proteinase K;

[0055] (3) 1 bottle of extract, containing phenol, chloroform and isoamyl alcohol, described phenol: chloroform: the volume ratio of isoamyl alcohol is 25: 24: 1;

[0056] (4) 1 bottle of TE buffer solution, containing 0.1-0.5mol / L Tris-HCl (pH7.0) buffer solution, 50mmol / LEDTA;

[0057] (5) 1 tube of fluorescent PCR reaction solution, containing 1~2U hot start Taq enzyme, 3~5.5mmol / L Mg 2+ , 100~200μmol / L dNTPs, 1×PCR buffer, 1×SYBR Green I solution, 100~300nmol / L upstream and downstream primers, 1×PCR buffer contains 0.2~0.3mmol / L ...

Embodiment 2

[0082] Refer to Example 1 for the fluorescent real-time quantitative PCR detection kit provided in this example for the detection of Gull-type bacteria in Hong Kong.

[0083] The present embodiment utilizes above-mentioned fluorescent real-time quantitative PCR kit to detect the method for Hong Kong gull-type bacteria in the intestinal tract of sweet-scented osmanthus fish, comprising the following steps:

[0084]a) Put the sweet-scented osmanthus fish to death, put it on the dissecting tray, disinfect the body surface with 75% alcohol cotton balls, take out the fish intestines completely with a scalpel and scissors, and handle them carefully to avoid intestinal rupture, resulting in the omission of intestinal contents For contamination, remove the fat adhering to the intestines with a camera, dissect under aseptic conditions, take 1-2g of samples into a sterilized 5mL Eppendorf tube, cut the tissue in the tube with scissors until uniform, add 4mL of sterile water, fully oscill...

Embodiment 3

[0099] Refer to Example 1 for the fluorescent real-time quantitative PCR detection kit provided in this example for the detection of Gull-type bacteria in Hong Kong.

[0100] The present embodiment utilizes the above-mentioned fluorescent real-time quantitative PCR kit to detect the method for Hong Kong gull-type bacteria in the intestinal tract of grass carp carp, comprising the following steps:

[0101] a) Put the grass carp to death, put it on the dissection tray, disinfect the body surface with 75% alcohol cotton balls, take out the fish intestines with a scalpel and scissors, and handle them carefully to avoid intestinal rupture, which may cause omission of intestinal contents Contamination, remove the fat adhering to the intestine with a camera, dissect under aseptic conditions, take 0.5-2g sample into a sterilized 5mL Eppendorf tube, cut the tissue in the tube with scissors until uniform, add 4mL sterile water, fully oscillate to suspend, Centrifuge at 4000rpm in a cent...

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Abstract

The invention provides a kit for fluorescence real-time quantitative PCR detection of Laribacter hongkongensis and a detection method thereof. The kit contains separation agents of lysis solution, protease K, extraction solution, TE buffer solution, fluorescence PCR reaction solution, ROX dye, positive control solution and negative control solution. The detection method comprises the following steps: preliminary treatment is carried out on a sample to obtain a sample PCR template; the fluorescence PCR reaction solution, the sample PCR template, the positive control solution and the negative control solution or the inner reference dye are added into a PCR reaction tube to carry out an amplification reaction; and whether the Laribacter hongkongensis exists in the intestinal tract of a freshwater fish or not is quantitatively detected through analyzing the Ct values and a dissolution kinetic curve. The method is simple and fast, and has favorable specificity and high sensitivity, great social and economic benefits for improving the edible security of freshwater products and very high practical value.

Description

technical field [0001] The invention relates to a detection kit and a detection method for pathogenic bacteria in freshwater fish intestines, in particular to a fluorescent real-time quantitative PCR detection kit and a detection method for Hong Kong seagull-type bacteria. Background technique [0002] Laribacter hongkongensis belongs to Proteobacteria β subclass, Neisseriaceae (Neisseriaceae). Professor Yuan Guoyong and Associate Professor Hu Zhaoyi from the Department of Microbiology of the School of Medicine first isolated it from the blood and chest juice of a liver cirrhosis patient who was admitted to the hospital due to high fever and shortness of breath. A series of biochemical analysis and molecular biology tests showed that the phenotype and genotype characteristics of the bacterium were different from all known bacteria, and belonged to a newly discovered genus of bacteria, which was originally named HKU1. With the further research on the bacteria, different stra...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64C12R1/01
Inventor 李来好杨贤庆岑剑伟郝淑贤石红吴燕燕魏涯刁石强周婉君陈胜军戚勃马海霞黄卉
Owner SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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