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Vibrio parahaemolyticus isothermal amplification detection kit and detection method

A technology of constant temperature amplification detection and hemolytic vibrio, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, resistance to vector-borne diseases, etc. and other problems, to achieve the effect of fast response, simple steps and high sensitivity

Inactive Publication Date: 2015-04-29
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional bacterial culture method mainly relies on experimental methods such as morphological discrimination, biochemical reaction and serological agglutination, which has the disadvantages of being greatly affected by factors such as culture conditions and reagents, low positive rate, and long detection time.
In recent years, the use of fluorescent quantitative PCR technology has shortened the detection time of Vibrio parahaemolyticus to about 2 hours, but due to the need for expensive fluorescent quantitative PCR equipment, it is difficult to widely use it at the grassroots level

Method used

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  • Vibrio parahaemolyticus isothermal amplification detection kit and detection method
  • Vibrio parahaemolyticus isothermal amplification detection kit and detection method
  • Vibrio parahaemolyticus isothermal amplification detection kit and detection method

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Embodiment 1

[0039] Composition and preparation of embodiment 1 kit of the present invention

[0040] a) DNA extraction reagent (preferably Bacterial Genomic DNA Extraction Kit from Takara Company (product number DV810A);

[0041] b) Constant temperature PCR amplification reaction solution for Vibrio parahaemolyticus nucleic acid: two peripheral primers (10pmol), two probes (20pmol) and two cross primers (40pmol), 1×Thermol buffer, MgSO 4(6mmol), dNTPs solution (0.4mmol each), Bst DNA polymerase (8U) and sterile double distilled water, the total reaction volume is 25μl;

[0042] The forward peripheral primer is: 5′-TCATCTAAAACAATGTTATAGCCA-3′;

[0043] The reverse peripheral primer is: 5'-GGTTTGGTTTTCTTGCGT-3';

[0044] Forward 5' end Biotin labeled probe 5'-Biotin-CTCTTCTTGTAATGGTTATTG-3';

[0045] Reverse 3' end FitC labeled probe 5'-Fitc-CCAACCTTATCACCAGAAATGG-3';

[0046] Amplify the forward primer:

[0047] 5'-CGGGAGTAATGCAGTTAATAGTGTGTTTTTCAATGTGCTTGGGT-3';

[0048] Amplificati...

Embodiment 2

[0057] Embodiment 2 detects the concrete method of vibrio parahaemolyticus nucleic acid with kit of the present invention

[0058] a) Using the DNA extraction solution in the Vibrio parahaemolyticus constant temperature amplification detection kit to extract DNA from the specimen to be tested.

[0059] b) Take sample DNA as a template and add it to a PCR tube containing Vibrio parahaemolyticus constant temperature PCR amplification reaction solution, in which 5 μl of sample DNA, 20 μl of reaction solution, 60 ° C amplification reaction for 35 minutes; positive and negative control PCR Positive and negative templates were added to the tubes separately.

[0060] c) Put the reacted PCR tube into the nucleic acid test strip anti-pollution detection device (No. 3 device of Hangzhou Ustar Biotechnology Co., Ltd.) for detection, and interpret the result after 2 minutes. When the sample contains the nucleic acid of Vibrio parahaemolyticus, the detection line of the test strip is posi...

Embodiment 3

[0062] Embodiment 3 detects the specificity of vibrio parahaemolyticus with kit of the present invention

[0063] Detect Salmonella typhimurium CMCC 50013, Shigella flexneri 2bATCC 12022, Vibrio parahaemolyticus ATCC 17802, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa according to the method of Example 2 ATCC 27853, Pseudomonas Shigella ATCC 14029, Listeria monocytogenes CMCC 54003 and Vibrio parahaemolyticus detection kit positive control substance, the result shows that detection kit Vibrio parahaemolyticus nucleic acid with the present invention has very strong Specificity, except for Vibrio parahaemolyticus ATCC 17802 and the positive control of the kit, all other strains were negative.

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Abstract

The invention discloses a Vibrio parahaemolyticus isothermal amplification detection kit and detection method. The kit comprises a DNA (deoxyribonucleic acid) extraction reagent, an isothermal PCR (polymerase chain reaction) amplification reaction solution, a positive control and a negative control. The detection kit has the advantages of high specificity and high sensitivity. The nucleic acid amplification reaction is kept at one constant temperature, the isothermal amplification only takes 35 minutes, the nucleic acid test strip detection result takes 1-2 minutes, and the whole detection process from sample reception to result acquisition only takes 1 hour or so; and the whole reaction process only needs one isothermal apparatus, and thus, is especially suitable for on-site quick detection and elimination of poisoning of marine products and other foods.

Description

(1) Technical field [0001] The invention relates to a constant temperature amplification rapid detection technology for nucleic acid of vibrio parahaemolyticus, which is suitable for qualitative detection of vibrio parahaemolyticus. (2) Background technology [0002] Vibrio parahaemolyticus is a marine bacterium widely distributed in coastal areas. In recent years, according to the report of the National Foodborne Disease Surveillance Network, Vibrio parahaemolyticus is the main pathogen causing foodborne diseases. Since 1998, the number of poisonings caused by Vibrio parahaemolyticus has shown a significant upward trend, ranking first among various microbial foodborne diseases, seriously affecting the food safety of our people and the development of tourism. How to prevent and quickly deal with food poisoning caused by Vibrio parahaemolyticus, the development of rapid detection technology has attracted the attention of governments and health departments at all levels. The ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6844C12Q2531/119C12Q2545/113Y02A50/30
Inventor 徐昌平卢亦愚黄世旺冯燕陈寅高见
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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