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Loop-mediated isothermal amplification (LAMP) kit for rapidly detecting vibrio parahaemolyticus

A technology of Vibrio hemolyticus and kits, applied in the field of LAMP kits, can solve the problems of poor specificity, low sensitivity, false positive application, etc., and achieve the effects of fast detection speed, high repeatability and good specificity

Inactive Publication Date: 2013-01-09
WUHAN ZHENFU PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The traditional culture method for detection of Vibrio parahaemolyticus requires separation, screening, biochemical identification, and serological identification if necessary, which generally takes 4 to 6 days, which is time-consuming and laborious, and has the disadvantages of low sensitivity, poor specificity, false positives, time-consuming and laborious, etc.
Various conventional PCR methods have the advantages of strong sensitivity, high specificity, simplicity, and rapidity. There are many reports on the detection of Vibrio parahaemolyticus. The equipment and equipment limit its application in grass-roots units

Method used

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  • Loop-mediated isothermal amplification (LAMP) kit for rapidly detecting vibrio parahaemolyticus
  • Loop-mediated isothermal amplification (LAMP) kit for rapidly detecting vibrio parahaemolyticus
  • Loop-mediated isothermal amplification (LAMP) kit for rapidly detecting vibrio parahaemolyticus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] 1. The composition and preparation of the new visual rapid detection Vibrio parahaemolyticus LAMP kit

[0072] a) LAMP reaction mixture

[0073] The LAMP reaction mixture consists of 2.5 μl of LAMP 10×buffer, 2.0 μl of 10 μmol / L internal primers FIP and BIP, 1.0 μl of 10 μmol / L external primers F3 and B3, 3.0 μl of 10 mmol / L dNTPs, 50 mmol / LMgSO 4 3.0 μl, 5.0mol / L betaine, 4.5 μl, Bst DNA polymerase large fragment 1.0 μl, the total volume of the reaction solution is 20 μl.

[0074] Wherein the primer sequence is as follows:

[0075] Upstream outer primer F3: 5'-ACGCCTCTGCTAATGAGGT-3';

[0076] Downstream outer primer B3: 5′-GAGATTCCGCTGGGTTTGT-3′;

[0077] Upstream internal primer FIP: 5′-GCGCTTCTGGTTCAACGATTGCAGAAACGATCGTAGAGCCGT-3′;

[0078] Downstream internal primer BIP: 5′-TGAATCCTTGGATTCCACGCGTGCAGTACGCAAATCGGTAGT-3′;

[0079] b) Standard positive template

[0080] The standard positive template pMD18-T-toxR is a pMD18-T-toxR vector composed of a 328 base pa...

Embodiment 2

[0090] 1. The composition and preparation of the new rapid visual detection Vibrio parahaemolyticus LAMP kit is different from the kit described in Example 1 in that the specific primer sequences are different. The primer sequences of this kit are as follows:

[0091] Upstream outer primer F3: 5'-CGCCTCTGCTAATGAGGTAG-3';

[0092] Downstream outer primer B3: 5′-GAGATTCCGCTGGGTTTGT-3′;

[0093] Upstream inner primer FIP: 5′-TGGCGCTTCTGGTTCAACGATTAAACGATCGTAGAGCCGTCT-3′;

[0094] Downstream internal primer BIP: 5′-TGTGGCTTCTGCTGTGAATCCTGCAGTACGCAAATCGGTAGT-3′;

[0095] 2. The method for detecting Vibrio parahaemolyticus using the PCR kit based on the loop-mediated isothermal amplification technology to quickly detect Vibrio parahaemolyticus in industrial foods is exactly the same as the method described in Example 1, and the test was repeated 3 times, and the obtained The detection results are the same, the experimental group has precipitation, and the control group has no prec...

Embodiment 3

[0097] 1. The composition and preparation of the new rapid visual detection Vibrio parahaemolyticus LAMP kit is different from the kit described in Example 1 in that the specific primer sequences are different. The primer sequences of this kit are as follows:

[0098] Upstream outer primer F3: 5'-AGAAACGATCGTAGAGCCGT-3';

[0099] Downstream outer primer B3: 5′-GAGATTCCGCTGGGTTTGT-3′;

[0100] Upstream inner primer FIP: 5′-AGCCACAGGTGCTTTTTCAGGTCGACGACTTCTGACGCAAT-3′;

[0101] Downstream internal primer BIP: 5′-TGAATCCTTGGATTCCACGCGTGCAGTACGCAAATCGGTAGT-3′;

[0102] 2. The method for detecting Vibrio parahaemolyticus using the PCR kit based on the loop-mediated isothermal amplification technology to quickly detect Vibrio parahaemolyticus in industrial foods is exactly the same as the method described in Example 1, and the test was repeated 3 times, and the obtained The detection results are the same, the experimental group has precipitation, and the control group has no preci...

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Abstract

The invention discloses a loop-mediated isothermal amplification (LAMP) kit for rapidly detecting vibrio parahaemolyticus. The LAMP kit is composed of LAMP reaction liquid, a standard positive template and a negative quality control standard substance. The LAMP reaction liquid contains a Bst DNA polymerase big fragment, a primer, LAMP10*buffer, dNTPs solution, MgSO4 solution and glycine betaine. The primer is divided into a forward primer and a reverse primer. The LAMP kit has the advantages of being good in specificity, high in sensitivity, rapid and convenient, high in repeatability, capable of judging results by using eyes and the like, can conduct rapid qualitative detection on vibrio parahaemolyticus in industrial foods, and can replace continuously-used traditional culture method and serological diagnosis method.

Description

technical field [0001] The invention relates to a LAMP kit for rapid detection of Vibrio parahaemolyticus, which belongs to the field of food detection. Background technique [0002] Vibrio parahaemolyticus (also known as Vibrio Parahaemolyticus) is a Gram-negative polymorphic bacillus or slightly curved Vibrio. This bacterium is halophilic and acid-phobic, and cannot grow on salt-free medium. It reproduces rapidly in 3% to 6% saline, and every 8 to 9 minutes is a cycle, and it stops growing when it is lower than 0.5% or higher than 8% saline. It will die in 1-3 minutes in vinegar, inactivate by heating at 56°C for 5-10 minutes, and die in 1% hydrochloric acid in 5 minutes. Vibrio parahaemolyticus food poisoning, also known as halophilic bacteria food poisoning, is caused by eating food containing this bacteria, mainly from seafood or salt pickled products, common ones are crabs, squid, jellyfish, fish, yellow mud snails, etc. , followed by eggs, meat or vegetables. Clini...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCY02A50/30
Inventor 王业富郑虎卢晅
Owner WUHAN ZHENFU PHARMA CO LTD
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