LAMP kit for rapid detection of Shigella

A Shigella and kit technology, applied in the field of LAMP kits for rapid detection of Shigella, can solve the problems of poor specificity, low sensitivity, false positive applications, etc., and achieve fast detection speed, high repeatability, good specific effect

Inactive Publication Date: 2013-01-02
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 Shigella requires isolation, screening, biochemical identification, and if necessary, serological identification, 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 Shigella detection. The equipment and equipment limit its application in grass-roots units

Method used

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  • LAMP kit for rapid detection of Shigella
  • LAMP kit for rapid detection of Shigella
  • LAMP kit for rapid detection of Shigella

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] 1. The composition and preparation of the new rapid visual detection Shigella LAMP kit, reagent composition:

[0081] a) LAMP reaction mixture

[0082] 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.

[0083] Wherein the primer sequence is as follows:

[0084] Upstream outer primer F3: 5'-TCTGGAGGACATTGCCCG-3';

[0085] Downstream outer primer B3: 5′-GCTTCTGACCATGGCTTCG-3′;

[0086] Upstream inner primer FIP: 5′-

[0087] GCATGGTCTGGAAGGCCAGGAAGTCAGAACTTCTCCATTTTGTGG-3′;

[0088] Downstream internal primer BIP: 5′-TCGCAGAGAAACTTCAGCTCTCCCGGAGGTCATTTGCTGTCAC-3′;

[0089] b) Standard positive template

[0090] The standard positive template pMD18-T-ipaH is a pMD18-T-ipaH vector compose...

Embodiment 2

[0100] 1. The composition and preparation of the new rapid visual detection Shigella LAMP kit, the difference from the kit described in Example 1 is that the specific primer sequences are different. The primer sequences of this kit are as follows:

[0101] Upstream outer primer F3: 5'-GGCAGGGAAATGTTCCGC-3';

[0102] Downstream outer primer B3: 5′-GCTTCTGACCATGGCTTCG-3′;

[0103] Upstream internal primer FIP: 5′-GGTCTGGAAGGCCAGGTAGACTTCTGGAGGACATTGCCCG-3′;

[0104] Downstream internal primer BIP: 5′-TGCTCGCAGAGAAACTTCAGCTTGCTGTCACTCCCGACAC-3′;

[0105] 2. use the described method based on ring-mediated isothermal amplification technology to quickly detect Shigella in industrial food PCR kits to detect Shigella is exactly the same as the method described in Example 1, repeat the test 3 times, and the obtained The detection results are the same, the experimental group has precipitation, and the control group has no precipitation (see attached image 3 ), which is consistent with...

Embodiment 3

[0107] 1. The composition and preparation of the new rapid visual detection Shigella LAMP kit, the difference from the kit described in Example 1 is that the specific primer sequences are different. The primer sequences of this kit are as follows:

[0108] Upstream outer primer F3: 5'-GCAGGGAAATGTTCCGCC-3';

[0109] Downstream outer primer B3: 5′-CTTCTGACCATGGCTTCGG-3′;

[0110] Upstream internal primer FIP: 5′-GGTCTGGAAGGCCAGGTAGACTGAGGACATTGCCCGGGATA-3′;

[0111] Downstream internal primer BIP: 5′-TGCTCGCAGAGAAACTTCAGCTAGGTCATTTGCTGTCACTCC-3′;

[0112] 2. use the described method based on ring-mediated isothermal amplification technology to quickly detect Shigella in industrial food PCR kits to detect Shigella is exactly the same as the method described in Example 1, repeat the test 3 times, and the obtained The detection results are the same, the experimental group has precipitation, and the control group has no precipitation (see attached Figure 4 ), which is consisten...

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Abstract

The invention discloses an LAMP (loop-mediated isothermal amplification) kit for rapid detection of Shigella. The kit is composed of an LAMP reaction solution, a standard positive template, and a negative quality control standard. The LAMP reaction solution contains a Bst DNA polymerase large fragment, primers, an LAMP10*buffer, a dNTPs solution, an MgSO4 solution and betaine. The primers include forward and reverse primers. The kit disclosed in the invention has the advantages of good specificity, high sensitivity, rapidness and convenience, high repeatability, and judgeable results obtained by naked eyes, etc., can perform rapid qualitative detection on Shigella in industrial food, and can be used to replace the continuously used traditional culture method and the serological diagnostic method.

Description

technical field [0001] The invention relates to a LAMP kit for rapid detection of Shigella, belonging to the field of food detection. Background technique [0002] Shigella (ShigellaCastellani and Chalmers) is a class of Gram-negative bacilli and is the most common pathogen of human bacillary dysentery, commonly known as Shigella. Bacillary dysentery is the most common intestinal infectious disease, with the most patients in summer and autumn. The source of infection is mainly patients and carriers, through oral infection through food and drinking water contaminated with Shigella. Humans are susceptible to Shigella, and 10 to 200 bacteria can cause 10 to 50% of volunteers to become ill. Generally speaking, bacillary dysentery caused by Shigella is severe. Acute bacillary dysentery is more common in children and can be caused by various types of Shigella. The onset is acute, often without abdominal pain and diarrhea, showing severe symptoms of systemic poisoning. Acute b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
Inventor 王业富郑虎卢晅
Owner WUHAN ZHENFU PHARMA CO LTD
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