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Quantitative PCR detecting kit and method for detecting vibrio parahaemolyticus thereof

A detection kit, a technology for Vibrio hemolyticus, applied in the determination/inspection of microorganisms, biochemical equipment and methods, resistance to vector-borne diseases, etc., can solve the problem of easy contamination, no molecular beacon probe, false positive and other problems, to achieve the effect of overcoming contamination, overcoming false positives, and good specificity

Inactive Publication Date: 2005-10-05
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But directly using conventional PCR detection kit to detect Vibrio parahaemolyticus, there are still following technical problems that are difficult to solve: 1, the reaction tube of conventional PCR detection kit does not contain molecular beacon probe, must " uncap operation "Transfer the PCR product, perform experiments such as agarose electrophoresis or Southern transfer hybridization to identify the PCR product, which is easy to contaminate, cause false positives, and is time-consuming (4 to 5 hours). The Ministry of Health has restricted its clinical use; 2. Conventional PCR detection kits do not contain standard products and cannot be used for quantitative detection of Vibrio parahaemolyticus

Method used

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  • Quantitative PCR detecting kit and method for detecting vibrio parahaemolyticus thereof
  • Quantitative PCR detecting kit and method for detecting vibrio parahaemolyticus thereof
  • Quantitative PCR detecting kit and method for detecting vibrio parahaemolyticus thereof

Examples

Experimental program
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Effect test

example 1

[0034] The quantitative PCR detection kit consists of the following 7 tubes:

[0035] (1) The reaction solution in the PCR reaction tube consists of the following components: 5 μl of 10×Buffer buffer, 25mM MgCl 2 3 μl of solution, 8 μl of 1.25 mM dNTP solution, 0.5 μl of 50 μM upstream primer, 0.5 μl of 50 μM downstream primer, 0.5 μl of 50 μM circular molecular beacon probe and 27 μl of deionized water;

[0036] (2) Sample treatment solution tube The treatment solution in the tube is composed of the following components: 0.5mol / LEDTA (PH8.0) 0.01ml, 1% SDS 0.25ml, 20mg / ml proteinase K 5μl and distilled water 0.235ml;

[0037] (3) 100 μl of recombinant positive plasmid DNA containing the following insert sequence in the positive control tube;

[0038] (4) Negative control tube containing 100 μl of recombinant negative plasmid DNA;

[0039] (5) 3 tubes of standard quality control, each containing 10 7 、10 6 、10 5 copy / ml recombinant positive plasmid DNA 100μl;

[0040] (6) ...

example 2

[0073] The components contained in the tubes of the positive control tube, negative control tube, standard product tube, polymerase tube and deionized water tube in the quantitative PCR detection kit are the same as those in Example 1, and the content of each tube depends on the specifications of the kit. It depends, that is, how many servings to make. The quantitative PCR detection kit described in Example 1 is designed for 20 people, and this implementation and the following examples can be deduced by analogy (common knowledge that those of ordinary skill in the art should know). The reaction solution in the tube of the PCR reaction tube is composed of the following components: 4 μl of 10×Buffer buffer solution, 25mM MgCl 2 2 μl of solution, 4 μl of 1.25 mM dNTP solution, 0.2 μl of 50 μM upstream primer, 0.2 μl of 50 μM downstream primer, 0.2 μl of 50 μM circular molecular beacon probe and 34.4 μl of deionized water; Composition: 0.5mol / LEDTA (PH8.0) 0.008ml, 1% SDS 0.20ml,...

example 3

[0077] The components and contents in the positive control tube, negative control tube, standard product tube, polymerase tube and deionized water tube in the quantitative PCR detection kit are the same as those in Example 1. The reaction solution in the tube of the PCR reaction tube is composed of the following components: 6 μl of 10×Buffer buffer solution, 25mM MgCl 2 4 μl of solution, 8 μl of 1.25 mM dNTP solution, 1.0 μl of 50 μM upstream primer, 1.0 μl of 50 μM downstream primer, 1.0 μl of 50 μM circular molecular beacon probe and 24 μl of deionized water; Composition: 0.5mol / LEDTA (PH8.0) 0.012ml, 1% SDS 0.30ml, 20mg / ml protease K 8μl and distilled water 0.250ml;

[0078] The preparation of the quantitative PCR detection kit and the method for detecting Vibrio parahaemolyticus can be implemented with reference to Example 1.

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Abstract

This invention relates to a fluorescence quantitative PCR reagent box and a method testing vibro parahaemolyticus. The reagent box is composed of PCR reacting agent tube, the tube of sample treatment, the tube of positive comparison, the tube of negative comparison, the tube of standard product, the tube of Taq DNA polyose and the tube of dehydronium. The testing procedures are: first collect and treat the pending tested sample, then implement molecular quantitative PCR test, according the CT values of positive and negative sample comparisons and the CT of tested samples to judge whether the sample is positive or negative, then record the fluorescence value and CT value of each sample, and get the standard curve from the computer to calculate the copy's number of vibro parahaemolyticus of each sample. This invention has sealed-loop operation, avoids the fake positive, and the reaction and hybridization is synchronous, and the testing time is short.

Description

Technical field [0001] The invention relates to a composition for measuring microorganisms, in particular to a fluorescent quantitative PCR kit and a method for detecting vibrio parahaemolyticus. technical background [0002] Vibrio parahaemolyticus (Vibrio parahaemolyticus, VP) is a halophilic bacterium belonging to the family Vibrio (Vibrio) and can easily cause food poisoning. The heat-resistant direct hemolytic toxin (TDH) produced by Vibrio parahaemolyticus is an important pathogenic factor, and the specific gene tdh is the main factor of food poisoning caused by Vibrio parahaemolyticus. [0003] At present, the routine detection methods of Vibrio parahaemolyticus in my country include Kanagawa phenomenon (KP) test, biochemical identification, serological reaction, nucleic acid hybridization and other technologies, but these tests or methods need to be enriched and cultured, and there are cumbersome operations and difficult It takes a long time (about 4 days) and so on....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCY02A50/30
Inventor 万成松
Owner SOUTHERN MEDICAL UNIVERSITY
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