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Quick bacterium examination kit and detection method thereof

A kit and bacteria technology, applied in the field of microbial detection, can solve problems such as no public reports found, and achieve the effects of easy operation, avoidance of use, and high sensitivity

Inactive Publication Date: 2010-05-12
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Polymerase Chain Reaction (PCR), referred to as PCR, is a molecular biology technique that uses a pair of specific primers to amplify the DNA fragments of specific microorganisms or bacteria. It can be regarded as special DNA replication outside the body. And through Agarose electrophoresis is used to detect target PCR products, which can be used to detect viruses, bacteria, and even molds and yeasts. However, due to the limitation of enzymes, the traditional PCR reaction requires at least one and a half hours of PCR cycles, which makes the entire detection time more than 2 hours. Hours.
[0008] Through document search, do not find the public report of the same document as the content of the present invention

Method used

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  • Quick bacterium examination kit and detection method thereof
  • Quick bacterium examination kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The rapid test kit of the present embodiment contains:

[0047] Cell pretreatment solution: 10mmol / L Tris-Cl, 1mmol / L EDTA, 0.05% TritonX-100, 0.08% bovine serum albumin, 0.02% Tween20 to adjust the pH to 8.0;

[0048] Reaction solution: 5x Phire Reaction Buffer (Finnzymes, Finland);

[0049] Complex enzyme: Phire Hot Start DNA Polymerase (Finnzymes, Finland) 2U / μL and iProof High Fidelity DNA Polymerase (BIO-RAD, USA) 2U / μL, the enzyme activity ratio of the two enzymes is 3:2.

Embodiment 2

[0050] Example 2 uses the kit of the present invention to detect various bacteria.

[0051] As a rapid test kit for bacteria, we hope that this method has wide applicability and can be widely used in the detection of various bacteria. Therefore, we selected three common food-borne pathogenic bacteria, described them according to biological classification, and divided the bacteria into Gram-positive and Gram-negative. Therefore, when selecting samples, this embodiment selects Gram-positive bacteria Listeria, Gram-negative bacteria Salmonella and Escherichia coli as representatives, and performs detection according to the method described in the kit. Take the kit of Example 1 as an example.

[0052] The specific operation is as follows:

[0053] 1. Take 50 μL of bacterial liquid in a centrifuge tube, centrifuge at 1000 rpm for 2 minutes, and remove the supernatant.

[0054] 2. Add 50 μL of cell pretreatment solution (the formula of cell pretreatment solution is: 10mmol / LTris-...

Embodiment 3

[0063] Example 3 Detection of the sensitivity of the present invention.

[0064] The overnight culture solution of Salmonella was diluted by 10 times with aseptic operation, colony counting was carried out, template was extracted according to the method of the kit of the present invention in Example 1, PCR electrophoresis detection was carried out, and the detection limit was calculated. Salmonella-specific primers were selected as primers for PCR detection. The target fragment amplified by the reaction was about 284bp. The base sequence of the Salmonella-specific primers was:

[0065] F 5′-TCATCG CAC CGT CAAAGGAAC C-3′

[0066] R 5′-GTG AAA TTA TCG CCA CGT TCG GGC AA-3′

[0067] The PCR reaction system, PCR reaction conditions and detection conditions are consistent with Example 1.

[0068] The results show that: electrophoresis detection found that when each mL of bacterial liquid contains 50 Salmonella, bands consistent with the theory can be amplified, and further diluti...

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Abstract

The invention provides a quick bacterium detection kit. In the kit, warm start enzyme and high-fidelity enzyme are taken as the complex enzyme of a PCR reaction system, and the enzyme activity ratio of the warm start enzyme to the high-fidelity enzyme is 1:1 to 3:2. The quick bacteria detection kit can qualitatively detect all bacteria. In the kit, the formulation of the complex enzyme is originally created, thus PCR reaction cycle time is greatly shortened, only one hour is needed from the preparation of a sample to the formation of an electrophoretogram, and bacterium detection time is greatly shortened. The kit is easy to operate and capable of avoiding the use of medicaments, such as organic reagents and the like, which are harmful to human body; and sensitivity to bacterium detection is 5.0*101 cfu / mL. The kit is low in cost, high in speed, strong in universality, suitable for the quick detection of various bacteria and good in application prospect.

Description

technical field [0001] The invention relates to a detection kit, in particular to a kit for rapid detection of bacteria using the PCR technique of compound enzymes and a detection method thereof, belonging to the field of microbial detection. Background technique [0002] People's living standards continue to improve, gradually moving from subsistence to well-off. People's requirements for food have changed from being able to afford enough food in the past to wishing to buy good-quality, hygienic and safe food. Food quality, sanitation and safety have become the main pursuit of consumers. Therefore, the problem of microbial contamination of food, especially the problem of bacterial contamination of food, has received corresponding attention. There is a possibility of bacterial contamination in all aspects of food production, processing, storage, transportation, and sales. Once contaminated, bacteria will be massive Food spoilage caused by reproduction, or food-borne infect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/01C12R1/42C12R1/19
Inventor 许文涛黄昆仑张南王龑罗云波梁志宏石慧
Owner CHINA AGRI UNIV