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A rapid detection kit for pathogenic microorganisms

A microbial and rapid technology, applied in the biological field, can solve problems such as difficulty in antibody preparation, false positive cross-reaction, and small amount of sample required

Inactive Publication Date: 2011-12-21
上海福盈资产管理有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, most of the existing rapid detection technologies for food-borne pathogens use immunological methods. This method is simple in operation, fast in detection speed, high in sensitivity, good in specificity, and requires a small amount of samples. For example, it is difficult to prepare monoclonal antibodies against pathogenic bacteria, the use of multiple antibodies is prone to cross-reactions, false positives, and only one or several pathogenic bacteria can be detected at a time.

Method used

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  • A rapid detection kit for pathogenic microorganisms
  • A rapid detection kit for pathogenic microorganisms
  • A rapid detection kit for pathogenic microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Embodiment 1, the design of primer

[0122] The target bacterial target gene to be detected was selected, its specific conserved sequence was determined by Blast analysis software, primers were designed by using Oligo 6 primer design software, and multiple sets of primers were designed for each detection bacterium to select the best primer combination. The selected super primers are shown in Table 2, and the selected and specific detection primer sequences for the target genes of food-borne microorganisms to be tested are shown in Table 2.

[0123] Table 2

[0124]

[0125]

Embodiment 2

[0126] Embodiment 2, multiple PCR detection method

[0127] (1) Nucleic acid extraction

[0128] Take the bacterial solution and centrifuge at 10000rpm for 5min, collect the bacterial pellet after centrifugation, and resuspend the bacterial pellet in 100μl sterile ddH 2 In O, break the cells in a boiling water bath for 15 minutes; shake and mix well, and centrifuge at 10,000 rpm for 5 minutes; take the supernatant as a nucleic acid template for PCR.

[0129] (2)PCR

[0130] Prepare the PCR reaction solution according to the ratio in Table 3.

[0131] table 3

[0132] Reagent

µL

wxya 2 o

14

mixed primer

6

Taq Master Mix

25

nucleic acid template

5

total capacity

50

[0133] In Table 3, the mixed primers contain target gene-specific detection primers (2 pairs of primers for each target gene) and super primers, wherein relative to any target gene, the amount of the forward super primer is a...

Embodiment 3

[0137] Embodiment 3, liquid phase chip detection

[0138] The procedure for coupling probes to microspheres is as follows:

[0139] (1) Take 0.5ml of naked bead solution and centrifuge at 12,000rpm for 3 minutes;

[0140] (2) Remove the supernatant, add 50 μL of 0.1M MES solution (pH 4.5, purchased from Sigma), vortex for 10 seconds, and centrifuge at 12,000 rpm for 3 minutes;

[0141] (3) Remove the supernatant, wash once with 0.1M MES solution (pH 4.5), shake and mix, add 2.5 μL of 100 pmol / L probe solution, and vortex for 10 seconds to mix;

[0142] (4) Prepare EDC solution with 0.1M MES solution (pH4.5) to make the concentration 10mg / mL. After preparation, take 2.5μL and add it to the mixture in step 3. Vortex for 10 seconds to mix well. Stand for 30 minutes under dark conditions;

[0143] (5) 2.5 μL of freshly prepared EDC solution (purchased from Sigma) was added, vortexed for 10 seconds to mix well, and left to stand for another 30 minutes at room temperature in the ...

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Abstract

The invention discloses a rapid detection method for microorganisms and a rapid detection kit for microorganisms. The method and reagent of the present invention can detect a variety of pathogenic bacteria in a very short period of time, and have the advantages of simple and fast operation, high sensitivity, strong specificity, low cost, small amount of sample required, high throughput, suitable for Features such as on-site screening can be used for rapid detection of food-borne poisoning and food safety, and overcome the shortcomings of current rapid microbial detection technology and equipment.

Description

technical field [0001] The invention belongs to the field of biotechnology; more specifically, the invention relates to a rapid detection kit for pathogenic microorganisms. Background technique [0002] In recent years, major food safety incidents have occurred frequently, which has aroused people's great attention. To fundamentally solve food safety problems, it is necessary to implement full-process management and monitoring of food production, processing, distribution and sales, which requires a large number of fast, convenient, accurate and sensitive food safety systems that can meet this requirement. Analytical detection technology. [0003] With the development of science and technology, food safety rapid analysis and detection methods play an increasingly important role in food hygiene inspection. From the perspective of long-term development, the development of immunology, molecular biology, computer technology and automation has greatly promoted the development of...

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 王慧王子良相丽吴松洁李井泉
Owner 上海福盈资产管理有限公司
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