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Food-originated pathogenic bactenium quick detection gene chip and its application

A technology for detecting food-borne pathogens and gene chips, applied in the field of detection, can solve the problems of inability to meet the needs of the detection work, false negative results, time-consuming and labor-intensive, etc.

Inactive Publication Date: 2004-10-13
INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional detection method has the following two deficiencies: one is that the operation is cumbersome, time-consuming and laborious; the other is that false positive or false negative results are easy to appear in the test results, and it is not easy to carry out quality control
However, these detection techniques can only detect one type of microorganism in one experiment, and there are many types of pathogenic bacteria in food, so they cannot meet the needs of actual detection work.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment one Preparation of gene chip for rapid detection of food-borne pathogenic bacteria

[0053] 1.1 Surface aldehyde treatment of carrier glass sheet:

[0054] Microscope slides were soaked overnight in chromic acid wash to remove impurities such as surface organic matter, then rinsed with distilled water and immersed in 25% ammonia overnight. Afterwards, the glass slide was immersed in 95% ethanol (pH to 4.5) containing aminopropyltrimethoxysilane for 20 minutes, ultrasonically cleaned with 95% ethanol for 30 minutes, ultrasonically cleaned with pure water for 20 minutes, and dried at 115° C. for 45 minutes. Finally, the slides were soaked in 5% glutaraldehyde solution for 50 minutes, then ultrasonically cleaned for 10 minutes, washed twice with water, and placed in a dry and dust-proof place for later use.

[0055] 1.2 Use the automatic spotting instrument to spot-coat the probe array on the glass slide:

[0056] The model of the manipulator of the biochip s...

Embodiment 2

[0070] Embodiment two : The present invention detects the application example of unknown bacterium

[0071] 2.1 Pick a ring of colonies grown on the culture medium, grind and mix them in 100 μl of pure water, boil for 10 minutes, centrifuge at 10,000 rpm for 1 minute, absorb 2 μl of the supernatant and add it to the PCR reaction mixture. The formula of the PCR reaction mixture is shown in Table 4.

[0072] Table 4: PCR Reaction Mix Recipe

[0073] Component Concentration Sample volume (μl)

[0074] wxya 2 0 - 37.3

[0075] 10×PCR Buffer (Mg2 + Plus) 10× 5.0

[0076] dNTP Mixture 2.5mmol / L 4.0

[0077] 1169U20 (Cy3 labeled) 5μmol / L 1.0

[0078] 1521L19 5μmol / L 0.5

[0079] TaKaRa Taq (5U / ml) 5U / μl 0.2

[0080] 2.2 Put the reaction tube into the PCR instrument (PE 2400), and set the cycle parameters: cycle parameters:

[0081] 94°C, 5min;

[0082] 94°C, 25sec; 55°C, 25sec; 72°C, 25sec; 40 cycles in total;

[0083] 72°C, 5min;

[0084] 100℃, 5min;

[0085] 4°C, 5m...

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PUM

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Abstract

The present invention provides a gene chip for quickly detecting pathogens from food source, discloses the preparation method of said gene chip and provides 26 oligonucleotide probe sequences for detection. Said gene chip can quickly, accurately and high-effectively detect and identify the class of the pathogens in food, and its detection range includes staphylococcus aureus, Shiga's bacillus, salmonella, colibacillus 0157, bacillus proteus, mononuclear hyperplastic listerella, enterocolitis yersinia, aeruginous pseudomonads, vibrio parahaemolyticus, vibrio cholerae, bacillus cereus, beta hemolytic streptococcus, coconut fermentation pseudomonads, boticin, vibrio jejuni and bacillus perfringens, etc.

Description

technical field [0001] The invention belongs to the technical field of detection, and relates to a gene chip (or DNA microarray), in particular to a gene chip for rapid detection of food-borne pathogenic bacteria. Background technique [0002] "Food is the paramount necessity of the people". Food is closely related to human survival and health. It is very important to ensure the safety and sanitation of food and prevent toxic and harmful substances from causing harm to the human body through food. Food may be affected and polluted by various factors in the environment during production, processing, storage, transportation and sales, among which microbial contamination is especially common. Food is contaminated by pathogenic bacteria, causing various foodborne diseases and even death. In order to judge whether food is safe, prevent and control the spread of food-borne infectious diseases, and quickly diagnose bacterial food poisoning, it is necessary to have a fast, accura...

Claims

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Application Information

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IPC IPC(8): C12Q1/04C12Q1/68
CPCY02A50/30
Inventor 李君文靳连群王新为晁福寰王升启
Owner INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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