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Frequent pathogenic bacteria multiple PCR rapid detecting kit for domestic animal meat and aquatic product and its detecting method

A detection kit and detection method technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as urgent time and large sample size, and achieve the effects of good specificity, quick, simple and economical diagnosis

Inactive Publication Date: 2009-07-01
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in food production and circulation, enterprises and government regulatory departments need to detect pathogenic bacteria on a large number of foods, the sample size is large, and the time is urgent. Therefore, PCR detection technology and general methods for single pathogenic bacteria obviously cannot meet the continuous development of these departments. needs

Method used

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  • Frequent pathogenic bacteria multiple PCR rapid detecting kit for domestic animal meat and aquatic product and its detecting method
  • Frequent pathogenic bacteria multiple PCR rapid detecting kit for domestic animal meat and aquatic product and its detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment one: the PCR detection of Salmonella

[0043] 1. Sample DNA was extracted by CTAB method. Take 2 g of the sample and place it in a sterilized Erlenmeyer flask filled with 10 mL of CTAB lysis buffer, and incubate with shaking at 65°C for 1 h. Add 10 μL of 20 mg / mL proteinase K and incubate at 65°C for 2 h with shaking, transfer 1 mL of the culture solution to a 1.5 mL Eppendorf tube, and centrifuge at 13,000 g for 10 min. Transfer 800 μl of supernatant to a new Eppendorf tube, then add 600 μL of chloroform, vortex the sample, and centrifuge at 13,000 g for 10 min. Aspirate 600 μL of the aqueous layer and transfer it to a new Eppendorf tube, then add 500 μL of isopropanol, let it stand at room temperature for 30 min, recover the DNA precipitate, and dissolve it in 50 μL of sterilized double-distilled water. The composition of CTAB lysis buffer: 2% CTAB, 1.4mol / L Nacl, 0.1mol / L Tris-Hcl, 20mmol / L EDTA, pH8.0.

[0044] 2. Primers:

[0045] Upstream: 5’-ATC GG...

Embodiment 2

[0050] Example 2: PCR detection of enterohaemorrhagic Escherichia coli

[0051] 1. The sample DNA was extracted using the CTAB method described in Example (1).

[0052] 2. Primers:

[0053] Upstream: 5’-CAC ACG GAG CTT ATA ATA TTC TGT-3’

[0054] Downstream: 5'-GAC ATC ATT TGA CTC ATT AAA-3'

[0055] 3. Amplification reaction system: 10×PCR buffer 2.5μL, 2mmol / L MgCl 2 , 200 μmol / L dNTP, 100 μmol / L primer, 2.5uTaq enzyme, 2 μL sample DNA, and 25 μL reaction system.

[0056] 4. Amplification reaction conditions: pre-denaturation at 94°C for 5 minutes; 30 cycles of denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, and extension at 70°C for 1 minute; extension at 72°C for 10 minutes; and storage at 4°C.

[0057] 5. Amplification reaction results: The amplified product was detected by 12g / L agarose gel electrophoresis and analyzed by UV I gel imaging system, and there was a band at 335bp.

Embodiment 3

[0058] Example 3: Simultaneous detection of Salmonella and enterohaemorrhagic Escherichia coli by double PCR

[0059] 1. Sample DNA was extracted using the CTAB method described in Example (1).

[0060] 2. Salmonella Primer: Upstream: 5'-ATC GGC GTT ATC CCT TTC TCT GGT G-3'

[0061] Downstream: 5’-ATG TTG TCC TGC CCC TGG TAA GAG A-3’; EHEC Primer: Upstream: 5’-CAC ACG GAG CTT ATA ATA TTC TGT-3’

[0062] Downstream: 5'-GAC ATC ATT TGA CTC ATT AAA-3', add the above two pairs of primers to the same PCR tube at the same time.

[0063] 3. Amplification reaction system: 10×PCR buffer 2.5μL, 3.0mmol / L MgCl 2 , 250μmol / L dNTPeach, the primer concentrations are: Salmonella 40nmol / L, EHEC 80nmol / L, 1.3U Taq enzyme, sample DNA 2μL, reaction system 25μL.

[0064] 4. Amplification reaction conditions: use touchdown PCR method. ① Pre-denaturation at 94°C for 5 minutes; ② Denaturation at 94°C for 1 minute; ③ Annealing for 2 minutes, from 63°C to 55°C, 2 cycles for each drop of 1°C; ④ Ext...

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Abstract

The invention relates to a multiplex PCR rapid detection kit for common pathogenic bacteria in livestock and poultry meat and aquatic products, which comprises 10×PCR buffer, MgCl2, dNTP, primers and DNA polymerase. The detection method includes: extracting the template of the sample to be tested; adding 10×PCR buffer, MgCl2, dNTP, primers, DNA polymerase, template of the sample to be tested and ddH2O water into the PCR thin-walled tube, mixing it and placing it on the PCR instrument for amplification. increase; electrophoresis amplification products in the electrophoresis equipment, record the results; analyze and judge the results. The present invention has better specificity, and can complete the detection of enterohemorrhagic Escherichia coli, Listeria monocytogenes, Salmonella and Vibrio parahaemolyticus within 8 to 9 hours, which is better than conventional bacteriological diagnosis. Quick, easy and economical.

Description

[Affiliated technical field] [0001] The invention relates to a multiple PCR rapid detection kit and detection method for rapid detection of common pathogenic bacteria in livestock and poultry meat and aquatic products, belonging to the field of biotechnology. [Background technique] [0002] In the past few decades, the incidence of food-borne diseases caused by eating food contaminated by Salmonella, Campylobacter jejuni, and enterohemorrhagic Escherichia coli has remained high. About 1 / 3 of the people in developed countries suffer from food-borne diseases every year There are about 76 million cases of food-borne diseases in the United States every year, which has aroused the attention of the international community on food safety, especially the problem of harmful microorganisms in food. Factors such as changes in food production patterns and dietary patterns, the increase in consumers susceptible to foodborne pathogens, and the increased demand for livestock, poultry meat ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 吴清平杨小鹃张菊梅阙绍辉陈素云
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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