Multi-PCR fast detecting reagent kit for salmonella and bacillus coli 0157 in livestock and poultry meat and testing method thereof

A detection kit and Salmonella detection technology, applied in biochemical equipment and methods, microorganism-based methods, microorganism measurement/inspection, etc., can solve the problems of low specificity, difficulty in confirming or excluding Escherichia coli O157 contamination, and achieve The effect of good specificity and good application prospect

Inactive Publication Date: 2008-08-20
GUANGDONG INST OF MICROORGANISM
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among the Escherichia coli O157 PCR detection methods reported at home and abroad, the four genes stx1, stx2, eaeA, and hlyA are most commonly used as target genes, but studies have found that different types of diarrhea-causing E. coli have the same gene fragments. It is not unique to Escherichia coli O157, therefore, the specificity of single detection of the above gene fragments is not high, and it is difficult to confirm or exclude Escherichia coli O157 contamination

Method used

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  • Multi-PCR fast detecting reagent kit for salmonella and bacillus coli 0157 in livestock and poultry meat and testing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Primer specific detection

[0025] According to the designed 2 pairs of specific PCR primers, the negative and positive results of 27 common pathogenic bacteria were tested in Table 2. The rfbE gene was detected in 5 strains of E. coli O157, the above genes were not detected in other serotypes of E. coli, and the above-mentioned specific amplification bands were not seen in all non-E. coli strains; invA genes were detected in 10 strains of Salmonella. The results show that the two pairs of primers are only specific to their target organisms.

[0026] Table 2 Primer specific electrophoresis results

[0027] Strain

[0028] E.coli

[0029] Note: NCTC (United Kingdom National Collection of Type Cultures), CMCC (National Center for Medical Culture of Collections), ATCC (American Type Culture Collection, American Standard Culture Collection) Center), GDCIQ (Guangdong Entry-Exit Inspection and Quarantine Bureau, Guangdong Entry-Exit Inspection and Q...

Embodiment 2

[0030] Example 2: Detection of primer sensitivity

[0031] The standard strain S.typhi CMCC50115, E.coli O157 NCTC12900 culture solution (plate count results were 2.4 × 10 9 cfu / mL and 2.2×10 9cfu / mL) was mixed in equal amounts, and then 10 times of gradient dilutions were made, and then each gradient dilution was inoculated into 90 mL nutrient broth culture broth containing 10 g minced pork. After culturing for 4 hours, DNA was extracted for multiplex PCR detection. The result is shown in Figure 1. The highest detectable dilution concentration is the highest detection sensitivity of multiplex PCR in the sample. The result shows that the highest detectable dilution is 10 -7 , The concentrations of the two bacteria that can be detected are: 2.4×10 2 cfu / mL Salmonella and 2.2×10 2 cfu / mL Escherichia coli O157.

Embodiment 3

[0032] Example Three: Detection of Salmonella and Escherichia coli O157 in pork using the multiplex PCR rapid detection kit of the present invention

[0033] 1. Sample pretreatment

[0034] Using aseptic operation, randomly sample 10 g, add it to 90 mL nutrient broth culture solution, and incubate at 37°C for 4 hours.

[0035] 2. Sample DNA extraction

[0036] 1) 1.5mL bacterial solution, centrifuged at 8000r / min for 10min, and remove the supernatant; 2) Add 567μL of TE to suspend the pellet, and add 30μL of 100g / L SDS, 3μL of 20mg / mL proteinase K, mix well, and incubate at 37°C for 1h; 3) Add 100μL of 5mol / L NaCl and mix well; 4) Add 80μL of CTAB / NaCl solution, mix well, and incubate at 65°C for 20min; 5) Use an equal volume of phenol: chloroform: isoamyl alcohol (25: 24:1, v: v: v) Extraction, centrifuge at 10000r / min for 10 minutes, transfer the supernatant to a clean centrifuge tube; 6) Extract with an equal volume of chloroform: isoamyl alcohol (24:1, v: v), take the supernat...

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Abstract

The invention relates to a microbial detection device and a detection method of meat of livestock and poultry. The salmonella and Escherichia coli O157 multiple PCR rapid detection kit of the meat of livestock and poultry includes: 10 multiplied by PCR buffer, 1.0 to 3.0mmol / L of MgCl2, 240 Mumol / L of dNTP each, 200nmol / L of salmonella primer, 200nmol / L of Escherichia coli O157 primer and 1.3 to 3.0U of Taq enzyme. The microbial detection device has better specificity, which can complete the simultaneous detection of the salmonella and the Escherichia coli O157 within 8 to 9h; compared with the conventional bacteriology, the diagnosis is rapid, convenient and economical, thus providing a powerful technical means for the diagnosis of pathogen in a food sample and having good application prospect.

Description

【Technical Field】 [0001] The invention relates to a detection device and a detection method for microorganisms in livestock and poultry meat. 【Background technique】 [0002] In the past few decades, the incidence of foodborne diseases caused by eating foods contaminated by Salmonella, Campylobacter jejuni and E. coli O157 has remained high. In developed countries, about one-third of people suffer from foodborne diseases each year. The United States There are about 76 million patients with food-borne diseases every year, which arouses the attention of the international community to food safety, especially the problem of harmful microorganisms in food. Changes in food production patterns and dietary patterns, an increase in consumers who are susceptible to food-borne pathogens, and increasing demand for livestock and poultry meat in developing countries are important reasons for the increase in the incidence of food-borne diseases. Therefore, increasing the inspection and quarantin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/42C12R1/19
Inventor 张菊梅吴清平杨小鹃郭伟鹏
Owner GUANGDONG INST OF MICROORGANISM
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