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Quadruple real-time fluorescence PCR method for detecting four types of pathogenic bacteria

A pathogenic bacteria and fluorescence technology, applied in biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc., can solve the problems of complex operation steps, uneven testing personnel ability, etc., and achieve economical operation. time, improving detection accuracy, and the effect of short detection time

Inactive Publication Date: 2017-11-10
GUIZHOU PROVINCIAL PRODUCT QUALITY SUPERVISION AND INSPECTION INSTITUTE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These patents are similar to academic articles, with a wide variety of reagents, complex operation steps, and high requirements for the skills of experimenters, and are more suitable for scientific research; The ability of personnel is uneven, and higher requirements are put forward for reagent cost, operation simplicity, method standardization, etc.

Method used

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  • Quadruple real-time fluorescence PCR method for detecting four types of pathogenic bacteria
  • Quadruple real-time fluorescence PCR method for detecting four types of pathogenic bacteria
  • Quadruple real-time fluorescence PCR method for detecting four types of pathogenic bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 self-made sample

[0042] Sample preparation: buy milk powder in a large local supermarket, according to its instructions, take an appropriate amount of milk powder and dissolve it in 100mL of sterilized ultrapure water to make reconstituted milk, and make 3 parts respectively.

[0043] ① Take the first part as the test sample: Inoculate the reconstituted milk with standard strains such as St.aureus (CICC21648), Sa.enterica (CICC 21490), L.monocytogenes (CICC 21633), V.parahemolyticus (CICC21617) and homogenize .

[0044] ②Use the second portion as a negative control: the reconstituted milk was sequentially inoculated with Escherichia coli O157:H7 (Enterohemorrhage E.coli O157:H7, CICC 21530), Shigella Sonnei (CICC 21535 ), Enterobacter sakazakii (Bntorobatesakazakii, CICC 21560), Bacillus cereus (CICC 10876), Campylobacter jejuni (Campylobacter jejuni, CICC 33291) and other standard strains were homogenized.

[0045] ③ Take the third portion as the spike...

Embodiment 4

[0096] Example 4 Enterprise Entrusted Sample --- Detection of Quick-frozen Dumplings

[0097] The list of ingredients includes: wheat flour, water, pork, fungus, mushrooms, onions, soybean protein, soy sauce, dried shrimp, sesame oil, refined vegetable oil, salt, sugar, starch, spices, monosodium glutamate. Grind the dumplings to be tested and take 25g to 225mL of 7.5% sodium chloride broth, buffered peptone water, Listeria broth LB 1 , sodium chloride alkaline peptone water, cultured at 36°C for 18h, 36°C for 8h, 30°C for 24h, and 36°C for 8h to enrich the bacteria. The 4 enrichment solutions were mixed homogeneously.

[0098] Using an outsourced DNA extraction kit (TOYOBO -Genome-) to extract DNA from the mixed enrichment solution, the DNA purity is OD 260 / OD 280 =1.81, the DNA concentration was 263.29 ng / μL, and stored at -20°C. Afterwards, the DNA was diluted to 100ng / μL for detection. The primer solution, the probe solution reagent and the positive control were the...

Embodiment 5

[0110] Example 5 Individual entrusted samples --- detection of oysters

[0111] Shred the oyster meat to be tested and take 25g to 225mL of 7.5% sodium chloride broth, buffered peptone water, and Listeria broth LB 1 , sodium chloride alkaline peptone water, cultured at 36°C for 18h, 36°C for 8h, 30°C for 24h, and 36°C for 8h to enrich the bacteria. The 4 enrichment solutions were mixed homogeneously.

[0112] Use an outsourced DNA extraction kit (name: Shanghai Sangong Bacteria Genomic DNA Rapid Extraction Kit) to extract DNA from the mixed enrichment solution, and the DNA purity is OD 260 / OD 280 =1.81, the DNA concentration was 277.27ng / μL, and stored at -20°C. Afterwards, the DNA was diluted to 100ng / μL for detection. The primer solution, probe solution reagent and positive control were the main components of the kit described in this patent. The negative control was plant-derived DNA, and the blank control was triple-distilled water. The fluorescent PCR reagent is Shan...

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PUM

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Abstract

The invention discloses a quadruple fluorescence PCR method for detecting four types of pathogenic bacteria. The four types of pathogenic bacteria are staphylococcus aureus, salmonella enterica, listeria monocytogenes and vibrio parahemolyticus. Four elements, namely sample DNA, a pair of universal primers and four probes, fluorescence PCR premix liquid and positive control are adopted in the method. The method comprises the following steps: designing the primers and the probes in accordance with 16S rRNA genes of the four types of bacteria, implementing amplification on a target sequence and exciting corresponding fluorescence signals; and preparing a kit from the to-be-detected sample DNA in accordance with a reagent formula and amplification conditions of the fluorescence PCR amplification; and specifically, the method comprises the following steps: establishing a quadruple fluorescence PCR primer system for detecting the four types of bacteria; establishing the quadruple fluorescence PCR kit for detecting the four types of bacteria; and determining the quadruple fluorescence PCR identification method for detecting the four types of bacteria. The method provided by the invention has the advantages of being low in standard error, short in detection time, and being capable of simultaneously detecting a plurality of target genes and reducing reagent cost; and the method is applicable to detection of pathogenic bacteria in food and cosmetics.

Description

technical field [0001] The invention relates to an identification method including genes, a detection method of pathogenic bacteria in food and cosmetics, in particular to a detection method of pathogenic bacteria in instant food. Background technique [0002] People depend on food, and food safety is one of the basic pillars of social harmony. In terms of food and cosmetics, especially in the field of ready-to-eat food, the detection of pathogenic bacteria is one of the important indicators of food safety. Referring to decades of accumulated experience in disease control and prevention, currently in various product implementation standards and food and drug supervision and sampling rules at all levels, most of the pathogenic bacteria detection items in ready-to-eat foods are Staphylococcus aureus and Salmonella; The main component is the detection of increased Listeria monocytogenes in livestock and poultry products; the main component is the detection of increased Vibrio ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12Q1/10C12Q1/04
CPCC12Q1/686C12Q2537/143C12Q2561/113Y02A50/30
Inventor 孙端方李春宇田志强董睿寻思颖肖莉陈梅
Owner GUIZHOU PROVINCIAL PRODUCT QUALITY SUPERVISION AND INSPECTION INSTITUTE
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