Method for detecting food-borne pathogenic bacteria by using nucleic acid test strip based on hyper-branched rolling cycle amplification and kit

A technology of nucleic acid test strips and rolling circle amplification, applied in the field of kits for the detection of Listeria monocytogenes and Salmonella, which can solve the problems of complex probe design, time-consuming and laborious, and expensive monoclonal antibodies

Inactive Publication Date: 2012-12-12
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, the detection methods of food-borne pathogens mainly rely on isolation culture and biochemical identification, which is time-consuming and labor-intensive.
Nucleic acid-based detection methods with higher sensitivity and better specificity can be directly used for bacterial detection without pre-cultivation or shortened incubation time, but they require temperature cycling and expensive equipment, such as polymer chain enzyme reaction (PCR). ) and fluorescence detection technology based on nucleic acid amplification
With the development of colloidal gold diagnostic technology, a colloidal gold immune test strip with fast detection speed, good specificity, high sensitivity, simple equipment, low cost and simple operation has been widely used in the field of biomedicine, especially in medical testing. application, the successful commercial application of early pregnancy test strips is a proof, but the monoclonal antibodies used in colloidal gold immune test strips are also relatively expensive, and the type of an

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  • Method for detecting food-borne pathogenic bacteria by using nucleic acid test strip based on hyper-branched rolling cycle amplification and kit
  • Method for detecting food-borne pathogenic bacteria by using nucleic acid test strip based on hyper-branched rolling cycle amplification and kit
  • Method for detecting food-borne pathogenic bacteria by using nucleic acid test strip based on hyper-branched rolling cycle amplification and kit

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Experimental program
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Effect test

Embodiment 1

[0102] 1. Extraction and digestion of DNA from Listeria monocytogenes

[0103] Genomic DNA of Listeria monocytogenes (strain number CMCC54007, purchased from Guangzhou Institute of Microbiology) was extracted using TIANamp Bacteria DNA kit. The conserved sequence of Listeria monocytogenes is the expression of lysin O gene (hlyA, NCBI number: AF253320), and the genomic DNA of Listeria monocytogenes is digested with EcoT14Ⅰ (StyⅠ) enzyme and Bsp1286Ⅰ (SduⅠ) enzyme.

[0104] 20μL Bsp1286Ⅰ digestion reaction system:

[0105]

[0106] Reaction conditions: 60°C for 1h→95°C for 10min;

[0107] 40μL EcoT14Ⅰ digestion reaction system:

[0108]

[0109] Reaction conditions: 60°C for 1 h→95°C for 10 min to obtain the hlyA target fragment, its sequence: 5'-TCTCCGCCTGCAAGTCCTAAGACGCCAATCGAAAAGAAACACGC-3'.

[0110] 2. Primer and probe design

[0111] Design a 5' phosphorylated padlock probe based on the hlyA target sequence, which is used to connect with the target sequence to for...

Embodiment 2

[0142] Genomic DNA of Salmonella (strain number CMCC50040, purchased from Guangzhou Institute of Microbiology) was extracted using TIANamp Bacteria DNA kit, according to the conserved sequence in the Salmonella invA gene sequence (GenBank EU348367) published by NCBI, and compared using blast in NCBI, Taq Ⅰ (restriction site is TCGA) and Alu Ⅰ (restriction site is AGCT) were used for enzyme digestion reaction to obtain invA target fragment, its sequence is: 5'-GTCTCTACAGAGACCGTACCGTTGACTTGTGCCGAAGAGCCGGC-3'.

[0143] Design a 5' phosphorylated padlock probe according to the invA target fragment sequence, the padlock probe sequence is: 5'-phosphate-TCAACGGTACGGTCTCTGTAGAGACGGGATTAGGTTACTGCGATTAGCACAAGCACCAAGAGCAACTACACGAATTCCGGCTCTTCGGCACAAG-3'; Part of it is complementary to the target sequence, and the middle two parts are the binding regions of primer 1 and primer 2 respectively. According to the principle of hyperbranched rolling circle amplification, padlock probes designed ...

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Abstract

The invention discloses a method for detecting food-borne pathogenic bacteria by using a colloidal gold nucleic acid test strip based on hyper-branched rolling cycle amplification and a kit for detecting Listeria monocytogenes and Salmonella, belonging to the technical field of biological detect. The method comprises the following main steps: (1) performing extraction and enzyme digestion on the DNA (deoxyribonucleic acid) of food-borne pathogenic bacteria; (2) designing an amplification primer and a nucleic acid probe sequence; (3) performing hyper-branched rolling cycle amplification reaction; (4) annealing and hybridizing; (5) preparing a nano gold probe; (6) preparing a colloidal gold nucleic acid test strip; and (7) detecting a sample. When the sample contains a target gene segment, both a T line and a C line of the colloidal gold nucleic acid test strip are formed into red lines; and if the sample does not contain the target gene segment, only the C line of the colloidal gold nucleic acid test strip is formed into a red line. According to the invention, food-borne pathogenic bacteria in the food sample can be detected quickly, specifically, sensitively, qualitatively and quantitatively; and the invention is simple in probe design, short in operation steps and favorable for popularization.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a method for detecting food pathogenic bacteria based on a hyperbranched rolling circle amplification colloidal gold nucleic acid test strip and a kit for detecting Listeria monocytogenes and Salmonella. Background technique [0002] In recent years, food-borne pathogens have caused more and more food safety incidents worldwide due to their infectivity and pathogenicity. With the continuous improvement of people's living standards and education levels, food safety is becoming more and more People pay more and more attention to it, and the problem of food pathogenic bacteria has correspondingly attracted more attention. The rapid, highly specific and highly sensitive method for detecting food pathogens can not only quickly diagnose the cause of the disease, but also prevent the occurrence of intestinal infectious diseases and food poisoning in real life. [0003] At ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12Q1/10
CPCY02A50/30
Inventor 周小明刘宏星邢达
Owner SOUTH CHINA NORMAL UNIVERSITY
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