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

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 ...

AI Technical Summary

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 antibody needs to be adjusted for different test samples, which is not suitable for broad-spectrum detection

Recently, a nucleic acid test strip based on nucleic acid detection has developed rapidly. It is combined with a constant temperature amplification method for detection. There is a loop-mediated constant temperature amplification nucleic acid test strip (LAMP-NALFTS), which is based on nucleic acid sequence Nucleic acid test strips (NASBA-NALFTS), cross primer constant temperature amplification nucleic acid test strips, cycle probe warm amplification nucleic acid test strips (CPT-NALFTS), recombinant polymerase warm amplification nucleic acid test strips (RPA- NALFTS), Strand Displacement Constant Temperature Amplification Nucleic Acid Test Strip (SDA-NALFTS) and other methods have been reported, but the probe design is generally complicated, the detection sensitivity is relatively low, and false positives are prone to occur, and in foodborne pathogenic There are fewer applications in the field of microbiological detection

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