Magnetic capture-dual real-time fluorescent PCR detection method for Campylobacter jejuni and Salmonella
A technology for Campylobacter jejuni and Salmonella, which is applied in the field of inspection and quarantine, can solve the problems of high false positive results, can not be used as a detection basis, occurrence and other problems, and achieves the effects of good specificity, improved bacterial detection rate, and improved accuracy.
Inactive Publication Date: 2016-05-25
SOUTH CHINA AGRI UNIV
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Problems solved by technology
[0004] Some simple methods such as PCR and ELISA often have high false positive results, so the results cannot be used as the final detection basis
Method used
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Embodiment 1
[0055] Example 1 Magnetic capture-double real-time fluorescent PCR detection method for Campylobacter jejuni and Salmonella
[0056] (1) Preparation of Salmonella immunomagnetic beads and Campylobacter jejuni immunomagnetic beads
[0057] 1) Prepare Salmonella antibody and Campylobacter jejuni antibody respectively;
[0058] The Salmonella antibody was selected from the diagnostic serum of Salmonella A-F group (Ningbo Tianrun Biopharmaceutical Co., Ltd.); the preparation of the Campylobacter jejuni antibody was as follows:
[0059] a. Preparation of Campylobacter jejuni antigen
[0060] Under sterile conditions, a small amount of Campylobacter jejuni strains ATCC33291, NCTC11168, and ZC8 frozen at -80°C were picked and inoculated on Columbia blood agar at 42°C under microaerophilic conditions for 36-48 hours. After continuous culture and passage for three times, the status of the revived Campylobacter jejuni was observed.
[0061] Add an appropriate amount of PBS (pH7.4) to...
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The invention discloses a magnetic trapping-dual real-time fluorescent PCR (Polymerase Chain Reaction) detection method for campylobacter jejuni and salmonella. According to the method, antiserum and magnetic microbeads are used for the preparation of campylobacter jejuni and salmonella immune magnetic beads, and the immune magnetic beads are used for directly trapping target bacteria in detection samples without enrichment culture; according to the detection method disclosed by the invention, specific primers and detection probes for conserved genes of the campylobacter jejuni and salmonella are respectively designed by utilizing the dual real-time fluorescent PCR method, and one-time real-time fluorescent PCR amplification is used for judging whether the samples are contaminated by the campylobacter jejuni and salmonella or not; the magnetic trapping-dual real-time fluorescent PCR detection method disclosed by the invention is easy and feasible, can be completed in one working day, and is good in specificity and high in sensitivity, thus being a rapid technical method which is applicable to the fields of inspection and quarantine, disease prevention, animal product safety inspection and the like.
Description
technical field [0001] The invention belongs to the field of inspection and quarantine, and relates to a dual real-time fluorescent PCR detection method, in particular to a method for magnetic capture-double real-time fluorescent PCR detection of Campylobacter jejuni and Salmonella by using immunomagnetic bead technology. Background technique [0002] Campylobacter jejuni and Salmonella are common pathogenic bacteria that cause bacterial food poisoning. They often contaminate animal foods such as meat, milk, and poultry. Eating contaminated food can cause acute gastroenteritis symptoms such as diarrhea and vomiting, and severe cases can cause death. Campylobacter jejuni is a zoonotic pathogen that has been widely recognized in the world for more than ten years. The World Health Organization has listed the disease as one of the most common foodborne infectious diseases. Campylobacter jejuni infection is also associated with human Guillain-Barré syndrome and other diseases wit...
Claims
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IPC IPC(8): C12Q1/68C12Q1/04C12R1/42C12R1/01
CPCC12Q1/6804C12Q2537/143C12Q2563/143C12Q2531/113
Inventor 罗开健高瑞娟
Owner SOUTH CHINA AGRI UNIV