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Primer group for detecting EHEC (enterohaemorrhagic escherichia coli) O157:H7 with LAMP (loop-mediated isothermal amplification) and detection method

A technology for Escherichia coli and enterohemorrhage, which is applied in the fields of animal health and food inspection, can solve the problems of cumbersome reaction program setting, and achieve the effect of good detection specificity and high sensitivity

Inactive Publication Date: 2017-05-17
COMPREHENSIVE INSPECTION & QUARANTINE TECH CENT NINGXIA ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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Problems solved by technology

Traditional PCR technology is inseparable from the time-consuming and energy-consuming process of repeated heating and cooling, and the reaction program setting is also cumbersome.

Method used

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  • Primer group for detecting EHEC (enterohaemorrhagic escherichia coli) O157:H7 with LAMP (loop-mediated isothermal amplification) and detection method
  • Primer group for detecting EHEC (enterohaemorrhagic escherichia coli) O157:H7 with LAMP (loop-mediated isothermal amplification) and detection method
  • Primer group for detecting EHEC (enterohaemorrhagic escherichia coli) O157:H7 with LAMP (loop-mediated isothermal amplification) and detection method

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Embodiment Construction

[0037] The test methods used in the following descriptions are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0038] 1. Primer design

[0039] In accordance with the principles of LAMP primer design, multiple sequence alignments were performed based on the genome sequence of the EHEC O157:H7 antigen gene rfbE in the GenBank database to find a highly conserved region as the target of amplification, and then 4 sets of primers were designed by the molecular biology software LAMPD Designer5. Purification by HPLC. The synthesized primers were diluted with ultrapure water to a 100 pmol / pL solution, and stored at -20°C for future use. The primer sequences are listed in Table 1.

[0040] Table 1 Primers for LAMP detection of EHEC O157:H7

[0041]

[0042] 2. Establishment of testing standard methods

[0043]The Bacterial Genomic DNA Rapid Extraction Kit is a product...

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Abstract

The invention belongs to the technical field of animal health and food inspection and relates to a primer group for detecting EHEC (enterohaemorrhagic escherichia coli) O157:H7 with LAMP (loop-mediated isothermal amplification) and a detection method. The primer group for LAMP detection comprises four specific primers including a forward inner primer FIP:SEQ ID NO.1, a reverse inner primer BIP:SEQ ID NO.2, a forward outer primer F3:SEQ ID NO.3 and a reverse outer primer B3:SEQ ID NO.4. The detection method comprises the following steps: 1, extracting a DNA of a to-be-detected sample; 2, preparing an LAMP detection reaction system; 3, performing an LAMP reaction with the DNA extracted in the step 1 as a template and identifying a result with a real-time turbidity method. The method is not limited by culture conditions, a target nucleic acid can be detected within 50 min, qualitative detection sensitivity can reach 3.45*10<4> CFU / mL and is 10 times higher than that obtained with common PCR, the reaction result is easy to observe, the cost is low, and rapid screening results can be provided for supervision and inspection of market products and emergency handling by supervision departments.

Description

[0001] Technical field: [0002] The invention relates to the technical fields of animal hygiene and food inspection, in particular to a LAMP detection primer set and a detection method for detecting enterohaemorrhagic Escherichia coli O157:H7. [0003] Background technique: [0004] Enterohaemorrhagic Escherichia coli (Enterohaemorrhagic Escherichia coli, EHEC) O157:H7 was first discovered in the United States in 1982, EHEC O157:H7 is the main serotype of Escherichia coli, the World Health Organization in 1997 listed it as a new foodborne pathogens. EHEC O157:H7 is mainly transmitted through the digestive tract, and clinically can cause hemorrhagic enteritis, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura and other diseases. The sporadic and outbreak epidemic cases caused by EHEC O157:H7 continue to occur around the world. In 1996, an epidemic occurred in Osaka, Japan, with more than 10,000 patients and 11 deaths. From 1999 to 2000, multiple outbreaks occurred...

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/19
CPCC12Q1/6844C12Q1/689C12Q2531/119
Inventor 郝俊虎郭澍强杨旭光李萍陈林
Owner COMPREHENSIVE INSPECTION & QUARANTINE TECH CENT NINGXIA ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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