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Loop-mediated isothermal amplification (LAMP) primer pair of bacillus cereus and detection method

A Bacillus cereus, ring-mediated isothermal technology, applied in the field of molecular biology testing of bacteria, can solve problems such as lack of pertinence and inability to determine toxin-producing pathogenic strains

Inactive Publication Date: 2010-09-15
WUHAN POLYTECHNIC UNIVERSITY
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  • Description
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  • Application Information

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Problems solved by technology

In the patent application number 200810152850.8, LAMP detection for the 16S-23S rRNA region coding sequence of Bacillus cereus has been involved, because all Bacillus cereus contain this sequence, including some probiotic strains of Bacillus cereus , so it has a broad-spectrum detection of Bacillus cereus, but it lacks pertinence, and it is impossible to determine the pathogenic strains that produce toxins

Method used

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  • Loop-mediated isothermal amplification (LAMP) primer pair of bacillus cereus and detection method
  • Loop-mediated isothermal amplification (LAMP) primer pair of bacillus cereus and detection method
  • Loop-mediated isothermal amplification (LAMP) primer pair of bacillus cereus and detection method

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Embodiment 1

[0235] Material

[0236] 1. Bacterial strains: non-toxic Bacillus cereus strains, non-toxic Bacillus cereus, toxic Bacillus cereus

[0237] 2. Reagents: Bst DNA polymerase, dNTP, TaqI endonuclease, SYBR Green, agar powder, ethidium bromide, DNA Marker, LAMP primer synthesis

[0238] 3. Instruments: centrifuge, water bath, electrophoresis apparatus / tank, gel imaging system or UV light box

[0239] method

[0240] 1. Bacterial DNA Extraction

[0241] (1) Take 100 μL of pure bacterial culture and centrifuge at 12000 r / min for 5 min, discard the supernatant.

[0242] (2) Add 100 μL of sterile water and mix well, lyse in a water bath at 100°C for 10 minutes, and ice-bath for 2 minutes.

[0243] (3) Centrifuge at 12000 r / min at 4°C for 5 minutes, store the supernatant at -20°C or take 4.0 μL of the supernatant for LAMP amplification.

[0244] 2. LAMP primer design

[0245] Access the Genbank network database to query the cesA / B gene sequence of Bacillus cereus pCER270, select ...

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Abstract

The invention relates to a primer pair and a method required by loop-mediated isothermal amplification (LAMP) detection of bacillus cereus causing vomit food poisoning. The primer pair can be used for preparing an LAMP detection kit which can be used for the rapid detection of the bacillus cereus expressing vomitoxin. The specific solution scheme comprises the step of designing a plurality of groups of LAMP inside and outside primer pairs including the whole target sequence by using a cesA / B gene with plasmid pCERP, carried by the vomit bacillus cereus as a target sequence. The invention comprises a vomit bacillus cereus cesA / B gene specificity LAMP primer pair, optimized reaction conditions, LAMP, minimum detection limit measurement and positive result identification. The LAMP method adopted by the vomit bacillus cereus has the advantages of high efficiency and rapid operation, high sensitivity and specificity, convenience, practicability and the like.

Description

technical field [0001] The present invention relates to the molecular biology examination technology of bacterium, specifically for the pathogenic bacillus cereus that expresses vomitoxin, the specific LAMP primer pair needed for loop-mediated isothermal amplification (LAMP) is designed, especially pathogenicity A loop-mediated isothermal amplification assay for Bacillus cereus. Background technique [0002] Bacillus cereus is widely distributed in nature and is common in many raw and cooked foods. When the number of Bacillus cereus contained in the food exceeds 10 6 Bacillus cereus contamination in milk powder reaches 100 per gram, and the incidence rate of food poisoning is higher than 60%. Food poisoning caused by Bacillus cereus can be divided into diarrhea syndrome and vomiting syndrome clinically. The diarrhea-type syndrome is caused by the diarrhea toxin produced by the bacteria, which can be inactivated in 5 minutes at 56°C, and is sensitive to trypsin, so it can ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/085
Inventor 刘志国李琦房国梁付云洁
Owner WUHAN POLYTECHNIC UNIVERSITY
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