Standard strain of Bacillus cereus containing specific molecular target and its detection and application
A Bacillus cereus, molecular target technology, applied in the field of molecular target genes and PCR amplification primers, can solve the problems of few strains, can not reflect the transmission characteristics well, lack and other problems, and achieve the effect of accurate identification
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Embodiment 1
[0036] The isolation and cultivation of embodiment 1 bacillus cereus standard bacterial strain
[0037] The collected food samples were thoroughly shredded under aseptic conditions, 25g of the sample was weighed, put into a sterile homogenizing cup filled with 225mL of PBS buffer solution or normal saline, and the rotary blade homogenizer was used at 8000 r / min~10000r / min homogenize for 1min~2min, oscillate and mix well, and use it as a 1:10 sample homogeneous solution. Draw 1mL of 1:10 sample homogeneous solution, add it into the dilution tube filled with 9mL PBS or normal saline, and mix well to make 1:100 sample homogeneous solution. Select the sample homogeneous solution (including the original solution) of the above three dilutions, transfer to three MYP agar plates with inoculum amounts of 0.3mL, 0.3mL, and 0.4mL, and then spread the entire plate with a sterile L stick, and let it stand for 10min. If the sample solution is not easy to absorb, place the plate in an in...
Embodiment 2
[0042] Embodiment 2 Bacillus cereus standard strain characteristic sequence analysis
[0043] Carry out specific sequence analysis to the 5 strains of bacillus cereus that embodiment 1 obtains, concrete implementation steps are as follows:
[0044] (1) Genome extraction
[0045] Collect bacteria by centrifugation, add lysozyme and glass beads to remove cell wall at the same time, add protease and lysate to lyse the bacteria, add ethanol to mix and put DNA on the column, wash to remove impurities, add eluent to elute DNA. After the DNA is eluted, store it in a -40°C refrigerator for later use.
[0046] (2) Whole genome sequencing
[0047] The DNA samples of the extracted standard strains were sequenced using the Thermo Fisher Scientific Ion S5 genome sequencing platform, and the sequence files obtained from the sequencing were assembled using the Spades software, and the genome sequences obtained after assembly were annotated using the Prokka software.
[0048] (3) Pan-gen...
Embodiment 3
[0069] Physiological and biochemical characteristics analysis of embodiment 3 bacillus cereus standard strain
[0070] 1. Biochemical identification results of standard strains
[0071] Biochemical identification was carried out according to GB 4789.14-2014 "National Food Safety Standard Food Microbiological Examination of Bacillus cereus", and a single purely cultured colony was picked for catalase test, kinetic test, nitrate reduction test, and lysozyme resistance Test, V-P test, glucose utilization (anaerobic) test, root growth test, hemolysis test, protein toxin crystallization test, yolk reaction test, casein decomposition test. Using Bacillus cereus standard strain (ATCC14579), Bacillus mycoides standard strain (ATCC10206) and Bacillus thuringiensis standard strain (ATCC10792) as controls to carry out simultaneous experiments, the biochemical identification results are as follows (Table 7)
[0072] Table 7 Record table of biochemical identification results of standard...
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