A kind of purposes of celb gene
A use and gene technology, applied in the field of CelB gene, can solve the problems of immunological methods such as easy contamination, low accuracy, and serious cross-reaction, and achieve broad application prospects and market value, simple operation, and good specificity Effect
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Embodiment 1
[0058] Example 1 Specific target gene screening and primer design
[0059] 1. Local BLAST analysis
[0060] Download the non-redundant nucleic acid database (about 60G) from NCBI to construct the target database;
[0061] The whole genome sequence of Klebsiella pneumoniae downloaded from NCBI (http: / / www.ncbi.nlm.nih.gov / genome / ) as retrieval data;
[0062] Perform a local BLAST search of the whole genome and the target database to obtain the comparison results of each sequence fragment of the strain and the database;
[0063] Screen the search results to obtain potential specific sequences, and perform subsequent screening.
[0064] 2. Specific target gene screening
[0065] Using the online BLAST function, use 2 strategies to confirm its strain specificity and strain identification availability;
[0066] The first strategy is to exclude Klebsiella pneumoniae during BLAST, and the results show that those without any similar sequences or the potential specific genes with s...
Embodiment 2
[0082] The collection and pretreatment of embodiment 2 environmental samples
[0083] 1. Collection of environmental samples
[0084] Wet a sterile cotton swab with sterile physiological saline, wipe the surface of the sample to be tested (about 3cm×5cm), immerse it in a sterile centrifuge tube containing 1mL of enrichment medium, stir gently, discard the cotton swab, and put the sterile Take the centrifuge tube back to the laboratory for experimentation. If it is a water sample, draw the water directly with a sterile tip or straw, and drop it into a sterile centrifuge tube containing 1mL of enrichment medium for enrichment.
[0085] 2. Pre-enrichment culture of environmental samples
[0086] In order to improve the detection rate, pre-enrichment culture was carried out in a shaking incubator at 120 rpm at 37°C for 4 hours, and the rotation speed was 120 rpm.
[0087] 3. Bacterial culture
[0088] Take 100 μL of the pre-enriched culture solution and add it to a 5 mL glass t...
Embodiment 3
[0089] Example 3 extracts the sample DNA to be tested
[0090] (1) Use the bacterial genomic DNA mini-extraction kit of Beijing Zhuangmeng International Biogene Technology Co., Ltd. for extraction and recovery, the steps are as follows:
[0091]1) Take 2 mL of overnight cultured bacterial solution, put it in a centrifuge, centrifuge at 12000 rpm for 1.5 min, discard the supernatant, add 500 μL of cell suspension to the centrifuge tube in the previous step, blow and mix the solution, put it into the pre-heated tube In a water bath at 37°C for 60 minutes, turn it upside down 3-4 times during the period, centrifuge at 12,000 rpm for 2 minutes, discard the supernatant, slowly add 225 μL of buffer A directly above the tube, and mix by pipetting;
[0092] 2) Add 10 μL of RNase A solution to the above tube, shake for 15 seconds, place at room temperature for 5 minutes, add 10 μL of proteinase K, mix by pipetting, then add 25 μL of lysis buffer S to the centrifuge tube, wait for mixin...
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