Nucleotide specific for bacillus coli O125 type O-antigen
A technology of Escherichia coli and nucleotides, which can be used in the fields of antibody medical components, microbe-based methods, and microbiological determination/testing, which can solve problems such as false positives
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Embodiment 1
[0052] Example 1: Genome extraction: Escherichia coli O125 was cultured overnight at 37° C. in 5 mL of LB medium, and the cells were collected by centrifugation. The cells were resuspended with 500 μl of 50 mM Tris-HCl (pH 8.0) and 10 μl of 0.4 MEDTA, incubated at 37° C. for 20 minutes, and then 10 μl of 10 mg / mL lysozyme was added for further incubation for 20 minutes. Then add 3 μl 20 mg / mL proteinase K, 15 μl 10% SDS, incubate at 50° C. for 2 hours, then add 3 μl 10 mg / mL RNase, and incubate at 65° C. for 30 minutes. Add an equal volume of phenol to extract the mixture, take the supernatant and extract twice with an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) mixed solution, take the supernatant and extract with an equal volume of ether to remove For residual phenol, the supernatant was precipitated with 2 times the volume of ethanol, the DNA was rolled out with glass wool and washed with 70% ethanol, and finally the DNA was resuspended in 30 μl TE. Genom...
Embodiment 2
[0053] Example 2: Amplification of the O-antigen gene cluster in Escherichia coli O125 by PCR:
[0054] Using the genome of Escherichia coli O125 as a template, its O-antigen gene cluster was amplified by Long PCR. First, design upstream primers (#w1-1098-ATT GGT AGC TGT AAG CCA AGG GCG GTA GCG T) based on the JUMPStart sequence often found in the promoter region of the O-antigen gene cluster, and then design according to the gnd gene downstream of the O-antigen gene cluster Downstream primer (#1524-TAG TCG CGT GNCCCT GGA TTA AGT TCG C); use Boehringer Mannheim’s Expand LongTemplate PCR method to amplify the O-antigen gene cluster, and the PCR reaction program is as follows: pre-denaturation at 94°C for 2 minutes; then 94°C Denaturation for 10 seconds, annealing at 55°C for 15 seconds, and extension at 68°C for 15 minutes, for 30 cycles. Finally, continue extending at 68° C. for 7 minutes to obtain PCR products, and use 0.8% agarose gel electrophoresis to detect the size and ...
Embodiment 3
[0055] Embodiment 3: construct O-antigen gene cluster library:
[0056] The first is the acquisition of the ligation product: use the modified Novagen DNaseI shot gun method to construct the O-antigen gene cluster library. The reaction system is 300ng PCR purified product, 0.9μl 0.1M MnCl 2 , 1 μl of 1 mg / mL DNaseI diluted 1:2000, and the reaction was carried out at room temperature. Digest for 10 minutes to concentrate the size of DNA fragments between 1.5kb-3kb, then add 2μl 0.1M EDTA to stop the reaction. Combine 4 tubes of the same reaction system, extract once with an equal volume of phenol, once with an equal volume of a mixed solution of phenol:chloroform:isoamyl alcohol (25:24:1), and once again with an equal volume of diethyl ether Finally, the DNA was precipitated with 2.5 times the volume of absolute ethanol, washed with 70% ethanol, and finally resuspended in 18 μl of water. Then add 2.5 μl dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP, 10mMdATP), 1.25μl 100mM DTT and 5 units...
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