Nucleotide sequence specific for O-antigen of E.coli 076 type
A technology of nucleotide sequence and Escherichia coli, which is applied in the determination/testing of microorganisms, sugar derivatives, biochemical equipment and methods, etc., and can solve problems such as false positives
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Embodiment 1
[0044] Embodiment 1: the extraction of genome:
[0045] Escherichia coli O76 was cultured overnight at 37°C in 5 mL of LB medium, and the cells were collected by centrifugation. The cells were resuspended with 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA, incubated at 37°C for 20 minutes, and then 10ul 10mg / ml lysozyme was added to continue the incubation for 20 minutes. Then add 3ul 20mg / ml proteinase K, 15ul 10% SDS, incubate at 50°C for 2 hours, then add 3ul 10mg / 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 used to precipitate DNA with 2 times the volume of ethanol, and the DNA was rolled out with glass wool and washed with 70% ethanol, and finally the DNA was resuspended in 30ul T...
Embodiment 2
[0046] Example 2: Amplification of the O-antigen gene cluster in Escherichia coli O76 by PCR:
[0047]The O-antigen gene cluster was amplified by Long PCR using the genome of Escherichia coli O76 as a template. First, design the upstream primer (#1523-ATT GTG GCT GCA GGG ATC AAA GAA AT) based on the JumpStart sequence often found in the promoter region of the O-antigen gene cluster, and then design the downstream primer (# 1524-TAG TCG CGT GNG CCT GGA TTA AGT TCGC); use Boehringer Mannheim’s Expand Long Template 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 denaturation at 94°C for 10 seconds , 60 ° C annealing for 15 seconds, 68 ° C extension for 15 minutes, so that 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 specificity of the PCR products. Combine 5 tubes of long PCR products...
Embodiment 3
[0048] Embodiment 3: construct O-antigen gene cluster library:
[0049] 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 was 300ng PCR purified product, 0.9ul 0.1M MnCl2, 1ul 1:2000 diluted 1mg / ml DNaseI, and the reaction was carried out at room temperature. Digest for 10 minutes to concentrate the DNA fragment size between 1.5kb-3kb, then add 2ul 0.1M EDTA to terminate 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 18ul of water. Then add 2.5ul dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP, 10mMdATP), 1.25ul 100mM DTT and 5 units of T4 D...
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