Composition and kit for detecting vibrio cholerae and detection method
A composition and a technology for testing samples, which are applied in biochemical equipment and methods, measurement/testing of microorganisms, and resistance to vector-borne diseases, etc., can solve the problems of increased testing costs and the inability to completely eliminate the risk of pathogenicity, etc. Achieve the effect of reducing biological hazards, saving manpower and time
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Embodiment 1
[0042] Embodiment 1 adopts the detection of common PCR method
[0043] Extraction of Bacterial Genomic DNA
[0044] The genomic DNA of each bacteria was extracted (MiniBEST kit from TAKARA Company). Utilize ultraviolet spectrophotometer to measure the purity and concentration of each bacterial genome DNA, O1 and O139 Vibrio cholerae genome DNA is diluted to 1×10 with TE buffer5 pg / μl to 1×10 -3 pg / μl gradient standard, the genome DNA of other 19 kinds of other intestinal pathogenic bacteria or common pathogenic bacteria in hospital infection was diluted to 5×10 with TE buffer 4 pg / μl, all kinds of genomic DNA were aliquoted in small quantities and stored at -20°C for future use.
[0045] Construction and preparation of plasmid standards
[0046] The target plasmid was constructed by TA cloning technique. After the molecular weight of the amplified fragment was confirmed by electrophoresis, the PCR products of rfb-O1, rfb-O139 and ctxA specific sequences were connected to t...
Embodiment 2
[0049] Embodiment 2 adopts fluorescence real-time quantitative PCR reaction method to detect
[0050] Using the same bacterial genomic DNA and plasmid standard as in the examples, the difference is that the PCR method used is fluorescent real-time quantitative PCR, and the three probes designed and synthesized above are used.
[0051] Real-time fluorescent triple PCR reaction conditions
[0052] The reaction system is 25 μl, take 2.5 μl of 10×PCR buffer, 2.0 μl of each dNTP of 2.5 mmol / L, 0.5 μl of each 25 μmol / L primer, 0.5 μl of each 5 μmol / L probe, 1 U of Blend Taq plus DNA polymerase, template DNA 10ul, add sterilized water to 25μl of the final system. PCR cycle parameters: pre-denaturation at 94°C for 2min, denaturation at 94°C for 10s, extension at 60°C for 30s, a total of 45 cycles of reaction. After the amplification was completed, the data was analyzed under the same conditions to determine the Ct value of each sample.
[0053] The evaluation of the detection sensi...
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