Quadruple fluorescence quantitive PCR typing detection method for common human papillomavirus infection
A human papillomavirus and fluorescence quantitative technology, which is applied in the direction of fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of simultaneous detection of multiple subtypes, expensive reagents, complex technology, etc. , to achieve the effects of monitoring clinical curative effect, improving detection sensitivity, and simple and fast operation
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[0083] 1. Construction of type four HPV target gene plasmid cloning vector
[0084] Using the T-A vector cloning scheme, the PCR products of the four target genes were electrophoresed to confirm the molecular weight of the amplified fragments, and then the amplified fragments were cloned into the PMD-19T plasmid and ligated overnight at 16°C. The ligation product was transformed into competent Escherichia coli DH5α, positive colonies were screened on LB medium (containing Amp100g / ml), and the plasmid was recovered, extracted, purified, and recombined with the plasmid mini-extraction kit. Sequencing verification by the biological company, the target plasmid whose sequence is completely correct after Blast is quantified with a UV spectrophotometer, and diluted to 10 with 1 × TE (pH8.0) buffer 10 copies / μl as a storage solution and stored at -20°C for future use.
[0085]2. DNA template extraction: Dissolve the secretion swab specimen with physiological saline, put the tissue bl...
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