Primer pair, probe and kit used for quantitative detection total DNAs of HIV-1 and capable of covering multiple subtypes of HIV-1
An HIV-1, detection kit technology, applied in recombinant DNA technology, microbial assay/test, DNA/RNA fragment, etc., can solve problems such as high cost, lack of clinical data on genotype and subtype coverage, and cumbersome operations. High sensitivity and specificity, overcoming tedious and cost-increasing effects
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[0042] Embodiment 1 Construction of the detection kit of the present invention
[0043] (1) Construction of HIV DNA Quantitative Standards
[0044] The HIV gene and part of the CD3 internal reference gene were respectively connected to the cloning vector by T-A cloning, and the two genes were connected in series to the same plasmid vector through enzyme digestion and ligation reactions to construct a standard containing both the HIV target gene and the internal reference gene.
[0045] The specific implementation is as follows: the amplified fragment of HIV LTR gene 554bp (nt356~nt890, HXB2) and the amplified fragment of human CD3 gene 812bp (nt460~nt1251, NC_000011.10) were simultaneously connected to the same pMD20- On the T vector vector (Takara, Code No.6028), positive plasmids containing two target gene fragments were identified by PCR, enzyme digestion and ligation. Extract the plasmid and measure its concentration, and calculate the plasmid copy number according to the...
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