Fluorescent PCR kit for qualitative detection of HLA-B*1502 gene subtypes
A HLA-B, qualitative detection technology, applied in fluorescence/phosphorescence, microbial determination/inspection, biochemical equipment and methods, etc., to achieve the effects of improved sensitivity and specificity, intuitive results, and high sensitivity
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Embodiment 1
[0032] Embodiment 1: the preparation of kit
[0033] 1. Design and synthesis of primers and probes
[0034]Using Primer Express 2.0, for exon 2 and exon 3 of HLA-B*1502 and the intron region in the middle, studies have shown that the current HLA-B genotyping is mainly concentrated in this region (Hughes AL, Nei M .Pattern of nucleotide substitution at MHC class I loci reveals overdominant selection. Nature, 1988, 335(6186): 167-170; Hughes AL, Yeager M. Natural selection at major histo-compatibility complex loci of vertebrats. Annu Rev Genet, 1998 , 32: 415-435; Gao X, Nelson GW, Karacki P, Martin MP, Phair J, Kaslow R, Goedert JJ, Buchbinder S, Hoots K, Vlahov D, O′Brien SJ, Carrington M. Effect of a single amino acid change in MHC class I molecules on the rate of progression-sion to AIDS.N Engl J Med, 2001, 344(22): 1668-1675; Xu Junping, Deng Zhihui, Zou Hongyan, etc., Chinese Han individuals HLA-A, - Determination of the full-length sequence of the B gene and the polymor...
Embodiment 2
[0068] Embodiment 2: the use of kit
[0069] 1. Sample detection
[0070] The positive control and negative control were respectively taken as DNA templates, and added to UNG enzyme, Taq polymerase, and fluorescent quantitative reaction solution containing specific PCR primers and specific fluorescent probes to form a PCR reaction system. In the positive control substance, the HLA-B*1502 plasmid standard substance (5.0 × 10 6 copy / microliter), as a standard for qualitative determination of samples.
[0071] The main components of the system are as follows:
[0072]
[0073] 2. Reaction procedure
[0074] Set the fluorescence detection channel for collecting FAM and VIC fluorescence signals, put the reaction tube into the fluorescence PCR instrument (ABI7500) to start amplification, the reaction procedure is as follows:
[0075] Table 3. PCR reaction program
[0076]
[0077] 3. Result judgment
[0078] The Ct value (cycle number) in the baseline range is 6-15 or au...
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