Specific primer and kit for detecting gene polymorphism of cardiovascular disease drugs by virtue of liquid-phase chip technique
A liquid phase chip, specific technology, applied in the field of biotechnology and medicine, can solve the problems of unsuitable for clinical promotion, expensive testing equipment, low flexibility, etc., achieve great clinical application value, good sensitivity and stability, avoid sample pollution effect
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Embodiment 1
[0070] Example 1. Primer set for detection of cardiovascular drugs-related genes by liquid chip technology
[0071] Design of primers and probes:
[0072] According to the rs numbers of the SNPs published on NCBI, the nucleotide sequences near the 15 mutation points were searched in Genbank, and Primer 6.0 primer design software was used to design specific primers for amplifying the following common cardiovascular drug-related target gene loci: CYP2C9 C430T polymorphism rs1799853 of gene, A1075C polymorphism rs1057910 of CYP2C9 gene, G1639A polymorphism rs9923231 of VKORC1 gene, C1347T polymorphism rs2108622 of CYP4F2 gene, G681A polymorphism of CYP2C19 gene rs4244285, G636A polymorphism of CYP2 Sex rs4986893, C806T polymorphism rs12248560 of CYP2C19 gene, Q192R polymorphism rs662 of PON1 gene, C2850T polymorphism rs16947 of CYP2D6 gene, C100T polymorphism rs1065852 of CYP2D6 gene, G1165C polymorphism of ADRB1 gene rs1801253, DH2 gene Primers for the G504A polymorphism rs671 ...
Embodiment 2
[0085] Example 2. Kit for simultaneous detection of polymorphisms of multiple cardiovascular drugs-related target genes using liquid chip technology of the present invention
[0086] The kit of the present invention includes:
[0087] 1. The primers in Table 1 and Table 2 designed in Example 1.
[0088] 2. 30 MagPlex-Tag microspheres coupled with addressing probe sequences capable of complementary pairing with tag sequences in ASPE extension primers. The 30 kinds of microsphere numbers and the anti-Tag sequences coupled to the microspheres are shown in Table 3:
[0089] Table 3. 30 MagPlex-Tag microspheres coupled with addressing probe sequences (anti-Tag sequences)
[0090]
[0091]
[0092] The selected 30 kinds of MagPlex-Tag microspheres were purchased from Luminex Company in the United States at a concentration of 2.5 × 10 6 pcs / ml, diluted to 2500 pcs per microsphere when used, wherein the addressing probe sequence is coupled to the microsphere and is complement...
Embodiment 3
[0094] Example 3. Detection of common cardiovascular drug-related target gene polymorphisms using the primer set and kit of the present invention
[0095] In this example, the primer set of Example 1 and the kit of Example 2 were used to detect 15 target gene polymorphisms of common cardiovascular drugs.
[0096] The specific experimental steps are as follows:
[0097] 1. Multiplex PCR amplification reaction: A total of 14 whole blood samples (blood samples from Dongguan Houjie Hospital) were extracted. According to the experimental requirements, another negative control was set up to extract DNA. Use the specific amplification primers in Table 1 of Example 1 to prepare a multiplex PCR reaction system (50 μl):
[0098] Element
Dosage (μl)
gDNA
50ng (2μl)
10x PCR reaction buffer
5
Taq TM HotStar
0.25
dNTPs (2.5mM each)
4
Upstream and downstream primers (10μM)
0.5 each
Deionized water
30.75
[00...
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