Kit for detecting multi-site variation of Gilbert syndrome UGT1A1 gene
A detection kit and multi-site mutation technology, which is applied in the field of medical inspection and detection, achieves the effects of fast speed, simple operation, and compensating for poor resolution and repeatability
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Embodiment 1
[0028] Example 1 Detects the design of primers and unlabeled probes for the four mutation sites of UGT1A1 gene p.G71R, p.P229Q, p.Y486D and A(TA)7TAA
[0029] The sequence of the UGT1A1 gene was obtained from GenBank, and specific amplification primers were designed using the NCBI primer online design software Primer-BLAST. The product amplified by the primers must contain each mutation site to be detected, and the mutation site should be slightly near the 5' end of the amplification product. The probe is a nucleic acid sequence of about 30 nucleotides covering the base of the mutation site to be detected. The GC content of the nucleic acid sequence should be lower than 45% as much as possible, and the 3' end of the sequence should be amino-modified to avoid extension . According to this principle, through screening and testing, four sets of primer pairs and non-labeled probes that can effectively detect the four mutation sites of p. The sequences are shown in Table 1. All ...
Embodiment 2
[0030] Example 2 Detection of UGT1A1 gene p.G71R, p.P229Q, p.Y486D and A(TA)7TAA four mutation sites of the composition of the unlabeled probe HRM detection kit
[0031] The detection kit includes primer pairs and probes designed in Example 1 for unlabeled probe HRM detection for the four mutation sites p.G71R, p.P229Q, p.Y486D and A(TA)7TAA of UGT1A1 respectively. Specifically, the primers and probes are shown in Table 1. The wild-type gene sequences of the four mutation sites are respectively SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16 shown in the sequence table; the four mutation sites The mutant gene sequences are respectively SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20 shown in the sequence listing. The above primers and probes are subpackaged.
[0032] The detection kit also includes the reagents and enzymes needed to complete the HRM reaction, for example, using commercially purchased reagents 2×HRM master mix, 25mM MgCl 2 and d...
Embodiment 3
[0033] Example 3 Repeatability experiment of unlabeled probe HRM detection of UGT1A1 gene p.G71R, p.P229Q and p.Y486D mutation sites
[0034] The specific process includes the following steps:
[0035] 1. Experimental samples
[0036] clinical specimen
[0037] 2. Repeatability test
[0038] 1. Intra-group repeatability
[0039] According to the aforementioned experimental system and experimental procedures, the detection was repeated three times in one experiment, and the Tm values and ΔTm values of different genotypes are shown in the table below.
[0040] 2. Repeatability between groups
[0041] According to the aforementioned experimental system and experimental procedures, five consecutive tests were carried out, and the Tm values and ΔTm values of different genotypes are shown in Table 4.
[0042] Table 4 Repeatability of unlabeled probe HRM detection
[0043]
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