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Method and kit for detecting human arthrolithiasis-related gene mutation sites

A technology for detection kits and detection methods, applied in the direction of microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of low sensitivity, cumbersome detection steps, and low efficiency of direct sequencing methods, and achieve high cost performance and low cost , the effect of less cost

Pending Publication Date: 2018-12-11
NINGBO UNIV
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Problems solved by technology

[0004] The current method for detecting ABCG2 and SLC2A9 mutations is DNA direct sequencing. However, the sensitivity of direct sequencing is low, only 20-30%, and the detection steps are cumbersome and inefficient. It takes at least 2-3 days to produce results; When the amount is scarce, direct sequencing can lead to a large number of missed detections and false negatives

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  • Method and kit for detecting human arthrolithiasis-related gene mutation sites
  • Method and kit for detecting human arthrolithiasis-related gene mutation sites
  • Method and kit for detecting human arthrolithiasis-related gene mutation sites

Examples

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Embodiment 1

[0117] Example 1: Detection of human ABCG2 and SLC2A9 genes using nucleic acid mass spectrometry detection method

[0118] 1. Human Genomic DNA Extraction

[0119] (1) Genomic DNA extraction is performed on the patient's blood sample in a biological safety cabinet. Extraction experiments were performed using Blood&CellCulture DNA Mini Kit (Qiagen Cat No: 13323).

[0120] (2) Primer design

[0121] SNP site primers were designed for human ABCG2 and SLC2A9 genes, and specific PCR primers and single base extension primers were designed for this SNP, respectively. The sequences of the designed PCR primers are shown in Table 1.

[0122] Table 1

[0123]

[0124] (3) Specific PCR reaction

[0125] The target fragments containing ABCG2 and SLC2A9 genes were amplified by specific PCR technology, and the amplification reaction was prepared according to the reaction system in Table 2. After the reaction system was prepared, the amplification was performed according to the reactio...

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Abstract

The invention relates to a method and kit for detecting human arthrolithiasis-related gene mutation sites. The detection method comprises the following steps: (1) designing specific primers aiming atABCG2 and SLC2A9 genes; (2) performing specific polymerase chain reaction (PCR) amplification so as to obtain target fragments containing ABCG2 and SLC2A9 genes; (3) performing restrictive enzyme disgestion on PCR product fragments; (4) performing single base extension; (5) performing desalting purification treatment; and (6) then detecting and analyzing sequences of target genes ABCG2 and SLC2A9genes by use of a nucleic acid velocitron. The method for detecting the human arthrolithiasis-related gene mutation sites is high in detection accuracy, high in experiment repeatability, great in fluxand low in cost. The invention also provides a kit for for detecting human arthrolithiasis-related gene mutation sites. The kit comprises a specific primer pair for amplifying ABCG2 and SLC2A9 genes.The kit for for detecting human arthrolithiasis-related gene mutation sites is capable of simplifying experimental procedures and has the advantages of short manual operation time, low difficulty andhigh experiment automation degree.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a detection method and a kit for a human gout-related gene mutation site. Background technique [0002] Gout is a metabolic disease caused by purine metabolism disorder and renal tubule-derived uric acid excretion disorder, which causes damage to joints and certain organs. It is directly related to the disease, especially acute characteristic arthritis and chronic tophi disease, mainly including acute onset arthritis, tophi formation, tophi chronic arthritis, urate nephropathy and uric acid urolithiasis. Joint disability and renal insufficiency develop. Gout is often accompanied by abdominal obesity, hyperlipidemia, hypertension, type 2 diabetes and cardiovascular disease. Due to the improvement of living standards and the change of diet structure, the incidence of gout is increasing year by year. The prevalence of gout in Europe and the United States is 1.4-3.9%, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6872C12Q1/6806
CPCC12Q1/6806C12Q1/6872C12Q1/6883C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2521/301C12Q2533/101C12Q2565/627
Inventor 王文忠胡军田军龙陈苏平卜云璇
Owner NINGBO UNIV
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