Quick and efficient detection method of mutation sites of genes associated with corneal dystrophy

Abstract

The invention discloses a quick and efficient detection method of mutation sites of genes associated with corneal dystrophy. The method comprises the steps of firstly, preparation of a mutational DNA template and design of primers, wherein TGFBI gene fragments containing four mutation sites are artificially synthesized respectively and used for detecting the detection efficiencies of the mutation sites; meanwhile, specific amplification primers aiming at normal sites and the mutation sites are designed respectively, corresponding reverse primers are designed, three primers are composited to form a primer premix liquid, and in addition, the 5' ends of two forward primers are connected with different primer detection sequences respectively and used for fluorescence detection; secondly, establishment of a PCR detection system; thirdly, result interpretation. Compared with the prior art, the technology has the advantages of being accurate and reliable in detection result, higher in flux, flexible in experimental design, convenient and efficient in operation, fast in detection speed, easy for popularization and low in cost.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and specifically, detects four mutation sites related to corneal dystrophy through a method of combining quantitative PCR and allele-specific amplification. Background technique [0002] Corneal dystrophies (CDS) is a rare disease with hereditary and bilateral primary pathological changes. Clinically, this type of disease is usually due to the cloudiness of insoluble material deposition in the corneal stroma, which further leads to visual impairment. Clinical medical research has found that various pathological features of corneal dystrophy are related to the secreted protein gene induced by transforming growth factor β (transforming growth factor β-induced, TGFBI). At present, more than 50 different mutation sites in the TGFBI gene have been found to be associated with various types of corneal dystrophy, including granular cornea dystrophy type I (granular cornea dystrophy type I, GCD1),...

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Problems solved by technology

Each method has its own advantages and disadvantages. For example, the PCR-direct sequencing method has low throughput, slow speed, and low sensitivity (especially when detecting somatic mutations in tumor tissues, when the proportion of target gene mutations in the tissue is less than 20 %, false negative results may occur); although mass spectrometry has a high throughput, the experiment is not flexible enough, requires special reagents and instruments, and is not easy to popularize; PCR-gene chip method, PCR-electrophoresis analysis, PCR- Both restriction fragment length polymorphism and in situ hybridization (ISH) methods require further processing of PCR products, which are not efficient, relatively cumbersome to operate, and prone to false positive or false negative results; The type is accurate, the operation is simple and fast, but the throughput of this method is not high, the cost of the probe is high, and the detection cost of a single locus is related to the sample size. The smaller the sample size, the higher the cost; allele-specific PCR method can be used for Detecting various types of SNPs has the advantage of high sensitivity and is especially suitable for detecting somatic mutations in tumor tissues. The disadvantage is that the false positive rate is high

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