Amplification primer and detection method for pkd2 gene mutation detection

Abstract

The invention belongs to a technology of medical in vitro diagnosis, and in particular relates to an amplification primer for detecting mutation of autosomal dominant polycystic kidney disease-causing gene PKD2 as well as a detection method. The amplification primer, in accordance with the sequence information of the PKD2 gene, designs four pairs of PCR primers; the PKD2 is subjected to targeted amplification by virtue of long-chain PCR; and an amplification region includes all exon sequences and most intron sequences, as well as partial 5' untranslated region sequences and 3' untranslated region sequences. According to the method, by combining long-chain PCR targeted amplification to the newest developed next generation of sequencing technology, a targeted sequencing process is simplified, the scope and flux of detecting the PKD2 gene mutation are improved, a detection cycle is shortened and cost is reduced.

Description

technical field [0001] The invention belongs to medical in vitro diagnostic technology, and specifically relates to an amplification primer and a detection method for detecting the mutation of autosomal dominant polycystic kidney disease pathogenic gene PKD2. Background technique [0002] Adult polycystic kidney disease (PKD) is the most common hereditary kidney disease, with an incidence of about 1 / 400 to 1 / 1000 in the population, mostly autosomal dominant inheritance, and a small part autosomal recessive inheritance. Autosomal dominant polycystic kidney disease (ADPKD) mainly manifests as multiple progressive vesicles in both kidneys, causing structural and functional damage to the kidneys, and is the most common end-stage renal disease (ESRD) Genetic etiology accounts for about 10% of all ESRD, and can also involve multiple organs throughout the body, such as liver cysts, hypertension, and intracranial aneurysms. [0003] Genetic studies have shown that ADPKD is mainly c...

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Problems solved by technology

The methods that have been reported to be applied to the detection of PKD2 gene mutations include PCR-single-strand conformation polymorphism (referred to as PCR-SSCP) method, denaturing high-performance liquid chromatography (referred to as DHPLC) method and Sanger sequencing method, etc., among which PCR-SSCP and DHPLC Both belong to mutation screening methods, which can improve the efficiency of mutation detection, but there are many influencing factors in the two methods, the results are not stable enough, and the accuracy is low

Sanger sequencing is the main method for PKD2 gene mutation detection. 17 pairs of primers are often used to amplify the 15 exons of PKD2. Exon 1 needs 3 pairs of primers for segmental amplification due to its high GC content. Introns 2 to 15 each require a pair of primers. Although this method is the gold standard for PKD2 gene mutation detection, Sanger sequencing has a large workload, low efficiency, and low throughput, and it cannot detect mutations within introns

Due to the large number of exons in the PKD2 gene, the identified PKD2 gene mutations are scattered in various regions of the gene, lacking obvious mutation hotspots, and the above methods have the disadvantages of low mutation detection rate and long time consumption.

In addition, US7083915B2 also discloses the PKD2 gene and its use, and CN1434130A discloses a detection kit for autosomal dominant polycystic kidney disease. low problem

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