Primer combinations, methods and kits for constructing multiple hemophilia targeting libraries based on high-throughput sequencing

Abstract

The invention discloses a primer combination, a method and a kit for constructing multiple hemophilia targeting libraries based on high-throughput sequencing. The base sequence of the primer group ofthe present invention is SEQ ID No. 1 to SEQ ID No. 450, Overridden F8, F9, F11, vWF, F2, F7 and SRY are all exons of 7 genes, The splicing region, partial promoter region and intron region, 128 target regions and 225 pairs of primers were used to detect gene mutations in hemophilia A, hemophilia B, hemophilia C, von Willebrand disease, factor II deficiency and factor VII deficiency simultaneously. The invention can be applied to detection and screening of pathogenic sites of congenital hemorrhagic diseases such as hemophilia, screening of family members and scientific research on corresponding pathogenic mutations. The method is simple, accurate and time-consuming, and greatly saves the time cost and labor cost of Sanger sequencing gene by gene segment by segment detection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer combination, a method and a kit for constructing various hemophilia targeting libraries based on high-throughput sequencing. Background technique [0002] Hemophilia is a group of congenital bleeding disorders with hereditary coagulation dysfunction. Its common features are the deficiency of certain coagulation factors, the generation of active thromboplastin, prolonged coagulation time, and a lifelong tendency to bleeding after minor trauma. Severe patients "Spontaneous" bleeding can occur without obvious trauma. On May 11, 2018, five departments including the National Health Commission jointly formulated the "First Batch of Rare Disease List", and hemophilia was included in it. Large-scale epidemiological surveys show that the incidence of hemophilia is 15-20 / 100,000, and it is the most common disease among congenital bleeding disorders. According to literature reports, ...

AI Technical Summary

Problems solved by technology

Therefore, the heterogeneity and individual differences of gene mutations determine that the gene mutation detection of hemophilia and congenital hemorrhagic disease needs to cover all exons and intron splicing regions of the disease-causing gene in order to be accurate and comprehensive. Covering and identifying its pathogenic mutations without omission, simply detecting a few hot mutation sites of a gene will result in a large number of missed detections

At present, there is no genetic testing kit for simultaneous detection and identification of multiple hemophilias in the market.

[0009] At present, IS-PCR is used to detect F8 gene inversion, and Sanger sequencing is used to detect single hemophilia point mutations and indels. This method can only detect certain hotspot mutations of a single gene at a time, and it is difficult to detect Potential disease-causing sites and new mutation sites of the entire gene are all detected, and more than 60% of false negatives may occur. Differential diagnosis needs to be detected one by one gene by one site, and the operation is cumbersome, time-consuming and labor-intensive

With the advancement of technology, whole-exome high-throughput sequencing has realized the simultaneous detection of multiple gene variations, but whole-exome high-throughput sequencing is expensive, and it is very wasteful to detect a small number of single-gene diseases. Due to the huge size of the human genome and the limitations of the whole exome sequencing technology itself, there will be a small number of genes that cannot be covered and randomly missed

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