Primer combination, method and kit for constructing targeted library of multiple inherited metabolic liver diseases based on high-throughput sequencing

Abstract

The invention discloses a primer combination, method and kit for constructing a targeted library of multiple inherited metabolic liver diseases based on high-throughput sequencing. The base sequence of the primer combination is shown as SEQ ID No. 1 to SEQ ID No. 2245, and the primer combination covers total 703 exons and cutting regions of 43 genes such as SERPINA1, G6PC, SLC37A4, AGL and the like, and is capable of detecting 41 sub-type gene variation loci of totally 20 inherited metabolic liver diseases comprising alpha-1-antitrypsin deficiency, glycogen storage disease, citrullinemia, aminosuccinuria, hemochromatosis, porphyrinopathy, cystic fibrosis, bile acid synthesis defect, Gaucher disease, hereditary fructose intolerance, cholesterol ester storage disease, Gilbert syndrome, Dubinsyndrome, Rotor syndrome, progressive familial intrahepatic choleatasia and the like. The primer combination disclosed by the invention is simple in operation, high in accuracy and low in time consumption, and the time cost and manual cost used for detecting genes and fragments one by one during Sanger sequencing can be greatly saved.

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 multiple genetic metabolic liver disease targeting libraries based on high-throughput sequencing. Background technique [0002] Inherited metabolic liver disease (IMLD) is a type of liver metabolic disorder caused by inherited enzyme defects. There are many kinds of inherited metabolic liver diseases. Currently, more than 600 kinds of genetic metabolic liver diseases have been identified, mainly including carbohydrate metabolism diseases, amino acid metabolism diseases, fatty acid metabolism diseases, organic acid metabolism diseases, mitochondrial liver diseases, lysosomal diseases, excessive Oxisome diseases, metal metabolism disorders and α1-antitrypsin deficiency and other 9 categories. As far as a single disease is concerned, the incidence rate is not high, but the overall incidence rate cannot be ignored. Hereditary me...

AI Technical Summary

Problems solved by technology

Therefore, the heterogeneity and individual differences of gene mutations determine that the detection of genetic metabolic liver disease gene mutations needs to cover all exons and intron splicing regions of the disease-causing gene in order to be able to identify accurately, fully and without omission For its pathogenic mutations, simply detecting several hotspot mutation sites of a certain gene will result in a large number of missed detections

At present, there is no genetic testing kit for simultaneous detection of multiple genetic metabolic liver diseases on the market.

[0005] At present, Sanger sequencing is mostly used at home and abroad to detect a single genetic metabolic liver disease. This method can only detect a few hotspot mutations of a gene at a time, and it is difficult to detect potential pathogenic sites and new mutation sites of the entire gene. If all are detected, there may be more than 50% false negatives, and only one gene can be detected at a time. When the direction of suspected diagnosis is vague, it needs to be detected one by one gene by one site, and the operation is cumbersome, time-consuming and labor-intensive

At present, only the method disclosed in the CN 106957901 A document can be retrieved in China to simultaneously detect multiple genetic metabolic liver diseases. This method uses PCR-sequencing method to sequence PCR reactions one by one, and can only detect hyperbilirubinemia A small number of hotspot mutations in , Wilson's disease, and / or hemochromatosis

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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