Noninvasive antepartum fetal beta-thalassemia gene mutation detection library building method, detection method and kit

Abstract

The invention discloses a noninvasive antepartum fetal beta-thalassemia gene mutation detection library building method, a detection method and a kit. In the library building method, a specific connector is connected onto a maternal peripheral blood free DNA (deoxyribonucleic acid) fragment; then, a connector connection product pre-amplification product is divided into two parts; two rounds of specific amplification are performed by respectively and independently using forward primers and reverse primers aiming at target sites; the target sites can be enriched at high specificity; the amplification specificity of the primers can be obviously improved. The forward and reverse primer groups of a plurality of SNP (single nucleotide polymorphism) sites used for calculating the fetal free DNA proportion are respectively used for performing two-round specific amplification; the fetal free DNA proportion can be efficiently and accurately calculated. The library is subjected to sequence testing; the beta-thalassemia gene mutation can be accurately and effectively detected; the result is identical to the result of the amniocentesis detection and typing; the safety, the noninvasion and the high efficiency are obviously superior to those of the amniocentesis detection.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a method for constructing a non-invasive prenatal fetal β-thalassemia gene mutation detection library, a detection method and a kit. Background technique [0002] Thalassemia (referred to as thalassemia) is one of the most common genetic diseases in the world. It is a hemolytic anemia caused by an imbalance in the synthesis rate of α, β-globin peptide chains due to human gene mutation or deletion. The two common types of thalassemia are α-thalassemia and β-thalassemia, α-thalassemia-related genes are HBA2 and HBA1, and β-thalassemia-related genes are HBB. Thalassemia is concentrated in tropical and subtropical regions, mostly in Mediterranean countries, followed by the Middle East, India, Pakistan, Southeast Asia, southern China and North Africa. The United States is an immigrant country, and its incidence is also relatively high. The provinces south of the Yangtze River ...

AI Technical Summary

Problems solved by technology

However, these detection methods based on single-site PCR technology have certain limitations, such as the low content of plasma DNA and the characteristics of high fragmentation, which may cause false negatives in PCR.

This method can only detect the presence of specific paternal mutations that are different from the mother, but cannot further determine the presence or absence of maternal mutations

Whole-genome sequencing or target region capture sequencing of cell-free DNA in pregnant women's plasma based on high-throughput sequencing technology can more accurately detect fetal thalassemia genes, but the cost of whole-genome sequencing and the complexity of analysis methods require parental monomers. Types, etc. limit the clinical application; target region capture sequencing can only realize the detection of fetal α due to its low capture efficiency of the target region SEA Missing the distinction of homozygous type, and there is also the possibility of mistyping

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