Development Of An ABO Genotyping Method And Its Preliminary Application Of Prenatal Diagnosis Of Hemolytic Disease Of Newborn

ABO blood group system is the first genetic marker system of blood found in the history of mankind. As early as1990, Yamamoto et al had reported on the complete sequence of the ABO blood group gene, which was built up laid the foundation for molecular detection of ABO blood group genotypes. Most current clinical detection are serological methods, which able to identify whether there is blood group antigens on red blood cells, however, it cannot determine the blood group genotype. In recent years, with the development of molecular biology and experimental techniques, the study of ABO blood group genotypes has widely carried out. To make the ABO blood group genotype can better help for the clinical diagnosis. Herein, two ABO blood group genotyping methods have been established, which will serve better and be used to the early diagnosis of hemolytic disease of the newborn.Firstly, an ABO blood group genotyping method was optimized and established, which was based on the amplification refractory mutation system polymerase chain reaction (ARMS-PCR). By using one-step PCR reaction, this method can be identified seven common ABO blood group genotype subtype (A1, A1Ⅴ, A2,B, O1, O1Ⅴ and O2),28subtypes of genotype combinations, the overall process from genomic DNA purification to obtain the genotype is about3-4hours. In addition, another ABO blood group genotyping method was optimized and established based on loop-mediated isothermal amplification (LAMP). This method through an isothermal reaction, can be identified six kinds of common ABO blood group genotype combinations (AA, AO, BB, BO,OO and AB), the overall process from genomic DNA purification to obtain the genotype is about1-2hours. Meanwhile, according to the genetic law of ABO blood group, the above ABO blood group genotyping methods were used in the early diagnosis of hemolytic disease of the newborn, and evaluated by DNA sequencing, and the results were100%match. Compared with LAMP method, ARMS-PCR method is low cost, easy operation, it is more suitable for general use in molecular testing laboratory or clinical; LAMP method is simple, fast, but the cost of reagent is relatively higher, it is more suitable for large-scale and rapid diagnosis requires laboratory or hospital use.