Establishment of genotyping method for fetal ABO group from pregnant maternal peripheral blood

This study was aimed to establish a genotyping method to detect ABO group gene of fetus from peripheral blood of pregnant women for prenatal diagnosis of hemolytic disease of newborn(HDN) resulting from ABO blood group incompatibility.4 pairs of primers were designed according to ABO blood group gene DNA and mRNA sequences.20 plasma DNA samples from healthy donors were extracted and amplified to explore the best conditions for plasma DNA extraction and PCR amplification.The O group plasma DNA was mixed with A group or B group plasmas by the ratios of 1:1,2:1,4:1,8:1,10:1,20:1,40:1,100:1 to simulate the status of mixed ABO gene from pregnant maternal blood and to establish the mixed blood group ABO genotyping technology.The pregnant maternal blood samples with more than 30 weeks of gestation were selected for detecting the fetal ABO blood group genotype.The blood samples should be taken as possible as after birth for identification of ABO blood group and evaluation of sensitivity and accuracy of fetal ABO blood group genotyping technology through peripheral blood of pregnant women.The results indicated that the minimal amount of template DNA from single blood plasma for accuracy identification was at least about 0.625 ng,the DNA amount extracted from 500 μl of plasma could meet the requirement for PCR amplification.When the proportion of O group plasma DNA in mixed plasma DNA was ≤10,the non-O group gene could be accurately detected.Among 14 peripheral blood samples from O-group pregnant women,the non-O group gene was amplified in 9 samples;the non-O group gene was not amplified in 5 samples.The identification of peripheral blood ABO group for 5 newborns using serologic method showed that the A group 3 cases,B group 2 cases,O group 1 case,which consisted with genotyping results with consistant rate 100%.It is concluded that in middle and late pregnancy the fetal ABO group gene can be detected accurately by means of established fetal ABO group gene extraction and typing technology,that provides some guidances for the prenatal diagnosis and prevention of HDN.