Application Of DdPCR In Noninvasive Prenatal Detection Of Fetal Hemolysis And Periodic Paralysis

DdPCR is a quantitative nucleic acid detection method based on Poisson distribution calculation.It has the characteristics of high sensitivity and strong specificity.At the same time,ddPCR has obvious advantages in the detection of rare mutation points.With the development of maternal plasma fetal free DNA extraction in recent years,the noninvasive prenatal detection technology based on it is becoming more and more mature.At present,a variety of non-invasive prenatal detection methods have been established,such as whole genome sequencing,fluorescence quantitative PCR,etc,but they still cannot meet the clinical requirements of"early diagnosis and early detection"in prenatal diagnosis.This study established a digital PCR method for diagnosing hemolysis of rare blood group and single gene disease in early pregnancy.At the same time,we establish a method to detect the frequency of population blood group gene by using the characteristics of precise quantitative ddPCR.The establishment of the above methods is not only beneficial to the diagnosis of hemolytic diseases,but also provides guidance for the management of blood banks and guarantees for the supply of blood for subsequent treatment.Methods:1.Cell free fetal DNA isolated from 26 healthy single pregnant women at different gestational stages was tested with QX200 Droplet Digital PCR.Results compared with fetal genotypes.2.SLC44A2 and Do genotypes of all DNA samples were detected by TaqMan real-time PCR with a specific probe,and the results were consistent with Sanger sequencing and detection results.3.DNA samples purified from 20 blood pools containing a total of 1000 donors in northwest China were subjected to ddPCR to detect three blood groups gene frequency.4.The alleles of SCN4A gene c.2111C>T were categorized by ddPCR.5.Different genotypes and genomic DNA proportions were mixed to simulate the circulating free cell DNA of fetuses at different gestational weeks to simulate the actual situation of pregnancy.ddPCR was used to detect simulated fetal DNA under simulated conditions.6.Fetal genotypes were predicted by detecting free fetal DNA in plasma of pregnant women from mutant families.The genotypes of fetal cells in amniotic fluid were tested by Sanger sequencing.Results:1.The blood groups of MNS,Kidd and CTL2 of 26 pregnant fetuses were detected successfully by multiplex ddPCR.The ddPCR results were consistent with the Sanger sequencing results of 26 fetal blood samples after birth.2.SLC44A2 and Do genotypes of all DNA samples were detected by TaqMan real-time PCR with a specific probe,and the results were consistent with Sanger sequencing and detection results.ddPCR can accurately detect the proportion of alleles SLC44A2.This allows the frequency of SLC44A2 in the population to be accurately inferred.3.The frequencies of four rare blood group genes detected by digital PCR were 9.30%for S,90.70%for s,48.43%for Jk~a,51.57%for Jk~b,66.57%for HNA-3A,33.43%for HNA-3B,11.28%for DO*A and 88.72%for DO*B,respectively.These results are similar to the gene frequencies found in the normal population,suggesting that it is feasible to use digital PCR to calculate the frequency of blood-type related genes in the population.4.Through digital PCR detection of simulated fetal free DNA,we successfully detected only 3%fetal free DNA in maternal plasma.At the same time,we successfully predicted the mutation of c.2111C>T site in SLC44A2 at 17 weeks of gestation in the family.The results were consistent with the sequencing results of amniotic fluid.Conclusions:1.Multiple ddPCR technology can be used to predict rare blood types of 5-week-old fetuses,which will promote the development of non-invasive prenatal testing of different blood types.2.Sanger sequencing results confirmed that this new method for detecting SLC44A2 and DO alleles has high sensitivity and specificity.3.The method was established to detect the rare HNA-3B and DO~a antigens in the blood pool by using the designed probe and predicted the corresponding gene frequencies by digital PCR.According to the results of ddPCR,which can accurately quantify gene frequencies,the gene frequencies of four rare blood groups in 1,000 people can be inferred.4.DdPCR can be used to detect single-gene hereditary diseases in early pregnancy.