Establishment And Optimization Of Prenatal Screening Method For Down's Syndrome Based On Padlock Probe-Rolling Circle Amplification (PLP-RCA)

Background and Objective : Down Syndrome(DS)is a common incurable chromosomal disorder that can only be prevented by birth prenatal intervention.The traditional prenatal screening method has a high false positive rate.Rapid prenatal diagnosis method,its traumatic sampling method has a certain risk of injury to pregnant women and fetuses.Non-invasive DNA prenatal screening method with the help of mature sequencing companies,costly and low popularity in remote areas,therefore,safe and effective prenatal screening for DS is essential.Maternal fetal free nucleic acid is the best sample for non-invasive prenatal diagnosis,however,due to its low content and interference from maternal background,it is necessary to detect it with high sensitivity and specificity to achieve prenatal screening for DS.This study is based on the ability of single-nucleotide polymorphism(SNP)genotyping and quantification based on padlock probe-rolling circle amplification(PLP-RCA).It is proposed to establish a high sensitivity and specificity of the PLP-RCA.Used for prenatal screening of DS to provide a new approach to prenatal screening for non-invasive DS.Methods:(1)According to the quantitative method of fetal specific gene heterozygous "RNA-SNP" allele ratio,select the SNP of fetal specific gene with high heterozygosity rate.The conditions of PLP-RCA were optimized,and the quantitative relationship between target molecule and endpoint fluorescence was detected to realize the SNP allelic genotyping and ratio quantification.(2)The improved linear-after-the-exponential PCR(imLATEPCR)method is used to amplify the target molecule to generate single-stranded DNA,and the signal-amplified target molecule is used to obtain amplification products of PLP-RCA.Combined with imLATE-PCR,the sensitivity of the PLP-RCA was improved though signal amplification.(3)The synthetic target molecules were used to prepare simulated samples of normal and DS patients,and the simulated samples were screened by fluorescence quantitative PLPRCA.The synthetic target molecules were used to prepare plasma simulated samples of normal and DS patients.The simulated environment of the clinical sample was simulated,and the plasma simulated sample was screened by fluorescence quantitative PLP-RCA.Results:(1)The site rs1044598,rs7717 and rs59066201 on fetal-specific genes of chromosome 21 with high heterozygous rates,the theoretical coverage of the three SNPs in the population reached 84.7%.The padlock probe of three SNPs was designed,Optimizing the system of the PLP-RCA achieved the genotyping of SNPs on fetal specific gene.The quantitative PLP-RCA analysis showed that the linear relationship between the target molecule with twice the concentration difference and the endpoint fluorescence was better,the target molecule of 1 nM can be detected,and the ratio of SNP alleles on the fetal specific gene is quantified.(2)The excess primer and limiting primer of the three SNPs for the imLATE-PCR were designed,and the ssDNA products were obtained after amplification.The linear relationship between the amplified product with twice the concentration difference and the endpoint fluorescence was better,the target molecule of 1 pM can be detected,and the sensitivity of the PLP-RCA is improved.(3)The quantitative PLP-RCA was used to detect the simulated samples of normal people and DS patients.The ratio of heterozygous SNP alleles in normal human simulated samples was 1:1,and the ratio of heterozygous SNP alleles in DS patients simulated sample was 2:1 or 1:2.This method enables the detection of simulated samples with DS.Conclusion : In this study,the reaction system of the PLP-RCA was optimized,and a method for specifically detecting fetal specific gene SNPs was established,which was used to genotype and quantify SNPs on fetal specific genes.The sensitivity of the PLP-RCA is improved by increasing the improved linear-after-the-exponential PCR.The screening of Down's syndrome simulated samples was initially implemented,which laid the foundation for the next phase of clinical sample testing.