Abstract: Objective This study is to establish a novel non-invasive testing method to detect fetal RHD blood group genes of cell free fetal DNA (cff-DNA) from peripheral blood of pregnant women, and to analyze the feasibility and accuracy of the method. Methods RhD-negative pregnant women who were enrolled in our medical center from August 2023 to August 2024 were used as the study subjects, and 5~8 mL of peripheral blood was collected from pregnant women, firstly, the mother's RHD genotype was detected, and the genomic DNA in the peripheral blood of the pregnant women was extracted by the adsorption column method, and the genomic DNA in the peripheral blood of the pregnant women was extracted by the sequence specific primer polymerase chain reaction (Polymerase Chain Reaction). Reaction-Sequence Specific Primer (PCR-SSP) method was used to detect the RHD genotypes of mothers, and pregnant women who met the inclusion criteria were screened out; then cff-DNA in the plasma of pregnant women was extracted by the magnetic bead method, and the extracted cff-DNA was first subjected to quality control testing, and cff-DNA was processed using methylation-sensitive restriction endonuclease, and RHD genotypes were amplified after enzymatic digestion. DNA, methylation-sensitive restriction endonuclease was used to treat cff-DNA, and the RASSF1A and β-actin genes were amplified after digestion to ensure that cff-DNA of fetal origin was extracted; then the fetal RHD blood group gene test was performed, and the nested Taqman probe was used in the real-time quantitative polymerase chain reaction (qPCR) to detect the cff-DNA.The qPCR method was used to amplify exons 4, 7 and 10 of RHD to determine the fetal RhD blood group; finally, the accuracy of the fetal cff-DNA RHD blood group gene test was verified, and the newborns' blood group after birth was tracked, which was used as a gold standard control to determine the accuracy of the maternal peripheral blood cff-DNA RHD blood group gene test. Results The samples from four RhD-negative pregnant women were collected for mother's RHD genotyping, and the results showed that three cases were RHD total deletion, and one case was RHD-CE (2-9), which all met the inclusion criteria; cff-DNA obtained from pregnant women's peripheral blood was enzymatically digested and then verified by quality control, and all of them were positive for the RASSF1A gene, negative for β-actin gene, and all of them were extracted into cff- DNA; using cff-DNA to detect fetal RHD blood group genes, the results showed that exons 4, 7 and 10 of RHD were positive in four cases, and the fetal RhD blood group was judged to be RhD positive; tracing back to four newborns, the newborn blood group serology results were RhD positive. Conclusion This new method of maternal peripheral blood cff-DNA detection of fetal RHD blood group genes is feasible, and the test results are consistent with the blood type of the newborn after birth, and its important for the early diagnosis of RhD-associated HDFN and the application of anti-D immune globulin in RhD-negative pregnant women.

Key words: Cell-free Fetal DNA, Fetal RHD Blood Group Gene, RhD negative, Hemolytic Disease of the Newborn, Nested PCR