Prenatal Screening And Establishing A Rapid Diagnosis Technique For Fetal Aneuploidies By Multiplex Real-time Fluorescence Relative Quantitative Polymerase Chain Reaction

Objective1. To explore the foreign software of prenatal screening for Down's syndrome in China.2. To establish a multiplex Real-time fluorescence relative quantitative PCR method for diagnosis of Down's and Edward's syndrome.MethodsBy the time-resolved fluoroimmunoassay system, the matemal serum PAPP-A and free ?-hCG of 1517 pregnant women were measured in the first trimester, while the maternal serum AFP and free ?-hCG of 4858 pregnant women were measured in the second trimester,the NT of the first trimester fetus were measured also.Then the first trimester data were evaluated by the software of The First Trimester Screening Program offerred by The Fetal Medicine Foundation (FMF), the second trimester data were evaluated by the software of LifeCycle offerred by PerkinElmer company.Using the technique of multiplex Real-time fluorescence relative quantitative PCR, the sequence of the amyloid precursor protein(APP) gene in the Down's region of chromosome 21 and of the thymidylate synthetase(TYMS) gene on chromosome 18 were amplified in the same tube. 36 blood samples from normal persons (Group A),15 amniotic fluid samples from normal pregnant women (Group B), 21 samples from Down's patients (Group C) and 6 samples from Edward's patients (Group D) were detected.The four group of△CT values were analyzed, we established the△CT value of the normal range: -0.93 ~ -0.03 (Mean土3 SD) in this study, then 182 unknown samples were tested by this method.Results1. 1517 pregnant women were screened in the first-trimester, and 40 cases were regarded as high-risk pregnant women, the False positive rate (FR) is 2.6%,and detected one Down's syndrome; 4858 pregnant women were screened in the second-trimester, and 209 cases were regarded as high-risk pregnant women. Seven of the 4858 pregnant had Down's syndrome, and 5 of them had positive screening result. The false positive rate and detective rate for Down's syndrome were 4.2% and 71.4%, respectively.2. The mean△CT values of the four groups were -0.48土0.15,-0.49土0.12,-1.26土0.17 and 0.25士0.12 respectively. The△CT value from Group B was no different from Group A(P>0.05), the△CT value from Group C was dramatically different from Group A(P<0.01), the△CT value from Group D was dramatically different from Group A(P<0.01), and no overlapping was shown. The technique of multiplex real-time quantitative PCR can differentiate 21,18 trisomy syndrome and normal karyotype samples, its result is consistent with the conventional cytogenetics.3. We have established the△CT value of the normal range: -0.93 ~ -0.03 (Mean土3 SD), the sensibility is 100% and the specificity is 98.3%. Conclusion1. The foreign software of prenatal screening for Down's syndrome is also applicable to Chinese population.2. The false positive rate (FPR) of first trimester prenatal screening is lower, it avoids more pregnant to have puncture and reduces the miscarriage rate.3. We establish a multiplex Real-time fluorescence relative quantitative PCR method for diagnosis of Down's and Edward's syndrome, this rapid prenatal diagnosis technique is simple, fast, low cost, large flux and with the clinical value.4. Rapid and accurate prenatal diagnosis was established to prevent the birth of Down's syndrome and Edward's syndrome through the first trimester prenatal screening.