Rapid Prenatal Diagnosis Of Aneuploidy Using Multiplex Fluorescence STR-PCR

IntroductionPCR (Polymerase chain reaction) had been applied in prenatal diagnosis for almost 20 years. Its effectiveness and efficiency has been well-acknowledged as the wide application of PCR in diagnosis. There is even the tendency to replace karyotype analysis in prenatal diagnosis (1,2,3). The PCR results will be shown in as short as 24-36 hours after amniotic fluid, villus or fetal tissue collection which could either release the anxiety of pregnant woman and her family with a chromosomal normal fetus or terminate the chromosomal abnormal fetus.Many researchers have studied the rapid aneuploidy detection in prenatal diagnosis. However, there are very few studies on the choice and the different combinations of primers in multiplex STR-PCR (Short tendem repeat PCR). In this thesis, we tried to explore the application of STR-PCRs to detect chromosomal anomalies in human chromosome 13,18 and 21. We successfully designed two pairs of STR-PCR primers, Ml and M2 respectively, which are able to diagnose Down’s syndrome by amplification of amniotic fluid cell DNA. The STR-PCR result is compared with karyotype analysis and the relevance ratio is the number. We have provided a new and feasible method for Down’s syndrome prenatal diagnosis.Part I Research of 50 samples on new STR marks by QF-PCR Objective The objective is to assess the performance of new short tandem repeat markers①13S796, D13S800、D13S886、D18S877、D18S851、D18S847、D18S819、D21S11L、D21S1409、D21S1809、D21S2052、D21S1994、D21S1446、 D21S1435、D21S11、D21S1432、D21S1442) in diagnosis quantitative fluorescence PCR (QF-PCR) to detect the number of chromosome 13,18 and 21.Methods 50 normal blood white cell samples were used for QF-PCR. The heterozygosity (H) and polymorphism information content (PIC) was calculated by SPSS12.0. The result of QF-PCR was used to diagnose the number of chromosome 13,18 and 21.Result The heterozygosity ranged 0.7-0.87, PIC ranged 0.63-0.84 in chromosome 21. The heterozygosity ranged 0.6-0.74, PIC ranged 0.65-0.7 in chromosome 13. The heterozygosity ranged 0.69-0.81, PIC ranged 0.68-0.8 in chromosome 18. Detection rate of diploid in chromosome 21, 13 and 18 was 100%.Conclusion The new STR sites contained high heterozygosity and could be applied in QF-PCR diagnosis for aneuploidy abnormality in chromosome 13,18 and 21.PartⅡApplication of the new STR site markers in diagnosis of 13,18 and 21 aneuploidy syndrome.Objective To determine the detection rate of different combinations of STR markers in QF-PCR diagnosis for 13,18 and 21 aneuploidy syndrome.Methods Blood white cell samples from twenty 13,18 and 21 chromosomal abnormal patients were tested by QF-PCR using different combinations of new STR markers.Result In 21-trisomic samples, the relevance ratio is 87.5%,86.7%, 93.75% and 100% by two, three, four and five STR markers respectively.Conclusion QF-PCR using the new STR markers can be used to test the numerical chromosomal abnormality in chromosomes 21. PartⅢApplication of multiplex QF-PCR in prenatal diagnosis of Down’s syndromeObjective To establish a rapid prenatal diagnosis of Down’s syndrome using QF-PCR analysis of amniotic fluid cells.Methods DNA was isolated from amniotic fluid cells and multiplex QF-PCR using different combinations of STR markers was applied to analyze the ploidy status of chromosomes 13,18 and 21. The affected samples were re-analyzed with polymorphic markers for chromosome 21. All samples were confirmed by karyotype analysis.Result Among the 34 amniotic samples, two samples were unsuitable for QF-PCR analysis because of the visible presence of blood. One sample was failed in both by QF-PCR and cytogenetic analysis. Twenty-nine samples were normal and two samples were trisomy 21.The detection ratio was 100%. The false negative rate was 0.Conclusion QF-PCR analysis is one of the effective rapid aneuploidy detection methods in prenatal diagnosis.