Single cell four-plex nested fluorescent PCR for hemophilia A genetic diagnosis and gender determination / 中华检验医学杂志

ABSTRACT

Objective To develop a rapid and efficient single cell PCR method for the non-invasive prenatal hemophilia A genetic diagnosis.Methods Six STR markers (DXS15,DXS9901,G6PD, DXS1073,DXS1108 and F8Civsl3) closely linked to F Ⅷ gene were examined in 118 healthy individuals and 12 hemophilia A families by muhi-plex fluorescent PCR.Three markers with the higher heterozygote rate and diagnostic rate were chosen and combined with amelogenin gene to establish the four-plex nested fluorescent PCR on single lymphocyte level.Results The single lymphocyte amplification rates of Amelogenin,DXS15,FSCivsl3 and DXSI073 were 94.3%,91.4%,100% and 100% in the normal male,and 100% ,97.1% ,97.1% and 97.1% in the normal female's respectively.The female was heterozygous in DXS1073 and DXS15.However,no allele dropout was found in both markers.Neither the false negative nor the false positive amplification was observed.Conclusion The single cell four-plex nested fluorescent PCR method established in this study is convenient and efficient,and will be hopefully employed in the clinical non-invasive prenatal genetic diagnosis for hemophilia A.