Ludwig M, Baumann P, Günes A, Gembruch U, Diedrich K
Klinik für Frauenheilkunde und Geburtshilfe, Medizinischen Universität Lübeck.
Zentralbl Gynakol. 1998;120(9):454-7.
This study intended to develop a molecular biological method for the determination of the rhesus blood type through the genome of amniocytes. Using micropipettes amniocytes were isolated under a stereomicroscope from amniotic fluid samples. Single or several (> or = 10) amniocytes, respectively, were pipetted into 0.2 ml vials each, and were lysated at 65 degrees C for 15 min using an alkaline solution making the DNA accessible to polymerase chain reaction. Thereafter, the rhesus D gene as well as the CE gene were amplified with nested primers in two subsequent PCR. Finally, the amplification product was electrophoretically analyzed using agarose gel. When single aminocytes were analysed the amplification rate was 64% for the rhesus D gene, and 80% for the rhesus CE gene; "allele specific non amplification" was observed in 24% of cases. With 10 or more amniocytes per vial the amplification rate was 100%. This method permits to reliably predict the fetal rh-type at low costs within about 4 hours.
本研究旨在开发一种通过羊膜细胞基因组测定恒河猴血型的分子生物学方法。使用微量移液器在体视显微镜下从羊水样本中分离羊膜细胞。分别将单个或几个(≥10个)羊膜细胞移入每个0.2 ml小瓶中,并使用碱性溶液在65℃下裂解15分钟,使DNA可用于聚合酶链反应。此后,在两个连续的PCR中使用巢式引物扩增恒河猴D基因和CE基因。最后,使用琼脂糖凝胶对扩增产物进行电泳分析。当分析单个羊膜细胞时,恒河猴D基因的扩增率为64%,恒河猴CE基因的扩增率为80%;在24%的病例中观察到“等位基因特异性无扩增”。每个小瓶中有10个或更多羊膜细胞时,扩增率为100%。该方法能够以低成本在约4小时内可靠地预测胎儿的恒河猴血型。