Findlay I, Ray P, Quirke P, Rutherford A, Lilford R
Institute of Epidemiology and Health Services Research, University of Leeds, UK.
Hum Reprod. 1995 Jun;10(6):1609-18. doi: 10.1093/humrep/10.6.1609.
Previously the diagnosis of sex and cystic fibrosis status has been studied on single cells using the polymerase chain reaction (PCR). It has been suggested that allelic drop-out (PCR failure of one allele) and/or preferential amplification (hypo-amplification of one allele) may contribute to poor reliability and misdiagnosis, although this remains controversial as some reports suggest that allelic drop-out does not occur. We investigated an improved method of diagnosing sex and cystic fibrosis in single cells using a new technology (fluorescent PCR) to determine the base level of PCR artefacts (allelic drop-out and preferential amplification) which, in combination with improved sensitivity, should improve PCR reliability and accuracy. Fluorescent PCR gives high reliability (approximately 97%) and accuracy rates (approximately 97%) in somatic cells for both sex and cystic fibrosis diagnosis and its lower detection threshold allows allelic drop-out and preferential amplification to be easily distinguished. We also achieved high reliability and accuracy in diagnosing cystic fibrosis in human blastomeres. This study confirms earlier reports of both allelic drop-out and preferential amplification in single cell analysis. We demonstrate that both allelic drop-out and preferential amplification occur in somatic cells and suggest these are separate phenomena. Preferential amplification appeared common in single cell PCR while allelic drop-out apparently occurred at random in each allele. Preferential amplification was mainly amplification of the larger allele. We suggest that some inaccuracy/misdiagnosis may be due to both preferential amplification as well as allelic drop-out. Other findings were variability in drop-out between PCR and that amplification of signals from human blastomeres may be linked to embryo quality. We suggest that allelic drop-out is dependent on the number of cells within the sample.
此前，已利用聚合酶链反应（PCR）对单个细胞的性别和囊性纤维化状态进行诊断研究。有人提出，等位基因脱扣（一个等位基因的PCR失败）和/或优先扩增（一个等位基因的低扩增）可能导致可靠性差和误诊，尽管这仍存在争议，因为一些报告表明等位基因脱扣不会发生。我们研究了一种利用新技术（荧光PCR）诊断单个细胞性别和囊性纤维化的改进方法，以确定PCR假象（等位基因脱扣和优先扩增）的基线水平，结合提高的灵敏度，应能提高PCR的可靠性和准确性。荧光PCR在体细胞的性别和囊性纤维化诊断中具有较高的可靠性（约97%）和准确率（约97%），其较低的检测阈值使等位基因脱扣和优先扩增易于区分。我们在诊断人类卵裂球的囊性纤维化方面也实现了高可靠性和准确性。本研究证实了早期关于单细胞分析中等位基因脱扣和优先扩增的报告。我们证明等位基因脱扣和优先扩增都发生在体细胞中，并表明它们是不同的现象。优先扩增在单细胞PCR中似乎很常见，而等位基因脱扣显然在每个等位基因中随机发生。优先扩增主要是较大等位基因的扩增。我们认为一些不准确/误诊可能是由于优先扩增以及等位基因脱扣。其他发现包括PCR之间脱扣的变异性，以及来自人类卵裂球的信号扩增可能与胚胎质量有关。我们认为等位基因脱扣取决于样本中的细胞数量。
