Department of Obstetrics and Gynaecology, Chinese University of Hong Kong, Hong Kong Special Administrative Region.
Clinical Genetic Service, Department of Health, Hong Kong Special Administrative Region.
Clin Chim Acta. 2016 May 1;456:137-143. doi: 10.1016/j.cca.2016.02.027. Epub 2016 Mar 3.
Sizing of FMR1 trinucleotide repeats in the clinical laboratory requires the use of capillary sequencer by PCR, or by a labor intensive measurement using Southern blot method. Our aim was to validate an accurate and robust PCR assay for quantification of CGG repeats.
We performed an analytical and clinical validation of a new PCR-based method that utilizes a low-cost capillary electrophoresis instrument and the FragilEase™ reagent kit. First, analytical performance was demonstrated on 12 Coriell reference samples comprising normal through full mutations. Subsequently, a cohort of 112 archived clinical DNA samples, enriched for premutation and full mutations, was analyzed.
All samples were amplified successfully. Quantification of repeat numbers was interpreted by the use of standards with known repeats. Twenty-five full-mutation samples were successfully amplified with the largest allele size measured at 1380 repeats. The repeat numbers from the new assay were concordant with those obtained with the reference method. The intra-assay (CV<2.5%) and inter-assay imprecision was within 1 CGG repeat.
This new PCR-based method is reproducible and capable of identifying all Fragile X alleles. It is an accurate and robust method that facilitates Fragile X testing in a broader spectrum of clinical laboratories.
在临床实验室中，对 FMR1 三核苷酸重复序列进行定序需要使用毛细管测序仪进行 PCR 扩增，或者使用Southern blot 方法进行繁琐的测量。我们的目的是验证一种用于 CGG 重复定量的准确而稳健的 PCR 检测方法。
我们对一种新的基于 PCR 的方法进行了分析和临床验证，该方法利用了低成本的毛细管电泳仪器和 FragilEase™ 试剂试剂盒。首先，在包含正常至完全突变的 12 个 Coriell 参考样本上证明了分析性能。随后，分析了 112 个存档的临床 DNA 样本，这些样本富含前突变和完全突变。
所有样本均成功扩增。通过使用具有已知重复次数的标准品来解释重复次数的定量结果。成功扩增了 25 个完全突变样本，最大等位基因大小测量值为 1380 个重复。新检测方法的重复次数与参考方法获得的重复次数一致。该检测方法的内（<2.5%）和间（<1 CGG 重复）的精密度均在 1 个 CGG 重复之内。
这种新的基于 PCR 的方法具有可重复性，能够识别所有脆性 X 等位基因。它是一种准确而稳健的方法，可在更广泛的临床实验室中进行脆性 X 检测。
