School of Chemistry, College of Science, University of Lincoln, Lincolnshire, UK.
IDna Genetics Limited, Scottow Enterprise Park, Norfolk, UK.
J Forensic Sci. 2022 Sep;67(5):1971-1978. doi: 10.1111/1556-4029.15084. Epub 2022 Jun 23.
The efficiency of reduced volume PCR amplification was studied using the VeriFiler™ Express PCR Amplification Kit. Full (25 μL) and reduced (5 μL) volumes were tested in parallel to identify any differences in template DNA sensitivity and other electropherogram parameters. Both volumes produced full DNA profiles down to 0.08 ng/μL DNA concentration at 26 PCR cycles; however, reduced volume produced higher peak heights due to increased signal intensities. Significant difference (p-value ≤ 0.05) in heterozygote peak height ratios was observed between both volumes, where the reduced volume threshold was lowered to 0.6 to accommodate all data points. However, no significant difference (p-value > 0.05) was identified in the stutter ratios between both volumes. The analytical threshold for reduced volume was also determined to be 150 RFU with the presence of template DNA in PCR amplification. When the optimized reduced volume parameters were tested on DNA extracted from buccal swab samples using Prep-n-Go™ Buffer, good quality DNA profiles were produced. Overall, the reduced volume not only showed better results compared to the full volume, but also enable more samples to be processed with a PCR amplification kit, thus reduced the cost.
本研究使用 VeriFiler™ Express PCR 扩增试剂盒评估了降体积 PCR 扩增的效率。同时平行测试全量（25μL）和降量（5μL）两种反应体系，以确定模板 DNA 灵敏度和其他电泳图谱参数是否存在差异。在 26 个 PCR 循环中，两种反应体系均能在 0.08ng/μL 浓度的 DNA 时得到完整的 DNA 图谱；然而，降量体系由于信号强度增加，产生了更高的峰高。两种体系的杂合峰高比存在显著差异（p 值≤0.05），其中降量体系的阈值降低至 0.6 以容纳所有数据点。但是，两种体系的峰簇比无显著差异（p 值>0.05）。当存在 PCR 扩增模板 DNA 时，降量体系的分析阈值也确定为 150 RFU。当使用 Prep-n-Go™ 缓冲液从口腔拭子样本中提取的 DNA 对优化的降量参数进行测试时，产生了良好的 DNA 图谱质量。总的来说，与全量体系相比，降量体系不仅结果更好，而且能够使用更少的 PCR 扩增试剂盒处理更多的样本，从而降低了成本。
