Keefe Brayleigh, Foley Megan, Novroski Nicole, Oldoni Fabio
Arcadia University, 450 S Easton Rd, Glenside, PA, 19038, USA.
The George Washington University, 2100 Foxhall Rd, Washington, DC, 20007, USA.
Int J Legal Med. 2025 Sep;139(5):2077-2086. doi: 10.1007/s00414-025-03495-1. Epub 2025 Apr 21.
Capillary electrophoresis (CE) is the conventional method for separating and detecting amplified DNA fragments for forensic human identification (HID). The Applied Biosystems SeqStudio Genetic Analyzer, referred to herein as the SeqStudio, is the most recent CE platform introduced for forensic purposes. The SeqStudio includes an all-in-one reagent cartridge containing four capillaries. Validating a CE platform requires consideration of injection time, sensitivity and reproducibility, and mixture performance. The evaluation of the SeqStudio was performed using a standard polymerase chain reaction (PCR)-CE workflow. Injection time, sensitivity, and mixture studies were performed to determine the optimal parameters for reproducible and reliable data. The injection time study yielded a general decline in the relative florescence units (RFU) as injection time decreased from ten to one second. The sensitivity study showed no locus or allelic dropout from one nanogram (ng) to 250 picogram (pg) DNA input at the one second injection time, however, when not considering the TH01 locus, the sensitivity was further reduced to 62.5pg. The two-person and three-person mixture studies demonstrated that the SeqStudio is capable of detecting a large range of DNA mixture ratios. The evaluation serves as a model for best practices and supports the use of the SeqStudio for HID.
毛细管电泳（CE）是用于法医人类身份识别（HID）中分离和检测扩增DNA片段的传统方法。应用生物系统公司的SeqStudio基因分析仪（本文简称为SeqStudio）是最近推出的用于法医目的的CE平台。SeqStudio包括一个包含四根毛细管的一体化试剂盒。验证一个CE平台需要考虑进样时间、灵敏度和重现性以及混合性能。使用标准聚合酶链反应（PCR）-CE工作流程对SeqStudio进行评估。进行进样时间、灵敏度和混合研究以确定可产生可重现且可靠数据的最佳参数。进样时间研究表明，随着进样时间从十秒减少到一秒，相对荧光单位（RFU）普遍下降。灵敏度研究显示，在一秒进样时间下，从一纳克（ng）到250皮克（pg）的DNA输入量均未出现位点或等位基因丢失，然而，在不考虑TH01位点时，灵敏度进一步降至62.5pg。两人和三人混合样本研究表明，SeqStudio能够检测大范围的DNA混合比例。该评估作为最佳实践的一个范例，并支持将SeqStudio用于HID。
