Developmental validation of an efficient differential separation method incorporating the i‐sep^®DL spin column with high sperm DNA recovery for the processing of sexual assault samples

Abstract

The differential separation method is key to recovering a DNA profile of the sperm donor from sexual assault samples. However, low numbers of spermatozoa from the perpetrator are often swamped by the victim's epithelial cells or lost during the separation process, with the separation process labor-intensive, time-consuming, and operator-dependent. The self-sealing filter of the i-sep ® DL spin column allows direct lysis of the substrate throughout the differential separation process while preventing intact sperm cells from passing through, maximizing DNA recovery, and separation of non-sperm and sperm cells present. This study investigated the efficacy of a modified differential separation method, which incorporated the i-sep ® DL spin column in comparison with the conventional pellet-based differential separation method. Using semen dilution series and mock post-coital samples, the sensitivity, reproducibility, repeatability, and efficiency of sperm DNA recovery of the pellet-based differential to the i-sep ® method were evaluated side by side. The i-sep ® differential method was more sensitive in capturing sperm fraction DNA, with informative semen donor alleles detected from high dilutions of semen inputs where the pellet method has been unsuccessful. The i-sep ® differential method reduces manual handling, generating repeatable, and reproducible results between operators. Re-extraction of samples previously processed by the pellet or i-sep ® differential method showed that the pellet method failed to recover 15-88% of sperm fraction DNA, while the i-sep ® differential method was able to recover >99% in the initial extraction. The i-sep ® method is robust for processing sexual assault samples, overcoming the challenges of sperm DNA losses encountered by pellet-based methods.

Advances in the use of nanomaterials for nucleic acid detection in point-of-care testing devices: A review

“…To reduce interference material in the sample, and to avoid influence on the determination of the result, sample purification is an important step that cannot be ignored. Currently, for the extraction and purification of NA, common methods are magnetic bead-based, column-based, and phenol-chloroform-based ( Mou et al, 2019 ; Wang S et al, 2021 ; White et al, 2022 ). In the early days of POCT devices, few researchers considered the project of integrating sample preprocessing into POCT devices.…”