Design, optimisation and preliminary validation of a human specific loop-mediated amplification assay for the rapid detection of human DNA at forensic crime scenes

Abstract

The identification of samples at a crime scene which require forensic DNA typing has been the focus of recent research interest. We propose a simple, but sensitive analysis system which can be deployed at a crime scene to identify crime scene stains as human or non-human. The proposed system uses the isothermal amplification of DNA in a rapid assay format, which returns results in as little as 30min from sampling. The assay system runs on the Genie II device, a proven in-field detection system which could be deployed at a crime scene. The results presented here demonstrate that the system was sufficiently specific and sensitive and was able to detect the presence of human blood, semen and saliva on mock forensic samples.

Evaluation of molecular inhibitors of loop-mediated isothermal amplification (LAMP)

“…2 ), but it did slightly decrease the amplicon signal in a concentration-dependent manner. Complete inhibition of the reaction was observed at 25 µM, which is on the same order of magnitude as the concentration that causes complete inhibition of qPCR (80 µM) 36 and is consistent with a previous report 17 for LAMP (> 20 µM). It has been shown before that hematin collisional quenches fluorescence of dyes such as ROX and sequesters magnesium from polymerases inhibiting the polymerase 36 .…”

“…Complete product (not fluorescence) inhibition was observed at 9 µg/mL, which is on a similar order of magnitude to concentrations that inhibit qPCR (1000 ng per 20 µL; 50 µg/mL; reactions supplemented with BSA that helps prevent amplification inhibition) 45 . Notably, we observed LAMP inhibition at lower concentrations than previous reports (inhibition not observed at even 20 µg/mL) 17 . The reasons for this could include that the previous report used a proprietary polymerase and buffer mix for LAMP (Optigene; cat.…”

“…Yet we still lack a good understanding of the key small molecules within these media that drive LAMP inhibition. Over 20 specific small molecule PCR inhibitors have been tested and their mechanisms have been evaluated 15 , 16 , but for LAMP, to the best of our knowledge, only a few inhibitors (humic acid 17 , indigo dye 17 , hematin 17 and urea 11 , 18 ) have been tested over a very narrow three-point concentration ranges 11 , 17 or with proprietary reaction formulations 17 , 18 . This is vital as on-the-field DNA and RNA samples may not come from fresh biological media and are often found as “touch” or dried samples on natural matrices such as soil or leather products with vastly different inhibitor concentrations.…”

Defining end user requirements for a field-based molecular detection system for wildlife forensic investigations

“…Analysis shows that participants believe that once introduced, the impact of the field-based intervention would be positive ( Table 3) [19,28,29]. This has often been in due to the high demands in law enforcement to analyse more DNA samples faster at less expense to increase the speed of casework resolution [13,30,31].…”

Duplex loop-mediated isothermal amplification assay for simultaneous detection of human and human male DNA

“…Thus, we hypothesized that its rapidity and simplicity make it suitable for screening human and human male DNA. Previous studies have reported the LAMP assays targeting human DNA for forensic use [ 5 – 7 ]. Another study has reported a male DNA-specific LAMP assay that can detect as low as 1 pg of male DNA within 20 min [ 8 ].…”