Short tandem repeat (STR) genotyping can provide individual-specific DNA profiles for forensic personal identification, with PCR followed by capillary electrophoresis (CE) serving as the standard methodology. However, PCR-derived stutter peaks, resulting from replication slippage during high-temperature extension and repeated thermal cycling, interfere with DNA profile interpretation for mixed samples from multi-individual. Specifically, alleles from the minor contributor can be obscured by stutter peaks from the major contributor, thereby complicating mixture profiles interpretation. In this study, recombinase polymerase amplification (RPA), a low-temperature isothermal alternative to PCR, is introduced to mitigate the stutter peak interference. Furthermore, gold nanoparticles (AuNPs) are integrated into RPA to suppress nonspecific artifacts observed in multi-individual mixed samples. The investigation of D21S11 genotyping demonstrates that although the stutter ratio increases with rising temperature for amplification (from 25 °C to 40 °C) and decreasing input DNA dosage (from 1 ng to 25 pg), RPA consistently maintains a stutter ratio significantly lower than that in PCR. Meanwhile, the RPA shows high tolerance to inhibitors, obtaining complete genotype under 5 mM of EDTA, 250 ng/µL of humic acid, and 100 µM of hemoglobin. No inhibition is observed from tannic acid. Besides, for D6S1043 genotyping using two-individual mixture, the allele of minor contributor can be successfully detected at the ratio as low as 1:29, a scenario where PCR-based analysis is often confounded by stutter masking. Although nonspecific artifacts are observed at the 1:29 ratio, the addition of AuNPs effectively eliminates them, yielding cleaner and more interpretable profiles. To evaluate the broad applicability of RPA assay, a panel of STR loci with varying lengths of core repeat unit is selected, including trinucleotide repeat loci (D12ATA63 and D18S853), tetranucleotide repeat loci (D21S11 and D13S317), and pentanucleotide repeat loci (Penta D and Penta E). For all tested loci, complete genotypes and correct STR profiles are obtained, and stutter peaks generated by RPA are either entirely absent or present at significantly lower levels than those produced by PCR. Furthermore, a quadruplex RPA is successfully developed and highlights its multiplexing potential, though further optimization is required for higher-order multiplex panels to meet the requirements of forensic analysis. This study establishes an RPA-AuNP assay for STR genotyping that effectively minimizes stutter and enhances the interpretability of mixture profiles, presenting a promising method for processing challenging forensic samples with low DNA content or complex mixtures.

Authors

• Wenjing Hu (School of Forensic Medicine, Shanxi Medical University, China)
• Huyun Zhou (School of Forensic Medicine, Shanxi Medical University, China)
• Mengyang Zhao (School of Forensic Medicine, Shanxi Medical University, China)
• Jingjing Xu (School of Forensic Medicine, Shanxi Medical University, China)
• Fenglong Yang (School of Forensic Medicine, Shanxi Medical University, China)
• Xiaonan Liu (School of Forensic Medicine, Shanxi Medical University, China)
• Jiangwei Yan (School of Forensic Medicine, Shanxi Medical University, China)