College of Science & Engineering, Flinders University, Adelaide 5042, Australia.
College of Science & Engineering, Flinders University, Adelaide 5042, Australia; Forensic Science SA, GPO Box 2790, Adelaide 5001, Australia.
Forensic Sci Int. 2022 Jul;336:111314. doi: 10.1016/j.forsciint.2022.111314. Epub 2022 Apr 25.
Profiling of DNA associated with illicit drug packages and paraphernalia is a common investigative tool. In addition, research is being conducted regarding the analysis of trace DNA present within illicit drugs and on capsules. The application of trace DNA analysis to illicit drugs has the potential to identify individuals involved in their manufacture and distribution. However, the inhibitory effects of illicit drugs and related compounds on downstream DNA analysis has not yet been investigated. If drug-induced polymerase chain reaction (PCR) inhibition occurs, the quality or informativeness of the resultant DNA profile may be impacted. In this study, the effects of a range of drugs, diluents, adulterants, and synthetic precursors on both quantitative PCR (qPCR) data and short tandem repeat (STR) DNA profiling results were examined. Twenty-two compounds representative of drug compounds and adulterants which may be encountered in drug seizures were spiked with 1 ng/μL and 0.05 ng/μL of control DNA and underwent DNA quantification using Quantifiler™ Trio. A subset of 13 compounds, including the majority that indicated potential inhibition in Quantifiler™ Trio, underwent STR profiling with VeriFiler™ Plus to determine if inhibition also occurred at this stage. The effect of diluting the DNA extract on the extent of inhibition of STR profiling was also investigated. Internal PCR controls within the qPCR were not a reliable indicator of inhibition, although suppression of the short and long autosomal fragments was observed in the presence of many compounds, and four compounds gave inconclusive results. STR internal quality controls indicated inhibition in 5 of the 13 compounds, however, profiles were affected by the presence of 11 of the 13 compounds in various ways such as a decreased average relative fluorescence units (RFU), drop out of certain alleles (some based on allele size range of locus) leading to a decreased likelihood ratio (LR), an increase in the proportion of stutter peaks and the presence of split or shoulder peaks. All profiles improved following a dilution of the compound in the PCR and allowing the generation of LR values in excess of 1 × 10, indicating inhibition occurred rather than DNA degradation. The data obtained show that removal of some of these compounds is required through an effective DNA extraction process for successful downstream trace DNA profiling. Upon successful PCR, the resultant DNA profiles provide the opportunity for opening new investigative avenues for law enforcement agencies.
分析与非法毒品包裹和用具相关的 DNA 是一种常见的调查工具。此外，人们还在研究分析存在于非法毒品和胶囊中的痕量 DNA。微量 DNA 分析在非法毒品中的应用有可能识别参与其制造和分销的个人。然而，尚未研究非法毒品和相关化合物对下游 DNA 分析的抑制作用。如果药物诱导的聚合酶链反应 (PCR) 抑制发生，则所得 DNA 图谱的质量或信息量可能会受到影响。在这项研究中，研究了一系列药物、稀释剂、掺杂物和合成前体对定量 PCR (qPCR) 数据和短串联重复 (STR) DNA 分析结果的影响。从毒品缉获中可能遇到的代表药物化合物和掺杂物的 22 种化合物与 1ng/μL 和 0.05ng/μL 的对照 DNA 混合，并用 Quantifiler™ Trio 进行 DNA 定量。包括在 Quantifiler™ Trio 中显示出潜在抑制作用的大多数化合物在内的 13 种化合物的子集进行了 STR 分析，用 VeriFiler™ Plus 确定在这一阶段是否也发生了抑制作用。还研究了稀释 DNA 提取物对 STR 分析抑制程度的影响。qPCR 中的内部 PCR 对照并不是抑制的可靠指标，尽管在存在许多化合物的情况下观察到短和长常染色体片段的抑制，并且有四种化合物给出了不确定的结果。STR 内部质量控制表明，13 种化合物中的 5 种存在抑制作用，然而，由于 13 种化合物中的 11 种以各种方式影响了图谱，例如平均相对荧光单位 (RFU) 下降，某些等位基因丢失（某些基于基因座的等位基因大小范围）导致可能性比率 (LR) 下降，峰突增的比例增加以及存在分裂或肩峰。在将化合物稀释到 PCR 中并允许产生超过 1×10 的 LR 值后，所有图谱都得到了改善，这表明发生了抑制而不是 DNA 降解。获得的数据表明，需要通过有效的 DNA 提取过程去除其中一些化合物，以便成功进行下游痕量 DNA 分析。成功进行 PCR 后，所得 DNA 图谱为执法机构提供了开辟新调查途径的机会。
