A panel of PCR-inhibitory reference materials for quality evaluation of multiplex STR analysis kits

doi:10.1016/j.fsigss.2015.09.126

Forensic Science International: Genetics Supplement Series

Volume 5, December 2015, Pages e317-e319

https://doi.org/10.1016/j.fsigss.2015.09.126 Get rights and content

Abstract

PCR inhibition is a critical parameter in forensic DNA analysis. Substances interfering with amplification of short tandem repeats (STRs) may generate partial and/or ambiguous DNA profiles. We present a strategy for developing a broad panel of PCR-inhibitory reference materials (RMs), representing common casework samples. Our panel, including solutions prepared from for example cigarettes, chewing gum and soil, is a tool for in-house validation and lot testing of STR systems. PowerPlex ESX 16 Fast System tolerated high levels of some RMs, but several substances caused amplification problems. Humic acid had a negative effect on amelogenin, moist snuff hindered amplification of longer fragments, and chewing gum caused generally lowered allele peak heights. Applying a broad panel of RMs ensures that a wide range of inhibitory substances are tested, giving an improved understanding of inhibitor tolerance and effects.

Introduction

PCR inhibition is a critical parameter in forensic DNA analysis [1]. Substances interfering with amplification of short tandem repeats (STRs) may generate partial and/or ambiguous DNA profiles [2]. PCR inhibitors can be partly removed by purification procedures, but this inevitably leads to DNA loss [3].

For forensic DNA laboratories it is vital to understand the effects that common inhibitory substances have on their STR analysis system. In developmental validation of STR systems, the manufacturers generally evaluate PCR inhibition by applying a few pure substances such as humic acid and hematin [4], [5]. Crime scene samples are more diverse and heterogeneous, creating a need for complementary testing. We present a strategy for developing a broad panel of PCR-inhibitory reference materials (RMs), representing common casework samples. Our panel, including solutions prepared from for example cigarettes, chewing gum, moist snuff and soil, is a tool for in-house validation and lot testing of STR systems.

Section snippets

Materials and methods

PCR-inhibitory RMs were prepared by leaching different materials in Super-Q water (SQ) to elute water-soluble PCR-inhibitory compounds, to mimic the situation in DNA extraction. Supernatants were used as DNA-free RMs. Cigarettes: tipping paper was cut and vortexed in 1 mL SQ. Chewing gum: gums were crashed with a mortar, and vortexed in 1 mL SQ. Moist snuff: snuff bags were leached in 1 mL SQ. Soil: soil was mixed with SQ (20% w/w) and shake-incubated for one hour. Sediment: lake sediment was

Results and discussion

Different inhibitory effects were observed for the various RMs (Fig. 1). ESX Fast was tolerant to the tested cigarette solution (Table 1). All other RMs affected amplification negatively, giving generally lowered allele peak heights. Soil and moist snuff also had negative effects on longer markers (D18S51/D3S1358 ratio from 0 to 0.20). DTT, FA and HA (≥2 μg) affected amelogenin amplification (amelogenin/D3S1358 ratio from 0 to 0.17). FA and sediment caused similar inhibitory effects: for the

Conclusions

We present a strategy for preparing a broad panel of PCR-inhibitory RMs for in-house validation and lot testing of STR analysis systems. The prepared RMs contain known PCR inhibitors such as cellulose, tobacco derivatives and humic substances [6]. The different inhibitory effects observed show the importance of evaluating PCR inhibition with a broad range of casework-like RMs. The RM panel makes it possible to test STR systems in a realistic, controlled and repeatable manner, improving the

Conflict of interest

None.

Acknowledgements

We thank Elin Nilsson and Laila Noppa, Swedish Defence Research Agency, for providing the lake sediment reference material.

References (6)

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