- Email: [email protected]
The Investigator® ESSplex Plus Kit: Fast, sensitive, and robust amplification of the European Standard Set of loci
Forensic Science International: Genetics Supplement Series 3 (2011) e407–e408
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
The Investigator1 ESSplex Plus Kit: Fast, sensitive, and robust amplification of the European Standard Set of loci Mario Scherer *, Daniel Mu¨ller, Sebastian Begemann, Britta Steeger, Sarah Pakulla, Melanie Breitbach, Stefan Cornelius, Lars Bochmann, Anke Prochnow, Thomas Schnibbe, Holger Engel QIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 August 2011 Accepted 15 September 2011
Forensic DNA laboratories need to provide results in a very short time. Therefore, in addition to crucial quality parameters (e.g., sensitivity and robustness), speed is an increasingly important feature of STR PCR assays. QIAGEN has developed the Investigator ESSplex Plus Kit, which combines the features necessary for fast and reliable analysis of demanding forensic samples. Based on our fast-cycling multiplex PCR technology, we have introduced a novel reaction mix that enables a standard 30 cycle amplification in 90 min, setting a new level of speed in STR analysis. Well-balanced full profiles are reliably obtained with 125 pg of DNA template. Even a single genomic DNA copy gives rise to peak heights easily detectable with commonly used analysis thresholds. The assay is very robust and can tolerate PCR inhibitors of up to 200 ng/ml humic acid or 1000 mM hematin without allelic dropout, higher than any other kit on the market. The Investigator ESSplex Plus Kit provides a clean baseline, without artifacts that may interfere with the analysis of low-copy-number samples. These features help to reduce the number of samples that require reanalysis and contribute to more streamlined and efficient laboratory workflow. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: European Standard Set of loci Forensic Inhibitor
1. Introduction
2. Materials and methods
Forensic casework samples often are challenging and obtaining a DNA profile at first attempt may fail for several reasons. (i) Samples frequently contain minute amounts of DNA, (ii) potential inhibitors of PCR reactions are present, or (iii) DNA became compromised due to degradation. Recent developments help laboratories to address these challenges by optimizing their workflows. Sample extraction methods have been improved in terms of sensitivity and inhibitor removal. Real-time PCR based quantification assays allow to accurately measure even very low concentrations of DNA and indicate the presence of inhibitors (Di Pasquale et al., this issue). In order to increase successful amplification from degraded samples, the ENFSI/EDNAP groups suggested the inclusion of new STR markers with small PCR products to build a new European Standard Set of STR loci [1]. Here we describe the highly sensitive, robust and fast Investigator ESSplex Plus Kit that covers the fifteen markers of the new standard.
The Investigator ESSplex Plus Kit was used following manufacturer’s instructions. All of the electropherograms shown were generated on an Applied Biosystems1 ABI 3500 Genetic Analyzer. Samples were injected for 8 s at 3 kV. GeneAmp1 PCR System 9700 with Gold-plated Silver block was used for amplification. Data were analyzed using the QIAGEN Investigator IDproof Mixture or Applied Biosystems GeneMapper ID-X software.
* Corresponding author. Tel.: +49 0 2103 29 16362; fax: +49 0 2103 29 26362. E-mail address: [email protected] (M. Scherer). 1875-1768/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2011.09.065
3. Results The Investigator ESSplex Plus PCR Kit makes use of QIAGEN’s multiplex PCR and fast cycling technology, enabling a simple, rapid, and robust PCR cycling protocol. The standard 30 cycle amplification protocol is completed in 90 min. Dilution series of control DNA XY5 were used to evaluate the sensitivity of the assay. Full profiles were reliably obtained from 125 pg template DNA. Partial profiles with good signal to noise ratios were obtained down to 8 pg DNA. No-template controls show levels of background fluorescence without any visible dye artifacts (Fig. 1). The robustness of the assay was tested with simulated inhibition using model substances mimicking inhibitors typical for forensic casework samples. Results are summarized in Fig. 2. Allelic drop outs were only observed at very high inhibitor
e408
M. Scherer et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e407–e408
Fig. 1. Sensitivity study of the Investigator ESSplex Plus Kit. Different concentrations of Control DNA XY5 were used as a template. Samples were amplified on an ABI GeneAmp PCR System 9700 Thermal Cycler with gold-plated silver block. Analysis was performed on an AB Genetic Analyzer 3500, equipped with a 36 cm 8-capillary array and POP-4TM polymer. NTC: no-template control. Note that the y-axis scales were adjusted for best fit. Each sample was run in triplicates, representative data are shown.
Fig. 2. Overview of the Investigator ESSplex Plus inhibitor resistance. The assay was tested with six inhibitors (humic acid, hematin, tannic acid, indigo carmine, calcium, and collagen). 500 pg of Control DNA XY5 was used as template and PCR was performed under standard conditions in duplicates. 100 RFU was used as the threshold for allele calling. The colour scheme indicates the number of called versus expected alleles.
concentrations, e.g. 225 ng/ml humic acid or 1125 mM hematin. Split peaks caused by 1 bp PCR products were found for some markers in the presence of high concentrations of tannic acid or calcium. Maximum inhibitor concentrations used in this study are beyond levels frequently observed in real casework samples and are to our knowledge not tolerated by any other kit in the market.
for challenging samples. It provides a very fast generic PCR protocol suitable for any kind of sample that contributes to a significant time reduction from sample to result. Conflict of interest The authors are employed at QIAGEN GmbH.
4. Discussion The Investigator ESSplex Plus Kit combines high sensitivity and robustness towards inhibitors to increase first round success rates
Reference [1] Forensic Science International 163 (2006) 155–157.
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
The Investigator1 ESSplex Plus Kit: Fast, sensitive, and robust amplification of the European Standard Set of loci Mario Scherer *, Daniel Mu¨ller, Sebastian Begemann, Britta Steeger, Sarah Pakulla, Melanie Breitbach, Stefan Cornelius, Lars Bochmann, Anke Prochnow, Thomas Schnibbe, Holger Engel QIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 August 2011 Accepted 15 September 2011
Forensic DNA laboratories need to provide results in a very short time. Therefore, in addition to crucial quality parameters (e.g., sensitivity and robustness), speed is an increasingly important feature of STR PCR assays. QIAGEN has developed the Investigator ESSplex Plus Kit, which combines the features necessary for fast and reliable analysis of demanding forensic samples. Based on our fast-cycling multiplex PCR technology, we have introduced a novel reaction mix that enables a standard 30 cycle amplification in 90 min, setting a new level of speed in STR analysis. Well-balanced full profiles are reliably obtained with 125 pg of DNA template. Even a single genomic DNA copy gives rise to peak heights easily detectable with commonly used analysis thresholds. The assay is very robust and can tolerate PCR inhibitors of up to 200 ng/ml humic acid or 1000 mM hematin without allelic dropout, higher than any other kit on the market. The Investigator ESSplex Plus Kit provides a clean baseline, without artifacts that may interfere with the analysis of low-copy-number samples. These features help to reduce the number of samples that require reanalysis and contribute to more streamlined and efficient laboratory workflow. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: European Standard Set of loci Forensic Inhibitor
1. Introduction
2. Materials and methods
Forensic casework samples often are challenging and obtaining a DNA profile at first attempt may fail for several reasons. (i) Samples frequently contain minute amounts of DNA, (ii) potential inhibitors of PCR reactions are present, or (iii) DNA became compromised due to degradation. Recent developments help laboratories to address these challenges by optimizing their workflows. Sample extraction methods have been improved in terms of sensitivity and inhibitor removal. Real-time PCR based quantification assays allow to accurately measure even very low concentrations of DNA and indicate the presence of inhibitors (Di Pasquale et al., this issue). In order to increase successful amplification from degraded samples, the ENFSI/EDNAP groups suggested the inclusion of new STR markers with small PCR products to build a new European Standard Set of STR loci [1]. Here we describe the highly sensitive, robust and fast Investigator ESSplex Plus Kit that covers the fifteen markers of the new standard.
The Investigator ESSplex Plus Kit was used following manufacturer’s instructions. All of the electropherograms shown were generated on an Applied Biosystems1 ABI 3500 Genetic Analyzer. Samples were injected for 8 s at 3 kV. GeneAmp1 PCR System 9700 with Gold-plated Silver block was used for amplification. Data were analyzed using the QIAGEN Investigator IDproof Mixture or Applied Biosystems GeneMapper ID-X software.
* Corresponding author. Tel.: +49 0 2103 29 16362; fax: +49 0 2103 29 26362. E-mail address: [email protected] (M. Scherer). 1875-1768/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2011.09.065
3. Results The Investigator ESSplex Plus PCR Kit makes use of QIAGEN’s multiplex PCR and fast cycling technology, enabling a simple, rapid, and robust PCR cycling protocol. The standard 30 cycle amplification protocol is completed in 90 min. Dilution series of control DNA XY5 were used to evaluate the sensitivity of the assay. Full profiles were reliably obtained from 125 pg template DNA. Partial profiles with good signal to noise ratios were obtained down to 8 pg DNA. No-template controls show levels of background fluorescence without any visible dye artifacts (Fig. 1). The robustness of the assay was tested with simulated inhibition using model substances mimicking inhibitors typical for forensic casework samples. Results are summarized in Fig. 2. Allelic drop outs were only observed at very high inhibitor
e408
M. Scherer et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e407–e408
Fig. 1. Sensitivity study of the Investigator ESSplex Plus Kit. Different concentrations of Control DNA XY5 were used as a template. Samples were amplified on an ABI GeneAmp PCR System 9700 Thermal Cycler with gold-plated silver block. Analysis was performed on an AB Genetic Analyzer 3500, equipped with a 36 cm 8-capillary array and POP-4TM polymer. NTC: no-template control. Note that the y-axis scales were adjusted for best fit. Each sample was run in triplicates, representative data are shown.
Fig. 2. Overview of the Investigator ESSplex Plus inhibitor resistance. The assay was tested with six inhibitors (humic acid, hematin, tannic acid, indigo carmine, calcium, and collagen). 500 pg of Control DNA XY5 was used as template and PCR was performed under standard conditions in duplicates. 100 RFU was used as the threshold for allele calling. The colour scheme indicates the number of called versus expected alleles.
concentrations, e.g. 225 ng/ml humic acid or 1125 mM hematin. Split peaks caused by 1 bp PCR products were found for some markers in the presence of high concentrations of tannic acid or calcium. Maximum inhibitor concentrations used in this study are beyond levels frequently observed in real casework samples and are to our knowledge not tolerated by any other kit in the market.
for challenging samples. It provides a very fast generic PCR protocol suitable for any kind of sample that contributes to a significant time reduction from sample to result. Conflict of interest The authors are employed at QIAGEN GmbH.
4. Discussion The Investigator ESSplex Plus Kit combines high sensitivity and robustness towards inhibitors to increase first round success rates
Reference [1] Forensic Science International 163 (2006) 155–157.