- Email: [email protected]
Development of the Investigator® 26plex QS Kit: A New multiplex PCR Kit for Global STR analysis
Journal Pre-proof Development of the Investigator® 26plex QS Kit: A New Multiplex PCR Kit for Global STR Analysis Miroslav Vraneˇs, Margaretha K¨onig, Stefan Cornelius, Annika Kohns, Keith Elliott, Anke Prochnow, Mario Scherer
PII:
S1875-1768(19)30100-3
DOI:
https://doi.org/10.1016/j.fsigss.2019.10.174
Reference:
FSIGSS 1807
To appear in:
Forensic Science International: Genetics Supplement Series
Received Date:
13 September 2019
Accepted Date:
16 October 2019
Please cite this article as: Vraneˇs M, K¨onig M, Cornelius S, Kohns A, Elliott K, Prochnow A, Scherer M, Development of the Investigator® 26plex QS Kit: A New Multiplex PCR Kit for Global STR Analysis, Forensic Science International: Genetics Supplement Series (2019), doi: https://doi.org/10.1016/j.fsigss.2019.10.174
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Page 1 of 3
Development of the Investigator® 26plex QS Kit: A New Multiplex PCR Kit for Global STR Analysis Miroslav Vraneš, Margaretha König, Stefan Cornelius, Annika Kohns, Keith Elliott, Anke Prochnow and Mario Scherer QIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden, Germany
Abstract
ro of
STR assays for forensic casework must conform to national DNA standards. QIAGEN developed the Investigator 26plex QS Kit as an assay to co-amplify 25 markers, including the current Chinese, CODIS and European standard markers in one reaction. The kit is designed for purified DNA from casework and reference samples. The new Fast Reaction Mix 3.0 ensures not only robust and fast PCR amplification with improved inhibitor resistance and easy handling, but also provides enhanced robustness towards DNA overloading and stability. Highly sensitive, yielding rapid and reliable results from trace DNA, the kit is suitable for all forensic applications and paternity testing. It can also be used for direct amplification of reference samples like blood or buccal cells on FTA cards or swabs. Furthermore, with the inclusion of Penta E and Penta D in the marker set, the Investigator 26plex QS Kit offers very high discriminatory power. The assay uses a 6-dye technology to keep the amplicon length of the markers short and to avoid marker overlap. The kit contains QIAGEN’s Quality Sensor System (QS1 and QS2) as an internal PCR control. Keywords: Investigator 26plex QS Kit, Chinese National Database, Quality Sensor
-p
1. Introduction
2. Materials and methods
na
lP
re
Forensic casework samples are often challenging and obtaining a DNA profile at first attempt may fail for several reasons. Samples frequently contain minute amounts of DNA, potential inhibitors of PCR reactions are present, or DNA became compromised due to degradation. Furthermore, STR assays need to include different standard marker sets to be conform to national standards. QIAGEN has developed the Investigator 26plex QS kits which amplifies 25 markers of the Chinese, CODIS and European standard markers [1-6] and offers high discrimination power. The Investigator 26plex QS kit is specifically designed for analyzing challenging samples of low quality and quantity from casework. The kit contains QIAGEN’s Quality Sensor System (QS1 and QS2), an internal PCR control which is useful for evaluating amplification efficiency. It indicates if the reaction has worked in general and enables discrimination between the presence of inhibitors or DNA degradation as a cause for the typical ski slope effect observed in STR profiles of challenging samples. This information can be used to choose the most appropriate rework strategy.
3. Results
ur
The Investigator 26plex QS Kit was used following manufacturer’s instructions. All of the electropherograms shown were generated on an Applied Biosystems ABI 3500 Genetic Analyzer. GeneAmp PCR System 9700 with Gold-plated Silver block was used for amplification. Data were analyzed using the Applied Biosystems GeneMapper ID-X software.
Jo
The Investigator 26plex QS kit makes use of QIAGEN’s newest multiplex PCR and fast cycling technology implemented in the Fast Reaction Mix 3.0, enabling a simple, rapid, and robust PCR cycling protocol. The standard 30 cycle amplification protocol is completed in ~65 min. Dilution series of control DNA 9948 were used to evaluate the sensitivity of the assay. Profiles were well-balanced and partial profiles with good signal to noise ratios were obtained down to 8 pg DNA (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 concentrations. Maximum inhibitor concentrations used in this study are beyond levels frequently observed in real casework samples.
ro of
Page 2 of 3
na
lP
re
-p
Fig. 1. Sensitivity study. A Summary generated from a sensitivity study with four different DNA amounts from 500 pg down to 8 pg of input DNA 9948, amplified in 4 replicates according to the recommended protocol described in the kit handbook. Samples were run in a final volume of 25 μl/reaction and analyzed using an Applied Biosystems® 3500 XL Genetic Analyzer. B The corresponding electropherogram for 500 pg of DNA shows the 100% allele count.
Jo
4. Conclusion
ur
Fig. 2. Investigator 26plex QS Kit inhibitor tolerance. The assay was tested with 6 inhibitors (calcium, EtOH, Indigo carmine, hematin, humic acid and tannic acid). 500 pg of control DNA 9948 was used and PCR was performed under the standard conditions described in the kit handbook. 50 RFU was used as the threshold for allele calling. Green: Consistently full profile. Yellow: 75% of expected PCR products detected. Orange: 50% of expected PCR products detected. Red: Less than 50% of expected PCR products detected.
The Investigator 26plex QS Kit combines high sensitivity and robustness towards inhibitors to increase first round success rates 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 insight. 5. Role of funding The authors are employed at QIAGEN GmbH. This research was funded by QIAGEN GmbH and performed on QIAGEN premises in Hilden, Germany. 6. Conflict of interest
Page 3 of 3
Conflict of interest: Authors are QIAGEN employees. 7. References
Jo
ur
na
lP
re
-p
ro of
1. Hares, D.R. 2012 Expanding the CODIS core loci in the United States. Forensic Science International: Genetics 6(1), e52–e54. 2. Hares, D.R. 2012. Addendum to expanding the CODIS core loci in the United States. Forensic Science International: Genetics 6(5), e135. 3. Hares, D.R. 2015. Selection and implementation of expanded CODIS core loci in the United States. Forensic Science International: Genetics 17, 33–34. 4. Gill, P. et al. 2006. New multiplexes for Europe – amendments and clarification of strategic development. Forensic Science International 163, 155–157. 5. Gill, P. et al. 2006 The evolution of DNA databases – recommendations for new European STR loci. Forensic Science International 156, 242–244. 6. Chinese Institute of Forensic Science, Ministry of Public Security.
PII:
S1875-1768(19)30100-3
DOI:
https://doi.org/10.1016/j.fsigss.2019.10.174
Reference:
FSIGSS 1807
To appear in:
Forensic Science International: Genetics Supplement Series
Received Date:
13 September 2019
Accepted Date:
16 October 2019
Please cite this article as: Vraneˇs M, K¨onig M, Cornelius S, Kohns A, Elliott K, Prochnow A, Scherer M, Development of the Investigator® 26plex QS Kit: A New Multiplex PCR Kit for Global STR Analysis, Forensic Science International: Genetics Supplement Series (2019), doi: https://doi.org/10.1016/j.fsigss.2019.10.174
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Page 1 of 3
Development of the Investigator® 26plex QS Kit: A New Multiplex PCR Kit for Global STR Analysis Miroslav Vraneš, Margaretha König, Stefan Cornelius, Annika Kohns, Keith Elliott, Anke Prochnow and Mario Scherer QIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden, Germany
Abstract
ro of
STR assays for forensic casework must conform to national DNA standards. QIAGEN developed the Investigator 26plex QS Kit as an assay to co-amplify 25 markers, including the current Chinese, CODIS and European standard markers in one reaction. The kit is designed for purified DNA from casework and reference samples. The new Fast Reaction Mix 3.0 ensures not only robust and fast PCR amplification with improved inhibitor resistance and easy handling, but also provides enhanced robustness towards DNA overloading and stability. Highly sensitive, yielding rapid and reliable results from trace DNA, the kit is suitable for all forensic applications and paternity testing. It can also be used for direct amplification of reference samples like blood or buccal cells on FTA cards or swabs. Furthermore, with the inclusion of Penta E and Penta D in the marker set, the Investigator 26plex QS Kit offers very high discriminatory power. The assay uses a 6-dye technology to keep the amplicon length of the markers short and to avoid marker overlap. The kit contains QIAGEN’s Quality Sensor System (QS1 and QS2) as an internal PCR control. Keywords: Investigator 26plex QS Kit, Chinese National Database, Quality Sensor
-p
1. Introduction
2. Materials and methods
na
lP
re
Forensic casework samples are often challenging and obtaining a DNA profile at first attempt may fail for several reasons. Samples frequently contain minute amounts of DNA, potential inhibitors of PCR reactions are present, or DNA became compromised due to degradation. Furthermore, STR assays need to include different standard marker sets to be conform to national standards. QIAGEN has developed the Investigator 26plex QS kits which amplifies 25 markers of the Chinese, CODIS and European standard markers [1-6] and offers high discrimination power. The Investigator 26plex QS kit is specifically designed for analyzing challenging samples of low quality and quantity from casework. The kit contains QIAGEN’s Quality Sensor System (QS1 and QS2), an internal PCR control which is useful for evaluating amplification efficiency. It indicates if the reaction has worked in general and enables discrimination between the presence of inhibitors or DNA degradation as a cause for the typical ski slope effect observed in STR profiles of challenging samples. This information can be used to choose the most appropriate rework strategy.
3. Results
ur
The Investigator 26plex QS Kit was used following manufacturer’s instructions. All of the electropherograms shown were generated on an Applied Biosystems ABI 3500 Genetic Analyzer. GeneAmp PCR System 9700 with Gold-plated Silver block was used for amplification. Data were analyzed using the Applied Biosystems GeneMapper ID-X software.
Jo
The Investigator 26plex QS kit makes use of QIAGEN’s newest multiplex PCR and fast cycling technology implemented in the Fast Reaction Mix 3.0, enabling a simple, rapid, and robust PCR cycling protocol. The standard 30 cycle amplification protocol is completed in ~65 min. Dilution series of control DNA 9948 were used to evaluate the sensitivity of the assay. Profiles were well-balanced and partial profiles with good signal to noise ratios were obtained down to 8 pg DNA (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 concentrations. Maximum inhibitor concentrations used in this study are beyond levels frequently observed in real casework samples.
ro of
Page 2 of 3
na
lP
re
-p
Fig. 1. Sensitivity study. A Summary generated from a sensitivity study with four different DNA amounts from 500 pg down to 8 pg of input DNA 9948, amplified in 4 replicates according to the recommended protocol described in the kit handbook. Samples were run in a final volume of 25 μl/reaction and analyzed using an Applied Biosystems® 3500 XL Genetic Analyzer. B The corresponding electropherogram for 500 pg of DNA shows the 100% allele count.
Jo
4. Conclusion
ur
Fig. 2. Investigator 26plex QS Kit inhibitor tolerance. The assay was tested with 6 inhibitors (calcium, EtOH, Indigo carmine, hematin, humic acid and tannic acid). 500 pg of control DNA 9948 was used and PCR was performed under the standard conditions described in the kit handbook. 50 RFU was used as the threshold for allele calling. Green: Consistently full profile. Yellow: 75% of expected PCR products detected. Orange: 50% of expected PCR products detected. Red: Less than 50% of expected PCR products detected.
The Investigator 26plex QS Kit combines high sensitivity and robustness towards inhibitors to increase first round success rates 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 insight. 5. Role of funding The authors are employed at QIAGEN GmbH. This research was funded by QIAGEN GmbH and performed on QIAGEN premises in Hilden, Germany. 6. Conflict of interest
Page 3 of 3
Conflict of interest: Authors are QIAGEN employees. 7. References
Jo
ur
na
lP
re
-p
ro of
1. Hares, D.R. 2012 Expanding the CODIS core loci in the United States. Forensic Science International: Genetics 6(1), e52–e54. 2. Hares, D.R. 2012. Addendum to expanding the CODIS core loci in the United States. Forensic Science International: Genetics 6(5), e135. 3. Hares, D.R. 2015. Selection and implementation of expanded CODIS core loci in the United States. Forensic Science International: Genetics 17, 33–34. 4. Gill, P. et al. 2006. New multiplexes for Europe – amendments and clarification of strategic development. Forensic Science International 163, 155–157. 5. Gill, P. et al. 2006 The evolution of DNA databases – recommendations for new European STR loci. Forensic Science International 156, 242–244. 6. Chinese Institute of Forensic Science, Ministry of Public Security.