Forensic evaluation of two nucleic acid extraction systems and validation of a RT-qPCR protocol for identification of SARS-CoV-2 in post-mortem nasopharyngeal swabs
•The INTCF has validated the molecular detection of SARS-CoV-2 for its forensic use.•Automated virus RNA extraction has greater relative efficiency than the manual one.•Molecular detection of SARS-CoV-2 with the kit tested is accurate and sensitive. The COVID-19 outbreak has represented a challenge...
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| Veröffentlicht in: | Forensic science international 2021-06, Vol.323, p.110775-110775, Article 110775 |
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| creator | Barrio, Pedro A. Fernández-Rodríguez, Amparo Martín, Pablo Fernández, Coro Fernández, Lourdes Alonso, Antonio |
| description | •The INTCF has validated the molecular detection of SARS-CoV-2 for its forensic use.•Automated virus RNA extraction has greater relative efficiency than the manual one.•Molecular detection of SARS-CoV-2 with the kit tested is accurate and sensitive.
The COVID-19 outbreak has represented a challenge for the international scientific community and particularly for forensic sciences. The lack of Coronavirus post-mortem testing led the National Institute of Toxicology and Forensic Sciences (INTCF) from Spain to verify the performance and utility of a quantitative reverse transcription PCR (RT-qPCR) clinical diagnosis protocol for SARS-CoV-2 detection (TaqPath™ COVID-19 CE-IVD RT-PCR Kit), to shed light on the cause of death (COD) in potentially COVID-19 cases in judicial autopsies. Two different RNA extraction methods were also tested (EZ1® DSP Virus Kit on the EZ1® Advanced XL robot versus MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit) regarding extraction efficiency, precision and contamination. RT-qPCR was evaluated for precision, specificity, limit of detection and concordance. Both the automated and the manual RNA extraction procedures showed good efficiency, but the automated virus extraction by bio-robot produced more reproducible results than the manual extraction. The SARS-CoV-2 RT-qPCR assay showed high sensitivity with a detection limit up to 10 copies/reaction and high specificity, as no cross-reactivity was detected between any of the 12 different RNA viruses tested, including three types of coronaviruses (SARS-CoV, NL63 and 229E). Reproducibility and repeatability of the studied method as well as concordance with other SARS-CoV-2 molecular detection protocols were also demonstrated. |
| doi_str_mv | 10.1016/j.forsciint.2021.110775 |
| format | Article |
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The COVID-19 outbreak has represented a challenge for the international scientific community and particularly for forensic sciences. The lack of Coronavirus post-mortem testing led the National Institute of Toxicology and Forensic Sciences (INTCF) from Spain to verify the performance and utility of a quantitative reverse transcription PCR (RT-qPCR) clinical diagnosis protocol for SARS-CoV-2 detection (TaqPath™ COVID-19 CE-IVD RT-PCR Kit), to shed light on the cause of death (COD) in potentially COVID-19 cases in judicial autopsies. Two different RNA extraction methods were also tested (EZ1® DSP Virus Kit on the EZ1® Advanced XL robot versus MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit) regarding extraction efficiency, precision and contamination. RT-qPCR was evaluated for precision, specificity, limit of detection and concordance. Both the automated and the manual RNA extraction procedures showed good efficiency, but the automated virus extraction by bio-robot produced more reproducible results than the manual extraction. The SARS-CoV-2 RT-qPCR assay showed high sensitivity with a detection limit up to 10 copies/reaction and high specificity, as no cross-reactivity was detected between any of the 12 different RNA viruses tested, including three types of coronaviruses (SARS-CoV, NL63 and 229E). Reproducibility and repeatability of the studied method as well as concordance with other SARS-CoV-2 molecular detection protocols were also demonstrated.</description><identifier>ISSN: 0379-0738</identifier><identifier>ISSN: 1872-6283</identifier><identifier>EISSN: 1872-6283</identifier><identifier>DOI: 10.1016/j.forsciint.2021.110775</identifier><identifier>PMID: 33866187</identifier><language>eng</language><publisher>Ireland: Elsevier B.V</publisher><subject>Acids ; Autopsies ; Autopsy ; Contamination ; Coronavirus Infectious Disease-19 (COVID-19) ; Coronaviruses ; COVID-19 ; Cross-reactivity ; Extraction procedures ; Forensic genetics ; Forensic microbiology ; Forensic science ; Forensic sciences ; Influenza ; Nucleic acids ; Polymerase chain reaction ; Quantitative reverse transcription PCR (RT-qPCR) ; Reproducibility ; Research Paper ; Reverse transcription ; Ribonucleic acid ; RNA ; RNA viruses ; Robots ; Severe acute respiratory syndrome ; Severe acute respiratory syndrome coronavirus 2 ; Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) ; Toxicology ; Validation ; Viral diseases ; Viruses</subject><ispartof>Forensic science international, 2021-06, Vol.323, p.110775-110775, Article 110775</ispartof><rights>2021 Elsevier B.V.</rights><rights>Copyright © 2021 Elsevier B.V. All rights reserved.</rights><rights>2021. Elsevier B.V.</rights><rights>2021 Elsevier B.V. All rights reserved. 2021 Elsevier B.V.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c503t-142b9f55c65d7da1085328121443b2d89e533c7ab27d3bf95294e7cb099aa2bb3</citedby><cites>FETCH-LOGICAL-c503t-142b9f55c65d7da1085328121443b2d89e533c7ab27d3bf95294e7cb099aa2bb3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.proquest.com/docview/2529230540?pq-origsite=primo$$EHTML$$P50$$Gproquest$$H</linktohtml><link.rule.ids>230,314,780,784,885,3550,27924,27925,45995,64385,64387,64389,72469</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/33866187$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Barrio, Pedro A.</creatorcontrib><creatorcontrib>Fernández-Rodríguez, Amparo</creatorcontrib><creatorcontrib>Martín, Pablo</creatorcontrib><creatorcontrib>Fernández, Coro</creatorcontrib><creatorcontrib>Fernández, Lourdes</creatorcontrib><creatorcontrib>Alonso, Antonio</creatorcontrib><title>Forensic evaluation of two nucleic acid extraction systems and validation of a RT-qPCR protocol for identification of SARS-CoV-2 in post-mortem nasopharyngeal swabs</title><title>Forensic science international</title><addtitle>Forensic Sci Int</addtitle><description>•The INTCF has validated the molecular detection of SARS-CoV-2 for its forensic use.•Automated virus RNA extraction has greater relative efficiency than the manual one.•Molecular detection of SARS-CoV-2 with the kit tested is accurate and sensitive.
The COVID-19 outbreak has represented a challenge for the international scientific community and particularly for forensic sciences. The lack of Coronavirus post-mortem testing led the National Institute of Toxicology and Forensic Sciences (INTCF) from Spain to verify the performance and utility of a quantitative reverse transcription PCR (RT-qPCR) clinical diagnosis protocol for SARS-CoV-2 detection (TaqPath™ COVID-19 CE-IVD RT-PCR Kit), to shed light on the cause of death (COD) in potentially COVID-19 cases in judicial autopsies. Two different RNA extraction methods were also tested (EZ1® DSP Virus Kit on the EZ1® Advanced XL robot versus MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit) regarding extraction efficiency, precision and contamination. RT-qPCR was evaluated for precision, specificity, limit of detection and concordance. Both the automated and the manual RNA extraction procedures showed good efficiency, but the automated virus extraction by bio-robot produced more reproducible results than the manual extraction. The SARS-CoV-2 RT-qPCR assay showed high sensitivity with a detection limit up to 10 copies/reaction and high specificity, as no cross-reactivity was detected between any of the 12 different RNA viruses tested, including three types of coronaviruses (SARS-CoV, NL63 and 229E). Reproducibility and repeatability of the studied method as well as concordance with other SARS-CoV-2 molecular detection protocols were also demonstrated.</description><subject>Acids</subject><subject>Autopsies</subject><subject>Autopsy</subject><subject>Contamination</subject><subject>Coronavirus Infectious Disease-19 (COVID-19)</subject><subject>Coronaviruses</subject><subject>COVID-19</subject><subject>Cross-reactivity</subject><subject>Extraction procedures</subject><subject>Forensic genetics</subject><subject>Forensic microbiology</subject><subject>Forensic science</subject><subject>Forensic sciences</subject><subject>Influenza</subject><subject>Nucleic acids</subject><subject>Polymerase chain reaction</subject><subject>Quantitative reverse transcription PCR (RT-qPCR)</subject><subject>Reproducibility</subject><subject>Research Paper</subject><subject>Reverse transcription</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>RNA viruses</subject><subject>Robots</subject><subject>Severe acute respiratory syndrome</subject><subject>Severe acute respiratory syndrome coronavirus 2</subject><subject>Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)</subject><subject>Toxicology</subject><subject>Validation</subject><subject>Viral 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chain reaction</topic><topic>Quantitative reverse transcription PCR (RT-qPCR)</topic><topic>Reproducibility</topic><topic>Research Paper</topic><topic>Reverse transcription</topic><topic>Ribonucleic acid</topic><topic>RNA</topic><topic>RNA viruses</topic><topic>Robots</topic><topic>Severe acute respiratory syndrome</topic><topic>Severe acute respiratory syndrome coronavirus 2</topic><topic>Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)</topic><topic>Toxicology</topic><topic>Validation</topic><topic>Viral diseases</topic><topic>Viruses</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Barrio, Pedro A.</creatorcontrib><creatorcontrib>Fernández-Rodríguez, Amparo</creatorcontrib><creatorcontrib>Martín, Pablo</creatorcontrib><creatorcontrib>Fernández, Coro</creatorcontrib><creatorcontrib>Fernández, Lourdes</creatorcontrib><creatorcontrib>Alonso, 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RT-qPCR protocol for identification of SARS-CoV-2 in post-mortem nasopharyngeal swabs</atitle><jtitle>Forensic science international</jtitle><addtitle>Forensic Sci Int</addtitle><date>2021-06-01</date><risdate>2021</risdate><volume>323</volume><spage>110775</spage><epage>110775</epage><pages>110775-110775</pages><artnum>110775</artnum><issn>0379-0738</issn><issn>1872-6283</issn><eissn>1872-6283</eissn><abstract>•The INTCF has validated the molecular detection of SARS-CoV-2 for its forensic use.•Automated virus RNA extraction has greater relative efficiency than the manual one.•Molecular detection of SARS-CoV-2 with the kit tested is accurate and sensitive.
The COVID-19 outbreak has represented a challenge for the international scientific community and particularly for forensic sciences. The lack of Coronavirus post-mortem testing led the National Institute of Toxicology and Forensic Sciences (INTCF) from Spain to verify the performance and utility of a quantitative reverse transcription PCR (RT-qPCR) clinical diagnosis protocol for SARS-CoV-2 detection (TaqPath™ COVID-19 CE-IVD RT-PCR Kit), to shed light on the cause of death (COD) in potentially COVID-19 cases in judicial autopsies. Two different RNA extraction methods were also tested (EZ1® DSP Virus Kit on the EZ1® Advanced XL robot versus MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit) regarding extraction efficiency, precision and contamination. RT-qPCR was evaluated for precision, specificity, limit of detection and concordance. Both the automated and the manual RNA extraction procedures showed good efficiency, but the automated virus extraction by bio-robot produced more reproducible results than the manual extraction. The SARS-CoV-2 RT-qPCR assay showed high sensitivity with a detection limit up to 10 copies/reaction and high specificity, as no cross-reactivity was detected between any of the 12 different RNA viruses tested, including three types of coronaviruses (SARS-CoV, NL63 and 229E). Reproducibility and repeatability of the studied method as well as concordance with other SARS-CoV-2 molecular detection protocols were also demonstrated.</abstract><cop>Ireland</cop><pub>Elsevier B.V</pub><pmid>33866187</pmid><doi>10.1016/j.forsciint.2021.110775</doi><tpages>1</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Forensic science international, 2021-06, Vol.323, p.110775-110775, Article 110775 |
| issn | 0379-0738 1872-6283 1872-6283 |
| language | eng |
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| source | Access via ScienceDirect (Elsevier); ProQuest Central UK/Ireland |
| subjects | Acids Autopsies Autopsy Contamination Coronavirus Infectious Disease-19 (COVID-19) Coronaviruses COVID-19 Cross-reactivity Extraction procedures Forensic genetics Forensic microbiology Forensic science Forensic sciences Influenza Nucleic acids Polymerase chain reaction Quantitative reverse transcription PCR (RT-qPCR) Reproducibility Research Paper Reverse transcription Ribonucleic acid RNA RNA viruses Robots Severe acute respiratory syndrome Severe acute respiratory syndrome coronavirus 2 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Toxicology Validation Viral diseases Viruses |
| title | Forensic evaluation of two nucleic acid extraction systems and validation of a RT-qPCR protocol for identification of SARS-CoV-2 in post-mortem nasopharyngeal swabs |
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