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

Pedro A Barrio; Amparo Fernández-Rodríguez; Pablo Martín; Coro Fernández; Lourdes Fernández; Antonio Alonso

doi:10.1016/j.forsciint.2021.110775

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

Forensic Sci Int. 2021 Apr 2:323:110775. doi: 10.1016/j.forsciint.2021.110775. Online ahead of print.

Authors

Pedro A Barrio¹, Amparo Fernández-Rodríguez², Pablo Martín², Coro Fernández³, Lourdes Fernández², Antonio Alonso⁴

Affiliations

¹ Biology Service, National Institute of Toxicology and Forensic Sciences, Department of Madrid, Spain. Electronic address: pedro.barrio@justicia.es.
² Biology Service, National Institute of Toxicology and Forensic Sciences, Department of Madrid, Spain.
³ Quality Assurance Service, National Institute of Toxicology and Forensic Sciences, Department of Madrid, Spain.
⁴ National Institute of Toxicology and Forensic Sciences, National Direction, Spain.

Abstract

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.

Keywords: Coronavirus Infectious Disease-19 (COVID-19); Forensic genetics; Forensic microbiology; Quantitative reverse transcription PCR (RT-qPCR); Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2); Validation.