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. (June 2021)

• Record Type: Journal Article
• Authors: Barrio, Pedro A.; Fernández-Rodríguez, Amparo; Martín, Pablo; Fernández, Coro; Fernández, Lourdes; Alonso, Antonio
• Is Part Of: Forensic science international. Volume 323(2021)
• Publication Date: 2021-06
• Subjects: Coronavirus Infectious Disease-19 (COVID-19) -- Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) -- Quantitative reverse transcription PCR (RT-qPCR) -- Validation -- Forensic microbiology -- Forensic genetics; Medical jurisprudence -- Periodicals; Chemistry, Forensic -- Periodicals; Forensic Medicine -- Periodicals; Médecine légale -- Périodiques; Chimie légale -- Périodiques; Gerechtelijke geneeskunde; Gerechtelijke chemie; Gerechtelijke psychiatrie; Chemistry, Forensic; Medical jurisprudence; Electronic journals; Periodicals; Electronic journals; 614.1
• DOI: 10.1016/j.forsciint.2021.110775
• Languages: English
• ISSNs: 0379-0738

Abstract

Highlights: 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. 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.