Post-Mortem Detection of SARS-CoV-2 RNA in Long-Buried Lung Samples
Abstract
:1. Introduction
2. Materials and Methods
2.1. Patients, Autopsies and Sample Collection
2.2. RNA Extraction
2.3. RNA Quantification and Reverse Transcription
2.4. RT-qPCR and ddPCR Molecular Analyses
2.5. End-Point PCR
2.6. Sequencing
2.7. Correlation Analysis
2.8. Statistical Analyses
3. Results
3.1. RT-qPCR and ddPCR SARS-CoV-2 Detection
3.2. Correlation Analysis
3.3. SARS-CoV-2 Sequencing
4. Discussion and Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Sample | ng/µL | ng for cDNA Synthesis |
---|---|---|
P1 | 8.96 | 44.80 |
P2 | 0.88 | 4.40 |
P3 | 1.41 | 7.06 |
P4 | 1.47 | 7.36 |
P5 | 0.94 | 4.72 |
P6 | 0.93 | 4.63 |
P7 | 2.05 | 10.24 |
P8 | 2.19 | 10.96 |
P9 | 7.04 | 35.20 |
P10 | 6.24 | 31.20 |
P11 | 5.04 | 25.20 |
P12 | 2.25 | 11.26 |
P13 | 1.10 | 5.49 |
P14 | 1.52 | 7.60 |
P15 | 3.00 | 15.01 |
P16 | 0.34 | 1.68 |
P17 | 46.63 | 233 |
P18 | 43.96 | 220 |
Gene | Primers | Oligonucleotide Sequence (5′→3′) | Label | Working Concentration | Amplicon Size bp | Used for | References |
---|---|---|---|---|---|---|---|
N | 2019-nCov_N-F | TTACAAACATTGGCCGCAAA | None | 20 μM | End-Point PCR | CDC, 2020 [10] | |
2019-nCov_N-R | GCGCGACATTCCGAAGAA | None | 20 μM | 66 | RT-qPCR | ||
2019-nCov_N-P | FAM-ACAATTTGCCCCCAGCGCTTCAG -BHQ1 | FAM, BHQ-1 | 5 μM | ddPCR | |||
Spike | 2019-nCov_Spike-F | CGGCCTTACTGTTTTGCCAC | None | 20 μM | 75 bp | Syber Green | Falzone et al., 2020 [12] |
2019-nCov_Spike-R | TGTACCCGCTAACAGTGCAG | None | 20 μM | RT-qPCR | |||
Spike/Orfa3 | 2019-nCov_Spike/Orfa3-F | TGAGCCAGTGCTCAAAGGAG | None | 20 μM | 195 | End-Point PCR | this paper |
2019-nCov_Spike/Orfa3-R | CGCCAACAATAAGCCATCCG | None | 20 μM | ||||
GAPDH | hGAPDH-F | CATGAGAAGTATGACAACAGCC | None | 20 μM | 115 | RT-qPCR | Carpenter et al., 2005 [14] |
hGAPDH-R | TGAGTCCTTCCACGATACC | None | 20 μM | ddPCR | |||
hGAPDH-P | FAM- AGCAATGCCTCCTGCACCACCAA -BHQ1 | FAM, BHQ-1 | 5 μM |
Sample ID | TaqMan RT-qPCR | ddPCR Absolute Quantification | TaqMan RT-qPCR | ddPCR Absolute Quantification | Spike Gene | |||
---|---|---|---|---|---|---|---|---|
GAPDH | GAPDH | N Gene | N Gene | SYBR RT-qPCR | Death Date | Autopsy Date | Burial Days | |
(Ct Value) | (Copies/μL Reaction) | (Ct Value) | (Copies/μL Reaction) | (Ct Value) | ||||
Decomposed Tissues | ||||||||
P1 | 26.6 | 6.0 | 28.9 | 10.2 | ND | 10/05/2020 | 11/06/2020 | 32 |
P2 | ND | 3.9 | 29.9 | 3.1 | ND | 02/05/2020 | 16/06/2020 | 45 |
P3 | ND | 2.3 | 28.5 | 160 | 28.98 | 20/04/2020 | 17/06/2020 | 58 |
P4 | ND | 3.0 | 30.8 | 9.6 | 29.1 | 03/05/2020 | 11/06/2020 | 39 |
P5 | ND | 3.1 | 29 | 6.4 | 30.1 | 18/04/2020 | 18/06/2020 | 61 |
P6 | ND | 2.8 | 29.2 | 5 | 30.9 | 15/04/2020 | 18/06/2020 | 64 |
P7 | ND | 3.1 | 27.4 | 169 | 25.9 | 29/04/2020 | 18/06/2020 | 50 |
P8 | 27.5 | 3.3 | 29.6 | 4.5 | 30.9 | 01/05/2020 | 16/06/2020 | 46 |
P9 | 25.4 | 2.5 | 17.2 | 50600 | 15.5 | 22/04/2020 | 17/06/2020 | 56 |
P10 | 26.4 | 3.0 | 24.5 | 410 | 19.4 | 24/04/2020 | 17/06/2020 | 54 |
P11 | 28.6 | 2.4 | 28.2 | 1.1 | 24.9 | 04/05/2020 | 11/06/2020 | 38 |
P12 | 31.6 | 2.5 | 29.8 | 1 | ND | 26/04/2020 | 16/06/2020 | 51 |
P13 | 28.9 | 1.9 | 30.2 | 3.2 | ND | 25/04/2020 | 16/06/2020 | 52 |
P14 | 26.6 | 3.5 | 21.3 | 1090 | 22.2 | 23/04/2020 | 17/06/2020 | 55 |
P15 | 28.5 | 3.0 | 27.5 | 135 | 28.2 | 18/05/2020 | 11/06/2020 | 24 |
P16 | 30.7 | 2.4 | 30.2 | 4.7 | 31.3 | 01/04/2020 | 18/06/2020 | 78 |
Fresh Cadaveric Tissue | ||||||||
P17 | 28.6 | 19.5 | 26.5 | 621 | 26.8 | |||
P18 | 24.0 | 14.1 | 29.3 | 12.2 | 35.2 | |||
Decomposed vs. Fresh Cadaveric Tissue | ||||||||
p-value | 0.578 | 0.121 | 0.888 | 0.363 | 0.2699 |
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Musso, N.; Falzone, L.; Stracquadanio, S.; Bongiorno, D.; Salerno, M.; Esposito, M.; Sessa, F.; Libra, M.; Stefani, S.; Pomara, C. Post-Mortem Detection of SARS-CoV-2 RNA in Long-Buried Lung Samples. Diagnostics 2021, 11, 1158. https://doi.org/10.3390/diagnostics11071158
Musso N, Falzone L, Stracquadanio S, Bongiorno D, Salerno M, Esposito M, Sessa F, Libra M, Stefani S, Pomara C. Post-Mortem Detection of SARS-CoV-2 RNA in Long-Buried Lung Samples. Diagnostics. 2021; 11(7):1158. https://doi.org/10.3390/diagnostics11071158
Chicago/Turabian StyleMusso, Nicolò, Luca Falzone, Stefano Stracquadanio, Dafne Bongiorno, Monica Salerno, Massimiliano Esposito, Francesco Sessa, Massimo Libra, Stefania Stefani, and Cristoforo Pomara. 2021. "Post-Mortem Detection of SARS-CoV-2 RNA in Long-Buried Lung Samples" Diagnostics 11, no. 7: 1158. https://doi.org/10.3390/diagnostics11071158
APA StyleMusso, N., Falzone, L., Stracquadanio, S., Bongiorno, D., Salerno, M., Esposito, M., Sessa, F., Libra, M., Stefani, S., & Pomara, C. (2021). Post-Mortem Detection of SARS-CoV-2 RNA in Long-Buried Lung Samples. Diagnostics, 11(7), 1158. https://doi.org/10.3390/diagnostics11071158