Evaluation of Pre-Analytical and Analytical Methods for Detecting SARS-CoV-2 in Municipal Wastewater Samples in Northern Italy
Abstract
:1. Introduction
2. Materials and Methods
2.1. Generation of Wastewater Samples Stock
2.2. Pre-Analytical Process: Concentration of Sewage Samples
- (1)
- Dextran and polyethylene glycol-6000 (PEG) two-phase separation according to the 2003 WHO Guidelines for Environmental Surveillance of Poliovirus protocol [14], omitting the chloroform treatment to preserve the integrity of the SARS-CoV-2 envelope, as described, firstly, by La Rosa, G. et al. [15]. Briefly, 250 mL of wastewater sample was centrifuged for 30 min at 4500× g to pellet the wastewater solids, retaining the pellet for further processing. The clarified wastewater was mixed with dextran and PEG-6000 (19.8 mL of 22% dextran, 143.5 mL 29% PEG 6000, 17.5 mL 5 N NaCl); after a constant agitation for 30 min using a horizontal shaker, the mixture was left to stand overnight at 4 °C in a separation funnel. The bottom layer and the interphase were then collected drop-wise; this concentrate was added to the wastewater solids [14].
- (2)
- (3)
- PEG-8000 precipitation of 250 mL of sewage, modified from Wu, F. et al. [16], as follows:
2.3. Pre-Analytical Process: RNA Extraction from Concentrated Sewage Samples
2.4. Analytical Process: Real-Time RT-PCR Assays
2.5. Pre-Analytical and Analytical Workflows
2.6. Evaluation of SARS-CoV-2 Recovery Efficiency
SARS-CoV-2 copies/µL seeded
2.7. Evaluation of SARS-CoV-2 RT-PCR Assays Efficiency
2.8. Data Analysis
3. Results
3.1. Performance of the Concentration Methods
3.2. SARS-CoV-2 Recovery Efficiency
3.3. Real-Time RT-PCR Efficiency
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Spiked Sample by SARS-CoV-2 Viral Load: 4.7 × 107 Copies/mL | Unknown Sample | |||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Volume of Sewage to be Concentrated | Methods of Concentration | Volume of Concentrating Sewage | RNA Extraction Kit | Extraction Input Elution Volume | RT-qPCR Kit | RT-PCR Instrument | Tournaround Time | Target | Positive Replicates | Mean Ct Value | SD Ct Value | ΔCt (Recoverded vs. Seeded) | Mean Recovery Efficiency (%) | Positive Replicates | Mean Ct Value | SD Ct Value |
250 mL | (A) Dextran and PEG-6000 two-phase separation | nearly 10 mL | QIAamp MinElute Virus Spin Kit (QIAGEN) | 400 μL 60 μL | AGPATH-ID ONE-STEP RT-PCR (Thermo Fisher) | Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher) | 16-h | N1 | 100% | 29.2 | 0.62 | 7.2 | 2% | 17% | 37.27 | / |
N3 | 100% | 30.4 | 0.85 | und | und | / | ||||||||||
ORF | 100% | 36.5 | 0.79 | und | und | / | ||||||||||
NucliSens EasyMag (bioMerieux) | 400 μL 100 μL | AGPATH-ID ONE-STEP RT-PCR (Thermo Fisher) | 16-h | N1 | 100% | 26.2 | 0.38 | 4.2 | 12% | 67% | 37.9 | 0.45 | ||||
N3 | 100% | 25.6 | 0.41 | und | und | / | ||||||||||
ORF | 100% | 28.9 | 0.23 | und | und | 0.02 | ||||||||||
NucliSens EasyMag (bioMerieux) | 4 mL 60 μL | AGPATH-ID ONE-STEP RT-PCR (Thermo Fisher) | 16-h | N1 | 100% | 27.6 | 0.25 | 5.6 | 5% | 33% | 38.2 | 0.02 | ||||
N3 | 100% | 27 | 0.22 | 67% | 37.6 | 0.2 | ||||||||||
ORF | 100% | 30.4 | 0.17 | und | und | / | ||||||||||
80 mL | (B) PEG-8000 precipitation without chloroform purification step | 1.5 mL | QIAamp MinElute Virus Spin Kit (QIAGEN) | 400 μL 60 μL | AGPATH-ID ONE-STEP RT-PCR (Thermo Fisher) | Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher) | 8-h | N1 | 100% | 20.1 | 0.13 | −1.9 | 76% | 100% | 33.1 | 0.4 |
N3 | 100% | 20.5 | 0.20 | 100% | 33.4 | 0.34 | ||||||||||
ORF | 100% | 24.6 | 0.30 | 100% | 37.5 | 0.75 | ||||||||||
NucliSens EasyMag (bioMerieux) | 500 μL 100 μL | AGPATH-ID ONE-STEP RT-PCR (Thermo Fisher) | 8-h | N1 | 100% | 21.4 | 0.38 | 0.6 | 31.4% | 100% | 34.1 | 0.22 | ||||
N3 | 100% | 19.7 | 0.31 | 100% | 32.6 | 0.19 | ||||||||||
ORF | 100% | 24.6 | 0.43 | 100% | 37.2 | 0.34 | ||||||||||
250 mL | (C) PEG-8000 precipitation with chloroform purification step | 6–10 mL | QIAamp MinElute Virus Spin Kit (QIAGEN) | 400 μL 60 μL | QScript XLT 1-Step RT-PCR ToughMix (QuantaBio) | CFX96 BioRad real-time PCR System (Biorad) | 8-h | N1 | 100% | 25.6 | 1.27 | 3.6 | 18% | 83% | 37.7 | 1.69 |
N3 | 100% | 26.9 | 1.05 | 67% | 36.8 | 0.46 | ||||||||||
ORF | 100% | 32.8 | 1.08 | und | und | / | ||||||||||
NucliSens EasyMag (bioMerieux) | 400 μL 100 μL | QScript XLT 1-Step RT-PCR ToughMix (QuantaBio) | 8-h | N1 | 100% | 29.2 | 0.33 | 7.2 | 2% | 50% | 38.7 | 0.3 | ||||
N3 | 100% | 29.5 | 0.43 | 67% | 37.4 | 0.01 | ||||||||||
ORF | 100% | 32.5 | 0.57 | und | und | / | ||||||||||
NucliSens EasyMag (bioMerieux) | 4 mL 60 μL | QScript XLT 1-Step RT-PCR ToughMix (QuantaBio) | 8-h | N1 | 100% | 29.3 | 0.5 | 7.3 | 1% | 17% | 38.03 | / | ||||
N3 | 100% | 26.1 | 0.38 | 67% | 37.9 | 0.46 | ||||||||||
ORF | 100% | 33.1 | 1.41 | und | und | / |
Target | Standard Curve | R2 | Efficiency | |
---|---|---|---|---|
AgPath-ID One-Step RT-PCR™ kit | N1 | y = −3.3863x + 37.009 | 0.9984 | 98.4% |
N3 | y = −3.3677x + 38.426 | 0.9982 | 98.2% | |
QScript XLT 1-Step RT-PCR ToughMix® | N1 | y = −3.279x + 39.076 | 0.9988 | 98.8% |
N3 | y = −3.3073x + 40.08 | 0.9995 | 99.5% | |
TaqMan™ Fast Virus 1-Step Master Mix | N1 | y = −3.7356x + 41.786 | 0.9985 | 85.2% |
N3 | y = −3.5652x + 38.426 | 0.9971 | 90.8% |
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Pellegrinelli, L.; Castiglioni, S.; Cocuzza, C.E.; Bertasi, B.; Primache, V.; Schiarea, S.; Salmoiraghi, G.; Franzetti, A.; Musumeci, R.; Tilola, M.; et al. Evaluation of Pre-Analytical and Analytical Methods for Detecting SARS-CoV-2 in Municipal Wastewater Samples in Northern Italy. Water 2022, 14, 833. https://doi.org/10.3390/w14050833
Pellegrinelli L, Castiglioni S, Cocuzza CE, Bertasi B, Primache V, Schiarea S, Salmoiraghi G, Franzetti A, Musumeci R, Tilola M, et al. Evaluation of Pre-Analytical and Analytical Methods for Detecting SARS-CoV-2 in Municipal Wastewater Samples in Northern Italy. Water. 2022; 14(5):833. https://doi.org/10.3390/w14050833
Chicago/Turabian StylePellegrinelli, Laura, Sara Castiglioni, Clementina E. Cocuzza, Barbara Bertasi, Valeria Primache, Silvia Schiarea, Giulia Salmoiraghi, Andrea Franzetti, Rosario Musumeci, Michela Tilola, and et al. 2022. "Evaluation of Pre-Analytical and Analytical Methods for Detecting SARS-CoV-2 in Municipal Wastewater Samples in Northern Italy" Water 14, no. 5: 833. https://doi.org/10.3390/w14050833