Applicability of Different Methods for Quantifying Virucidal Efficacy Using MENNO Florades and Tomato Brown Rugose Fruit Virus as an Example
Abstract
:1. Introduction
2. Results
2.1. Quantitative Double Antibody Sandwitch—ELISA (qELISA)
2.2. Quantification of ToBRFV Concentration via Infectivity (Local Lesion Assay)
2.3. RNA Extraction
2.4. End Point RT-PCR, Cloning, and Sequencing
2.5. Optimization of RT-qPCR
2.6. Quantification of ToBRFV Genomic RNA by RT-qPCR
3. Discussion
4. Materials and Methods
4.1. Virus Source
4.2. Quantitative ELISA
4.3. Local Lesion Tests and Models Used for Quantifications Based on the Bioassay
4.4. Disinfection Test
4.5. RNA Isolation and cDNA Synthesis
4.6. Primers and Probe Design
4.7. Cloning and Sequencing of the ToBRFV Partial Genome Used as Standard for RT-qPCR
4.8. Plasmid Standard Curve
4.9. In Vitro Transcription and RNA Standard Curve
4.10. Absolute RT-qPCR
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Sample Concentration or Name | Log of Virus Concentration | Mean Value of Reads | Log of Mean Values of Reads | Reacted | Calculated Concentration (mg/mL) | |
---|---|---|---|---|---|---|
Serially diluted ToBRFV particles to establish the standard curve for qELISA | 2,000,000 ng/mL | 6.3010 | 0.9045 | −0.0436 | positive * | 1.83 |
400,000 ng/mL | 5.6021 | 0.4274 | −0.3692 | positive | 0.47 | |
80,000 ng/mL | 4.9031 | 0.2674 | −0.5728 | positive | 0.085 | |
16,000 ng/mL | 4.2041 | 0.2456 | −0.6098 | positive | 0.017 | |
3200 ng/mL | 3.5052 | 0.1538 | −0.8130 | negative | NA ** | |
640 ng/mL | 2.8062 | 0.1031 | −0.9867 | negative | NA | |
128 ng/mL | 2.1072 | 0.1322 | −0.8788 | negative | NA | |
1,000,000 ng/mL | 6 | 0.8156 | −0.0885 | positive | 1.40 | |
100,000 ng/mL | 5 | 0.2922 | −0.5343 | positive | 0.11 | |
10,000 ng/mL | 4 | 0.1523 | −0.8173 | negative | NA | |
Germ carriers and plant sap loaded by 1 mg ToBRFV particles and treated with 4% MENNO Florades for 16 h | metal carrier untreated with MF | 0.0962 | −1.0168 | negative | NA | |
metal carrier disinfected with MF | 0.1045 | −0.9809 | negative | NA | ||
plastic carrier untreated with MF | 0.1012 | −1.9208 | negative | NA | ||
plastic carrier disinfected with MF | 0.1599 | −0.7962 | negative | NA | ||
plant sap untreated with MF | 0.0864 | −1.0635 | negative | NA | ||
plant sap disinfected with MF | 0.0829 | −1.0814 | negative | NA | ||
Inoculated plants with ToBRFV | N. benthamiana plant (1:10 diluted) | 0.8069 | −0.0932 | positive | 1.37 (13.7) *** | |
N. clevelandii plant (1:10 diluted) | 0.9091 | −0.0414 | positive | 1.85 (18.52) *** | ||
Negative control (N. clevelandii non-inoculated) | 0.1089 | −0.9630 | negative | NA | ||
Negative control (N. benthamiana non-inoculated) | 0.1013 | −0.9944 | negative | NA | ||
Substrate (p-Nitrophenyl phosphate) | 0.0738 | −1.1319 | NA | NA |
Virus Titer in Inoculum (mg/mL) | Calculated Virus Load in Inoculum or Remained Virus Load after Treatment with Disinfectant (mg/mL) Based on | |||
---|---|---|---|---|
The Kleczkowski Model [51] | The Growth Curve Model [49] | |||
2 | 87.130 | 2.0629 | 2.0267 | |
Virus titer in serially diluted ToBRFV isolate PV-1236 particles used as inoculum | 0.4 | 25.909 | 0.3367 | 0.3520 |
0.08 | 12.477 | 0.1167 | 0.1226 | |
0.016 | 2.616 | 0.0130 | 0.0128 | |
0.0032 | 1.128 | 0.0042 | 0.0038 | |
0.00064 | 0.493 | 0.0014 | 0.0012 | |
0.000128 | 0.030 | 0.0003 | 0.0002 | |
N. clevelandii | unknown | 85.9892 | 2.0218 | 1.9885 |
N. benthamiana | unknown | 41.0477 | 1.7765 | 1.7647 |
disinfected metal carrier ** | unknown | 0.00689 | 0.00001 | 0.00000 |
disinfected plastic carrier | unknown | 0.02001 | 0.0004 | 0.00045 |
disinfected sap | unknown | 0.09529 | 0.00019 | 0.00011 |
Model parameters *** | N = 11,064, λ = 2, = 5.16 | N= 1134.53, β = 20, γ = 1.64 | ||
2.40 | 1.60 |
Name | Sequence 5’ → 3’ | Start | End | Application |
---|---|---|---|---|
ToBRFV-1482-s | TAATCAGCAAGTTTAGTTTG | 1482 | 1501 | D, C |
ToBRFV-1677-as | TCAGTCACTAATCTATCGTG | 1677 | 1658 | D, C |
ToBRFV-4750-as | GGATCTTCTGAACTCTTCTA | 4750 | 4731 | C, I |
ToBRFV-4640-s | ATACATCATGACAGAGGGTG | 4640 | 4659 | C |
ToBRFV-6323-as | GCCTACGGATGTGTATGAAC | 6342 | 6323 | C |
ToBRFV-KpnI-4388-s | GTTTATGGTACCAGAGAAAGAG | 4389 | 4410 | C |
ToBRFV-HindIII-6153-as | CTCTAAGCTTACCATTGTAAACCGGATGCAC | 6173 | 6153 | C |
ToBRFV-CP-Eco47-s | CGTAGAGTAGATGACGCAACG | 6050 | 6070 | C |
ToBRFV-HindIII-3UTR-as | TATATAAGCTTGCATGCTGGGCCCCTACCGGGGGTTCCGGGGGAAT | 6393 | 6366 | C, I |
ToBRFV-2703-s | AAGCCACAAGAGATAATGTTCGTA | 2703 | 2726 | Q |
ToBRFV-2838-as | CAATTTCGCACAGAGACATAG | 2858 | 2838 | Q, I |
ToBRFV-2760L | 6FAM-CTGACAGCGTGTTCCTTTACCG-BHQ1 | 2773 | 2752 | Q |
Sample | Total RNA Used in cDNA (ng) | Ct1 ± (SD) 2 | Quantity ± (SD) | Calculated Virus Concentration | ||
---|---|---|---|---|---|---|
Copy No. per µg Total RNA | VP 3 (mg) per µg Total RNA | VP (mg) per 100 mg Tissue | ||||
0.1 mg virus particles (diluted 1:5000) 4 | 500 | 17.39 ± (0.087) | 2,730,724.75 ± (162233) | 2.7307 × 1010 | 0.0018 | 0.0056 |
0.5µg in vitro transcript RNA (diluted 1:5000) equal to 0.0126 mg particles 5 | 500 | 15.264 ± (0.077) | 6,738,004 ± (339,407.656) | 6.738 × 1010 | 0.0045 | NA 6 |
Inoculated N. clevelandii (diluted 1:50,000) | 500 | 22.793 ± (0.272) | 45,962.41 ± (8171.118) | 4.596 × 109 | 0.0003 | 0.0023 |
Inoculated N. benthamiana (diluted 1:5000) | 500 | 23.098 ± (0.333) | 37,752.36 ± (8331.31) | 3.775 × 109 | 0.0002 | 0.0013 |
N. clevelandii containing 0.1 mg ToBRFV particles (diluted 1:50,000) | 1000 | 21.644 ± (0.087) | 97,568.10 ± (5498.168) | 4.88 × 109 | 0.0003 | 0.0025 |
N. clevelandii containing 1 mg ToBRFV particles (diluted 1:50,000)(i.e., plant sap untreated with MF) | 1000 | 20.891 ± (0.068) | 160,767.54 ± (7322.144) | 8.03109 | 0.0005 | 0.0128 |
metal carrier untreated with MF | NA | |||||
metal carrier disinfected with MF | NA | |||||
plastic carrier untreated with MF | NA | |||||
plastic carrier disinfected with MF | NA | |||||
plant sap untreated with MF | NA |
Sample Concentration or Name | qELISA mg/mL | RT-qPCR mg/mL | Bioassay Calculated Virus Concentration (mg/mL) | |
---|---|---|---|---|
inoculum | 1 mg/mL | 1.37 | 0.0056 † | 1 ‡ |
Germ carriers and plant sap loaded by 1 mg ToBRFV particles and treated with 4% MENNO Florades (MF) for 16 h | metal carrier untreated with MF * | NA (negative) ** | NA (negative) *** | 0.95 |
metal carrier disinfected with MF | NA (negative) | NA (negative) | 0.000002 | |
plastic carrier untreated with MF | NA (negative) | NA (negative) | 0.93 | |
plastic carrier disinfected with MF | NA (negative) | NA (negative) | 0.00011 | |
plant sap untreated with MF | NA (negative) | NA (negative) | 1.003 | |
plant sap disinfected with MF | NA (negative) | NA (negative) | 0.00011 | |
Inoculated plants with ToBRFV | N. benthamiana plant | 13.7 | 0.0013 | 1.76–1.78 |
N. clevelandii plant | 18.5 | 0.0023 | 1.99–2.02 |
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Nourinejhad Zarghani, S.; Ehlers, J.; Monavari, M.; von Bargen, S.; Hamacher, J.; Büttner, C.; Bandte, M. Applicability of Different Methods for Quantifying Virucidal Efficacy Using MENNO Florades and Tomato Brown Rugose Fruit Virus as an Example. Plants 2023, 12, 894. https://doi.org/10.3390/plants12040894
Nourinejhad Zarghani S, Ehlers J, Monavari M, von Bargen S, Hamacher J, Büttner C, Bandte M. Applicability of Different Methods for Quantifying Virucidal Efficacy Using MENNO Florades and Tomato Brown Rugose Fruit Virus as an Example. Plants. 2023; 12(4):894. https://doi.org/10.3390/plants12040894
Chicago/Turabian StyleNourinejhad Zarghani, Shaheen, Jens Ehlers, Mehran Monavari, Susanne von Bargen, Joachim Hamacher, Carmen Büttner, and Martina Bandte. 2023. "Applicability of Different Methods for Quantifying Virucidal Efficacy Using MENNO Florades and Tomato Brown Rugose Fruit Virus as an Example" Plants 12, no. 4: 894. https://doi.org/10.3390/plants12040894