Improvement of a Real-Time Reverse Transcription–Polymerase Chain Reaction Assay for the Sensitive Detection of the F Gene of Avian Orthoavulavirus-1 (AOAV-1)
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Virus Strains and Field Samples
2.2. Primers and Probe
2.3. Generation of Recombinant Plasmids and Synthesis of Reference RNA Transcripts
2.4. Real Time RT-PCR Assays
2.5. Determination of Analytical Sensitivity
3. Results
3.1. Primer and Probe Modification
3.2. Analytical Sensitivity of the AOAV-1 Fmod rRT-PCR Assay
3.3. Detection of the F Gene of AOAV-1 in Field Samples
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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No. | Host | Country | Year | Genotype a | Gen Bank No. or Internal Identifier |
---|---|---|---|---|---|
1 | Pigeon (Columba livia domestica) | Belgium | 1998 | XXI.2 | JX901110.1 |
2 | Pigeon (Columba livia domestica) | Germany | 1998 | KJ736742.1 | |
3 | Pigeon (Columba livia domestica) | Ireland | 2004 | XXI.2 | JN986839.1 |
4 | Pigeon (Columba livia domestica) | Belgium | 2011 | JX901122.1 | |
5 | European turtle dove (Streptopelia turtur) | Italy | 2012 | KU377536.1 | |
6 | Pigeon (Columba livia domestica) | Italy | 2014 | KU377537.1 | |
7 | Pigeon (Columba livia domestica) | Italy | 2019 | VI.2.1.1.2.2 | 19VIR1689-2, PPMV-1 |
8 | Pigeon (Columba livia domestica) | Switzerland | 2021 | VI.2.1.1.2.2 | V82-2 |
9 | Pigeon (Columba livia domestica) | Switzerland | 2021 | VI.2.1.1.2.2 | V86-3 |
10 | Pigeon (Columba livia domestica) | Switzerland | 2021 | VI.2.1.1.2.2 | V90-1 |
11 | Pigeon (Columba livia domestica) | Ukraine | 2011 | XXI.1.1 | KJ914672 [11] |
12 | Chicken (Gallus gallus) | Netherlands | 1993 | VII.2 | JN986837.1 |
13 | Chicken (Gallus gallus) | USA | 1993 | XIX | AY562986.1 |
14 | Chicken (Gallus gallus) | Mexico | 1996 | V.1 | AY288999.1 |
15 | Chicken (Gallus gallus) | Italy | 2000 | VI.2.1.1.2.1 | 00VIR2875, APMV-1_2 |
16 | Chicken (Gallus gallus) | Italy | 2000 | XIII.1.1 | 00VIR3282, APMV-1_3 |
17 | Chicken (Gallus gallus) | Belgium | 2018 | MN547991.1 | |
18 | Chicken (Gallus gallus) | Botswana | 2019 | MT091063.1 | |
19 | NDV VG/GA vaccine strain | I.1.1 |
Name | Wise-Based F Gene 5′ → 3′ | Name | Modified (AOAV-1 Fmod) 5′ → 3′ | |
---|---|---|---|---|
For | F+4839 | TCCGGAGGATACAAGGGTCT | F+4839_m2 | TCCGCARGATACAAGGRTCT |
Probe | F+4894 | AAGCGTTTCTGTCTCCTTCCTCCA | NDF_FAM_m4 | AARCGYTTCTGYCTCCTTCCTCCHG |
Rev | F−4939 | AGCTGTTGCAACCCCAAG | F−4939_m3 | GMTGTYGCAACCCCAAGAG |
Target | ||||
---|---|---|---|---|
Parameter | Primer/Probe Set Genotype | PPMV-1 VI.2.1.1.2.2 | APMV-1_2 VI.2.1.1.2.1 | APMV-1_3 XIII.1.1 |
r2 | Modified (AOAV-1 Fmod) | 0.98 | 0.99 | 0.97 |
Wise-based F gene | 0.87 | 0.96 | 0.99 | |
E | Modified (AOAV-1 Fmod) | 92.2% | 100.2% | 96.3% |
Wise-based F gene | 153.3% | 172.7% | 83.7% | |
Detection limit a | Modified (AOAV-1 Fmod) | 102 | 102 | 102 |
Wise-based F gene | 103 | 103 | 104 |
No. | Pigeon No. | CT AOAV-1 Fmod | CT Wise-Based F Gene | CT M-Gene | No. | Pigeon No. | CT AOAV-1 Fmod | CT Wise-Based F Gene | CT M-Gene |
---|---|---|---|---|---|---|---|---|---|
1 | 0115 | 32.15 | nd | 32.50 | 21 | 0296 | 27.16 | 33.71 | 28.96 |
2 | 0116 | 34.15 | nd | 34.01 | 22 | 0297 a | 18.45 | 27.89 | 19.32 |
3 | 0147 a | 17.75 | 24.96 | 19.65 | 23 | 0305 | 23.82 | 29.84 | 25.77 |
4 | 0148 | 26.25 | 32.01 | 28.35 | 24 | 0306 a | 30.15 | nd | 33.14 |
5 | 0149 | 20.06 | 26.63 | 22.06 | 25 | 0311 | 21.50 | 26.70 | 22.85 |
6 | 0172 | 34.85 | nd | 35.20 | 26 | 0315 | 25.64 | nd | 26.82 |
7 | 0212 | 35.79 | nd | 38.41 | 27 | 0316 | 32.98 | nd | 34.08 |
8 | 0217 | 35.51 | nd | 38.83 | 28 | 0345 a | 27.69 | nd | 29.66 |
9 | 0218 | 18.48 | 25.89 | 20.45 | 29 | 0352 | 33.00 | nd | 36.54 |
10 | 0227 | 21.80 | 28.32 | 26.06 | 30 | 0355 | 30.80 | nd | 33.24 |
11 | 0228 a | 20.05 | 26.19 | 20.85 | 31 | 0359 a | 32.86 | nd | 34.11 |
12 | 0230 | 23.57 | 28.83 | 24.22 | 32 | 0360 | 35.28 | nd | 39.63 |
13 | 0234 | 34.22 | nd | 36.13 | 33 | 0366 | 36.93 | nd | 40.34 |
14 | 0236 | 35.99 | nd | 38.58 | 34 | 0368 | 33.87 | nd | 39.16 |
15 | 0259 | 34.48 | nd | 39.79 | 35 | 0369 | 34.88 | nd | 35.79 |
16 | 0261 a | 29.31 | nd | 30.58 | 36 | 0371 | 38.02 | nd | 41.50 |
17 | 0263 | 27.57 | 32.73 | 28.78 | 37 | 0374 | 33.85 | nd | 35.17 |
18 | 0286 | 33.76 | nd | 35.47 | 38 | 0377 | 31.96 | nd | 34.04 |
19 | 0289 | 34.17 | nd | 36.41 | 39 | 0379 | 32.19 | nd | 33.49 |
20 | 0293 | 33.62 | nd | 34.78 | 40 | 0382 a | 19.20 | nd | 19.42 |
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Bhande, P.; Sigrist, B.; Balke, L.; Albini, S.; Wolfrum, N. Improvement of a Real-Time Reverse Transcription–Polymerase Chain Reaction Assay for the Sensitive Detection of the F Gene of Avian Orthoavulavirus-1 (AOAV-1). Vet. Sci. 2023, 10, 223. https://doi.org/10.3390/vetsci10030223
Bhande P, Sigrist B, Balke L, Albini S, Wolfrum N. Improvement of a Real-Time Reverse Transcription–Polymerase Chain Reaction Assay for the Sensitive Detection of the F Gene of Avian Orthoavulavirus-1 (AOAV-1). Veterinary Sciences. 2023; 10(3):223. https://doi.org/10.3390/vetsci10030223
Chicago/Turabian StyleBhande, Prerana, Brigitte Sigrist, Linard Balke, Sarah Albini, and Nina Wolfrum. 2023. "Improvement of a Real-Time Reverse Transcription–Polymerase Chain Reaction Assay for the Sensitive Detection of the F Gene of Avian Orthoavulavirus-1 (AOAV-1)" Veterinary Sciences 10, no. 3: 223. https://doi.org/10.3390/vetsci10030223