Detection and Phylogenetic Analyses of Taura Syndrome Virus from Archived Davidson’s-Fixed Paraffin-Embedded Shrimp Tissue
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sample Selection
2.2. RNA Extraction and Quality Assessment
2.3. Detection of TSV through Real-Time RT-PCR (RT-qPCR)
2.4. Statistical Analysis
2.5. Detection of Eukaryotic Translation Elongation Factor 1 Alpha (EF-1α) through Real-Time RT-PCR
2.6. TSV Primer Design
2.7. Amplification of TSV by RT-PCR and Amplicon Sequencing
2.8. Phylogenetic Analysis
3. Results
3.1. RNA Quantity and Quality Assessment
3.2. Detection of TSV Using RT-qPCR
3.3. Detection of TSV Capsid Protein Genes (VP1 and VP2), RdRp and Internal Control Gene EF-1α by Conventional RT-PCR
3.4. Phylogenetic Analysis
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Data Availability
References
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Case No. | Norgen KitSample # | Qiagen KitSample # | Invitrogen KitSample # | Year | Origin | Species | Grade of Infection |
---|---|---|---|---|---|---|---|
1 | 1 | 58 | 73 | 2005 | Belize | L. vannamei | G3 |
2 | 2 | 59 | 74 | 2005 | Venezuela | L. vannamei | G3, G4 |
3 | 3 | 60 | 75 | 2005 | Thailand | P. monodon | G3, G4 |
4 | 4 | 61 | 76 | 2005 | Hawaii | L. vannamei | G2–4 |
5 | 5 | 62 | 77 | 2005 | Hawaii | L. vannamei | G3, G4 |
6 | 6 | 63 | 78 | 2005 | Venezuela | L. vannamei | G3 |
7 | 7 | 64 | 79 | 2005 | Thailand | P. monodon | G3 |
8 | 8 | 65 | 80 | 2005 | Belize | L. vannamei | G3 |
9 | 9 | 66 | 81 | 2005 | Hawaii | L. vannamei | G3, G4 |
10 | 10 | 67 | 82 | 2005 | Venezuela | L. vannamei | G3, G4 |
11 | 11 | 68 | 83 | 2005 | Belize | L. vannamei | G4 |
12 | 12 | 69 | 84 | 2005 | Thailand | L. vannamei | G4 |
13 | 13 | 70 | 85 | 2005 | Belize | L. vannamei | G4 |
14 | 14 | 71 | 86 | 2005 | Belize | L. vannamei | G3, G4 |
15 | 15 | 72 | 87 | 2005 | Venezuela | L. vannamei | G3, G4 |
16 | 44 | 30 | 16 | 2005 | Belize | L. vannamei | G4 |
17 | 45 | 31 | 17 | 2005 | Thailand | L. vannamei | G4 |
18 | 46 | 32 | 18 | 2005 | Venezuela | L. vannamei | G3, G4 |
19 | 47 | 33 | 19 | 2005 | Belize | L. vannamei | G4 |
20 | 48 | 34 | 20 | 2005 | Thailand | L. vannamei | G4 |
21 | 49 | 35 | 21 | 2005 | Venezuela | L. vannamei | G3 |
22 | 50 | 36 | 22 | 2005 | Hawaii | L. vannamei | G3, G4 |
23 | 51 | 37 | 23 | 2005 | Hawaii | L. vannamei | G3, G4 |
24 | 52 | 38 | 24 | 2005 | Venezuela | L. vannamei | G2–4 |
25 | 53 | 39 | 25 | 2005 | Hawaii | L. vannamei | G3 |
26 | 54 | 43 | 26 | 2005 | Thailand | L. vannamei | G3 |
27 | 55 | 42 | 27 | 2005 | Hawaii | L. vannamei | G3 |
28 | 56 | 40 | 28 | 2005 | Thailand | P. monodon | G3, G4 |
29 | 57 | 41 | 29 | 2005 | Thailand | P. monodon | G3, G4 |
Primer Name | Location in TSV Genome | Nucleotide Position in the Genome of TSV Reference Strain | Sequence (5′–3′) | Amplicon Size (bp) |
---|---|---|---|---|
TSV 1-F | VP1 | 7901 | GGCGTAGTGAGTAATGTAGCT | 137 |
TSV 1-R | VP1 | AGAGACAGGGGTACGCCATA | ||
TSV 2-F | VP1 | 7955 | ACGAAAGTCAACGCATATGAGA | 123 |
TSV 2-R | VP1 | AGGCACTGCAATTGTGGGAT | ||
TSV 3-F | VP1 | 8057 | GATCCCACAATTGCAGTGCC | 125 |
TSV 3-R | VP1 | AGAGACAGGGGTACGCCATA | ||
TSV 4-F | VP1 | 8155 | TGACACTCCTGATGCGCATG | 122 |
TSV 4-R | VP1 | CTAGACTAACTGGGGCAGCG | ||
TSV 5-F | VP1 | 8257 | CGCTGCCCCAGTTAGTCTAG | 122 |
TSV 5-R | VP1 | AGGGGAGATATTGCACCAGC | ||
TSV 6-F | VP1 | 8359 | GCTGGTGCAATATCTCCCCT | 150 |
TSV 6-R | VP1 | GGATCGTACACTCGCATCCA | ||
TSV 7-F | VP1 | 8414 | TCACAGATCATCGACATCTCACA | 124 |
TSV 7-R | VP1 | CACAATCTGCCGTGTACCCA | ||
TSV 8-F | VP1 | 8518 | TGGGTACACGGCAGATTGTG | 145 |
TSV 8-R | VP1 | AAGCGTACCTGGTTCAGCAA | ||
TSV 9-F | VP1 | 8643 | TTGCTGAACCAGGTACGCTT | 149 |
TSV 9-R | VP1 | TTCCCCCAAAGACACCTTCG | ||
TSV 10-F | VP1 | 8721 | CAGTAACGCGTGCTCCAGTA | 147 |
TSV 10-R | VP1 | GCAGTCCGGCATAAGCTAGT | ||
TSV 11-F | VP1 | 8834 | GGTGGAAGGCACAGACTAGC | 121 |
TSV 11-R | VP1 | CAAGAGTTGGAGCGCTGGTA | ||
TSV 12-F | VP1 | 8935 | TACCAGCGCTCCAACTCTTG | 150 |
TSV 12-R | VP1 | TCACCAATCGCTGCCATACT | ||
TSV 13-F | VP1 | 9031 | TGGTATTTCCGAGGAGACGT | 137 |
TSV 13-R | VP1 | TCACTGGAGCTTTGGACTCA | ||
TSV 14-F | VP1 | 9071 | GCAGCGATTGGTGAAGCTAC | 127 |
TSV 14-R | VP1 | TGACCACGGTATAGTTACCTGG | ||
TSV 15-F | VP2 | 6948 | TGCCTGCTAACCCAGTTGAA | 55 |
TSV 15-R | VP2 | AGTCCTCCACTGGTTGTTGT | ||
TSV 16-F | VP2 | 7117 | AGTCCAGGACCAAGCTCTCA | 119 |
TSV 16-R | VP2 | CTGTTGCAAGCTGTTCCTGC | ||
TSV 17-F | VP2 | 7233 | CAGAATTCAATCAGCCACAC | 131 |
TSV 17-R | VP2 | TACTCGTACAGTAACCTCGT | ||
TSV 18-F | RdRp | 5194 | CAATGGCCATTGGTTCCGTT | 111 |
TSV 18-R | RdRp | TATACAAGGTAGCGGGGGCT | ||
TSV 19-F | RdRp | 5576 | GTGGTTGGGCTCTGAGGAAT | 117 |
TSV 19-R | RdRp | GCCGCAAAAATACCCAAGCT | ||
TSV 20-F | RdRp | 6099 | AACCATTCTCAGCCTTCCGG | 105 |
TSV 20-R | RdRp | CCCGTTTTCTCGCTGAGCTA | ||
TSV 21-F | RdRp | 6529 | AAACAACGCGCATTGCTTCT | 101 |
TSV 21-R | RdRp | GTACCCTGCGTTCCTACACG | ||
TSV 9195F * | Intergenic region/Open Reading Frame (ORF) 2 | 9195 | TCAATGAGAGCTTGGTCC | 231 |
TSV 9992R * | Intergenic region/ORF 2 | AAGTAGACAGCCGCGCTT | ||
TSV 1004F ** | ORF1 | 1004 | TTGGGCACCCGACATT | 72 |
TSV 1075R ** | ORF1 | GGGAGCTTAAACTGGACACACTGT |
GenBank Accession Number | Geographical Origin | Year | Species |
---|---|---|---|
FJ876481 | Belize | 2009 | P. vannamei |
FJ876490 | Belize | 2009 | P. vannamei |
FJ876507 | Nicaragua | 2009 | P. vannamei |
FJ876516 | Nicaragua | 2009 | P. vannamei |
FJ876520 | Taiwan | 2009 | P. vannamei |
FJ876487 | Indonesia | 2009 | P. vannamei |
FJ876509 | China | 2009 | P. vannamei |
AY997025 | Thailand | 2005 | P. vannamei |
GQ359322 | Texas, USA | 2010 | P. vannamei |
GQ502201 | Texas, USA | 2010 | P. vannamei |
FJ876492 | Texas, USA | 2009 | P. vannamei |
FJ876469 | Hawaii | 2009 | P. vannamei |
AF277675 | Hawaii | 1994 | P. vannamei |
FJ876468 | Hawaii | 2009 | P. vannamei |
FJ876466 | Ecuador | 2009 | P. vannamei |
FJ876461 | Ecuador | 2009 | P. vannamei |
FJ876513 | Ecuador | 2009 | P. vannamei |
FJ876512 | Ecuador | 2009 | P. vannamei |
FJ7876493 | Mexico | 2009 | P. vannamei |
DQ000302 | Eritrea | 2006 | P. monodon |
FJ876495 | Eritrea | 2009 | P. vannamei |
FJ876508 | Aruba | 2009 | P. vannamei |
FJ876511 | Aruba | 2009 | P. vannamei |
FJ876503 | Venezuela | 2009 | P. vannamei |
DQ212790 | Venezuela | 2005 | P. vannamei |
FJ876502 | Venezuela | 2009 | P. vannamei |
JQ356858 | Saudi Arabia | 2012 | P. indicus |
JX094350 | Saudi Arabia | 2012 | P. indicus |
Extraction Kit | Mean RNA Concentration (ng/µL) | Mean 260/280 Ratio | Mean 260/230 Ratio | Mean Ct Value (TSV) | Mean Ct Value (EF-1α) |
---|---|---|---|---|---|
Norgen Biotek FFPE RNA Purification Kit | 254.90 ± 109.5 | 1.87 ± 0.05 | 1.49 ± 0.34 | 24.12 ± 3.4 * | 28.25 ± 2.3 |
Qiagen RNeasy FFPE Kit | 249.20 ± 179.5 | 1.91 ± 0.06 | 2.00 ± 0.14 | 22.90 ± 3.3 * | 28.49 ± 1.6 |
Invitrogen PureLink FFPE RNA Isolation Kit | 355.30 ± 207.8 | 1.86 ± 0.05 | 1.49 ± 0.27 | 27.18 ± 3.9 | 28.68 ± 1.7 |
Sample No. | Total Number of Amplicons Amplified from VP1 Gene (out of 14 Amplicons) | Total Number of Amplicons Amplified fromVP2 Gene (out of 3 Amplicons) | Total Number of Amplicons Amplified from RdRp Gene (out of 4 Amplicons) | Total Number Amplified (of 21 Amplicons) |
---|---|---|---|---|
1 | 8 | 3 | 3 | 14 |
2 | 6 | 3 | 3 | 12 |
3 | 0 | 2 | 1 | 3 |
4 | 14 | 3 | 4 | 21 |
5 | 11 | 3 | 3 | 17 |
6 | 12 | 3 | 3 | 18 |
7 | 0 | 2 | 0 | 2 |
8 | 9 | 3 | 3 | 15 |
9 | 3 | 3 | 1 | 7 |
10 | 14 | 3 | 4 | 21 |
11 | 6 | 3 | 3 | 12 |
12 | 7 | 3 | 2 | 12 |
13 | 3 | 3 | 3 | 8 |
14 | 14 | 3 | 4 | 21 |
15 | 13 | 3 | 3 | 19 |
16 | 10 | 3 | 4 | 17 |
17 | 8 | 3 | 0 | 11 |
18 | 12 | 3 | 3 | 18 |
19 | 3 | 3 | 3 | 9 |
20 | 6 | 3 | 4 | 13 |
21 | 12 | 3 | 3 | 18 |
22 | 7 | 3 | 3 | 13 |
23 | 4 | 3 | 0 | 7 |
24 | 4 | 3 | 1 | 8 |
26 | 5 | 3 | 2 | 10 |
27 | 14 | 3 | 4 | 21 |
28 | 0 | 2 | 0 | 2 |
29 | 3 | 3 | 1 | 7 |
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Ochoa, L.M.; Cruz-Flores, R.; Dhar, A.K. Detection and Phylogenetic Analyses of Taura Syndrome Virus from Archived Davidson’s-Fixed Paraffin-Embedded Shrimp Tissue. Viruses 2020, 12, 1030. https://doi.org/10.3390/v12091030
Ochoa LM, Cruz-Flores R, Dhar AK. Detection and Phylogenetic Analyses of Taura Syndrome Virus from Archived Davidson’s-Fixed Paraffin-Embedded Shrimp Tissue. Viruses. 2020; 12(9):1030. https://doi.org/10.3390/v12091030
Chicago/Turabian StyleOchoa, Lauren Marie, Roberto Cruz-Flores, and Arun K. Dhar. 2020. "Detection and Phylogenetic Analyses of Taura Syndrome Virus from Archived Davidson’s-Fixed Paraffin-Embedded Shrimp Tissue" Viruses 12, no. 9: 1030. https://doi.org/10.3390/v12091030