Acta Veterinaria et Zootechnica Sinica ›› 2024, Vol. 55 ›› Issue (8): 3590-3599.doi: 10.11843/j.issn.0366-6964.2024.08.030
• Preventive Veterinary Medicine • Previous Articles Next Articles
Lindan LÜ1(
), Hao MU2,3, Xia HU1, Mingni LIU2, Shaomei LI2, Xing LI2, Zhenhui SONG1,*(), Liu YANG2,3,*()
Received:2023-10-23
Online:2024-08-23
Published:2024-08-28
Contact:
Zhenhui SONG, Liu YANG
E-mail:276121248@qq.com;szh7678@swu.edu.cn;492691662@qq.com
CLC Number:
Lindan LÜ, Hao MU, Xia HU, Mingni LIU, Shaomei LI, Xing LI, Zhenhui SONG, Liu YANG. Establishment and Preliminary Application of RAA Assay for the Detection of Porcine Transmissible Gastroenteritis Virus based on S Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3590-3599.
Table 1
Basic RAA primer sequences"
| 编号 Number | 引物序列 Primer sequences | 引物组合 Primer pairing | 产物大小/bp Product size/bp |
| TGEV-F1 | 5′-GGTGTTAGGTGATTATTTTCCTACTGTACA-3′ | TGEV-F1/TGEV-R1 | 308 |
| TGEV-F2 | 5′-ACGGTTAAACGTAGTCGTTAATGGATACCC-3′ | TGEV-F2/TGEV-R1 | 164 |
| TGEV-F3 | 5′-CGGTTAAACGTAGTCGTTAATGGATACCCA-3′ | TGEV-F3/TGEV-R1 | 163 |
| TGEV-R1 | 5′-ACCTGCACTCACTACCCCAATTGCAAGTCA-3′ | TGEV-F1/TGEV-R2 | 309 |
| TGEV-R2 | 5′-AACCTGCACTCACTACCCCAATTGCAAGTC-3′ | TGEV-F2/TGEV-R2 | 165 |
| TGEV-F3/ TGEV-R2 | 164 |
Table 2
Fluorescent RAA probe and labeled primer sequences"
| 编号 Number | 探针和标记引物序列 Probe and labeled primer sequences |
| TGEV-PF1 | 5′-AACAACCCGCAATTTTAATTCTGCTGAAGGTGCT//THF//TTATATGCATTTGCAA-3′ (5′-端FAM修饰，3′-端C3 Spacer修饰，中间/THF/间隔及BHQ修饰) |
| TGEV-PF2 | 5′-AACAACCCGCAATTTTAATTCTGCTGAAGGTGCT//THF//TTATATGCATTTGCAA-3′ (5′-端FITC修饰，3′-端C3 Spacer修饰，中间/THF/间隔及BHQ修饰) |
| TGEV-PR1 | 5′-ACCTGCACTCACTACCCCAATTGCAAGTCA-3′(5′-端Biotin修饰) |
Fig. 1
The results of optimal primer pair, probe and probe addition amount A. Primer screening results (M. DL500 DNA marker; N. Negative control; 1-6. Primer pairs were F1/R1, F1/R2, F2/R1, F2/R2, F3/R1, F3/R2); B. Probe primer screening results (1. PF1/PR1; 2. PF2/PR1; 3. Positive control); C. Fluorescent RAA probe optimization results (1-3. The amount of probe primer was 1.0, 1.5, 2.0 μL; N. Negative control)"
Fig. 2
The results of temperature optimization A. Basic RAA temperature optimization results; B. Fluorescence RAA temperature optimization results; M. DL1000 DNA marker; 1-6. Amplification temperature at 37, 38, 39, 40, 41, 42 ℃; N. Negative control"
Fig. 3
The results of times optimization A. Basic RAA time optimization results; B. Fluorescence RAA time optimization results; M. DL1000 DNA marker; 1-5. Amplification time ranged from 30, 35, 40, 45, 50 min; N. Negative control"
Fig. 4
The results of sensitivity test A. The results of basic RAA sensitivity test (M. DL1000 DNA marker; 1-9. 10×gradient dilution plasmid standard concentration 1.34×108 copies ·μL-1-1.34×100 copies ·μL-1; N. Negative control); B. The results of fluorescence RAA sensitivity test (1-8. 10×gradient dilution plasmid standard concentration 1.34×107 copies ·μL-1-1.34×100 copies ·μL-1; N. Negative control)"
Fig. 5
The results of repeatability test A. The results of basic RAA repeatability test (M. DL1000 DNA marker; 1-3. The concentration of plasmid standard was 1.34×105 copies ·μL-1; N. Negative control); B. The results of fluorescence RAA repeatability test (1-3. The concentration of plasmid standard was 1.34×101 copies ·μL-1; N. Negative control)"
Fig. 6
The results of specificity test A. The results of basic RAA specificity test; B. The results of fluorescent RAA specificity test; M. DL1000 DNA marker; N. Negative control; 1. PRCV; 2. PCV2; 3. PKoV; 4. PEDV; 5. PRRSV; 6. PDCoV; 7. TGEV"
Fig. 7
The results of TGEV positive clinical samples test A. The results of fluorescent RAA assay test; B. The results of fluorescence quantitative PCR assay test; 1-6. The results of TGEV positive clinical samples"
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