Figure 1.
Screening of optimal primer pair using the RAA-AGE assay. M, DL2000 DNA Marker; 1, PPV-RAA-1; 2, PPV-RAA-1-negative control; 3, PPV-RAA-2; 4, PPV-RAA-2-negative control; 5, PPV-RAA-3; 6, PPV-RAA-3-negative control.
Figure 1.
Screening of optimal primer pair using the RAA-AGE assay. M, DL2000 DNA Marker; 1, PPV-RAA-1; 2, PPV-RAA-1-negative control; 3, PPV-RAA-2; 4, PPV-RAA-2-negative control; 5, PPV-RAA-3; 6, PPV-RAA-3-negative control.
Figure 2.
Establishment and optimization of the PPV RAA-LFD assay. (a) Establishment of the PPV RAA-LFD assay. N, ddH2O (negative control); PPV, PPV DNA template (positive control); C, control line; T, test line; (b) Optimization of reaction temperature. N, ddH2O (negative control); C, control line; T, test line; (c) Optimization of reaction temperature. N, ddH2O (negative control); C, control line; T, test line; (d) Optimization of reaction time. N, ddH2O (negative control); C, control line; T, test line.
Figure 2.
Establishment and optimization of the PPV RAA-LFD assay. (a) Establishment of the PPV RAA-LFD assay. N, ddH2O (negative control); PPV, PPV DNA template (positive control); C, control line; T, test line; (b) Optimization of reaction temperature. N, ddH2O (negative control); C, control line; T, test line; (c) Optimization of reaction temperature. N, ddH2O (negative control); C, control line; T, test line; (d) Optimization of reaction time. N, ddH2O (negative control); C, control line; T, test line.
Figure 3.
The specificity analysis of PPV RAA-LFD assay. N, ddH2O (negative control); PPV, porcine parvovirus; PCV2, porcine circovirus2; JEV, Japanese encephalitis virus; CSFV, classical swine fever virus; PRRSV, porcine reproductive and respiratory syndrome virus; PRV, pseudorabies virus; SVA, Senecavirus A; ASFV, African swine fever virus; C, control line; T, test line.
Figure 3.
The specificity analysis of PPV RAA-LFD assay. N, ddH2O (negative control); PPV, porcine parvovirus; PCV2, porcine circovirus2; JEV, Japanese encephalitis virus; CSFV, classical swine fever virus; PRRSV, porcine reproductive and respiratory syndrome virus; PRV, pseudorabies virus; SVA, Senecavirus A; ASFV, African swine fever virus; C, control line; T, test line.
Figure 4.
The sensitivity evaluation of the PPV RAA-LFD assay by detecting a ten-fold serially diluted recombinant plasmid pMD19-T-VP1 compared with the PPV RAA-AGE assay and the PPV PCR-AGE assay. (a) PPV RAA-LFD assay. N, negative control; C, control line; T, test line; (b) PPV RAA-AGE assay. M, DL2000 DNA Marker; N, negative control; (c) PPV PCR-AGE assay. M, DL2000 DNA Marker; N, negative control.
Figure 4.
The sensitivity evaluation of the PPV RAA-LFD assay by detecting a ten-fold serially diluted recombinant plasmid pMD19-T-VP1 compared with the PPV RAA-AGE assay and the PPV PCR-AGE assay. (a) PPV RAA-LFD assay. N, negative control; C, control line; T, test line; (b) PPV RAA-AGE assay. M, DL2000 DNA Marker; N, negative control; (c) PPV PCR-AGE assay. M, DL2000 DNA Marker; N, negative control.
Figure 5.
The sensitivity evaluation of the PPV RAA-LFD assay by detecting a ten-fold serially diluted PPV DNA compared with the PPV RAA-AGE assay and the PPV PCR-AGE assay. (a) PPV RAA-LFD assay. N, negative control; C, control line; T, test line; (b) PPV RAA-AGE assay. M, DL2000 DNA Marker; N, negative control; (c) PPV PCR-AGE assay. M, DL2000 DNA Marker; N, negative control.
Figure 5.
The sensitivity evaluation of the PPV RAA-LFD assay by detecting a ten-fold serially diluted PPV DNA compared with the PPV RAA-AGE assay and the PPV PCR-AGE assay. (a) PPV RAA-LFD assay. N, negative control; C, control line; T, test line; (b) PPV RAA-AGE assay. M, DL2000 DNA Marker; N, negative control; (c) PPV PCR-AGE assay. M, DL2000 DNA Marker; N, negative control.
Figure 6.
The sensitivity evaluation of the PPV RAA-LFD assay by detecting different titers of PPV compared with the RAA-AGE assay and the PCR-AGE assay. (a) PPV RAA-LFD assay. N, negative control; C, control line; T, test line; (b) PPV RAA-AGE assay. M, DL2000 DNA Marker; N, negative control; (c) PPV PCR-AGE assay. M, DL2000 DNA Marker; N, negative control.
Figure 6.
The sensitivity evaluation of the PPV RAA-LFD assay by detecting different titers of PPV compared with the RAA-AGE assay and the PCR-AGE assay. (a) PPV RAA-LFD assay. N, negative control; C, control line; T, test line; (b) PPV RAA-AGE assay. M, DL2000 DNA Marker; N, negative control; (c) PPV PCR-AGE assay. M, DL2000 DNA Marker; N, negative control.
Figure 7.
Clinical samples detection of the PPV RAA-LFD assay. (a) PPV RAA-LFD assay. N, negative control; P, positive control; C, control line; T, test line; (b) PPV PCR assay. M, DL2000 DNA Marker; N, negative control; P, positive control.
Figure 7.
Clinical samples detection of the PPV RAA-LFD assay. (a) PPV RAA-LFD assay. N, negative control; P, positive control; C, control line; T, test line; (b) PPV PCR assay. M, DL2000 DNA Marker; N, negative control; P, positive control.
Table 1.
Primers used for amplification of PPV VP1 gene fragment.
Table 1.
Primers used for amplification of PPV VP1 gene fragment.
Primers | Sequence (5′-3′) | Length (bp) | Gene |
---|
PPV-F1 | CACGCATCAAGACTCATACATC | 1226 | VP1 |
PPV-R1 | TCTGTATCAAGTTCTTTATCCCAT |
Table 2.
Primers for PCR assay.
Table 2.
Primers for PCR assay.
Primers | Sequence (5′-3′) | Length (bp) |
---|
PPV-F2 | CACAGAAGCAACAGCAATTAGG | 203 |
PPV-R2 | CTAGCTCTTGTGAAGATGTGG |
Table 3.
Selection of primers for RAA-AGE assay.
Table 3.
Selection of primers for RAA-AGE assay.
RAA Assay | Primers | Sequence (5′-3′) | Length (bp) |
---|
PPV-RAA-1 | PPV-RAA-F1 | ACACTGGACAATCACAACAAATAACAGACTCA | 340 |
PPV-RAA-R1 | CCTACCTGAGCTGGCCTAATTGCTGTTGCTTC |
PPV-RAA-2 | PPV-RAA-F2 | TACAGATATTACCTATCATGCACCAGAAAC | 274 |
PPV-RAA-R2 | CTGTGGTAGGTTCAGTTAGTAGTTTTGGAG |
PPV-RAA-3 | PPV-RAA-F3 | CATACATCTAAATATGCCAGAACACGAAAC | 354 |
PPV-RAA-R3 | GGTGTGTATGGAAGTGTGTTATTGGTGTCT |
Table 4.
Optimal primer pair and probe for RAA assay.
Table 4.
Optimal primer pair and probe for RAA assay.
Primers/Probe | Sequence (5′-3′) |
---|
PPV-RAA-F1 | ACACTGGACAATCACAACAAATAACAGACTCA |
PPV-RAA-R1 | Biotin-CCTACCTGAGCTGGCCTAATTGCTGTTGCTTC |
PPV-Probe | FAM-ACAGATCTCTAGGACTGCCTCCAAAACTAC-THF-AACTGAACCTACCAC-C3 spacer |
Table 5.
The detection limits of the RAA-LFD assay, the RAA-AGE assay, and the PCR-AGE assay by detecting a ten-fold serially diluted recombinant plasmid pMD19-T-VP1, PPV DNA, and virus with different titers, respectively.
Table 5.
The detection limits of the RAA-LFD assay, the RAA-AGE assay, and the PCR-AGE assay by detecting a ten-fold serially diluted recombinant plasmid pMD19-T-VP1, PPV DNA, and virus with different titers, respectively.
Template | PPV RAA-LFD | PPV RAA-AGE | PPV PCR-AGE |
---|
Recombinant plasmid (copies/μL) | 102 | 104 | 104 |
DNA (ng/μL) | 6.38 × 10−7 | 6.38 × 10−4 | 6.38 × 10−4 |
Virus titer (TCID50/mL) | 10−1 | 1 | 1 |
Table 6.
Detection results of PPV RAA-LFD assay and PPV PCR-AGE assay by detecting 38 suspected samples, simultaneously.
Table 6.
Detection results of PPV RAA-LFD assay and PPV PCR-AGE assay by detecting 38 suspected samples, simultaneously.
Method | PPV RAA-LFD | PPV PCR-AGE |
---|
Results | Positive | Negative | Positive | Negative |
11 | 27 | 11 | 27 |
Total samples | 38 | 38 |
Positive rate | 28.95% | 28.95% |