Acta Agriculturae Zhejiangensis ›› 2021, Vol. 33 ›› Issue (4): 618-631.DOI: 10.3969/j.issn.1004-1524.2021.04.07
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Establishment and application of multiplex PCR-capillary electrophoresis for 7 swine diseases
TU Teng1,2(
), YIN Qingqing1,2, ZHANG Pengfei1,2, WANG Yin1,2,*(), YANG Zexiao1,2, YAO Xueping1,2, LUO Yan1,2
- 1. College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
2. Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu 611130, China
-
Received:2020-05-22Online:2021-04-25Published:2021-04-25 -
Contact:WANG Yin
CLC Number:
Cite this article
TU Teng, YIN Qingqing, ZHANG Pengfei, WANG Yin, YANG Zexiao, YAO Xueping, LUO Yan. Establishment and application of multiplex PCR-capillary electrophoresis for 7 swine diseases[J]. Acta Agriculturae Zhejiangensis, 2021, 33(4): 618-631.
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| 病原 Pathogen | 引物 Primer sequences (5'-3') | 靶基因 Target gene | 大小 Size/bp |
|---|---|---|---|
| SS CSFV JEV PRRSV HPS PoRV SE | F:AGGTGACACTATAGAATAATAATCCGCCAGCAACAAC R:GTACGACTCACTATAGGGATCAGCACGCAATAAAGCAC F:AGGTGACACTATAGAATATGCCCGTTTGATACGAGTCC R:GTACGACTCACTATAGGGAGGTCTTTACCACTTCTGTCC F:AGGTGACACTATAGAATAGCAAACGACAAACCAACACTA R:GTACGACTCACTATAGGGACAATGCTTCCTTTCCCGA F:AGGTGACACTATAGAATAACCTCCAGATGCCGTTTGT R:GTACGACTCACTATAGGGAACAAGGTTTACCACTCCCT F:AGGTGACACTATAGAATAGGGATTAGGTAGTTGGTGGGG R:GTACGACTCACTATAGGGACTGTGACTAACGTCAATGTAATGCT F:AGGTGACACTATAGAATATATCCTACCGAAGCATCAACTC R:GTACGACTCACTATAGGGAAAGATTGTCCCATCGATATCCA F:AGGTGACACTATAGAATACCCTTTGCGAATAACATCC R:GTACGACTCACTATAGGGATTGGCTATGTGTTGCGGA | SLY E2 E M 16S VP7 Inva | 160 193 235 261 292 324 348 |
Table 1 Primers used in capillary electrophoresis multiplex PCR
| 病原 Pathogen | 引物 Primer sequences (5'-3') | 靶基因 Target gene | 大小 Size/bp |
|---|---|---|---|
| SS CSFV JEV PRRSV HPS PoRV SE | F:AGGTGACACTATAGAATAATAATCCGCCAGCAACAAC R:GTACGACTCACTATAGGGATCAGCACGCAATAAAGCAC F:AGGTGACACTATAGAATATGCCCGTTTGATACGAGTCC R:GTACGACTCACTATAGGGAGGTCTTTACCACTTCTGTCC F:AGGTGACACTATAGAATAGCAAACGACAAACCAACACTA R:GTACGACTCACTATAGGGACAATGCTTCCTTTCCCGA F:AGGTGACACTATAGAATAACCTCCAGATGCCGTTTGT R:GTACGACTCACTATAGGGAACAAGGTTTACCACTCCCT F:AGGTGACACTATAGAATAGGGATTAGGTAGTTGGTGGGG R:GTACGACTCACTATAGGGACTGTGACTAACGTCAATGTAATGCT F:AGGTGACACTATAGAATATATCCTACCGAAGCATCAACTC R:GTACGACTCACTATAGGGAAAGATTGTCCCATCGATATCCA F:AGGTGACACTATAGAATACCCTTTGCGAATAACATCC R:GTACGACTCACTATAGGGATTGGCTATGTGTTGCGGA | SLY E2 E M 16S VP7 Inva | 160 193 235 261 292 324 348 |
| 因素 Elements | 嵌合引物使用浓度Concentration of chimeric primers/(μmol·L-1) | ||||||
|---|---|---|---|---|---|---|---|
| SS | CSFV | JEV | PRRSV | HPS | PoRV | SE | |
| 实验1 Test 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| 实验2 Test 2 | 1 | 1.25 | 1 | 1 | 0.75 | 1.25 | 1.25 |
| 实验3 Test 3 | 1 | 1.5 | 1 | 1 | 0.75 | 1.5 | 1.5 |
| 实验4 Test 4 | 1.25 | 1 | 1 | 1 | 0.75 | 1.5 | 1.5 |
| 实验5 Test 5 | 1.25 | 1.25 | 1 | 1 | 0.75 | 1 | 1 |
| 实验6 Test 6 | 1.25 | 1.5 | 1 | 1 | 1 | 1.25 | 1.25 |
| 实验7 Test 7 | 1.5 | 1 | 1 | 1 | 0.75 | 1.25 | 1.5 |
| 实验8 Test 8 | 1.5 | 1.25 | 1 | 1 | 1 | 1.5 | 1 |
| 实验9 Test 9 | 1.5 | 1.5 | 1 | 1 | 0.75 | 1 | 1.25 |
| 实验10 Test 10 | 1 | 1 | 1 | 1 | 0.75 | 1.25 | 1 |
| 实验11 Test 11 | 1 | 1.25 | 1 | 1 | 0.75 | 1.5 | 1.25 |
| 实验12 Test 12 | 1 | 1.5 | 1 | 1 | 1 | 1 | 1.5 |
| 实验13 Test 13 | 1.25 | 1 | 1 | 1 | 1 | 1.5 | 1.25 |
| 实验14 Test 14 | 1.25 | 1.25 | 1 | 1 | 0.75 | 1 | 1.5 |
| 实验15 Test 15 | 1.25 | 1.5 | 1 | 1 | 0.75 | 1.25 | 1 |
| 实验16 Test 16 | 1.5 | 1 | 1 | 1 | 0.75 | 1 | 1.25 |
| 实验17 Test 17 | 1.5 | 1.25 | 1 | 1 | 1 | 1.25 | 1.5 |
| 实验18 Test 18 | 1.5 | 1.5 | 1 | 1 | 0.75 | 1.5 | 1 |
Table 2 Orthogonal experimental scheme of chimeric primer concentrations
| 因素 Elements | 嵌合引物使用浓度Concentration of chimeric primers/(μmol·L-1) | ||||||
|---|---|---|---|---|---|---|---|
| SS | CSFV | JEV | PRRSV | HPS | PoRV | SE | |
| 实验1 Test 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| 实验2 Test 2 | 1 | 1.25 | 1 | 1 | 0.75 | 1.25 | 1.25 |
| 实验3 Test 3 | 1 | 1.5 | 1 | 1 | 0.75 | 1.5 | 1.5 |
| 实验4 Test 4 | 1.25 | 1 | 1 | 1 | 0.75 | 1.5 | 1.5 |
| 实验5 Test 5 | 1.25 | 1.25 | 1 | 1 | 0.75 | 1 | 1 |
| 实验6 Test 6 | 1.25 | 1.5 | 1 | 1 | 1 | 1.25 | 1.25 |
| 实验7 Test 7 | 1.5 | 1 | 1 | 1 | 0.75 | 1.25 | 1.5 |
| 实验8 Test 8 | 1.5 | 1.25 | 1 | 1 | 1 | 1.5 | 1 |
| 实验9 Test 9 | 1.5 | 1.5 | 1 | 1 | 0.75 | 1 | 1.25 |
| 实验10 Test 10 | 1 | 1 | 1 | 1 | 0.75 | 1.25 | 1 |
| 实验11 Test 11 | 1 | 1.25 | 1 | 1 | 0.75 | 1.5 | 1.25 |
| 实验12 Test 12 | 1 | 1.5 | 1 | 1 | 1 | 1 | 1.5 |
| 实验13 Test 13 | 1.25 | 1 | 1 | 1 | 1 | 1.5 | 1.25 |
| 实验14 Test 14 | 1.25 | 1.25 | 1 | 1 | 0.75 | 1 | 1.5 |
| 实验15 Test 15 | 1.25 | 1.5 | 1 | 1 | 0.75 | 1.25 | 1 |
| 实验16 Test 16 | 1.5 | 1 | 1 | 1 | 0.75 | 1 | 1.25 |
| 实验17 Test 17 | 1.5 | 1.25 | 1 | 1 | 1 | 1.25 | 1.5 |
| 实验18 Test 18 | 1.5 | 1.5 | 1 | 1 | 0.75 | 1.5 | 1 |
| 分组 Specimen groups | 感染情况 Infection conditions | 数量 Total No. | 采集地点 Collecting place |
|---|---|---|---|
| 1 | JEV | 5 | 四川遂宁 Suining,Sichuan |
| 2 | CSFV | 10 | 四川浦江 Pujiang,Sichuan |
| 3 | PRRSV | 11 | 四川雅安 Yaan,Sichuan |
| 4 | HPS | 8 | 四川遂宁Suining,Sichuan |
| 5 | SS | 7 | 四川雅安Yaan,Sichuan |
| 6 | SE | 4 | 四川遂宁Suining,Sichuan |
| 7 | JEV+PRRSV | 3 | 四川浦江Pujiang,Sichuan |
| 8 | PRRSV+CSFV | 2 | 四川遂宁Suining,Sichuan |
| 9 | CSFV+HPS | 2 | 四川成都Chengdu,Sichuan |
| 10 | PRRSV+HPS | 4 | 四川雅安Yaan,Sichuan |
| 11 | SS+SE | 3 | 四川遂宁Suining,Sichuan |
| 12 | CSFV+HPS | 2 | 四川浦江Pujiang,Sichuan |
| 13 | CSFV+JEV+PRRSV | 1 | 四川遂宁Suining,Sichuan |
| 14 | SE+HPS+SS | 1 | 四川成都 Chengdu,Sichuan |
| 15 | Uninfected | 3 | 四川绵阳 Mianyang,Sichuan |
Table 3 Statement of Clinical specimens
| 分组 Specimen groups | 感染情况 Infection conditions | 数量 Total No. | 采集地点 Collecting place |
|---|---|---|---|
| 1 | JEV | 5 | 四川遂宁 Suining,Sichuan |
| 2 | CSFV | 10 | 四川浦江 Pujiang,Sichuan |
| 3 | PRRSV | 11 | 四川雅安 Yaan,Sichuan |
| 4 | HPS | 8 | 四川遂宁Suining,Sichuan |
| 5 | SS | 7 | 四川雅安Yaan,Sichuan |
| 6 | SE | 4 | 四川遂宁Suining,Sichuan |
| 7 | JEV+PRRSV | 3 | 四川浦江Pujiang,Sichuan |
| 8 | PRRSV+CSFV | 2 | 四川遂宁Suining,Sichuan |
| 9 | CSFV+HPS | 2 | 四川成都Chengdu,Sichuan |
| 10 | PRRSV+HPS | 4 | 四川雅安Yaan,Sichuan |
| 11 | SS+SE | 3 | 四川遂宁Suining,Sichuan |
| 12 | CSFV+HPS | 2 | 四川浦江Pujiang,Sichuan |
| 13 | CSFV+JEV+PRRSV | 1 | 四川遂宁Suining,Sichuan |
| 14 | SE+HPS+SS | 1 | 四川成都 Chengdu,Sichuan |
| 15 | Uninfected | 3 | 四川绵阳 Mianyang,Sichuan |
Fig.1 Verified results of specific chimeric primers M, DNA Marker B (100-600 bp); 1, SE; 2, PoRV; 3, HPS; 4, PRRSV; 5, JEV; 6, CSFV; 7, SS.
Fig.2 Results of capillary electrophoresis simplex PCR 1, SS; 2, CSFV; 3, JEV; 4, PRRSV; 5, HPS; 6, PoRV; 7, SE; 8, ddH2O as negative control; M, QIAxcel Size Marker(25-500 bp).
Fig.3 Result of capillary electrophoresis simplex PCR A-D indicated detection peak figures of SS, CSFV, JEV, PRRSV. The abscissas represented the bp numbers and the ordinates represented the relative intensity of fluorescence.
Fig.4 Result of capillary electrophoresis simplex PCR A-C indicated detection peak figures of HPS, PoRV, SE. D indicated detection peak figures of Size Marker as control. The abscissas represented the bp numbers and the ordinates represented the relative intensity of fluorescence.
Fig.5 Gel figure of capillary electrophoresis multiplex PCR annealing temperature test Lane 1, 50.7 ℃; Lane 2, 52.0 ℃; Lane 3, 53.9 ℃; Lane 4, 56.3 ℃; Lane 5, 58.3 ℃; Lane 6, 59.4 ℃; Lane 7, 60.0 ℃; Lane 8, Negative control; Lane M, QIAxcel Size Marker (25-500 bp).
Fig.6 Result of capillary electrophoresis multiplex PCR annealing temperature test
Fig.7 Results of capillary electrophoresis multiplex PCR sensitivity test Lane M, QIAxcel Size Marker (25-500 bp); Lane 1, 107 copies·μL-1; Lane 2, 106 copies·μL-1; Lane 3, 105 copies·μL-1; Lane 4, 104 copies·μL-1; Lane 5, 103 copies·μL-1; Lane 6, 102 copies·μL-1; Lane 7, 101 copies·μL-1; Lane 8, 100 copies·μL-1; Lane 9, 10-1 copies·μL-1; Lane 10, Negative control.
Fig.8 Results of capillary electrophoresis multiplex PCR specificity test Lane 1, Mixed template containing DNA/cDNA of 7 pathogens; Lane 2, HPS, PoRV and SE; Lane 3, SS and CSFV; Lane 4, CSFV and JEV; Lane 5, CSFV, PRRSV and HPS; Lane 6, SS, CSFV, PRRSV and HPS; Lane 7, CSFV and PRRSV; Lane 8, Negative control; Lane M, QIAxcel Size Marker (25- 500 bp); Lane 11, E. coli; Lane 12, PPV; Lane 13, ASFV; Lane 14, PEDV; Lane 15, TGEV.
Fig.9 Results of capillary electrophoresis multiplex PCR cross reactivity test A-C indicated cross reactivity peak figures of SS+CSFV, CSFV+JEV and CSFV+PRRSV. The abscissas represented the bp numbers and the ordinates represented the relative intensity of fluorescence.
Fig.10 Results of capillary electrophoresis multiplex PCR cross reactivity test A-D indicated cross reactivity peak figures of SS+CSFV+PRRSV+HPS, CSFV+PRRSV+HPS, HPS+PoRV+SE, HPS+SE. The abscissas represented the bp numbers and the ordinates represented the relative intensity of fluorescence.
Fig.11 Overlaided peak figure of repeatability test
| 分组 Specimen groups | 国标/行业检测结果Results identified by national standard/insdustry standard | 毛细管电泳多重PCR结果 Results of multiplex PCR-CE | ||||||
|---|---|---|---|---|---|---|---|---|
| SS | CSFV | JEV | PRRSV | HPS | PoRV | SE | ||
| 1 | - | - | +4 | - | - | - | - | +5 JEV |
| 2 | - | +9 | - | - | - | - | - | +10 CSFV |
| 3 | - | - | - | +10 | - | - | - | +11 PRRSV |
| 4 | - | - | - | - | +8 | - | - | +8 HPS |
| 5 | +7 | - | - | - | - | - | - | +7 SS |
| 6 | - | - | - | - | - | - | +4 | +4 SE |
| 7 | - | - | - | - | - | +3 | - | +3 PoRV |
| 7 | - | - | +3 | +3 | - | - | - | +3 JEV, PRRSV |
| 8 | - | +2 | - | +2 | - | - | - | +2 PRRSV, CSFV |
| 9 | - | +2 | - | - | +2 | - | - | +2 CSFV, HPS |
| 10 | - | - | - | +4 | +4 | - | - | +4 PRRSV, HPS |
| 11 | +3 | - | - | - | - | - | +3 | +3 SS,SE |
| 12 | +2 | - | - | - | - | +2 | - | +2 PoRV, HPS |
| 13 | - | +1 | +1 | +1 | - | - | - | +1 CSFV,JEV,PRRSV |
| 14 | +1 | - | - | - | +1 | - | +1 | +1 SE,HPS,SS |
| 3份阴性样品 | - | - | - | - | - | - | - | Negative |
| 3 negative samples | ||||||||
Table 4 Results of clinical specimens by capillary electrophoresis multiplex PCR
| 分组 Specimen groups | 国标/行业检测结果Results identified by national standard/insdustry standard | 毛细管电泳多重PCR结果 Results of multiplex PCR-CE | ||||||
|---|---|---|---|---|---|---|---|---|
| SS | CSFV | JEV | PRRSV | HPS | PoRV | SE | ||
| 1 | - | - | +4 | - | - | - | - | +5 JEV |
| 2 | - | +9 | - | - | - | - | - | +10 CSFV |
| 3 | - | - | - | +10 | - | - | - | +11 PRRSV |
| 4 | - | - | - | - | +8 | - | - | +8 HPS |
| 5 | +7 | - | - | - | - | - | - | +7 SS |
| 6 | - | - | - | - | - | - | +4 | +4 SE |
| 7 | - | - | - | - | - | +3 | - | +3 PoRV |
| 7 | - | - | +3 | +3 | - | - | - | +3 JEV, PRRSV |
| 8 | - | +2 | - | +2 | - | - | - | +2 PRRSV, CSFV |
| 9 | - | +2 | - | - | +2 | - | - | +2 CSFV, HPS |
| 10 | - | - | - | +4 | +4 | - | - | +4 PRRSV, HPS |
| 11 | +3 | - | - | - | - | - | +3 | +3 SS,SE |
| 12 | +2 | - | - | - | - | +2 | - | +2 PoRV, HPS |
| 13 | - | +1 | +1 | +1 | - | - | - | +1 CSFV,JEV,PRRSV |
| 14 | +1 | - | - | - | +1 | - | +1 | +1 SE,HPS,SS |
| 3份阴性样品 | - | - | - | - | - | - | - | Negative |
| 3 negative samples | ||||||||
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