Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (7): 117-124.doi: 10.13560/j.cnki.biotech.bull.1985.2023-1107
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TIAN Xing-miao1(), WANG Jian-lin1, GUO Lei2, SI Duo-duo1, GONG Zhen-xing1, LI Ji-dong1()
Received:
2023-11-23
Online:
2024-07-26
Published:
2024-07-30
Contact:
LI Ji-dong
E-mail:1076049403@qq.com;lijidongi@foxmail.com
TIAN Xing-miao, WANG Jian-lin, GUO Lei, SI Duo-duo, GONG Zhen-xing, LI Ji-dong. Establishment of Dual LFD-RPA Rapid Detection Method for Mycoplasma gallisepticum and Mycoplasma synoviae[J]. Biotechnology Bulletin, 2024, 40(7): 117-124.
引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 片段大小 Product length/bp |
---|---|---|
PCR-MG-F | GTGTTCTCGGGTGCTATTGCTC | 819 |
PCR-MG-R | TGGGGGCTGATTCATTCCTTGA | |
PCR-MS-F | TAGACCCTTAATCGAATTTTG | 506 |
PCR-MS-R | CTTTTGAGTATTCAGCATCTTC |
Table 1 PCR primers
引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 片段大小 Product length/bp |
---|---|---|
PCR-MG-F | GTGTTCTCGGGTGCTATTGCTC | 819 |
PCR-MG-R | TGGGGGCTGATTCATTCCTTGA | |
PCR-MS-F | TAGACCCTTAATCGAATTTTG | 506 |
PCR-MS-R | CTTTTGAGTATTCAGCATCTTC |
引物名称Primer name | 引物序列Primer sequence(5'-3') | 片段大小Product length/bp |
---|---|---|
RPA-MG-F1 | AGATTACCTCCGAACCCTGATTTTATCCAGT | 362 |
RPA-MG-R1 | AATTCTTTGTTGGGGATTAGGACCAAATTGC | |
RPA-MG-F2 | CCTCCGAACCCTGATTTTATCCAGTAGTG | 202 |
RPA-MG-R2 | GTTGGCTCAATCGCTTCTGTTTTATTTTGT | |
RPA-MG-F3 | ATTTGGTCCTAATCCCCAACAAAGAATTAACCC | 301 |
RPA-MG-R3 | ATTAAACCCACCTCCAGCTTTATTTCCCATCG | |
RPA-MS-F1 | AAAATTAAATTACTATTAGCAGCTAGTGCAGT | 130 |
RPA-MS-R1 | GGTTTTGAGGATTATCAGTATTTGGGTTTCCAG | |
RPA-MS-F2 | AATTAAATTACTATTAGCAGCTAGTGCAGT | 244 |
RPA-MS-R2 | TTCTGTTTTAGCAGCCTCTACAGGGTCAAC | |
RPA-MS-F3 | CCTGAACCAACACCTGGAAACCCAAATACTG | 167 |
RPA-MS-R3 | TTAGCTTCTGTTTTAGCAGCCTCTACAGGGTC |
Table 2 Basic RPA primers
引物名称Primer name | 引物序列Primer sequence(5'-3') | 片段大小Product length/bp |
---|---|---|
RPA-MG-F1 | AGATTACCTCCGAACCCTGATTTTATCCAGT | 362 |
RPA-MG-R1 | AATTCTTTGTTGGGGATTAGGACCAAATTGC | |
RPA-MG-F2 | CCTCCGAACCCTGATTTTATCCAGTAGTG | 202 |
RPA-MG-R2 | GTTGGCTCAATCGCTTCTGTTTTATTTTGT | |
RPA-MG-F3 | ATTTGGTCCTAATCCCCAACAAAGAATTAACCC | 301 |
RPA-MG-R3 | ATTAAACCCACCTCCAGCTTTATTTCCCATCG | |
RPA-MS-F1 | AAAATTAAATTACTATTAGCAGCTAGTGCAGT | 130 |
RPA-MS-R1 | GGTTTTGAGGATTATCAGTATTTGGGTTTCCAG | |
RPA-MS-F2 | AATTAAATTACTATTAGCAGCTAGTGCAGT | 244 |
RPA-MS-R2 | TTCTGTTTTAGCAGCCTCTACAGGGTCAAC | |
RPA-MS-F3 | CCTGAACCAACACCTGGAAACCCAAATACTG | 167 |
RPA-MS-R3 | TTAGCTTCTGTTTTAGCAGCCTCTACAGGGTC |
RPA-MG-F2/R2/μL | RPA-MS-F1/R1/μL |
---|---|
0.0 | 2.0 |
0.2 | 1.8 |
0.4 | 1.6 |
0.6 | 1.4 |
0.8 | 1.2 |
1.0 | 1.0 |
1.2 | 0.8 |
1.4 | 0.6 |
1.6 | 0.4 |
1.8 | 0.2 |
2.0 | 0.0 |
Table 3 Optimization of primer ratios for dual basic RPA
RPA-MG-F2/R2/μL | RPA-MS-F1/R1/μL |
---|---|
0.0 | 2.0 |
0.2 | 1.8 |
0.4 | 1.6 |
0.6 | 1.4 |
0.8 | 1.2 |
1.0 | 1.0 |
1.2 | 0.8 |
1.4 | 0.6 |
1.6 | 0.4 |
1.8 | 0.2 |
2.0 | 0.0 |
引物/探针名称 Primer/Probe name | 引物序列 Primer sequence(5'-3') |
---|---|
MG-LFD-RPA-F | CCTCCGAACCCTGATTTTATCCAGTAGTG |
MG-LFD-RPA-R | TAMRA-GTTGGCTCAATCGCTTCTGTTTTATTTTGT |
MG-LFD-RPA-Probe | Digoxin-AGAATGATGATCCAAGAACGTGAAGAACA CCA/idSp/AAGATGGTTGAATCC-C3-spacer |
MS-LFD-RPA-F | AAAATTAAATTACTATTAGCAGCTAGTGCAGT |
MS-LFD-RPA-R | Biotin-GGTTTTGAGGATTATCAGTATTTGGGTTTCCAG |
MS-LFD-RPA-Probe | FAM-TGCTCCTGCTGTTATAGCAATTTCATGTGGTGA /idSp/CAAACTCCAGCACCT-C3-spacer |
Table 4 LFD-RPA primers and probes
引物/探针名称 Primer/Probe name | 引物序列 Primer sequence(5'-3') |
---|---|
MG-LFD-RPA-F | CCTCCGAACCCTGATTTTATCCAGTAGTG |
MG-LFD-RPA-R | TAMRA-GTTGGCTCAATCGCTTCTGTTTTATTTTGT |
MG-LFD-RPA-Probe | Digoxin-AGAATGATGATCCAAGAACGTGAAGAACA CCA/idSp/AAGATGGTTGAATCC-C3-spacer |
MS-LFD-RPA-F | AAAATTAAATTACTATTAGCAGCTAGTGCAGT |
MS-LFD-RPA-R | Biotin-GGTTTTGAGGATTATCAGTATTTGGGTTTCCAG |
MS-LFD-RPA-Probe | FAM-TGCTCCTGCTGTTATAGCAATTTCATGTGGTGA /idSp/CAAACTCCAGCACCT-C3-spacer |
试剂Reagent | 体积Volume/μL |
---|---|
MG-LFD-RPA-F | 0.6 |
MG-LFD-RPA-R | 0.6 |
MG-LFD-RPA-Probe | 0.3 |
MS-LFD-RPA-F | 1.4 |
MS-LFD-RPA-R | 1.4 |
MS-LFD-RPA-Probe | 0.3 |
A buffer | 29.4 |
Template | 3(MG)+3(MS) |
ddH2O | 7.5 |
B buffer | 2.5 |
Table 5 LFD-RPA reaction system(50 μL)
试剂Reagent | 体积Volume/μL |
---|---|
MG-LFD-RPA-F | 0.6 |
MG-LFD-RPA-R | 0.6 |
MG-LFD-RPA-Probe | 0.3 |
MS-LFD-RPA-F | 1.4 |
MS-LFD-RPA-R | 1.4 |
MS-LFD-RPA-Probe | 0.3 |
A buffer | 29.4 |
Template | 3(MG)+3(MS) |
ddH2O | 7.5 |
B buffer | 2.5 |
引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 片段大小 Product length/bp |
---|---|---|
MG 16S rRNA F | GAGCTAATCTGTAAAGTTGGTC | 183 |
MG 16S rRNA R | GCTTCCTTGCGGTTAGCAAC | |
MS 16S rRNA F | GAGAAGCAAAATAGTGATATCA | 211 |
MS 16S rRNA R | CAGTCGTCTCCGAAGTTAACAA |
Table 6 OIE PCR primers
引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 片段大小 Product length/bp |
---|---|---|
MG 16S rRNA F | GAGCTAATCTGTAAAGTTGGTC | 183 |
MG 16S rRNA R | GCTTCCTTGCGGTTAGCAAC | |
MS 16S rRNA F | GAGAAGCAAAATAGTGATATCA | 211 |
MS 16S rRNA R | CAGTCGTCTCCGAAGTTAACAA |
Fig. 3 Primer selection of MG and MS basic RPA F1/R1, F2/R2, F3/R3: The first, second, and third primer pair, respectively;M: DL500 DNA marker; 1: 5 min; 2: 10 min; 3: 15 min; 4: 20 min; 5: 25 min; 6: 30 min
Fig. 4 Optimization of optimal primer ratios for MG and MS dual basic RPA M: DL500 DNA marker. 1-11: The ratios of RPA-MG-F2/R2 and RPA-MS-F1/R1 are 0∶2, 0.2∶1.8, 0.4∶1.6, 0.6∶1.4, 0.8∶1.2, 1∶1, 1.2∶0.8, 1.4∶0.6, 1.6∶0.4, 1.8∶0.2, 2∶0, respectively
Fig. 5 Establishment of dual LFD-RPA method with MG and MS A: Reaction time optimization. B: Reaction temperature optimization. C: Sensitivity test. D: Specificity test. E: Repeatability test(a: The first repeat; b: the second repetition; c: the third repetition). N: Negative control
样品名称 Sample name | LFD-RPA检测结果 LFD-RPA test result | OIE PCR检测结果 OIE PCR test result |
---|---|---|
MG阳性 | 37 | 33 |
MG阴性 | 83 | 87 |
MS阳性 | 52 | 50 |
MS阴性 | 68 | 70 |
Table 7 Comparative analysis of MG and MS dual LFD-RPA and OIE PCR detection methods
样品名称 Sample name | LFD-RPA检测结果 LFD-RPA test result | OIE PCR检测结果 OIE PCR test result |
---|---|---|
MG阳性 | 37 | 33 |
MG阴性 | 83 | 87 |
MS阳性 | 52 | 50 |
MS阴性 | 68 | 70 |
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