Rapid and Sensitive Detection of Streptococcus iniae in Trachinotus ovatus Based on Multienzyme Isothermal Rapid Amplification
Abstract
:1. Introduction
2. Results
2.1. Evaluation of MIRA Primers
2.2. Optimal Reaction Conditions
2.3. Specificity of Real-Time MIRA and MIRA-LFD Assays
2.4. Sensitivity of Real-Time MIRA and MIRA-LFD Assays
2.5. Reproducibility of Real-Time MIRA and MIRA-LFD Assays
2.6. Practicability of Real-Time MIRA and MIRA-LFD Assays
3. Discussion
4. Materials and Methods
4.1. Fish and Bacterial Strains
4.2. Genomic DNA Extraction
4.3. Design of Primers and Probes
4.4. Establishment of MIRA, Real-Time MIRA, and MIRA-LFD Assays
4.5. Specificity of Real-Time MIRA and MIRA-LFD Assays
4.6. Sensitivity of Real-Time MIRA and MIRA-LFD Assays
4.7. Reproducibility of Real-Time MIRA and MIRA-LFD Assays
4.8. Practicability of Real-Time MIRA and MIRA-LFD Assays
5. Conclusions
6. Patents
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Name | Sequence (5′–3′) | Location |
---|---|---|
simA-F1 | TAGAAGCGGCTAAGAAAGAAGCAGAAGAA | 923–951 |
simA-F2 | TGACTAAAGCATTAGAAGCGGCTAAGAAA | 911–939 |
simA-F3 | ACTAAAGCATTAGAAGCGGCTAAGAAAGA | 913–941 |
simA-F4 | TAAAGCATTAGAAGCGGCTAAGAAAGAAG | 915–943 |
simA-F5 | AAGCATTAGAAGCGGCTAAGAAAGAAGCAG | 917–946 |
simA-R1 | GTGCTTTCTCAAGGTCTTTTTCAAGTTCT | 1057–1029 |
simA-R2 | CTGCTTGTGCTTTCTCAAGGTCTTTTTCAA | 1063–1034 |
simA-R3 | TCCAATTCAGCTTTTGTTTCTGCTAGTTTA | 1112–1083 |
simA-R4 | TTTCCAATTCAGCTTTTGTTTCTGCTAGTT | 1114–1085 |
simA-R5 | CAATAGTTGCTTCAAGTTCTGCTTTTTCA | 1153–1125 |
Assay | Name | Sequence (5′–3′) |
---|---|---|
Real-time MIRA | exo-F | TAAAGCATTAGAAGCGGCTAAGAAAGAAG |
exo-R | CAATAGTTGCTTCAAGTTCTGCTTTTTCA | |
exo-P | TTTCCAATTCAGCTTTGTTTCTGCTAGT(i6FAMdT) (idSp)(iBHQ1dT)AGTTTCAAGGTCTTTAG | |
MIRA-LFD | nfo-F | TAAAGCATTAGAAGCGGCTAAGAAAGAAG |
nfo-R | (Biotin)CAATAGTTGCTTCAAGTTCTGCTTTTTCA | |
nfo-P | (FAM)TTTCCAATTCAGCTTGTTTCTGCTAGTT (idSp)TAGTTTCAAGGTCTTTAG(C3 Spacer) |
Name | Sequence (5′–3′) |
---|---|
PCR-F | TGAAGAGCTTGACAAACTAAATG |
PCR-R | ACTTGCTGTGAAGAATGGGTTA |
SilldP-F | ACACAGGTGAGCACGCTAAA |
SilldP-R | CGTCACCATCGTCTTGGTCA |
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Wang, Y.; Niu, J.; Sun, M.; Li, Z.; Wang, X.; He, Y.; Qi, J. Rapid and Sensitive Detection of Streptococcus iniae in Trachinotus ovatus Based on Multienzyme Isothermal Rapid Amplification. Int. J. Mol. Sci. 2023, 24, 7733. https://doi.org/10.3390/ijms24097733
Wang Y, Niu J, Sun M, Li Z, Wang X, He Y, Qi J. Rapid and Sensitive Detection of Streptococcus iniae in Trachinotus ovatus Based on Multienzyme Isothermal Rapid Amplification. International Journal of Molecular Sciences. 2023; 24(9):7733. https://doi.org/10.3390/ijms24097733
Chicago/Turabian StyleWang, Yifen, Jingjing Niu, Minmin Sun, Ziyi Li, Xiangyuan Wang, Yan He, and Jie Qi. 2023. "Rapid and Sensitive Detection of Streptococcus iniae in Trachinotus ovatus Based on Multienzyme Isothermal Rapid Amplification" International Journal of Molecular Sciences 24, no. 9: 7733. https://doi.org/10.3390/ijms24097733