Optimization and validation of a real-time PCR method for the simultaneous detection of Lactococcus garvieae and Streptococcus agalactiae in fish
Background: Lactococcus garvieae and Streptococcus agalactiae infections contribute to heavy losses in aquaculture farms worldwide. Currently, available pathogen diagnostic tools use biochemical and microbiological methods beleaguered by very low accuracy, reproducibility and specificity. Aim: To o...
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Plovdiv University Press
2025-01-01
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| Series: | Journal of BioScience and Biotechnology |
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| Online Access: | https://editorial.uni-plovdiv.bg/index.php/JBB/article/view/529/393 |
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| author | Taona Zinyakasa Farisai Chidzwondo Tatenda Makawa Sitokozile Sibanda Exnevia Gomo Tivapasi Musa Elizabeth Gori |
| author_facet | Taona Zinyakasa Farisai Chidzwondo Tatenda Makawa Sitokozile Sibanda Exnevia Gomo Tivapasi Musa Elizabeth Gori |
| author_sort | Taona Zinyakasa |
| collection | DOAJ |
| description | Background: Lactococcus garvieae and Streptococcus agalactiae infections contribute to heavy losses in aquaculture farms worldwide. Currently, available pathogen diagnostic tools use biochemical and microbiological methods beleaguered by very low accuracy, reproducibility and specificity.
Aim: To optimize and validate a rapid, sensitive and specific real-time PCR (qPCR) method for detecting L. garvieae and S. agalactiae in fish.
Methods: Pairs of Streptococcus-specific (IGS-s/IGS-a) and Lactococcus-specific (CAU12F/CAU15R) primers were tested for specificity and sensitivity in the qPCR. qPCR was carried out at different temperatures and primer concentrations. The optimal conditions were determined to be the temperature and primer concentration with the lowest CT values.
Results: For both primer sets, the optimal annealing temperature was 60oC, and the optimal primer concentration was 500 nM. The detection limit for L. garvieae was at dilution factor 10-3, with a mean CT value of 25.0, for S. agalactiae, 10-4 with a mean CT value of 29.8. The PCR efficiencies were 97% for L. garvieae and 91% for S. agalactiae, with linear slopes (R2 = 0.999). The assay demonstrated high repeatability and reproducibility.
Conclusion: The optimum conditions established for the qPCR method enable rapid, highly sensitive and specific diagnosis of L. garvieae and S. agalactiae infection in fish. |
| format | Article |
| id | doaj-art-45b33999bd4645d09e792315f3a3a67e |
| institution | Kabale University |
| issn | 1314-6238 1314-6246 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Plovdiv University Press |
| record_format | Article |
| series | Journal of BioScience and Biotechnology |
| spelling | doaj-art-45b33999bd4645d09e792315f3a3a67e2025-01-28T07:21:07ZengPlovdiv University PressJournal of BioScience and Biotechnology1314-62381314-62462025-01-0113211312310.69085/jbb20242113Optimization and validation of a real-time PCR method for the simultaneous detection of Lactococcus garvieae and Streptococcus agalactiae in fishTaona Zinyakasa0Farisai Chidzwondo1Tatenda Makawa2Sitokozile Sibanda3Exnevia Gomo4Tivapasi Musa5Elizabeth Gori6Veterinary Biosciences, Clinical Science Departments, University of Zimbabwe, 630 Churchill Avenue P.O. Box MP 167, Mount Pleasant, Harare, Zimbabwe; Biotechnology and Biochemistry Department, University of Zimbabwe, 630 Churchill Avenue P.O. Box MP 167, Mount Pleasant, Harare, ZimbabweBiotechnology and Biochemistry Department, University of Zimbabwe, 630 Churchill Avenue P.O. Box MP 167, Mount Pleasant, Harare, ZimbabweVeterinary Biosciences, Clinical Science Departments, University of Zimbabwe, 630 Churchill Avenue P.O. Box MP 167, Mount Pleasant, Harare, ZimbabweCentral Veterinary Laboratory, Department of Livestock and Veterinary Services,18 Borrowdale Road, Harare, ZimbabweFaculty of Medicine and Health Sciences, Box A178 Avondale, University of ZimbabweVeterinary Biosciences, Clinical Science Departments, University of Zimbabwe, 630 Churchill Avenue P.O. Box MP 167, Mount Pleasant, Harare, ZimbabweDepartment of Medical Biochemistry, Molecular Biology & Genetics, College of Medicine and Health Sciences-School of Medicine and Pharmacy, University of Rwanda, P.O. Box 117-Butare, RwandaBackground: Lactococcus garvieae and Streptococcus agalactiae infections contribute to heavy losses in aquaculture farms worldwide. Currently, available pathogen diagnostic tools use biochemical and microbiological methods beleaguered by very low accuracy, reproducibility and specificity. Aim: To optimize and validate a rapid, sensitive and specific real-time PCR (qPCR) method for detecting L. garvieae and S. agalactiae in fish. Methods: Pairs of Streptococcus-specific (IGS-s/IGS-a) and Lactococcus-specific (CAU12F/CAU15R) primers were tested for specificity and sensitivity in the qPCR. qPCR was carried out at different temperatures and primer concentrations. The optimal conditions were determined to be the temperature and primer concentration with the lowest CT values. Results: For both primer sets, the optimal annealing temperature was 60oC, and the optimal primer concentration was 500 nM. The detection limit for L. garvieae was at dilution factor 10-3, with a mean CT value of 25.0, for S. agalactiae, 10-4 with a mean CT value of 29.8. The PCR efficiencies were 97% for L. garvieae and 91% for S. agalactiae, with linear slopes (R2 = 0.999). The assay demonstrated high repeatability and reproducibility. Conclusion: The optimum conditions established for the qPCR method enable rapid, highly sensitive and specific diagnosis of L. garvieae and S. agalactiae infection in fish.https://editorial.uni-plovdiv.bg/index.php/JBB/article/view/529/393diagnostic toolqpcrzoonoticaquaculturespecificitysensitivity |
| spellingShingle | Taona Zinyakasa Farisai Chidzwondo Tatenda Makawa Sitokozile Sibanda Exnevia Gomo Tivapasi Musa Elizabeth Gori Optimization and validation of a real-time PCR method for the simultaneous detection of Lactococcus garvieae and Streptococcus agalactiae in fish Journal of BioScience and Biotechnology diagnostic tool qpcr zoonotic aquaculture specificity sensitivity |
| title | Optimization and validation of a real-time PCR method for the simultaneous detection of Lactococcus garvieae and Streptococcus agalactiae in fish |
| title_full | Optimization and validation of a real-time PCR method for the simultaneous detection of Lactococcus garvieae and Streptococcus agalactiae in fish |
| title_fullStr | Optimization and validation of a real-time PCR method for the simultaneous detection of Lactococcus garvieae and Streptococcus agalactiae in fish |
| title_full_unstemmed | Optimization and validation of a real-time PCR method for the simultaneous detection of Lactococcus garvieae and Streptococcus agalactiae in fish |
| title_short | Optimization and validation of a real-time PCR method for the simultaneous detection of Lactococcus garvieae and Streptococcus agalactiae in fish |
| title_sort | optimization and validation of a real time pcr method for the simultaneous detection of lactococcus garvieae and streptococcus agalactiae in fish |
| topic | diagnostic tool qpcr zoonotic aquaculture specificity sensitivity |
| url | https://editorial.uni-plovdiv.bg/index.php/JBB/article/view/529/393 |
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