RFLP를 이용한 Gyrodactylus salaris의 internal transcribed spacer(ITS) PCR 위양성 판별
Determination of false positives in PCR diagnostics based on the internal transcribed spacer (ITS) of Gyrodactylus salaris using RFLP
0
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
The World Organization for Animal Health (WOAH) recommends two protocols (ITS and COI) for conventional PCR of G. salaris diagnosis. However, ITS PCR protocol may yield false-positive results, leading to unnecessary countermeasures. It's difficult to distinguish between G. salaris and false-positive by similar amplicon size of PCR, since the amplicon size of ITS PCR in G. salaris and false-positive was 1,300 and 1,187 bp, respectively. The nucleotide sequences of ITS false-positive in rainbow trout is 99.7% identical to previously reported host genome sequences of rainbow trout (Oncorhynchus mykiss) and 95.3 to 89.1% identical to those of other salmonid fish species. To reduce false-positive PCR band, PCR was performed by the different annealing temperature, but PCR bands were still detected. In RFLP analysis by HaeIII, the PCR product of G. salaris was digested into four bands of 512, 399, 234 and 154 bp, while the false-positive was digested into seven bands of 297, 263, 242, 144, 93, 80 and 68 bp. In the RFLP patterns digested by HindIII, G. salaris showed two bands of 659 and 640 bp, while false-positive had one fragment of 1,187 bp without any digestion. Therefore, the RFLP method of ITS PCR with HaeIII and HindIII can be used for differentiation between G. salaris and false-positive. These results might provide important information on the im-provement of PCR diagnostic method of G. salaris.
Abstract
The World Organization for Animal Health (WOAH) recommends two protocols (ITS and COI) for conventional PCR of G. salaris diagnosis. However, ITS PCR protocol may yield false-positive results, leading to unnecessary countermeasures. It's difficult to distinguish between G. salaris and false-positive by similar amplicon size of PCR, since the amplicon size of ITS PCR in G. salaris and false-positive was 1,300 and 1,187 bp, respectively. The nucleotide sequences of ITS false-positive in rainbow trout is 99.7% identical to previously reported host genome sequences of rainbow trout (Oncorhynchus mykiss) and 95.3 to 89.1% identical to those of other salmonid fish species. To reduce false-positive PCR band, PCR was performed by the different annealing temperature, but PCR bands were still detected. In RFLP analysis by HaeIII, the PCR product of G. salaris was digested into four bands of 512, 399, 234 and 154 bp, while the false-positive was digested into seven bands of 297, 263, 242, 144, 93, 80 and 68 bp. In the RFLP patterns digested by HindIII, G. salaris showed two bands of 659 and 640 bp, while false-positive had one fragment of 1,187 bp without any digestion. Therefore, the RFLP method of ITS PCR with HaeIII and HindIII can be used for differentiation between G. salaris and false-positive. These results might provide important information on the im-provement of PCR diagnostic method of G. salaris.
참고문헌 (16)
- Appleby, C., "Population dynamics of Gyrodactylus salaris(Monogenea)infecting Atlantic salmon, Salmo salar, parr in the river Batnfjordselva, Norway", J. Parasitol, 83(1), pp.23~30 (1997)[DOI]
- Bakke, T. A., "The host specificity of Gyrodactylus salaris Malmberg(Platyhelminthes, Monogenea) : susceptibility of Oncorhynchus mykiss(Walbaum)under experimental conditions", J. Fish Biol, 39, pp.45~57 (1991)[DOI]
- Chong R.S.M., "Aquaculture Pathophysiology", pp.513~515 (2022), Academic Press
- Cunningham, C. O., "Species variation within the internal transcribed spacer(ITS)region of Gyrodactylus(Monogenea : Gyrodactylidae)ribosomal RNA genes", J. Parasitol, 83, pp.215~219 (1997)[DOI]
- Denholm, S. J., "Predicting the potential for natural recovery of Atlantic salmon (Salmo salar L.) populations following the introduction of Gyrodactylus salaris Malmberg, 1957(Monogenea)", PLoS One, 11(12), pp.0169168~ (2016)[DOI]
- Harris, P. D., "Nominal species of the genus Gyrodactylus von Nordmann 1832 (Monogenea: Gyrodactylidae), with a list of principal host species", Syst. Parasitol, 59(1), pp.1~27 (2004)[DOI]
- Jensen, A. J., "Site specificity of Gyrodactylus salaris Malmberg, 1957(Monogenea)on Atlantic Salmon(Salmo salar L)in the river Lakselva, northern Norway", Canadian Journal of Zoology, 70, pp.264~267 (1992)[DOI]
- Meinilä, M., "Primers for amplifying approximately 820 bp of highly polymorphic mitochondrial COI gene of Gyrodactylus salaris", Hereditas, 137, pp.72~74 (2002)[DOI]
- Mo, T. A., "Occurrence and seasonality of Gyrodactylus salaris and G. salmonis (Monogenea) on Arctic char (Salvelinus alpinus (L.)) in the Fustvatnet lake, Northern Norway", J. Fish Dis., 46(4), pp.395~403 (2023)[DOI]
- Olstad, K., "Unpredicted transmission strategy of Gyrodactylus salaris(Monogenea : Gyrodactylidae) : survival and infectivity of parasites on dead hosts", Parasitology, 133, pp.33~41 (2006)[DOI]
- Paladini, G., "Geographical distribution of Gyrodactylus salaris Malmberg, 1957 (Monogenea, Gyrodactylidae)", Parasit. Vectors, 1957(14), pp.34~ (2021)[DOI]
- Peeler, E. J., "Qualitative analysis of the risk of introducing Gyrodactylus salaris into the United Kingdom", Dis. Aquat. Org, 62, pp.103~113 (2004)[DOI]
- Peeler, E. J., "Demonstrating freedom from Gyrodactylus salaris(Monogenea : Gyrodactylidae)in farmed rainbow trout Oncorhynchus mykiss", Dis. Aquat. Org, 79, pp.47~56 (2008)[DOI]
- Pettersen, R. A., "Osmoregulatory disturbances in Atlantic salmon, Salmo salar L., caused by the monogenean Gyrodactylus salaris", J. Fish Dis, 36(1), pp.67~70 (2013)[DOI]
- World Organisation for Animal Health, "Manual of Diagnostic Tests for Aquatic Animals", ~ (2023)
- 국립수산물품질관리원, "수산생물병성감정지침서", ~ (2021)