Journal of fish pathology (한국어병학회지)

The Korean Society of Fish Pathology (한국어병학회)
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Determination of false positives in PCR diagnostics based on the internal transcribed spacer (ITS) of Gyrodactylus salaris using RFLP

RFLP를 이용한 Gyrodactylus salaris의 internal transcribed spacer(ITS) PCR 위양성 판별

  • Min Seong Kim (Division of Convergence on Marine Science, Korea Maritime and Ocean University) ;
  • Hee Jung Choi (Aquatic Disease Control Division, National Fishery Products Quality Management Service) ;
  • Ji-Min Jeong (Aquatic Disease Control Division, National Fishery Products Quality Management Service) ;
  • Mun-Gyeong Kwon (Aquatic Disease Control Division, National Fishery Products Quality Management Service) ;
  • Seong Don Hwang (Division of Convergence on Marine Science, Korea Maritime and Ocean University)
  • 김민성 (국립한국해양대학교 해양과학융합학부) ;
  • 최희정 (국립수산물품질관리원 수산방역과) ;
  • 정지민 (국립수산물품질관리원 수산방역과) ;
  • 권문경 (국립수산물품질관리원 수산방역과) ;
  • 황성돈 (국립한국해양대학교 해양과학융합학부)
  • Received : 2024.05.21
  • Accepted : 2024.06.04
  • Published : 2024.06.30
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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 improvement of PCR diagnostic method of G. salaris.

Keywords

Acknowledgement

이 논문은 국립수산물품질관리원(수산생물 검방역 관리 기술개발(NFQS2024001)의 지원에 의해 수행되었습니다.

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