Korean Journal of Veterinary Service (한국동물위생학회지)

The Korean Society of Veterinary Service (한국동물위생학회)
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One step multiplex RT-PCR preventing DNA carryover contamination for differential diagnosis of swine influenza viruses

DNA 교차 오염 방지 기능을 가진 돼지 인플루엔자바이러스 감별진단용 one-step multiplex RT-PCR 진단법

  • Kim, Hee-Jung (College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University) ;
  • Kim, Eun-Mi (College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University) ;
  • Shin, Yeun-Kyung (Virology Division, Animal and Plant Quarantine Agency) ;
  • Song, Jae-Young (Virology Division, Animal and Plant Quarantine Agency) ;
  • Kim, Seong-Hee (Disease Diagnostic Center, Animal and Plant Quarantine Agency) ;
  • Lee, Kyoung-Ki (Disease Diagnostic Center, Animal and Plant Quarantine Agency) ;
  • Lee, Myoung-Heon (Disease Diagnostic Center, Animal and Plant Quarantine Agency) ;
  • Kim, Young-Hwa (National Institute of Animal Science, R.D.A.) ;
  • Park, Jun-Cheol (National Institute of Animal Science, R.D.A.) ;
  • Yeo, Sang-Geon (College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University) ;
  • Park, Choi-Kyu (College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University)
  • 김희정 (경북대학교 수의과대학 & 수의전염병제어센터) ;
  • 김은미 (경북대학교 수의과대학 & 수의전염병제어센터) ;
  • 신연경 (농림축산검역본부 바이러스과) ;
  • 송재영 (농림축산검역본부 바이러스과) ;
  • 김성희 (농림축산검역본부 질병진단과) ;
  • 이경기 (농림축산검역본부 질병진단과) ;
  • 이명헌 (농림축산검역본부 질병진단과) ;
  • 김영화 (국립축산과학원) ;
  • 박준철 (국립축산과학원) ;
  • 여상건 (경북대학교 수의과대학 & 수의전염병제어센터) ;
  • 박최규 (경북대학교 수의과대학 & 수의전염병제어센터)
  • Received : 2014.10.31
  • Accepted : 2014.11.20
  • Published : 2014.12.30
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Abstract

In this study, we developed a cost and time saving one-step multiplex RT-PCR for the simultaneous detection and differentiation of swine influenza viruses (SIV) and 2009 pandemic influenza H1N1 virus (pH1N1). The one-step multiplex RT-PCR using four sets of primer was confirmed to be capable of detection of all SIV subtypes and differential diagnosis of major SIV subtype H1, H3 and pH1N1 on individual or mixed viral culture samples. The sensitivity of the multiplex RT-PCR was determined to be at least $2^{-6}$ $HA/25{\mu}L$ of the presented SIVs, providing sufficient efficacy for a routine SIV monitoring in diagnostic laboratories. In addition, compared with the conventional RT-PCR methods that cannot avoid the carryover DNA contamination, the developed RT-PCR applied with the uracil DNA glycosylase (UNG) system was proven to prevent a false positive reaction by carryover contamination of the pre-amplified DNA. In conclusion, the one-step RT-PCR with UNG system could be applicable to detect and differentiate of SIV from the viral cultures without worry of carryover DNA contamination in clinical laboratories.

Keywords

References

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