Korean Journal of Veterinary Service (한국동물위생학회지)

The Korean Society of Veterinary Service (한국동물위생학회)
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Single-tube nested reverse transcription-polymerase chain reaction for simultaneous detection of genotyping of porcine reproductive and respiratory syndrome virus without DNA carryover contamination

DNA 교차오염 방지기능이 있는 single-tube nested reverse transcription-polymerase chain reaction을 이용한 돼지생식기호흡기증후군바이러스 유전형 감별진단

  • Jeong, Pil-Soo (College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University) ;
  • Park, Su-Jin (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) ;
  • Park, Ji-Young (College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University) ;
  • Park, Yu-Ri (College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University) ;
  • Kang, Dae-Young (College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University) ;
  • Cha, Hyun-Ouk (NANOHELIX Inc.) ;
  • Lee, Kyoung-Ki (Animal Disease Diagnostic Division, Animal and Plant Quarantine Agency) ;
  • Kim, Seong-Hee (Animal Disease Diagnostic Division, Animal and Plant Quarantine Agency) ;
  • Park, Choi-Kyu (College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University)
  • 정필수 (경북대학교 수의과대학 & 수의전염병제어센터) ;
  • 박수진 (경북대학교 수의과대학 & 수의전염병제어센터) ;
  • 김은미 (경북대학교 수의과대학 & 수의전염병제어센터) ;
  • 박지영 (경북대학교 수의과대학 & 수의전염병제어센터) ;
  • 박유리 (경북대학교 수의과대학 & 수의전염병제어센터) ;
  • 강대영 (경북대학교 수의과대학 & 수의전염병제어센터) ;
  • 차현욱 ((주) 나노헬릭스) ;
  • 이경기 (농림축산검역본부 질병진단과) ;
  • 김성희 (농림축산검역본부 질병진단과) ;
  • 박최규 (경북대학교 수의과대학 & 수의전염병제어센터)
  • Received : 2016.05.30
  • Accepted : 2016.06.27
  • Published : 2016.06.30
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Abstract

In the study, we developed and evaluated a uracil N-glycosylase (UNG)-supplemented single-tube nested reverse transcription-polymerase chain reaction (UsnRT-PCR) assay that can carried out first-round RT-PCR and second-round nested PCR in a reaction tube without reaction tube opening and can simultaneously detect EU- and NA-PRRSV. The UsnRT-PCR confirmed to have a preventing ability of mis-amplification by contamination of pre-amplified PRRSV DNA from previous UsnRT-PCR. Primer specificities were evaluated with RNAs extracted from 8 viral strains and our results revealed that the primers had a high specificity for both genotypes of PRRSV. The sensitivity of the UsnRT-PCR was 0.1 $TCID_{50}$/0.1 mL for EU- or NA-PRRSV, respectively, which is comparable to that of previously reported real time RT-PCR (RRT-PCR). Clinical evaluation on 110 field samples (60 sera and 50 lung tissues) by the UsnRT-PCR and the RRT-PCR showed that detection rates of the UsnRT-PCR was 70% (77/110), and was relatively higher than that of the RRT-PCR (69.1%, 76/110). The percent positive or negative agreement of the UsnRT-PCR compared to RRT-PCR was 96.1% (73/76) or 90.9% (30/33), showing that the test results of both assays may be different for some clinical samples. Therefore, it is recommend that diagnostic laboratory workers use the two diagnostic assays for the correct diagnosis for the relevant samples in the swine disease diagnostic laboratories. In conclusion, the UsnRT-PCR assay can be applied for the rapid, and reliable diagnosis of PRRSV without concerns about preamplified DNA carryover contamination that can occurred in PCR process in the swine disease diagnostic laboratories.

Keywords

References

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