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KMID : 1023520140370040253
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Korean Journal of Veterinary Service
2014 Volume.37 No. 4 p.253 ~ p.261
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UNG-based direct polymerase chain reaction (udPCR) for the detection of porcine circovirus 2 (PCV2)
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Kim Eun-Mi
Park Choi-Kyu
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Abstract
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Porcine circovirus disease (PCVD) is a major problem of swine industry worldwide, and diagnosis of PCV2, causal agent of PCVD, has been doing in clinical laboratories of pig disease by polymerase chain reaction (PCR) methods. But the PCR analyses have a serious problem of misdiagnosis by contamination of DNA, in particular, from carryover contamination with previously amplified DNA or extracted DNA from field samples. In this study, an uracil DNA glycosylase (UNG)-based direct PCR (udPCR) without DNA extraction process and DNA carryover contamination was developed and evaluated on PCV2 culture and field pig samples. The sensitivity of the udPCR combined with dPCR and uPCR was same or better than that of the commercial PCR (cPCR) kit (Median diagnostics, Korea) on PCV2-positive serum, lymph node and lung samples of the pigs. In addition, the udPCR method confirmed to have a preventing ability of mis-amplification by contamination of pre-amplified PCV2 DNA from previous udPCR. In clinical application, 170 pig samples (86 tissues and 84 serum) were analysed by cPCR kit and resulted in 37% (63/170) of positive reaction, while the udPCR was able to detect the PCV2 DNA in 45.3% (77/170) with higher sensitivity than cPCR. In conclusion, the udPCR developed in the study is a time, labor and cost saving method for the detection of PCV2 and providing a preventing effect for DNA carryover contamination that can occurred in PCR process. Therefore, the udPCR assay could be an useful alternative method for the diagnosis of PCV2 in the swine disease diagnostic laboratories.
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KEYWORD
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Porcine circovirus 2 (PCV2), Uracil DNA glycosylase (UNG), Direct PCR (dPCR)
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