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http://dx.doi.org/10.7853/kjvs.2014.37.4.263

One step multiplex RT-PCR preventing DNA carryover contamination for differential diagnosis of swine influenza viruses  

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)
Publication Information
Korean Journal of Veterinary Service / v.37, no.4, 2014 , pp. 263-271 More about this Journal
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
Swine influenza viruse (SIV); Multiplex RT-PCR; Uracil DNA glycosylase (UNG); Differential diagnosis;
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