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KMID : 1023520160390020107
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Korean Journal of Veterinary Service
2016 Volume.39 No. 2 p.107 ~ p.116
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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
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Jeong Pil-Soo
Park Su-Jin
Kim Eun-Mi
Park Ji-Young
Park Yu-Ri
Kang Dae-Young
Park Choi-Kyu
Cha Hyun-Ouk
Lee Kyoung-Ki
Kim Sung-Hee
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Abstract
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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 TCID50/0.1 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.
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KEYWORD
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Porcine reproductive and respiratory syndrome virus, PRRSV, Single-tube nested reverse transcription-polymerase chain reaction, snRT-PCR, Uracil DNA glycosylase, UNG
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