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KMID : 1023520220450010001
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Korean Journal of Veterinary Service
2022 Volume.45 No. 1 p.1 ~ p.11
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Development of a real-time polymerase chain reaction assay for reliable detection of a novel porcine circovirus 4 with an endogenous internal positive control
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Kim Hye-Ryung
Park Jong-Hyun
Park Ji-Hoon
Kim Jong-Min
Baek Ji-Su
Kim Da-Young
Lyoo Young-S.
Park Choi-Kyu
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Abstract
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A novel porcine circovirus 4 (PCV4) was recently identified in Chinese and Korean pig herds. Al-though several conventional polymerase chain reaction (cPCR) and real-time PCR (qPCR) assays were used for PCV4 detection, more sensitive and reliable qPCR assay is needed that can simultane-ously detect PCV4 and internal positive control (IPC) to avoid false-negative results. In the present study, a duplex qPCR (dqPCR) assay was developed using primers/probe sets targeting the PCV4 Cap gene and pig (glyceraldehyde-3-phosphate dehydrogenase) GAPDH gene as an IPC. The de-veloped dqPCR assay was specifically detected PCV4 but not other PCVs and porcine pathogens, indicating that the newly designed primers/probe set is specific to the PCV4 Cap gene. Furthermore, GAPDH was stably amplified by the dqPCR in all tested viral and clinical samples containing pig cel-lular materials, indicating the high reliability of the dqPCR assay. The limit of detection of the assay 5 copies of the target PCV4 genes, but the sensitivity of the assay was higher than that of the previously described assays. The assay demonstrated high repeatability and reproducibility, with coefficients of intra-assay and inter-assay variation of less than 1.0%. Clinical evaluation using 102 diseased pig samples from 18 pig farms showed that PCV4 circulated in the Korean pig population. The detection rate of PCV4 obtained using the newly developed dqPCR was 26.5% (27/102), which was higher than that obtained using the previously described cPCR and TaqMan probe-based qPCR and similar to that obtained using the previously described SYBR Green-based qPCR. The dqPCR assay with IPC is highly specific, sensitive, and reliable for detecting PCV4 from clinical samples, and it will be useful for etiological diagnosis, epidemiological study, and control of the PCV4 infections.
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KEYWORD
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Capsid gene, Internal control, Porcine circovirus 4, Real-time PCR
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