Development of a real-time polymerase chain reaction assay for reliable detection of a novel porcine circovirus 4 with an endogenous internal positive control
1
조회
0
다운로드
3
인용
0
참고문헌
키워드
초록
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.
Abstract
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.
참고문헌 (17)
- Bustin SA, "The MIQE guidelines : minimum information for publication of quantitative real-time PCR experiments", Clin Chem, 55, pp.611~622 (2009)
- Chen N, "Development and application of a quadruplex real-time PCR assay for differential detection of porcine circoviruses(PCV1 to PCV4)in Jiangsu province of China from 2016 to 2020", Transbound Emerg Dis, 68, pp.1615~1624 (2021)
- Duvigneau JC, "Quantitative simultaneous multiplex real-time PCR for the detection of porcine cytokines", J Immunol methods, 306, pp.16~27 (2005)
- Ha Z, "Retrospective surveillance of porcine circovirus 4 in pigs in Inner Mongolia, China, from 2016 to 2018", Arch Virol, 166, pp.1951~1959 (2021)
- Hou CY, "Simultaneous detection and differentiation of porcine circovirus 3 and 4using a SYBR Green І-based duplex quantitative PCR assay", J Virol Methods, 293, pp.114152~ (2021)
- Kim DY, "Detection of a novel porcine circovirus 4 in Korean pig herds using a loop-mediated isothermal amplification assay", J Virol Methods, 299, pp.114350~ (2022)
- Kim HR., "Multiplex realtime polymerase chain reaction for the differential detection of porcine circovirus 2 and 3", J Virol Methods, 250, pp.11~16 (2017)
- Kwiecien R, "Concordance analysis : part 16 of a series on evaluation of scientific publications", Deutsches Ärzteblatt International, 108, pp.515~ (2011)
- Navarro E, "Real-time PCR detection chemistry", Clinica Chimica Acta, 439, pp.231~250 (2015)
- Nguyen VG, "Molecular-based detection, genetic characterization and phylogenetic analysis of porcine circovirus 4 from Korean domestic swine farms", Transbound Emerg Dis, 00, pp.1~11 (2021)
- 박최규, "Porcine circovirus2 국내 분리주의 유전적 특성", 대한수의학회지, 44(4), pp.571~579 (2004), 대한수의학회
- Rosario K, "Revisiting the taxonomy of the family Circoviridae : establishment of the genus Cyclovirus and removal of the genus Gyrovirus", Arch Virol, 162, pp.1447~1463 (2017)
- Sun W, "Detection and genetic characterization of porcine circovirus 4(PCV4)in Guangxi, China", Gene, 773, pp.145384~ (2021)
- Tajadini M, "Comparison of SYBR Green and TaqMan methods in quantitative real-time polymerase chain reaction analysis of four adenosine receptor subtypes", Adv Biomed Res, 3, pp.85~ (2014)
- Tian RB, "Molecular detection and phylogenetic analysis of porcine circovirus 4 in Henan and Shanxi Provinces of China", Transbound Emerg Dis, 68, pp.276~282 (2021)
- Zhang D, "Establishment of an SYBR Green-based real-time PCR assay for porcine circovirus type 4detection", J. Virol Methods, 285, pp.113963~ (2020)
- Zhang HH, "Novel circovirus species identified in farmed pigs designated as porcine circovirus 4, Hunan province, China", Transbound Emerg Dis, 67, pp.1057~1061 (2020)