Development of a real-time TaqMan PCR assay for the detection of porcine circovirus 4

Abstract

Introduction Porcine circovirus 4 (PCV4) was first discovered in 2019 in a herd of pigs with porcine respiratory disease, dermatitis and nephropathy syndrome in Hunan Province, China. It has subsequently been detected in other provinces and in South Korea. In consideration of the potential of the virus to cause an epidemic, rapid, sensitive, and specific detection of PCV4 is needed, as is the facilitation of further epidemiological research through elucidation of the whole genome of PCV4. This study had those two aims. Material and Methods Fifty-five blood samples, two pig tissue samples, nine saliva swabs and one semen sample which all originated from Sichuan province pig farms were analysed. The virus’ genome of 1,770 bp was synthesised artificially based on a Chinese reference strain and primers and probes for the ORF2 gene were designed. Then, the amplified target fragment was cloned into the pMD19-T vector and a series of diluted recombinant plasmids were used to generate a standard curve. An optimised real-time TaqMan PCR method was established. Results The results of this study showed that the established method is specific for PCV4 but not for other viruses, and has amplification efficiency of 99.6%, a regression squared value (R2) of 1.000 and a detection limit of 2.2×10 DNA copies. This method was shown to be analytically specific and sensitive with a low intra- and inter-assay coefficient of variation (<1.67 %). Of a total of 67 clinical samples tested using the established method, three were shown to be positive (4%). Conclusion This study confirms the existence of PCV4 in Sichuan and provides a promising alternative tool for rapid detection of PCV4.

Prevalence and co-infection status of three pathogenic porcine circoviruses (PCV2, PCV3, and PCV4) by a newly established triplex real-time polymerase chain reaction assay

“…For clinical evaluation of the newly developed tqPCR assay and investigation of prevalence and co-infection status of PCVs in Korean pig farms, 1476 clinical samples collected from 198 pig farms were tested using the tqPCR and each monoplex qPCR assays. The results were in 100% agreement with those of previously reported monoplex qPCR assays for PCV2, PCV3, and PCV4 (Kim et al, 2017;Chen et al, 2022), regardless of the tested sample type, with a κ value (95% CI) of 1 (1.00∼1.00), were 100% concordant with the tqPCR assay (Table 3). (39/198), respectively (Fig.…”

“…1∼3). The sensitivity of the developed tqPCR assay was <10 copies/μL for PCV2, PCV3, and PCV4, which was comparable to that of previously reported duplex PCR assay for PCV2 and PCV3 (Kim et al, 2017) and like those of SYBR Green (Zhang et al, 2020a;Hou et al, 2021) and TaqMan qPCR assays (Chen et al, 2022) for PCV4. To assess the intraassay repeatability and inter-assay reproducibility of the tqPCR, three different concentrations of each standard DNA were tested in triplicate on six different runs performed by two different operators on different days (Bustin et al, 2009).…”

“…To test specificity, the tqPCR assay was performed with total nucleic acids extracted from eight viral samples (PCV1, PCV2, PCV3, PCV4, type 1 and 2 PRRSV, CSFV, and PPV) and two non-infected cultured cells (ST and PK-15 cells) of porcine-origin as negative controls. The sensitivity of the tqPCR and its corresponding monoplex qPCR assays was determined in triplicate using serial dilutions (from 10 6 to 10 0 copies/μL) of each standard plasmid DNA containing the capsid genes of detection system (Bio-Rad) as previously described re-action conditions (Kim et al, 2017;Chen et al, 2022).…”

“…Since PCV4 is difficult to isolate in vitro using cell culture (Zhang et al, 2020b), molecular diagnostic assays, such as conventional and real-time PCR, are currently used to detect PCV4 in clinical samples. Conventional PCR and quantitative real-time PCR (qPCR) are used for the routine diagnosis of PCV4 because of their high specificity, sensitivity, and ability to detect and differentiate multiple targets simultaneously (Zhang et al, 2020a;Chen et al, 2021;Hou et al, 2021;Tian et al, 2021;Chen et al, 2022;Kim et al, 2022b). The clinical manifestations of PCV3 and PCV4 are similar to those of PCV2, and co-infection with PCV2, PCV3, and PCV4 has been confirmed in pig populations.…”

Colorimetric Kit for Rapid Porcine Circovirus 2 (PCV-2) Diagnosis

“…A TaqMan rtPCR assay was recently developed to help detecting porcine circovirus 4. Results shown that the established method was specific for PCV-4; it recorded 99.6% efficiency and detection limit of 2.2 × 10 DNA copies [ 11 ]. Liu et al developed a sensitive assay using droplet digital PCR (ddPCR) to detect PCV-3.…”