Establishment and preliminary application of an assay for the detection of porcine parvovirus in cells used for production

Abstract

Objective To establish an assay for the detection of porcine parvovirus (PPV) and to verify its application for monitoring cells used for production. Methods A pair of primers and one probe were designed according to the conserved sequence encoding non-structural protein 1 (NS1). Based on the designed primers, a real-time fluorescent quantitative PCR assay for the detection of PPV was developed. Several parameters including the linearity, precision, minimum detection limit and anti-interference of the established assay were evaluated. A stock of PPV strains was prepared by infecting swine testicle (ST) cells with PPV strains. An assay for the detection of PPV infection was developed by using ST cells as sensitive cells. A combined ST cell infection-PCR test was developed by combining the ST cell infection assay with the real-time fluorescent quantitative PCR assay. The sensitivity of ST cell infection-PCR test was analyzed. The cell samples used for production of biological products were detected by using the established assay. Results The real-time fluorescent quantitative PCR assay was specific for the detection of PPV without cross-reaction to other species of parvovirus virus, SV40 virus and other porcine viruses. The linear range of the assay was 1×109-1×104 copies/μl with a R2 value more than 0.98. The sensitivity of the real-time quantitative PCR assay was 1×104 copies/μl. Both of the intra- and inter-coefficient of variation (CV) were less than 5% in Ct values. The intra- and inter-CV in copies of detection were 5%-15% and 30%-40% respectively. The minimum detection limit of the real-time fluorescent quantitative PCR assay was 1CCID50/ml. The PPV strains were detected in cell samples with no interference. The sensitivity of ST cells infection-PCR test was 0.01CCID50/ml. All of the 22 cell samples were negative for PPV by using the real-time fluorescent quantitative PCR assay. Conclusion The real-time fluorescent quantitative PCR and the ST cell infection-PCR test for the detection of PPV in cells were established successfully. The application of the two assays was conducive to further enhance the safety of using cells for production and therapy. Key words: Porcine parvovirus (PPV); Real-time fluorescent quantitative PCR assay; Swine testicle (ST) cells; Infection assay; Infection-PCR assay