Establishment and preliminary application of an assay for the detection of Torque teno sus virus strains

Abstract

Objective To establish an assay for the detection of Torque teno sus virus (TTSuV) strains and to analyze its preliminary application to biologics. Methods Primers and probe were designed according to the conserved sequences. A fluorescent PCR assay for the detection of TTSuV strains was established. Several parameters including the specificity, linearity, accuracy, sensitivity and anti-interference of the established assay were verified. The fluorescent PCR assay was performed to detect the samples of porcine blood, cell substrate and rotavirus vaccine. The porcine blood samples positive for TTSuV strains were further genotyped. Results The established fluorescent PCR assay was confirmed to have high specificity as no cross-reactions with parvovirus virus of various species, SV40 and porcine circovirus strains were detected. The linear range of the assay was 1×109-1×103 copies/μl for TTSuV1 genotype and 1×109-1×102 copies/μl for TTSuV2 genotype with a R2 value more than 0.993. The sensitivity of the fluorescent PCR assay was 1×103 copies/μl for TTSuV1 genotype and 1×102 copies/μl for TTSuV2 genotype. The intra- and inter-CVs were both less than 7% in Ct values and less than 25% and 45% respectively in copies. No interference was found in the detection of TTSuV nucleic acids from cell samples. 8 out of 20 porcine blood samples were positive for TTSuV strains, among which one sample was positive for TTSuV1 genotype, four samples were positive for TTSuV2 genotype and the rest were positive for both TTSuV1 and TTSuV2 genotypes. Compared with the reference strain, strains genotyped as TTSuV1 and TTSuV2 were respectively shared 98%-99% and 98% homologies in sequences. All of the cell substrate and rotavirus vaccine samples detected by the fluorescent PCR assay were negative for TTSuV strains. Conclusion The fluorescent PCR assay for the detection of TTSuV was established successfully, the application of which would further improve the safety of biologics. Key words: Torque teno sus virus (TTSuV); Fluorescent PCR; Cells; Genotyping