Résumé
Objective To establish the method for extracting exogenous short DNA fragments of Schistosoma japonicum from urine samples, and to evaluate the efficiency of this method for extraction from urine samples treated with various methods. Methods The S. japonicum SjG28 gene fragment was selected as a target sequence, and the 81 bp short DNA fragment was amplified on the target sequence using PCR assay. Following characterization using sequencing, the short DNA fragment was added into the urine samples as an exogenous short DNA fragment. Primers and probes were designed with SjG28 as a target gene, to establish the real-time fluorescent quantitative PCR (qPCR) assay. The sensitivity of this qPCR assay was evaluated with exogenous short DNA fragments that were diluted at a 1:10 dilution ratio as the DNA template, and the specificity of the qPCR assay was evaluated with the genomic DNA of S. mansoni, S. haematobium, Babesia, Ancyiostoma duodenaie, Cionorchis sinensis, and Paragonimus westermani as DNA templates. Exogenous short DNA fragments were added into artificial and healthy volunteers’ urine samples, followed by pH adjustment, centrifugation and concentration, and the efficiency of extracting exogenous short DNA fragments from urine samples was compared with the QIAmp Viral RNA Mini Kit (Qiagen kit) and BIOG cfDNA easy kit (BIOG kit). Results An 81 bp small DNA fragment of S. japonicum was successfully prepared, and the lowest detection limit of the established qPCR assay was 100 copies/μL of the 81 bp small DNA fragment of S. japonicum. If the genomic DNA of S. japonicum, S. mansoni, S. haematobium, Babesia, A. duodenaie, C. sinensis, and P. westermani served as DNA templates, the qPCR assay only detected fluorescent signals with S. japonicum genomic DNA as the DNA template. If the pH values of artificial urine samples were adjusted to 5, 6, 7 and 8, the recovery rates were (49.12 ± 2.09)%, (84.52 ± 4.96)%, (89.38 ± 3.32)% and (87.82 ± 3.90)% for extracting the exogenous short DNA fragment of S. japonicum with the Qiagen kit, and were (2.30 ± 0.07)%, (8.11% ± 0.26)%, (13.35 ± 0.61)% and (20.82 ± 0.68)% with the BIOG kit, respectively (t = 38.702, 26.955, 39.042 and 29.571; all P values < 0.01). If the Qiagen kit was used for extracting the exogenous short DNA fragment from artificial urine samples, the lowest recovery rate was seen from urine samples with a pH value of 5 (all P values < 0.05), and there were no significant differences in the recovery rate from urine samples with pH values of 6, 7 and 8 (all P values > 0.05). Following centrifugation of artificial [(64.30 ± 1.00)% vs. (58.87 ± 0.26)%; t = 12.033, P < 0.05] and healthy volunteers’ urine samples [(31 165 ± 1 017) copies/μL vs. (28 471 ± 818) copies/μL; t = 23.164, P < 0.05]. In addition, concentration of artificial urine samples with the 10 kDa Centrifugal Filter and concentration of healthy volunteers’ urine samples with the 100 kDa Centrifugal Filter were both effective to increase the recovery of the Qiagen kit for extracting the exogenous short DNA fragment of S. japonicum (both P values < 0.01). Conclusions A method for extracting exogenous short DNA fragments of S. japonicum from urine samples has been successfully established, and the Qiagen kit has a high extraction efficiency. Adjustment of urine pH to 6 to 8 and concentration of healthy volunteers’ urine samples with the 100 kDa Centrifugal Filter are both effective to increase the efficiency of extracting exogenous short DNA fragments of S. japonicum.
Résumé
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 quanti-tative 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.