Development and Clinical Evaluation of a Direct Amplification Method to Diagnose Canine Parvovirus and Canine Distemper Viral Infections in Dogs without Nucleic Acid Extraction
Xuefeng Cao1, Guangneng Peng1, Xiaobin Gu1, Changliang He1, Guizhou Yue2, Jun Shi3,* and Zhijun Zhong1,*
Amplification curves (A, CPV; C, CDV) and standard curves (B, CPV; D, CDV) for CPV and CDV. Ten-fold serial dilutions of the CPV plasmid standard and the CDV plasmid standard were used for amplification, as indicated on the X-axis, and the corresponding cycle threshold (CT) values are presented on the Y-axis. A: 1–5: Equal dilutions of the CPV plasmid 10-1–10-5; C: 1-5: equal dilutions of the CDV plasmid 10-1–10-5.
Sensitivity of the real-time PCR/RT-PCR for the detection of CPV and CDV plasmid standards. Serial 10-fold plasmid dilutions (CPV: 7.44×108 copies·μL-1 to 7.44×100 copies·μL-1; CDV: 4.20×107 copies·μL-1 to 4.20×100 copies·μL-1) were plotted against the threshold cycle (Ct) values. A minimum of 7.44×101 copies of CPV (A) and 4.20×101 copies of CDV (B) were detected. 1–9: Equal dilutions of the CPV plasmid standard at 101–109; 12-19: equal dilutions of the CDV plasmid standard at 101–108; 10, 20: negative control; 11, 21: threshold line.
Sensitivity tests for real-time PCR and DARPM. Serial 10-fold dilutions of CPV DNA and CPV virus culture supernatant (CPV DNA: 1.39×108 copies·μL-1 to 1.39×100 copies·μL-1, CPV virus culture supernatant: 7.11×108 copies·μL-1 to 7.11×100 copies·μL-1) were detected. A minimum of 1.53×101 copies of CPV DNA and 6.70×101 copies of CPV virus culture supernatant were detected. A: 1–9: Equal dilutions of CPV DNA at 10-1–10-9; B: 1–9: CPV virus culture supernatant at 10-1–10-9.
Sensitivity tests for real-time RT-PCR and DARPM. Serial 10-fold dilutions of CDV cDNA and CDV virus culture supernatant (CDV cDNA: 6.23×107 copies·μL-1 to 6.23×100 copies·μL-1, CDV virus culture supernatant: 2.28×105 copies·μL-1 to 2.28×100 copies·μL-1) were detected. A minimum of 9.56×101 copies of CDV cDNA and 7.77×101 copies of CDV virus culture supernatant were detected. A: 1-8: Equal dilutions of CDV cDNA at 10-1–10-8, 9: negative control; B: 1–6: CDV virus culture supernatant at 10-1–10-6, 7: negative control.