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Development and Clinical Evaluation of a Direct Amplification Method to Diagnose Canine Parvovirus and Canine Distemper Viral Infections in Dogs without Nucleic Acid Extraction

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,*

1College of Veterinary Medicine, Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, China
2College of Science, Sichuan Agricultural University, Ya’an, Sichuan, 625014, China
3State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China

Xuefeng Cao and Guangneng Peng have contributed equally to this work.

*      Corresponding author: zhongzhijun488@126.com;
shijun215@tongji.edu.cn

Fig. 1.

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.

Fig. 2.

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.

Fig. 3.

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.

Fig. 4.

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.

Pakistan Journal of Zoology

October

Vol. 51, Iss. 5, Pages 1599-1997

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