Development and clinical evaluation of a direct amplification method to diagnose canine parvovirus and canine distemper viral infections in dogs without nucleic acid extraction

Abstract

To rapidly detect canine parvoviruses (CPV) or canine distemper virus (CDV) without nuclear acid extraction, we established a direct amplification TaqMan real-time (RT)-PCR method (DARPM) to detect CPV and CDV in clinical samples. We compared this new method against real-time PCR/(RT)-PCR and it showed no cross-reactivity with other pathogens. Sensitivity testing showed the minimum detection limits of real-time (RT)-PCR were 7.44×101 copies·μL-1 (CPV) and 4.20×101 copies·μL-1 (CDV). DARPM detection of CPV with DNA showed a minimum detectable of 1.53×101copies μL-1, while the minimum detectable amount from the virus culture supernatant was 6.70×101 copies μL-1. The minimum detectable copy numbers for CDV cDNA and for the virus culture supernatant were 9.56×101 copies μL-1 and 7.77×101 copies μL-1, respectively. To validate the accuracy of DARPM, 112 clinical samples and 97 clinical samples suspected of harboring CPV and CDV were tested. DARPM showed a 100% compliance rate with ordinary PCR and colloidal gold rapid detection methodology, while the coincidence rate for DARPM and the same method with DNA added was also 100%. Therefore, DARPM detects CPV and CDV without the need for pre-PCR nuclear acid extraction. Our results show that DARPM is a specific, sensitive, fast and powerful method for detecting CPV and CDV in clinical samples.