Development of one-step multiplex real-time PCR for the detection of CHV-1, CAdV-2, and CDV

Abstract

Canine Infectious Respiratory Disease Complex (CIRDC) is a highly contagious disease that frequently affects canine populations and has emerged as a global epidemic. It has been reported that CIRDC can have a serious impact on related life. Therefore, the rapid detection and differentiation of common viruses that cause CIRDC are essential. It is generally believed that CIRDC is mainly caused by infection of three pathogens: canine herpesvirus-1 (CHV-1), canine adenovirus-2 (CAdV-2), and canine distemper virus (CDV). In this study, we developed and validated a TaqMan probe-based multiplex real-time PCR method to detect and identify these three viruses simultaneously. We designed specific primers and probes, and optimized the concentrations of each reactant in the system. The method was found to have good sensitivity, specificity and stability, and had a limit of detection of 10² copies/μL, 10¹ copies/μL and 10¹ copies/μL for CHV-1, CAdV-2, and CDV, respectively. In addition, co-infection simulation experiments confirmed that the method worked effectively, even if the concentrations of multiple viruses in the sample were close to the limit of detection or the concentrations of different viruses were different. The method was used to detect 122 clinical samples, and the results showed that it was more sensitive and reliable than conventional singleplex PCR. Thus, the method developed in this study is suitable for the clinical monitoring of CIRDC and is of great significance for the prevention and management of respiratory diseases in canine populations.

Keywords: CIRDC; canine adenovirus-2; canine distemper virus; canine herpesvirus-1; multiplex real-time PCR.

Figure 1

Preparation of plasmid standards. (A,C,E) Amplification curves (X-axis: Cycle, Y-axis: Fluorescence) of CHV-1, CAdV-2, and CDV for each plasmid with concentrations of 1 × 10⁷ copies/μL to 1 × 10² copies/μL. (B,D,F) Standard curves of CHV-1, CAdV-2, and CDV plasmids.

Figure 2

Optimization of the multiplex real-time PCR system and establishment of standard curves. (A) Amplification curves (X-axis: Cycle, Y-axis: Fluorescence) of CHV-1, CAdV-2, and CDV for each plasmid with concentrations of 1 × 10⁷ copies/μL to 1 × 10² copies/μL in multiplex real-time PCR. (B) Standard curves of CHV-1, CAdV-2, and CDV plasmids in the multiplex real-time PCR assay.

Figure 3

(A–C) Amplification curves (X-axis: Cycle, Y-axis: Fluorescence) of 10-fold serial dilutions (1 × 10⁷–1 × 10⁰ copies/μL) of plasmid standards for CHV-1, CAdV-2, and CDV detected by the multiplex real-time PCR assay. (D) Three amplification curves represent samples positive for CHV-1, CAdV-2, and CDV detected by our multiplex real-time PCR assay; negatives include CPV, CCoV, CPIV, CAstV, ChPV, and the negative control.

Figure 4

Co-infection simulation experiments with two pathogens. (A,C,E) Amplification curves (X-axis: Cycle, Y-axis: Fluorescence) of CHV-1 + CAdV-2, CHV-1 + CDV, CAdV-2 + CDV at concentrations of 10 times the limit of detection. (B,D,F) Amplification curves of CHV-1 + CAdV-2, CHV-1 + CDV, CAdV-2 + CDV at the limit of detection. Two replicates were used per reaction.

Figure 5

Co-infection of all three pathogens at different concentrations. (A) The concentration of plasmid standards for CHV-1 was 1 × 10⁷ copies/μL, while the others were 10 times the limit of detection. (B) The concentration of plasmid standards for CAdV-2 was 1 × 10⁷ copies/μL, while the others were 10 times the limit of detection. (C) The concentration of plasmid standards for CDV was 1 × 10⁷ copies/μL, while the others were 10 times the limit of detection. Two replicates were used per reaction. X-axis: Cycle; Y-axis: Fluorescence.