Development of recombinase polymerase amplification assays for rapid and visual detection of canine distemper virus infecting giant panda

Abstract

Background: Canine distemper virus (CDV) is an enveloped negative-strand RNA virus that exhibits a high mutation rate and continuously expands the range of hosts. Notably, CDV has infected giant panda with spill over from viral reservoirs in canines. Giant pandas (Ailuropoda melanoleuca), especially captive pandas, are known to be susceptible to natural infection with CDV. The high fatality rate of CDV poses a serious threat to the safety of the giant panda population. However, vaccines or drugs for canine distemper in giant pandas have not been developed to date. Therefore, a rapid test that can achieve accurate onsite detection of CDV is important to enable the timely implementation of control measures. In this study, we established a nucleic acid visualization assay for targeting the CDV N gene by using combines reverse transcription recombinase polymerase amplification with a closed vertical flow visualization strip (RT-RPA-VF).

Results: The RT-RPA-VF assay does not require sophisticated equipment, and it was determined to provide rapid detection at 35 °C for 30 min, while the limit of detection was 5 × 10¹ copies/μl RNA transcripts and 10^0.5 TCID₅₀ ml^{- 1} viruses. The results showed that the assay was high specific to CDV and had no cross-reactivity with other viruses infecting the giant panda. Compared with RT-qPCR, RT-RPA-VF assay had a sensitivity of 100% and a specificity of 100% in 29 clinical samples. The coincidence rate between RT-RPA-VF and RT-qPCR was 100% (kappa = 1), indicating that the RT-RPA-VF assay possessed good diagnostic performance on clinical samples.

Conclusions: The RT-RPA-VF provides a novel alternative for the simple, sensitive, and specific identification of CDV and showed great potential for point of care diagnostics for captive and wild giant panda.

Keywords: Canine distemper virus; Giant panda; Nucleic acid visualization assay; Reverse transcription recombinase polymerase amplification.

Fig. 1

RT-RPA-VF assay limits of detection with CDV RNA extracted from strains that were serially diluted tenfold

Fig. 2

The specificity of the RT-RPA-VF assays against the CDV N gene

Fig. 3

Primers and probe specificity of the RT-RPA-VF assays for the CDV N gene

Fig. 4

Schematic illustration of RT-RPT-VF assay targeting the CDV N genes. The RT-RPA was performed at a constant temperature. Firstly, Viral RNA is transcribed into cDNA by transcriptase. Secondly, the recombinase, strand-displacing DNA polymerase, SSB (not shown) and primers initiated amplification reaction. Another enzyme, nfo can cleave THF site when the probe hybridizes to its target sequence, result in the departure of C3-spacer and probe extension. The tube was placed in a closed device of vertical flow visualization strip device for detecting the RT-RPA products