Establishment and comparison of RF-RAA and qPCR for rapid detection of Chinese soft-shelled turtle adenovirus (CSTAdV).

Abstract

Background: Chinese soft-shelled turtle adenovirus (CSTAdV) is a new virus discovered recently that infects farmed Chinese soft-shelled turtle. In order to investigate its epizootiology and meet the requirements of timely prevention and control, it is imperative to establish an efficient diagnostic assay for CSTAdV.

Results: In this study, on-site diagnostic real-time fluorescence Recombinase-aided amplification (RF-RAA) and real-time quantitative polymerase chain reaction (qPCR) detection methods were established based on the specific sequence of the viral DNA polymerase gene. The results showed that the sensitivity of the CSTAdV RF-RAA assay and qPCR assay was 1.0 × 10² copies/μL within 20 min at 42 °C, and 1.0 × 10¹ copies/μL in approximately 60 min for detecting plasmid pUC57-CSTAdV, respectively. Both the RF-RAA assay and the qPCR assay were highly specific for CSTAdV, with no cross-reaction with Soft-shelled turtle iridovirus, Trionyx sinensis hemorrhagic syndrome virus, Citrobacter freundii, Aeromonas veronii, Aeromonas hydrophila, Morganella morganii, and Vibrio cholerae. A total of 107 clinical specimens of Chinese soft-shelled turtle were tested by the RF-RAA and qPCR assays. The qPCR results were consistent with adenoviral consensus nested PCR published previously, whereas the RF-RAA assay exhibited diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of 95.45% and 100%, respectively. The findings suggest that the newly developed RF-RAA and qPCR assays exhibit high accuracy in detecting CSTAdV in clinical specimens.

Conclusions: Therefore, the RF-RAA and qPCR assays provide two novel alternatives for simple, sensitive and specific identification of CSTAdV for pathogen screening in the field and quantitative analysis in the laboratory.