Recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) designed for rapid detection of canine distemper virus

• Published in: Journal of Veterinary Medical Science Vol. 86; no. 5; pp. 584 - 591
• Main Authors: ZHANG, Shanshan, WANG, Chengyu, MENG, Keyin, LIU, Jun
• Format: Journal Article
• Language: English
• Published: Japan JAPANESE SOCIETY OF VETERINARY SCIENCE 01.01.2024; Japan Science and Technology Agency; The Japanese Society of Veterinary Science
• Subjects: Animals; Biotin; Canine distemper; canine distemper virus; Conserved sequence; Distemper - diagnosis; Distemper - virology; Distemper Virus, Canine - genetics; Distemper Virus, Canine - isolation & purification; Dogs; lateral flow dipstick; Nucleic Acid Amplification Techniques - methods; Nucleic Acid Amplification Techniques - veterinary; Recombinase; recombinase polymerase amplification; Recombinases - metabolism; Secretions; sensitivity; Sensitivity and Specificity; specificity; Tissue culture; Virology; specificity; lateral flow dipstick; sensitivity; canine distemper virus; recombinase polymerase amplification
• ISSN: 0916-7250; 1347-7439; 1347-7439
• DOI: 10.1292/jvms.23-0389

In the present study, recombinase polymerase amplification (RPA) was combined with the colloidal gold lateral flow dipstick (LFD) method to establish a new, stable, and efficient assay for the detection of canine distemper virus (CDV). We designed a set of specific primers labeled with biotin and a specific probe labeled with dSpacer and C3 spacer, according to the conserved region in the N-terminal gene sequence of CDV. The reaction conditions and systems were then optimized, and the sensitivity and specificity were analyzed for potential clinical application. The results showed that the RPA-LFD assay for CDV detection was successfully established. We also found that the temperature in a closed fist (35°C) is optimal for the RPA reaction. The optimal ratio of primer to probe was 2:1. The minimum detection limit of the RPA-LFD assay was 1 × 101 the median tissue culture infective dose (TCID50)/mL. Using this assay with samples from experimentally infected dogs, CDV was detected in nasal secretions, eye secretions, and blood on the fourth day post infection. In summary, this novel RPA-LFD assay for CDV detection is simple to use, and preliminary findings indicate its high specificity and sensitivity.