A Constant Temperature Fluorescent RPA Assay for Detecting Novel Muscovy Duck Parvovirus

Objective A constant temperature fluorescent RPA assay for detecting novel Muscovy duck parvovirus (N-MDPV) was developed.

Method  EXO fluorescent probes were specifically bound to the targeted conserved VP3 fragment of N-MDPV. RPA amplification primers were designed to amplify the segment by using recombinant enzyme polymerase. An assay for detecting N-MDPV was established with reaction time and temperature optimized and specificity, sensitivity, and accuracy tested on the collected nucleic acid of disease material in comparison with traditional PCR and virus isolation identification methods.

Result The optimized assay reaction temperature and time were 39 ℃ for 30 min to achieve a lowest sensitivity for nucleic acid detection at 10 fg·μL⁻¹. The nucleic acid of N-MDPV was specifically amplified without any cross reaction with those of duck adenovirus type 3, fowl adenovirus type 4, duck circovirus, duck plague virus, duck hepatitis virus, duck tembusu virus, and novel duck reovirus. Along with the conventional PCR and the virus isolation and identification methods, the newly developed assay detected the nucleic acid on 38 duck tissue specimens with a positive rate of 36.8% (14/38) in comparison to those at 36.8% (14/38) for the PCR and 31.6% (12/38) for the isolation and identification method. In addition, 100% coincidence rates of the assay and the two other methods on positive samples as well as of the assay and the PCR on the positive clinical samples were secured.

Conclusion  The new RPA method to rapidly and visually detect N-MDPV demonstrated to be highly specific, sensitive, and accurate. It was deemed appropriate for clinical applications.