title: Recombinase polymerase amplification combined with a lateral flow dipstick for discriminating between infectious Penaeus stylirostris densovirus and virus-related sequences in shrimp genome.
abstract: Penaeus stylirostris densovirus (PstDV) is an important shrimp pathogen that causes mortality in P. stylirostris and runt deformity syndrome (RDS) in Penaeus vannamei and Penaeus monodon. Recently, PstDV-related sequences were found in the genome of P. monodon and P. vannamei. This led to false positive results by PCR-based detection system. Here, a more efficient detection platform based on recombinase polymerase amplification (RPA) and a lateral flow dipstick (LFD) was developed for detecting PstDV. Under the optimal conditions, 30 min at 37°C for RPA followed by 5 min at room temperature for LFD, the protocol was 10 times more sensitive than the Saksmerphrome et al's interim 3-tube nested PCR and showed no cross-reaction with other shrimp viruses. It also reduced false positive results arising from viral inserts to ∼5% compared to 76-78% by the IQ2000™ nested PCR kit and the 309F/R PCR protocol currently recommended by World Organization for Animal Health (OIE) for PstDV detection. Together with simplicity and portability, the protocol serves as an alternative tool to PCR for primarily screening PstDV, which is suitable for both laboratory and field application.
authors.affiliation: School of Veterinary and Biomedical Sciences, James Cook University, Townsville, Queensland 4811, Australia.
authors.forename: Wansadaj
authors.initials: W
authors.lastname: Jaroenram
authors.affiliation: School of Veterinary and Biomedical Sciences, James Cook University, Townsville, Queensland 4811, Australia. Electronic address: Leigh.owens@jcu.edu.au.
authors.forename: Leigh
authors.initials: L
authors.lastname: Owens
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D003429:Cross Reactions; D004259:DNA-Directed DNA Polymerase; D017990:Densovirus / Q000235:genetics / Q000302:isolation & purification*; D005189:False Positive Reactions; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D033561:Penaeidae / Q000821:virology*; D045522:Recombinases; D012680:Sensitivity and Specificity; D013696:Temperature; D013995:Time
chemical_list: D045522:Recombinases; D004259:DNA-Directed DNA Polymerase
keywords: LFD; Penaeus stylirostris; PstDV; RPA; Recombinase polymerase amplification