title: Recombinase Polymerase Amplification Assay-A Simple, Fast and Cost-Effective Alternative to Real Time PCR for Specific Detection of Feline Herpesvirus-1.
abstract: Feline herpesvirus 1 (FHV-1), an enveloped dsDNA virus, is one of the major pathogens of feline upper respiratory tract disease (URTD) and ocular disease. Currently, polymerase chain reaction (PCR) remains the gold standard diagnostic tool for FHV-1 infection but is relatively expensive, requires well-equipped laboratories and is not suitable for field tests. Recombinase polymerase amplification (RPA), an isothermal gene amplification technology, has been explored for the molecular diagnosis of infectious diseases. In this study, an exo-RPA assay for FHV-1 detection was developed and validated. Primers targeting specifically the thymidine kinase (TK) gene of FHV-1 were designed. The RPA reaction was performed successfully at 39°C and the results were obtained within 20 min. Using different copy numbers of recombinant plasmid DNA that contains the TK gene as template, we showed the detection limit of exo-RPA was 102 copies DNA/reaction, the same as that of real time PCR. The exo-RPA assay did not cross-detect feline panleukopenia virus, feline calicivirus, bovine herpesvirus-1, pseudorabies virus or chlamydia psittaci, a panel of pathogens important in feline URTD or other viruses in Alphaherpesvirinae, demonstrating high specificity. The assay was validated by testing 120 nasal and ocular conjunctival swabs of cats, and the results were compared with those obtained with real-time PCR. Both assays provided the same testing results in the clinical samples. Compared with real time PCR, the exo-RPA assay uses less-complex equipment that is portable and the reaction is completed much faster. Additionally, commercial RPA reagents in vacuum-sealed pouches can tolerate temperatures up to room temperature for days without loss of activity, suitable for shipment and storage for field tests. Taken together, the exo-RPA assay is a simple, fast and cost-effective alternative to real time PCR, suitable for use in less advanced laboratories and for field detection of FHV-1 infection.
authors.affiliation: Inspection and Quarantine Technical Center of Hebei Entry-Exit Inspection and Quarantine Bureau, Xinhua District, Shijiazhuang, Hebei, China.
authors.forename: Jianchang
authors.initials: J
authors.lastname: Wang
authors.affiliation: Inspection and Quarantine Technical Center of Hebei Entry-Exit Inspection and Quarantine Bureau, Xinhua District, Shijiazhuang, Hebei, China.
authors.forename: Libing
authors.initials: L
authors.lastname: Liu
authors.affiliation: Inspection and Quarantine Technical Center of Hebei Entry-Exit Inspection and Quarantine Bureau, Xinhua District, Shijiazhuang, Hebei, China.
authors.forename: Jinfeng
authors.initials: J
authors.lastname: Wang
authors.affiliation: Inspection and Quarantine Technical Center of Hebei Entry-Exit Inspection and Quarantine Bureau, Xinhua District, Shijiazhuang, Hebei, China.
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Sun
authors.affiliation: College of Veterinary Medicine, Agricultural University of Hebei, Baoding, Hebei, China.
authors.forename: Wanzhe
authors.initials: W
authors.lastname: Yuan
mesh_terms: D000818:Animals; D017927:Calicivirus, Feline / Q000235:genetics / Q000302:isolation & purification*; D002371:Cat Diseases / Q000821:virology*; D002415:Cats; D003362:Cost-Benefit Analysis; D010957:Plasmids / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000191:economics* / Q000662:veterinary*; D045522:Recombinases / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature; D013937:Thymidine Kinase / Q000235:genetics
chemical_list: D045522:Recombinases; D013937:Thymidine Kinase