title: Reverse transcription recombinase polymerase amplification assay for the rapid detection of type 2 porcine reproductive and respiratory syndrome virus.
abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in pigs, and has tremendous negative economic impact on the swine industry worldwide. PRRSV is classified into the two distinct genotypes: type 1 and type 2, and most of the described PRRSV isolates in China are type 2. Rapid and sensitive detection of PRRSV is of great importance for the disease control and regional eradication programs. Recombinase polymerase amplification (RPA) has emerged as a novel isothermal amplification technology for the molecular diagnosis of infectious diseases. In this study, a fluorescence reverse transcription RPA (RT-RPA) assay was developed to detect the type 2 PRRSV using primers and exo probe specific for the viral nucleocapsid gene. The reaction was performed at 40°C within 20min. The RT-RPA assay could detect both the classical (C-PRRSV) and highly pathogenic PRRSV (HP-PRRSV), but there was no cross-reaction to other pathogens. Using the in vitro transcribed PRRSV RNA as template, the analytical sensitivity of RT-RPA was 690 copies. The assay performance was evaluated by testing 60 field samples and compared to real-time RT-PCR. The detection rate of RT-RPA was 86.6% (52/60), while the detection rate of real-time RT-PCR was 83.3% (50/60). This simple, rapid and reliable method could be potentially applied for rapid detection of PRRSV in point-of-care and rural areas.
authors.affiliation: Inspection and Quarantine Technical Center of Hebei Entry-Exit Inspection and Quarantine Bureau, 318 Heping West Rd., Shijiazhuang, Hebei 050051, China.
authors.forename: Jian-Chang
authors.initials: JC
authors.lastname: Wang
authors.affiliation: College of Veterinary Medicine, Agricultural University of Hebei, No. 38 Lingyusi Street, Baoding, Hebei 071001, China. Electronic address: yuanwanzhe@163.com.
authors.forename: Wan-Zhe
authors.initials: WZ
authors.lastname: Yuan
authors.affiliation: Hebei Animal Disease Control Center, 219 Alishan Street, Shijiazhuang, Hebei 050050, China.
authors.forename: Qing-An
authors.initials: QA
authors.lastname: Han
authors.affiliation: Inspection and Quarantine Technical Center of Hebei Entry-Exit Inspection and Quarantine Bureau, 318 Heping West Rd., Shijiazhuang, Hebei 050051, China.
authors.forename: Jin-Feng
authors.initials: JF
authors.lastname: Wang
authors.affiliation: Inspection and Quarantine Technical Center of Hebei Entry-Exit Inspection and Quarantine Bureau, 318 Heping West Rd., Shijiazhuang, Hebei 050051, China.
authors.forename: Li-Bing
authors.initials: LB
authors.lastname: Liu
mesh_terms: D000818:Animals; D002681:China; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D019318:Porcine Reproductive and Respiratory Syndrome / Q000175:diagnosis* / Q000821:virology; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics / Q000302:isolation & purification*; D045522:Recombinases / Q000378:metabolism*; D048348:Reverse Transcription; D012680:Sensitivity and Specificity; D013552:Swine; D013696:Temperature; D013997:Time Factors; D014730:Veterinary Medicine / Q000379:methods
chemical_list: D045522:Recombinases
keywords: Molecular diagnosis; PRRSV; RPA; Real time PCR