title: Rapid detection of foot-and-mouth disease virus, influenza A virus and classical swine fever virus by high-speed real-time RT-PCR.
abstract: High sensitivity, minor risk of cross-contamination and in particular the rapid reaction time make quantitative real-time polymerase chain reaction (qPCR) assays well suited for outbreak investigations as well as for monitoring epidemics of pathogens. In this study qPCR assays for three highly contagious animal diseases, namely foot-and-mouth-disease (FMD), influenza A (IA) and classical swine fever (CSF) have been developed. Furthermore, an amplification control targeting 18S ribosomal RNA was included. Each assay was validated with samples from infected animals using three different standard qPCR-machines in two thermal profiles: one standard and one high-speed approach, respectively. The high-speed PCR assays allowed the reliable diagnosis of FMD, influenza A and CSF in less than 28 min with an analytical sensitivity of at least 200 genome copies/μl in every case, with slight differences regarding reaction time and sensitivity for the individual PCR-cycler instruments. Therefore, the newly established rapid RT-PCR systems will be a valuable method for the monitoring and control of these three important viruses and will be a robust option for the development of novel molecular pen-side tests.
authors.affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Suedufer 10, 17493 Greifswald-Insel Riems, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Wernike
authors.forename: Martin
authors.initials: M
authors.lastname: Beer
authors.forename: Bernd
authors.initials: B
authors.lastname: Hoffmann
mesh_terms: D000818:Animals; D006692:Classical Swine Fever Virus / Q000235:genetics / Q000302:isolation & purification*; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics / Q000302:isolation & purification*; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification*; D025202:Molecular Diagnostic Techniques / Q000379:methods* / Q000592:standards; D012337:RNA, Ribosomal, 18S / Q000032:analysis / Q000235:genetics; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000821:virology; D013997:Time Factors; D014730:Veterinary Medicine / Q000379:methods; D014773:Virology / Q000379:methods / Q000592:standards
chemical_list: D012337:RNA, Ribosomal, 18S; D012367:RNA, Viral
keywords: Classical swine fever; Foot-and-mouth disease; High-speed PCR; Influenza A; Real-time PCR