title: Development of a primer-probe energy transfer based real-time PCR for the detection of Swine influenza virus.
abstract: Swine influenza virus (SIV) causes a contagious and requiring official notification disease of pigs and humans. In this study, a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay based on primer-probe energy transfer (PriProET) for the detection of SIV RNA was developed. The assay uses matrix gene-specific primers and an Oregon Green-labeled fluorescent probe and was employed for the detection of SIV in clinical samples to identify outbreaks and to monitor the prevalence of disease. The PriProET technology was used to obtain a probe melting profile for confirmation of the specific product amplification. The assay is specific for influenza virus with a sensitivity of detection limit of approximately 10 copies of RNA by PCR. Based on serial dilutions of SIV, the detection limit of the assay was approximately 0.003 TCID(50)/ml for H1N1 A/Swine/Poland/KPR9/2004 virus. The PriProET RT-PCR was suitable for the detection of SIV RNA isolated directly from clinical samples. The assay detected SIV RNA in pre-clinical swab samples as early as 2 days post-infection (dpi). The PriProET RT-PCR assay is an alternative to the existing diagnostic assays and could have enhanced applicability for clinical diagnosis.
authors.affiliation: The National Veterinary Research Institute, 24-100 Puławy, 57 al Partyzantow, Poland. andrzej.kowalczyk@piwet.pulawy.pl
authors.forename: Andrzej
authors.initials: A
authors.lastname: Kowalczyk
authors.forename: Iwona
authors.initials: I
authors.lastname: Markowska-Daniel
authors.forename: Thomas Bruun
authors.initials: TB
authors.lastname: Rasmussen
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D004735:Energy Transfer; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification*; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics; D009976:Orthomyxoviridae Infections / Q000175:diagnosis* / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000821:virology; D014730:Veterinary Medicine / Q000379:methods*; D014773:Virology / Q000379:methods*
chemical_list: D017931:DNA Primers; D015345:Oligonucleotide Probes