Development of a one-step real-time quantitative PCR assay based on primer-probe energy transfer for the detection of porcine reproductive and respiratory syndrome virus.
A one-step real-time RT-PCR method has been developed for the simultaneous detection of both genotypes of porcine reproductive and respiratory syndrome virus (PRRSV). The assay is based on primer-probe energy transfer, and the most important advantage of this is the relative tolerance towards mutations in the target-probe region. The primers and the probe were designed using an alignment of 235 Type 1 (including all subtypes) and Type 2 PRRSV strains. According to the alignment, multiple degenerations were included in the forward and reverse primers to enable the detection of all PRRSV strains deposited in the GenBank. Specificity was tested using 37 different PRRSV strains and eight other swine pathogen viruses. The detection limit was approximately 10 copies of RNA prepared from the Lelystad virus, a European Subtype 3 virus (Belarus strain Soz-8), and an American vaccine virus (Ingelvac MLV). One TCID(50) was the detection limit in the case of the cell cultured Lelystad virus and an American wild type isolate, respectively. The melting point analysis revealed melting point decrease, but no significant sensitivity and signal loss in the presence of numerous (up to five) target-probe mismatches, indicating the capability of tolerating even more mutations. The method was suitable for the detection and quantitation of phylogenetically divergent strains and can serve as a robust, high throughput tool for molecular diagnosis of the PRRSV.
Balka
Gyula
G
Department of Pathology and Forensic Veterinary Medicine, Faculty of Veterinary Science, Szent István University, Budapest, Hungary. balka.gyula@aotk.szie.hu
Hornyák
Akos
A
Bálint
Adám
A
Benyeda
Zsófia
Z
Rusvai
Miklós
M
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D019318:Porcine Reproductive and Respiratory Syndrome / Q000175:diagnosis*; D019316:Porcine respiratory and reproductive syndrome virus / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013552:Swine
D017931:DNA Primers; D015345:Oligonucleotide Probes