PriProET based melting point analyses on PRRSV positive field samples.
A one-step real time RT-PCR method has previously been developed for the simultaneous detection of both genotypes of porcine reproductive and respiratory syndrome virus (PRRSV). For further evaluation of the assay and a detailed characterization of the probe binding sites a collection of 24 PRRSV positive field samples from Hungary, Serbia, Austria, a highly pathogenic strain from Bhutan and commercially available MLV vaccine strains were collected and sequenced from the terminal part of ORF6 to the 3' end UTR. The regions that were targeted by the probe were analyzed in detail, and their sequences were compared to that of the probe. Each sample showed a positive result with the PriProET assay, and the samples that showed nucleotide mismatches on the probe binding region had shifted melting points compared to the perfectly matching Lelystad strain. Based on the melting temperatures the strains were classified into 8 groups ranging from 62.4°C to 75.5°C. The samples with the lowest melting temperatures were Type I strains which had less mismatches on the probe binding site than Type II strains. However, these mutations were closer to the 3' end of the probe. It can be speculated that mismatches near the 5' end of the probe had lower influence on the melting temperature.
Balka
Gyula
G
Department of Pathology and Forensic Veterinary Medicine, Faculty of Veterinary Science, Szent István University, István u. 2, H-1078 Budapest, Hungary. balka.gyula@aotk.szie.hu
Hornyák
Akos
A
Dán
Adám
A
Ladinig
Andrea
A
Biksi
Imre
I
Rusvai
Miklós
M
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics*; D004735:Energy Transfer; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D044366:Transition Temperature
D017931:DNA Primers; D004279:DNA, Viral