Direct detection of the porcine reproductive and respiratory syndrome (PRRS) virus by reverse polymerase chain reaction (RT-PCR).
A method for direct detection of the porcine reproductive and respiratory syndrome (PRRS) virus was developed, based on reverse transcription of the viral RNA coupled to DNA amplification by polymerase chain reaction. A set of primers was designed from Lelystad virus sequence within ORF 7 encoding nucleocapsid protein. From seven Spanish field isolated strains the 312 bp amplified fragment was cloned and sequenced. Alignment with Lelystad virus sequence revealed a 96-97% homology. A maximum sensitivity of 6.7 TCID50 was achieved with the reported procedure in experimentally infected swine alveolar macrophages cultures. The sensitivity obtained in crude clinical samples from experimentally infected 3-weeks old pigs was approximately 10(2) TCID50. High specificity for the PRRS virus was demonstrated for the method, as none of the seven common swine virus assayed rendered DNA amplification product.
Suárez
P
P
Departamento de Patología Animal I, Universidad Complutense de Madrid, Facultad de Veterinaria, Spain.
Zardoya
R
R
Prieto
C
C
Solana
A
A
Tabarés
E
E
Bautista
J M
JM
Castro
J M
JM
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D008264:Macrophages / Q000382:microbiology; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction / Q000379:methods*; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase; D012689:Sequence Homology, Nucleic Acid; D013030:Spain; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis*
D017931:DNA Primers; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase