Detection and differentiation of pathogenicity of avian paramyxovirus serotype 1 (APMV-1) from field cases using one-step real-time RT-PCR.
A real-time fluorescent RT-PCR assay was developed to amplify avian paramyxovirus serotype 1 (APMV-1)-specific nucleic acid fragments from field samples. Subsequent restriction endonuclease analysis (REA) using BglI was carried out to type strains according to their virulence. Primer sequences were used to amplify a 202 base-pair fragment, encompassing the fusion protein cleavage site, in a one-step RT-PCR test for detection of a range of field cases and reference strains of APMV-1. Subsequent restriction endonuclease analysis of the amplified fragments enabled differentiation of low virulent lentogenic field and vaccine strains from more virulent mesogenic and velogenic field strains of APMV-1, including pigeon PMV-1. In 2004, seven cases of pigeon PMV-1 in Northern Ireland were diagnosed and differentiated more rapidly using the fluorescent RT-PCR assay when compared with the use of virus isolation. We report the development and application of a one-step real-time RT-PCR test coupled with REA as a fast, specific method for both the detection and typing of APMV-1 from field samples.
Creelan
J L
JL
Veterinary Sciences Division, Department of Agriculture and Rural Development, Belfast, Northern Ireland, United Kingdom. Julie.Creelan@dardni.gov.uk
McCullough
S J
SJ
D000818:Animals; D002645:Chickens / Q000821:virology*; D009521:Newcastle Disease / Q000175:diagnosis* / Q000821:virology*; D009522:Newcastle disease virus / Q000302:isolation & purification* / Q000472:pathogenicity*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D014774:Virulence / Q000235:genetics