Multiplex Amplification Refractory Mutation System Polymerase Chain Reaction (ARMS-PCR) for diagnosis of natural infection with canine distemper virus.
BACKGROUND
Canine distemper virus (CDV) is present worldwide and produces a lethal systemic infection of wild and domestic Canidae. Pre-existing antibodies acquired from vaccination or previous CDV infection might interfere the interpretation of a serologic diagnosis method. In addition, due to the high similarity of nucleic acid sequences between wild-type CDV and the new vaccine strain, current PCR derived methods cannot be applied for the definite confirmation of CD infection. Hence, it is worthy of developing a simple and rapid nucleotide-based assay for differentiation of wild-type CDV which is a cause of disease from attenuated CDVs after vaccination. High frequency variations have been found in the region spanning from the 3'-untranslated region (UTR) of the matrix (M) gene to the fusion (F) gene (designated M-F UTR) in a few CDV strains. To establish a differential diagnosis assay, an amplification refractory mutation analysis was established based on the highly variable region on M-F UTR and F regions.


RESULTS
Sequences of frequent polymorphisms were found scattered throughout the M-F UTR region; the identity of nucleic acid between local strains and vaccine strains ranged from 82.5% to 93.8%. A track of AAA residue located 35 nucleotides downstream from F gene start codon highly conserved in three vaccine strains were replaced with TGC in the local strains; that severed as target sequences for deign of discrimination primers. The method established in the present study successfully differentiated seven Taiwanese CDV field isolates, all belonging to the Asia-1 lineage, from vaccine strains.


CONCLUSIONS
The method described herein would be useful for several clinical applications, such as confirmation of nature CDV infection, evaluation of vaccination status and verification of the circulating viral genotypes.
Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, 250 Kou Kuang Road, Taichung 402, Taiwan.
Songkhla
S
Chulakasian
Min-Shiuh
MS
Lee
Chi-Young
CY
Wang
Shyan-Song
SS
Chiou
Kuan-Hsun
KH
Lin
Fong-Yuan
FY
Lin
Tien-Huan
TH
Hsu
Min-Liang
ML
Wong
Tien-Jye
TJ
Chang
Wei-Li
WL
Hsu
D054458:Amplified Fragment Length Polymorphism Analysis; D000818:Animals; D004216:Distemper / Q000175:diagnosis*; D004217:Distemper Virus, Canine / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D004285:Dogs; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA; D014765:Viral Vaccines / Q000627:therapeutic use
D014765:Viral Vaccines