Development of a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction assay for the differential diagnosis of Feline leukemia virus vaccine and wild strains.
A multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) was developed for the differential diagnosis of Feline leukemia virus (FeLV) vaccine and wild-type strains based on a point mutation between the vaccine strain (S) and the wild-type strain (T) located in the p27 gene. This system was further upgraded to obtain a real-time ARMS RT-PCR (ARMS qRT-PCR) with a high-resolution melt analysis (HRMA) platform. The genotyping of various strains of FeLV was determined by comparing the HRMA curves with the defined wild-type FeLV (strain TW1), and the results were expressed as a percentage confidence. The detection limits of ARMS RT-PCR and ARMS qRT-PCR combined with HRMA were 100 and 1 copies of transcribed FeLV RNA per 0.5 ml of sample, respectively. No false-positive results were obtained with 6 unrelated pathogens and 1 feline cell line. Twelve FeLV Taiwan strains were correctly identified using ARMS qRT-PCR combined with HRMA. The genotypes of the strains matched the defined FeLV wild-type strain genotype with at least 91.17% confidence. A higher degree of sequence polymorphism was found throughout the p27 gene compared with the long terminal repeat region. In conclusion, the current study describes the phylogenetic relationship of the FeLV Taiwan strains and demonstrates that the developed ARMS RT-PCR assay is able to be used to detect the replication of a vaccine strain that has not been properly inactivated, thus acting as a safety check for the quality of FeLV vaccines.
Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan (Ho, Chung, Kuo, Wang).|Department of Veterinary Medicine, College of Agriculture, National Chiayi University, Chiayi, Taiwan (Chan, Yang).|Department of Biological Sciences, College of Science, National Sun Yat-sen University, Kaoshiung, Taiwan (Chiang).
Chia-Fang
CF
Ho
Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan (Ho, Chung, Kuo, Wang).|Department of Veterinary Medicine, College of Agriculture, National Chiayi University, Chiayi, Taiwan (Chan, Yang).|Department of Biological Sciences, College of Science, National Sun Yat-sen University, Kaoshiung, Taiwan (Chiang).
Kun-Wei
KW
Chan
Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan (Ho, Chung, Kuo, Wang).|Department of Veterinary Medicine, College of Agriculture, National Chiayi University, Chiayi, Taiwan (Chan, Yang).|Department of Biological Sciences, College of Science, National Sun Yat-sen University, Kaoshiung, Taiwan (Chiang).
Wei-Cheng
WC
Yang
Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan (Ho, Chung, Kuo, Wang).|Department of Veterinary Medicine, College of Agriculture, National Chiayi University, Chiayi, Taiwan (Chan, Yang).|Department of Biological Sciences, College of Science, National Sun Yat-sen University, Kaoshiung, Taiwan (Chiang).
Yu-Chung
YC
Chiang
Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan (Ho, Chung, Kuo, Wang).|Department of Veterinary Medicine, College of Agriculture, National Chiayi University, Chiayi, Taiwan (Chan, Yang).|Department of Biological Sciences, College of Science, National Sun Yat-sen University, Kaoshiung, Taiwan (Chiang).
Yang-Tsung
YT
Chung
Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan (Ho, Chung, Kuo, Wang).|Department of Veterinary Medicine, College of Agriculture, National Chiayi University, Chiayi, Taiwan (Chan, Yang).|Department of Biological Sciences, College of Science, National Sun Yat-sen University, Kaoshiung, Taiwan (Chiang).
James
J
Kuo
Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan (Ho, Chung, Kuo, Wang).|Department of Veterinary Medicine, College of Agriculture, National Chiayi University, Chiayi, Taiwan (Chan, Yang).|Department of Biological Sciences, College of Science, National Sun Yat-sen University, Kaoshiung, Taiwan (Chiang).
Chi-Young
CY
Wang
D000818:Animals; D002371:Cat Diseases / Q000821:virology*; D002415:Cats; D005256:Leukemia Virus, Feline / Q000145:classification* / Q000235:genetics; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D017354:Point Mutation; D015682:Retroviridae Proteins, Oncogenic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D017423:Sequence Analysis, RNA; D020079:Terminal Repeat Sequences / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics; D014765:Viral Vaccines / Q000235:genetics
D015682:Retroviridae Proteins, Oncogenic; D014764:Viral Proteins; D014765:Viral Vaccines; C045569:feline leukemia virus vaccine
Feline leukemia virus; multiplex amplification refractory mutation system; polymerase chain reaction; vaccine