title: The genotyping of infectious bronchitis virus in Taiwan by a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction.
abstract: Infectious bronchitis virus (IBV; Avian coronavirus) causes acute respiratory and reproductive and urogenital diseases in chickens. Following sequence alignment of IBV strains, a combination of selective primer sets was designed to individually amplify the IBV wild-type and vaccine strains using a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) approach. This system was shown to discriminate the IBV wild-type and vaccine strains. Moreover, an ARMS real-time RT-PCR (ARMS qRT-PCR) was combined with a high-resolution analysis (HRMA) to establish a melt curve analysis program. The specificity of the ARMS RT-PCR and the ARMS qRT-PCR was verified using unrelated avian viruses. Different melting temperatures and distinct normalized and shifted melting curve patterns for the IBV Mass, IBV H120, IBV TW-I, and IBV TW-II strains were detected. The new assays were used on samples of lung and trachea as well as virus from allantoic fluid and cell culture. In addition to being able to detect the presence of IBV vaccine and wild-type strains by ARMS RT-PCR, the IBV Mass, IBV H120, IBV TW-I, and IBV TW-II strains were distinguished using ARMS qRT-PCR by their melting temperatures and by HRMA. These approaches have acceptable sensitivities and specificities and therefore should be able to serve as options when carrying out differential diagnosis of IBV in Taiwan and China.
authors.affiliation: Department of Veterinary Medicine, College of Veterinary Medicine (Huang, Ho, Shien, Wang), National Chung Hsing University, Taichung, TaiwanGraduate Institute of Life Sciences (Liu), National Chung Hsing University, Taichung, TaiwanDepartment of Veterinary Medicine, College of Agriculture, National Chiayi University, Chiayi, Taiwan (Chan)Animal Health Research Institute, Council of Agriculture, Executive Yuan, Taiwan (Cheng).
authors.forename: Shr-Wei
authors.initials: SW
authors.lastname: Huang
authors.affiliation: Department of Veterinary Medicine, College of Veterinary Medicine (Huang, Ho, Shien, Wang), National Chung Hsing University, Taichung, TaiwanGraduate Institute of Life Sciences (Liu), National Chung Hsing University, Taichung, TaiwanDepartment of Veterinary Medicine, College of Agriculture, National Chiayi University, Chiayi, Taiwan (Chan)Animal Health Research Institute, Council of Agriculture, Executive Yuan, Taiwan (Cheng).
authors.forename: Chia-Fang
authors.initials: CF
authors.lastname: Ho
authors.affiliation: Department of Veterinary Medicine, College of Veterinary Medicine (Huang, Ho, Shien, Wang), National Chung Hsing University, Taichung, TaiwanGraduate Institute of Life Sciences (Liu), National Chung Hsing University, Taichung, TaiwanDepartment of Veterinary Medicine, College of Agriculture, National Chiayi University, Chiayi, Taiwan (Chan)Animal Health Research Institute, Council of Agriculture, Executive Yuan, Taiwan (Cheng).
authors.forename: Kun-Wei
authors.initials: KW
authors.lastname: Chan
authors.affiliation: Department of Veterinary Medicine, College of Veterinary Medicine (Huang, Ho, Shien, Wang), National Chung Hsing University, Taichung, TaiwanGraduate Institute of Life Sciences (Liu), National Chung Hsing University, Taichung, TaiwanDepartment of Veterinary Medicine, College of Agriculture, National Chiayi University, Chiayi, Taiwan (Chan)Animal Health Research Institute, Council of Agriculture, Executive Yuan, Taiwan (Cheng).
authors.forename: Min-Chung
authors.initials: MC
authors.lastname: Cheng
authors.affiliation: Department of Veterinary Medicine, College of Veterinary Medicine (Huang, Ho, Shien, Wang), National Chung Hsing University, Taichung, TaiwanGraduate Institute of Life Sciences (Liu), National Chung Hsing University, Taichung, TaiwanDepartment of Veterinary Medicine, College of Agriculture, National Chiayi University, Chiayi, Taiwan (Chan)Animal Health Research Institute, Council of Agriculture, Executive Yuan, Taiwan (Cheng).
authors.forename: Jui-Hung
authors.initials: JH
authors.lastname: Shien
authors.affiliation: Department of Veterinary Medicine, College of Veterinary Medicine (Huang, Ho, Shien, Wang), National Chung Hsing University, Taichung, TaiwanGraduate Institute of Life Sciences (Liu), National Chung Hsing University, Taichung, TaiwanDepartment of Veterinary Medicine, College of Agriculture, National Chiayi University, Chiayi, Taiwan (Chan)Animal Health Research Institute, Council of Agriculture, Executive Yuan, Taiwan (Cheng).
authors.forename: Hung-Jen
authors.initials: HJ
authors.lastname: Liu
authors.affiliation: Department of Veterinary Medicine, College of Veterinary Medicine (Huang, Ho, Shien, Wang), National Chung Hsing University, Taichung, TaiwanGraduate Institute of Life Sciences (Liu), National Chung Hsing University, Taichung, TaiwanDepartment of Veterinary Medicine, College of Agriculture, National Chiayi University, Chiayi, Taiwan (Chan)Animal Health Research Institute, Council of Agriculture, Executive Yuan, Taiwan (Cheng) cyoungwang@dragon.nchu.edu.tw.
authors.forename: Chi-Young
authors.initials: CY
authors.lastname: Wang
mesh_terms: D000818:Animals; D002642:Chick Embryo; D002645:Chickens; D002681:China; D018352:Coronavirus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D005838:Genotype; D001351:Infectious bronchitis virus / Q000235:genetics* / Q000302:isolation & purification; D060885:Multiplex Polymerase Chain Reaction / Q000662:veterinary*; D011201:Poultry Diseases / Q000175:diagnosis / Q000821:virology*; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary*; D012680:Sensitivity and Specificity; D013624:Taiwan
keywords: Infectious bronchitis virus; Taiwan; multiplex amplification refractory mutation system; vaccine