Redesign of primer and application of the reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism test to the DE072 strain of infectious bronchitis virus.
Diagnosis of the DE072 strain of infectious bronchitis virus (IBV) by the reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) serotype identification test was not possible because the primer used in the RT-PCR did not amplify the S1 gene of the DE072 strain. The 3' end of the polymerase gene and the 5' end of the S2 gene of the DE072 strain were sequenced and compared with the forward and reverse RT-PCR primers, respectively. A 2-bp mismatch at the 3' end of the reverse primer was found. On the basis of these data, a degenerate primer that could amplify the S1 gene of the DE072 strain as well as eight other serotypes of the virus was synthesized. In addition, we were able to differentiate the DE072 strain from all of the other IBV strains examined by RFLP analysis of the RT-PCR product.
Lee
C W
CW
Department of Avian Medicine, College of Veterinary Medicine, University of Georgia, Athens 30602-4875, USA.
Hilt
D A
DA
Jackwood
M W
MW
D000818:Animals; D001483:Base Sequence; D002642:Chick Embryo; D018352:Coronavirus Infections / Q000175:diagnosis / Q000662:veterinary*; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D001351:Infectious bronchitis virus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D012150:Polymorphism, Restriction Fragment Length; D011201:Poultry Diseases / Q000175:diagnosis* / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary*; D012703:Serotyping; D013045:Species Specificity; D014481:United States
D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases