FURTHER DEVELOPMENT AND USE OF A MOLECULAR SEROTYPE IDENTIFICATION TEST FOR INFECTIOUS-BRONCHITIS VIRUS

Previously, we developed a rapid serotype identification test for infe ctious bronchitis virus (IBV) that utilizes the reverse transcriptase- polymerase chain reaction (RT-PCR) and restriction fragment length pol ymorphism analysis. The RT-PCR is used to amplify the S1 gene from RNA extracted from the virus grown in eggs. Restriction enzyme digestion and electrophoresis of that PCR product is used to determine the serot ype of the virus. The purpose of this study was threefold. First, usin g a modified 5' PCR primer, we altered the procedures of our rapid ser otype identification test and amplified the S1 gene of IBV in tracheal swabs collected from specific-pathogen-free leghorn chickens experime ntally inoculated with the Arkansas or Mass 41 serotypes of IBV. Direc t amplification of IBV in tracheal swabs eliminates the need to isolat e the virus in eggs. Second, we attempted to amplify inactivated IBV i n allantoic fluid, possibly allowing us to obtain and determine the se rotype of isolates originating from outside the U.S.A. Virus inactivat ed by formalin (0.1% final concentration) could not be amplified by th e RT-PCR procedure, but heat-inactivated IBV (56 C for 15 min) was suc cessfully amplified. Third, we developed an internal control for the R T-PCR test by synthesizing RNA runoff transcripts of a cloned truncate d S1 gene. The truncated S1 RNA transcripts were added to the RT-PCR r eaction and a 1031-bp product was amplified, which could be distinguis hed from the coamplified S1 gene from viral RNA. The internal RNA cont rol reduces the possibility of obtaining false-negative results in the RT-PCR test.