Further development and use of a molecular serotype identification test for infectious bronchitis virus

Previously, we developed a rapid serotype identification test for infectious bronchitis virus (IBV) that utilizes the reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism 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 serotype of the virus. The purpose of this study was threefold. First, using a modified 5' PCR primer, we altered the procedures of our rapid serotype identification test and amplified the S1 gene of IBV in tracheal swabs collected from specific-pathogen-free leghorn chickens experimentally inoculated with the Arkansas or Mass 41 serotypes of IBV. Direct amplification of IBV in tracheal swabs eliminates the need to isolate the virus in eggs. Second, we attempted to amplify inactivated IBV in allantoic fluid, possibly allowing us to obtain and determine the serotype of isolates originating from outside the U.S.A. Virus inactivated by formalin (0.1% final concentration) could not be amplified by the RT-PCR procedure, but heat-inactivated IBV (56 C for 15 min) was successfully amplified. Third, we developed an internal control for the RT-PCR test by synthesizing RNA runoff transcripts of a cloned truncated S1 gene. The truncated S1 RNA transcripts were added to the RT-PCR reaction and a 1031-bp product was amplified, which could be distinguished from the coamplified S1 gene from viral RNA. The internal RNA control reduces the possibility of obtaining false-negative results in the RT-PCR test.