Redesign of primer and application of the reverse transcriptase-polymerasechain 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 r everse transcriptase-polymerase chain reaction (RT-PCR) and restriction fra gment length polymorphism (RFLP) serotype identification test was not possi ble because the primer used in the RT-PCR did not amplify the S1 gene of th e 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 r everse RT-PCR primers, respectively. A 2-bp mismatch at the 3' end of the r everse primer was found. On the basis of these data, a degenerate primer th at could amplify the S1 gene of the DE072 strain as well as eight other ser otypes of the virus was synthesized. In addition, we were able to different iate the DE072 strain from all of the other IBV strains examined by RFLP an alysis of the RT-PCR product.