Universal oligonucleotides for the detection of infectious bronchitis virus by the polymerase chain reaction

Abstract

The universality of seven pairs of oligonucleotides for detection of the coronavirus infectious bronchitis virus (IBV) by reverse-transcription polymerase chain reaction (RT-PCR) was examined using 41 isolates of IBV collected over five decades from Europe, Japan and the USA. Oligonucleotides specific for sequences within the S2 region of the spike (S) gene (Lin et al., 1991a) and nucleocapsid (N) gene (Zwaagstra et al., 1992) gave the appropriate products with all 41 isolates. Oligonu-cleotide pair UTR1 - UTR2 +, corresponding to sequences within the 3 untranslated region (UTR) of the genome, also gave the predicted product with all the isolates. Oligonucleotide pair UTR3 - /UTR4 + was internal to oligonucleotides UTR1 - /UTR2 + and was used in a nested-set arrangement for greater specificity and sensitivity, giving the correct product with the 39 isolates examined. Oligonucleotide pair S1Unil - /S1Uni2 + was used to produce a 1.6 kb cDNA, corresponding to most of the S1 region of the S gene, with 24/24 isolates tested. This oligonucleotide pair was less suited than the others for routine detection of IBV but is recommended for the amplification of the S1 region of the S gene of new isolates for subsequent analysis. Other oligonucleotide pairs, yielding cDNA corresponding to the variable region of the IBV genome where genes 3 and 4 (M) overlap, were selected to be largely specific for Massachusetts serotype isolates, in the context of European strains. RT-PCR analysis using these oligonucleotide pairs showed that a number of field isolate preparations also contained a small amount of Massachusetts serotype virus, probably of vaccine origin and indicative of low level persistent infection. These results suggest that any strain of IBV is likely to be detectable by RT-PCR with at least one of our primer pairs.