“…This constant arising of new IB variants in different countries [8,18] is usually pointed as the main cause of vaccine failures in the field and constitutes a great challenge for the molecular direct diagnosis of IBV.Variants of IBV are mutants evolving from parental strains of this virus that differ from strains considered classic, such as those from Massachusetts group, regarding genotype, pathotype, antigenicity and/or immunogenicity. Additionally, IBV mutants are constantly subject to host immune selection and only strains with high antigenic variation are able to evade immune responses induced by vaccines and to survive in the population of hosts, leading to the development of clinical disease [25].Protection studies between commercial vaccines and IBV variants, especially those using Massachusetts (Mass) vaccines, which is the most common attenuated available vaccine, have demonstrated at least partial cross-protection by some authors [9,10,12,14,34], while another studies have not found cross-protection [7,35].Therefore, the rapid detection of IBV infection in poultry flocks and its classification between Mass and variants is considered as major challenge, which requires use of appropriate diagnostic methods. To overcome this issue, several rapid assays have been developed focusing on detection and/or characterization of S1 gene of IBV, including direct gene sequencing, RT-PCR methods with serotype-specific primers, and RT-PCR/RFLP, have become widely used, because their results correlated well with the virusserotyping [19,21,22,33].…”