A pancoronavirus RT-PCR assay for detection of all known coronaviruses

The recent discoveries of novel human coronaviruses, including the coronavirus causing SARS, and the previously unrecognized human coronaviruses HCoV-NL63 and HCoV-HKU1, indicate that the family Coronaviridae harbors more members than was previously assumed. All human coronaviruses characterized at present are associated with respiratory illnesses, ranging from mild common colds to more severe lower respiratory tract infections. Since the etiology of a relatively large percentage of respiratory tract diseases remains unidentified, it is possible that for a certain number of these illnesses, a yet unknown viral causative agent may be found. Screening for the presence of novel coronaviruses requires the use of a method that can detect all coronaviruses known at present. In this chapter, we describe a pancoronavirus degenerate primer-based method that allows the detection of all known and possibly unknown coronaviruses by RT-PCR amplification and sequencing of a 251-bp fragment of the coronavirus polymerase gene.