Comprehensive Detection and Identification of Seven Animal Coronaviruses and Human Respiratory Coronavirus 229E with a Microarray Hybridization Assay

Abstract

Based on microarray hybridization, a diagnostic test for coronavirus infection was developed using eight coronavirus strains: canine coronavirus (CCoV), feline infectious peritonitis virus (FIPV), feline coronavirus (FCoV), bovine coronavirus (BCoV), porcine respiratory coronavirus (PRCoV), turkey enteritis coronavirus (TCoV), transmissible gastroenteritis virus (TGEV), and human respiratory coronavirus (HRCoV). Up to 104 cDNA clones of eight viruses were obtained by reverse transcription PCR with different pairs of primers designed for each virus and a pair of universal primers designed for the RNA polymerase gene of coronavirus. Total RNAs extracted from virus were reverse transcribed, followed by multi-PCR amplification and labeled with Cy3-dCTP. All labeled cDNAs and prepared gene chips were subjected to specific hybridization. The results showed that extensive cross-reaction existed between CCoV, FCoV, FIPV, TGEV and PRCoV, while there was no cross-reaction between BCoV, TCoV and HRCoV. The ultimate specific gene chip was developed with DNA fragments reamplified from the chosen recombinant plasmids without cross-reaction between different coronaviruses. The hybridization results showed that this gene chip could specifically identify and distinguish the eight coronaviruses and the sensitivity of the chip may be 1,000× more sensitive than PCR, indicating that it can be used for the diagnosis of eight coronavirus infections at the same time.