ABSTRACT
Turkey coronaviral enteritis caused by turkey coronavirus (TCoV) continues to infect turkey flocks, resulting in significant economic loss. Determining and understanding genetic relationships among different TCoV isolates or strains is important for controlling the disease. Using two-step RT-PCR assays that amplify the full length of TCoV spike (S) gene, TCoV isolates can be sequenced, analyzed, and genotyped. Described in this chapter is the protocol on PCR amplification and sequencing analysis of full-length TCoV S gene. Such protocol is useful in molecular epidemiology for establishing an effective strategy to control the transmission of TCoV among turkey flocks. Copyright © Springer Science+Business Media New York 2016
ABSTRACT
A multiplex polymerase chain reaction (PCR) method for differential detection of turkey coronavirus (TCoV), infectious bronchitis virus (IBV), and bovine coronavirus (BCoV) is presented in this chapter. Primers are designed from the conserved or variable regions of nucleocapsid (N) or spike (S) protein genes of TCoV, IBV, and BCoV and used in the same PCR reaction. Reverse transcription followed by PCR reaction is used to amplify a portion of N or S gene of the corresponding coronaviruses. Two PCR products, a 356-bp band corresponding to N gene and a 727-bp band corresponding to S gene, are obtained for TCoV. In contrast, one PCR product of 356 bp corresponding to a fragment of N gene is obtained for IBV strains and one PCR product of 568 bp corresponding to a fragment of S gene is obtained for BCoV. Copyright © Springer Science+Business Media New York 2016.
ABSTRACT
Turkey coronavirus (TCoV) infection continues to threaten turkey industry. Because specific treatment and effective vaccination program are not available, rapid and cost-effective detection of antibodies to TCoV infection is an important control measure to monitor the disease status in the fields. Two antibody-capture enzyme-linked immunosorbent assay (ELISA) procedures for detection of antibodies to TCoV are outlined in this chapter. One ELISA method uses chicken infectious bronchitis coronavirus (IBV) as the coating antigen based on antigenic cross-reactivity between TCoV and IBV. The other method relies on a recombinant TCoV nucleocapsid protein. Both methods are useful for serological diagnosis of TCoV infection in the turkey flocks. Copyright © Springer Science+Business Media New York 2016.
ABSTRACT
Expression and purification of turkey coronavirus (TCoV) nucleocapsid (N) protein from a prokaryotic expression system as histidine-tagged fusion protein are presented in this chapter. Expression of histidine-tagged fusion N protein with a molecular mass of 57 kDa is induced with isopropyl beta-D-1-thiogalactopyranoside (IPTG). The expressed N protein inclusion body is extracted and purified by chromatography on nickel-agarose column to near homogeneity. The protein recovery can be 10 mg from 100 ml of bacterial culture. The purified N protein is a superior source of TCoV antigen for antibody-capture ELISA for detection of antibodies to TCoV. Copyright © Springer Science+Business Media New York 2016.