key: cord-017894-8iahlshj authors: Loa, Chien Chang; Wu, Ching Ching; Lin, Tsang Long title: A Multiplex Polymerase Chain Reaction for Differential Detection of Turkey Coronavirus from Chicken Infectious Bronchitis Virus and Bovine Coronavirus date: 2015-09-10 journal: Animal Coronaviruses DOI: 10.1007/978-1-4939-3414-0_12 sha: doc_id: 17894 cord_uid: 8iahlshj 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. Turkey coronavirus (TCoV) contributed to signifi cant economic losses and remains as a serious threat to the turkey producers. Turkey coronaviral enteritis in areas with high concentrations of turkeys on a year-round basis is not easily eliminated and is encountered frequently in turkey poults [ 1 ] . Accurate and rapid method for diagnosis of TCoV infection is the key to effective control of the disease. Turkey coronavirus belongs to the family Coronaviridae, which is a group of enveloped, positive-stranded RNA viruses that infect a wide range of mammalian and avian species. The major structural proteins of coronavirus include phosphorylated nucleocapsid (N) protein, peplomeric spike (S) glycoprotein, and transmembrane or membrane (M) glycoprotein. Spike protein contributes to the distinctive peplomers on the viral surface and contains neutralizing and group-specifi c epitopes. Spike protein is highly variable among different coronaviruses while M and N proteins are more conserved among coronaviruses between different antigenic groups [ 2 ] . There is a close antigenic and genomic relationship between TCoV and infectious bronchitis virus (IBV) according to studies of immunofl uorescent antibody assay ( IFA ), enzyme-linked immunosorbent assay ( ELISA ), and sequence analysis in our and other laboratories [ 3 -8 ] . In addition, bovine coronavirus (BCoV) was demonstrated to cause experimental enteric infection in turkey [ 9 ] . Therefore, close relationship between TCoV and BCoV was previously reported and TCoV was placed in an antigenic group as BCoV [ 10 , 11 ] . Although the sequence data revealed divergence of S genes among TCoV, IBV, and BCoV [ 12 -14 ] , there is still a need to detect and differentiate them accurately and quickly. Polymerase chain reaction ( PCR ) assay has been an important approach for detecting many veterinary important microorganisms with the distinct advantages of high sensitivity and specifi city. This chapter describes a multiplex PCR assay to detect and differentiate TCoV, IBV, and BCoV in a single reaction [ 15 ] . Elmer Centers Corp., Norwalk, CT, USA). 6. Ethidium bromide, 0.5 μg/ml. Stomacher with fi vefold volume of PBS solution. 2. The intestinal homogenates are clarifi ed by centrifugation at 1500 × g for 10 min. The supernatants containing TCoV are used as virus source for preparation of RNA templates for reverse transcription (RT)-PCR reaction. 3. Two hundred microliters of above supernatants are mixed with 1 ml of RNApure reagent and incubated on ice for 10 min ( see Note 2 ). 4. Add 180 μl of chloroform, mix the mixture, and vortex vigorously for 10 s ( see Note 3 ). 5. Centrifuge at 13,000 × g for 10 min at 4 °C. Carefully take the upper aqueous phase into a clean tube and mix with equal volume of cold isopropanol by vortexing vigorously for 30 s. Incubate on ice for 10 min. 6. Centrifuge at 13,000 × g for 10 min at 4 °C. Carefully discard the supernatant without disturbing RNA pellet. 7. Wash RNA pellet with 1 ml of cold 70 % ethanol. Incubate on ice for 5 min. 2. The suggested ratio of RNApure reagent to sample is 10:1. Excess amount of RNApure reagent has no negative impact. The lower ratio (5:1) in this step is intended to obtain higher concentration of viral RNA in the fi nal supernatants. If the upper aqueous phase after centrifugation at step 5 is more than half of the total volume, there is not enough RNApure reagent added. The appropriate reagent amount may be adjusted. Chloroform is applied at 150 μl for every ml of lysate. 3. The sample mixture with chloroform at this step can be stored at −70 °C or lower than −70 °C before proceeding to the next step. 7. For routine practice in the laboratory for large number of samples, 25 μl reaction can be applied with reduced cost of reagents. TCoV positive: two PCR products, a 356-bp band, and a 727bp band. IBV positive: one PCR product of 356 bp. BCoV positive: one PCR product of 568 bp. Negative: no PCR product. One limitation of this multiplex PCR should be noted. When both TCoV and IBV are present in the sample, the IBVpositive PCR product at 356 bp is overlapping with one of the two TCoV PCR products. The result of two PCR product bands at 356 and 727 bp refl ects a confi rmed diagnosis of TCoV positive but does not rule out the presence of IBV. This concern is alleviated by tissue tropism of IBV. Turkey enteric infection by chicken respiratory IBV has never been reported. Accordingly, the presence of IBV in turkey intestines (the test sample) is not likely. Any such concern should be further evaluated by a separate PCR specifi c for IBV [ 16 ] . On the other hand, IBV-positive result is able to exclude the presence of TCoV. Coronaviral enteritis of turkeys (blue comb disease) Coronavirus immunogens Antigenic characterization of a turkey coronavirus identifi ed in poult enteritis and mortality syndrome-affected turkeys Phylogenetic analysis of a highly conserved region of the polymerase gene from 11 coronaviruses and development of a consensus polymerase chain reaction assay Sequence analysis of the matrix/nucleocapsid gene region of turkey coronavirus Sequence analysis of the turkey coronavirus nucleocapsid protein gene and 3′ untranslated region identifi es the virus as a close relative of infectious bronchitis virus Detection of antibody to turkey coronavirus by antibodycapture enzyme-linked immunosorbent assay utilizing infectious bronchitis virus antigen Nucleocapsid protein gene sequence analysis reveals close genomic relationship between turkey coronavirus and avian infectious bronchitis virus Experimental bovine coronavirus in turkey poults and young chickens Antigenic and genomic relationships among turkey and bovine enteric coronaviruses Sequence analysis of the turkey enteric coronavirus nucleocapsid and membrane protein genes: a close genomic relationship with bovine coronavirus Complete sequences of 3′end coding region for structural protein genes of turkey coronavirus Comparison of 3′ end encoding regions of turkey coronavirus isolates from Indiana, North Carolina, and Minnesota Complete nucleotide sequence of polyprotein gene 1 and genome organization of turkey coronavirus Differential detection of turkey coronavirus, infectious bronchitis virus, and bovine coronavirus by a multiplex polymerase chain reaction Redesign of primers and application of the reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism test to the DE072 strain of infectious bronchitis virus The protocol "A multiplex polymerase chain reaction for differential detection of turkey coronavirus from chicken infectious bronchitis virus and bovine coronavirus" outlined in this chapter had been successfully carried out in the authors' studies on molecular diagnostics and molecular virology of turkey coronavirus infection in turkeys. Those studies were in part fi nancially supported by USDA, North Carolina Poultry Federation, and/or Indiana Department of Agriculture and technically assisted by Drs. Tom Brien and David Hermes, Mr. Tom Hooper, and Ms. Donna Schrader for clinical and diagnostic investigation, virus isolation and propagation, and animal experimentation.