Differential detection of turkey coronavirus, infectious bronchitis virus, and bovine coronavirus by a multiplex polymerase chain reaction

Abstract

The objective of the present study was to develop a multiplex polymerase chain reaction (PCR) method for differential detection of turkey coronavirus (TCoV), infectious bronchitis coronavirus (IBV), and bovine coronavirus (BCoV). Primers were designed from conserved or variable regions of nucleocapsid (N) or spike (S) protein gene among TCoV, IBV, and BCoV and used in the same PCR reaction. Reverse transcription followed by the PCR reaction was used to amplify a portion of N or S gene of the corresponding coronaviruses. The PCR products were detected on agarose gel stained with ethidium bromide. Two PCR products, a 356-bp band corresponding to N gene and a 727-bp band corresponding to S gene, were obtained for TCoV isolates. In contrast, one PCR product of 356 bp corresponding to a fragment of N gene was obtained for IBV strains and one PCR product of 568 bp corresponding to a fragment of S gene was obtained for BCoV. There were no PCR products with the same primers for Newcastle disease virus, Marek's disease virus, turkey pox virus, pigeon pox virus, fowl pox virus, reovirus, infectious bursal disease virus, enterovirus, astrovirus, Salmonella enterica, Escherichia coli, and Mycoplasma gallisepticum. Performance of the assay with serially diluted RNA demonstrated that the multiplex PCR could detect 4.8x10(-3) microg of TCoV RNA, 4.6x10(-4) microg of IBV RNA, and 8.0x10(-2) microg of BCoV RNA. These results indicated that the multiplex PCR as established in the present study is a rapid, sensitive, and specific method for differential detection of TCoV, IBV, and BCoV in a single PCR reaction.

Fig. 1

Agarose gel electrophoresis of products amplified by reverse transcription-multiplex polymerase chain reaction. Lane 1 = molecular weight marker (100 bp ladder), lane 2 = bovine coronavirus, lane 3 = infectious bronchitis coronavirus strain Mass 41, lane 4 = turkey coronavirus Indiana isolate 540, and lane 5 = negative control without template.

Fig. 2

Specificity of reverse transcription-multiplex polymerase chain reaction. Lane 1 = molecular weight marker (100 bp ladder), lane 2 = turkey coronavirus (TCoV) prototype isolate ATCC, lane 3 = TCoV 2580, lane 4 = TCoV 1440, lane 5 = TCoV 284, lane 6 = TCoV 1038, lane 7 = infectious bronchitis coronavirus (IBV) strain CA, lane 8 = IBV Mass 41, lane 9 = infectious bursal disease virus, lane 10 = Newcastle disease virus, lane 11 = Marek's disease virus, lane 12 = astrovirus, lane 13 = enterovirus, lane 14 = Salmonella enterica, lane 15 = Escherichia coli, and lane 16 = negative control without template.

Fig. 3

Sensitivity of reverse transcription-multiplex polymerase chain reaction for (A) turkey coronavirus RNA at 4.8, 4.8 × 10⁻¹, 4.8 × 10⁻², 4.8 × 10^-3, or 4.8 × 10^-4 μg from lanes 2 to 6, respectively. Lane 1 = molecular weight marker (100 bp ladder) and lane 7 = negative control without template. (B) bovine coronavirus RNA at 8.0 × 10⁻¹, 8.0 × 10⁻², 8.0 × 10⁻³, 8.0 × 10⁻⁴, or 8.0 × 10⁻⁵ μg from lanes 9 to 13, respectively. Lane 8 = molecular weight marker (100 bp ladder) and lane 14 = negative control without template. (C) Infectious bronchitis coronavirus RNA at 4.6, 4.6 × 10⁻¹, 4.6 × 10⁻², 4.6 × 10⁻³, 4.6 × 10⁻⁴, or 4.6 × 10⁻⁵ μg from lanes 2 to 7, respectively. Lane 1 = molecular weight marker (100 bp ladder) and lane 8 = negative control without template.

Fig. 4

Specificity of primers NewS1oligo5′ and Degenerate3′. Lane 1 = molecular weight marker (100 bp ladder), lane 2 = infectious bronchitis coronavirus (IBV) strain Mass 41, lane 3 = IBV Ark, lane 4 = IBV Conn, lane 5 = turkey coronavirus (TCoV) prototype isolate ATCC, lane 6 = TCoV 540, lane 7 = TCoV 1440, lane 8 = bovine coronavirus (BCoV) strain Nebraska, lane 9 = BCoV isolate 4758, and lane 10 = negative control without template.