Rapid differentiation of avian infectious bronchitis virus isolates by sample to residual ratio quantitation using real-time reverse transcriptase-polymerase chain reaction

Abstract

A rapid diagnostic assay for differentiating avian infectious bronchitis virus (IBV) isolates was developed. The basis of the assay is the cleavage of target RNA by RNase H mediated by sequence-specific chimeric oligonucleotides followed by sample to residual ratio quantitation (SRRQ) using RRT-PCR. Four serotype-specific chimeric oligonucleotides were designed, one each for the Massachusetts, Connecticut, Arkansas, and Delaware/Georgia 98 serotypes, and tested for their ability to mediate specific cleavage of target RNA from known homologous and heterologous strains of IBV. Specific cleavage of target RNAs by each chimeric oligonucleotide was verified using agarose gel analysis and RRT-PCR. There were no non-specific cleavage products. Eight different IBV strains representing seven serotypes were tested and each chimeric oligonucleotide mediated cleavage of target RNA only from strains within the serotype that the chimeric was designed against. The SRRQ assay was evaluated on 15 samples without prior knowledge of their grouping and correctly identified the serotype of each sample. The assay is rapid; six samples can be tested in approximately 4 h. In addition, the primer set amplifies all IBV RNAs tested to date and provides a built in control for detecting IBV whether it is typeable or not.

Fig. 1

Design of specific chimeric oligonucleotides for the Massachusetts and Arkansas serotypes. (A) An S1 gene sequence alignment of IBV strains belonging to the Massachusetts and Arkansas serotypes was performed for the region flanked by the primer set NewS1OLIGO5′ and M41L328. Only nucleotides 42–204 (ATG start site = 1) are shown. The sequences targeted by the anti-Massachusetts and anti-Arkansas chimeric oligonucleotides are boxed in black. Dots indicate nucleotides that are identical to the majority sequence, while letters indicate nucleotides that differ from the majority sequence. (B) The structure of a representative chimeric oligonucleotide hybridized to a complimentary strand of RNA. The solid black line with letters is the target RNA. Below is the chimeric oligonucleotide (uppercase = DNA bases, lowercase = 2′-O-Me RNA bases). The arrow denotes the site at which RNase H will cleave the strand of target RNA (Bogdanova et al., 1995, Yu and Steitz, 1997).

Fig. 2

Native agarose gel analysis of Massachusetts 41 S1 runoff RNA cleavage as mediated by RNase H and chimeric oligonucleotides specific for strains in the Massachusetts, Arkansas, Connecticut, and Delaware/Georgia 98 serotypes. Lane 1 = RNA ladder, sizes from top to bottom are 9000, 7000, 5000, 3000, 2000, 1000, and 500 bases (New England Biolabs, Beverly, MA); lane 2 = uncleaved Massachusetts 41 S1 runoff RNA; lane 3 = Massachusetts 41 S1 runoff RNA incubated with anti-Massachusetts chimeric oligonucleotide and RNase H; lane 4 = same as lane 3 except, anti-Arkansas chimeric oligonucleotide used; lane 5 = same as lane 3, except anti-Connecticut chimeric oligonucleotide used; lane 6 = same as lane 3, except anti-Delaware chimeric oligonucleotide used. Arrows indicate cleavage products of ∼1500 and 300 bases.

Fig. 3

Representative sample to residual ratio quantification (SRRQ) amplification graphs and data analysis. For each graph, the x-axis represents the PCR cycle number and the y-axis represents the fluorescence measured during each PCR cycle. The cycle threshold (C_t) for each sample was calculated using the second derivative maximum option. Amplification signal in the negative control (water) samples represents primer dimer and was verified by melting curve and agarose gel analysis (data not shown). (A) SRRQ for IBV strain Arkansas 99 (sample A = anti-Connecticut, C_t = 24.82; sample B = anti-Massachusetts, C_t = 24.15; sample C = anti-Delaware, C_t = 24.90; sample D = no chimeric, C_t = 24.98; sample E = anti-Arkansas, C_t = 28.57). (B) SRRQ for IBV strain CAV 56b (sample A = anti-Massachusetts, C_t = 20.85; sample B = anti-Arkansas, C_t = 21.34; sample C = anti-Connecticut, C_t = 20.59; sample D = anti-Delaware, C_t = 21.57; sample E = no chimeric, C_t = 21.07).