Analytical verification of a PCR assay for identification of Bordetella avium

Abstract

Bordetella avium is the etiologic agent of turkey coryza or bordetellosis, a respiratory disease responsible for substantial economic losses to the turkey industry. At present, identification of this bacterium relies on isolation and biochemical testing. Although a PCR for the detection of B. avium was proposed a number of years ago, lack of analytical verification precludes its use as a diagnostic tool. Furthermore, a number of details pertaining to the reaction conditions used are missing or unclear. In the present study we have identified an optimal set of PCR conditions for use with the previously described primer pair and determined the limit of detection under these conditions to be approximately 20 pg. Assay sensitivity is 100%, based on an analysis of 72 B. avium isolates from diverse geographic locations and covering a time span of at least 25 years. Evaluation of a separate group of 87 bacterial isolates from poultry, comprising both gram-positive and gram-negative commensals and pathogens representing 11 genera, demonstrated an assay specificity of 98.8%. Reproducibility is 100% using either purified genomic DNA or boiled cell lysates less than 3 days old. Sequence analysis of the B. avium PCR amplicons identified only three occasional sequence polymorphisms. These data indicate the B. avium PCR assay can provide clinically significant results.

FIG. 1.

Effect of DMSO, MgCl₂, and primer concentrations on the yield of the N-avium/C-avium amplicon. A) One hundred picograms of B. avium chromosomal DNA was amplified in the presence of the DMSO, MgCl₂, and primer concentrations indicated. B) One hundred twenty picograms of B. avium chromosomal DNA was amplified in the presence of 5% DMSO, 0.5 μM primers, and the MgCl₂ concentration indicated. Other conditions were as described in the text.

FIG. 2.

Effect of different cycling conditions on the yield of the N-avium/C-avium amplicon. Two hundred sixty picograms of B. avium chromosomal DNA was amplified using the program indicated, as outlined in Table 2. Other conditions were as described in the text.

FIG. 3.

Limit of detection for the N-avium/C-avium amplicon. A) One hundred picograms of purified B. avium DNA was used as the template in optimized PCRs as described in the text, with an annealing temperature of 50°C (lane 1), 52°C (lane 2), or 54°C (lane 3). B) Optimized PCRs using an annealing temperature of 52°C and 40 pg (lane 1), 20 pg (lane 2), or 15 pg (lane 3) of purified B. avium DNA.

FIG. 4.

Specificity of the N-avium/C-avium primer set. Representative results obtained from PCRs with the N-avium/C-avium primer set (A) or the univ16S-3/univ16S-4 primer set (B). Lanes: 1, marker; 2, S. hyicus 14-4A; 3, S. hyicus 1-C; 4. Staphylococcus aureus; 5, Staphylococcus epidermis; 6, Enterococcus cecorum; 7, Micrococcus luteus; 8, Escherichia coli; 9, Acinetobacter baumannii; 10, P. multocida; 11. Ornithobacterium rhinotracheale; 12, B. hinzii.

FIG. 5.

DNA sequence variants identified in the region amplified by the N-avium/C-avium primer pair. Dashes indicate conserved bases, asterisks indicate deleted bases, and substitutions are indicated by the appropriate letter, compared to the sequence defined as variant A. The sequence originally reported for this amplicon by Savelkoul et al. (19) was derived from GenBank accession no. X74117.