Simple Algorithm for Identification of Bordetella pertussis Pertactin Gene Variants

Abstract

Studies performed in several countries have demonstrated the recent emergence and subsequent dominance of circulating Bordetella pertussis strains harboring pertactin and pertussis toxin variants not included in pertussis vaccines. Determination of the pertactin gene variants is commonly performed using a time-consuming and expensive sequence analysis. We developed a simple and reliable pertactin typing algorithm suitable for large-scale screening. The assay correctly identified all pertactin alleles in representative strains. The typing of 231 clinical strains of B. pertussis routinely isolated in Belgium showed that this algorithm was adequate to identify less-frequent prn types like prn9 and prn11.

FIG. 1.

(A) Polymorphism in the prn gene region 1. The 11 known sequences are aligned. Dots represent identical bases; dashes represent deleted bases. The nucleotide numbers correspond to those of prn1 sequence accession number AJ011091 (GenBank database [http://www.ncbi.nlm.nih.gov/entrez]). (B) Primers prnC and prnE were designed to recognize a region of two constitutive GGAVP repeats and the overlap between GGAVP and GGFGP, respectively. They were used in two separate reactions with the prnD primer that hybridizes with a region downstream of region 1, as shown in panel C. (C) The amplification of isolates with primer pairs prnC-prnD and prnE-prnD allowed us to determine the total number of repeating units (GGAVP and GGFGP) and the number of GGFGP repeating units, respectively (as at right). n. a., no amplification.

FIG. 2.

Ethidium bromide-stained 3% LSI MP agarose (Life Science International) gel containing B. pertussis DNA amplified with primer pairs prnC-prnD (A) and prnE-prnD (B) from strains with known prn types.

FIG. 3.

Workflow for typing prn alleles. The sequence-specific PCR method using the primer pairs prnC-prnD and prnE-prnD allows the efficient identification of the major types prn2 and prn3. A further identification of the minor types prn1 and prn4 to prn11 can then be performed by a single nucleotide polymorphism determination method (SNP) by either real-time PCR or sequencing.

FIG. 4.

(A) Amplification profiles of real-time PCR with SYBR Green I. Curves 1 and 2, amplification profile for a representative prn1 strain with primer pairs QJF3-QJR1 and QJF4-QJR1, respectively; curves 3 and 4, amplification profile for a representative prn7 strain with primer pairs QJF3-QJR1 and QJF4-QJR1, respectively. (B) Melting curves from the specific amplification assays shown in panel A.

FIG. 5.

Ethidium bromide-stained 3% LSI MP agarose (Life Science International) gel containing B. pertussis DNA amplified with primer pairs prnC-prnD (A) and prnE-prnD (B).