Abstract
BackgroundThe genetic composition of the bacterium causing whooping cough, Bordetella pertussis, has been investigated using microarray studies in order to examine potential genetic contributors to the disease re-emergence in the past decade. Regions of difference (RDs) have been previously identified as clusters of genes flanked by insertion sequences which are variably present in different sets of isolates, and have also been shown to be potential markers of B. pertussis evolution.This study used microarray data to identify and select a panel of RDs; primers and probes for these RDs were then designed to test for the presence or absence of these regions in a novel and less expensive multiplex PCR-based reverse line blot (mPCR/RLB) assay. By comparing the presence or absence of RDs, we aimed to determine the genomic variability of a diverse collection of B. pertussis strains and how they have changed over time.ResultsA B. pertussis specific mPCR/RLB using 43 genes representing 30 RDs, was developed and used to characterise a set of 42 B. pertussis isolates. When mapped against the previously identified evolutionary relationships of the strains, the losses of two RDs - BP0910A - BP00930 and BP1948-BP1962 - were found to be associated with significant events in B. pertussis history: the loss of BP0910A - BP00930 coincided with introduction of whole cell vaccines in the 1950s while that of BP1948-BP1962 occurred after the introduction of acellular vaccines. The loss of BP1948-BP1962 also coincided with expansion of the most recent B. pertussis strains.ConclusionsThe mPCR/RLB assay offers an inexpensive and fast method of determining the gene content of B. pertussis strains and also confirms that gene losses are an ongoing feature of B. pertussis evolution.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-0500-7-727) contains supplementary material, which is available to authorized users.
Highlights
The genetic composition of the bacterium causing whooping cough, Bordetella pertussis, has been investigated using microarray studies in order to examine potential genetic contributors to the disease re-emergence in the past decade
Development and application of multiplex PCR/ reverse line blot to detect selected regions of difference In this study, multiple PCR targets were combined in an mPCR/RLB assay to simultaneously identify and differentiate B. pertussis isolates based on patterns of gene loss
An initial mPCR/RLB was performed using B. pertussis Tohama I because it is a completely sequenced and well-annotated historic isolate collected in 1954 [2] which has been often used as a vaccine production strain
Summary
The genetic composition of the bacterium causing whooping cough, Bordetella pertussis, has been investigated using microarray studies in order to examine potential genetic contributors to the disease re-emergence in the past decade. The assay allows multiple isolates to be processed in a single blot and, by using a multiplex approach, more that 40 targets can be probed simultaneously without increasing the number of individual PCRs [6]. This technique has been successfully applied to genotyping of bacterial pathogens of public health importance, such as Streptococcus agalactiae [7], Streptococcus pneumoniae [8], uropathogenic Escherichia coli [9], Staphylococcus aureus [10], methicillin resistant Staphylococcus aureus [11] as well as different viral pathogens including human papilloma virus (HPV) [12] and human adenoviruses [13]
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