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Single-Copy Gene Targets

An insertion sequence is an example of a multi-copy gene target within the bacterial genome. There are also some single-copy gene targets within B. pertussis that can be used for detection. Three examples of single-copy targets for B. pertussis include pertussis toxin, pertactin, and filamentous hemagglutinin (FHA), which are all major virulence factors (adhesins) for B. pertussis.
Pertussis toxin is coded by a multi-subunit protein complex. PCR assays focus on the promoter region of the pertussis toxin operon, which is well-characterized and is already used as a target for diagnostic PCR assays. This genetic region is not specific to B. pertussis as it can also be found in the genome of B. parapertussis and B. bronchiseptica. However, these other species contain mutations in their operon sequence, which prevent the expression of the actual toxin. These mutations provide a way to differentiate between the three species of Bordetella using melting temperatures in real-time PCR.
Pertactin is another major virulence factor for B. pertussis. Pertactin is an outer-membrane protein that promotes adherence to ciliated epithelial cells of the respiratory tract. A reverse transcription-PCR (RT-PCR) assay with the pertactin gene sequence as a target has shown some promise in the correct detection of B. pertussis. However, some cross-reactivity with B. bronchiseptica forces the assay to be used in conjunction with other diagnostic testing.
FHA is a large, filamentous protein that helps strengthen attachment of the bacteria to the ciliated epithelial cells in the upper respiratory tract. As with the other single-copy targets, FHA is also not specific to B. pertussis and must be differentiated from B. parapertussis and B. bronchiseptica, which also contain the genetic sequence to produce the FHA protein.