Detection and Differentiation of Bordetella spp. by Real-Time PCR

Abstract

Molecular detection of Bordetella pertussis DNA is a sensitive and specific method for the rapid diagnosis of pertussis. In this study, a new molecular assay for the detection and differentiation of Bordetella spp. based on automated DNA extraction and real-time PCR was evaluated. The analytical sensitivity of the new assay was determined by Probit analysis of serial dilutions of both cloned PCR products IS481 and IS1001 and cell suspensions of B. pertussis, B. parapertussis, and B. bronchiseptica. The specificity was analyzed by testing a number of pathogens producing respiratory infections. Moreover, a total of 92 clinical samples were investigated. The results were compared to those obtained by an in-house assay based on manual DNA extraction, followed by real-time PCR and detection of IS481. The analytical sensitivity of the new assay for the detection of IS481 and IS1001 was determined to be 2.2 and 1.2 genome equivalents/l, respectively. The analytical sensitivity for the detection of B. pertussis, B. parapertussis, and B. bronchiseptica was determined to be 1.6, 1.0, and 2.7 genome equivalents/l, respectively. When clinical specimens were tested with the new assay, 46 of 92 were found to be positive for Bordetella DNA. With the in-house assay, 45 samples tested positive. The new molecular assay proved to be suitable for the rapid diagnosis of pertussis in the routine diagnostic laboratory.Bordetella pertussis is the agent responsible for producing pertussis, also called whooping cough. B. parapertussis, B. bronchiseptica, and B. holmesii may also cause a pertussis-like respiratory disease but with a milder clinical course (7). Pertussis occurs worldwide, with an increased incidence among young unvaccinated infants. Neonates and elderly patients may show an atypical course of disease (2, 4). A rapid and reliable diagnostic method is thus essential for appropriate treatment and prophylaxis. Culture has been considered the gold standard for detection of B. pertussis but often lacks sensitivity, and a minimum of 4 days may be required to obtain results (1).Today, PCR has been shown to be a sensitive and specific method for the rapid diagnosis of pertussis (3,6,11). In the Bordetella genome, there are repetitive insertion sequences present in a high copy number that may be useful for the amplification of different bacterial strains. Insertion sequences include IS481 in B. pertussis, B. holmesii, and, occasionally B. bronchiseptica, and IS1001 in B. parapertussis and, occasionally, B. bronchiseptica and B. holmesii. These sequences have been reported as target sequences in several PCR protocols (5,10,13,15).In the present study, the new artus Bordetella LC PCR Kit (QIAGEN Hamburg GmbH, Germany), which includes a multiplex PCR system consisting of four independent PCRs for qualitative detection and differentiation of B. pertussis, B. parapertussis, B. bronchiseptica, and the heterologous internal control (IC), was evaluated. After we tested these methods for analytical sensitivity and specificity, clinical speci...

High frequency of pertussis in older children and adolescents with prolonged cough in Turkey

“…Polymerase chain reaction testing is an important tool for timely diagnosis of pertussis and is widely used for diagnosis of pertussis since 2001 23 . PCR is a molecular technique used to detect DNA sequences of the B. pertussis bacterium, and unlike culture, does not require viable bacteria present in the specimen 24 .…”

“…PCR is a rapid test and has excellent sensitivity 18 . For PCR, the optimal timing for specimen collection is the first 3 weeks of cough, but bordetella DNA may be detected by PCR for 4 weeks after cough onset 18,23 . After the fourth week of illness, the amount of bacterial DNA rapidly diminishes, which increases the risk of obtaining falsely-negative results.…”

Towards Improved Accuracy of Bordetella pertussis Nucleic Acid Amplification Tests

“…This will be discussed in more detail later in this minireview. IS1001 is found in B. parapertussis at approximately 20 copies per genome (35) and has been used in multiplex PCR with IS481 to detect and differentiate B. pertussis and B. parapertussis (14,16,29,31). IS1001 is also present in isolates of B. bronchiseptica (4 of 73 human-derived isolates tested by Tatti et al [31] and 26 of 38 animal-derived isolates tested by van der Zee et al [34]) at 1 to 7 copies per genome (34 (Table 2).…”

“…It is also produced by B. parapertussis and B. bronchiseptica. A realtime PCR assay that detects these three Bordetella species, followed by postamplification differentiation of filamentous hemagglutinin amplicons by melting curve analysis, was recently described (14). A PCR assay targeting the Bordetella adenylate cyclase gene was described by Douglas et al (4).…”

Detection of Bordetella avium by TaqMan real-time PCR in tracheal swabs from wildlife birds

“…In the literature there are reports about molecular diagnosis of bacterial infections including Bordetella sp. (Register et al 2005, Koidl et al 2007). The described methods requires multiplication of bacteria before the DNA extraction, so if the sample contains bacteria, it should be alive.…”