Analysis of a repetitive DNA sequence from Bordetella pertussis and its application to the diagnosis of pertussis using the polymerase chain reaction

Abstract

A tandemly repeated 1,046-base-pair (bp) ClaI DNA fragment from Bordetella pertussis was cloned into Escherichia coli by using the vector pUC19. This fragment, when isolated, hybridized strongly to DNA from all 100 clinical isolates of B. pertussis tested. It was shown to have homology to single-copy sequences in Bordetella bronchiseptica but not Bordetella parapertussis and did not hybridize to lysate blots of a wide range of other bacteria, including members of the closely related genera Pasteurella, Alcaligenes, and Haemophilus. The 1,046-bp fragment was sequenced, and complementary synthetic oligonucleotides flanking a 153-bp region within the repeated element were used as primers for specific amplification of this region using the polymerase chain reaction (PCR). This procedure was then applied to the rapid (5-h) detection of B. pertussis in nasopharyngeal secretions collected from 332 children with suspected pertussis. The test yielded positive results in a total of 98 samples, compared with 66 for culture and 33 for direct immunofluorescence (IF). All of the IF-positive samples were PCR positive, as were 63 of the samples from which B. pertussis was eventually cultured. Two hundred thirty-one specimens which were negative by IF and culture were also negative in the PCR assay. However, 33 culture- and IF-negative specimens were positive by PCR assay. Several of these specimens were collected from close contacts of culture-proven pertussis patients, were follow-up specimens from such patients, or were from patients with serological evidence of pertussis and therefore may be true-rather than false-positives.

Infant Pertussis and Household Transmission in Korea

“…Laboratory tests were performed at the Vaccine Bio Institute (VBI) of The Catholic University of Korea. It is determined as the criteria for laboratory-confirmed pertussis case when the subjects is applicable to one of the following criteria: 1) positive result of B. pertussis on culture of nasopharyngeal aspirates (NPA) or swab; this sample was collected and cultured on Regan-Lowe culture medium at 37°C for more than 1 week; 2) positive result of B. pertussis in PCR or real-time PCR (RT-PCR) of NPA or swab; PCR was done by the method Glare et al reported (16), and RT-PCR was done by modified method of Reischl U. Colleagues manual (17); 3) positive serology which defined as pertussis toxin (PT) antibody in a single serum sample that was higher than cut-off value (24 EU/mL) of enzymelinked immunosorbent assay (ELISA) kit (IBL, Hamburg, Germany) or a 4-fold increased change in anti-PT antibody between acute-phase and convalescent-phase serum.…”

ERIC sequences: a novel family of repetitive elements in the genomes of Escherichia coli, Salmonella typhimurium and other enterobacteria

“…(Eisenach etai. 1990), Bordetella pertussis (Glare et al. 1990), Rhodomicrobium vannielli (Russell and Mann, 1986), Agrobacterium spp, and Rhizobium spp.…”

Characterization of two Achromobacter xylosoxidans isolates from patients with pertussis-like symptoms

“…A direct nucleic acid extraction was performed from the nasopharyngeal samples using the High Pure Template Preparation Kit (Roche Applied Science, Mannheim, Germany) following the manufacturer's instructions, and pertussis status was confirmed by PCR. Thus, both 191 bp of the ptxA gene and 145 bp of the IS418 were amplified using primers described elsewhere [16,17] . To confirm the correctness of the amplifications the PCR products were recovered, purified (SpinPrepTM Gel DNA Kit, San Diego, USA) and sequenced (Macrogen, Seoul, Korea).…”