Semiquantitative Polymerase Chain Reaction Enzyme Immunoassay for the Diagnosis of Pertussis

Abstract

The two most commonly used targets for diagnosis of pertussis by the polymerase chain reaction have been the pertussis toxin promoter and the repeated insertion sequence IS481. A comparative assessment of these primers was performed on routinely collected nasopharyngeal swabs, stored at -20 C, using novel semiquantitative enzyme immunoassays. Both sets of primers behaved similarly with bacterial suspensions, and the 17 culture-positive nasopharyngeal swabs were also positive with the pertussis toxin promoter primers, with one exception, which had been subject to prolonged storage. Significantly more of the 69 culture-negative swabs were positive with the pertussis toxin promoter primers (n = 36) than with the IS481 primers (n = 18). To determine the effect of inhibitors, a comparative assessment of three primer pairs against human DNA (beta-globin and glyceraldehyde-3-phosphate dehydrogenase) was also performed.

Evaluation of Real-Time PCR for Detection of and Discrimination between Bordetella pertussis , Bordetella parapertussis , and Bordetella holmesii for Clinical Diagnosis

“…In a comparison of different PCR assays used in pertussis vaccine studies, all assays provided an increased sensitivity for detection of pertussis cases by at least 70% in comparison to culture (23). Various PCR protocols have been developed that target different regions of the genome: insertion sequences IS481 and IS1001 (4,6,13,15,26,30), the pertussis toxin promoter region (6,15,20,26), the porin gene (5), and the adenylate cyclase gene (3). The achievements of the PCR assays targeting these different genes have not been widely compared.…”

External Quality Assessment for Molecular Detection of Bordetella pertussis in European Laboratories

“…These unexpected false-positive reactions for the majority of the participating laboratories suggest the presence of a homologous sequence in the B. bronchiseptica strain used. (13,14,18,25,28,37,43,45,46). More recently, some laboratories introduced real-time PCR and have demonstrated the reliability of this technique as well as the shorter turnaround time (1, 4, 6, 12, 21, 22, 39, 42).…”

“…Numerous applications of extraction, amplification, and detection methods have been investigated (13,14,18,25,28,37,43,45,46). An improved sensitivity over that of culture was observed in all instances.…”

Prevalence and Sequence Variants of IS 481 in Bordetella bronchiseptica : Implications for IS 481 -Based Detection of Bordetella pertussis

“…On the basis of a comparison of the B. bronchiseptica IS481 sequences with those of IS481 primers used for conventional PCR identification of B. pertussis (2,3,9,10,12,13,16,18,20,21,23,25,37,45,46,47,53), it seems likely that misidentification of IS481-containing strains of B. bronchiseptica would occur. Many of the suggested primers are 100% identical to the B. bronchiseptica sequence reported here, while others have a few base substitutions which may still permit amplification.…”