Nested PCR optimized for detection of Bordetella pertussis in clinical nasopharyngeal samples

Abstract

Several genes and sequences in Bordetella pertussis have been used as targets in diagnostic PCR assays. A previously developed single-step PCR assay for the detection of B. pertussis was based on an insertion sequence, IS480, that is present in about 70 to 80 copies in each genome. The diagnostic sensitivity, specificity, and reliability of this assay with aspirated and heat-treated samples from the nasopharynx of patients and their contacts was improved by the use of a nested PCR configuration. The nested PCR assay produced a 205-bp fragment with all of the 115 B. pertussis strains tested and was negative with all strains belonging to other Bordetella species (n = 44) as well as other bacteria commonly found in the upper respiratory tract (n = 115). The diagnostic value of the assay was verified by giving positive results for B. pertussis in all the 51 nasopharyngeal aspirates from culture-positive patients. The assay also detected 18 positive aspirates from a total of 196 culture-negative patients. A confirmatory cleavage of the 205-bp nested PCR product by MvaI gave in all cases two bands of 88 and 117 bp. In conclusion, this newly developed nested PCR assay was shown to be reasonably fast and uncomplicated, with an optimal sensitivity and a high degree of specificity for the diagnosis of B. pertussis in aspirated nasopharyngeal samples processed simply by heat treatment. The detection level in the nested PCR was about 10 bacteria per ml, or seven to eight insertion sequence copies per 10 microliters of boiled sample.