A. Backman et al., NESTED PCR OPTIMIZED FOR DETECTION OF BORDETELLA-PERTUSSIS IN CLINICAL NASOPHARYNGEAL SAMPLES, Journal of clinical microbiology, 32(10), 1994, pp. 2544-2548
Several genes and sequences in Bordetella pertussis have been used as
targets in diagnostic PCR assays. A previously developed single-step P
CR assay for the detection of B. pertussis was based on an insertion s
equence, IS480, that is present in about 70 to 80 copies in each genom
e. The diagnostic sensitivity, specificity, and reliability of this as
say with aspirated and heat-treated samples from the nasopharynx of pa
tients and their contacts was improved by the use of a nested PCR conf
iguration. 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 bacte
ria commonly found in the upper respiratory tract (n = 115). The diagn
ostic value of the assay was verified by giving positive results for B
. pertussis in all the 51 nasopharyngeal aspirates from culture-positi
ve patients. The assay also detected 18 positive aspirates from a tota
l 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 1
17 bp. In conclusion, this newly developed nested PCR assay was shown
to be reasonably fast and uncomplicated, with an optimal sensitivity a
nd a high degree of specificity for the diagnosis of B. pertussis in a
spirated nasopharyngeal samples processed simply by heat treatment. Th
e detection level in the nested PCR was about 10 bacteria per ml, or s
even to eight insertion sequence copies per 10 mu l of boiled sample.