NESTED PCR OPTIMIZED FOR DETECTION OF BORDETELLA-PERTUSSIS IN CLINICAL NASOPHARYNGEAL SAMPLES

Citation
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
Citations number
26
Categorie Soggetti
Microbiology
ISSN journal
00951137
Volume
32
Issue
10
Year of publication
1994
Pages
2544 - 2548
Database
ISI
SICI code
0095-1137(1994)32:10<2544:NPOFDO>2.0.ZU;2-C
Abstract
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.