Identification of Bordetella pertussis infection by shared-primer PCR.
A shared-primer PCR method for the detection of infection was developed by using primers derived from DNA sequences upstream of the structural genes for the porin proteins of Bordetella pertussis and Bordetella parapertussis. This method resulted in a 159-bp PCR product specific for B. pertussis and a 121-bp DNA fragment specific for B. parapertussis and allowed for the simultaneous detection of these pathogens. The PCR procedure was shown to be very specific since no PCR product was obtained from 36 non-Bordetella bacterial DNAs. Nasopharyngeal aspirates (NPAs) from children suspected of having pertussis were evaluated by the PCR method, culture, and the Chinese hamster ovary (CHO) cell assay, which detects pertussis toxin. B. pertussis was cultured from 119 of 205 NPAs assayed, and the presence of pertussis toxin was detected in 69 of the NPAs by the CHO cell assay. When ethidium bromide staining was used to detect PCR products, 100 NPAs gave positive results by shared-primer PCR; 94 of these NPAs were also positive by culture. The result indicated a sensitivity of 79% for PCR when culture was used as the standard. The sensitivity of PCR was increased to 95% when a digoxigenin immunoblot system was used. An additional 20 NPAs from patients with suspected pertussis that were culture negative also gave positive results by PCR. The specific and sensitive PCR method described here should be useful for both the clinical diagnosis of pertussis and case identification in vaccine trials.
Li
Z
Z
Division of Bacterial Products, U.S. Food and Drug Administration, Bethesda, Maryland 20892.
Jansen
D L
DL
Finn
T M
TM
Halperin
S A
SA
Kasina
A
A
O'Connor
S P
SP
Aoyama
T
T
Manclark
C R
CR
Brennan
M J
MJ
D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D001884:Bordetella / Q000235:genetics; D001886:Bordetella pertussis / Q000235:genetics* / Q000302:isolation & purification; D016466:CHO Cells; D002648:Child; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005069:Evaluation Studies as Topic; D005798:Genes, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D009305:Nasopharynx / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D018272:Porins / Q000235:genetics; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014917:Whooping Cough / Q000175:diagnosis*
D017931:DNA Primers; D004269:DNA, Bacterial; D018272:Porins