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Identification of Bordetella pertussis infection by shared-primer PCR
1Division of Bacterial Products, U.S. Food and Drug Administration, Bethesda, Maryland 20892.
Abstract:
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.
Insights
A new shared-primer PCR method accurately detects Bordetella pertussis and Bordetella parapertussis infections. This sensitive assay aids in diagnosing pertussis and identifying cases in vaccine trials.
Area of Science:
- Microbiology
- Molecular Biology
- Infectious Diseases
Background:
- Pertussis, caused by Bordetella pertussis and Bordetella parapertussis, remains a significant public health concern.
- Accurate and rapid diagnostic methods are crucial for effective disease control and management.
Purpose of the Study:
- To develop and evaluate a shared-primer polymerase chain reaction (PCR) method for the simultaneous detection of Bordetella pertussis and Bordetella parapertussis.
- To assess the specificity and sensitivity of the developed PCR assay compared to traditional culture and Chinese hamster ovary (CHO) cell assays.
Main Methods:
- A shared-primer PCR assay was designed using DNA sequences upstream of the porin protein genes.
- The method was tested for specificity against 36 non-Bordetella bacterial DNAs.
- Nasopharyngeal aspirates (NPAs) from children with suspected pertussis were analyzed using PCR, culture, and the CHO cell assay.
Main Results:
- The PCR method produced specific DNA fragments of 159 bp for B. pertussis and 121 bp for B. parapertussis.
- The assay demonstrated high specificity, with no cross-reactivity observed.
- Using ethidium bromide staining, PCR showed 79% sensitivity compared to culture; this increased to 95% with a digoxigenin immunoblot system.
- PCR identified additional positive cases among culture-negative samples.
Conclusions:
- The developed shared-primer PCR method is a specific and sensitive tool for detecting Bordetella pertussis and Bordetella parapertussis.
- This PCR assay offers a valuable improvement for clinical diagnosis of pertussis and epidemiological surveillance in vaccine trials.