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Nested PCR optimized for detection of Bordetella pertussis in clinical nasopharyngeal samples
A Bäckman1, B Johansson, P Olcén
1Department of Clinical Microbiology and Immunology, Orebro Medical Center Hospital, Sweden.
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.
Insights
A new nested PCR method effectively detects Bordetella pertussis (whooping cough) using the IS480 insertion sequence. This highly sensitive and specific assay improves diagnosis from nasopharyngeal samples.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Bordetella pertussis causes whooping cough, necessitating accurate diagnostic tools.
- Existing PCR assays for B. pertussis detection have limitations in sensitivity and specificity.
- The IS480 insertion sequence is a validated target for B. pertussis identification.
Purpose of the Study:
- To develop and evaluate an improved nested PCR assay for sensitive and specific detection of B. pertussis.
- To assess the diagnostic performance of the assay using clinical nasopharyngeal samples.
- To optimize sample processing for rapid Bordetella pertussis diagnosis.
Main Methods:
- A nested PCR assay targeting the IS480 insertion sequence of B. pertussis was developed.
- The assay was tested with characterized strains of B. pertussis, other Bordetella species, and common respiratory bacteria.
- Clinical nasopharyngeal aspirates from culture-positive and culture-negative patients were analyzed.
- PCR products were confirmed by MvaI restriction enzyme digestion.
Main Results:
- The nested PCR assay detected all 115 B. pertussis strains tested, showing 100% specificity against other Bordetella species (44 strains) and common respiratory bacteria (115 strains).
- The assay correctly identified B. pertussis in all 51 culture-positive nasopharyngeal aspirates.
- It detected B. pertussis in 18 out of 196 culture-negative patients, indicating enhanced sensitivity.
- The detection limit was approximately 10 bacteria/mL, with a 205-bp fragment confirmed by MvaI digestion.
Conclusions:
- The developed nested PCR assay offers a fast, uncomplicated, and highly sensitive method for diagnosing B. pertussis.
- The assay demonstrates excellent specificity, making it reliable for clinical use with simple heat-treated nasopharyngeal samples.
- This improved diagnostic approach aids in the timely identification of whooping cough cases.