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Bordetella pertussis PCR: simultaneous targeting of signature sequences
Xuan Qin1, David K Turgeon, Brian P Ingersoll
1Microbiology Laboratory, Department of Laboratories and Pathology, CH-37, Children's Hospital and Regional Medical Center, Seattle, Washington 98105, USA. xqin@chmc.org
Abstract:
The absence of analytical controls for polymerase chain reaction (PCR)-based diagnostic tests for Bordetella pertussis limits their clinical utility. In this study, multiplex PCR simultaneously targeted two specific Bordetella pertussis sequences, the chromosomal repeated insertion sequence IS481 (IS) and the pertussis toxin promoter region (PT). A multi-target hybridization-EIA (Hyb-EIA) method in a 96-well microtiter-plate format was used to detect amplicons. Forty-seven (15%) of the 318 nasopharygeal specimens tested positive for at least one DNA target of B. pertussis by PCR, including the 10 known positive samples by culture and/or direct fluorescent antibody (DFA). Forty-six of the 47 PCR positive samples were considered positive for B. pertussis using the consensus interpretation criteria. Simultaneous detection of multiple chromosomal regions may identify false-positive and -negative results due to analytical variations or potential sequence polymorphism, and uncover a wider range of pathogenic strains.
Insights
This study developed a multiplex polymerase chain reaction (PCR) assay for Bordetella pertussis detection. The new method improves diagnostic accuracy by simultaneously targeting multiple DNA sequences, enhancing reliability in clinical settings.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Polymerase chain reaction (PCR)-based diagnostic tests for Bordetella pertussis lack analytical controls, limiting their clinical utility.
- Accurate detection of Bordetella pertussis is crucial for diagnosing and managing whooping cough.
Purpose of the Study:
- To develop and validate a multiplex PCR assay for Bordetella pertussis detection.
- To improve the reliability and accuracy of Bordetella pertussis diagnostics by incorporating analytical controls.
Main Methods:
- Multiplex PCR simultaneously targeted two Bordetella pertussis DNA sequences: IS481 and the pertussis toxin promoter region (PT).
- A multi-target hybridization-EIA (Hyb-EIA) method in a 96-well microtiter-plate format was used for amplicon detection.
- 318 nasopharyngeal specimens were tested.
Main Results:
- Forty-seven (15%) of 318 specimens tested positive for at least one B. pertussis DNA target.
- Forty-six of the 47 PCR-positive samples were confirmed positive using consensus interpretation criteria.
- The assay identified 10 samples previously positive by culture and/or DFA.
Conclusions:
- Simultaneous detection of multiple chromosomal regions enhances the identification of false-positive and -negative results.
- This multiplex PCR approach offers improved analytical control and broader detection of pathogenic Bordetella pertussis strains.
- The developed method has the potential to increase the clinical utility of PCR-based diagnostics for pertussis.