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Amplification of DNA by the polymerase chain reaction for the efficient diagnosis of pertussis
P Olcén1, A Bäckman, B Johansson
1Department of Clinical Microbiology and Immunology, Orebro Medical Center Hospital, Sweden.
Abstract:
The standard diagnostic methods for pertussis have several shortcomings. With the increased knowledge of the Bordetella pertussis genome a specific and conserved DNA sequence, present in about 70-80 copies in each genome, was selected for amplification with the polymerase chain reaction (PCR) technique in order to evaluate its diagnostic potential in children with suspected pertussis. The 400 basepair DNA sequence chosen was present and amplified in all 112 B. pertussis strains and in no other bacterial species examined. The specificity of the amplified material was documented by restriction enzyme cleavage. In nasopharyngeal aspirates a B. pertussis specific PCR product was visualized in 19/25 culture positive and in 5/50 culture negative children. In conclusion the present PCR assay for B. pertussis can be clinically useful and permit a specific diagnosis within 1 day after sampling. Further studies are requested to document its sensitivity, specificity and predictive value for positive and negative results.
Insights
A new polymerase chain reaction (PCR) assay accurately detects Bordetella pertussis DNA. This method offers a rapid and specific diagnostic tool for pertussis in children, improving upon traditional methods.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Pediatric Infectious Diseases
Background:
- Standard pertussis diagnostic methods have limitations.
- Advancements in Bordetella pertussis genomics enable novel diagnostic approaches.
Purpose of the Study:
- To evaluate the diagnostic potential of a specific DNA sequence amplification using polymerase chain reaction (PCR) for pertussis.
- To assess the utility of PCR in identifying Bordetella pertussis in children with suspected pertussis.
Main Methods:
- Selected a conserved 400 basepair DNA sequence from the Bordetella pertussis genome for PCR amplification.
- Tested PCR assay specificity against 112 B. pertussis strains and other bacterial species.
- Analyzed nasopharyngeal aspirates from children using the developed PCR assay.
Main Results:
- The chosen DNA sequence was amplified in all 112 B. pertussis strains and no other bacterial species.
- PCR detected B. pertussis DNA in 19/25 culture-positive and 5/50 culture-negative children's samples.
- Restriction enzyme cleavage confirmed the specificity of the amplified DNA.
Conclusions:
- The developed PCR assay is a clinically useful tool for specific pertussis diagnosis.
- This PCR method allows for diagnosis within one day of sample collection.
- Further research is needed to fully establish sensitivity, specificity, and predictive values.