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High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR
Published on: November 28, 2016
Detection of Bordetella pertussis in clinical specimens by PCR and a microtiter plate-based DNA hybridization assay
S Nelson1, A Matlow, C McDowell
1Department of Pediatric Laboratory Medicine, Hospital for Sick Children, Toronto, Ontario, Canada. suenelson@mailhub.sickkids.on.ca
Abstract:
In order to improve detection of Bordetella pertussis in nasopharyngeal aspirates (NPAs) in our laboratory, a PCR-based assay was optimized, and a study was designed (i) to compare results obtained by PCR to those obtained by culture and (ii) to evaluate a novel microtiter plate-based DNA hybridization assay (PCR-plate) by comparing it to agarose gel electrophoresis (PCR-gel) for detection of the PCR product. DNA for the PCR was extracted with a guanidine thiocyanate buffer and used in a PCR mixture containing primers directed against a reiterated gene sequence in B. pertussis (Q. He, J. Mertsola, H. Soini, M. Skurnik, O. Ruuskanen, and M. K. Viljanen, J. Clin, Microbiol. 31:642-645, 1993). Of 96 NPAs submitted from a targeted study group, 23 were positive by culture, 27 were positive by PCR-gel, and 31 were positive by PCR-plate. All culture-positive specimens were also positive by PCR. Of nine patients with culture-negative-PCR-positive results, six had discharge diagnoses of pertussis. Thus, PCR with plate-based product detection is a sensitive method for the laboratory detection of B. pertussis in NPAs. Additional advantages of the plate assay include rapidity, objectivity in reading results, specificity, and the capability of being adapted to a high-volume, automated system.
Insights
A new PCR assay improves Bordetella pertussis detection in nasopharyngeal aspirates (NPAs). The PCR-plate method is more sensitive than culture and PCR-gel, offering rapid, objective results for pertussis diagnosis.
Area of Science:
- Medical Laboratory Science
- Microbiology
- Molecular Diagnostics
Background:
- Improving the laboratory detection of Bordetella pertussis is crucial for timely diagnosis and treatment of pertussis.
- Traditional culture methods can be slow and have variable sensitivity.
- Molecular methods like PCR offer potential for faster and more sensitive detection.
Purpose of the Study:
- To optimize a PCR assay for Bordetella pertussis detection in NPAs.
- To compare PCR results with traditional culture methods.
- To evaluate a novel microtiter plate-based DNA hybridization assay (PCR-plate) against agarose gel electrophoresis (PCR-gel).
Main Methods:
- DNA extraction from NPAs using guanidine thiocyanate buffer.
- PCR amplification using primers targeting a reiterated gene sequence in B. pertussis.
- Detection of PCR products using agarose gel electrophoresis (PCR-gel) and a microtiter plate-based hybridization assay (PCR-plate).
Main Results:
- Out of 96 NPAs, 23 were positive by culture, 27 by PCR-gel, and 31 by PCR-plate.
- All culture-positive specimens were also PCR-positive.
- The PCR-plate assay identified more positive cases than PCR-gel and culture, including six culture-negative cases with a pertussis diagnosis.
Conclusions:
- PCR with microtiter plate-based detection is a sensitive and effective method for laboratory detection of Bordetella pertussis in NPAs.
- The PCR-plate assay offers advantages in rapidity, objectivity, specificity, and adaptability to high-volume automated systems.
- This optimized PCR approach enhances the diagnostic capability for pertussis, aiding clinical management.
