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The rationale and method for constructing internal control DNA used in pertussis polymerase chain reaction
F M Müller1, N Schnitzler, O Cloot
1Children's Hospital, Institute for Medical Microbiology, University of Aachen, Germany.
Diagnostic Microbiology and Infectious Disease
|October 9, 1998
Summary
Internal control DNA in polymerase chain reaction (PCR) detects PCR failure in Bordetella pertussis and parapertussis testing. This method is highly recommended for accurate identification from clinical samples.
Area of Science:
- Microbiology
- Molecular Biology
- Clinical Diagnostics
Background:
- Polymerase chain reaction (PCR) is crucial for detecting bacterial pathogens.
- Ensuring PCR assay reliability is essential for accurate clinical diagnostics.
- Internal controls are vital for identifying PCR inhibition or failure.
Purpose of the Study:
- To develop and evaluate internal control DNAs (ICD I and ICD II) for PCR detection of Bordetella pertussis and Bordetella parapertussis.
- To assess the utility of internal control DNA in identifying PCR failures in clinical samples.
Main Methods:
- Two coamplification internal control DNAs (ICD I and ICD II) were produced using overlap extension and a PCR MIMIC construction kit.
- ICD II was prospectively evaluated in 360 patients with suspected pertussis.
- Nasopharyngeal swabs were analyzed using PCR with the internal control.
Main Results:
- The internal control (ICD II) was positive in 88% of 360 nasopharyngeal swabs.
- PCR failure was indicated by negative internal controls in 12% of samples.
- Phenol-chloroform extraction improved internal control detection in an additional 10 samples.
Conclusions:
- Internal control DNA is a reliable method for detecting PCR failure in Bordetella species detection.
- The use of internal controls is highly recommended for accurate PCR-based identification of B. pertussis and B. parapertussis.
- This approach enhances the reliability of molecular diagnostics for pertussis and related infections.