The rationale and method for constructing internal control DNA used in pertussis polymerase chain reaction.
The inclusion of an appropriate internal control DNA in polymerase chain reaction (PCR) is a rapid and simple method for the detection of PCR failure. Two PCR coamplification internal control DNAs (ICD I and ICD II) with the same primer-binding sequences as the target DNA for the detection of Bordetella pertussis and Bordetella parapertussis were produced using an overlap extension technique and a PCR MIMIC construction kit, respectively. The ICD II was further evaluated in a prospective clinical study in 360 patients with a clinical diagnosis of pertussis. From 360 nasopharyngeal swabs the internal control was positive in 318 (88%) samples, but was negative in 42 (12%). After phenol-chloroform extraction an additional 10 internal controls became positive. For the detection of PCR failure, the use of internal control DNA is highly recommended for PCR-based identification of B. pertussis and B. parapertussis organisms from nasopharyngeal swabs and aspirates.
Children's Hospital, Institute for Medical Microbiology, University of Aachen, Germany.
F M
FM
Müller
N
N
Schnitzler
O
O
Cloot
P
P
Kockelkorn
G
G
Haase
Z
Z
Li
D001884:Bordetella / Q000235:genetics / Q000302:isolation & purification*; D001886:Bordetella pertussis / Q000235:genetics / Q000302:isolation & purification*; D002648:Child; D017931:DNA Primers; D005069:Evaluation Studies as Topic; D005188:False Negative Reactions; D006801:Humans; D009305:Nasopharynx / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011446:Prospective Studies; D014917:Whooping Cough / Q000175:diagnosis*
D017931:DNA Primers