A principle of quality assessment using a competitive polymerase chain reaction assay for the detection of Chlamydia trachomatis in cervical specimens.
The polymerase chain reaction (PCR)-based identification of Chlamydia trachomatis in clinical specimens should include a built-in control to assess the quality of the whole assay from DNA isolation to detection. For this purpose we established a competitive PCR assay with the following design. A 215 base-pair DNA fragment from a Chlamydia trachomatis plasmid sequence was amplified in a polymerase chain reaction. An internal control DNA was coamplified in the same reaction. The differentiation between the amplified C. trachomatis DNA and the internal control is based on hybridisation against two different probes using Enzymun Test DNA detection (Boehringer Mannheim). The internal control capture probe recognizes an additional 20 base-pair DNA sequence in the competitor DNA construct. Cervical swabs from 65 C. trachomatis positive patients were used. We examined the influence of different DNA isolation methods on the sensitivity of the assay. Detection of C. trachomatis from positive cervical swabs was compared using PCR and competitive PCR assays. The advantage of the competitive assay was a better assessment of reduced sensitivity arising from inhibitory effects or mistakes during the DNA preparation or amplification.
Institut für Klinische Chemie I, Medizinischen Hochschule Hannover, Germany.
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R
Lichtinghagen
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R
Glaubitz
D002584:Cervix Uteri / Q000737:chemistry / Q000382:microbiology*; D002692:Chlamydia trachomatis / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D004926:Escherichia coli; D005260:Female; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*
D004269:DNA, Bacterial