Objectives: To develop and evaluate the use of a real-time quadriplex PCR for the diagnosis of lymphogranuloma venereum (LGV) and non-LGV chlamydial infections using rectal swab specimens.
Methods: The design of the real-time quadriplex PCR assay incorporates an LGV-specific, a non-LGV-specific target sequence, the C. trachomatis plasmid target, and the human RNase P gene as an internal control. The performance of the real-time quadriplex PCR assay was compared to our previously reported real-time duplex PCR assay whose LGV diagnosis was an indirect method and based on exclusion.
Results: A very good agreement (85 of 89 specimens, 95.5%) was found between the two real-time multiplex PCR assays for the detection of C. trachomatis DNA (kappa value = 0.93, 95% CI: 0.86-0.99). Both assays identified 34 LGV, 35 non-LGV C. trachomatis, and 16 negative specimens. Of two specimens that tested non-LGV by the real-time duplex PCR; one was found to be a mixed infection, and the other was positive only for plasmid and RNase P targets by the quadriplex PCR. Two additional specimens that had equivocal results for non-LGV by the duplex PCR also tested positive only for plasmid target and human DNA control by the quadriplex PCR. In addition, six specimens that tested negative by the duplex PCR assay were found to be invalid (containing PCR inhibitors or without RNase P DNA) by the quadriplex PCR.
Conclusions: We have developed a real-time quadriplex PCR assay that is capable of detecting LGV, non-LGV, or mixed infections simultaneously in rectal specimens. The assay also contains a supplemental amplification target for the confirmation of C. trachomatis infection as well as a human DNA control for monitoring sample adequacy and PCR inhibition.
- C. trachomatis
- lymphogranuloma venereum
- real-time quadriplex PCR
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