Inhibition of amplification of Chlamydia trachomatis plasmid DNA by the ligase chain reaction associated with female urines
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OBJECTIVE: To determine the rate of inhibition of ligase chain reaction (LCR) amplification of Chlamydia trachomatis plasmid DNA in urines from pregnant and non-pregnant women, to correlate inhibitors with urinary substances through urinalysis, and to explore methods of removing inhibitors from urine. METHODS: First-void urines (FVUs) from 287 non-pregnant and 101 pregnant women (15--40 years of age) in Hamilton, Canada were tested for urinary factors by urinalysis and for C. trachomatis by LCR. A sample of each urine was 'spiked' with a positive control containing approximately one infectious unit of C. trachomatis L2 serovar. RESULTS: Complete inhibition of DNA amplification was observed in nine of 287 (3.1%) urines from non-pregnant women and in one of 101 (1%) who were pregnant. Partial inhibition (> 20% reduction in signal) was observed in an additional five of 388 (1.3%) urines. Nine urines contained C. trachomatis plasmid DNA, but none of these contained inhibitors of LCR. By multivariate analysis, the only substances in urine which were significantly and independently related to LCR inhibition were nitrites. Holding the urines at 4C or minus sign70C overnight reduced the numbers which were inhibitory by approximately 50%. Diluting the urines 1:10 or extracting with phenol/chloroform removed all inhibition and retained amplifiable DNA. CONCLUSIONS: We conclude that urinary inhibitors of C. trachomatis DNA amplification by LCR are minimal and appear to be related to the presence of nitrites. We anticipate that most would disappear in transit or during processing and that a positive control for every urine is not warranted, considering the added cost and work associated with duplicate testing.
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