Novel Internal Controls For Real-Time PCR Assays

In the 19 years since the first descriptions of the PCR (1), nucleic acid amplification methods have made the transition from research to clinical laboratories. Molecular diagnostics are now firmly established as part of laboratory medicine, with applications in genetics, oncology, pharmacology, and infectious disease. Routine diagnostic applications of these methods have been made possible by the thoughtful use of controls coupled with laboratory practices intended to reduce false-positive and -negative results (2)(3)(4).Nucleic acid amplification assays are prone to inhibition by a variety of substances found in clinical samples. Although the identities and biochemical mechanisms of action of many inhibitors remain unclear, bile salts and complex polysaccharides in feces (5), heme in blood (6), and urea in urine(7) have all been shown to inhibit PCR, probably through interference with the binding and/or polymerization activity of DNA polymerases. Carryover of reagents used for isolation of nucleic acids from clinical specimens can also inhibit amplification reactions. Other causes of false-negative results include target nucleic acid degradation, sample processing errors, thermal cycler malfunction, and in reverse transcription-PCR, failure of the reverse transcription step.A simple approach to detect inhibitors is to add the target nucleic acid to a separate aliquot of the sample after processing. Addition of the external control to a separate reaction, however, doubles the cost of the assay, and the approach may be unworkable if batch sizes are large. Moreover, addition of the control after sample processing is complete cannot detect sporadic …