Use of an RT-PCR internal control to evaluate viral removal

Abstract We constructed an internal control based on the poliovirus genome and cloned it into a transcription plasmid to obtain a competitor RNA distinguishable by its size from the wild-type PCR product. A known number of copies was introduced into sewage samples before nucleic acid extraction in order to evaluate the reliability of the extraction step and amplification. The final gel was analysed with a densitometer and the number of viral RNA copies present in the sewage was calculated. In vitro experiments were carried Out with this internal control with peracetic acid and UV to estimate decontamination efficiency. A very high concentration of peracetic acid was required to eliminate viruses with a moderate UV dose to obtain a negative PCR. This study demonstrates that competitor RNA is useful both in controlling the different reaction steps (extraction and amplification) and inhibitor detection and in providing a quantitative estimation of contamination.