Comparison of three different techniques for the isolation of viral RNA in sputum

Abstract Background Respiratory infections are a major cause of morbidity and mortality worldwide. A high percentage of all respiratory tract infections are caused by RNA viruses. Real-time PCR is a highly sensitive method for the detection of respiratory viruses in clinical samples. A good RNA isolation protocol is of high importance, since RNA is more unstable than DNA and many clinical samples contain RNAses. Objectives To evaluate the performance of three different RNA extraction protocols for the extraction of respiratory viral RNA from sputum samples obtained from patients with the suspicion of a viral respiratory tract infection. Study design A total of 50 sputum samples, PCR positive for a respiratory RNA virus, were used for viral RNA isolation with the phenol/chloroform method, RTP ® DNA/RNA virus mini kit and the automated MagNa Pure LC (MPLC) extraction system. After isolation, real-time PCR was performed for the detection of viral RNA in the sputum samples. Results The MPLC extraction increased the detection probability from 82% (phenol/chloroform) and 86% (RTP ® DNA/RNA virus mini kit) to 94%. In 16% the RTP ® DNA/RNA virus mini kit resulted in lower C t values compared to the phenol/chloroform method, while in 32% the phenol/chloroform resulted in lower C t values. Conclusions The extraction of viral RNA performed with the MPLC extraction method was superior to the extraction with the RTP ® DNA/RNA virus mini kit and to the extraction with phenol/chloroform. In general, there was no difference in the detection of viral RNA between the phenol/chloroform extraction method and the RTP ® DNA/RNA virus mini kit.