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(R) 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(R) DNA/RNA virus mini kit) to 94%. In 16% the RTP(R) DNA/RNA virus mini kit resulted in lower Ct values compared to the phenol/chloroform method, while in 32% the phenol/chloroform resulted in lower Ct values. CONCLUSIONS: The extraction of viral RNA performed with the MPLC extraction method was superior to the extraction with the RTP(R) 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(R) DNA/RNA virus mini kit.
|Number of pages||5|
|Journal||Journal of Clinical Virology|
|Publication status||Published - Oct 2014|
- RNA isolation
- Respiratory infections
- COMMUNITY-ACQUIRED PNEUMONIA
- EXTRACTION METHODS