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Comparison of three different techniques for the isolation of viral RNA in sputum
M J Vanspauwen1, P F G Wolffs1, F M E Franssen2
1Department of Medical Microbiology, Maastricht University Medical Centre, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands; CAPHRI School, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands.
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 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(®) 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.
