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

M.J. Vanspauwen, P.F.G. Wolffs, F.M.E. Franssen, C.A. Bruggeman, E.F.M. Wouters, C.F.M. Linssen

Research output: Contribution to journalArticleAcademicpeer-review

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(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.
Original languageEnglish
Pages (from-to)265-269
Number of pages5
JournalJournal of Clinical Virology
Volume61
Issue number2
DOIs
Publication statusPublished - Oct 2014

Keywords

  • RNA isolation
  • Virus
  • Respiratory infections
  • COMMUNITY-ACQUIRED PNEUMONIA
  • POLYMERASE-CHAIN-REACTION
  • EXTRACTION METHODS
  • DNA
  • INFECTION
  • SERUM
  • DIAGNOSIS

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