title: 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.
  
  
  OBJECTIVE
  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.
  
  
  METHODS
  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.
authors.affiliation: Department 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. Electronic address: marijke_vanspauwen@hotmail.com.
authors.forename: M J
authors.initials: MJ
authors.lastname: Vanspauwen
authors.affiliation: Department 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.
authors.forename: P F G
authors.initials: PF
authors.lastname: Wolffs
authors.affiliation: Department of Respiratory Medicine, Maastricht University Medical Centre, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands; CIRO, Centre of Expertise for Chronic Organ Failure, P.O. Box 4009, 6080 AA Haelen, The Netherlands.
authors.forename: F M E
authors.initials: FM
authors.lastname: Franssen
authors.affiliation: Department 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.
authors.forename: C A
authors.initials: CA
authors.lastname: Bruggeman
authors.affiliation: Department of Respiratory Medicine, Maastricht University Medical Centre, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands; CIRO, Centre of Expertise for Chronic Organ Failure, P.O. Box 4009, 6080 AA Haelen, The Netherlands.
authors.forename: E F M
authors.initials: EF
authors.lastname: Wouters
authors.affiliation: Department 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.
authors.forename: C F M
authors.initials: CF
authors.lastname: Linssen
mesh_terms: D006801:Humans; D012327:RNA Virus Infections / Q000175:diagnosis / Q000821:virology; D012367:RNA, Viral / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012141:Respiratory Tract Infections / Q000175:diagnosis / Q000821:virology; D012189:Retrospective Studies; D013048:Specimen Handling / Q000379:methods*; D013183:Sputum / Q000821:virology*
chemical_list: D012367:RNA, Viral
keywords: RNA isolation; Respiratory infections; Virus