RNA extraction for quantitative enterovirus RT-PCR: comparison of three methods.
Quantification of virus-like RNA sequences in biological fluids, like serum and cerebrospinal fluid, requires an RNA extraction method that is both reproducible and fast. Three RNA extraction methods were tested on enteroviruses: (1) the acid guanidine thiocyanate-phenol/chloroform (AGPC) method; (2) a method based on differential precipitation of the RNA and (3) a 'bind-wash-elute' system based on silica-gel membrane binding. The latter two methods yielded a comparable detection limit as measured by RT-PCR ELISA. The detection limit for the AGPC method was 10 times higher. The relative standard deviation for the bind-wash-elute method (3%) was superior to that of the other methods tested (both 20%) and provides a reliable and fast method to extract (viral) RNA from biological fluids for quantification by RT-PCR.
Verheyden
Bart
B
Departement Microbiologie en Hygiëne, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium.
Thielemans
Anne
A
Rombaut
Bart
B
Kronenberger
Peter
P
D004770:Enterovirus / Q000737:chemistry / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D012313:RNA / Q000032:analysis*; D012328:RNA Viruses / Q000737:chemistry / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
D012313:RNA