Comparative evaluation of four large-volume RNA extraction kits in the isolation of viral RNA from water samples

The quality of the RNA extraction system plays a crucial role for the detection of viruses in water or environmental samples. In the present study we investigated the detection limit, the efficiency and the presence of eventually co-extracted inhibitors by comparing four commercially available large scale ( greater than or equal to 1 ml) viral RNA extraction methods. (QIAamp Viral RNA Mini Kit in combination with preconcentration by Centricon YM-100 [Centricon-QIAamp], QIAamp UltraSens Virus Kit, NucliSens Isolation Kit and NucleoSpin RNA Virus F). A 1 ml 50 mM glycine (pH 8.0) containing 1% beef extract was spiked with different concentrations of poliovirus vaccine strains, extracted by the four methods and analysed by RT-nested PCR or RT-quantitative LightCycler(TM) PCR. Eight replicates were analysed for each concentration on different days. The positive cut-off point was determined to be at 0.25 CCID50 per ml (Centricon-QIAamp), 1.46 CCID50 per ml (UltraSens), 0.4 CCID50 per ml (NucliSens) and 3.03 CCID50 per ml (NucleoSpin). Quantitative analysis (LightCycler(TM)) of a high-titer sample showed significant differences between the efficiencies of the four extraction methods examined. The efficiencies of the extraction methods were normalized to the NucliSens method as follows: (71% Centricon-QIAamp, 18% UltraSens, 100% NucliSens and 23% NucleoSpin). In addition, spiked negative controls did show significant differences, indicating a co-extraction of inhibitors. Compared with the non-inhibited positive control the inhibitions were 21, 37, 27 and 68% for the Centricon-QIAamp, UltraSens, NucliSens and NucleoSpin methods, respectively. Taken together, these findings indicate that of the four evaluated extraction methods both the NucliSens and Centricon-QIAamp are best suited to extract viral RNA from water samples previously concentrated and have shown to be very sensitive, efficient and robust methods. (C) 2002 Elsevier Science B.V. All rights reserved.