Purpose: To compare the ability of four nucleic acid extraction kits to extract and purify MS2 bacteriophage RNA from (1) green onion eluates, and (2) concentrated eluates prepared with and without QIAGEN QIAshredder pre-treatment, with detection by RT-PCR.
Methods: QIAGEN QIAamp Viral RNA Mini Kit, QIAGEN QIAamp UltraSens Virus Kit, MOBIO UltraClean Tissue & Cells RNA Isolation Kit, and Ambion MagMAX Viral RNA Isolation Kit were evaluated for their ability to extract and purify RNA from MS2 phage in eluates made from pulsifying or shaking 50 g green onion pieces with 50 ml glycine-NaCl buffer (0.75M/0.15M, pH 7.6) at 103 to 100 PFU/ml or in ultracentrifuged eluates at 40 PFU/ml, with or without QIAGEN QIAshredder processing prior to extraction to remove additional inhibitors. MS2 RNAs were amplified using a real-time Taqman probe-based RT-PCR assay along with a non-competitive internal amplification control (IAC).
Results: The MS2 positives from five samples inoculated at 103, 102, 101, and 100 PFU/ml was, respectively: 4, 4, 2, 2 (QIAamp); 5, 5, 1, 2 (UltraSens); 5, 5, 2, 2 (UltraClean); and 5, 4, 2, 2 (MagMax). Compared to a ‘no matrix’ control, the IAC assay indicated that the greatest inhibition resulted from using MagMax followed by UltraSens, QIAamp, and MoBio. The MS2 positives from five 200X-concentrated samples, ‘without’ or ‘with’ QIAshredder pre-treatment, was as follows: QIAamp: 5, 5; UltraSens: 5, 5; MOBIO UltraClean: 4, 5; and MagMAX: 1, 5, respectively. QIAshredder treatment does not appear to be needed for inhibitor removal with the QIAamp kit, whereas inhibition was greatly reduced using the MagMax kit, and inconsistent results were seen for both the UltraSens and MoBio kits.
Significance: These data will assist with selection of appropriate methods for extraction and purification of nucleic acids from enteric virus-contaminated green onions.