Comparison of RNA extraction kits for the purification and detection of an enteric virus surrogate on green onions via RT-PCR
Introduction
Human enteric viruses, including noroviruses (NoV), hepatitis A virus (HAV) and rotavirus (RV), contribute greatly to the burden of foodborne disease in the U.S. (Scallan et al., 2011, Koopmans and Duizer, 2004). The major risk factor for enteric viral illnesses is the consumption of raw or insufficiently cooked shellfish (Le Guyader et al., 2008, Power and Collins, 1989, Shieh et al., 2007). Additionally, fruit, such as minimally processed berries (Calder et al., 2003, Le Guyader et al., 2004, Niu et al., 1992), and vegetables including ready-to-eat salads, lettuce, and green onions, have also been reported as the cause of foodborne outbreaks (Dentinger et al., 2001, Long et al., 2002, Rosenblum et al., 1990). An outbreak of hepatitis A virus in western Pennsylvania due to the ingestion of contaminated green onions resulted in three deaths among a total of 601 cases (Wheeler et al., 2005).
Detection of viral contamination of food or water has been problematic for several reasons. The viruses of greatest concern, HAV and NoV, do not replicate in the environment, water, or food, thus viral loads present in food samples are typically much lower than those found in clinical samples, and the detection methods need to be sensitive (Butot et al., 2007, Sair et al., 2002a). Classical detection of human enteric virus from food concentrates is based on mammalian cell culture assays; nevertheless, the fundamental drawback of this method is that, besides being costly, laborious and time-consuming, some enteric viruses grow poorly (HAV) or not at all (NoV) in cultured cells (Downes and Ito, 2001, Fong and Lipp, 2005, Haramoto et al., 2004, Jaykus, 2000). For these reasons, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) has been utilized as a rapid, sensitive and reliable tool for the detection and quantification of HAV, NoV and other enteric viruses in food samples (Casas et al., 2007, Costafreda et al., 2006, Dubois et al., 2006, Jothikumar et al., 2005, Shan et al., 2005).
The application of real-time RT-PCR for enteric RNA virus detection in food is hindered by two challenges. The first is the need to concentrate low levels of viruses from complex food matrices into the small volumes used in real-time RT-PCR analysis (Butot et al., 2007), which is possible through the use of concentration techniques such as ultracentrifugation. The second issue is the need to remove or overcome inhibition of the real-time RT-PCR, which may originate from constituents of bacterial cells and non-target DNA; unintentional contamination with reagents, containers, or disposables during reaction preparation; or from food constituents including organic and phenolic compounds, glycogen, fats, and calcium compounds (Wilson, 1997) co-extracted with viral nucleic acids (Butot et al., 2007, Croci et al., 2008).
Sample processing for fresh produce is one of the critical steps requiring evaluation to determine the techniques which allow the best recovery of viral RNA without carryover of inhibitory compounds into real-time RT-PCR. A range of extraction protocols have been employed for extracting enteric viral RNA and simultaneously removing or inactivating potential inhibitors from complex food matrices prior to RT-PCR (Butot et al., 2007, Fong and Lipp, 2005, Lampel et al., 2000, Sair et al., 2002b); however, many of them depend on the use of complex home-made reagents or on procedures that are not commercially available (Bianchi et al., 2011). A wide variety of commercial kits for the rapid isolation and purification of nucleic acids from different sources offer a reliable and reproducible means of obtaining enteric viral RNA for detection by RT-PCR. Most of these kits are based on guanidinium lysis, followed by capture of nucleic acids on a column or bead of silica (Bosch et al., 2011). Several publications have evaluated and compared various kits’ abilities for extraction of virus RNA from different matrices such as water (Burgener et al., 2003), strawberries (Bianchi et al., 2011) or fecal specimens (Hale et al., 1996), but none has evaluated the ability to recover viral RNA from green onion washes.
The aim of this study was to evaluate the ability of four commercially available RNA extraction and purification kits with and without additional homogenization treatment for recover RNA from MS2 coliphage, a surrogate for the enteric viruses, in green onions. The four chosen kits were Qiagen QIAamp Viral RNA Mini and QIAamp UltraSens Virus kits, MoBio UltraClean Tissue and Cells RNA Isolation kit and the Ambion MagMAX Viral RNA Isolation kit. Three of the kits were silica membrane spin-column kits which differed by kit size (mini- or large-volume), and recommended input material. The MagMAX kit was paramagnetic bead-based, which was thought to be an advantage for isolation of RNA from highly concentrated solutions potentially containing particulates. Detection of MS2 from green onion wash concentrates inoculated at 40 pfu/g and from green onion pieces inoculated at lower levels (5–20) pfu/g was evaluated with and without pre-treatment using Qiagen Qiashredder homogenizer units.
Section snippets
Experimental design
This study used a two-stage approach. Initially, four kits were compared for their ability to purify MS2 phage RNA from inoculated concentrated washes produced from five lots (A–E) of green onions. Eight individual concentrated wash samples for each onion lot were prepared and two were extracted using each kit with and without pre-homogenization using QIAshredder units (Fig. 1). Duplicate amplifications of each preparation were used for detection of the MS2 phage and Internal Amplification
Comparison of RNA extraction kits for the detection of MS2 coliphage from concentrated green onion washes
Detection from concentrated washes inoculated at 40 pfu/g was possible from 5/5 lots for the QIAamp Mini and UltraClean kits, 4/5 for the UltraSens, and 1/5 for the MagMAX kit (Table 1). Interestingly, comparison of the average Ct values for the reagent positive control reactions for each kit indicates that the best cycle thresholds would be expected from the MagMAX kit (31.81 ± 1.08 cycles) if there was no onion matrix inhibition present. The other three kits tested had average reagent positive
Discussion
The FDA BAM sample preparation method for detection of hepatitis A virus from green onions (Williams-Woods et al., 2014) used in this study is advantageous in that it attempts to increase the level of sensitivity by extracting virions from a large sample size (50 g) and concentrating it into a relatively small volume which can be RNA-extracted in its entirety and eluted into a small volume (50–80 μl). While this concentration helps ensure detection of the virions, it also has the potential to
Conclusion
Overall, the dual use of the MS2 and IAC assays in this study highlights the variability of inhibitors present in different lots of green onions and the necessity for removing these compounds prior to detection of the target by real-time RT-PCR. Though the formats for the silica membrane–based kits differed slightly in intended input type and volume, they were found to consistently reduce PCR inhibitors to levels which would allow detection of low numbers of MS2 from highly concentrated green
Acknowledgements
This study was supported in part by grant number 5U01FD003801 from the U. S. Food and Drug Administration to the Institute for Food Safety and Health of the Illinois Institute of Technology and a FDA Chief Scientist Challenge grant. The authors would like to thanks Drs. Mary Tortorello, Richard McDonald and Jason Wan for critical review of this manuscript.
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