Comparison of RNA extraction kits for the purification and detection of an enteric virus surrogate on green onions via RT-PCR.
Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) offers a rapid and sensitive molecular method for detection of enteric viruses. Unfortunately, these assays are often hampered by the low virus titre found in foods and PCR inhibition due to matrix carryover during RNA extraction. Four commercial RNA extraction kits (Qiagen's QIAamp Viral RNA Mini and UltraSens Virus kits, MoBio UltraClean Tissue & Cells RNA Isolation kit, and Ambion MagMAX Viral RNA Isolation kit) were evaluated for their ability to extract and purify MS2 bacteriophage RNA, an enteric virus surrogate, from inoculated green onions, a food which has been associated with viral gastroenteritis outbreaks. Inoculated green onion wash concentrates and green onion pieces with and without Qiagen QIAshredder homogenization were assayed in the kit comparison. MS2 detection and PCR inhibition were evaluated using a duplex real-time RT-PCR for MS2 and an exogenous internal amplification control (IAC) assay. Without homogenization, MS2 inoculated at 40pfu/g was detected in at least 4 lots of green onion wash concentrates using the silica-membrane spin-column kits. Inhibition was a factor for the magnetic silica-based MagMAX kit, which resulted in detection of MS2 in 1 of 5. Addition of QIAshredder homogenization prior to extraction did not adversely affect the silica-membrane kit results but improved the MS2 detection by MagMAX to 5 of 5 lots. Use of a 1:10 dilution of primary RNA extracts also improved detection. The QIAamp Viral RNA Mini and MagMAX kits were further compared for detection of MS2 from green onion pieces inoculated at 20 and 5pfu/g. Using homogenization, the MagMAX kit detected 20pfu/g in only 1 of 2 green onion lots, whereas the QIAamp Viral RNA kit detected 2 of 2 lots at 5 pfu/g without homogenization.
Institute for Food Safety and Health, Illinois Institute of Technology, 6502 S. Archer Road, Bedford Park, IL 60501, United States. Electronic address: rhona.xry@hotmail.com.
Ruoyang
R
Xu
U. S. Food and Drug Administration, 6502 S. Archer Road, Bedford Park, IL 60501, United States. Electronic address: carol.shieh@fda.hhs.gov.
Y Carol
YC
Shieh
U. S. Food and Drug Administration, 6502 S. Archer Road, Bedford Park, IL 60501, United States. Electronic address: diana.stewart@fda.hhs.gov.
Diana S
DS
Stewart
D006801:Humans; D017909:Levivirus / Q000235:genetics / Q000302:isolation & purification*; D008280:Magnetics; D019697:Onions / Q000821:virology*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D011933:Reagent Kits, Diagnostic; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012822:Silicon Dioxide; D019562:Viral Load; D014780:Viruses / Q000235:genetics / Q000302:isolation & purification
D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; D012822:Silicon Dioxide
Enteric virus; Green onions; MS2 coliphage; RNA extraction