Application of Armored RNA to Compare Four Norovirus RNA Extraction Methods in Oysters

Abstract:

Norovirus (NoVs) is the most prevalent worldwide foodborne pathogen and causes acute viral gastroenteritis. NoVs are transmitted mainly via the fecal–oral route and by person-to-person contact. It is thought that the majority of NoVs infections are caused by the consumption of contaminated food; the ingestion of contaminated oysters is the primary cause of foodborne NoVs infection since oyster digestive diverticula accumulate viral particles from seawater via filter feeding. Real time RT-PCR is commonly used to detect NoVs RNA in oysters; however, these assays are often hampered by the low viral titer in oysters and PCR inhibition due to matrix carryover during RNA extraction. Extraction is a critical step for obtaining sufficient high-quality viral RNA for amplification; however, studies comparing and evaluating NoVs RNA extraction and detection methods in shellfish are often limited by the lack of standard samples with clear quantitative values and a lack of biosafety hazards. In this study, four RNA extraction methods (TRIzol reagent, Viral RNA Kit, High Pure Viral Nucleic Acid Kit, and Column Virus RNAOUT Kit) were used on oyster digestive gland homogenate samples and artificial freeze-dried samples contaminated with NoVs armored RNA (3.01010 copies/sample) as a reference material. RNA extracted by the four methods was analyzed by real time RT-PCR and quantified using previously established standard curves. For the homogenized samples, the TRIzol method had the highest recovery rate (6.8± 0.89)% and was significantly higher than that by the Viral RNA Kit (4.51±2.28)%. The recovery rates of these two methods were both significantly higher than those by the High Pure Viral Nucleic Acid Kit (0.24±0.05)% and Column Virus RNAOUT kit [(0.11±0.02)%, P˂0.05]. For the freeze-dried samples, the Viral RNA Kit had the highest recovery rate (8.71±0.17)% and was significantly higher than that by the TRIzol method (7.12±0.64)%. The recovery rates of these two methods were both significantly higher than those of the High Pure Viral Nucleic Acid Kit (0.33±0.12)% and Column Virus RNAOUT kit [(0.06± 0.01)%, P˂0.05]. This study indicated that the TRIzol method and Viral RNA Kit could extract target RNA from oyster digestive gland homogenate samples with an ideal recovery rate; moreover, armored RNA could serve as a good reference material for comparing RNA extraction methods.