装甲RNA在牡蛎中诺如病毒4种RNA提取方法比较研究中的应用

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

中文关键词: 诺如病毒  装甲RNA  牡蛎  RNA提取方法  比较

英文关键词: Norovirus  Armored RNA  Oyster  RNA extraction method  Comparison

中文摘要:

      RNA提取是诺如病毒检测的关键步骤，而目前贝类中诺如病毒RNA提取、检测方法的比较与评价常囿于缺乏量值明确、无生物安全隐患的标准样品作为参考依据。本研究将前期制备的 GⅡ型诺如病毒装甲RNA (3.0×1010拷贝)作为标准样品，人工污染牡蛎(Ostrea gigas tnunb)消化腺匀浆物，用4种常见RNA提取方法：TRIzol试剂、Viral RNA Kit、High Pure Viral Nucleic Acid Kit、柱式病毒RNAOUT试剂盒，分别提取RNA，经实时荧光RT-PCR检测后，利用标准曲线进行定量分析，分别计算4种方法对装甲RNA的回收率。结果显示，对于匀浆样本，TRIzol法对装甲RNA的回收率最高(6.80±0.89)%，显著高于Viral RNA Kit (4.51±2.28)%，二者的回收率又显著高于High Pure Viral Nucleic Acid Kit (0.24±0.05)%与柱式病毒RNAOUT试剂盒(0.11±0.02)% (P˂0.05)；对于冻干样本，Viral RNA Kit对装甲RNA的回收率最高(8.71±0.17)%，显著高于TRIzol试剂(7.12±0.64)%，二者的回收率又显著高于High Pure Viral Nucleic Acid Kit (0.33±0.12)%与柱式病毒RNAOUT试剂盒(0.06±0.01)% (P˂0.05)。研究表明，TRIzol试剂与Viral RNA Kit对牡蛎消化腺样本中人工添加的装甲RNA均有良好的回收效果，同时也提示装甲RNA可作为一种良好的标准样品用于不同RNA提取试剂盒方法的评价与比较研究。

英文摘要:

      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.