Division for Microbiology and Production, National Food Institute, Technical University of Denmark, Kemitorvet, Building 204, 2800, Kgs. Lyngby, Denmark.
Division for Food and Feed Safety, Danish Veterinary and Food Administration, Stationsparken 31-33, 2600, Glostrup, Denmark.
Food Environ Virol. 2020 Sep;12(3):226-239. doi: 10.1007/s12560-020-09437-x. Epub 2020 Jul 10.
Detection of norovirus (NoV) and hepatitis A virus (HAV) on fruits and vegetables using current standard methodologies can be inefficient. Method optimisation focussing on ease, rapidity and increased viral RNA recovery is needed for efficient reverse transcription (RT)-qPCR detection of viruses. A simple and quick direct lysis method for RNA extraction was optimised (method A) to achieve increased viral RNA recovery and minimised RT-qPCR inhibition by increasing the volume of lysis buffer and inclusion of pectinase, Plant RNA Isolation Aid and OneStep PCR Inhibitor Removal Kit. Method A and an internal method structurally comparable to the ISO 15216 standard (method B) were compared for their efficiencies to recover viral RNA from the process controls, mengovirus (MC) and murine norovirus (MNV), spiked in 13 types of fruits, vegetables, compound foods or seeds/nuts. All extracts (> 61) were also analysed for RT-qPCR inhibition and for natural contamination of NoV and HAV. The overall mean extraction efficiencies of MC and MNV were 36 ± 31 and 44 ± 38%, respectively, for method A and 9 ± 16 and 5 ± 11%, respectively, for method B. Inhibition of RT-qPCR amplification of RNA from NoV genogroup (G)I, NoV GII, and HAV ranged from 5 ± 10 to 13 ± 14% for method A and 34 ± 36 to 48 ± 40% for method B. NoV GII was detected in samples of strawberries and seaweed processed by both methods. In conclusion, the new direct lysis method showed an overall better performance compared to the modified ISO 15216 standard and should be validated for implementation in analysis of viruses in foods of plant origin.
使用当前的标准方法检测水果和蔬菜中的诺如病毒（NoV）和甲型肝炎病毒（HAV）可能效率不高。需要针对简便、快速和提高病毒 RNA 回收率进行方法优化，以便通过逆转录（RT）-qPCR 高效检测病毒。优化了一种简单快速的直接裂解方法用于 RNA 提取（方法 A），通过增加裂解缓冲液的体积并加入果胶酶、植物 RNA 分离辅助剂和一步法 PCR 抑制剂去除试剂盒，以提高病毒 RNA 的回收率并减少 RT-qPCR 抑制。方法 A 和与 ISO 15216 标准结构上可比的内部方法（方法 B）用于比较从过程对照物（MC）和鼠诺如病毒（MNV）中回收病毒 RNA 的效率，MC 和 MNV 分别用 13 种水果、蔬菜、复合食品或种子/坚果进行了加标。所有提取物（>61）也进行了 RT-qPCR 抑制分析和 NoV 和 HAV 的天然污染分析。对于方法 A，MC 和 MNV 的总体平均提取效率分别为 36±31%和 44±38%，对于方法 B，MC 和 MNV 的总体平均提取效率分别为 9±16%和 5±11%。方法 A 中 RT-qPCR 扩增 RNA 对 NoV 基因组（G）I、NoV GII 和 HAV 的抑制率为 5±10%至 13±14%，方法 B 中为 34±36%至 48±40%。用两种方法均在加工草莓和海藻的样品中检测到了 NoV GII。总之，与修改后的 ISO 15216 标准相比，新的直接裂解方法总体性能更好，应在分析植物源性食品中的病毒时进行验证。
