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 (MC₀) 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 MC₀ 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.

中文翻译：

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一种用于水果和蔬菜上食源性病毒的RNA提取和检测的优化直接裂解方法。

使用当前的标准方法检测水果和蔬菜上的诺如病毒（NoV）和甲型肝炎病毒（HAV）可能效率不高。要使病毒有效地进行逆转录（RT）-qPCR检测，就需要进行方法优化，重点是简便，快速和提高病毒RNA回收率。优化了一种简单快速的直接RNA提取裂解方法（方法A），以增加病毒RNA回收率并通过增加裂解缓冲液的体积和添加果胶酶，植物RNA分离助剂和OneStep PCR抑制剂去除试剂盒来最小化RT-qPCR抑制。比较了方法A和在结构上与ISO 15216标准相当的内部方法（方法B）在从过程对照芒果病毒中回收病毒RNA的效率（MC ₀）和鼠诺如病毒（MNV），掺入13种水果，蔬菜，复合食品或种子/坚果中。还分析了所有提取物（> 61）的RT-qPCR抑制作用以及NoV和HAV的自然污染情况。MC ₀的总体平均提取效率方法A和方法MNV分别为36±31％和44±38％，方法B分别为9±16和5±11％。抑制来自NoV基因组（G）I，NoV的RNA的RT-qPCR扩增GII和方法A的HAV范围为5±10至13±14％，方法B的HAV范围为34±36至48±40％。在两种方法处理的草莓和海藻样品中均检测到NoV GII。总之，与修改后的ISO 15216标准相比，新的直接裂解方法显示出总体上更好的性能，应进行验证以用于植物来源食品中病毒的分析。