Optimization of PMAxx pretreatment to distinguish between human norovirus with intact and altered capsids in shellfish and sewage samples
Optimization of PMAxx pretreatment to distinguish between human norovirus with intact and altered capsids in shellfish and sewage samples
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DOI：
10.1016/j.ijfoodmicro.2017.11.011
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发表时间：
2018-02-02
影响因子：
5.4
通讯作者：
Sanchez, Gloria
中科院分区：
文献类型：
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作者：
Randazzo, Walter;Khezri, Mohammad;Sanchez, Gloria
人类诺如病毒对贝类的污染是一个严重的健康和经济问题。最近，基于RT-qPCR定量检测贝类中HuNoVs的ISO程序已经发布，但这些程序无法区分灭活病毒和潜在传染性病毒。本研究的目的是优化PMAxx预处理，以更好地区分贝类和污水中完整和热处理的HuNoVs。为此，以100 μ M PMAxx和0.5% Triton孵育30 min，双光活化为最佳条件，对热灭活（99℃孵育5 min）的HuNoV GI和GII人工接种贻贝、牡蛎和贝。这一预处理将蛤贝和贻贝中热失活的HuNoV GII信号降低了100倍。此外，该预处理降低了牡蛎中热失活的HuNoV GI和GII在1和1.5 log之间的信号。通过PMAxx-Triton预处理，还评估了HuNoV GI和GII在PBS、污水和生物积累牡蛎中的热失活情况。结果显示，在72℃和95℃处理15分钟后，PBS中观察到的对照和pmaxx处理样品的减少有显著差异。污水中，在72℃和95℃加热后，hnov GI的RT-qPCR信号被PMAxx预处理完全去除，而hnov GII的RT-qPCR信号仅在95℃时被完全消除。最后，PMAxx- triton预处理应用于自然污染的污水和牡蛎，结果在污水和牡蛎样品中定量的大部分HuNoV基因组（17个样本中有12个）对应于未损坏的衣壳。尽管这一过程可能仍然高估了传染性，但PMAxx-Triton预处理代表了更好地解释复杂基质（如污水和贝类）中完整HuNoVs的定量的一步，并且它肯定可以包含在基于RT-qPCR的程序中。
Shellfish contamination by human noroviruses (HuNoVs) is a serious health and economic problem. Recently an ISO procedure based on RT-qPCR for the quantitative detection of HuNoVs in shellfish has been issued, but these procedures cannot discriminate between inactivated and potentially infectious viruses. The aim of the present study was to optimize a pretreatment using PMAxx to better discriminate between intact and heat-treated HuNoVs in shellfish and sewage. To this end, the optimal conditions (30 min incubation with 100 mu M of PMAxx and 0.5% of Triton, and double photoactivation) were applied to mussels, oysters and cockles artificially inoculated with thermally-inactivated (99 degrees C for 5 min) HuNoV GI and GII. This pretreatment reduced the signal of thermally-inactivated HuNoV GI in cockles and HuNoV GII in mussels by > 3 log. Additionally, this pretreatment reduced the signal of thermally-inactivated HuNoV GI and GII between 1 and 1.5 log in oysters. Thermal inactivation of HuNoV GI and GII in PBS, sewage and bioaccumulated oysters was also evaluated by the PMAxx-Triton pretreatment. Results showed significant differences between reductions observed in the control and PMAxx-treated samples in PBS following treatment at 72 and 95 degrees C for 15 min. In sewage, the RT-qPCR signal of HuNoV GI was completely removed by the PMAxx pretreatment after heating at 72 and 95 degrees C, while the RT-qPCR signal for HuNoV GII was completely eliminated only at 95 degrees C.Finally, the PMAxx-Triton pretreatment was applied to naturally contaminated sewage and oysters, resulting in most of the HuNoV genomes quantified in sewage and oyster samples (12 out of 17) corresponding to undamaged capsids. Although this procedure may still overestimate infectivity, the PMAxx-Triton pretreatment represents a step forward to better interpret the quantification of intact HuNoVs in complex matrices, such as sewage and shellfish, and it could certainly be included in the procedures based on RT-qPCR.