Detection of noroviruses in bivalve shellfish is difficult because of the low concentration of norovirus and the presence of reverse transcription (RT)-PCR inhibitors. This study aimed to assess the presence of noroviruses in oysters extracted using a proteinase K extraction (ISO 15216 method) and an adsorption–elution method. Seventy oyster samples were extracted using the two extraction methods and evaluated using RT-nested PCR. The results showed norovirus detection rates at an equal frequency of 28.6%, of which a total of 48 (68.6%) samples had corresponding positive or negative results, while there were 22 (31.4%) samples with discrepant results. Norovirus genogroup (G)I, GII, and mixed GI and GII were detected in 20%, 4.3%, and 4.3% of samples, respectively, by the proteinase K extraction method, which comprised of GI.2, GI.5b, GI.6b, GII.4, and GII.17 genotypes. With the adsorption–elution method noroviruses were detected in 17.1%, 8.6%, and 2.9% of samples, respectively, which comprised of GI.2, GII.2, GII.4, and GII.17 genotypes. All norovirus-positive oyster samples were further estimated for genome copy number using RT-quantitative PCR. The oyster samples processed using the adsorption–elution method contained norovirus GI of 3.36 × 10¹–1.06 × 10⁵ RNA copies/g of digestive tissues and GII of 1.29 × 10³–1.62 × 10⁴ RNA copies/g. Only GII (2.20 × 10¹ and 7.83 × 10¹ RNA copies/g) could be quantified in samples prepared using the proteinase K extraction method. The results demonstrate the different performance of the two sample-processing methods, and suggest the use of either extraction method in combination with RT-nested PCR for molecular surveillance of norovirus genotypes in oysters.

中文翻译：

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使用两种不同的提取方法监测牡蛎中的诺如病毒。

由于低浓度的诺如病毒和反转录（RT）-PCR抑制剂的存在，很难检测双壳贝类中的诺如病毒。这项研究旨在评估用蛋白酶K提取（ISO 15216方法）和吸附-洗脱方法提取的牡蛎中诺如病毒的存在。使用两种提取方法提取了70个牡蛎样品，并使用RT巢式PCR进行了评估。结果显示，诺如病毒的检出率在相同频率下为28.6％，其中共有48个（68.6％）样品具有相应的阳性或阴性结果，而有22个（31.4％）样品具有不同的结果。通过蛋白酶K提取法分别检测到诺如病毒基因组（G）I，GII和混合GI和GII，分别由GI.2，GI.5b，GI组成的蛋白酶K提取方法检测到。 .6b，GII.4，和GII.17基因型。用吸附-洗脱法分别检测到由GI.2，GII.2，GII.4和GII.17基因型组成的样品中的诺如病毒分别占17.1％，8.6％和2.9％。使用RT定量PCR进一步估计所有诺如病毒阳性牡蛎样品的基因组拷贝数。用吸附-洗脱方法处理的牡蛎样品中含有诺维克病毒GI为3.36×10^{1 –} 1.06×10 ⁵ RNA拷贝/克的消化组织和GII为1.29×10 ³ –1.62×10 ⁴ RNA拷贝/克。使用蛋白酶K提取方法制备的样品中仅GII（2.20×10 ¹和7.83×10 ¹ RNA拷贝/ g）可以定量。结果证明了两种样品处理方法的不同性能，并建议将两种提取方法与RT巢式PCR结合用于牡蛎中诺如病毒基因型的分子监测。