牡蛎中诺如病毒基因多样的研究对病毒的流行传播和疫情防控具有十分重要的意义,二代测序技术(next generation sequencing,NGS)的扩增子测序具有高通量、高准确性等优势,被广泛应用于基因组学、转录组学和表观基因组学等领域。为评估扩增子测序技术对牡蛎中诺如病毒多样性研究的可行性,采用经典的巢式反转录PCR(reverse transcription-PCR, RT-PCR)方法对人为污染诺如病毒的牡蛎样品进行检测并将产物进行扩增子测序,对病毒基因型覆盖度和灵敏度进行分析。结果显示,在覆盖度方面,扩增子测序技术能够检测出人为添加到牡蛎中的6种GⅡ型诺如病毒,覆盖率达到100%;在灵敏度方面,对人为污染有不同数量诺如病毒的牡蛎进行检测及测序分析,证实扩增子测序的灵敏性较高,低于50个病毒粒子也能检测出。综上,扩增子测序技术提高了牡蛎中诺如病毒检测的准确性以及基因型的丰富度。
The exploration of norovirus genetic diversity within oysters is of paramount importance in understanding the virus’s epidemic spread and devising effective prevention and control strategies.Amplicon sequencing, a next-generation sequencing (NGS) technique, offers significant advantages, such as high throughput and accuracy, commonly applied in genomics, transcriptomics, and epigenomics.This study aims to assess the viability of amplicon sequencing technology for probing norovirus diversity in oysters.To evaluate its efficacy, oyster samples were intentionally contaminated with norovirus underwent amplicon sequencing using the classical nested RT-PCR method.The analysis aimed to ascertain the viral genotype coverage and sensitivity of this approach.Results demonstrated that amplicon sequencing successfully detected all six GⅡ noroviruses present in oysters, achieving 100% coverage.Remarkably, the technique′s sensitivity was underscored through the detection and sequencing analysis of oysters contaminated with varying amounts of noroviruses, confirming its ability to identify less than 50 viral particles.In summary, amplicon sequencing technology markedly enhances the accuracy and genotypic richness of norovirus detection in oysters.
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