Development of an RNA Extraction Protocol for Norovirus from Raw Oysters and Detection by qRT-PCR and Droplet-Digital RT-PCR

Abstract

Foodborne viruses such as norovirus and hepatitis A virus cause frequent outbreaks associated with the consumption of raw or undercooked oysters. Viral particles are bioaccumulated in the oyster’s digestive glands, making RNA extraction and RT-PCR detection difficult due to the complex nature of the food matrix and the presence of RT-PCR inhibitors. Herein, we have developed a viral RNA extraction protocol from raw oysters using murine norovirus (MNV) as a surrogate for human noroviruses. The method combines lysis in Tri-Reagent reagent, followed by RNA extraction using Direct-Zol purification columns and lithium chloride precipitation. Viral load quantification was performed by both qRT-PCR and droplet-digital RT-PCR. We have demonstrated that this method can efficiently remove RT-PCR inhibitors, and is sensitive enough to reliably detect viral contamination at 25 PFU/0.2 g. We have also compared the efficiency of this method with the ISO 15216-1:2017 method and Method E developed by Quang and colleagues, and observed significantly higher efficiency compared with the ISO 15216-1 method and comparable efficiency with Method E, with less steps, and shorter hands-on time.

Amplicon-Based High-Throughput Sequencing Method for Genotypic Characterization of Norovirus in Oysters

“…This supported the variation in the correlation between input material and obtained HTS reads from spiked and naturally contaminated samples ( Table 3 ). Literature suggests this can partially be overcome by the choice of RNA extraction method, i.e., phenol-chloroform extraction with ethanol purification ( 12 , 58 ), but yield is an important consideration in environmentally contaminated samples ( 58 , 59 ). Despite this observation, bioaccumulation experiments with fragmented norovirus RNA and clinical samples established that the intact virus was preferentially bioaccumulated over fragments of viral RNA and could survive up to 2 weeks ( 60 ).…”

Occurrence of carbapenem-resistant hypervirulent Klebsiella pneumoniae in oysters in Egypt: a significant public health issue

“…The external valves of oysters were thoroughly washed with sterile water and aseptically opened, and the digestive tissues were dissected, cleaned, and finely chopped to a paste-like consistency to ensure the uniformity of the starting material [ 30 ].…”

Comprehensive analysis of predominant pathogenic bacteria and viruses in seafood products

“…HuNoV, an exceptionally contagious RNA calicivirus, is responsible for inducing infections in humans (Lanrewaju et al., 2022). A substantial portion of human NoV outbreaks, approximately 48%, are attributed to contaminated shellfish consumption (Plante et al., 2021). NoV infections are recognized as a leading cause of nonbacterial gastroenteritis, frequently linked to the consumption of partially cooked, raw shellfish, particularly oysters (Elbashir et al., 2018; Okumus, 2023; Woods et al., 2016).…”