Quantification of hepatitis E virus in raw pork livers using droplet digital RT-PCR. (February 2023)

• Record Type: Journal Article
• Authors: Wang, Kairui; Liu, Libing; Wang, Jinfeng; Sun, Xiaoxia; Han, Qingan; Zhou, Cang; Xu, Xiangdong; Wang, Jianchang
• Is Part Of: Food microbiology. Volume 109(2023)
• Publication Date: 2023-02
• Subjects: Hepatitis E virus -- Droplet digital PCR -- Undercooked pork -- Nested RT-PCR -- Phylogenetic analysis; Food Microbiology -- Periodicals; Aliments -- Microbiologie -- Périodiques; Food -- Microbiology; Periodicals; Food -- Microbiology -- Periodicals; Food contamination -- Periodicals; 664.001579
• DOI: 10.1016/j.fm.2022.104114
• Languages: English
• ISSNs: 0740-0020

Abstract

Abstract: Hepatitis E virus (HEV) is the causative agent of hepatitis E. Some of the rise in hepatitis E infection in China may be linked to undercooked pork. In this study, we established a reverse transcription droplet digital PCR (RT-ddPCR) method to detect HEV in raw pork livers. The detection limit of the assay for HEV RNA was as low as 1.81 copies/μL. The suggested approach was validated on 14 samples, demonstrating greater sensitivity, specificity, and anti-interference performance features than RT-qPCR. Furthermore, we amplified the partial ORF2 gene by nested RT-PCR and sequenced for the HEV RNA positive samples. The prevalence of HEV in all collected samples was 2.24% (14/626), and the viral load was between 8.0 copies/μL and 8975 copies/μL. Specifically, the virus was detected in 10.62% (12/113) of the samples collected from the bio-safety disposal centers for dead livestock and poultry, in 0.67% (2/300) of the samples collected from the slaughterhouses, and none of the samples collected from the retail markets was HEV RNA positive. The subsequent phylogenetic analysis revealed that all HEV isolates belonged to the subtype 4d, which is one of the most common subtypes in northern China. Graphical abstract: Image 1 Highlights: The quantitative detection of HEV RNA by a RT-ddPCR assay was developed. The limit of detection of the assay for HEV RNA was 1.81 copies/μL (95% CI: 1.41 copies/μL to 3.22 copies/μL). A total of 626 raw pork livers were analyzed, and the prevalence of HEV was 2.24% (14/626). All the HEV isolates belonged to the genotype 4d.