关键词: 食源性病毒, 多联装甲RNA, Qβ噬菌体, 阳性质控样品, 实时荧光定量逆转录-聚合酶链式反应

Abstract: This study was undertaken to construct multiplex armored RNA (AR-MulV) containing the target RNAs of norovirus (NoV), hepatitis A virus (HAV), rotavirus (RV), and astrovirus (AstV) based on Qβ bacteriophage for foodborne virus detection. The armored RNA was purified and quantified as well. The recombinant plasmid was efficiently expressed in Escherichia coli and the molecular mass of the product was estimated to be 14.1 kDa on SDS-PAGE. The purified AR-MulV was found to be free of any unwanted protein or residual plasmid and it was observed as virus like particles with good structural and morphological integrity 25 nm in diameter under electron microscopy. The target RNAs of NoV (GI and GII), HAV, RV and AstV in the AR-MulV were quantified as (1.24 ± 0.2) × 107, (2.54 ± 0.6) × 107, (2.24 ± 0.3) × 107, (2.96 ± 0.5) × 107 and (3.19 ± 0.4) × 107 copies/μL respectively. The multiplex armored RNA based on Qβ bacteriophage could serve as a good positive control material candidate for use in the simultaneous detection of 4 foodborne viruses. Moreover, this study provides a new strategy for preparing positive reference material for use in the development of a multiplex real-time RT-PCR assay for foodborne viruses.

Key words: foodborne viruses, multiplex armored RNA, Qβ bacteriophage, positive control material, real-time RT-PCR