含4种食源性病毒检测靶标多联装甲RNA的制备、纯化与定值

基于Qβ噬菌体装甲RNA制备平台构建同时含有诺如病毒(norovirus,NoV)、甲肝病毒(hepatitis Avirus,HAV)、轮状病毒(rotavirus,RV)、星状病毒(astrovirus,AstV)检测靶标RNA的多联装甲RNA(multiplexarmoredRNA,AR-MulV),并进行纯化与初步定值。结果表明,重组质粒在大肠杆菌中成功表达,表达产物大小约为14.1 kDa;制备的AR-MulV经纯化后无杂蛋白与残留重组质粒,电镜下可见大量结构形态完整的病毒样颗粒,大小约为25 nm。初步定值结果显示,AR-MulV中GⅠ型NoV、GⅡ型NoV、HAV、RV和AstV检测靶标RNA的含量分别为(1.24±0.2)×10~7、(2.54±0.6)×10~7、(2.24±0.3)×10~7、(2.96±0.5)×10~7 copies/μL和(3.19±0.4)×10~7 copies/μL。本研究基于Qβ噬菌体装甲RNA制备平台成功制备同时包含4种食源性病毒标准方法检测靶标的多联装甲RNA,为食源性病毒的分子检测以及多重实时荧光定量逆转录-聚合酶链式反应阳性质控样品的研发提供新思路。

Construction,Purification and Quantification of Multiplex Armored RNA Containing Targets for Detection of 4 Foodborne Viruses

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.