Use of a robotic RNA purification protocol based on the NucliSens ® easyMAG™ for real-time RT-PCR detection of hepatitis A virus in bottled water

Abstract

Hepatitis A virus (HAV) infection is the leading cause of acute viral hepatitis throughout the world. An important part of viral control is rapid detection of HAV in drinking water contaminated with feces. One critical step in HAV detection methods is sample preparation. The objective of this study was to test the efficacy of different approaches to extracting RNA from HAV-inoculated bottled water. The optimal method is based on viral concentration by filtration on membrane filters and elution of adsorbed viruses from filters before RNA extraction and RT-PCR amplification. In this approach, the commercially available NucliSens ® easyMAG™ bio-robot (Biomérieux) performs viral RNA purification with silica magnetic beads, which mediate purification of nucleic acids by binding them and allowing other substances to be washed away. A new rapid simplified NucliSens ® easyMAG™-based approach is described and compared with the classical NucliSens ® easyMAG™ approach and with manual silica-based spin column purifications (Qiagen). The limit of detection (LOD) with the new rapid simplified NucliSens ® easyMAG™ approach was about 1 PFU/1.5 L against about 100 PFU/1.5 L using conventional sample treatments that included a concentration step using ultra-filtration.