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.
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 1PFU/1.5L against about 100PFU/1.5L using conventional sample treatments that included a concentration step using ultra-filtration.
Perelle
Sylvie
S
Agence Française de Sécurité Sanitaire des Aliments (AFSSA), 94706 Maisons-Alfort cedex, France. s.perelle@afssa.fr
Cavellini
Laetitia
L
Burger
Christian
C
Blaise-Boisseau
Sandra
S
Hennechart-Collette
Catherine
C
Merle
Ghislaine
G
Fach
Patrick
P
D005374:Filtration / Q000379:methods; D030041:Hepatitis A virus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012371:Robotics / Q000379:methods*; D012680:Sensitivity and Specificity; D014871:Water Microbiology
D012367:RNA, Viral