Development of an Extraction and Concentration Procedure for the Detection of Enteric Viruses in Soil

Introduction: Enteric viruses, including Norovirus, rotavirus and hepatitis A viruses have been recognized as being the leading cause of non-bacterial gastroenteritis and infectious hepatitis outbreaks around the world. Understanding the prevalence and persistence of enteric viruses in environmental samples is an important issue for the effective control of these infections. Commercial extraction kits developed for soil allow the treatment of very small volumes (0.5 g – 5 g) and viruses are generally in low concentration in the environment.

Purpose: The aim of this study was to develop an efficient extraction and concentration procedure for enteric viruses from larger samples.

Methods: 25 g of soil (black earth, loamy and sandy soils) were spiked at various concentrations between 10⁵ and 10² PFU/g of murine norovirus (MNV) and feline calicivirus (FCV) used as model for enteric viruses. The soil samples were mixed with different buffers, additives (PVPP, activated carbon) and concentrated on ultra-filtration units. Viral genetic material was extracted by different commercial kits and conventional RT-PCR and Taqman real-time RT-PCR systems were used for detection.

Results: Different combinations of buffers, additives, extraction kits were tested and the more efficient procedure to remove PCR inhibitors was composed of MEM buffer, PVPP and the Dynal beads extraction. This procedure has allowed the detection of 10² PFU/g of MNV and 10³ PFU/g of FCV in the three types of soil samples.

Significance: This method allows the concentration of MNV and FCV viral particles from 25 g of three different kinds of soil frequently used for fresh products culture. This method can be applied for the detection by molecular methods of other unculturable viral species, such as Norovirus, responsible of foodborne diseases, and to increase our knowledge on their persistence in soil and its impact on viral contamination of fresh horticultural products.