New Micro-amount of Virion Enrichment Technique (MiVET) to detect influenza A virus in the duck faeces.

Abstract

Transboundary animal diseases, including highly pathogenic avian influenza, cause vast economic losses throughout the world. While it is important to identify the sources and propagation routes of the spread, such strategies are often hindered by incomplete epidemiological evidence. Isolation/detection of micro-amounts of pathogens from environmental samples is rarely successful due to the very low contamination level. This paper describes the development of the micro-amount of virion enrichment technique (MiVET), a simple and highly sensitive method that combines the use of a complex comprising a polyclonal antibody and protein G-coated magnetic beads for virion capture, and simple sodium dodecyl benzenesulfonate (SDBS) elution for low volume samples. The performance of the MiVET was evaluated using avian influenza A viruses (AIVs) in artificially spiked samples by real-time reverse transcription polymerase chain reaction (rRT-PCR). Four AIVs, H3N2, H4N2, H5N2 and H7N7, were used to artificially spike 50ml of phosphate-buffered saline (PBS) and 1ml of 10%-25% duck faecal supernatants. The MiVET system successfully concentrated AIVs in both PBS and faecal samples with at least 2 and 1 log greater efficacy, respectively, than conventional RNA extraction methods. The MiVET could be completed in <30min from the beginning of sample preparation to final RNA extraction. The MiVET effectively prevented the effects of inhibitors in faecal samples, and did not require special equipment. This is the first report of this novel type of system, which is expected to be useful for the detection of micro-amounts of various veterinary and human viruses to elucidate their circulation dynamics in the environment, and for rapid and sensitive diagnosis with greater detection power.