Viral elution and concentration method for detection of influenza A viruses in mud by real-time RT-PCR

► A method for the detection of influenza viruses from large mud samples was optimized. ► H5N1 viruses were detected in mud samples collected during outbreak investigation in Cambodia. ► Because of the lack of scientific knowledge on many aspects of the ecology and environmental properties of HPAIVs, in particular H5N1, this method will allow understanding the year-by-year perpetuation of the virus and its survival in the environment. The role of environmental reservoirs in avian influenza virus (AIV) transmission has been investigated during AIV-associated outbreaks. To date, no method has been defined for detection of AIV from mud samples. A procedure using elution and polyethylene glycol (PEG) concentration steps was designed to detect AIV by RT-PCR from 42 g of raw mud, corresponding to 30 g of the solid fraction of mud. RNA was recovered with MagMAX AI/ND Viral RNA Isolation kit (Ambion, Austin, TX). Three elution buffers were studied and viral recoveries higher than 29% were yielded by elution with a 10% beef extract solution (pH 7). The overall method showed that, under some conditions, virus was not detectable in PEG samples, whereas viruses were detected in the elution fractions. PCR curves were improved significantly by running the amplification reaction with a mixture containing a PCR additive for inhibitor removal, such as T4 gene 32 protein (Gp32), although PCR inhibitors from mud were removed partially from PEG samples. A theoretical detection threshold of 5 × 10 5 RNA copies of H5N1 virus per 30 g of solid mud could be obtained by elution. The overall method has proved successful for detecting H5N1 virus contamination of mud specimens collected during outbreak investigations of avian influenza in Cambodia.