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Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field
Published on: August 2, 2011
Direct detection of highly pathogenic avian influenza A/H5N1 virus from mud specimens
Srey Viseth Horm1, Nathalie Deboosere, Ramona A Gutiérrez
1Institut Pasteur in Cambodia, Réseau International des Instituts Pasteur, Virology Unit, 5 Monivong Blvd, PO Box 983, Phnom Penh, Cambodia.
Abstract:
Contaminated mud and soil may play roles as reservoirs and sources of transmission for avian influenza A virus. However, the persistence of highly pathogenic avian influenza (HPAI) H5N1 virus in soil or mud has not been well documented, and specific methods of H5N1 virus detection in mud and soil specimens have not been described. The aim of this work was to evaluate the capacities of five different commercial kits and one elution-concentration technique to extract nucleic acids from H5N1 virus and to detect infectious viral particles in experimentally infected mud specimens. The viral RNA detection thresholds for the QIAamp kit, Trizol LS and the MagNA Pure LC kit were 5 × 10(2)RNA copies per gram of mud. Trizol reagent and the RNA PowerSoil™ kit were unsuccessful in recovering any viral RNA from mud. When the elution-concentration technique was performed prior to nucleic acid extraction, the performance of the MagNA Pure kit increased to a level that allowed the detection of H5N1 nucleic acids in naturally contaminated environmental samples that had previously tested negative after direct extraction using commercial kits. The levels of detection of infectious virus after inoculation into embryonated eggs were higher in concentrates than in eluates.
Insights
Detecting avian influenza A virus (HPAI H5N1) in mud requires effective nucleic acid extraction. The MagNA Pure LC kit, with an elution-concentration technique, showed superior performance for H5N1 virus detection in environmental samples.
Area of Science:
- Environmental microbiology
- Virology
- Molecular diagnostics
Background:
- Avian influenza A virus, particularly highly pathogenic avian influenza (HPAI) H5N1, can persist in environmental matrices like mud and soil.
- Mud and soil may act as reservoirs and transmission sources for HPAI H5N1.
- Limited data exists on HPAI H5N1 persistence in soil/mud and validated detection methods.
Purpose of the Study:
- To evaluate commercial kits and an elution-concentration technique for H5N1 virus nucleic acid extraction from experimentally infected mud.
- To assess the detection limits of different methods for H5N1 virus in mud specimens.
- To determine the efficacy of these methods for detecting infectious H5N1 virus particles.
Main Methods:
- Comparative evaluation of five commercial nucleic acid extraction kits (QIAamp, Trizol LS, MagNA Pure LC, Trizol reagent, RNA PowerSoil™ kit).
- Assessment of an elution-concentration technique prior to nucleic acid extraction.
- Quantification of viral RNA detection thresholds (RNA copies/gram of mud).
- Detection of infectious virus via inoculation into embryonated eggs.
Main Results:
- The QIAamp, Trizol LS, and MagNA Pure LC kits had detection thresholds of 5 × 10^2 RNA copies/gram.
- Trizol reagent and RNA PowerSoil™ kit failed to recover viral RNA from mud.
- The elution-concentration technique significantly enhanced MagNA Pure LC kit performance, enabling detection in previously negative environmental samples.
- Concentrates yielded higher levels of infectious virus detection compared to eluates.
Conclusions:
- The MagNA Pure LC kit combined with an elution-concentration technique is effective for H5N1 virus detection in mud.
- Certain commercial kits are unsuitable for H5N1 virus RNA recovery from mud.
- Optimized extraction methods are crucial for detecting HPAI H5N1 in environmental samples.
