Open Access Highly Accessed Research article

A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control

Livia Di Trani1*, Barbara Bedini1, Isabella Donatelli2, Laura Campitelli2, Barbara Chiappini1, Maria Alessandra De Marco3, Mauro Delogu4, Canio Buonavoglia5 and Gabriele Vaccari1

  • * Corresponding author: Livia Di Trani ditrani@iss.it

  • † Equal contributors

Author Affiliations

1 Department of Food Safety and Veterinary Public Health, Istituto Superiore di Sanità; V.le Regina Elena 299, 00161 Rome, Italy

2 Department of Parasitic, Immune-Mediated and Infectious Diseases, Istituto Superiore di Sanità; V.le Regina Elena 299,00161 Rome, Italy

3 National Wildlife Institute, Italy; Via Ca' Fornacetta 9, 40064 Ozzano Emilia (BO) Italy

4 Department of Veterinary Public Health and Animal Pathology, University of Bologna,; Via Tolara di Sopra 50, 40064 Ozzano Emilia (BO), Italy

5 Department of Animal Health and Well-Being, Faculty of Veterinary Medicine of Bari, Prov. Per Casamassima Valenzano,70010 Bari, Italy

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BMC Infectious Diseases 2006, 6:87  doi:10.1186/1471-2334-6-87

Published: 25 May 2006

Abstract

Background

Avian influenza viruses (AIVs) are endemic in wild birds and their introduction and conversion to highly pathogenic avian influenza virus in domestic poultry is a cause of serious economic losses as well as a risk for potential transmission to humans. The ability to rapidly recognise AIVs in biological specimens is critical for limiting further spread of the disease in poultry. The advent of molecular methods such as real time polymerase chain reaction has allowed improvement of detection methods currently used in laboratories, although not all of these methods include an Internal Positive Control (IPC) to monitor for false negative results.

Therefore we developed a one-step reverse transcription real time PCR (RRT-PCR) with a Minor Groove Binder (MGB) probe for the detection of different subtypes of AIVs. This technique also includes an IPC.

Methods

RRT-PCR was developed using an improved TaqMan technology with a MGB probe to detect AI from reference viruses. Primers and probe were designed based on the matrix gene sequences from most animal and human A influenza virus subtypes. The specificity of RRT-PCR was assessed by detecting influenza A virus isolates belonging to subtypes from H1–H13 isolated in avian, human, swine and equine hosts. The analytical sensitivity of the RRT-PCR assay was determined using serial dilutions of in vitro transcribed matrix gene RNA. The use of a rodent RNA as an IPC in order not to reduce the efficiency of the assay was adopted.

Results

The RRT-PCR assay is capable to detect all tested influenza A viruses. The detection limit of the assay was shown to be between 5 and 50 RNA copies per reaction and the standard curve demonstrated a linear range from 5 to 5 × 108 copies as well as excellent reproducibility. The analytical sensitivity of the assay is 10–100 times higher than conventional RT-PCR.

Conclusion

The high sensitivity, rapidity, reproducibility and specificity of the AIV RRT-PCR with the use of IPC to monitor for false negative results can make this method suitable for diagnosis and for the evaluation of viral load in field specimens.