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Rapid Detection of Avian Influenza Virus Using One Tube Sybr Green I Based Real-Time Polymerase Chain Reaction


A one step SYBR Green I based real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay for the detection of avian influenza virus subtypes was developed. The assay was optimized using primers that amplified the conserved region of nucleocapsid (NP) gene and serially diluted RNA of AIV subtype H3N8. The specificity of the amplification was confirmed by melting curves analysis. The melting temperatures (Tm) of amplicons were between 82.8oC to 84oC. The assay was also able to detect other AIV subtypes such as H5 and H9. The sensitivity of the assay compared to the standard embryonated eggs inoculation method is currently been evaluated.


Highly pathogenic avian influenza virus (HPAI) is a highly contagious viral disease causes severe disease in domestic poultry including chickens and turkeys. Avian influenza viral genome consists of 8 segments (ranging from 890 to 2341 nucleotides) encode for 8 different genes, haemagglutinin (H) gene, neuraminidase (N) gene, matrix (M) gene, nonstructural (NS) gene, nucleoprotein (NP) gene, and three polymerase (PA, PB1 and PB2) genes (Hoffmann et al., 2001). The surfaces proteins include hemagglutinin (H) and neuraminidase (N) play important role for the production of protective immune response (Collins et al, 2002). In addition, characterization of AIV isolates is based on the H and N proteins. At present, 15 H and 9 N subtypes are recognized.

Several routine diagnostic methods are available for detection and subtyping of influenza A virus including virus isolation, haemagglutination and haemagglutination inhibition tests. However the use of molecular technique such as RT-PCR to detect viruses can facilitate the rapid identification and genetic characterization of AIV.

Materials and Methods

AIV subtypes H3N8 was provided by Veterinary Research Institute, Ipoh, Perak. The virus was propagated in 11 day-old specific-pathogen-free (SPF) embryonating chicken eggs by allantoic sac route. The eggs were incubated at 37oC for 5 days. Allantoic fluid was collected and tested for presence of haemagglutinin using haemaglutination (HA) test. The viral RNA was extracted from allantoic fluid using TRI Reagent ® LS (Molecular Research Centre, INC) as described by the manufacturer. A set of primers that amplified a 218 bp conserved regions of NP gene was designed. The primers were designed based on the conserved regions of all the 15 H genes of AIV subtypes. The RT-PCR was performed using a one-step Access RT-PCR system (Promega, USA). Optimization and evaluation on the performances of SYBR Green 1 based real-time PCR for the detection and differentiation of AIV and data analysis was performed using Opticon 2 DNA Engine (MJ Research, USA).

Result and Discussion

Haemagglutination test showed a positive agglutination result on allantoic fluid obtained from inoculated eggs. RNA extracted from the inoculated allantoic fluids show specific amplification with threshold (Ct) value ranging from 14 to 25. The specificity of the amplification was confirmed by melting curve analysis. The melting temperatures (Tm) of amplicons were between 82.8oC to 84oC.

The real-time RTPCR was chosen as an alternative method for AIV detection because it offers advantages over conventional RTPCR. Currently the sensitivity of the assay compared to the conventional egg inoculation work is being evaluated. The assay was also able to detect other AIV subtypes including H5 and H9. The ability of the assay to detect other AIV subtypes is yet to be evaluated. Nevertheless, the assay offers a rapid, sensitive and accurate assay for detection of AIV compared to the conventional PCR detection method.


1. Collins R.A, L.S Ko, K.L So, T. Ellis, L.T. Lau, A.C.H Yu. (2002). Detection of highly pathogenic and low pathogenic avian influenza subtype H5 (Eurasian lineage) using NASBA. Journal of Virology Methods 103: 213-215.

2. Hoffmann, E. Stech, J., Guan, Y., Webster R.G., and Perez D.R. (2001). Universal primer set of the full-length amplification of all influenza virus. Arch. Virol. 146: 2275-2289.


Faculty of Veterinary Medicine, Universiti Putra Malaysia