APPLICATION OF BIOLOGICAL AND MOLECULAR TECHNIQUES TO THE DIAGNOSIS AND CONTROL OF AVIAN INFLUENZA AND OTHER EMERGING POULTRY PATHOGENS
Location: Exotic and Emerging Avian Viral Diseases Research Unit
Title: Further improvement and validation of MagMAX-96 AI/ND viral RNA isolation for efficient removal of RT-PCR inhibitors from cloacal swabs and tissues for rapid diagnosis of avian influenza virus by RT reverse transcription PCR
Submitted to: American Association of Veterinary Laboratory Diagnosticians
Publication Type: Proceedings
Publication Acceptance Date: September 27, 2007
Publication Date: October 18, 2007
Citation: Das, A., Spackman, E., Pantin Jackwood, M.J., Swayne, D.E., Suarez, D.L. 2007. Further improvement and validation of MagMAX-96 AI/ND viral RNA isolation for efficient removal of RT-PCR inhibitors from cloacal swabs and tissues for rapid diagnosis of avian influenza virus by RT reverse transcription PCR. In: Proceedings of the 50th American Association of Veterinary Laboratory Diagnosticians Conference, October 18-24, 2007, Reno, Nevada, p.32.
Interpretive Summary: Avian influenza virus is naturally found in wild aquatic birds including shore birds, ducks, swan and geese where their presence does not cause any disease. These viruses grow and multiply mostly in the guts of wild birds and are easily transmitted into other birds including domestic birds such as chickens and turkeys through direct contacts or fecal discharges. Therefore monitoring AIV in fecal samples from wild birds has been common strategy AI surveillance. The standard protocol used for detection of AIV in fecal samples has been extraction of RNA followed by amplification of the RNA via RRT-PCR. One of the major problems associated with the detection of AI in fecal samples has been presence of PCR inhibitors which block amplification of the RNA resulting false negative results. Fecal samples with highly pathogenic AIV such as H5N1, if remains undetected, could threat potential outbreaks and economic losses.
In this report we used a modified protocol using the MagMAX-96 AI/ND RNA isolation kit for extraction of RNA from fecal samples of wild birds spiked with AIV and from different tissues of experimental chickens and ducks infected with highly pathogenic H5N1. The limit of detection of AIV by RRT-PCR in either sample type was found to be more sensitive with the RNA extracted by the modified MagMAX protocol than the standard MagMAX protocol.
Real time RT-PCR (RRT-PCR) is a high throughput molecular diagnostic test used for rapid detection of avian influenza virus (AIV) in clinical samples. However the performance of RRT-PCR can be adversely affected by RT-PCR inhibitors present in the sample. The tested commercial RNA extraction kits did not remove all RT-PCR inhibitors from clinical samples from cloacal swabs and tissues. In this study we used a modified MagMAX-96 AI/ND viral RNA isolation kit (MagMAX, Ambion) for extraction of RNA from cloacal swabs and tissues. RRT-PCR was carried out in the presence of an internal positive control to detect inhibitors in the sample.
Cloacal swabs from 2668 wild birds of Alaska and South America were analyzed by RRT-PCR after the RNA was extracted by the standard and modified MagMAX protocols. With the RNA extracted by the standard MagMAX protocol 403 (15.1%) samples tested positive for AIV, 2167 (81.22%) samples tested negative for AIV, and 491 (18.40%) samples had evidence for PCR inhibitors (false negatives). Further analysis of 433 of the samples with inhibition with the modified MagMAX protocol indicated 36 (8.3%) of the samples tested were positive for AIV, 412 (95%) samples tested negative for AIV and only 21 (4.8%) samples tested still had evidence of RRT-PCR inhibitors.
For tissues the RNA was purified using a Trizol-MagMAX hybrid protocol in which the RNA was extracted by Trizol LS and purified using both standard and modified MagMAX protocols. Tissues of breast (n=28), thigh (n=28), and heart (n=28) from chickens infected with high pathogenicity (HP) Asian H5N1 AIV and those of brain (n=16), lung (n=18), spleen (n=11), heart, (n=9) muscle (n=11), and kidney (n=6) from HP H5N1 infected ducks were analyzed by RRT-PCR after the RNA extracted by the above protocols. The average Cts corresponding to the virus in tissues from infected chickens and ducks were between 0.5 and 3.0 higher with the RNA extracted by the standard MagMAX protocol than the modified MagMAX protocol. The results show more efficient removal of RT-PCR inhibitors from both cloacal swabs and tissues by the modified MagMAX RNA extraction protocol, which ensures high quality of the RNA for accurate detection of AIV by RRT-PCR.