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Title: Removal of real-time reverse transcription polymerase chain reaction (RT-PCR) inhibitors associated with cloacal swab samples and tissues for improved diagnosis of avian influenza virus by RT-PCR

item Das, Amaresh
item Spackman, Erica
item Pantin Jackwood, Mary
item Suarez, David

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/17/2009
Publication Date: 11/1/2009
Citation: Das, A., Spackman, E., Pantin Jackwood, M.J., Suarez, D.L. 2009. Removal of real-time reverse transcription polymerase chain reaction (RT-PCR) inhibitors associated with cloacal swab samples and tissues for improved diagnosis of avian influenza virus by RT-PCR. Journal of Veterinary Diagnostic Investigation. 21:771-778.

Interpretive Summary: Avian influenza virus can infect a wide variety of birds and mammals. The early and rapid diagnosis of infection can greatly aid in the control of the virus. Recently the use of real-time reverse transcription-polymerase chain reaction (PCR) as a diagnostic test has provided for rapid and sensitive diagnosis of the virus from many types of clinical samples. The PCR test amplifies purified ribonucleic acid (RNA) to levels that are easily detected with a specialized PCR machine. However not all samples work the same with this technology, and the presence of inhibitors in fecal samples has been shown to be a problem. This paper describes the comparison of different methods to purify RNA for use in the PCR test, and it was found that a simple modification of an existing commercial test greatly improved test results with fecal samples. This new method was shown to work in experimental and field samples and should improve testing in the field.

Technical Abstract: Real time reverse transcriptase polymerase chain reaction (RRT-PCR) is routinely used for the rapid detection of Avian Influenza virus (AIV) in clinical samples. The usefulness of diagnostic RRT-PCR can be limited, in part, by the inhibitory substances present in some clinical specimens, which can reduce or block polymerase chain reaction (PCR) amplification. Most commercial ribonucleic acid (RNA) extraction kits have limited capacity to remove inhibitors from clinical samples. In this study we used a modified commercial protocol (MagMAX, Ambion) in which a high salt wash with 2M NaCl -2mM ethylenediaminetetraacetic acid (EDTA) was shown to significantly improve the ability of the kit to remove inhibitors from cloacal swabs and tissues. RRT-PCR was carried out in the presence of an internal positive control (IPC) to detect inhibitors present in the purified RNA. Cloacal swabs from wild birds were analyzed by RRT-PCR comparing RNA extracted with the standard (MagMAX-S) and a modified MagMAX (MagMAX-M) protocol. Of the 2668 samples extracted with the MagMAX-S protocol, 403 (15.1%) samples tested positive for AIV, 2,167 (81.22%) samples tested negative for AIV, and 491 (18.40%) samples tested negative for both AIV and IPC, indicating presence of inhibitor(s) in the latter samples. The 433 AIV and IPC negative samples were further analyzed using the MagMAX-M protocol for RNA extraction, and 36 (8.3%) samples were positive for AIV, 412 (95%) samples negative for AIV and only 21 (4.8%) samples tested negative for both AIV and IPC by RRT-PCR. The modified protocol was also tested for RNA extraction from tissues using a MagMAX-Trizol hybrid protocol as previously described (refe rence 14). Tissues of breast (n=28), thigh (n=28) and heart (n=28) from chickens and those of brain (n=16), lung (n=18), spleen (n=11), heart, (n=9) muscle (n=11) and kidney (n=6) from ducks experimentally infected with high pathogenicity (HP) Asian H5N1 AIV were analyzed by RRT-PCR and the limit of detection of the virus was improved by 0.5 to 3.0 Ct units with the RNA extracted by the MagMAX-M protocol. The M-MagMAX protocol reported in this study can be useful to extract high quality RNA for accurate detection of AIV from cloacal swabs and tissues by RRT-PCR.