Submitted to: Journal of Clinical Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 6/25/2006
Publication Date: 9/1/2006
Citation: Das, A., Spackman, E., Senne, D., Pedersen, J., Suarez, D.L. 2006. Development of an internal positive control for rapid diagnosis of avian influenza virus infections by real-time reverse-transcription-PCR with lyophilized reagents. Journal of Clinical Microbiology. 44(9):3065-3073. Interpretive Summary: Avian influenza virus is found normally in wild birds. It can also infect other species, including chickens, turkeys, domestic ducks, swine, and horses. Sometimes this can result in a severe disease outbreak. The key to controlling these outbreaks is rapid diagnosis and response. The use of real time RT-PCR for the detection of avian influenza has been extremely beneficial because it can provide both rapid and sensitive test results. In an effort to assure the best results, our study looked at adding a control to the test that provides assurance that the test was done properly. Also the internal control can determine if something is interfering with the proper performance of the test. The diagnostic test was also simplified by putting several of the reagents into a bead format. This simplifies performing the test and allows greater assurance that all the components of the test are working properly. Both the internal control and the bead reagents are incremental improvements of the test that will aid in the diagnosis of avian influenza from infected birds.
Technical Abstract: Real time RT-PCR (RRT-PCR) has been widely adopted as a diagnostic screening test for Avian Influenza (AI) in the U.S. However, concerns about false negative results due to inhibition or human error have been an issue. We developed an internal positive control (IPC) in the nucleic acid amplification step to help ensure the accuracy of the diagnostic test results for RT-PCR and RRT-PCR. The IPC was 228 bases long in vitro transcribed RNA from a DNA template. The IPC was designed to have the same binding sites for the forward and reverse primers of the AI matrix gene as the target amplicon, but it had a unique internal sequence not found in the target genome that was used for the probe site. The amplification of the viral RNA and the IPC by RRT-PCR were monitored with two different fluorescent probes in a multiplex format, one specific for the AI matrix gene and the other for the IPC. A minimal amount of the IPC was used per reaction to reduce the potential for decreased sensitivity by competition. The test was further simplified with the use of lyophilized bead reagents for the detection of AI RNA in clinical samples. The RRT-PCR with the bead reagents was more sensitive than the conventional wet reagents for the detection of AI RNA from clinical samples. Additionally, different types of diagnostic samples were spiked with AI virus and compared for evidence of PCR inhibitors after RNA extraction with Trizol. Blood, kidney, lungs, spleen, intestine and cloacal swabs all had inhibitory substances, but allantoic fluid from chicken eggs, serum and tracheal swabs did not. The accuracy of the RRT-PCR test results with the lyophilized beads on 32 cloacal and 66 tracheal swabs from experimental birds inoculated with AI was compared with virus isolation (VI) on embryonating chicken eggs. There was 97-100% agreement of the RRT-PCR test results with VI for tracheal swabs, and 81% agreement with VI for cloacal swabs, indicating a high level of accuracy of the RRT-PCR assay in detecting AIV in clinical specimens. The same IPC in the form of Armored RNA was also used to monitor the extraction of viral RNA and subsequent detection by RRT-PCR.