طراحی کنترل مثبت داخلی ‭( Internal Positive Control) ‬برای آزمایش های شناسایی ویروس انفلوانزا‭A ‬با روش ‭RT-PCR‬

The H5NI virus (Orthomyxoviridea,a family, genus Influenza A causes severe and acute disease not only in poultry, but also in humanbeings.On of the common procedures for virus detection is RT-PCR but likewise other techniques,quality control of the steps involved, is critical to ensure the accuracy of results. Using Internal Positive Control (IPC) helps the monitoring of the PCR assays and the risk of obtaining false negative results. The IPC (-300bp) was designed to have the same binding site for the forward and reverse primers of the H5 HA gene as the target amplicon (-540bp) so it can be amplify competitively with target and with the same pair of primers. The PCR fTagment (300bp) was then cloned into a accurate vector and transformed using proper E.coli cells. After plasmid extraction, the plasmid was serially diluted to define the suitable amount of IPC in reaction.Finally RT-PCR was performed using internal positive control and specimens which were used formerly in reaction excluding IPC. The concentration of IPC was defined to be 0/37 ngul 1- .There was no difference in the results of the former RT PCR and the one which includes internal positive control (40 specimens, 34 negative and 6 positive samples were uses in the assay including IPC). Although there was no difference between the result of the former RT-PCR and the new one,IPC should be omitted in reactions to monitor false negative results due to PCR failure caused by poor techniques, malfunction of thermal cycler, poor polymerase activity or presence of inhibitory substances in the samples