The no template control reactions should not exhibit fl uorescence growth curves that cross the threshold line. Although the real-time RT-PCR is highly sensitive and specifi c assay for novel H1N1 virus detection, the limitations include need of trained personnel for assay set up and result interpretation, false negative results which may occur if inadequate numbers of organisms are present in the specimen due to improper collection, transport, handling or excess of DNA/RNA template in the reaction and initial cost of machine. (Table 1) (Fig. 2) .