Comparison of a nucleoprotein gene based RT-PCR with real time RT-PCR for diagnosis of avian influenza in clinical specimens

A nucleoprotein (NP) gene based reverse transcription polymerase chain reaction (npRT-PCR) assay was developed in our laboratory which could detect 35.09% of the experimental samples negative for virus isolation in first passage but positive by third passage. Reducing the reaction volume to 12.5 μl did not alter the test sensitivity and the results did not vary when duplicate samples were run in a different thermal cycler. The positive and negative agreements of this test in clinical specimens were compared with a matrix gene based real time RT-PCR with virus isolation as standard. A total of 516 clinical specimens including tissues, swabs and feces submitted from various States of India as part of active surveillance for avian influenza were tested by npRT-PCR, RRT-PCR and virus isolation in 9–11 day old embryonated specific pathogen free chicken eggs. The positive and negative agreements of npRT-PCR with virus isolation were found to be 0.909 ± 0.022 and 0.980 ± 0.004 respectively and that of RRT-PCR with virus isolation were 0.902 ± 0.023 and 0.977 ± 0.005 respectively. Since the positive and negative agreements of both npRT-PCR and RRT-PCR tests were similar, we suggest that this test can be used by peripheral veterinary laboratories that do not have real time PCR facility for active surveillance of AIV.