Application of Q-Gene software for the quantitation on Nipah virus in experimental animals using real-time PCR

L.I. Pritchard1, Y. Kaku2, G. Crameri, B.T. Eaton, D.B. Boyle, AAHL CSIRO; 2, National Institute of Animal Health, Toyko, Japan

Nipah virus is a newly recognized zoonotic virus, which has caused disease in animals and in humans. The virus was discovered in Malaysia in 1999 as the cause of a large outbreak of encephalitis in which 105 people died and 1.1 million pigs were culled. Epidemiological studies indicate that the virus crossed species from fruit bats to pigs and then subsequently to humans. We developed a specific fluorogenic (5’ nuclease probe) Taqman real-time PCR assay for the diagnosis and detection of Nipah virus in experimental animal and diagnostic samples. The application of this assay for molecular diagnosis reduced the test duration to 4 hours from receipt of sample and was found to be ten-fold more sensitive than a conventional, heminested RT-PCR. Tests against a range of Paramyxoviruses, including the closely related Hendra virus showed the assay specifically detected Nipah virus. The limit of detection was approximately 1.4 TCID/50 units / ml. Q-Gene (Muller et al., 2002) is a Microsoft® Excel®-based software application coded in Visual Basic, which manages and expedites the planning, performance, and evaluation of quantitative real-time PCR experiments, as well as the mathematical and statistical analysis, storage, and graphical presentation of the data. The Q-Gene software application is a tool to cope with complex quantitative real-time PCR experiments at a high-throughput scale and considerably expedites and rationalizes the experimental setup, data analysis, and data management while ensuring highest reproducibility. In this work, we focus on the mathematical evaluation and analysis of the data generated by the Nipah virus quantitative real-time PCR, the calculation of the final results, the propagation of experimental variation of the measured values to the final results, and the statistical analysis.