Sensitivity and specificity of one-tube qRT-PCR for detection of Nipah virus RNA. DNA fragments obtained from the RT-PCR were visualized in ethidium bromide-stained agarose gel (a). Input Nipah virus RNA in equivalent log PFU is indicated above the lanes. RNA extracted from mock-infected Vero cells and the Nipah virus Armored RNA® served as the negative (neg) and positive (pos) controls, respectively. Lane (M) consisted of DNA molecular mass marker. Amplification plot of the SYBR® Green I dye-based qRT-PCR assay were obtained from tenfold serial diluted Nipah virus RNA (1 × 106 to 1 PFU) as indicated in (b). RNA extracted from mock-infected Vero cells was used as the negative control (NTC). The standard curve for the qRT-PCR (c) was generated using the same dilution series of Nipah virus RNA as the amplification plot. Correlation between log PFU/μL of infectious virus against total copy number of Nipah virus RNA (log RNA copy/μL) obtained from the qRT-PCR is shown in (d). Specificity of the assay was assessed and the difference in the melting temperature of the amplified DNA of Nipah virus (thick arrow) and Hendra virus (thin arrow) is indicated in the melting curve analysis (e).