Duplex nested RT-PCR for detection of Nipah virus RNA from urine specimens of bats

A method for duplex nested RT-PCR (nRT-PCR) with internal control (IC) for the detection of Nipah virus RNA is described. Incorporation of IC RNA distinguished false and true negative results. The extrinsic RNA was added directly to the PCR master mix and co-amplified with virus specific RNA in a duplex reaction to determine the presence of PCR inhibitor. Limit of detection was affected minimally when IC was added. Of 53 pooled urine samples collected from fruit bats (Pteropus lylei), 16 were validated by the presence of IC band on gel electrophoresis. Seven of these were also Nipah virus RNA positive. The remaining 37 samples were considered invalid. Twenty-two urine samples became valid after dilution of 1:5 and re-examined; two were Nipah virus RNA positive. These nine positive results were confirmed by sequencing of heminested PCR products. The result indicated that at least two different Nipah strains circulated in this bat species from Thailand. This method should be useful for surveillance for Nipah virus infection in animals in a country where a biosecurity level (BSL) 4 laboratory is not available. PCR inhibitors were present in a significant number of bat urine samples. The technique described in this study should improve reliability of surveillance statistics.