One-step real time RT-PCR assay for SRBSDV genomic RNA was determined by using 10-fold serial dilutions of the RNA standards ranging from 5.0 × 1010 to 5.0 × 104 copies/reaction (Fig. 1a,b) to ascertain the detection limits of the one-step real time RT-PCR method and the linearity of the assay. Ct-values learn more were measured and plotted against the known

copy numbers of the standard sample. The standard curve covered a linear range of seven orders of magnitude. The slope (−3.317) and the correlation coefficient (R2 = 0.996) of the standard curve showed that this assay could be used to quantify target RNA in infected rice tissue. Following amplification, a melting curve analysis was performed to verify the correct product by its specific melting temperature. Melting curve with IQ 5 optical system software Version 2.0 showed that SRBSDV S9 gene specific amplicon melts at 78°C (77.5–78.5°C). The dissociation plots (Fig. 2) showing

the SRBSDV specific melting temperature (Tm = 78°C) revealed the one-step real time RT-PCR was specific for SRBSDV. The results of specificity further verify that the primers were absolutely specific for SRBSDV. The viral RNA standards (Fig. 3, lane 1) and total RNA extracted from rice leaf infected with SRBSDV Gefitinib (Fig. 3, lanes 2–9) could be easily detected and quantified. In contrast, the rice leaf tissue carrying RBSDV (Fig. 3, lanes

10–11) was not detectable. In order to evaluate the sensitivity between one-step real time RT-PCR assay and RT-PCR in SRBSDV detection, a series of 10-fold dilutions of standard ssRNA ranging from 6.4 × 1010 to 64 copies were tested using click here the two detection techniques. Positive one-step real time RT-PCR amplifications were observed up to dilutions of 64 copies (Fig. 4a), while in the RT-PCR, product amplification was seen up to dilutions of 6.4 × 103 copies, as indicated by the presence of 141 bp amplicon after agarose gel electrophoresis (Fig. 4b). The negative control did not show a consistent or detectable product yield by either assay. Comparing the results, the one-step real time RT-PCR assay was 100 times more sensitive than the RT-PCR for SRBSDV detection. The disease caused by SRBSDV has recently became one of the most damaging rice crop disease in Southern China and Vietnam and led to significant economic loss (Zhang et al. 2008; Zhou et al. 2008, 2012). Rice plants infected with SRBSDV show no symptoms in the latent period of infection and is difficult to diagnose at an early stage, but is very destructive at a late stage. Therefore, these diseases need to be monitored and diagnosed at their early stages for effective mitigation of loss and risk assessment of infected rice paddy field (Hoang et al. 2011; Zhou et al. 2012; Zhang et al. 2013).