Development of a one-step immunocapture real-time RT-PCR assay for the detection of barley stripe mosaic virus strains in barley seedlings

Abstract

Summary. -A one-step immunocapture real-time RT-PCR (IC-real-time RT-PCR) was developed for efficient detection of barley stripe mosaic virus (BSMV) in barley seedlings. The novel detection system was designed using a primer set targeting the conserved region in the triple gene block 2 (TGB2) to expand its capacity to detect all BSMV strains. This assay was evaluated for its efficiency in detecting BSMV in barley seedlings. Using the immunocapture sample preparation, real-time RT-PCR was able to detect BSMV in samples, which were indicated as negative by ELISA. The sensitivity of detection in the real-time RT-PCR was as low as 50 fg/µl of total viral RNA under optimal reaction conditions. This level of sensitivity indicated that the one-step IC-realtime RT-PCR developed in the present study could be used for routine plant and seed health assays.

A Comparison of Ultrastructural Changes of Barley Cells Infected with Mild and Aggressive Isolates of Barley stripe mosaic virus

“…The presence of the virus in inoculated plants was confirmed by the DAS-ELISA test (Clark & Adams 1977), using a polyclonal antiserum produced at the Institute of Plant Protection in Pozna in cooperation with Dr. T. Malinowski from the Institute of Horticulture in Skierniewice. Additionally, BSMV-De-M and BSMV-M infection was confirmed by RT-PCR or IC real-time RT-PCR assays using primers amplifying the TGB2 region (Zarzy ska et al 2014). …”

“…These phenomena might have great impact on diagnostic of the virus. It has been previously shown that due to the very low concentration of mild isolates in infected plants the application of a more sophisticated method than ELISA assay is necessary (Zarzy ska et al 2014).…”

Distribution of Wheat-Infecting Viruses and Genetic Variability of Wheat Streak Mosaic Virus and Barley Stripe Mosaic Virus in Kazakhstan

“…The initial screening was performed using previously established detection primers ( Table 1 ): two sets of primers for WSMV [ 43 , 62 ], and one of each for BSMV [ 59 ], TriMV [ 43 ], and HPWMoV [ 43 ]. However, the majority of samples showing strong symptoms of viral infection were negative for all viruses, or produced amplicons outside of target sizes ( Figure S1 ).…”

“…PCR screening for WSMV, BSMV, TriMV, and HPWMoV was performed according to the protocols described in the respective works [ 43 , 59 , 62 ]. For the newly developed primers, PCR was conducted in 25 μL of reaction mix containing 10 mM of each primer and 2 μL of cDNA as a template for amplification.…”

“…Multiplex RT-PCR has been successfully applied for the screening and quantification of WSMV and TriMV [ 56 , 57 ], and RT-qPCR has been shown to be twice and thrice more sensitive than ELISA in the detection of TriMV and HPWMoV, respectively [ 58 ]. For BSMV, Zarzyńska et al demonstrated the limitations of ELISA as a diagnostic tool: it could not detect mild infections with moderate virus titers, while RT-PCR [ 59 ] and LAMP [ 60 ] were more sensitive. RT-qPCR is a valuable tool in studying virus gene expression and virus–host interactions [ 6 ].…”