A reverse-transcription loop-mediated isothermal amplification （RT-LAMP） method was established for the detection of wheat streak mosaic virus （WSMV）. Ac-cording to the conservative regions of the genes that encod...A reverse-transcription loop-mediated isothermal amplification （RT-LAMP） method was established for the detection of wheat streak mosaic virus （WSMV）. Ac-cording to the conservative regions of the genes that encode the coat protein of WSMV, 2 pairs of primers were designed. Final y, the 1st pair of primers was select-ed through the specificity test. The sensitivity test showed the sensitivity of RT-LAMP method was 10 times higher than that of RT-PCR. In addition, the amplifica-tion of target gene could be judged visual y from the presence of fluorescence （cal-cein） in the final reaction system. The RT-LAMP method, established in this study, was rapid, easy, specific and sensitive. Moreover, it did not require sophisticated equip-ment. The RT-LAMP was suitable for the rapid detection of WSMV.展开更多
Wheat streak mosaic virus（WSMV）has become a re-emerging pathogen in cereal crops in the Czech Republic.WSMV was first reported in the former Czechoslovakia in the early 1980s,and then no record of the virus was docu...Wheat streak mosaic virus（WSMV）has become a re-emerging pathogen in cereal crops in the Czech Republic.WSMV was first reported in the former Czechoslovakia in the early 1980s,and then no record of the virus was documented until 2009.The incidence of the virus was recorded in recent years in several winter wheat fields and many grass species.Here,we surveyed the incidence of WSMV in cereal crops.The results demonstrated the existence of the virus in winter wheat and volunteer wheat during each year of the monitoring period,which spanned from 2013–2016.Although the range of infected samples was low（6.4%of the total tested samples）,a high incidence of well-distributed virus was recorded.In at least six fields,the virus reached severe and potentially epidemic levels.In accordance with our previous report detailing WSMV infection of native grasses,we tested several grass species commonly grown in the Czech Republic.We found that some grass species acted as experimental hosts and possible reservoirs of the virus;these included Anthoxanthum odoratum（sweet vernal grass）,Arrhenatherum elatius（false oat-grass）,Lolium multiflorum（Italian rye-grass）,Bromus japonicus（Japanese chess）,Echinochloa crus-galli（barnyard grass）,Holcus lanatus（meadow soft grass）and Holcus mollis（creeping soft grass）.Some of these grass species are also important weeds of cereals,which may be the potential source of WSMV infection in cereal crops.Several widely used winter wheat cultivars were tested in the field after artificial inoculation with WSMV to evaluate virus titre by RT-qPCR.Overall,the tested cultivars had a low virus titre,which is associated with mild disease symptoms and may provide a good level of crop resistance to WSMV.展开更多
文摘A reverse-transcription loop-mediated isothermal amplification （RT-LAMP） method was established for the detection of wheat streak mosaic virus （WSMV）. Ac-cording to the conservative regions of the genes that encode the coat protein of WSMV, 2 pairs of primers were designed. Final y, the 1st pair of primers was select-ed through the specificity test. The sensitivity test showed the sensitivity of RT-LAMP method was 10 times higher than that of RT-PCR. In addition, the amplifica-tion of target gene could be judged visual y from the presence of fluorescence （cal-cein） in the final reaction system. The RT-LAMP method, established in this study, was rapid, easy, specific and sensitive. Moreover, it did not require sophisticated equip-ment. The RT-LAMP was suitable for the rapid detection of WSMV.
文摘为给小麦线条花叶病毒(Wheat streak mosaic virus,WSMV)的灵敏、稳定、快速检测提供依据,根据该病毒不同分离株外壳蛋白(Coat protein,CP)基因的保守序列,设计了特异性引物与Taq Man MGB荧光探针,建立了WSMV的实时荧光RT-PCR检测方法。结果表明,本研究设计的Taq Man MGB荧光探针对WSMV的检测具有很好的特异性,解决了因病毒不同分离物之间基因组变异较大、难以找到长片段保守序列来设计探针的困难。该方法无需任何PCR后处理,基因污染风险小。它与双抗体夹心酶联免疫检测方法相比,灵敏度提高1 000倍,具有快速、灵敏和高特异性的优点,适合于WSMV的快速检测。
基金supported by the grants from the Ministry of Agriculture, Czech Republic from projects QJ1230159 (50%) and RO0415 (50%)
文摘Wheat streak mosaic virus（WSMV）has become a re-emerging pathogen in cereal crops in the Czech Republic.WSMV was first reported in the former Czechoslovakia in the early 1980s,and then no record of the virus was documented until 2009.The incidence of the virus was recorded in recent years in several winter wheat fields and many grass species.Here,we surveyed the incidence of WSMV in cereal crops.The results demonstrated the existence of the virus in winter wheat and volunteer wheat during each year of the monitoring period,which spanned from 2013–2016.Although the range of infected samples was low（6.4%of the total tested samples）,a high incidence of well-distributed virus was recorded.In at least six fields,the virus reached severe and potentially epidemic levels.In accordance with our previous report detailing WSMV infection of native grasses,we tested several grass species commonly grown in the Czech Republic.We found that some grass species acted as experimental hosts and possible reservoirs of the virus;these included Anthoxanthum odoratum（sweet vernal grass）,Arrhenatherum elatius（false oat-grass）,Lolium multiflorum（Italian rye-grass）,Bromus japonicus（Japanese chess）,Echinochloa crus-galli（barnyard grass）,Holcus lanatus（meadow soft grass）and Holcus mollis（creeping soft grass）.Some of these grass species are also important weeds of cereals,which may be the potential source of WSMV infection in cereal crops.Several widely used winter wheat cultivars were tested in the field after artificial inoculation with WSMV to evaluate virus titre by RT-qPCR.Overall,the tested cultivars had a low virus titre,which is associated with mild disease symptoms and may provide a good level of crop resistance to WSMV.
