Development of Polyclonal Antibodies and a Serological-Based Reverse-Transcription Loop-Mediated Isothermal Amplification (S-RT-LAMP) Assay for Rice Black-Streaked Dwarf Virus Detection in Both Rice and Small Brown Planthopper

开发用于检测水稻黑条矮缩病毒的多克隆抗体和基于血清学的逆转录环介导等温扩增(S-RT-LAMP)检测方法,该方法适用于水稻和小褐飞虱。

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Abstract

Rice black-streaked dwarf virus (RBSDV) infects rice and maize, and seriously affects rice yields in main rice-producing areas. It can be transmitted via small brown planthopper (SBPH: Laodelphax striatellus Fallén). To more rapidly, sensitively, and highly throughput diagnose RBSDV in the wild condition, we first purified the recombinant His-CP(RBSDV) protein, and prepared the polyclonal antibodies against the His-CP(RBSDV) protein (PAb-CP(RBSDV)). Based on the PAb-CP(RBSDV), we developed a series of serological detections, such as Western blot, an enzyme-linked immunosorbent assay (ELISA), and a dot immunoblotting assay (DIBA). Furthermore, we developed a serological-based reverse-transcription loop-mediated isothermal amplification assay (S-RT-LAMP) that could accurately detect RBSDV in the wild. Briefly, the viral genomic dsRNA together with viral CP were precipitated by co-immunoprecipitation using the PAb-CP(RBSDV), then the binding RNAs were crudely isolated and used for RT-LAMP diagnosis. Using the prepared PAb-CP(RBSDV), four serology-based detection methods were established to specifically detect RBSDV-infected rice plants or SBPHs in the wild. The method of S-RT-LAMP has also been developed to specifically, high-throughput, and likely detect RBSDV in rice seedlings and SBPHs simultaneously. The antiserum prepared here laid the foundation for the rapid and efficient detection of RBSDV-infected field samples, which will benefit for determination of the virulence rate of the transmission vector SBPH and outbreak and epidemic prediction of RBSDV in a rice production area.

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