Rapid and Specific Detection of Burkholderia glumae in Rice Seed by Real-Time Bio-PCR Using Species-Specific Primers Based on an rhs Family Gene.

Abstract

In this study, we developed a reliable, quick, and accurate quantitative polymerase chain reaction (qPCR) assay to detect grain rot caused by Burkholderia glumae in rice seed. The control of bacterial grain rot is difficult, and the only practical methods for disease management rely on the use of pathogen-free seed, appropriate culture practices, and resistant cultivars. Therefore, the specific detection of this pathogen in seed is essential for effective control of the disease. However, other Burkholderia spp. are also detected by currently available molecular and serological methods. In this study, we exploited the available genome sequence information in public databases to develop specific PCR primers for accurate diagnosis of B. glumae. An SYBR Green real-time PCR primer set was designed based on the rhs family gene (YD repeat protein) of B. glumae BGR1 because these genes are structurally diverse. The specificity of the primers was evaluated using purified DNA from 5 isolates of B. glumae, 6 different species of Burkholderia, and 18 other reference pathogenic bacteria. The assay was able to detect at least one genome equivalent of cloned amplified target DNA using purified DNA or 1 CFU per reaction when using calibrated cell suspension. This method is rapid and reliable and has great potential for analyzing large numbers of samples.