PCR-based sensitive and specific detection of Pectobacterium atrosepticum using primers based on Rhs family gene sequences

The present study describes bio-polymerase chain reaction (PCR) assays to detect bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae in rice. Successful control of X. oryzae. pv. oryzae requires a specific and reliable diagnostic tool. However, other X. oryzae pathovars are detected by currently available molecular and serological methods. In this study, SYBR Green real-time and conventional PCR primer sets were designed based on an rhs family gene of X. oryzae pv. oryzae KACC10331 because these genes are structurally diverse. The specificity of the primers was evaluated using purified DNA from 11 isolates of two X. oryzae pathovars, 21 other Xanthomonas species, and 4 other reference phytopathogenic bacteria and fungi. The assay was also able to detect at least two genome equivalents of cloned amplified target DNA using purified DNA. Thus, the SYBR Green real-time PCR-based method can be used for the rapid and specific detection of X. oryzae pv. oryzae and will potentially simplify and facilitate diagnosis and monitoring of this pathogen and guide plant disease management.

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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

Plant Disease ◽

10.1094/pdis-03-11-0235 ◽

2012 ◽

Vol 96 (4) ◽

pp. 577-580 ◽

Cited By ~ 11

Author(s):

Byoung Kyu Kim ◽

Min Seok Cho ◽

Myeong Ho Kim ◽

Hyeon Jin Choi ◽

Man Jung Kang ◽

...

Keyword(s):

Real Time ◽

Pathogenic Bacteria ◽

Quantitative Polymerase Chain Reaction ◽

Pcr Primers ◽

Effective Control ◽

Sequence Information ◽

Rice Seed ◽

Specific Detection ◽

Burkholderia Glumae ◽

Family Gene

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.

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