The aim of the present study was to develop a noble and specific marker for a quantitative polymerase chain reaction (PCR) assay for the species-specific detection of Pseudomonas aeruginosa based on the O-antigen acetylase gene. It is an important challenge to characterize populations of the bacterium P. aeruginosa, an opportunist by virtue of its physiological and genetic adaptability. However, molecular and serological methods currently available for sensitive and specific detection of P. aeruginosa are by no means satisfactory because there have been critical defects in the diagnosis and identification of P. aeruginosa strains in that these assays also detect other Pseudomonas species, or do not obtain amplified products from P. aeruginosa strains. Therefore, a primer set was designed based on the O-antigen acetylase gene of P. aeruginosa PA01 because it has been known that this gene is structurally diverse among species. The specificity of the primer set was evaluated using genomic DNA from six isolates of P. aeruginosa, 18 different species of Pseudomonas, and 23 other reference pathogenic bacteria. The primer set used in the PCR assay amplified a 232-bp amplicon for only six P. aeruginosa strains. The assay was also able to detect at least 1.41 × 10(3) copies/μl of cloned amplified target DNA using purified DNA, or 2.7 × 10(2) colony-forming unit per reaction when using calibrated cell suspension. In conclusion, this assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of water with a low level or latent infection of P. aeruginosa.
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.
The aim of this study was to develop a quantitative polymerase chain reaction (qPCR) assay for specific detection of Pectobacterium wasabiae using a primer pair based on the YD repeat protein gene for amplification of a 140-bp DNA fragment from infected wasabi (Wasabia japonica), a member of the crucifer family. The soft rot caused by P. wasabiae is an emerging disease that is present in many wasabi-producing areas. However, specific and reliable methods for identifying the pathogen are not available. Therefore, a qPCR primer set for accurate diagnosis of P. wasabiae was developed from publically available genome sequences. A SYBR Green qPCR primer set was designed based on a YD repeat protein gene of P. wasabiae WPP163 because it is known that this gene is structurally diverse among species, pathovars, or subspecies. The specificity of the primer set was evaluated using genomic DNA from 5 isolates of P. wasabiae, 5 different species of Pectobacterium, and 16 other pathogenic reference bacteria. The primer set used in the PCR assay successfully amplified a 140-bp amplicon for all five P. wasabiae strains. No amplification was obtained from 29 other pathogenic bacteria. The assay was also able to detect at least two genomic DNA, or 3 CFU per reaction, when using calibrated cell suspension.
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