Dong Suk Park

Improved PCR for identification of Pseudomonas aeruginosa

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 × 103 copies/μl of cloned amplified target DNA using purified DNA, or 2.7 × 102 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.

Sensitive and Specific Detection of Xanthomonas oryzae pv. oryzae by Real-Time Bio-PCR Using Pathovar-Specific Primers Based on an rhs Family Gene

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.

Quantitative Real-Time Polymerase Chain Reaction Assay for Detection of Pectobacterium wasabiae Using YD Repeat Protein Gene-Based Primers

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.

The influence of red pepper powder on the density of Weissella koreensis during kimchi fermentation.

Weissella koreensis is a psychrophilic bacterium that is the dominant species found in kimchi and exhibits anti-obesity effects via its production of ornithine. In this study, we mined the genome of W. koreensis KACC15510 to identify species-specific genes that can serve as new targets for the detection and quantification of W. koreensis in kimchi. A specific polymerase chain reaction (PCR) primer set for the membrane protein-encoding gene of W. koreensis KACC15510 was designed and investigated to quantify its sensitivity and specificity for detecting the bacterium in kimchi. The specificity of the primer set was evaluated using genomic DNA from eight isolates of W. koreensis, 11 different species of Weissella and 13 other reference lactic acid bacterium (LAB) strains. In addition, red pepper powder was observed to strongly influence the density of W. koreensis during kimchi fermentation.

Sensitive and specific detection of phaseolotoxigenic and nontoxigenic strains of Pseudomonas syringae pv. phaseolicola by TaqMan real-time PCR using site-specific recombinase gene sequences

Pseudomonas syringae pv. phaseolicola, the causative agent of halo blight, is the most important bacterial pathogen of bean. Both nontoxigenic (Tox(-)) and toxigenic (Tox+) strains of this pathogen cause halo blight in beans. However, nontoxigenic strains cannot be detected by currently available molecular and serological tools. In this study, a TaqMan probe and primer set were designed based on the phage integrase family site-specific recombinase of P. s. pv. phaseolicola 1448A because it is known that most site-specific recombinases are structurally and functionally diverse. The specificity of the probe and primers was evaluated using purified DNA from 29 isolates of 3 different pathovars of P. syringae. The probe and primer set were able to detect Tox(-) and Tox+ isolates of P. s. pv. Phaseolicola, but no other phytopathogenic bacteria. The assay was also able to detect at least 5 genome equivalents of cloned amplified target DNA, using purified DNA, or 7 colony forming unit (CFU) per reaction when using calibrated cell suspensions. Thus, the TaqMan real-time PCR-based method can be used for the rapid detection of both types of P. s. pv. Phaseolicola, and will potentially simplify and facilitate the diagnosis and monitoring of this pathogen, and guide plant disease management.

Red pepper powder is a crucial factor that influences the ontogeny of Weissella cibaria during kimchi fermentation.

Weissella cibaria has been found in Korean kimchi and other sources, including fermented foods, Greek salami, Spanish sausages, and animal and human excrement. W. cibaria was recently reported to show anticancer, immunomodulatory, anti-inflammatory and antioxidant properties. Nevertheless, fundamental ecological succession studies are required to scientifically confirm the probiotic action of W. cibaria under various conditions, such as fermentation. Therefore, in the present study, we mined the W. cibaria KACC11862 genome in search of species-specific genes to use as new PCR targets for the detection and quantification of W. cibaria in kimchi. The sensitivity and specificity of the identified primer set from the putative outer membrane protein gene for the detection of W. cibaria KACC11862 in kimchi were analysed. Primer set specificity was evaluated using genomic DNA from eight W. cibaria isolates, 10 different species of Weissella and 13 other reference lactic acid bacteria (LAB) strains. Interestingly, by using the qPCR assay developed herein, we found that red pepper powder markedly affects the ontogeny of W. cibaria during kimchi fermentation.

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

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.

Insertional Transposon Mutagenesis of Xanthomonas oryzae pv. oryzae KXO85 by Electroporation

The bacterial leaf blight, which is caused by Xanthomonas oryzae pv. oryzae, is the most damaging and intractable disease of rice. To identify the genes involved in the virulence mechanism of transposon Tn5 complex, which possesses a linearized transposon and transposase, was successfully introduced into X. oryzae pv. oryzae by electroporation. The transposon mutants were selected and confirm the presence of transposition in X. oryzae pv. oryzae by the PCR amplification of transposon fragments and the Southern hybridization using these mutants. Furthermore, transposon insertion sites in the mutant bacterial chromosome were determined by direct genomic DNA sequencing using transposon-specific primers with ABI 3100 Genetic Analyzer. Efficiency of transposition was influenced mostly by the competence status of X. oryzae pv. oryzae cells and the conditions of electroporation. These results indicated that the insertion mutagenesis strategy could be applied to define function of uncharacterized genes in X. oryzae pv. oryzae.

Rapid and Specific Detection of Acidovorax avenae subsp. citrulli Using SYBR Green-Based Real-Time PCR Amplification of the YD-Repeat Protein Gene.

The aim of this study was to develop a SYBR Green-based real-time PCR assay for the rapid, specific, and sensitive detection of Acidovorax avenae subsp. citrulli, which causes bacterial fruit blotch (BFB), a serious disease of cucurbit plants. The molecular and serological methods currently available for the detection of this pathogen are insufficiently sensitive and specific. Thus, a novel SYBR Green-based real-time PCR assay targeting the YD-repeat protein gene of A. avenae subsp. citrulli was developed. The specificity of the primer set was evaluated using DNA purified from 6 isolates of A. avenae subsp. citrulli, 7 other Acidovorax species, and 22 of non-targeted strains, including pathogens and non-pathogens. The AC158F/R primer set amplified a single band of the expected size from genomic DNA obtained from the A. avenae subsp. citrulli strains but not from the genomic DNA of other Acidovorax species, including that of other bacterial genera. Using this assay, it was possible to detect at least one genomeequivalents of the cloned amplified target DNA using 5 × 10(0) fg/μl of purified genomic DNA per reaction or using a calibrated cell suspension, with 6.5 colony-forming units per reaction being employed. In addition, this assay is a highly sensitive and reliable method for identifying and quantifying the target pathogen in infected samples that does not require DNA extraction. Therefore, we suggest that this approach is suitable for the rapid and efficient diagnosis of A. avenae subsp. citrulli contaminations of seed lots and plants.

A quantitative and direct PCR assay for the subspecies-specific detection of Clavibacter michiganensis subsp. michiganensis based on a ferredoxin reductase gene.

The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis is the causal agent of canker disease in tomato. Because it is very important to control newly introduced inoculum sources from commercial materials, the specific detection of this pathogen in seeds and seedlings is essential for effective disease control. In this study, a novel and efficient assay for the detection and quantitation of C. michiganensis subsp. michiganensis in symptomless tomato and red pepper seeds was developed. A pair of polymerase chain reaction (PCR) primers (Cmm141F/R) was designed to amplify a specific 141 bp fragment on the basis of a ferredoxin reductase gene of C. michiganensis subsp. michiganensis NCPPB 382. The specificity of the primer set was evaluated using purified DNA from 16 isolates of five C. michiganensis subspecies, one other Clavibacter species, and 17 other reference bacteria. The primer set amplified a single band of expected size from the genomic DNA obtained from the C. michiganensis subsp. michiganensis strains but not from the other C. michiganensis subspecies or from other Clavibacter species. The detection limit was a single cloned copy of the ferredoxin reductase gene of C. michiganensis subsp. michiganensis. In conclusion, this quantitative direct PCR assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of seeds and seedlings with a low level or latent infection of C. michiganensis subsp. michiganensis.