• Journal of Microbiology
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• v.45 no.1
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• pp.21-28
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• 2007

In order to search for specific genotypes related to this unique phenotype, we used whole genomic DNA microarray to characterize the genomic diversity of Helicobacter pylori (H. pylori) strains isolated from clinical patients in China. The open reading frame (ORF) fragments on our microarray were generated by PCR using gene-specific primers. Genomic DNA of H. pylori 26695 and J99 were used as templates. Thirty-four H. pylori isolates were obtained from patients in Shanghai. Results were judged based on In(x) transformed and normalized Cy3/Cy5 ratios. Our microarray included 1882 DNA fragments corresponding to 1636 ORFs of both sequenced H. pylori strains. Cluster analysis, revealed two diverse regions in the H. pylori genome that were not present in other isolates. Among the 1636 genes, 1091 (66.7%) were common to all H. pylori strains, representing the functional core of the genome. Most of the genes found in the H. pylori functional core were responsible for metabolism, cellular processes, transcription and biosynthesis of amino acids, functions that are essential to H. pylori's growth and colonization in its host. In contrast, 522 (31.9%) genes were strain-specific genes that were missing from at least one strain of H. pylori. Strain-specific genes primarily included restriction modification system components, transposase genes, hypothetical proteins and outer membrane proteins. These strain-specific genes may aid the bacteria under specific circumstances during their long-term infection in genetically diverse hosts. Our results suggest 34 H. pylori clinical strains have extensive genomic diversity. Core genes and strain-specific genes both play essential roles in H. pylori propagation and pathogenesis. Our microarray experiment may help select relatively significant genes for further research on the pathogenicity of H. pylori and development of a vaccine for H. pylori.

• Biomedical Science Letters
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• v.4 no.1
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• pp.43-56
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• 1998

A multiplex PCR method was designed by employing primers specific for the eaeA gene, conserved sequences of Shiga-like toxins (SLT-I.II), and the 60-MDa plasmid of enterohemorrhagic E. coli (EHEC) O157:H7 strain. A set of six synthetic oligonucleotide primers derived from sequences of the SLT-I.II, eaeA, and 60-MDa plasmid genes of E. coli O157:H7 were used in a multiplex PCR amplification procedure to detect these genes in the same enteric pathogens. In two enterohemorrhagic E. coli O157:H7 (ATCC 35150, ATCC 43894) reference strains, PCR products of 317bps (eaeA), 228bps (SLT-I.II), and 167bps (60-MDa plasmid) were successfully amplified simultaneously in a single reaction. However, the specific PCR products were not amplified in control strains of other enteric bacteria. The sensitivity of the multiplex PCR assay for detection of the SLT-I.II, eaeA, and 60-MDa plasmid genes of E. coli O157:H7 was found to be 2.5$\times$10$^{6}$ of bacteria in diarrheal stool to amplify all three bands. The multiplex PCR technology will allow large-scale screening of many clinical specimens or contaminated foods, and will be a very useful method for the detection of a wide range of microorganisms present in the environment, including EHEC O157:H7 in various types of specimens. The multiplex PCR assay has the potential to be used as a specific and rapid method for clinical diagnosis of disease caused by EHEC O157:H7.

• The Plant Pathology Journal
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• v.15 no.3
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• pp.137-143
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• 1999

A novel chromosomal gene tagging technique using a specific fragment of the fatty acid desaturase-like open reading frame (des-like ORF) from the tox-argK gene cluster of Pseudomonas syringae pv. phaseolicola was developed to identify Xanthomonas spp.released into the environment as biocontrol agents. X. campestris pv. convolvuli FB-635, a pathogen of Convolvulus arvensis L., (bindweed), was chosen as the organism in which to develop and test the system. A 0.52 kb DES fragment amplified from P. syringae pv. phaseolicola C-199 was inserted into pGX15, a cosmid clone containing a 10.3 kb Eco RI-HindIII fragment derived from the xanthomonadin biosynthetic gene cluster contained in plasmid pIG102, to create a pigG::DES insertion. The 10.8 kb EcoRI-BamHI fragment carrying the pigG:: DES insertion was cloned into pLAFR3 to generate pLXP22. pLXP22 was then conjugated into X. campestris pv. convolvuli FB-635 and the pigG::DES insertion integrated into the bacterial chromosome by marker exchange. Rifampicin resistant, tetracycline sensitive, starch hydrolyzing, white colonies were used to differentiate the marked strain from yellow pigmented wild-type ones. PCR primers specific for the unique DES fragment were used for direct detection of the marked strain. Result showed the marked strain could be detected at very low levels even in the presence of high levels of other closely related or competitive bacteria. This PCR-based DES-tagging system provides a rapid and specific tool for directly monitoring the dispersal and persistence of Xanthomonas spp.released into the environment.

• Biomedical Science Letters
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• v.13 no.1
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• pp.33-38
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• 2007

The aim of this work was to validate a rapid and an accurate method for detecting Mycobacterium leprae in clinical specimens using nested PCR targeting M. leprae-specific repetitive element (RLEP) sequence. The primers were derived from the RLEP sequence which yield a 272 bp outer product and a 230 bp inner product. The specificity and the sensitivity of the nested PCR were compared with those of single PCR for detecting M. leprae using DNAs isolated from reference strain and various species of Mycobacterium. The results showed that the sensitivity of the nested PCR was about 100 to 1,000 times higher than that of the single PCR and also showed that both the single and the nested PCR were highly specific to M. leprae. Subsequently, the usefulness of the single and nested PCR was evaluated with clinical samples isolated from leprosy patients. The number of positive detections by the single and the nested PCR with a total of 20 specimens from leprosy patients were 9 (45%) and 20 (100%), respectively. The results clearly showed that nested PCR has highest sensitivity in detecting M. leprae from clinical specimens. Therefore, nested primers targeting RLEP sequence developed in this study seems to be useful to detect the presence of M. leprae.

• Research in Plant Disease
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• v.10 no.4
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• pp.310-312
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• 2004

Pseudomonas syringae pv. actinidiae is the causative agent of bacterial canker in kiwifruit. A nested PCR detection method that uses primers designed from the cfl gene, involved in production of the phytotoxin coronatine, was applied on soil samples. These primers yielded 665 and 310-bp fragments in consecutive PCR amplification step with DNA from soil inoculated with Korean strain of P. syringae pv. actinidiae. This system was applied to survey soil samples from a kiwifruit orchard destroyed by bacterial canker. A specific 310-bp PCR product was obtained from all six samples of soil tested.

• Research in Plant Disease
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• v.18 no.2
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• pp.129-132
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• 2012

Citrus bacterial canker is an economically important disease affecting citrus production in many citrusgrowing areas and several pathotypes have been recognized within the Xanthomonas pathogens causing canker. In view of the containment of the disease, accurate identification of the causal bacterium is important. In this study, triplex PCR method was developed by using the previously reported primers. Two groups of primer combination, such as, one group including primers 2/3, J-pth1/J-pth2 and XACF/XACR, and another group 2/3, J-pth1/J-pth2 and Xac01/Xac02, were suitable for the detection and differentiation of X. a. pv. citri $A^w$, X. a. pv. aurantifolii B and C, and X. a. pv. citrumelo E strains. Moreover, the primer combination of Xac01 and J-pth2 promised us to use as a specific primer set to detect X. a. pv. citrumelo E strain. The PCR methods developed in this study could be used for the rapid differentiation of Xanthomonas pathotypes of citrus.

• Parasites, Hosts and Diseases
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• v.39 no.1
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• pp.43-48
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• 2001

We have studied the genetic differences among four isolates of Trichinella including a new strain of Trichinella spiralis (ISS 623) recently found from a human case who took a badger in Korea. Because they have a different host origin and came from geographically separated regions, we supposed the genetic pattern of the isolates might be different as had been previously reported. It was analysed by PCR-RFLP analysis of the rDNA repeat that can readily distinguish a species or strain from others. Isolated genomic DNA of each isolate of Trichinella larvae was amplified with ITSl specific primers and digested with restriction endonucleases. The PCR product of ITSl was confirmed using Southern blot analysis to be a 910 Up fragment. The restriction fragments of each isolate had variable patterns when it was digested with Rsa I only. According to the RFLP patterns, the estimated genetic divergence between each isolate was different. In conclusion, four isolates of Thichinella including a new strain of T. spiralis obtained from a Korean patient may have genetic differences in the ITSl region and the Shanghai isolate was genetically more similar to the Japanese unknown isolate than others in the ITSl region.

• Journal of Microbiology and Biotechnology
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• v.23 no.3
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• pp.357-363
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• 2013

The identification of bacteriophage proteins on the surface of Lactobacillus rhamnosus Pen was performed by LC-MS/MS analysis. Among the identified proteins, we found a phage-derived major tail protein, two major head proteins, a portal protein, and a host specificity protein. Electron microscopy of a cell surface extract revealed the presence of phage particles in the analyzed samples. The partial sequence of genes encoding the major tail protein for all tested L. rhamnosus strains was determined with specific primers designed in this study. Next, RT-PCR analysis allowed detection of the expression of the major tail protein gene in L. rhamnosus strain Pen at all stages of bacterial growth. The transcription of genes encoding the major tail protein was also proved for other L. rhamnosus strains used in this study. The present work demonstrates the spontanous release of prophage-encoded particles by a commercial probiotic L. rhamnosus strain, which did not significantly affect the bacterial growth of the analyzed strain.

• The Korean Journal of Mycology
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• v.38 no.1
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• pp.34-39
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• 2010

We have attempted to verify 30 strains of Hypsizigus marmoreus from various mushroom stocks in Korea using random amplified polymorphic DNA (RAPD) methodology. Chromosomal DNAs of them were extracted and subjected to PCR analyses with 3 random primers. Each PCR produced approximately 30 distinct PCR bands with the size from 200 bp to 3000 bp. A dendrogram was acquired using the unweighted pair-group method with arithmetic average (UPGMA) clustering methodology on the basis of the DNA band pattern. The analysis revealed that 30 strains of H. marmoreus were clustered into two distinct clusters. Cluster 1 contained 3 subgroups while the cluster 2 consisted of rather diverse strains. Interestingly, Hm3-10, a wild strain collected from Deog-Yu mountain, was not included in either clusters, indicative of uniqueness of this strain. We nextly attempted to develop strain-specific DNA markers to verify a specific strain. A unique band in the RAPD gel lane of Hm0-4 was extracted and its sequence was determined. PCR with a primer set from the determined sequence revealed that the primer set gave a 250 bp DNA band only for Hm0-4, indicating that this approach works well for the strain-specific identification of H. marmoreus.

• Korean Journal of Veterinary Research
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• v.38 no.2
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• pp.304-313
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• 1998

Sixteen Korean field transmissible gastroenteritis viruses (TGEVs) were isolated using swine testicular cell (STC) and the genomic diversity of them was analyzed. All TGEV isolates produced a typical cytopathic effect in STC and were confirmed as TGEV by immunofluorescence assay using monoclonal antibody against TGEV and PCR using TGEV specific primers. RNAs from TGEV field isolates and vaccine TGEV were extracted and amplified by RT and PCR. The RT-PCR products were digested with selected restriction enzymes and analyzed RFLP patterns. The N-terminal end region of S gene and ORF 3 and 3-1 genes of TGEV amplified by TGEV specific primer pairs seemed to be conserved. Most specific variations were detected in S gene amplified by TGEV 4/6 primer pairs which includes antigenic sites A and D. When the PCR products were treated with Sau3AI and Ssp I, Bvac(vaccine strain), field isolates 133 and 347 were differentiated from Miller and Purdue types. In the case of D5 field isolates, it was classified into Purdue type by Sau 3AI but classified into independent TGEV by Ssp I. Two different TGEV strains from D2 sample were confirmed by plaque purification and RT-PCR-RFLP analysis. To investigate the change occurring in TGEV genome after serial passage, the TGEV P44 strain was passaged through STC. There were specific changes in S gene and a large deletion was observed in ORF 3 and 3-1 genes. These studies showed that a distinct difference in genome exists among TGEV field isolates.