• Korean Journal of Veterinary Research
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• v.38 no.1
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• pp.173-181
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• 1998

Esherichia coli O157 : H7의 slt I, slt II, uid A 및 eaeA 4종 유전자를 동시에 검출하기 위한 multiplex PCR 기법을 확립하고 쇠고기중 직접 E coli O157 : H7 검출시험을 실시하였다. 4 set의 primers를 이용한 multiplex PCR 기법으로 31종의 장내세균에 대한 특이성을 조사한 결과 E coli O157 : H7 에서 1,087bp (eae A), 584bp (slt II), 348bp (slt I) 또는 252bp (uid A)크기의 DNA를 동시에 특이적으로 검출할 수 있었다. E coli O157 : H7 15주는 모두 uid A 및 eae A 유전자가 동시에 검출되었고, 다른 장내세균에서는 검출되지 않았다. slt I 또는 slt II 유전자를 가지고 있는 E coli 표준균주 24종을 이용하여 multiplex PCR 기법과 Vero cell cytotoxicity assay을 비교검사한 결과 베로톡신 산생능과 PCR법의 결과는 일치하였다. mutiplex PCR 기법의 쇠고기중 검출한계는 modified EC(mEC)에서 증균없이는 E coli O157 : H7균 $10^4cells/g$ 이상에서 검출이 가능하였으나 mEC에 1차 증균후 modified TSB 증균하였을 경우에는 10cells/g이하까지도 검출이 가능하였다. 개발된 multiplex PCR 기법을 쇠고기 40종에 직접 적용한 결과 E coli O157 : H7은 검출되지 않았으나 slt I 및 slt II유전자를 가지고 있는 E coli 4종이 검출되었으며, 이들의 혈청형은 O6, O112, O115 및 O139 였다. 이 연구에서 개발된 multiplex PCR은 쇠고기중 E coli O157 : H7을 신속하고 특이적으로 검출하는데 사용할 수 있을 것으로 사료된다.

• 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.

• Journal of Microbiology
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• v.37 no.3
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• pp.168-174
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• 1999

Escherichia coli O157 is an important pathogenic organism which causes diarrhea, haemorrhagic colitis, and haemolytic ureamic syndrome (HUS) in human. E. coli O157 KNIH317 was isolated form patients suffering with HUS in Korea. We designed a primer set for cloning shiga-like toxin (slt) gene. The amplified PCR product was used to Southern and colony hybridization as a probe. As a result, we cloned 4.5-kb KpnI fragment containing the slt gene encoding shiga-like toxin from chromosomal DNA of E. coli O157 KNIH317. This recombinant plasmid was named pOVT45. E. coli XL1-Blue harboring pOVT45 showed cytotoxicity in Vero cells. We sequenced the slt gene of this strain. The A-subunit gene of the slt was composed of 960 base pairs with ATG initiation codon and TAA terminationcodon. The B-subunit was composed of 270 base paris with ATG initiation codon and TGA termination codon. Nucleotide sequence comparison of the slt gene exhibited 100%, 98.4%, 93.7%, and 93.7% identity with that of shiga-like toxin type II (sltII) of E. coli bacteriophage 933W, variant slt of E. coli, slt of E. coli, and variant sltII of E. coli, respectively. From these results, it was concluded that the cloned slt gene belongs to SltII family and that the strain used in this study may be a lysogeny of E. coli bcteriphage 933W.

• Korean Journal of Veterinary Research
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• v.39 no.2
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• pp.338-342
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• 1999

Twenty three strains of Escherichia (E) coli O157 : H7 isolated from Korea, Japan, USA were analyzed by pulsed-field gel electrophoresis (PFGE) of XbaI-digested chromosomal DNA and multiplex polymerase chain reaction. Various PFGE patterns of E. coli O157 : H7 were found on the same farm. Most of the E, coli O157 : H7 strains had shiga-like toxin (slt) II gene only (43.5%) or both slt I and slt II genes(30.4%). eaeA gene was highly conserved in the E. coli O157 : H7. There was no correlation between PFGE and slt gene patterns. The results indicate that various genotypes of E. coli O157 : H7 have spread throughout the country and genomic DNA patterns generated by PFGE are highly specific for different strains and have significant value in epidemiologic investigations of infectious disease outbreaks.

• Weed & Turfgrass Science
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• v.1 no.4
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• pp.57-63
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• 2012

This study was carried our to examine the genetic relationship of 13 commercial turfgrass cultivars using Random Amplified Polymorphic DNA to provide genetic informations more efficient golf course management. Analysis of 56 random hexamer primers generated 13 to 54 polymorphic bands among the 13 cultivars with an average of 30.7 bands per primer. The results of cluster analysis based on RAPDs revealed that three major variety groups: Group I - 'Shadow II', 'Aurora Gold', 'Little Bighorn Blue', 'PennA-1', and 'PennA-4'; Group II - 'Midnight II', 'Prosperity', 'Moon light SLT', 'Bright star SLT', and 'Silver dollar'; and Group III - 'Olympic Gold', 'Silver Star', and 'Tar Heel II'. The genetic similarity coefficients among 13 turfgrass cultivars ranged from 0.039 to 1.0 with highest coefficient in Group III. Studies on morphological characters and the effective molecular markers such as sequence characterized amplified regions are further needed to identify relationships and genetic diversities within species and among species.