• Journal of Microbiology and Biotechnology
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• 제25권10호
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• pp.1621-1628
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• 2015

Chlamydia possesses a conserved 7.5 kb plasmid that is known to play an important role in chlamydial pathogenesis, since some chlamydial organisms lacking the plasmid are attenuated. The chlamydial transformation system developed recently required the use of plasmid-free organisms. Thus, the generation and identification of plasmid-free organisms represent a key step in understanding chlamydial pathogenic mechanisms. A tricolor immunofluorescence assay for simultaneously detecting the plasmid-encoded Pgp3 and whole organisms plus DNA staining was used to screen C. muridarum organisms selected with novobiocin. PCR was used to detect the plasmid genes. Next-generation sequencing was then used to sequence the genomes of plasmid-free C. muridarum candidates and the parental C. muridarum Nigg strain. We generated five independent clones of plasmid-free C. muridarum organisms by using a combination of novobiocin treatment and screening plaque-purified clones with anti-Pgp3 antibody. The clones were confirmed to lack plasmid genes by PCR analysis. No GlgA protein or glycogen accumulation was detected in cells infected with the plasmid-free clones. More importantly, whole-genome sequencing characterization of the plasmid-free C. muridarum organism and the parental C. muridarum Nigg strain revealed no additional mutations other than loss of the plasmid in the plasmid-free C. muridarum organism. Thus, the Pgp3-based immunofluorescence assay has allowed us to identify authentic plasmid-free organisms that are useful for further investigating chlamydial pathogenic mechanisms.

• 대한미생물학회지
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• 제34권6호
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• pp.513-518
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• 1999

A total of 113 Vibrio sp. strains were examined for plasmid content which were subjected to digestion with restriction enzymes. About the 55% Vibrio spp. have the plasmid more than one. Most of these plasmid various derivatives ranged from $2.4\;kb{\sim}23\;kb$, especially two strains of V. mimicus and one strain of V. furnissii carried one high-molecular weight plasmid (molecular weight ranging between $70\;kb{\sim}100\;kb$). Results of restriction analysis for plasmid of this three strains were by no means the rule. For detection of tdh and ctx gene, the virulence factor involved in the pathogenesis, we carried out the TDH and CT assay, PCR amplification, and hybridization. A total 11 strains were produced TDH, involved in 9 strains of V. parahaemolyticus and 1 strain of V. alginolyticus from clinical isolates and 1 strains of V. mimicus from environmental isolates. In the experiments of tdh gene detection, in all, 3 strains of V. parahaemolyticus from clinical isolates and 2 strains from environmental isolates could be successfully amplified in 400 bp by PCR. The PCR results were consistent with DNA hybridization tests. In the experiments of CT assay, in all, 3 strains of V. cholerae from clinical isolate and 1 strain of V. cholerae from environmental isolates were observed CT-producing. These CT-producing strains amplified in 302 bp by PCR for the detection of ctx gene. All CT-producing strains hybridized with digoxigenin-labeled DNA probe, while CT-negative strains did not hybridize. Also hybridization tests results for detection of ctx gene consistent with PCR.

• 대한의생명과학회지
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• 제4권1호
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• pp.43-56
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• 1998

최근 전세계적으로 문제가 되고 있는 장출형성 대장균 O157:H7을 분리배양 및 동정 없이 바로 시료를 분석하여 신속하게 검출하기 위한 다중 중합효소 연쇄반응 (multiplex PCR) 기법을 확립하고, 이 기법을 이용하여 국내 분리 균주 중에서 SLT-I.II, eaeA, 60-MDa plasmid gene을 가지고 있는 대장균을 유전자 수준에서 검출하고자 하였다. 장출혈성 대장균 O157:H7이 가진 SLT-I.II, 60-MDa plasmid 유전자들에 대한 특이 oligonucleotide primers (MK1'-MK2', NAE19-NAE20, MFSIF-MFSIR)를 함께 동시에 반응 완충액에 넣어 다중 중합효소 연쇄반응을 시행한 결과 317bp (eaeA), 228bp (SLT-I.II), 167bp (60-MDa plasmid)의 PCR 증폭 DNA생성물을 표준균주 (E. coli ATCC 35150)에서는 확인할 수 있었지만, 기타 다른 병원성 장내세균 13세균 13균주에서는 band를 확인할 수 없었다. 한편 다중 중합효소 연쇄반응의 template DNA 추출 방법에 따른 PCR 결과를 비교하였다. 각각의 DNA 추출 방법 중 boiling lysis 방법이 신속하고 간편하여 장출혈성 대장균 O157:H7에 의한 식중독의 임상진단에 다중 중합효소 연쇄반응 (multiplex PCR) 적용하는 데에는 boiling lysis법을 이용하는 것이 가장 적합한 방법으로 확인되었다.

• 대한수의학회지
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• 제28권1호
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• pp.59-65
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• 1988

This paper deals with the 0 groups of citrate utilizing variants of Escherichia coli ($Cit^+$ E. coli) isolated from cattle, the production of colicin, hemolysin, K99 antigen, heat stable enterotoxin, and the isolation of plasmid DNA. Among 42 $Cit^+$ E. coli, 12 strains were 020, 9 strains 08, 5 strains 045, 3 strains 0115, 1 strain 064, 1 strain 0139 and remaining strains(11) were untypable. Thirty-nine(81.3%) out of 48 $Cit^+$ E. coli were produced colicin and 13(27.0%) were produced hemolysin. Of 12 $Cit^+$ E. coli bearing K99 antigen, 6(50.0%) were produced heat stable enterotoxin. In gel electrophoresis for the isolation of plasmid DNA, the number of plasmids varied from 1 to 7 in 10 $Cit^+$ E. coli. It's molecular weight ranged from 2 to 50 Mdalton, and 50 Mdalton plasmid was commonly existed in all strains.

• 한국미생물·생명공학회지
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• 제16권5호
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• pp.421-426
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• 1988

한강의 지천으로부터 채취한 시료에서 29종의 아닐린 분해 균주를 분리선발하였다. 분리 균주중 10, 균주에 대하여 동정 시험하여 본 결과 9균주는 Pseudomonas 속이었고 한 균주는 Acinetobacter 속이었다. 전체 29균주중 plasmid를 갖고 있는 균주는 모두 5종이었으며 이들 5균주중 4균주는 meta 경로를 통하여 아닐린을 분해 이용하였고 나머지 한 균주는 ortho 경로를 통하여 아닐린을 분해하였다. meta 경로 이용 균주의 하나인 Pseudomonas acidovorans 4A1은 230kb 크기의 plasmid를 갖고 있었으며, 접합실험과 curing 실험을 통하여 이 plasmid에 아닐린 분해 유전 정보가 수록되었을 가능성을 확인할 수 있었다.

• 약학회지
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• 제45권1호
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• pp.71-77
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• 2001

We recently developed a high efficiency expression vectors pCK, which drives a high level of gene expression in the skeletal muscles of mice. In this study, we investigated the pharmacokinetics and biodistribution of pCK-VEGF expressing human VEGF165 after intravenous or intramuscular administration. The quantity of pCK-VEGF in the tissues of mice was measured by the PCR method which has a detection limit of approximately 1 pg of the exogenously added plasmid. In the case of intravenous administration, the half life of the pCK-VEGF plasmid in the bloodstream was 1.68 min. After intra-muscular administration, the half life of pCK-VEGF plasmid in the bloodstream was 6.78 min. At 90 min post-administration, 30% of the injected pCK-VEGF was found at the site of injection, where it persisted for up to 8 hours. Less than 1.6% of the injected pCK-VEGF plasmid DNA was detected in highly vascularized tissues such as the lung, kidney; and liver at 90 min post-administration, but the plasmid was undetectable at later time points. These results suggested that intramuscularly administrated pCK-VEGF persisted for longer periods of time in muscles than in other tissues and that direct intra-muscular injection of pCK-VEGF might be useful for local therapeutic angiogenesis.

• Parasites, Hosts and Diseases
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• 제60권3호
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• pp.201-205
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• 2022

Babesia microti is one of the most common causative agents of babesiosis. A sensitive and rapid detection is necessary for screening potentially infected individuals. In this study, B. microti cytochrome c oxidase subunit I (cox1) was selected as the target gene, multiple primers were designed, and optimized by a recombinase-aided amplification (RAA) assay. The optimal primers and probe were labeled with fluorescein. The sensitivity of fluorescent RAA (fRAA) was evaluated using gradient diluents of the cox1 recombinant plasmid and genomic DNA extracted from whole blood of B. microti infected mice. The specificity of fRAA was assessed by other transfusion transmitted parasites. The analytical sensitivity of the fRAA assay was 10 copies of recombinant plasmid per reaction and 10 fg/µl B. microti genomic DNA. No cross-reaction with any other blood-transmitted parasites was observed. Our results demonstrated that the fRAA assay would be rapid, sensitive, and specific for the detection of B. microti.

• 한국미생물·생명공학회지
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• 제27권4호
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• pp.278-285
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• 1999

In order to use heat shock promoter for the detection of toxic substances, dnaK promoter was amplified from E. coli genomic DNA by using a polymerase chain reaction(PCR) followed by sequencing and sub-cloning into the multi-cloning site of the plasmid, pUCD615. The pUCD615 is a broad-host-range vector containing promoterless lux operon originated from V.fischeri. The recombinant plasmid was transfered to E. coli DH5$\alpha$ through electroporation. The recombinant E. coli showed several patterns of bioluminescent responses to ethanol stress. The bioluminescent E. coli also showed responses to other toxic substances including FeK3(CN)6, CdCl2, p-nitrophenol and HgCl2. The increases of RLU(Relative Light Unit) were observed at 100ppm of FeK3(CN)6, 10ppm and 100ppm and 100ppm of CdCl2, 1ppm of 10ppm of p-nitrophenol and at 1ppm of HgCl2.

• Preventive Nutrition and Food Science
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• 제17권4호
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• pp.274-279
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• 2012

For the quantitative analysis of genetically modified (GM) maize in processed foods, primer sets and probes based on the 35S promoter (p35S), nopaline synthase terminator (tNOS), p35S-hsp70 intron, and zSSIIb gene encoding starch synthase II for intrinsic control were designed. Polymerase chain reaction (PCR) products (80~101 bp) were specifically amplified and the primer sets targeting the smaller regions (80 or 81 bp) were more sensitive than those targeting the larger regions (94 or 101 bp). Particularly, the primer set 35F1-R1 for p35S targeting 81 bp of sequence was even more sensitive than that targeting 101 bp of sequence by a 3-log scale. The target DNA fragments were also specifically amplified from all GM labeled food samples except for one item we tested when 35F1-R1 primer set was applied. A reference plasmid pGMmaize (3 kb) including the smaller PCR products for p35S, tNOS, p35S-hsp70 intron, and the zSSIIb gene was constructed for real-time PCR (RT-PCR). The linearity of standard curves was confirmed by using diluents ranging from $2{\times}10^1{\sim}10^5$ copies of pGMmaize and the $R^2$ values ranged from 0.999~1.000. In the RT-PCR, the detection limit using the novel primer/probe sets was 5 pg of genomic DNA from MON810 line indicating that the primer sets targeting the smaller regions (80 or 81 bp) could be used for highly sensitive detection of foreign DNA fragments from GM maize in processed foods.

• Journal of Veterinary Science
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• 제23권4호
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• pp.51.1-51.10
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• 2022

Background: Due to the unavailability of an effective vaccine or antiviral drug against the African swine fever virus (ASFV), rapid diagnosis methods are needed to prevent highly contagious African swine fever. Objectives: The objective of this study was to establish the ladder-shape melting temperature isothermal amplification (LMTIA) assay for the detection of ASFV. Methods: LMTIA primers were designed with the p72 gene of ASFV as the target, and plasmid pUC57 was used to clone the gene. The LMTIA reaction system was optimized with the plasmid as the positive control, and the performance of the LMTIA assay was compared with that of the commercial real-time polymerase chain reaction (PCR) kit in terms of sensitivity and detection rate using 200 serum samples. Results: Our results showed that the LMTIA assay could detect the 10⁴ dilution of DNA extracted from the positive reference serum sample, which was the same as that of the commercial real-time PCR kit. The coincidence rate between the two assays was 100%. Conclusions: The LMTIA assay had high sensitivity, good detection, and simple operation. Thus, it is suitable for facilitating preliminary and cost-effective surveillance for the prevention and control of ASFV.