• Genomics & Informatics
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• v.19 no.3
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• pp.30.1-30.8
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• 2021

Salmonella species are among the major pathogens that cause foodborne illness outbreaks. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of Salmonella species. We designed LAMP primers targeting the hilA gene as a universal marker of Salmonella species. A total of seven Salmonella species strains and 11 non-Salmonella pathogen strains from eight different genera were used in this study. All Salmonella strains showed positive amplification signals with the Salmonella LAMP assay; however, there was no non-specific amplification signal for the non-Salmonella strains. The detection limit was 100 femtograms (20 copies per reaction), which was ~1,000 times more sensitive than the detection limits of the conventional polymerase chain reaction (PCR) assay (100 pg). The reaction time for a positive amplification signal was less than 20 minutes, which was less than one-third the time taken while using conventional PCR. In conclusion, our Salmonella LAMP assay accurately detected Salmonella species with a higher degree of sensitivity and greater rapidity than the conventional PCR assay, and it may be suitable for point-of-care testing in the field.

• Journal of Microbiology and Biotechnology
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• v.20 no.11
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• pp.1563-1570
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• 2010

To construct a highly sensitive detection system for endocrine disruptors (EDs), we have compared the activity of promoters with the n-alkane-inducible cytochrome P450 gene (ALK1), isocitrate lyase gene (ICL1), ribosomal protein S7 gene (RPS7), and the translation elongation factor-1${\alpha}$ gene (TEF1) for the heterologous gene in Yarrowia lipolytica. The promoters were introduced into the upstream of the lacZ or hERa reporter genes, respectively, and the activity was evaluated by ${\beta}$-galactosidase assay for lacZ and Western blot analysis for hER${\alpha}$. The expression analysis revealed that the ALK1 and ICL1 promoters were induced by n-decane and by EtOH, respectively. The constitutive promoter of RPS7 and TEF1 showed mostly a high level of expression in the presence of glucose and glycerol, respectively. In particular, the TEF1 promoter showed the highest ${\beta}$-galactosidase activity and a significant signal by Western blotting with the anti-estrogen receptor, compared with the other promoters. Moreover, the detection system was constructed with promoters linked to the upstream of the expression vector for the hER${\alpha}$ gene transformed into the Y. lipolytica with a chromosome-integrated lacZ reporter gene under the control of estrogen response elements (EREs). It was indicated that a combination of pTEF1p-hER${\alpha}$ and CXAU1-2XERE was the most effective system for the $E_2$-dependent induction of the ${\beta}$-galactosidase activity. This system showed the highest ${\beta}$-galactosidase activity at $10^{-6}\;M\;E_2$, and the activity could be detected at even the concentration of $10^{-10}\;M\;E_2$. As a result, we have constructed a strongly sensitive detection system with Y. lipolitica to evaluate recognized/suspected ED chemicals, such as natural/synthetic hormones, pesticides, and commercial chemicals. The results demonstrate the utility, sensitivity, and reproducibility of the system for identifying and characterizing environmental estrogens.

• Journal of Marine Bioscience and Biotechnology
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• v.2 no.4
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• pp.263-266
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• 2007

Vibrio vulnificusis a causative agent of serious diseases in humans resulting from the contact of wound with seawater or consumption of raw seafood. Several studies aimed at detecting V. vulnificus have targeted vvh as a representative virulence toxin gene belonging to the bacterium. In this study, we targeted the rpoS gene, a general stress regulator, to detect V. vulnificus. PCR specificity was identified by amplification of 8 V. vulnificus templates and by the loss of a PCR product with 36 non-V. vulnificus strains. The PCR assay had the 273-bp fragment and the sensitivity of 10 pg DNA from V. vulnificus. SYBR Green I-based real-time PCR assay targeting the rpoS gene showed a melting temperature of approximately $84^{\circ}C$ for V. vulnificus strains. The minimum level of detection by real-time PCR was 2 pg of purified genomic DNA, or $10^3$ V. vulnificus cells from pure cultured broth and $10^3$ cells in 1g of oyster tissue homogenates. These data indicate that real-time PCR is a sensitive, species-specific, and rapid method for detecting this bacterium using the rpoS gene in pure cultures and in infected oyster tissues.

• Journal of Oral Medicine and Pain
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• v.20 no.2
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• pp.515-528
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• 1995

The human genomic deoxyribonucleic acid(DNA) was extracted from the pulp, dentin of 22 teeth by clelex, phenol methods. Samples of the tooth-derived DNA were amplified by polymerase chain reaction(PCR), electrophosed for sex determination by detection of X-Y homologus amelogenin gene and D1S80 locus detection The following results have been achieved. 1. Chelex and phenol method are effective to sex determination in the pulp and dentin 2. Chelex method is not suitable for detection of D1S80 locus. 3. Concentration and purity of DNA for teeth using chelex method is lower than using phenol method. From the above investigation, chelex method is simple, rapid for sex determination, but it is not suitable for detection of VNTRs.

• Korean Journal of Environmental Biology
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• v.23 no.3 s.59
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• pp.269-274
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• 2005

The 2D/E gel analysis for polypeptide expression reflecting I-18 C gene (early-ecdysterone inducible gene) has conducted the emerged C. riparius adults from larval phase exposure to tebufenozide acting as an ecdysteroidal molting hormone. Control group, the amount of ORE II of the I-18 C gene was larger than that of ORE I of this gene. After treatments, ORE I of the I-18 C gene was overexpressed as the polypeptide, whereas ORF II of this gene was expressed as the polypeptide and was clearly reduced expression. Accordingly, we consider that tebufenozide exhibited endocrine disruptions related processing of ecdysteroid receptor protein reflecting ORF II of I-18 C gene. Also, earlier emergence day was related overexpressed polypeptide reflecting ORE I of I-18 C gene. In this study result, tebufenozide induced changing of physiological condition, and then polypeptide expression reflecting early-ecdysterone inducible I-18 C gene was different between control group and exposure group.

• Journal of Microbiology and Biotechnology
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• v.9 no.2
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• pp.184-190
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• 1999

The use of the polymerase chain reaction (PCR) method was described using two sets of primers based on the ceuN gene (JEJ 1 and JEJ 2) which encodes a protein involved in siderophore transport and 16S rRNA gene (pA and pB) for the sensitive and specific detection of enteropathogen Campylobacter jejuni. Six oligonucleotides were utilized in an amplification experiment and PCR products of predicted sizes were generated from whole cells and boiled cell lysates at the same intensity. Two sets of the primer pairs, JEJ and pAB, were specific enough for all C. jejuni strains tested for the direct use of whole cells without DNA extraction or lysis steps. In the PCR using the pAB primer pair, the detection limit, as determined by the ethidium bromide staining of the amplification products on agarose gels, was at the level of $10^1$ bacteria cells or less in both the pure culture and artificially inoculated milk and chicken enrichment samples, whereas the detection limit with the JEJ primer pair was relatively low, i.e. $10^3$ cells or more in the same PCR samples. The PCR method using either a primer JEJ or pAB was both repeatable and specific for the detection of C. jejuni in food. This method is simply completed within 4 h.

• KSII Transactions on Internet and Information Systems (TIIS)
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• v.4 no.6
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• pp.1273-1293
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• 2010

Immune-inspired intrusion detection is a promising technology for network security, and well known for its diversity, adaptation, self-tolerance, etc. However, scalability and coverage are two major drawbacks of the immune-inspired intrusion detection systems (IIDSes). In this paper, we propose an IIDS framework, named GEP-IIDS, with improved basic system elements to address these two problems. First, an additional bio-inspired technique, gene expression programming (GEP), is introduced in detector (corresponding to detection rules) representation. In addition, inspired by the avidity model of immunology, new avidity/affinity functions taking the priority of attributes into account are given. Based on the above two improved elements, we also propose a novel immune algorithm that is capable of integrating two bio-inspired mechanisms (i.e., negative selection and positive selection) by using a balance factor. Finally, a pruning algorithm is given to reduce redundant detectors that consume footprint and detection time but do not contribute to improving performance. Our experimental results show the feasibility and effectiveness of our solution to handle the scalability and coverage problems of IIDS.

• Journal of Microbiology and Biotechnology
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• v.30 no.5
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• pp.662-667
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• 2020

Epidermal growth factor receptor (EGFR) mutations are not only genetic markers for diagnosis but also biomarkers of clinical-response against tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC). Among the EGFR mutations, the in-frame deletion mutation in EGFR exon 19 kinase domain (EGFR exon 19-del) is the most frequent mutation, accounting for about 45% of EGFR mutations in NSCLCs. Development of sensitive method for detecting the EGFR mutation is highly required to make a better screening for drug-response in the treatment of NSCLC patients. Here, we developed a fluorometric tandem gene amplification assay for sensitive detection of low-abundance EGFR exon 19-del mutant genomic DNA. The method consists of pre-amplification with PCR, thermal cycling of ligation by Taq ligase, and subsequent rolling circle amplification (RCA). PCR-amplified DNA from genomic DNA samples was used as splint DNA to conjugate both ends of linear padlock DNA, generating circular padlock DNA template for RCA. Long stretches of ssDNA harboring multiple copies of G-quadruplex structure was generated in RCA and detected by thioflavin T (ThT) fluorescence, which is specifically intercalated into the G-quadruplex, emitting strong fluorescence. Sensitivity of tandem gene amplification assay for detection of the EGFR exon 19-del from gDNA was as low as 3.6 pg, and mutant gDNA present in the pooled normal plasma was readily detected as low as 1% fraction. Hence, fluorometric detection of low-abundance EGFR exon 19 deletion mutation using tandem gene amplification may be applicable to clinical diagnosis of NSCLC patients with appropriate TKI treatment.

• Korean Journal of Veterinary Service
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• v.30 no.4
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• pp.557-562
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• 2007

This study was carried out to discriminate Hanwoo from the milking and hybrid cattle by detection of MC1R gene related to bovine hair color. One hundred sixty six samples were collected from the abattoir (n = 106) and local market (n = 60). The beef from abattoir were originated from Hanwoo (n=27), Holstein (n=29), Hybrid (n=45) and imported cattle (n=5), respectively. The beef from market consisted of Hanwoo (n=36), Holstein (n=7) and imported ones (n=17). Commercialized screening kit (Kogenebiotec, Korea) was used for MC1R gene analysis. As a result, Hanwoo was discriminated from Holstein. However, 9 of 45 hybrid and 11 of 22 imported beef samples were indistinguishable from Hanwoo. It could be explained by second generation of crossing of Hanwoo with Holstein or the cattle with silver or yellow hair. This results suggest that additional tests as well as MC1R gene detection be needed to confirm Hanwoo beef among cattle beef.

• Korean Journal of Veterinary Research
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• v.50 no.4
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• pp.331-333
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• 2010

Moribund albino catfish, Clarias sp., displayed from an indoor private commercial aquarium were submitted in the laboratory for diagnostic examination. Dense culture of bacteria was recovered from the kidney and was characterized using Vitek System 2 and showed 98% probability to Aeromonas (A.) hydrophila. PCR result showed positive using A. hydrophila extracellular hemolysin gene ahh1 (130 bp) and aerolysin gene aerA (309 bp). The 16S rRNA gene was identical and exhibited 97% sequence similarity with the other known isolates of A. hydrophila available in the GenBank. In this paper, we reported the isolation and molecular detection of A. hydrophila from an albino catfish.