• Journal of Microbiology and Biotechnology
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• v.30 no.5
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• pp.681-688
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• 2020

Bacterial cell-based biosensors, or whole-cell bioreporters (WCBs), are an alternative tool for the quantification of hazardous materials. Most WCBs share similar working mechanisms. In brief, the recognition of a target by sensing domains induces a biological event, such as changes in protein conformation or gene expression, providing a basis for quantification. WCBs targeting heavy metal(loid)s employ metalloregulators as sensing domains and control the expression of genes in the presence of target metal(loid) ions, but the diversity of targets, specificity, and sensitivity of these WCBs are limited. In this study, we genetically engineered the metal-binding loop (MBL) of ZntR, which controls the znt-operon in Escherichia coli. In the MBL of ZntR, three Cys sites interact with metal ions. Based on the crystal structure of ZntR, MBL sequences were modified by site-directed mutagenesis. As a result, the metal-sensing properties of WCBs differed depending on amino acid sequences and the new selectivity to Cr or Pb was observed. Although there is room for improvement, our results support the use of currently available WCBs as a platform to generate new WCBs to target other environmental pollutants including metal(loid)s.

• Journal of Biosystems Engineering
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• v.39 no.2
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• pp.111-114
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• 2014

Purpose: This review aims to introduce aptamers and the methods of its development to improve the sensitivity and selectivity to target bacteria. In this review, we have highlighted current developments and directions in the pathogen detection based on aptamers. Background: Aptamers, the specific nucleic acid sequences, can bind to targets with high affinity and specificity. Some of researches on the use of aptamers for the detection of pathogen have been reported in recent years. Aptamers have more applicability than antibodies for the development of pathogen detection using biosensor; such as easy to synthesis and labeling, lack of immunogenicity, and a low cost of production. However, only few reports on the development and use of aptamers for the detection of pathogen have been published. Review: Aptamers specific to pathogen are obtained by whole-cell systematic evolution of ligands by exponential enrichment (SELEX) process. SELEX process is composed of screening random oligonucleotide bound with target cells, multiple separation and amplification of nucleic acids, final identification of the best sequences. For improving those affinity and selectivity to target bacteria, optimization of multiple separating process to remove unbounded oligonucleotides from aptamer candidates and sorting process by flow cytometry are required.

• Korean Journal of Veterinary Research
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• v.13 no.1
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• pp.23-30
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• 1973

Thin layer, paper and column chromatography were compared for the separation, detection and quantitation of three kinds of estrogen in urine of dairy cows. While thin layer chromatography utilizing silica gel was better for the detection of estrogens, column chromatography using celite 545 was preferable. Spectrophotometry was compared with fluorometry for determination of estrone, estradiol-17 ${\beta}$ and estriol eluted by paper chromatography and column chromatography. Optical density of three standard estrogens showed almost same curve at maximum absorption wave length of 230 and $282m{\mu}$. However, the former showed a higher peak. In fluorometry, the fluorescence intensity of estrone and estradiol-17 ${\beta}$ were rather strong, when the estrogens were dissolved in sulfuric acid, and showed higher sensitivity than that of the spectrophotometry. However, in the case of estriol was exceptional.

• Applied Chemistry for Engineering
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• v.20 no.6
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• pp.581-585
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• 2009

Diabetes is caused by the lack of insulin secretion or the reduction in its sensitivity. The recent treatments for diabetes do not fulfill the requirements for the maintaining of normal blood glucose level. The most frequently used treatment for type I diabetes is an insulin injection. However, the insulin injection often involves the life-threatening risk and the low compliance of patients. Accordingly, extensive studies have been done to overcome the shortcomings of the injection. A representative approach is to employ self-regulated insulin delivery systems. This report includes the principles of glucose-responsive release in the self-regulated insulin delivery systems.

• Journal of fish pathology
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• v.27 no.2
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• pp.99-105
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• 2014

Biosensors consist of biochemical recognition agents like antibodies immobilized on the surfaces of transducers that change the recognition into a measurable electronic signal. Here we report a piezoelectric immunosensor made to detect Vibrio vulnificus. A 9MHz AT-cut piezoelectric wafer attached with two gold electrodes of 5mm diameter was used as the transducer of the QCM biosensor with a reproducibility of ${\pm}0.1Hz$ in frequency response. We have tried different approaches to immobilize antibody on the sensor chip. Concerning the orientation of antibody for the best antigen binding capacity, the antibody was immobilized by specific binding to protein G or by cross-linking through hydrazine. In addition, protein G was cross-linked on glutaraldehyde activated immine layer (PEI) or EDC/NHS activated sulfide monolayer (MPA). PEI was found to be more effective to immobilize protein G following glutaraldehyde activation than MPA. However, hydrazine chip showed a better capability to immobilize more IgG than protein G chip and a higher sensitivity. The sensor system was able to detect V. vulnificus in dose dependent manner and was able to detect bacterial cells within 5 minutes by monitoring frequency shifts in real time. The detection limit can be improved by preincubation to enrich the bacterial cell number.

• The Journal of Korean Society of Virology
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• v.27 no.1
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• pp.49-57
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• 1997

We developed a sensitive, nested reverse transcription-polymerase chain reaction (RT-PCR) to detect Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses in animal tissues. Total RNA was extracted from blood, lung or kidney samples of experimentally-infected hamsters by using the guanidine isothiocyanate buffer-acid phenol-chloroform method. Genus-reactive outer primers were derived from the consensus region of the G1 gene sequences of several hantaviruses. Serotype-specific primers were selected within the region amplified by the outer primers. To examine the sensitivity and specificity of the test, we diluted known quantities of Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses in human or hamster immune sera before performing the nested RT-PCR. We could detect as little as 1 pfu of virus, even in the presence of high-titer neutralizing antibodies, and the serotype-specific primers amplified only homologous serotype viruses. RT-PCR with these primers demonstrated virus in the blood of experimentally-infected hamsters as early as four days to as late as 30 days after infection. A comparison of a standard immunofluorescent antibody screening test (IFAT) to nested RT-PCR with RNA extracted from lung or kidney tissues of the hamsters, demonstrated that RT-PCR to be more sensitive for identifying viruses in these tissues.

• Korean Journal of Veterinary Pathology
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• v.6 no.1
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• pp.1-5
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• 2002

A1celaphine herpesvirus 1 (AHV-1) is a causative agent of malignant catarrhal fever which is a fatal and a lymphoproliferative syndrome. AHV-1 is a gamma herpesvirus, which induces frequent latent infection and often difficult to detect its antigens or specific nucleic acids because of its low viral copies in the infected tissues. A new method, in situ PCR, is developed for the detection of AHV-1 nucleic acid in this study. Target sequences of AHV-1 open reading frame 50 gene were detected within AHV-1 infected MDBK cells. As compare with other molecular biological methods for the detection of AHV-1, in situ PCR was found to be more sensitive than in situ hybridization and to be less sensitive than nested PCR. However, nested PCR cannot afford to observe and differentiate AHV-1 infected cells. In situ PCR amplifies a target sequence within cells that can be visualized microscopically with increased sensitivity compared to detection by in situ hybridization. In situ PCR has wide applications for sensitive localization of low copy AHV-1 viral sequences within cells to investigate the role of viruses in a variety of clinical conditions and also provide the rapid, sensitive, and specific detection of AHV-1 infection.

• Korean Journal of Veterinary Research
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• v.20 no.2
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• pp.79-84
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• 1980

One hundred and eighteen cultures of Gram-negative rods isolated from cases of clinical bovine mastitis during lactation were examined for distribution of specific types, and activity of several antimicrobial agents to the isolates was determined by two-fold tube dilution method employing sterile whole milk as fluid medium. Of the isolates, 59.2% were Escherichia coli. Most of the remaining isolates were Klebsiella pneumoniae and Enterobacter aerogenes. Most Gram-negative rods(89.8%) were isolated from acute local and chronic mastitis. The cases of peracute systemic form with a marked symptoms of toxemia were associated with Escherichia coli and Klebsiella pneumoniae. The minimal lethal concentrations (MLC) of gentamicin and oxolinic acid in sterile whole milk were 16-128 times higher than the MLC obtained in trypticase soy broth (TSB), while the MLC of ampicillin and tetracycline in milk increased 2-4 times compared with TSB. Of the drugs tested, gentamicin was the most active antibiotics with MLC of $100{\mu}g/ml$ in sterile whole milk against all of Gram-negative rods isolated from bovine udder infections.

• Analytical Science and Technology
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• v.8 no.4
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• pp.887-894
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• 1995

Toward the development of universal, sensitive, and convenient method of DNA (or RNA) detection, two kinds of electrochemically active DNA ligands. acridine - viologen and oligonucleotide - ferrocene conjugate, were prepared. Thermodynamic and electrochemical study revealed that these probes bound strongly to DNA, and showed a typical cyclic voltammograms, indicating a potential for use as a reversible electrochemical labelling agent for DNA. Especially, using the electrochemically active oligonucleotide, we have been able to demonstrate the detection of DNA at femtomole levels by HPLC equipped with ordinary electrochemical detector (ECD). These results lead to the conclusion that the redox-active probes are very useful for the microanalysis of nucleic acid due to the stabilily of the complexes, high detection sensitivity, and wide applicability to the target structures (single- and double strands) and sequences.

• Korean Journal of Clinical Laboratory Science
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• v.46 no.4
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• pp.140-142
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• 2014

To evaluate the recovery rates to increase toxigenic C. difficile, the selective enrichment broth culture methods were compared with commonly used cytotoxin assays and toxigenic culture. First, the enrichment culture, using the selective medium broth for 2 to 5 days, was performed and then, toxigenic C. difficile was confirmed by C. difficile toxin gene-specific PCR after being cultured on C. difficile selective agar. The sensitivity of C. difficile from the enrichment culture (100%) was higher than that of C. difficile selective agar culture (93.8%), while positive predictive values (PPV) were low; 72.7% (16/22) and 88.2% (15/17). PPV of the enrichment culture are not high. Recently, combinations of C. difficile selective agar culture, C. difficile A & B assays, glutamate dehydrogenase, and nucleic acid amplification method are widely used. The enrichment culture was disadvantageous in PPV, turn-around time, and cost. So, what we performed is not considered as a common method of diagnosis of C. difficile-associated diarrhea.