• 한국식품위생안전성학회지
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• 제34권3호
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• pp.290-295
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• 2019

최근 한국에서 발생한 Salmonella로 인한 식중독 사고는 2018년 9월 학교급식에서 제공된 초콜릿 무스 케이크가 원인이 되었다. 이 연구의 목적은 Salmonella Typhimurium이 인위적으로 접종된 무스케이크와 티라미수에서 3M Molecular Detection Assay 2 - Salmonella와 식품공전에 등재된 방법인 분리배지와 real-time PCR을 비교하는 것이었다. 무스케이크 2종과 티라미수 2종 25 g에 225 mL BPW를 넣고 $37^{\circ}C$에서 24시간 동안 증균 배양하였다. 배양 후, 3M Molecular Detection Assay 2 - Salmonella, 분리배지 그리고 real-time PCR로 분석하였다. 초콜릿 무스 케이크를 제외하고 3가지 방법은 유사한 결과를 보였다. 초콜릿 무스 케이크에서 분리배지와 3M Molecular Detection Assay 2 - Salmonella는 모든 접종수준에서 동일한 결과를 나타낸 반면 real-time PCR은 $10^4CFU/25g$ 수준에서 1번의 양성결과를 제외하고 모두 검출되지 않았다. 초콜릿 무스에 S. Typhimurium을 $10^2CFU/25g$ 수준으로 접종하였을때, real-time PCR를 이용한 검출은 15%에서는 부분적인 음성을 나타냈고, 20-100% 함량의 초콜릿 무스에서는 모두 음성이었다. Real-time PCR로는 chocolate이 15% 이상 함유된 식품에서의 Salmonella균 검출이 불가능하였지만, LMAP 기반의 3M Molecular Detection Assay 2으로는 chocolate 농도에 관계없이 검출이 가능하였다.

• Toxicological Research
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• 제16권3호
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• pp.201-209
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• 2000

In association with the international validation program to establish a rodent uterotrophic assay, we conducted preliminary uterotrophic assay proposed by GECD using immature female rats. In the present study, oral and subcutaneous routes were chosen to compare the effects of estrogenic com-pounds in the two dosing regimens. The reference compound ethinyl estradiol (EE) and the antagonist ZM189154(ZM) were administered by gavage or subcutaneously (s.c.) to immature female SD rats from 20 to 22 days of age. For each study, sixty-six female rats were randomly assigned to eleven groups: Untreated control, EE 0,0.01, 0.03, 0.1, 0.3, 1.0,3.0 and 10.0 $\mu\textrm{g}$/kg, EE 3.0 $\mu\textrm{g}$/kg(gavage)/0.3 $\mu\textrm{g}$/kg(s.c) & ZM 0.1 mg/kg, and EE 3.0 $\mu\textrm{g}$/kg(gavage)/0.3 $\mu\textrm{g}$/kg (s.c) & ZM 1.0 mg/kg. There were no treatment-related changes in clinical signs, body weights, food consumption, and necropsy findings in any groups of two studies. The wet and blotted uterus weights increased dose-dependently. Histopathological examination revealed that diameter of uterine duct, height of uterine luminal epithelium. and height oj vaginal epithelium increased dose-dependently. The proliferating cell nuclear antigen (PCNA) immunoreactive cells were increased in number dose-dependently. The estrogenic effects observed in the present studies occurred at $\geq$ 0.3 $\mu\textrm{g}$/kg of oral dose and $\geq$ 0.1 $\mu\textrm{g}$/kg of s.c. dose. An antagonistic effect of ZM against EE was found in both uterus weight and histopathological parameters. From the results obtained, it can be concluded that dose-dependence of the uterotrophic assay using EE and ZM was well demonstrated by gavage and subcutaneous administration and that the estrogenic effects of EE by s.c. dose were higher than those by gavage administration. In addition, blotted uterus weight was more sensitive than wet uterus weight and vaginal epithelial height was found to be the most sensitive parameter among the parameters examined.

• 셀메드
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• 제3권1호
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• pp.9.1-9.10
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• 2013

In traditional systems of medicine including homeopathy, the Condurango extract (Con) is often used to cure stomach cancer mainly, without having any scientific validation of its anti-cancer ability. Con has therefore been tested against non-small-cell lung cancer cells (NSCLC) A549 and NCI-H522 (H522) known to contain the KRAS mutation, making them resistant to most chemotherapeutic agents. As cancer cells generally defy cytotoxicity developed by chemopreventive agents and escape cell death, any drug showing the capability of preferentially killing cancer cells through apoptosis is worth consideration for judicious application. A549 and H522 cells were exposed to $0.35{\mu}g/{\mu}l$ and $0.25{\mu}g/{\mu}l$ of Con, respectively, for 48 h and analysed based on various protocols associated with apoptosis and DNA damage, such as MTT assay to determine cell viability, LDH assay, DNA fragmentation assay, comet assay, and microscopical examinations of DNA binding fluorescence stains like DAPI, Hoechst 33258 and acridine orange/ethidium bromide to determine the extent of DNA damage made in drug-treated and untreated cells and the results compared. Changes in mitochondrial membrane potential and the generation of reactive oxygen species were also documented through standard techniques. Con killed almost 50% of the cancer cells but spared normal cells significantly. Fluorescence studies revealed increased DNA nick formation and depolarized membrane potentials after drug treatment in both cell types. Caspase-3 expression levels confirmed the apoptosis-inducing potential of Con in both the NSCLC lines. Thus, overall results suggest considerable anticancer potential of Con against NSCLC in vitro, validating its use against lung cancer by practitioners of traditional medicine including homeopathy.

• Toxicological Research
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• 제24권1호
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• pp.37-44
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• 2008

The in vitro cell transformation assays (CTA) were performed using BALB/3T3 murine fibroblasts and HaCaT human keratinocytes in order to evaluate concordance between both in vitro CTAs and carcinogenicity with compounds differing in their genotoxic and carcinogenic potential. Six test articles were evaluated, two each from three classes of compounds: genotoxic carcinogens (2-amino-5-nitrophenol and 4-nitroquinoline-N-oxide), genotoxic noncarcinogens (8-hydroxyquinoline and benzyl alcohol), and nongenotoxic carcinogens (methyl carbamate and N-nitrosodiphenylamine). Any foci of size $\geq$2 mm regardless of invasiveness and piling was scored as positive in CTA with BALB/3T3. As expected, four carcinogens regardless of their genotoxicity had positive outcomes in two-stage CTA using BALB/3T3 cells. However, of the two genotoxic noncarcinogens, benzyl alcohol was positive CTA finding. We concluded that, of the 6 chemicals tested, the sensitivity for BALB/3T3 system was reasonably high, being 100%. The respective specificity for BALB/3T3 assay was 50%. We also investigated the correlation between results of BALB/3T3 assay and results from HaCaT assay in order to develop a reliable human cell transformation assay. However, evaluation of staining at later time points beyond the confluency stage did not yield further assessable data because most of HaCaT cells were detached after $2{\sim}3$ days of confluency. Thus, after test article treatment, HaCaT cells were split before massive cell death began. In this modified protocol for this HaCaT system, growing attached colonies were counted instead of transformed foci 3 weeks since last subculture. Compared to BALB/3T3 assay, HaCaT assay showed moderate low sensitivity and high specificity. Despite these differences in specificity and sensitivity, both cell systems did exhibit same good concordance between in vitro CTA and rodent carcinogenicity findings (overall 83% concordant results). At present the major weakness of these in vitro CTA is lack of validation for regulatory acceptance and use. Thus, more controlled studies will be needed in order to be better able to assess and quantitatively estimate in vitro CTA data.

• Fisheries and Aquatic Sciences
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• 제19권1호
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• pp.3.1-3.6
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• 2016

Phlorotannins are reported to have diverse biological properties. However, no analytical methods for the standardization of phlorotannin preparations have been reported. Herein, we developed and validated an analytical method for the determination of dieckol in phlorotannin extracts (PRT) using high-performance liquid chromatography (HPLC). The optimum HPLC conditions consisted of a Supelco Discovery C18 column stationary phase, a mobile phase (A: 15 % HPLC grade methanol in deionized water, B: methanol), UV detection at 230 nm, and a flow rate of 0.7 mL/min. The optimized chromatographic conditions were validated and exhibited good specificity and linearity ($R^2$ > 0.9994-1.0000). The recoveries were in the range of 100.9-102.3 %. The method had good intermediate (%RSD 1.2) and intra-day (%RSD 0.4-1.7) assay precisions. This HPLC method had good accuracy and quality in the determination of dieckol in PRT.

• 생약학회지
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• 제54권1호
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• pp.38-43
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• 2023

A simple and accurate assay was developed for the quantitative analysis of 1-deoxynojirimycin (1-DNJ) derived from the silkworm (Bombyx mori). Normal-phase high-performance liquid chromatography coupled with an evaporative light scattering detector (HPLC-ELSD) and a hydrophilic interaction liquid chromatography column was used. Various parameters were applied to optimize the analysis method. The limits of detection and quantification of 1-DNJ were 2.97 × 10^-3 and 9.00 × 10^-3 mg/mL, respectively. The calibration curve showed good linearity results. The concentration range and the r² value were 0.0625-1.0 mg/mL and 0.9997, respectively. The accuracy test demonstrated a significantly high recovery rate (89.95-103.22%). The relative standard deviation was ≤ 1.00%. Thus, a method for the accurate identification and quantitative analysis of 1-DNJ in silkworms was developed. Moreover, in this procedure, the process of derivatization of 1-DNJ, which was required in previous experiments, could be eliminated. This technique may be actively utilized for the development of pharmaceuticals and health functional foods using 1-DNJ.

• 대한의생명과학회지
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• 제29권4호
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• pp.220-230
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• 2023

Identification of the pathogens causing infection is important in terms of patient's health management and infection control. Synovial fluids could be used as clinical samples to detect causative pathogens of prosthetic joint infections (PJIs) using molecular diagnostic assays, therefore, normalization and validation of clinical samples are necessary. Microbial culture is considered the gold standard for all infections, including PJIs. Recently, molecular diagnostic methods have been developed to overcome the limitation of microbial culture. Therefore, guideline for validating clinical samples to provide reliable results of molecular diagnostic assays for infectious diseases is required in clinical field. The present study aimed to develop an accurate validating method of synovial fluid clinical samples using GAPDH gene as an internal control to perform the quantitative PCR TaqMan probe assay to detect pathogens causing PJIs.

• 한국식품저장유통학회지
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• 제30권3호
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• pp.383-394
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• 2023

The ultra-fast quantitative real-time polymerase chain reaction (qPCR) assay was developed and validated to differentiate the morphologically similar ones, Oncorhynchus keta and Oncorhynchus mykiss. Species-specific primers were designed for the COI genes of mtDNA. The species-specific primers designed for O. keta and O. mykiss were selectively amplified by O. keta and O. mykiss DNA, respectively. The sensitivity of O. keta and O. mykiss primers was 1 ng/μL. Quantitative testing showed that the results met the 'Guidelines on Standard Procedures for Preparing Analysis Method such as Food' proposed by the Ministry of Food and Drug Safety. The qPCR method developed and validated in this study for identifying O. keta and O. mykiss has advantages such as speed and field applicability. Therefore, this method is expected to help control forgery and alteration of raw materials in the seafood industry.

• KSBB Journal
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• 제18권4호
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• pp.255-260
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• 2003

현재는 재조합 Erythropoietin의 생물학적 활성을 mouse를 이용한 in vivo bioassay로 실시하고 있으나, 이 방법의 여러가서 불편함으로 인하여, 이를 대체하기 위해 Ba/F3 세포주를 이용한 in vitro assay 방법을 확립하였으나, 위에 상술한 바와 같이 in vitro assay는 erythropoietin의 당분포에 의한 차이를 보이지 않게 때문에, 이를 보완하기 위해서는 in vivo bio-activity와 정량적인 상관관계가 있는 당단백질의 isoform 분석법인 Capillary zone electrophoresis (CZE) 와 sialic acid 함량 결과를 동시에 분석해야 했다. 위의 결과 sialic acid 함량은 erythropoietin 원액에서 10 mol/mol,EPO 이상의 sialic acid 함량을 가져야 되며, CZE 결과는 재조합 erythropoietin이 isoform 분포가 isoform 3의 경우 5.594～0.593％, isoform 4의 경우 31.598～11.704％, isoform 5의 경우 37.033～29.301％, isoform 6의 경우 27.837～18.807％, isoform 7의 경우 17.085～7.824％, isoform 8의 경우 7.642～1.964％을 보여줌과 동시에 isoform 4의 면적은 isoform 5의 면적보다 항상 작아야한다. 이상 두 가지 시험결과와 in vitro assay 결과를 combine해서 in vivo assay를 대체할 수 있다는 좋은 실험적 data를 얻었으므로 이상 위의 3가지 분석법을 활용한 combined in vitro bioassay 법의 기초를 확립하였다.

• Journal of Pharmaceutical Investigation
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• 제37권3호
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• pp.179-186
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• 2007

A simple, sensitive and selective liquid chromatographic/tandem mass spectrometric method (LC-MS/MS) was developed and validated for doxifluridine and 5-fluorouracil (5-FU) quantification in dog heparinized plasma. Sample preparation was based on liquid-liquid extraction using a mixture of isopropanol/ethyl acetate (1/9 v/v) to extract doxifluridine, 5-FU and 5-chlorouracil (5-CU, an internal standard) from plasma. Chromatography was performed on a C-18 analytical column and the retention times were 2.7, 1.5 and 1.7 min for doxifluridine, 5-FU and 5-CU, respectively with shorter analysis time within 5 min than previously reported methods. The ionization was optimized using ESI negative mode and selectivity was achieved by tandem mass spectrometric analysis by multiple reaction monitoring (MRM) using the transformations of m/z 244.8>107.6, 129.0>42.0 and 144.9>42.1 for doxifluridine, 5-FU and 5-CU, respectively. The achieved low limit of quantification was 20.0 ng/mL and the assay exhibited linear range of 20-2000 ng/mL ($R^2>0.99957$ for doxifluridine and $R^2>0.99857$ for 5-FU), using $100{\mu}L$ of plasma. Accuracy and precision of quality control samples for both doxifluridine and 5-FU met KFDA and FDA Guidance criteria of 15% for accuracy with coefficients of variation less than 15%. This method demonstrated adequate sensitivity, specificity, accuracy, precision and stability to support the simultaneous analysis of doxifluridine and 5-FU in dog plasma samples in pharmacokinetic and bioequivalence studies.