• Title/Summary/Keyword: Limit of Detection

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Species-specific Marker Development for Environmental DNA Assay of Endangered Bull-head Torrent Catfish, Liobagrus obesus (멸종위기어류 퉁사리의 환경 DNA 분석을 위한 종 특이 마커 개발)

  • Yun, Bong Han;Kim, Yong Hwi;Sung, Mu Sung;Han, Ho-Seop;Han, Jeong-Ho;Bang, In-Chul
    • Korean Journal of Ichthyology
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    • v.34 no.3
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    • pp.208-217
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    • 2022
  • We wanted to develop a real-time PCR assay capable of detecting Liobagrus obesus in environmental DNA (eDNA) extracted from freshwater samples using a pair of species-specific primers and probe for the endangered fish, L. obesus. The species-specific primers and probe were designed in consideration of single nucleotide polymorphisms between 65 species of freshwater fish living in the Republic of Korea within the cytochrome b (cytb) gene of mitochondrial DNA. The species-specific primers and probe, in the real-time PCR assay, showed high specificity as only the L. obesus genomic DNA (gDNA) was found to be positive in the specificity verification using 65 species gDNA of freshwater fish in the Republic of Korea. In addition, in the detection limit analysis using the serial dilution concentrations of L. obesus gDNA, it was found that it was possible to detect up to 0.2 pg, showing high sensitivity. Afterwards, using the species-specific primers and probe, real-time PCR assay was performed on freshwater samples obtained from 8 stations in the mid-upper basin of Geum River. As a result, the cytb gene of L. obesus was detected in total 5 stations including all 3 stations where this species was collected at the time of field survey. Therefore, the species-specific primers and probe developed in present study, and the real-time PCR assay using them, can accurately detect the cytb gene of L. obesus from eDNA samples, which can be utilized to monitor the existing habitats of this species and to discover potential new habitats.

An Analytical Validation of the GenesWellTM BCT Multigene Prognostic Test in Patients with Early Breast Cancer (조기 유방암 환자를 위한 다지표 예후 예측 검사 GenesWellTM BCT의 분석적 성능 시험)

  • Kim, Jee-Eun;Kang, Byeong-il;Bae, Seung-Min;Han, Saebom;Jun, Areum;Han, Jinil;Cho, Min-ah;Choi, Yoon-La;Lee, Jong-Heun;Moon, Young-Ho
    • Korean Journal of Clinical Laboratory Science
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    • v.49 no.2
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    • pp.79-87
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    • 2017
  • GenesWell$^{TM}$ BCT is a 12-gene test suggesting the prognostic risk score (BCT Score) for distant metastasis within the first 10 years in early breast cancer patients with hormone receptor-positive, HER2-negative, and pN0~1 tumors. In this study, we validated the analytical performance of GenesWell$^{TM}$ BCT. Gene expression values were measured by a one-step, real-time qPCR, using RNA extracted from FFPE specimens of early breast cancer patients. Limit of Blank, Limit of Detection, and dynamic range for each of the 12 genes were assessed by serially diluted RNA pools. The analytical precision and specificity were evaluated by three different RNA samples representing low risk group, high risk group, and near-cutoff group in accordance with their BCT Scores. GenesWell$^{TM}$ BCT could detect gene expression of each of the 12 genes from less than $1ng/{\mu}L$ of RNA. Repeatability and reproducibility across multiple testing sites resulted in 100% and 98.3% consistencies of risk classification, respectively. Moreover, it was confirmed that the potential interference substances does not affect the risk classification of the test. The findings demonstrate that GenesWell$^{TM}$ BCT have high analytical performance with over 95% consistency for risk classification.

Study on Improvement in Reliability of Measurement and Analysis for Polycyclic Aromatic Hydrocarbons in the Atmosphere (대기 중 다환방향족탄화수소(PAHs)의 측정.분석 신뢰도 향상에 관한 고찰)

  • Lee, Min-Do;Lim, Yong-Jae;Lee, Sang-Uk;Kong, Boo-Joo;Lee, Sang-Deok;Han, Jin-Seok
    • Journal of Korean Society for Atmospheric Environment
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    • v.23 no.5
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    • pp.515-525
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    • 2007
  • In this study, various techniques for measurement and analysis of PAHs in the ambient air were verified in order to select a more reliable method. Sampling and analysis of PAHs were done by the EPA TO-13a method. QA/QC of the measurement was conducted to minimize errors in sampling and analyzing processes. The linearity of calibration curve of the PAH standards was good ($R^2{\geq}0.99$). Audit accuracy was evaluated using 5 internal standards of PAHs ($Naphthalene-d_8,\;Acenaphthene-d_{10},\;Phenanthrene-d_{10},\;Chrysene-d_{12},\;Perylene-d_{12}$). Relative standard deviations of the internal standard of the PAHs were ranged from 6.22% for $acenaphthene-d_{10}$ to 8.11% for $chrysene-d_{12}$. To evaluate the surrogate recoveries, two field surrogate standards of PAHs ($fluoranthene-d_{10},\;benzo(a)pyrene-d_{12}$) and two extract surrogate standards of the PAHs ($fluorene-d_{10},\;pyrene-d_{12}$) were spiked into all samples before field sampling and sample extraction, respectively. Recoveries of field the surrogate standards ranged from $80.4{\pm}12.2%$ for $fluoranthene-d_{10}$ to $66.2{\pm}12.8%$ for $benzo(a)pyrene-d_{12}$. Extraction recoveries of the surrogate standards ranged from $70.4{\pm}10.2%$ for $fluorene-d_{10}$ to $77.6{\pm}10.8%$ for $pyrene-d_{10}$. The detection limit of benzo(a)pyrene among 16 PAHs standards for quantitation was 20 pg.

EGFR Analysis in Cytologic Samples of Lung Adenocarcinoma by Microdissection (미세 절제에 의한 폐 선암 세포 검체에서 EGFR 분석)

  • Han, Jeong Yeon;Lee, Hoon Taek;Oh, Seo Young
    • Korean Journal of Clinical Laboratory Science
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    • v.47 no.3
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    • pp.125-131
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    • 2015
  • The discovery of activating mutations in EGFR in a subset of lung adenocarcinomas was a major advance in our understanding of lung adenocarcinoma biology, and has led to groundbreaking studies that have demonstrated the efficacy of tyrosine kinase inhibitor therapy. Cytologic specimen procedures have become increasingly popular for obtaining diagnostic material in lung carcinomas. However, frequently the small amount of material or sparseness of tumor cells obtained from cytologic preparations limit the number of specialized studies, such as mutation analysis, that can be performed. In this study we used microdissection to isolate small numbers of tumor cells to assess for EGFR mutations from 76 cytological smear slides of patients with lung adenocarcinomas. We compared our results with previous molecular assays that had been performed on either surgical or cytology specimens as part of the patient's initial clinical work-up. Not only were we able to detect the identical EGFR mutation through the pyrosequencing, but we were also able to consistently detect the mutation from as few as 25 microdissected tumor cells. Furthermore, isolating a purer population of tumor cells resulted in increased sensitivity of mutation detection as we were able to detect mutations from microdissection-enriched cases. Therefore, microdissection can not only significantly increase the number of lung adenocarcinoma patients that can be screened for EGFR mutations, but can also facilitate the use of cytologic samples in the newly emerging field of molecular-based personalized therapies.

Analysis of Butyltin Compounds and New Antifouling Agents in the Southwestern Korean Tidal flats (한국연안의 갯벌 중에 유기주석화합물 및 새로운 방오도료제의 분석)

  • Lee, Seong-Eon;You, Jae-Bum;Park, Jae-Hong;Lee, Yong-Woo;Won, Ho-Shik;Lee, Dong-Sup
    • Journal of the Korean Society for Marine Environment & Energy
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    • v.11 no.1
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    • pp.1-12
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    • 2008
  • The prohibition of usage of tributyltin (TBT) compounds and the legal usage of new antifouling agents have changed the Korean costal environments in recent. 39 sampling sites of southwestern tidal flats were chosen in order to investigate the concentration of antifouling agents, and results in 2006 were compared with previous results in 1998. The concentrations of TBT compounds in most of sites except Incheon (It1) have been drastically decreased. Interestingly, In Jebudo (Jt2), Mokpo(MOt4) and Suncheon(SUt3) sites were detected as below the limit of detection and it is because of the legal restriction of TBT compounds. However, in most of the sampling sites in Korea, new antifouling agents, viz. Irgarol 1051, Dichlofluanid and Chlorothalonil, were detected. In particular, Irgarol 1051 was detected with high concentrations. In Jebudo (Jt4), a high concentration of Irgarol 1051 of 159.45 ng $g^{-1}$(dry wt) was detected. We were able to observe that the concentration of TBT compounds are has gradually been reduced whereas the new major antifouling agents are easily detected in most Korean tidal flats.

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Relation of cyst counts with numbers of total nuclei of Pneumocystis carinii in rats (흰쥐 주폐포자충에 있어서 도말표본상 포낭의 수와 핵의 총 수와의 상관성)

  • Hong, Seong-Tae;Yu, Ji-Suk;Lee, Mi-Jeong
    • Parasites, Hosts and Diseases
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    • v.32 no.3
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    • pp.171-176
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    • 1994
  • Wistar rats were induced of Pneumowstis cayinii infection by injection with methyl-prednisolone to correlate the cyst counts and numbers of nuclei. Seven sections of the lungs were examined by impression smears and also whole lung homogenates were screened for nucleus counting for each rat. At the first week of the experiment, all of the Impression smears except one were cyst negative but trophic forms were counted around 106. At the third week, the cysts appeared one per 20 Immersion oil lens fields. The nuclei were on the order of 107 at this period, and this amount of Pc is regarded as the limit of cyst detection on Impression smears. When the nuclei were over 109 in the lungs, the cysts were counted about 50 in 20 microscopic fields. The organisms were distributed in the lungs without any predilection focus. The present data suggest that the trophic forms, proliferate first and the cysts appear later in the lungs.

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Current Status of Standard Diagnostics and Treatment for Malaria, Tuberculosis, and Hepatitis in Myanmar (미얀마의 말라리아, 결핵 및 간염의 표준 진단법 및 치료법 현황)

  • Han, Eun-Taek;Lee, Jong-Seok;Cheong, Jae-Hun;Chang, Chulhun L.;Nyunt, Myat Htut;Aung, Wah Wah;Kyaw, Yi Yi;Thant, Kyaw Zin
    • Laboratory Medicine Online
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    • v.7 no.3
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    • pp.94-102
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    • 2017
  • Malaria, tuberculosis, and hepatitis are common and notorious infectious diseases in Myanmar. Despite intensive efforts to control these diseases, their prevalence remains high. For malaria, which is a vector-borne disease, a remarkable success in the reduction of new cases has been achieved. However, the annual number of tuberculosis cases has increased over the last few decades, and the prevalence of chronic viral hepatitis infection has been high in Myanmar and other nearby countries. Early detection and prompt treatment are crucial to control these diseases. We have devoted our research efforts to understanding the status of these infectious diseases and working towards their eventual elimination for the last four years with the support of the Korea International Cooperation Agency. In the modern era, an infection that develops in one geographical area can spread globally because national borders do not effectively limit disease transmission. Our efforts to understand the status of infectious diseases in Myanmar will benefit not only Myanmar but also neighboring countries such as Korea.

The Topical Absorption of Ketoprofen from Gels and Plaster in Human Volunteers (케토프로펜 겔제와 플라스터제의 피부 흡수 비교)

  • Gang, Won-Gu;Lee, Chang-Hyeon;U, Jong-Su;Gwon, Gwang-Il
    • YAKHAK HOEJI
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    • v.42 no.1
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    • pp.25-30
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    • 1998
  • This study was designed to compare the absorption fraction and extent of ketoprofen gels and a matrix typed ketoprofen plaster patch. 3g (90mg as ketoprofen) of the two gels whi ch has oleohydrogel or hydrogel as a base, respectively, and 3 pieces of plaster patches (90mg as ketoprofen) were, applied in the area of 210$cm^2$ on forearm in 12 volunteers by cross over design. Blood samples were collected serially up to 24 hours and the plasma concentrations of ketoprofen were analyzed by HPLC using flurbiprofen as an internal standard. The detection limit of the assay was 1ng/ml of ketoprofen in plasma. The pharmacokinetic parameters (e.g. $AUC_{24hr}$, $AUMC_{24hr}$, MRT, Fraction Absorbed) were calculated from the plasma concentrations time data of each volunteer. The oleo-hydrogel showed significantly higher absorption fraction and extent of ketoprofen than the current hydrogel. The mean plasma concentrations of the oleo-hydrogel were increased to 98.46${\pm}$23.15ng/ml by 6 hour after application, and increased futher to 100.61${\pm}$18.65ng/ml at 24 hour. On the other hand, those of the hydrogel were increased 17.61${\pm}$18.65ng/ml at 5 hour to 34.68${\pm}$9.65ng/ml at 24 hour gradually. Therefore the plasma concentrations of oleo-hydrogel at each measured time were 3~7 times greater than those of the hydrogel with statistical significance. The $AUC_{24hr}$ (1797.26${\pm}$52.09ng.h/ml) of the oleo-hydrogel was 3.5 times greater (P<0.05) than that (516.17${\pm}$104.52ng.h/ml) of the hydrogel. The plaster patches showed higher bioavailability ($AUC_{24hr}$ 2877.37${\pm}$578.27ng.h/ml) than the olea-hydrogel ($AUC_{24hr}$ 1797.26${\pm}$52.09ng.h/ml) without statistical significance. But the absorption fraction of the oleo-hydrogel was rather higher than that of the plaster patches during the first 6 hours after administration. These results suggest that newly developed ketoprofen gel which is used oleo-hydrogel as a base would show excellent skin permeation on topical application for the corresponding clinical indications and could be absorbed as well as plaster patches.

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TLC, HPTLC FINGERPRINTING AND ACUTE ORAL TOXICITY EVALUATION OF HABB-E-AZARAQI: A NUX-VOMICA-BASED TRADITIONAL UNANI FORMULATION

  • Ara, Shabnam Anjum;Viquar, Uzma;Zakir, Mohammed;Husain, Gulam Mohammed;Naikodi, Mohammed Abdul Rasheed;Urooj, Mohd;Kazmi, Munawwar Husain
    • CELLMED
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    • v.11 no.3
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    • pp.13.1-13.9
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    • 2021
  • Background and Objective: Nux-vomica based traditional Unani formulation, Habb-e-Azaraqi (HAZ) is an important drug used by Unani physicians since several decades. It possesses Muqawwi-i-A'sab (nervine tonic), Muharrik-i-A'sab (nervine stimulant) properties and is an effective treatment option for diseases like Laqwa (facial palsy), Falij (paralysis), Niqris (gout) and Waja'al-Mafasil (arthritis) etc. The aim of the study is to access and provide information of HAZ for its TLC, HPTLC Fingerprinting defining its clear qualitative perspective and acute oral toxicity evaluation for its safety assessment which was not done earlier, thus contributing in the field of research. Materials and Methods: The chief ingredient, nux-vomica was detoxified as per method mentioned in Unani Pharmacopeia before its use in formulation. TLC and HPTLC was developed under four detection system i.e., UV 366nm, UV 254nm, exposure to iodine vapours and after derivatization with anisaldehyde sulphuric acid. Acute toxicity studies were performed as per OECD Guidelines 425 at a limit dose of 2000 mg/kg. Observations were done for signs of toxicity, body weight, and feed consumption at regular intervals followed by haematological and biochemistry evaluation. Results: The generated data proved the authenticity and established the TLC and HPTLC profile of the formulation. Acute toxicity revealed no significant differences in HAZ-treated animals with respect to body weight gain, feed consumption, haematology, clinical biochemistry evaluation. No significant gross pathological observation was noticed in necropsy. Conclusion: Data of the present study is substantial and scientific proof of HAZ in terms of standardization and toxicity study that can be utilize in future research activities.

Analysis of Volatile Organic Compounds in Sediments Using HS-GC/MS - Confirmation of Matrix Effects in External and Internal Standard Methods - (HS-GC/MS를 이용한 퇴적물 중 휘발성유기화합물 분석 - 외부 및 내부표준방법에서 매질영향 확인 -)

  • Shin, Myoung-Chul;Jung, Da-som;Noh, Hye-ran;Yu, Soon-ju;Seo, Yong-Chan;Lee, Bo-Mi
    • Journal of Korean Society on Water Environment
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    • v.37 no.6
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    • pp.510-519
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    • 2021
  • Volatile Organic Compounds (VOCs) in sediments, which can cause human health problems, have been monitored in Korea since 2014. Measured VOC concentrations can be affected by matrix type and the volatility of target substances. In this study, (1) VOCs volatility and the influence of matrix interference were confirmed, and (2) internal standards (IS) method was applied to improve analytical method. For these purposes, method detection limit (MDL), calibration linearity, precision and accuracy of VOCs were compared in various matrices using the IS. Some of VOCs in sediments showed different peak areas and reduced rates compared to water matrix. It was suggested that adsorption properties of sediments hindered the migration to vapor during heat pretreatment in headspace method. A calibration curve was created in clean sand. Recovery rates for the calibration curve method and IS applying method were 64.1~83.1% and 99.1~119.3%, respectively. Relative standard deviations ranged from 11.1% to 21.6% for the calibration curve method and those for IS ranged 4.7% to 13.7%. In case of real sediment, calibration curve and 1,2-Dichlorobenzene-d4 (ODCB) among IS were not suitable. The average recovery rate of Fluorobenzene (FBZ) increased by 56.4% and Relative Standard Deviation (RSD) by 4.7%. However, the recovery rate was increased in the samples with large values of igniting intensity. This study confirmed that influence of the matrix of VOCs in sediment, and addition of IS materials improved precision and accuracy. Although IS corrects volatilization and adsorption, it is recommended that more than two types of IS should be added rather than single.