• Title/Summary/Keyword: Molecular Detection

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Optimization of ultra-fast convection polymerase chain reaction conditions for pathogen detection with nucleic acid lateral flow immunoassay

  • Kim, Tae-Hoon;Hwang, Hyun Jin;Kim, Jeong Hee
    • International Journal of Oral Biology
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    • v.44 no.1
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    • pp.8-13
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    • 2019
  • Recently, the importance of on-site detection of pathogens has drawn attention in the field of molecular diagnostics. Unlike in a laboratory environment, on-site detection of pathogens is performed under limited resources. In this study, we tried to optimize the experimental conditions for on-site detection of pathogens using a combination of ultra-fast convection polymerase chain reaction (cPCR), which does not require regular electricity, and nucleic acid lateral flow (NALF) immunoassay. Salmonella species was used as the model pathogen. DNA was amplified within 21 minutes (equivalent to 30 cycles of polymerase chain reaction) using ultra-fast cPCR, and the amplified DNA was detected within approximately 5 minutes using NALF immunoassay with nucleic acid detection (NAD) cassettes. In order to avoid false-positive results with NAD cassettes, we reduced the primer concentration or ultra-fast cPCR run time. For singleplex ultra-fast cPCR, the primer concentration needed to be lowered to $3{\mu}M$ or the run time needed to be reduced to 14 minutes. For duplex ultra-fast cPCR, $2{\mu}M$ of each primer set needed to be used or the run time needed to be reduced to 14 minutes. Under the conditions optimized in this study, the combination of ultra-fast cPCR and NALF immunoassay can be applied to on-site detection of pathogens. The combination can be easily applied to the detection of oral pathogens.

Characterization and RT-PCR Detection of dsRNA Mycoviruses from the Oyster Mushroom, Pleurotus ostreatus

  • Seo, Jang-Kyun;Lim, Won-Seok;Jeong, Ji-Hye;Yoo, Young-Bok;Yie, Se-Won;Kim, Kook-Hyung
    • The Plant Pathology Journal
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    • v.20 no.3
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    • pp.200-205
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    • 2004
  • The partial nucleotide sequences of the genomic dsRNA mycovirus infecting Pleurotus ostreatus isolates ASI2223 and Suhan were determined and compared with those of mycoviruses belonging to partitiviruses and totiviruses. Partial nucleotide sequences of the purified dsRNA from ASI2223 and Suhan showed RNA-dependent RNA polymerase sequences that are closely related to those of partitiviruses, including Fusarium poae virus 1, Fusarium solani virus, Rhizoctoniasolani virus, Discula destructiva virus 2, and Oyster mushroom isometric virus 2. Specific primers were designed for RT-PCR detection of dsRNA viruses from the P. ostreatus isolate ASI2223 and Suhan. Two virus specific primer sets were found to specifically detect each virus among six sets of designed oligonucleotide primers. Collectively, these results suggest that dsRNA mycoviruses from P. ostreatus isolates ASI2223 and Suhan belong to the family Partitiviridae, although, they are not the same virus species. Our results also suggest that these virus-specific primer sets can be employed for the specific detection of each viral sequence in infected tissues.

Anticancer Activity of Atractylodes lancea (Thunb.) DC in a Hamster Model and Application of PET-CT for Early Detection and Monitoring Progression of Cholangiocarcinoma

  • Plengsuriyakarn, Tullayakorn;Matsuda, Naoki;Karbwang, Juntra;Viyanant, Vithoon;Hirayama, Kenji;Na-Bangchang, Kesara
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.15
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    • pp.6279-6284
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    • 2015
  • Opisthorchis viverrini (OV)-induced cholangiocarcinoma (CCA) is an important cancer in the Great Mekong region, particularly in Thailand. Limitations of treatment options and the lack of an effective diagnostic tool for early detection of CCA are major concerns for the control of this type of cancer. The aim of the study was to investigate anti-CCA activity of the ethanolic extract of Atractylodes lancea (Thunb.) DC., and the applicability of positron emission tomography-computed tomography (PET-CT) as a tool for detection and monitoring the progression of CCA in Opisthorchis viverrini (OV)/dimethylnitrosamine (DMN)-induced CCA hamsters. Male Syrian hamsters were used for toxicity tests and anti-CCA activity evaluation. Development of CCA was induced by initial feeding of 50 metacercariae of OV, followed by drinking water containing 12.5 ppm of DMN in hamsters. The ethanolic extract of A. lancea (Thunb.) DC. was administered orally for 30 days. PET-CT was performed every 4 weeks after initiation of CCA using 18F-fluorodeoxyglucose ($^{18}F-FDG$). Results from the present study suggest that the ethanolic extract of A. lancea (Thunb.) DC. rhizome exhibited promising anti-CCA activity and safety profile in the OV/DMN-induced hamster model. To successfully apply PET-CT as a tool for early detection of tumor development and progression, modification of radiolabeling approach is required to improve its specificity for CCA cells.

Detection of the BCR/abl Gene Rearrangement by Reverse Transcriptase Based Polymerase Chain Reaction

  • Lee, Kyung-Ok;Park, Young-Suk;Kim, Yong-Woo;Han, Jung-A;Kim, Yoon-Jung
    • BMB Reports
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    • v.29 no.3
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    • pp.241-247
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    • 1996
  • The Philadelphia (Ph) chromosome is the single most intensively studied chromosome alteration characterizing a human malignancy. The specific genetic alteration of chronic myelogenous leukemia (CML) is the formation of the BCR/abl fusion gene in leukemic cells. The presence of the BCR/abl gene has important diagnostic and prognostic implications in CML. The detection of BCR/abl transcripts by reverse transcriptase based polymerase chain reaction (RT-PCR) was investigated in patients with CML in whom the Ph chromosome abnormality was documented by cytogenetic analysis. In a total of 68 CML patient cases, the Ph chromosome was found in 53 cases (77.9%) by cytogenetic analysis. On the other hand, sixty two cases (91.2%) were detected to have BCR/abl gene rearrangement Of these, b3a2 was 44 cases (64.7%) and b2a2 was 17 cases (25,0%). There was one case with both b3a2 and b2a2 (1.5%). Of the fifteen cases of Ph chromosome negative by cytogenetic anlaysis, the BCR/abl gene was observed in nine cases, The results of BCR/abl fusion gene confirmed by the direct sequencing method correlated well with PCR analysis, The amplified PCR products were detected by $1{\times}10^{-5}$ dilutions. In conclusion, PCR technique is sensitive, rapid and relatively simple for a laboratory test in detecting the BCR/abl fusion gene with CML regardless of the result of cytogenetic analysis.

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