• Title/Summary/Keyword: 등온증폭법

Search Result 23, Processing Time 0.022 seconds

Detection of Hepatitis B Virus by LAMP and DHPLC (등온증폭반응법과 변성 고성능 액체 크로마토그래피를 이용한 B형 간염 바이러스의 검출)

  • Ahn, Young-Chang;Seo, Jae-Won;Choi, Jae-Gu;Jang, Won-Cheoul
    • Journal of the Korean Chemical Society
    • /
    • v.55 no.2
    • /
    • pp.262-267
    • /
    • 2011
  • The denaturing high performance liquid chromatography(DHPLC) with fluorescence detector assay is very useful tool for detecting nucleic acids. Furthermore, loop-mediated isothermal amplification(LAMP) constitutes a potentially valuable tool for rapid diagnosis of pathogenic microorganisms. In this study, we evaluated the specificity, detection limit, and sensitivity of a LAMP method and DHPLC method for rapid detection of the hepatitis b virus(HBV). As a result, the LAMP assay reported here has the advantage of rapid detection whereas, DHPLC assay has more sensitivity than other assays. These findings suggest that LAMP and DHPLC assay may be good tool for rapid diagnosis of clinical HBV infection.

Detection for Methicillin Resistant Staphylococcus aureus in Using Bio-Chip Based Loop Mediated Isothermal Amplification Assay (칩 기반 등온증폭법을 이용한 약제 내성 포도상구균의 검출)

  • Cho, Min-Ho;Jang, Won-Cheoul;Choi, Jae-Gu
    • Journal of the Korean Chemical Society
    • /
    • v.57 no.1
    • /
    • pp.81-87
    • /
    • 2013
  • Staphylococcus aureus is the most important pathogen in nosocomial infections, including bloodstream infections. Prompt identification of S. aureus from blood cultures and detection of methicillin resistance are essential in cases of suspected sepsis. We have studied a new method for the sequence-specific visual detection of minute amounts of nucleic acids using intercalating reaction by addition of SYBR Green to amplicons of LAMP, and it's a unique gene amplification method in which DNA can be isothermally amplified using only one enzyme. Staphylococcus-LAMP, which targets the spa gene, encoding S.aureus-specific protein A, and the mecA gene, encoding penicillin-binding protein-2' for methicillin resistance, detected MRSA and MRSE. In this study, by using LAMP assay, I detected for Staphylococcus aureus and Staphylococcus epidermidis concentration in the clinical sample. The detection of Staphylococcus aureus and Staphylococcus epidermidis was tested by using serial 10-fold dilutions standard solution. I have accurate detected the limit of detection, sensitity, specificity and reproducibility of the assay. The Bio-chip based LAMP assay allowed easy, rapid, accurate and sensitive detection of infection with Staphylococcus and especially applicable in a resource-limited situation.

Detection of Salmonella Using the Loop Mediated Isothermal Amplification and Real-time PCR (등온 증폭법과 Real-time PCR을 이용한 Salmonella 검출)

  • Ahn, Young-Chang;Cho, Min-Ho;Yoon, Il-Kyu;Jung, Duck-Hyun;Lee, Eun-Young;Kim, Jin-Ho;Jang, Won-Cheoul
    • Journal of the Korean Chemical Society
    • /
    • v.54 no.2
    • /
    • pp.215-221
    • /
    • 2010
  • Salmonella is an important food-and water-borne pathogen associated with acute gastrointestinal illnesses around the world. The most common serotypes isolated from humans are Salmonella enterica serotype Typhimurium (S. Typhimurium) and S. Enteritidis. Traditional detection methods for Salmonella are based on cultures using selective media and characterization of suspicious colonies by biochemical and serological tests. These methods are generally time-consuming and not so highly sensitive. Recently, the Loop Mediated Isothermal Amplification and real-time PCR has been used as a highly sensitive, specific, and rapid test for the presence of pathogenic bacteria. In this study, a LAMP and real-time PCR was used to detect S. Typhimurium and S. Enteritidis. We selected target genes, which were the in invA and a randomly cloned sequence specific for the genus Salmonella. With LAMP and real-time PCR, random sequence was detected from Salmonella spp, invA were detected from all strain of S. Typhimurium and S. Enteritidis. This assay indicate that the specificity, sensitivity and rapid of the LAMP and real-time PCR make them potentially valuable tools for detection of S. Typhimurium and S. Enteritidis.

Detection of Mycobacterium Tuberculosis by Loop-Mediated Isothermal Amplification Assay (등온 증폭법을 이용한 결핵균의 빠른 검출 시스템 개발)

  • Ahn, Young-Chang;Nam, Youn-Hyoung;Park, Su-Min;Cho, Min-Ho;Seo, Jae-Won;Yoon, Il-Kyu;Park, Yong-Hyun;Jang, Won-Cheoul
    • Journal of the Korean Chemical Society
    • /
    • v.52 no.3
    • /
    • pp.273-280
    • /
    • 2008
  • Mycobacterium tuberculosis (MTB) remains a major worldwide public health problem. In recent years, the incidence of MTB has been rising. Rapid and reliable diagnosis of Mycobacterium tuberculosis is essential to initiate correct treatment, avoid severe complications, and prevent transmission. LAMP was used to develop a rapid and sensitive laboratory diagnostic system for the MTB. In this research, the loop-mediated isothermal amplification method (LAMP) that amplifies DNA with high specificity and rapidity at an isothermal condition was evaluated for rapid detection of MTB. Undiluted DNA (2.10 × 106 copy/mL), 10-1, 10-2, 10-3, 10-4, 10-5 and 10-6 (copy/mL) of MTB DNA were amplified by PCR and LAMP to determine the sensitivity of the assay. At results, the LAMP assay reported here has the advantages of rapid amplification, high sensitivity, and high specificity and will be useful for rapid and reliable clinical diagnosis of MTB in hospital clinical laboratory.

A Rapid and Simple Detection Assay for Rice Bacterial Leaf Blight by Recombinase Polymerase Amplification (벼 흰잎마름병의 신속하고 간편한 진단을 위한 Recombinase Polymerase Amplification 등온증폭법)

  • Kim, Shinhwa;Lee, Bong Choon;Kim, Hyun Ju;Choi, Soo Yeon;Seo, Su Jwa;Kim, Sang-Min
    • Research in Plant Disease
    • /
    • v.26 no.4
    • /
    • pp.195-201
    • /
    • 2020
  • Rice bacterial leaf blight (BLB) by Xanthomonas oryzae pv. oryzae (Xoo) is considered to be one of the major rice diseases steadily occurring around the rice-producing countries. In this study, we developed a recombinase polymerase amplification (RPA) assay for the rapid, convenient and specific diagnosis of Xoo by targeting Xoo-specific transposase A gene. As the target gene can be amplified in 10 min without DNA extraction process and special equipment for temperature control, RPA for BLB can be useful and practical component for on-site diagnosis.

Development of a Loop-mediated Isothermal Amplification Detection Assay for Verticillium dahliae Infection in Chrysanthemum (국화에 발생하는 반쪽시들음병균 Verticillium dahliae 검출용 등온 증폭법 개발)

  • Back, Chang-Gi;Park, Mi-Jeong;Han, Kyung-Sook;Park, Jong-Han
    • The Korean Journal of Mycology
    • /
    • v.47 no.4
    • /
    • pp.437-441
    • /
    • 2019
  • Verticillium wilt disease is caused by a fungal plant pathogen Verticillium dahliae, which attacks commercial crops such as chrysanthemum. The conventional methods so far used to identify this fungal pathogen require high expertise and are time-consuming. Therefore, in this study, we developed an assay for the rapid and specific detection of V. dahliae infection using loop-mediated isothermal amplification (LAMP) method. For this assay, four primers for LAMP were designed for targeting cellulose-growth-specific protein partial mRNA gene in Verticillium dahliae. Under standard condition, the optimum reaction temperature for amplification is around 60 ℃ within 60 minutes. This LAMP assay was designed to amplify only present in V. dahliae. When this LAMP assay applied to the DNAs for four other soil-borne fungi and host plants, no amplification was detected. Therefore, this LAMP assay we developed for V. dahliae is expected to do detection at the early stage of its infection. The fast and reliable detection method will allow us to develop effective management system to monitor and control infection of this pathogen in chrysanthemum plant.

Loop-mediated Isothermal Amplification (LAMP) for Detection of Streptococcus parauberis (Loop-mediated isothermal amplification (LAMP)법을 이용한 Streptococcus parauberis 의 신속 진단)

  • Moon, Kyung-Mi;Kim, Dong-Hwi;Heo, Moon-Soo
    • Journal of Life Science
    • /
    • v.24 no.4
    • /
    • pp.428-436
    • /
    • 2014
  • Loop-mediated isothermal amplification (LAMP) technique relies on autocycling strand displacement DNA sysnthesis without template denaturation steps under isothermal conditions. LAMP has more advantages than modern PCR, as it requires only basic laboratory equipment like an isothermal water bath, oven, and heating cabinet. Hence, in this study, five random primers were designed with Streptococcus parauberis, shikimate kinase Arok gene sequences (Genbank accession number: CP0024711). Two primers were selected based on the ladder pattern. Other optimum reaction conditions like temperature, time, and sensitivity were established and confirmed with conventional SYBR-green PCR. Results confirmed that the designed random primers were species specific, without any non-target DNA amplification under isothermal conditions. Hence, this study suggests that LAMP techniques could be used in the diagnosis of fish pathogen, specifically S. parauberis.

Diagnostic Method for the Detection of JC Polyomavirus Using Loop-mediated Isothermal Amplification (등온증폭법을 이용한 고감도 JC polyomaviruses 진단법 개발)

  • Cho, Kyu Bong
    • Korean Journal of Clinical Laboratory Science
    • /
    • v.51 no.4
    • /
    • pp.414-419
    • /
    • 2019
  • JC polyomavirus (JCPyV) is a human pathogenic virus belonging to the family Polyomaviridae, a viral group containing dsDNA nucleic acid. A recent recommendation is to apply the presence of JCPyV as a fecal indicator for water contamination in environments like sewage, and techniques to monitor JCPyV in water are being proposed. To date, the conventional PCR system has been applied as a diagnostic method for detecting JCPyV. There is a need for a more rapid and sensitive JCPyV diagnostic detection method in clinical and environmental samples. In this study, we developed a loop-mediated isothermal amplification (LAMP) primer set for the detection of JCPyV. Our results indicate that the LAMP method using a specific primer set shows about 10-fold higher detection sensitivity than the conventional PCR system. The effectiveness of the LAMP method developed in this study has been validated by PCR product digestion using the HaeIII restriction enzyme. We, therefore, propose that the LAMP method using a specific primer set can be applied as a rapid and sensitive detection method for monitoring JCPyV in clinical and environmental samples.

Rapid, Simultaneous Detection of Various Biological Toxin Genes Using Multiplex Reverse Transcription Loop-Mediated Isothermal Amplification(RT-LAMP) (다중 역전사-루프매개등온증폭법(RT-LAMP)를 이용한 생물 독소 유전자 신속 진단법)

  • Seungho Lee;Chanho Chung;Sehun Gu;Jungeun Kim;Hyeongseok Yun;Daesang Lee;Gyeunghaeng Hur;Donghyun Song
    • Journal of the Korea Institute of Military Science and Technology
    • /
    • v.27 no.4
    • /
    • pp.516-527
    • /
    • 2024
  • Rapid, early, accurate detection and identification of the various pathogenic agents associated with the development of biological weapons is critical in preventing loss of life and limiting the impact of these organisms when used against civilian or military targets. The aim of this study was to produce a system for the simple, rapid, accurate and simultaneous detection and identification of Ricin, Botulinum toxin B and Staphylococcal enterotoxin B as a proof of principle for developing field appropriate reverse transcription loop-mediated isothermal amplification systems for the accurate identification of potential biological threats. These systems were designed to facilitate the identification of potential threats even in remote or resource-limited locations.