12,121 research outputs found

    Detection of Pseudomonas fluorescens from broth, water and infected tissues by loop-mediated isothermal amplification (LAMP) method

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    Loop mediated isothermal amplification is rapid, highly sensitive and specifically developed method for detection of bacterial infections. AprX gene for alkaline metalloprotease of Pseudomonas fluorescens was used to design four primers and loop mediated isothermal amplification (LAMP) conditions were standardized for amplification of DNA. LAMP primers successfully amplified P. fluorescens from DNA and bacterial cells taken directly from broth, water and infected tissues with high specificity and sensitivity (10 pg) under isothermal condition at 61°C.Key words: Pseudomonas fluorescens, loop mediated isothermal amplification (LAMP), rapid, simple, specificity, sensitivity

    Microfluidic method for rapid turbidimetric detection of the DNA of Mycobacterium tuberculosis using loop-mediated isothermal amplification in capillary tubes

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    We describe a microfluidic method for rapid isothermal turbidimetric detection of the DNA of Mycobacterium tuberculosis. Loop-mediated isothermal amplification is accomplished in capillary tubes for amplifying DNA in less than 15 min, and sensitivity and specificity were compared to conventional loop-mediated isothermal amplification (LAMP). The method can detect as little as 1 pg mL−1 DNA in a sample. Results obtained with clinical specimens indicated 90 % sensitivity and 95 % specificity for microfluidic LAMP in comparison to culture methods. No interference occurred due to the presence of nonspecific DNAs. The findings demonstrate the power of the new microfluidic LAMP test for rapid molecular detection of microorganisms even when using bare eyes. © 2014, Springer-Verlag Wien

    Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification

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    Isothermal amplification assays, such as loop-mediated isothermal amplification (LAMP), show great utility for the development of rapid diagnostics for infectious diseases because they have high sensitivity, pathogen-specificity and potential for implementation at the point of care. However, elimination of non-specific amplification remains a key challenge for the optimization of LAMP assays. Here, using chlamydia DNA as a clinically relevant target and high-throughput sequencing as an analytical tool, we investigate a potential mechanism of non-specific amplification. We then develop a real-time digital LAMP (dLAMP) with high-resolution melting temperature (HRM) analysis and use this single-molecule approach to analyze approximately 1.2 million amplification events. We show that single-molecule HRM provides insight into specific and non-specific amplification in LAMP that are difficult to deduce from bulk measurements. We use real-time dLAMP with HRM to evaluate differences between polymerase enzymes, the impact of assay parameters (e.g. time, rate or florescence intensity), and the effect background human DNA. By differentiating true and false positives, HRM enables determination of the optimal assay and analysis parameters that leads to the lowest limit of detection (LOD) in a digital isothermal amplification assay

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    The development of loop-mediated isothermal amplification targeting alpha-tubulin DNA for the rapid detection of Plasmodium viva

    Capillary-based multiplexed isothermal nucleic acid-based test for sexually transmitted diseases in patients

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    We demonstrate a multiplexed loop mediated isothermal amplification (LAMP) assay for infectious disease diagnostics, where the analytical process flow of target pathogens genomic DNA is performed manually by moving magnetic beads through a series of plugs in a capillary. Heat is provided by a water bath and the results read by the naked eye, enabling applications in low resource settings

    Electric LAMP: Virtual Loop-Mediated Isothermal AMPlification

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    We present eLAMP, a PERL script, with Tk graphical interface, that electronically simulates Loop-mediated AMPlification (LAMP) allowing users to efficiently test putative LAMP primers on a set of target sequences. eLAMP can match primers to templates using either exact (via builtin PERL regular expressions) or approximate matching (via the tre-agrep library). Performance was tested on 40 whole genome sequences of Staphylococcus. eLAMP correctly predicted that the two tested primer sets would amplify from S. aureus genomes and not amplify from other Staphylococcus species. Open source (GNU Public License) PERL scripts are available for download from the New York Botanical Garden’s website

    Lack of correlation between reaction speed and analytical sensitivity in isothermal amplification reveals the value of digital methods for optimization: validation using digital real-time RT-LAMP

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    In this paper, we asked if it is possible to identify the best primers and reaction conditions based on improvements in reaction speed when optimizing isothermal reactions. We used digital single-molecule, real-time analyses of both speed and efficiency of isothermal amplification reactions, which revealed that improvements in the speed of isothermal amplification reactions did not always correlate with improvements in digital efficiency (the fraction of molecules that amplify) or with analytical sensitivity. However, we observed that the speeds of amplification for single-molecule (in a digital device) and multi-molecule (e.g. in a PCR well plate) formats always correlated for the same conditions. Also, digital efficiency correlated with the analytical sensitivity of the same reaction performed in a multi-molecule format. Our finding was supported experimentally with examples of primer design, the use or exclusion of loop primers in different combinations, and the use of different enzyme mixtures in one-step reverse-transcription loop-mediated amplification (RT-LAMP). Our results show that measuring the digital efficiency of amplification of single-template molecules allows quick, reliable comparisons of the analytical sensitivity of reactions under any two tested conditions, independent of the speeds of the isothermal amplification reactions

    Loop-Mediated isothermal amplification in human Cytomegalovirus diagnostic

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    The pathogenicity and the consequences of human cytomegalovirus (HCMV) infection in immunocompromised persons are reported in the literature and, HCM Viral DNA detection is the reliable indicator of infection especially to study its reactivation after organ transplantation in the recipient patients. Congenital infections in pregnant women cause opportunistic diseases of the central nervous system in new-born.  HCMV increases suffering and death in patients with chronic diseases like diabetes, tuberculosis, hepatitis C or patients with immunodeficient HIV/AIDS. Diagnostic methods are present in medical analysis laboratories either by immunofluorescence, Chemiluminescence, immunochromatography, western blot or by molecular biology techniques such as PCR are used to detect qualitatively and/or quantitatively IgG and IgM antibodies in the organism using serum or plasma as samples. The follow-up and treatment of patients are carried out in case the clinical data correlate with specific pathologies and symptoms, thus, the analysis of cytomegalovirus by PCR is requested to be sure of the cause of this pathology and to eliminate other doubts because this virus shares genetic and pathological similarity with Epstein-Barr virus (EBV), Varicella-Zoster virus (VZV) and Herpes Simplex Virus (HSV) as they are all of the same family of the Herpesviridae. The viral load quantified by PCR is a key element for monitoring and treatment. Molecular biology researchers have developed different PCR amplification techniques to answer the question of sensitivity, efficiency, and speed of this technique, and more recently in 2000, Notomi and the EIKEN Chemical Co. Ltd. developed a method of amplification under isothermal conditions named LAMP (Loop-mediated isothermal amplification) which combines speed and easy access for all laboratories that want to use only the less expensive equipment like those used with real-time PCR, later this technique was validated by the World Health Organization (WHO) and the organization recommended it to be used as an alternative in research and diagnostics. In this bibliographic study, we will exploit the contribution of the LAMP method in the diagnosis of human cytomegalovirus

    Deteksi Vibrio harveyi dengan Metode Amplifikasi DNA pada Gen toxR

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    Abstrak -Infeksi hewan akuakultur oleh Vibrio harveyi dapat menyebabkan kematian serta kerugian ekonomi. Kemampuan untuk mendeteksi bakteri tersebut secara dini dapat mencegah penyebarannya dalam akuakultur. Penelitian ini bertujuan untuk mengembangkan metode untuk mendeteksi Vibrio harveyi melalui amplifikasi gen toxR. Amplifikasi DNA dilakukan dengan dua metode, yakni amplifikasi isotermal termediasi loop (loop-mediated isothermal amplification, LAMP) dan reaksi berantai polimerase (PCR). Amplifikasi menggunakan metode LAMP menunjukan perlu dilakukan optimasi protokol maupun desain primer untuk mencegah perolehan hasil false positive. Amplifikasi menggunakan metode PCR menghasilkan produk berukuran 229 pasang basa yang spesifik pada Vibrio harveyi dengan batas deteksi hingga 0,526 ng.µL-1 (setara 2,09 × 106 CFU.mL-1). Kata kunci: Akuakultur, LAMP, PCR, toxR, Vibrio harveyi Abstract -Vibrio harveyi infection in aquacultures may cause death and economical loss. Rapid detection of this bacteria may prevent its dispersal in aquacultures. The goal of this research was to develop method in detection of Vibrio harveyi via amplification of toxR gene. DNA amplification was carried out with two methods, loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). Amplification with LAMP suggest optimization of either protocol or primer design was needed to prevents false positive results. Amplification with PCR yields 229 bp-length product specific to Vibrio harveyi with detection limit up to 0.526 ng.µL-1 (equals to 2.09 × 106 CFU.mL-1). Keywords: Aquaculture, LAMP, PCR, toxR, Vibrio harvey

    Biosensors Based on Isothermal DNA Amplification for Bacterial Detection in Food Safety and Environmental Monitoring

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    The easy and rapid spread of bacterial contamination and the risk it poses to human health makes evident the need for analytical methods alternative to conventional time-consuming laboratory-based techniques for bacterial detection. To tackle this demand, biosensors based on isothermal DNA amplification methods have emerged, which avoid the need for thermal cycling, thus facilitating their integration into small and low-cost devices for in situ monitoring. This review focuses on the breakthroughs made on biosensors based on isothermal amplification methods for the detection of bacteria in the field of food safety and environmental monitoring. Optical and electrochemical biosensors based on loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), helicase dependent amplification (HDA), strand displacement amplification (SDA), and isothermal strand displacement polymerisation (ISDPR) are described, and an overview of their current advantages and limitations is provided. Although further efforts are required to harness the potential of these emerging analytical techniques, the coalescence of the different isothermal amplification techniques with the wide variety of biosensing detection strategies provides multiple possibilities for the efficient detection of bacteria far beyond the laboratory bench.info:eu-repo/semantics/publishedVersio
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