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Sample records for helicase-dependent isothermal amplification

  1. Isothermal DNA amplification in vitro: the helicase-dependent amplification system.

    PubMed

    Jeong, Yong-Joo; Park, Kkothanahreum; Kim, Dong-Eun

    2009-10-01

    Since the development of polymerase chain reaction, amplification of nucleic acids has emerged as an elemental tool for molecular biology, genomics, and biotechnology. Amplification methods often use temperature cycling to exponentially amplify nucleic acids; however, isothermal amplification methods have also been developed, which do not require heating the double-stranded nucleic acid to dissociate the synthesized products from templates. Among the several methods used for isothermal DNA amplification, the helicase-dependent amplification (HDA) is discussed in this review with an emphasis on the reconstituted DNA replication system. Since DNA helicase can unwind the double-stranded DNA without the need for heating, the HDA system provides a very useful tool to amplify DNA in vitro under isothermal conditions with a simplified reaction scheme. This review describes components and detailed aspects of current HDA systems using Escherichia coli UvrD helicase and T7 bacteriophage gp4 helicase with consideration of the processivity and efficiency of DNA amplification. PMID:19629390

  2. Direct detection of nasal Staphylococcus aureus carriage via helicase-dependent isothermal amplification and chip hybridization

    PubMed Central

    2012-01-01

    Background The bacterium Staphylococcus aureus constitutes one of the most important causes of nosocomial infections. One out of every three individuals naturally carries S. aureus in their anterior nares, and nasal carriage is associated with a significantly higher infection rate in hospital settings. Nasal carriage can be either persistent or intermittent, and it is the persistent carriers who, as a group, are at the highest risk of infection and who have the highest nasal S. aureus cell counts. Prophylactic decolonization of S. aureus from patients’ noses is known to reduce the incidence of postsurgical infections, and there is a clear rationale for rapid identification of nasal S. aureus carriers among hospital patients. Findings A molecular diagnostic assay was developed which is based on helicase-dependent target amplification and amplicon detection by chip hybridization to a chip surface, producing a visible readout. Nasal swabs from 70 subjects were used to compare the molecular assay against culturing on “CHROMagar Staph aureus” agar plates. The overall relative sensitivity was 89%, and the relative specificity was 94%. The sensitivity rose to 100% when excluding low-count subjects (<100 S. aureus colony-forming units per swab). Conclusions This molecular assay is much faster than direct culture and has sensitivity that is appropriate for identification of high-count (>100 S. aureus colony-forming units per swab) nasal S. aureus carriers who are at greatest risk for nosocomial infections. PMID:22882800

  3. Helicase-Dependent Isothermal Amplification of DNA and RNA by Using Self-Avoiding Molecular Recognition Systems.

    PubMed

    Yang, Zunyi; McLendon, Chris; Hutter, Daniel; Bradley, Kevin M; Hoshika, Shuichi; Frye, Carole B; Benner, Steven A

    2015-06-15

    Assays that detect DNA or RNA (xNA) are highly sensitive, as small amounts of xNA can be amplified by PCR. Unfortunately, PCR is inconvenient in low-resource environments, and requires equipment and power that might not be available in these environments. Isothermal procedures, which avoid thermal cycling, are often confounded by primer dimers, off-target priming, and other artifacts. Here, we show how a "self avoiding molecular recognition system" (SAMRS) eliminates these artifacts and gives clean amplicons in a helicase-dependent isothermal amplification (SAMRS-HDA). We also show that incorporating SAMRS into the 3'-ends of primers facilitates the design and screening of primers for HDA assays. Finally, we show that SAMRS-HDA can be twofold multiplexed, difficult to achieve with HDA using standard primers. Thus, SAMRS-HDA is a more versatile approach than standard HDA, with a broader applicability for xNA-targeted diagnostics and research. PMID:25953623

  4. Rapid detection of Staphylococcus aureus in dairy and meat foods by combination of capture with silica-coated magnetic nanoparticles and thermophilic helicase-dependent isothermal amplification.

    PubMed

    Chen, Xingxing; Wu, Xiaoli; Gan, Min; Xu, Feng; He, Lihua; Yang, Dong; Xu, Hengyi; Shah, Nagendra P; Wei, Hua

    2015-03-01

    Staphylococcus aureus is one of the main pathogens in dairy and meat products; therefore, developing a highly sensitive and rapid method for its detection is necessary. In this study, a quantitative detection method for Staph. aureus was developed using silica-coated magnetic nanoparticles and thermophilic helicase-dependent isothermal amplification. First, genomic DNA was extracted from lysed bacteria using silica-coated magnetic nanoparticles and amplified using thermophilic helicase-dependent isothermal amplification. After adding the nucleic-acid dye SYBR Green I to the amplicons, the fluorescence intensity was observed using a UV lamp or recorded using a fluorescence spectrophotometer. This detection system had a detection limit of 5×10(0) cfu/mL in pure culture and milk-powder samples and 5×10(1) cfu/mL in pork samples using a UV light in less than 2h. In addition, a good linear relationship was obtained between fluorescence intensity and bacterial concentrations ranging from 10(2) to 10(4) cfu/mL under optimal conditions. Furthermore, the results from contaminated milk powder and pork samples suggested that the detection system could be used for the quantitative analysis of Staph. aureus and applied potentially to the food industry for the detection of this pathogen. PMID:25547304

  5. Characterization of a thermostable UvrD helicase and its participation in helicase-dependent amplification.

    PubMed

    An, Lixin; Tang, Wen; Ranalli, Tamara A; Kim, Hyun-Jin; Wytiaz, Jamie; Kong, Huimin

    2005-08-12

    Helicase-dependent amplification (HDA) is an isothermal in vitro DNA amplification method based upon the coordinated actions of helicases to separate double-stranded DNA and DNA polymerases to synthesize DNA. Previously, a mesophilic form of HDA (mHDA) utilizing the Escherichia coli UvrD helicase, DNA polymerase I Klenow fragment, two accessory proteins, MutL and single-stranded DNA-binding protein (SSB), was developed (1). In an effort to improve the specificity and performance of HDA, we have cloned and purified a thermostable UvrD helicase (Tte-UvrD) and the mutL homolog (Tte-MutL) from Thermoanaerobacter tengcongensis. Characterization of the Tte-UvrD helicase shows that it is stable and active from 45 to 65 degrees C. We have found that the Tte-UvrD helicase unwinds blunt-ended DNA duplexes as well as substrates possessing 3'- or 5'-ssDNA tails. Tte-UvrD was used to develop athermophilichelicase-dependent amplification (tHDA) system to selectively amplify target sequences at 60-65 degrees C. The tHDA system is more efficient than mHDA, displaying heightened amplification sensitivity without the need for the MutL and SSB accessory proteins. Amplification independent of MutL corresponds with studies demonstrating that maximal Tte-UvrD helicase activity does not require the mutL homolog. The tHDA system allows for rapid amplification and detection of targets present in genomic DNA. The expeditious nature and simplistic design of the tHDA platform makes the technology ideal for use in diagnostic applications requiring rapid identification of organisms at the point-of-need. PMID:15955821

  6. Targeting helicase-dependent amplification products with an electrochemical genosensor for reliable and sensitive screening of genetically modified organisms.

    PubMed

    Moura-Melo, Suely; Miranda-Castro, Rebeca; de-Los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo J; Dos Santos Junior, J Ribeiro; da Silva Fonseca, Rosana A; Lobo-Castañón, Maria Jesús

    2015-08-18

    Cultivation of genetically modified organisms (GMOs) and their use in food and feed is constantly expanding; thus, the question of informing consumers about their presence in food has proven of significant interest. The development of sensitive, rapid, robust, and reliable methods for the detection of GMOs is crucial for proper food labeling. In response, we have experimentally characterized the helicase-dependent isothermal amplification (HDA) and sequence-specific detection of a transgene from the Cauliflower Mosaic Virus 35S Promoter (CaMV35S), inserted into most transgenic plants. HDA is one of the simplest approaches for DNA amplification, emulating the bacterial replication machinery, and resembling PCR but under isothermal conditions. However, it usually suffers from a lack of selectivity, which is due to the accumulation of spurious amplification products. To improve the selectivity of HDA, which makes the detection of amplification products more reliable, we have developed an electrochemical platform targeting the central sequence of HDA copies of the transgene. A binary monolayer architecture is built onto a thin gold film where, upon the formation of perfect nucleic acid duplexes with the amplification products, these are enzyme-labeled and electrochemically transduced. The resulting combined system increases genosensor detectability up to 10(6)-fold, allowing Yes/No detection of GMOs with a limit of detection of ∼30 copies of the CaMV35S genomic DNA. A set of general utility rules in the design of genosensors for detection of HDA amplicons, which may assist in the development of point-of-care tests, is also included. The method provides a versatile tool for detecting nucleic acids with extremely low abundance not only for food safety control but also in the diagnostics and environmental control areas. PMID:26198403

  7. Enhancing helicase-dependent amplification by fusing the helicase with the DNA polymerase.

    PubMed

    Motré, Aurélie; Li, Ying; Kong, Huimin

    2008-08-15

    In this study, we have engineered a new bifunctional protein named "helimerase", by physically linking Thermoanaerobacter tengcongensis UvrD helicase (TteUvrD) and Bacillus stearothermophilus DNA polymerase I Large Fragment (Bstpol) using a coiled-coil. TteUvrD is fused with one part of the coiled-coil, WinZip-A2 (WZA2), through a linker L1 and possesses a Maltose Binding Protein (MBP) tag at the N terminal end. Bstpol is fused with the other part of the coiled-coil, WinZip-B1 (WZB1), through the same linker and possesses a His tag at the N terminal end. Therefore, the two fusion proteins MBP-WZA2-L1-TteUvrD and His-WZB1-L1-Bstpol composed the helimerase. We showed that this complex could be formed in vivo as well as in vitro and possessed specific activities of both TteUvrD and Bstpol. Moreover, we demonstrated that significantly longer fragments could be amplified by the helimerase in Helicase-Dependent Amplification (HDA) reactions instead of using TteUvrD and Bstpol proteins that do not form a complex. PMID:18556147

  8. Bioanalytical applications of isothermal nucleic acid amplification techniques.

    PubMed

    Deng, Huimin; Gao, Zhiqiang

    2015-01-01

    The most popular in vitro nucleic acid amplification techniques like polymerase chain reaction (PCR) including real-time PCR are costly and require thermocycling, rendering them unsuitable for uses at point-of-care. Highly efficient in vitro nucleic acid amplification techniques using simple, portable and low-cost instruments are crucial in disease diagnosis, mutation detection and biodefense. Toward this goal, isothermal amplification techniques that represent a group of attractive in vitro nucleic acid amplification techniques for bioanalysis have been developed. Unlike PCR where polymerases are easily deactivated by thermally labile constituents in a sample, some of the isothermal nucleic acid amplification techniques, such as helicase-dependent amplification and nucleic acid sequence-based amplification, enable the detection of bioanalytes with much simplified protocols and with minimal sample preparations since the entire amplification processes are performed isothermally. This review focuses on the isothermal nucleic acid amplification techniques and their applications in bioanalytical chemistry. Starting off from their amplification mechanisms and significant properties, the adoption of isothermal amplification techniques in bioanalytical chemistry and their future perspectives are discussed. Representative examples illustrating the performance and advantages of each isothermal amplification technique are discussed along with some discussion on the advantages and disadvantages of each technique. PMID:25467448

  9. Isothermal Amplification of Nucleic Acids.

    PubMed

    Zhao, Yongxi; Chen, Feng; Li, Qian; Wang, Lihua; Fan, Chunhai

    2015-11-25

    Isothermal amplification of nucleic acids is a simple process that rapidly and efficiently accumulates nucleic acid sequences at constant temperature. Since the early 1990s, various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). These isothermal amplification methods have been used for biosensing targets such as DNA, RNA, cells, proteins, small molecules, and ions. The applications of these techniques for in situ or intracellular bioimaging and sequencing have been amply demonstrated. Amplicons produced by isothermal amplification methods have also been utilized to construct versatile nucleic acid nanomaterials for promising applications in biomedicine, bioimaging, and biosensing. The integration of isothermal amplification into microsystems or portable devices improves nucleic acid-based on-site assays and confers high sensitivity. Single-cell and single-molecule analyses have also been implemented based on integrated microfluidic systems. In this review, we provide a comprehensive overview of the isothermal amplification of nucleic acids encompassing work published in the past two decades. First, different isothermal amplification techniques are classified into three types based on reaction kinetics. Then, we summarize the applications of isothermal amplification in bioanalysis, diagnostics, nanotechnology, materials science, and device integration. Finally, several challenges and perspectives in the field are discussed. PMID:26551336

  10. Zero-Background Helicase-Dependent Amplification and Its Application to Reliable Assay of Telomerase Activity in Cancer Cell by Eliminating Primer-Dimer Artifacts.

    PubMed

    Chen, Feng; Zhang, Dexin; Zhang, Qing; Zuo, Xiaolei; Fan, Chunhai; Zhao, Yongxi

    2016-06-16

    Primer-dimer artifacts resulting from unintended template-independent primer-primer interactions often hinder the specific amplification of nucleic acids. We demonstrate, for the first time, zero-background helicase-dependent amplification (HDA), with low concentrations of both ATP and dNTPs. This strategy achieved the reliable evaluation of telomerase activity in cancer cells by eliminating primer-dimer artifacts, which have plagued many previous methods with reduced specificity. We found that the performance of the telomerase assay by zero-background HDA was negatively affected by highly concentrated cellular proteins. This inhibitory effect is attributed to the binding of DNA templates to proteins, thus making them unavailable for polymerases. However, gold nanoparticles were demonstrated to highly attenuate such inhibition by abundant proteins, and to enhance the assay sensitivity and reliability when the reaction was performed with concentrated cell extracts. PMID:26690725

  11. Isothermal amplification of insect DNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The loop-mediated isothermal amplification of DNA (LAMP) method can amplify a target DNA sequence at a constant temperature in about 1 hour. LAMP has broad application in agriculture and medicine because of the need for rapid and inexpensive diagnoses. The power of LAMP is being used by researchers ...

  12. Isothermal Amplification of Insect DNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The loop-mediated isothermal amplification of DNA (LAMP) method can amplify a target DNA sequence at a constant temperature in about one hour. LAMP has broad application in agriculture and medicine because of the need for rapid and inexpensive diagnoses. LAMP eliminates the need for temperature cycl...

  13. Polyethersulfone improves isothermal nucleic acid amplification compared to current paper-based diagnostics.

    PubMed

    Linnes, J C; Rodriguez, N M; Liu, L; Klapperich, C M

    2016-04-01

    Devices based on rapid, paper-based, isothermal nucleic acid amplification techniques have recently emerged with the potential to fill a growing need for highly sensitive point-of-care diagnostics throughout the world. As this field develops, such devices will require optimized materials that promote amplification and sample preparation. Herein, we systematically investigated isothermal nucleic acid amplification in materials currently used in rapid diagnostics (cellulose paper, glass fiber, and nitrocellulose) and two additional porous membranes with upstream sample preparation capabilities (polyethersulfone and polycarbonate). We compared amplification efficiency from four separate DNA and RNA targets (Bordetella pertussis, Chlamydia trachomatis, Neisseria gonorrhoeae, and Influenza A H1N1) within these materials using two different isothermal amplification schemes, helicase dependent amplification (tHDA) and loop-mediated isothermal amplification (LAMP), and traditional PCR. We found that the current paper-based diagnostic membranes inhibited nucleic acid amplification when compared to membrane-free controls; however, polyethersulfone allowed for efficient amplification in both LAMP and tHDA reactions. Further, observing the performance of traditional PCR amplification within these membranes was not predicative of their effects on in situ LAMP and tHDA. Polyethersulfone is a new material for paper-based nucleic acid amplification, yet provides an optimal support for rapid molecular diagnostics for point-of-care applications. PMID:26906904

  14. Polyethersulfone improves isothermal nucleic acid amplification compared to current paper-based diagnostics

    PubMed Central

    Linnes, J. C.; Rodriguez, N. M.; Liu, L.

    2016-01-01

    Devices based on rapid, paper-based, isothermal nucleic acid amplification techniques have recently emerged with the potential to fill a growing need for highly sensitive point-of-care diagnostics throughout the world. As this field develops, such devices will require optimized materials that promote amplification and sample preparation. Herein, we systematically investigated isothermal nucleic acid amplification in materials currently used in rapid diagnostics (cellulose paper, glass fiber, and nitrocellulose) and two additional porous membranes with upstream sample preparation capabilities (polyethersulfone and polycarbonate). We compared amplification efficiency from four separate DNA and RNA targets (Bordetella pertussis, Chlamydia trachomatis, Neisseria gonorrhoeae, and Influenza A H1N1) within these materials using two different isothermal amplification schemes, helicase dependent amplification (tHDA) and loop-mediated isothermal amplification (LAMP), and traditional PCR. We found that the current paper-based diagnostic membranes inhibited nucleic acid amplification when compared to membrane-free controls; however, polyethersulfone allowed for efficient amplification in both LAMP and tHDA reactions. Further, observing the performance of traditional PCR amplification within these membranes was not predicative of their effects on in situ LAMP and tHDA. Polyethersulfone is a new material for paper-based nucleic acid amplification, yet provides an optimal support for rapid molecular diagnostics for point-of-care applications. PMID:26906904

  15. Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.

    PubMed

    Huang, Shichu; Do, Jaephil; Mahalanabis, Madhumita; Fan, Andy; Zhao, Lei; Jepeal, Lisa; Singh, Satish K; Klapperich, Catherine M

    2013-01-01

    In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile) DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA) to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10(-2) pg of C. difficile DNA) while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health. PMID:23555883

  16. Detection of the 35S promoter in transgenic maize via various isothermal amplification techniques: a practical approach.

    PubMed

    Zahradnik, Celine; Kolm, Claudia; Martzy, Roland; Mach, Robert L; Krska, Rudolf; Farnleitner, Andreas H; Brunner, Kurt

    2014-11-01

    In 2003 the European Commission introduced a 0.9% threshold for food and feed products containing genetically modified organism (GMO)-derived components. For commodities containing GMO contents higher than this threshold, labelling is mandatory. To provide a DNA-based rapid and simple detection method suitable for high-throughput screening of GMOs, several isothermal amplification approaches for the 35S promoter were tested: strand displacement amplification, nicking-enzyme amplification reaction, rolling circle amplification, loop-mediated isothermal amplification (LAMP) and helicase-dependent amplification (HDA). The assays developed were tested for specificity in order to distinguish between samples containing genetically modified (GM) maize and non-GM maize. For those assays capable of this discrimination, tests were performed to determine the lower limit of detection. A false-negative rate was determined to rule out whether GMO-positive samples were incorrectly classified as GMO-negative. A robustness test was performed to show reliable detection independent from the instrument used for amplification. The analysis of three GM maize lines showed that only LAMP and HDA were able to differentiate between the GMOs MON810, NK603, and Bt11 and non-GM maize. Furthermore, with the HDA assay it was possible to realize a detection limit as low as 0.5%. A false-negative rate of only 5% for 1% GM maize for all three maize lines shows that HDA has the potential to be used as an alternative strategy for the detection of transgenic maize. All results obtained with the LAMP and HDA assays were compared with the results obtained with a previously reported real-time PCR assay for the 35S promoter in transgenic maize. This study presents two new screening assays for detection of the 35S promoter in transgenic maize by applying the isothermal amplification approaches HDA and LAMP. PMID:24880871

  17. Loop-Mediated Isothermal Amplification (LAMP) Signature Identification Software

    Energy Science and Technology Software Center (ESTSC)

    2009-03-17

    This is an extendable open-source Loop-mediated isothermal AMPlification (LAMP) signature design program called LAVA (LAMP Assay Versatile Analysis). LAVA was created in response to limitations of existing LAMP signature programs.

  18. The development of nanostructure assisted isothermal amplification in biosensors.

    PubMed

    Duan, Ruixue; Lou, Xiaoding; Xia, Fan

    2016-03-21

    Developing simple and inexpensive methods to ultrasensitively detect biomarkers is important for medical diagnosis, food analysis and environmental security. In recent years, isothermal amplifications with sensitivity, high speed, specificity, accuracy, and automation have been designed based on interdisciplinary approaches among chemistry, biology, and materials science. In this article, we summarize the advances in nanostructure assisted isothermal amplification in the past two decades for the detection of commercial biomarkers, or biomarkers extracted from cultured cells or patient samples. This article has been divided into three parts according to the ratio of target-to-signal probe in the detection strategy, namely, the N : N amplification ratio, the 1 : N amplification ratio, and the 1 : N(2) amplification ratio. PMID:26812957

  19. Coupled isothermal polynucleotide amplification and translation system

    NASA Technical Reports Server (NTRS)

    Joyce, Gerald F. (Inventor)

    1998-01-01

    A cell-free system for polynucleotide amplification and translation is disclosed. Also disclosed are methods for using the system and a composition which allows the various components of the system to function under a common set of reaction conditions.

  20. Isothermal DNA amplification strategies for duplex microorganism detection.

    PubMed

    Santiago-Felipe, Sara; Tortajada-Genaro, Luis Antonio; Morais, Sergi; Puchades, Rosa; Maquieira, Ángel

    2015-05-01

    A valid solution for micro-analytical systems is the selection of a compatible amplification reaction with a simple, highly-integrated efficient design that allows the detection of multiple genomic targets. Two approaches under isothermal conditions are presented: recombinase polymerase amplification (RPA) and multiple displacement amplification (MDA). Both methods were applied to a duplex assay specific for Salmonella spp. and Cronobacter spp., with excellent amplification yields (0.2-8.6 · 10(8) fold). The proposed approaches were successfully compared to conventional PCR and tested for the milk sample analysis as a microarray format on a compact disc (support and driver). Satisfactory results were obtained in terms of resistance to inhibition, selectivity, sensitivity (10(1)-10(2)CFU/mL) and reproducibility (below 12.5%). The methods studied are efficient and cost-effective, with a high potential to automate microorganisms detection by integrated analytical systems working at a constant low temperature. PMID:25529713

  1. Sequence dependence of isothermal DNA amplification via EXPAR

    PubMed Central

    Qian, Jifeng; Ferguson, Tanya M.; Shinde, Deepali N.; Ramírez-Borrero, Alissa J.; Hintze, Arend; Adami, Christoph; Niemz, Angelika

    2012-01-01

    Isothermal nucleic acid amplification is becoming increasingly important for molecular diagnostics. Therefore, new computational tools are needed to facilitate assay design. In the isothermal EXPonential Amplification Reaction (EXPAR), template sequences with similar thermodynamic characteristics perform very differently. To understand what causes this variability, we characterized the performance of 384 template sequences, and used this data to develop two computational methods to predict EXPAR template performance based on sequence: a position weight matrix approach with support vector machine classifier, and RELIEF attribute evaluation with Naïve Bayes classification. The methods identified well and poorly performing EXPAR templates with 67–70% sensitivity and 77–80% specificity. We combined these methods into a computational tool that can accelerate new assay design by ruling out likely poor performers. Furthermore, our data suggest that variability in template performance is linked to specific sequence motifs. Cytidine, a pyrimidine base, is over-represented in certain positions of well-performing templates. Guanosine and adenosine, both purine bases, are over-represented in similar regions of poorly performing templates, frequently as GA or AG dimers. Since polymerases have a higher affinity for purine oligonucleotides, polymerase binding to GA-rich regions of a single-stranded DNA template may promote non-specific amplification in EXPAR and other nucleic acid amplification reactions. PMID:22416064

  2. Loop mediated isothermal amplification: An innovative gene amplification technique for animal diseases

    PubMed Central

    Sahoo, Pravas Ranjan; Sethy, Kamadev; Mohapatra, Swagat; Panda, Debasis

    2016-01-01

    India being a developing country mainly depends on livestock sector for its economy. However, nowadays, there is emergence and reemergence of more transboundary animal diseases. The existing diagnostic techniques are not so quick and with less specificity. To reduce the economy loss, there should be a development of rapid, reliable, robust diagnostic technique, which can work with high degree of sensitivity and specificity. Loop mediated isothermal amplification assay is a rapid gene amplification technique that amplifies nucleic acid under an isothermal condition with a set of designed primers spanning eight distinct sequences of the target. This assay can be used as an emerging powerful, innovative gene amplification diagnostic tool against various pathogens of livestock diseases. This review is to highlight the basic concept and methodology of this assay in livestock disease. PMID:27284221

  3. Loop mediated isothermal amplification: An innovative gene amplification technique for animal diseases.

    PubMed

    Sahoo, Pravas Ranjan; Sethy, Kamadev; Mohapatra, Swagat; Panda, Debasis

    2016-05-01

    India being a developing country mainly depends on livestock sector for its economy. However, nowadays, there is emergence and reemergence of more transboundary animal diseases. The existing diagnostic techniques are not so quick and with less specificity. To reduce the economy loss, there should be a development of rapid, reliable, robust diagnostic technique, which can work with high degree of sensitivity and specificity. Loop mediated isothermal amplification assay is a rapid gene amplification technique that amplifies nucleic acid under an isothermal condition with a set of designed primers spanning eight distinct sequences of the target. This assay can be used as an emerging powerful, innovative gene amplification diagnostic tool against various pathogens of livestock diseases. This review is to highlight the basic concept and methodology of this assay in livestock disease. PMID:27284221

  4. Isothermal amplified detection of DNA and RNA.

    PubMed

    Yan, Lei; Zhou, Jie; Zheng, Yue; Gamson, Adam S; Roembke, Benjamin T; Nakayama, Shizuka; Sintim, Herman O

    2014-05-01

    This review highlights various methods that can be used for a sensitive detection of nucleic acids without using thermal cycling procedures, as is done in PCR or LCR. Topics included are nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), loop-mediated amplification (LAMP), Invader assay, rolling circle amplification (RCA), signal mediated amplification of RNA technology (SMART), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), nicking endonuclease signal amplification (NESA) and nicking endonuclease assisted nanoparticle activation (NENNA), exonuclease-aided target recycling, Junction or Y-probes, split DNAZyme and deoxyribozyme amplification strategies, template-directed chemical reactions that lead to amplified signals, non-covalent DNA catalytic reactions, hybridization chain reactions (HCR) and detection via the self-assembly of DNA probes to give supramolecular structures. The majority of these isothermal amplification methods can detect DNA or RNA in complex biological matrices and have great potential for use at point-of-care. PMID:24643211

  5. Diagnosis of brugian filariasis by loop-mediated isothermal amplification.

    PubMed

    Poole, Catherine B; Tanner, Nathan A; Zhang, Yinhua; Evans, Thomas C; Carlow, Clotilde K S

    2012-01-01

    In this study we developed and evaluated a Brugia Hha I repeat loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Brugia genomic DNA. Amplification was detected using turbidity or fluorescence as readouts. Reactions generated a turbidity threshold value or a clear visual positive within 30 minutes using purified genomic DNA equivalent to one microfilaria. Similar results were obtained using DNA isolated from blood samples containing B. malayi microfilariae. Amplification was specific to B. malayi and B. timori, as no turbidity was observed using DNA from the related filarial parasites Wuchereria bancrofti, Onchocerca volvulus or Dirofilaria immitis, or from human or mosquito. Furthermore, the assay was most robust using a new strand-displacing DNA polymerase termed Bst 2.0 compared to wild-type Bst DNA polymerase, large fragment. The results indicate that the Brugia Hha I repeat LAMP assay is rapid, sensitive and Brugia-specific with the potential to be developed further as a field tool for diagnosis and mapping of brugian filariasis. PMID:23272258

  6. Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification

    PubMed Central

    Poole, Catherine B.; Tanner, Nathan A.; Zhang, Yinhua; Evans, Thomas C.; Carlow, Clotilde K. S.

    2012-01-01

    In this study we developed and evaluated a Brugia Hha I repeat loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Brugia genomic DNA. Amplification was detected using turbidity or fluorescence as readouts. Reactions generated a turbidity threshold value or a clear visual positive within 30 minutes using purified genomic DNA equivalent to one microfilaria. Similar results were obtained using DNA isolated from blood samples containing B. malayi microfilariae. Amplification was specific to B. malayi and B. timori, as no turbidity was observed using DNA from the related filarial parasites Wuchereria bancrofti, Onchocerca volvulus or Dirofilaria immitis, or from human or mosquito. Furthermore, the assay was most robust using a new strand-displacing DNA polymerase termed Bst 2.0 compared to wild-type Bst DNA polymerase, large fragment. The results indicate that the Brugia Hha I repeat LAMP assay is rapid, sensitive and Brugia-specific with the potential to be developed further as a field tool for diagnosis and mapping of brugian filariasis. PMID:23272258

  7. Rapid isothermal detection of Phytophthora species on plant samples using recombinase polymerase amplification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recently several isothermal amplification techniques have been developed that are extremely tolerant towards inhibitors present in many plant extracts. Recombinase polymerase amplification (RPA) assays for the genus Phytophthora have been developed which provide a simple and rapid method to macerate...

  8. Microfluidic Continuous Flow Digital Loop-Mediated Isothermal Amplification (LAMP)

    PubMed Central

    Rane, Tushar D.; Chen, Liben; Zec, Helena C.; Wang, Tza-Huei

    2015-01-01

    Digital nucleic acid detection is rapidly becoming a popular technique for ultra-sensitive and quantitative detection of nucleic acid molecules in a wide range of biomedical studies. Digital polymerase chain reaction (PCR) remains the most popular way of conducting digital nucleic acid detection. However, due to the need for thermocycling, digital PCR is difficult to implement in a streamlined manner on a single microfluidic device, leading to complex fragmented workflows and multiple separate devices and instruments. Loop-mediated isothermal amplification (LAMP) is an excellent isothermal alternative to PCR with potentially better specificity than PCR through the use of multiple primer sets for a nucleic acid target. Here we report a microfluidic droplet device implementing all the steps required for digital nucleic acid detection including droplet generation, incubation and in-line detection for digital LAMP. As compared to microchamber or droplet array-based digital assays, continuous flow operation of this device eliminates the constraints on the number of total reactions by the footprint of the device and the analysis throughput by the time for lengthy incubation and transfers of materials between instruments. PMID:25431886

  9. A phaseguided passive batch microfluidic mixing chamber for isothermal amplification.

    PubMed

    Hakenberg, Sydney; Hügle, Matthias; Weidmann, Manfred; Hufert, Frank; Dame, Gregory; Urban, Gerald A

    2012-11-01

    With a view to developing a rapid pathogen detection system utilizing isothermal nucleic acid amplification, the necessary micro-mixing step is innovatively implemented on a chip. Passive laminar flow mixing of two 6.5 μl batches differing in viscosity is performed within a microfluidic chamber. This is achieved with a novel chip space-saving phaseguide design which allows, for the first time, the complete integration of a passive mixing structure into a target chamber. Sequential filling of batches prior to mixing is demonstrated. Simulation predicts a reduction of diffusive mixing time from hours down to one minute. A simple and low-cost fabrication method is used which combines dry film resist technology and direct wafer bonding. Finally, an isothermal nucleic acid detection assay is successfully implemented where fluorescence results are measured directly from the chip after a one minute mixing sequence. In combination with our previous work, this opens up the way towards a fully integrated pathogen detection system in a lab-on-a-chip format. PMID:22952055

  10. Quadratic isothermal amplification for the detection of microRNA.

    PubMed

    Duan, Ruixue; Zuo, Xiaolei; Wang, Shutao; Quan, Xiyun; Chen, Dongliang; Chen, Zhifei; Jiang, Lei; Fan, Chunhai; Xia, Fan

    2014-03-01

    This protocol describes an isothermal amplification approach for ultrasensitive detection of specific microRNAs (miRNAs). It achieves this level of sensitivity through quadratic amplification of the target oligonucleotide by using a Bst DNA polymerase-induced strand-displacement reaction and a lambda exonuclease-aided recycling reaction. First, the target miRNA binds to a specifically designed molecular beacon, causing it to become a fluorescence emitter. A primer then binds to the activated beacon, and Bst polymerase initiates the synthesis of a double-stranded DNA segment templated on the molecular beacon. This causes the concomitant release of the target miRNA from the beacon--the first round of 'recycling'. Second, the duplex beacon thus produced is a suitable substrate for a nicking enzyme present in solution. After the duplex beacon is nicked, the lambda exonuclease digests the beacon and releases the DNA single strand just synthesized, which is complementary to the molecular beacon, inducing the second round of recycling. The miRNA detection limit of this protocol is 10 fmol at 37 °C and 1 amol at 4 °C. This approach also affords high selectivity when applied to miRNA extracted from MCF-7 and PC3 cell lines and even from breast cancer tissue samples. Upon isolation of miRNA, the detection process can be completed in ∼2 h. PMID:24525753

  11. Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification

    PubMed Central

    Mauk, Michael G.; Liu, Changchun; Song, Jinzhao; Bau, Haim H.

    2015-01-01

    Microfluidic components and systems for rapid (<60 min), low-cost, convenient, field-deployable sequence-specific nucleic acid-based amplification tests (NAATs) are described. A microfluidic point-of-care (POC) diagnostics test to quantify HIV viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of “lab on a chip” NAAT devices. A portable, miniaturized POC NAAT with performance comparable to conventional PCR (polymerase-chain reaction)-based tests in clinical laboratories can be realized with a disposable, palm-sized, plastic microfluidic chip in which: (1) nucleic acids (NAs) are extracted from relatively large (~mL) volume sample lysates using an embedded porous silica glass fiber or cellulose binding phase (“membrane”) to capture sample NAs in a flow-through, filtration mode; (2) NAs captured on the membrane are isothermally (~65 °C) amplified; (3) amplicon production is monitored by real-time fluorescence detection, such as with a smartphone CCD camera serving as a low-cost detector; and (4) paraffin-encapsulated, lyophilized reagents for temperature-activated release are pre-stored in the chip. Limits of Detection (LOD) better than 103 virons/sample can be achieved. A modified chip with conduits hosting a diffusion-mode amplification process provides a simple visual indicator to readily quantify sample NA template. In addition, a companion microfluidic device for extracting plasma from whole blood without a centrifuge, generating cell-free plasma for chip-based molecular diagnostics, is described. Extensions to a myriad of related applications including, for example, food testing, cancer screening, and insect genotyping are briefly surveyed.

  12. Hendra virus detection using Loop-Mediated Isothermal Amplification.

    PubMed

    Foord, Adam J; Middleton, Deborah; Heine, Hans G

    2012-04-01

    Hendra virus (HeV) is a zoonotic paramyxovirus endemic in Australian Pteropus bats (fruit bats or flying foxes). Although bats appear to be unaffected by the virus, HeV can spread from fruit bats to horses, causing severe disease. Human infection results from close contact with the blood, body fluids and tissues of infected horses. HeV is a biosecurity level 4 (BSL-4) pathogen, with a high case-fatality rate in humans and horses. Current assays for HeV detection require complex instrumentation and are generally time consuming. The aim of this study was to develop a Loop-Mediated Isothermal Amplification (LAMP) assay to detect nucleic acid from all known HeV strains in horses without the requirement for complex laboratory equipment. A LAMP assay targeting a conserved region of the HeV P-gene was combined with a Lateral Flow Device (LFD) for detection of amplified product. All HeV isolates, the original HeV isolated in 1994 as well as the most recent isolates from 2011 were detected. Analytical sensitivity and specificity of the HeV-LAMP assay was equal to a TaqMan assay developed previously. Significantly, these assays detected HeV in horses before clinical signs were observed. The combined LAMP-LFD procedure is a sensitive method suitable for HeV diagnosis in a resource-limited situation or where rapid test results are critical. PMID:22327143

  13. Isothermal amplification detection of nucleic acids by a double-nicked beacon.

    PubMed

    Shi, Chao; Zhou, Meiling; Pan, Mei; Zhong, Guilin; Ma, Cuiping

    2016-03-01

    Isothermal and rapid amplification detection of nucleic acids is an important technology in environmental monitoring, foodborne pathogen detection, and point-of-care clinical diagnostics. Here we have developed a novel method of isothermal signal amplification for single-stranded DNA (ssDNA) detection. The ssDNA target could be used as an initiator, coupled with a double-nicked molecular beacon, to originate amplification cycles, achieving cascade signal amplification. In addition, the method showed good specificity and strong anti-jamming capability. Overall, it is a one-pot and isothermal strand displacement amplification method without the requirement of a stepwise procedure, which greatly simplifies the experimental procedure and decreases the probability of contamination of samples. With its advantages, the method would be very useful to detect nucleic acids in point-of-care or field use. PMID:26706801

  14. Brief review of monitoring methods for loop-mediated isothermal amplification (LAMP).

    PubMed

    Zhang, Xuzhi; Lowe, Stuart B; Gooding, John Justin

    2014-11-15

    The loop-mediated isothermal amplification (LAMP) technique has the potential to revolutionize molecular biology because it allows DNA amplification under isothermal conditions and is highly compatible with point-of-care analysis. To achieve efficient genetic analysis of samples, the method of real-time or endpoint determination selected to monitor the biochemical reaction is of great importance. In this paper we briefly review progress in the development of monitoring methods for LAMP. PMID:24949822

  15. Two methods for increased specificity and sensitivity in loop-mediated isothermal amplification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The technique of loop-mediated isothermal amplification (LAMP) utilizes 4 (or 6) primers targeting 6 (or 8) regions within a fairly small segment of a genome for amplification, with concentration higher than that used in traditional PCR methods. The high concentrations of primers used leads to an in...

  16. Real-time quantitative nicking endonuclease-mediated isothermal amplification with small molecular beacons.

    PubMed

    Xu, Wentao; Wang, Chenguang; Zhu, Pengyu; Guo, Tianxiao; Xu, Yuancong; Huang, Kunlun; Luo, Yunbo

    2016-04-21

    Techniques of isothermal amplification have recently made great strides, and have generated significant interest in the field of point-of-care detection. Nicking endonuclease-mediated isothermal amplification (NEMA) is an example of simple isothermal technology. In this paper, a real-time quantitative nicking endonuclease-mediated isothermal amplification with small molecular beacons (SMB-NEMA) of improved specificity and sensitivity is described. First, we optimized the prohibition of de novo synthesis by choosing Nt·BstNBI endonuclease. Second, the whole genome was successfully amplified with Nt·BstNBI (6 U), betaine (1 M) and trehalose (60 mM) for the first time. Third, we achieved 10 pg sensitivity for the first time after adding a small molecular beacon that spontaneously undergoes a conformational change when hybridizing to target, and the practical test validated the assay's application. The small molecular beacon has a similar melting temperature to the reaction temperature, but is approximately 10 bp shorter than the length of a traditional molecular beacon. A new threshold regulation was also established for isothermal conditions. Finally, we established a thermodynamic model for designing small molecular beacons. This multistate model is more correct than the traditional algorithm. This theoretical and practical basis will help us to monitor SMB-NEMA in a quantitative way. In summary, our SMB-NEMA method allows the simple, specific and sensitive assessment of isothermal DNA quantification. PMID:27027375

  17. Isothermal Amplification Methods for the Detection of Nucleic Acids in Microfluidic Devices

    PubMed Central

    Zanoli, Laura Maria; Spoto, Giuseppe

    2012-01-01

    Diagnostic tools for biomolecular detection need to fulfill specific requirements in terms of sensitivity, selectivity and high-throughput in order to widen their applicability and to minimize the cost of the assay. The nucleic acid amplification is a key step in DNA detection assays. It contributes to improving the assay sensitivity by enabling the detection of a limited number of target molecules. The use of microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device. The vast majority of miniaturized systems for nucleic acid analysis exploit the polymerase chain reaction (PCR) amplification method, which requires repeated cycles of three or two temperature-dependent steps during the amplification of the nucleic acid target sequence. In contrast, low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features. Here, the use of miniaturized analysis systems using isothermal amplification reactions for the nucleic acid amplification will be discussed. PMID:25587397

  18. Development of rapid isothermal amplification assays for Phytophthora species from plant tissue

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several isothermal amplification techniques recently have been developed that are tolerant of inhibitors present in many plant extracts, which can reduce the need for obtaining purified DNA for running diagnostic assays. One such commercially available technique that has similarities with real time ...

  19. LAMP (Loop-mediated isothermal amplification of DNA) - A technique for biotype discrimination in Bemisia tabaci

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Loop-mediated isothermal amplification of DNA (LAMP) can amplify a target DNA sequence at a constant temperature in about 1 hour. LAMP technology has great potential for agricultural applications because of the need for rapid and inexpensive diagnoses. Assays based on LAMP technology are well suited...

  20. Sensitive and specific detection of Yersinia pseudotuberculosis by loop-mediated isothermal amplification.

    PubMed

    Horisaka, Tomoko; Fujita, Kayoko; Iwata, Taketoshi; Nakadai, Aya; Okatani, Alexandre T; Horikita, Tetsuya; Taniguchi, Takahide; Honda, Eiichi; Yokomizo, Yuichi; Hayashidani, Hideki

    2004-11-01

    We developed a loop-mediated isothermal amplification method able to detect Yersinia pseudotuberculosis strains in 30 min by using six primers designed by targeting the inv gene. This method is more sensitive than PCR and might be a useful tool for detecting and identifying Y. pseudotuberculosis. PMID:15528740

  1. Three isothermal amplification techniques for rapid identification of Cladophialophora carrionii, an agent of human chromoblastomycosis.

    PubMed

    Deng, Shuwen; de Hoog, G Sybren; Pan, Weihua; Chen, Min; van den Ende, A H G Gerrits; Yang, Liyue; Sun, Jiufeng; Najafzadeh, Mohammad Javad; Liao, Wanqing; Li, Ruoyu

    2014-10-01

    In this study, we developed rapid and sensitive assays for the detection of Cladophialophora carrionii, a common agent of human chromoblastomycosis. The isothermal techniques evaluated were rolling-circle amplification (RCA), multiplex ligation-dependent probe amplification (MLPA), and loop-mediated isothermal amplification (LAMP). The probes for RCA and MLPA were designed with target sequences in the rDNA internal transcribed spacer gene (ITS) region, and LAMP primers were designed using the elongation factor 1α gene (EF1); these probes and primers specifically amplified DNA of isolates of the species. The three techniques were sufficiently specific and sensitive for discriminating target DNA of C. carrionii from that of related Cladophialophora species and other agents of chromoblastomycosis. RCA, MLPA, and LAMP are advantageous in their reliability and ease of operation compared to standard PCR and conventional methods. PMID:25009046

  2. A Simple, Low-Cost Platform for Real-Time Isothermal Nucleic Acid Amplification

    PubMed Central

    Craw, Pascal; Mackay, Ruth E.; Naveenathayalan, Angel; Hudson, Chris; Branavan, Manoharanehru; Sadiq, S. Tariq; Balachandran, Wamadeva

    2015-01-01

    Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays have resulted in a range of solutions for nucleic acid amplification tests suited for point of care and field use. However, miniaturisation of instrumentation for such assays has not seen such rapid advances and fluorescence based assays still depend on complex, bulky and expensive optics such as fluorescence microscopes, photomultiplier tubes and sensitive lens assemblies. In this work we demonstrate a robust, low cost platform for isothermal nucleic acid amplification on a microfluidic device. Using easily obtainable materials and commercial off-the-shelf components, we show real time fluorescence detection using a low cost photodiode and operational amplifier without need for lenses. Temperature regulation on the device is achieved using a heater fabricated with standard printed circuit board fabrication methods. These facile construction methods allow fabrications at a cost compatible with widespread deployment to resource poor settings. PMID:26389913

  3. A Simple, Low-Cost Platform for Real-Time Isothermal Nucleic Acid Amplification.

    PubMed

    Craw, Pascal; Mackay, Ruth E; Naveenathayalan, Angel; Hudson, Chris; Branavan, Manoharanehru; Sadiq, S Tariq; Balachandran, Wamadeva

    2015-01-01

    Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays have resulted in a range of solutions for nucleic acid amplification tests suited for point of care and field use. However, miniaturisation of instrumentation for such assays has not seen such rapid advances and fluorescence based assays still depend on complex, bulky and expensive optics such as fluorescence microscopes, photomultiplier tubes and sensitive lens assemblies. In this work we demonstrate a robust, low cost platform for isothermal nucleic acid amplification on a microfluidic device. Using easily obtainable materials and commercial off-the-shelf components, we show real time fluorescence detection using a low cost photodiode and operational amplifier without need for lenses. Temperature regulation on the device is achieved using a heater fabricated with standard printed circuit board fabrication methods. These facile construction methods allow fabrications at a cost compatible with widespread deployment to resource poor settings. PMID:26389913

  4. Isothermal solid-phase amplification system for detection of Yersinia pestis.

    PubMed

    Mayboroda, Olena; Gonzalez Benito, Angel; Sabaté del Rio, Jonathan; Svobodova, Marketa; Julich, Sandra; Tomaso, Herbert; O'Sullivan, Ciara K; Katakis, Ioanis

    2016-01-01

    DNA amplification is required for most molecular diagnostic applications, but conventional polymerase chain reaction (PCR) has disadvantages for field testing. Isothermal amplification techniques are being developed to respond to this problem. One of them is the recombinase polymerase amplification (RPA) that operates at isothermal conditions without sacrificing specificity and sensitivity in easy-to-use formats. In this work, RPA was used for the optical detection of solid-phase amplification of the potential biowarfare agent Yersinia pestis. Thiolated forward primers were immobilized on the surface of maleimide-activated microtitre plates for the quantitative detection of synthetic and genomic DNA, with elongation occurring only in the presence of the specific template DNA and solution phase reverse primers. Quantitative detection was achieved via the use of biotinylated reverse primers and post-amplification addition of streptavidin-HRP conjugate. The overall time of amplification and detection was less than 1 h at a constant temperature of 37 °C. Single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) sequences were detected, achieving detection limits of 4.04*10(-13) and 3.14*10(-16) M, respectively. The system demonstrated high specificity with negligible responses to non-specific targets. PMID:26563112

  5. A novel thermostable polymerase for RNA and DNA loop-mediated isothermal amplification (LAMP).

    PubMed

    Chander, Yogesh; Koelbl, Jim; Puckett, Jamie; Moser, Michael J; Klingele, Audrey J; Liles, Mark R; Carrias, Abel; Mead, David A; Schoenfeld, Thomas W

    2014-01-01

    Meeting the goal of providing point of care (POC) tests for molecular detection of pathogens in low resource settings places stringent demands on all aspects of the technology. OmniAmp DNA polymerase (Pol) is a thermostable viral enzyme that enables true POC use in clinics or in the field by overcoming important barriers to isothermal amplification. In this paper, we describe the multiple advantages of OmniAmp Pol as an isothermal amplification enzyme and provide examples of its use in loop-mediated isothermal amplification (LAMP) for pathogen detection. The inherent reverse transcriptase activity of OmniAmp Pol allows single enzyme detection of RNA targets in RT-LAMP. Common methods of nucleic acid amplification are highly susceptible to sample contaminants, necessitating elaborate nucleic acid purification protocols that are incompatible with POC or field use. OmniAmp Pol was found to be less inhibited by whole blood components typical in certain crude sample preparations. Moreover, the thermostability of the enzyme compared to alternative DNA polymerases (Bst) and reverse transcriptases allows pretreatment of complete reaction mixes immediately prior to amplification, which facilitates amplification of highly structured genome regions. Compared to Bst, OmniAmp Pol has a faster time to result, particularly with more dilute templates. Molecular diagnostics in field settings can be challenging due to the lack of refrigeration. The stability of OmniAmp Pol is compatible with a dry format that enables long term storage at ambient temperatures. A final requirement for field operability is compatibility with either commonly available instruments or, in other cases, a simple, inexpensive, portable detection mode requiring minimal training or power. Detection of amplification products is shown using lateral flow strips and analysis on a real-time PCR instrument. Results of this study show that OmniAmp Pol is ideally suited for low resource molecular detection of

  6. A Novel Real-Time DNA Detection System for Loop-Mediated Isothermal Amplification Method

    NASA Astrophysics Data System (ADS)

    Kakugawa, Koji; Yamada, Kenji; Maeda, Hiroshi; Takashiba, Shougo

    We developed a novel real-time DNA detection system for loop-mediated isothermal amplification (LAMP) method. Our prototype was composed of a thermostatic chamber, a hole slide glass, LED and a web camera. The reaction mixture was injected into the slide glass hole and the LAMP reaction was carried out at 63°C for 2 hours. To observe the DNA amplification, we monitored the fluorescence intensity of SYBR Green I that was excited by the blue LED. The captured BMP images were analyzed by NIH Image J software. The DNA amplification and amplification monitoring experiment was successful. Furthermore, quantitative accuracy was evaluated based on real-time PCR. The reaction time correlates well with the DNA concentration. These results indicate the successful development of a novel real-time DNA detection system for LAMP method.

  7. Evaluation of a Loop-Mediated Isothermal Amplification Assay for Diagnosis of Clostridium difficile Infections▿

    PubMed Central

    Lalande, Valérie; Barrault, Laurence; Wadel, Sophie; Eckert, Catherine; Petit, Jean-Claude; Barbut, Frédéric

    2011-01-01

    A new assay (illumigene C. difficile; Meridian Bioscience), based on the original loop-mediated isothermal amplification (LAMP) assay, was evaluated with 472 unformed stools from patients suspected of Clostridium difficile infection. Compared to the toxigenic culture, the sensitivity, specificity, and positive and negative predictive values were 91.8, 99.1, 91.8, and 99.1% for the illumigene C. difficile assay and 69.4, 100, 100, and 96.6% for the cytotoxicity assay, respectively. PMID:21525213

  8. Evaluation of a loop-mediated isothermal amplification assay for diagnosis of Clostridium difficile infections.

    PubMed

    Lalande, Valérie; Barrault, Laurence; Wadel, Sophie; Eckert, Catherine; Petit, Jean-Claude; Barbut, Frédéric

    2011-07-01

    A new assay (illumigene C. difficile; Meridian Bioscience), based on the original loop-mediated isothermal amplification (LAMP) assay, was evaluated with 472 unformed stools from patients suspected of Clostridium difficile infection. Compared to the toxigenic culture, the sensitivity, specificity, and positive and negative predictive values were 91.8, 99.1, 91.8, and 99.1% for the illumigene C. difficile assay and 69.4, 100, 100, and 96.6% for the cytotoxicity assay, respectively. PMID:21525213

  9. Chemiluminescence imaging for microRNA detection based on cascade exponential isothermal amplification machinery.

    PubMed

    Xu, Yongjie; Li, Dandan; Cheng, Wei; Hu, Rong; Sang, Ye; Yin, Yibing; Ding, Shijia; Ju, Huangxian

    2016-09-14

    A novel G-quadruplex DNAzyme-driven chemiluminescence (CL) imaging method was developed for ultrasensitive and specific detection of miRNA based on the cascade exponential isothermal amplification reaction (EXPAR) machinery. A structurally tailored hairpin probe switch was designed to selectively recognise miRNA and form hybridisation products to trigger polymerase and nicking enzyme machinery, resulting in the generation of product I, which was complementary to a region of the functional linear template. Then, the response of the functional linear template to the generated product I further activated the exponential isothermal amplification machinery, leading to synthesis of numerous horseradish peroxidase mimicking DNAzyme units for CL signal transduction. The amplification paradigm generated a linear response from 10 fM to 100 pM, with a low detection limit of 2.91 fM, and enabled discrimination of target miRNA from a single-base mismatched target. The developed biosensing platform demonstrated the advantages of isothermal, homogeneous, visual detection for miRNA assays, offering a promising tool for clinical diagnosis. PMID:27566360

  10. A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers.

    PubMed

    Du, Yan; Hughes, Randall A; Bhadra, Sanchita; Jiang, Yu Sherry; Ellington, Andrew D; Li, Bingling

    2015-01-01

    Strand exchange nucleic acid circuitry can be used to transduce isothermal nucleic acid amplification products into signals that can be readable on an off-the-shelf glucometer. Loop-mediated isothermal amplification (LAMP) is limited by the accumulation of non-specific products, but nucleic acid circuitry can be used to probe and distinguish specific amplicons. By combining this high temperature isothermal amplification method with a thermostable invertase, we can directly transduce Middle-East respiratory syndrome coronavirus and Zaire Ebolavirus templates into glucose signals, with a sensitivity as low as 20-100 copies/μl, equating to atto-molar (or low zepto-mole). Virus from cell lysates and synthetic templates could be readily amplified and detected even in sputum or saliva. An OR gate that coordinately triggered on viral amplicons further guaranteed fail-safe virus detection. The method describes has potential for accelerating point-of-care applications, in that biological samples could be applied to a transducer that would then directly interface with an off-the-shelf, approved medical device. PMID:26050646

  11. A novel multiplex isothermal amplification method for rapid detection and identification of viruses

    PubMed Central

    Nyan, Dougbeh-Chris; Swinson, Kevin L.

    2015-01-01

    A rapid multiplex isothermal amplification assay has been developed for detection and identification of multiple blood-borne viruses that infect millions of people world-wide. These infections may lead to chronic diseases or death if not diagnosed and treated in a timely manner. Sets of virus-specific oligonucleotides and oligofluorophores were designed and used in a reverse-transcription loop-mediated multiplexed isothermal amplification reaction for detection and gel electrophoretic identification of human Immunodeficiency virus (HIV), hepatitis-B virus (HBV), hepatitis-C virus (HCV), hepatitis-E virus (HEV), dengue virus (DENV), and West Nile (WNV) virus infection in blood plasma. Amplification was catalyzed with two thermostable enzymes for 30–60 minutes under isothermal condition, utilizing a simple digital heat source. Electrophoretic analysis of amplified products demonstrated simultaneous detection of 6 viruses that were distinctly identified by unique ladder-like banding patterns. Naked-eye fluorescent visualization of amplicons revealed intensely fluorescing products that indicated positive detection. The test demonstrated a 97% sensitivity and a 100% specificity, with no cross-reaction with other viruses observed. This portable detection tool may have clinical and field utility in the developing and developed world settings. This may enable rapid diagnosis and identification of viruses for targeted therapeutic intervention and prevention of disease transmission. PMID:26643761

  12. A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers

    NASA Astrophysics Data System (ADS)

    Du, Yan; Hughes, Randall A.; Bhadra, Sanchita; Jiang, Yu Sherry; Ellington, Andrew D.; Li, Bingling

    2015-06-01

    Strand exchange nucleic acid circuitry can be used to transduce isothermal nucleic acid amplification products into signals that can be readable on an off-the-shelf glucometer. Loop-mediated isothermal amplification (LAMP) is limited by the accumulation of non-specific products, but nucleic acid circuitry can be used to probe and distinguish specific amplicons. By combining this high temperature isothermal amplification method with a thermostable invertase, we can directly transduce Middle-East respiratory syndrome coronavirus and Zaire Ebolavirus templates into glucose signals, with a sensitivity as low as 20-100 copies/μl, equating to atto-molar (or low zepto-mole). Virus from cell lysates and synthetic templates could be readily amplified and detected even in sputum or saliva. An OR gate that coordinately triggered on viral amplicons further guaranteed fail-safe virus detection. The method describes has potential for accelerating point-of-care applications, in that biological samples could be applied to a transducer that would then directly interface with an off-the-shelf, approved medical device.

  13. A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers

    PubMed Central

    Du, Yan; Hughes, Randall A.; Bhadra, Sanchita; Jiang, Yu Sherry; Ellington, Andrew D.; Li, Bingling

    2015-01-01

    Strand exchange nucleic acid circuitry can be used to transduce isothermal nucleic acid amplification products into signals that can be readable on an off-the-shelf glucometer. Loop-mediated isothermal amplification (LAMP) is limited by the accumulation of non-specific products, but nucleic acid circuitry can be used to probe and distinguish specific amplicons. By combining this high temperature isothermal amplification method with a thermostable invertase, we can directly transduce Middle-East respiratory syndrome coronavirus and Zaire Ebolavirus templates into glucose signals, with a sensitivity as low as 20–100 copies/μl, equating to atto-molar (or low zepto-mole). Virus from cell lysates and synthetic templates could be readily amplified and detected even in sputum or saliva. An OR gate that coordinately triggered on viral amplicons further guaranteed fail-safe virus detection. The method describes has potential for accelerating point-of-care applications, in that biological samples could be applied to a transducer that would then directly interface with an off-the-shelf, approved medical device. PMID:26050646

  14. Simple System for Isothermal DNA Amplification Coupled to Lateral Flow Detection

    PubMed Central

    Roskos, Kristina; Hickerson, Anna I.; Lu, Hsiang-Wei; Ferguson, Tanya M.; Shinde, Deepali N.; Klaue, Yvonne; Niemz, Angelika

    2013-01-01

    Infectious disease diagnosis in point-of-care settings can be greatly improved through integrated, automated nucleic acid testing devices. We have developed an early prototype for a low-cost system which executes isothermal DNA amplification coupled to nucleic acid lateral flow (NALF) detection in a mesofluidic cartridge attached to a portable instrument. Fluid handling inside the cartridge is facilitated through one-way passive valves, flexible pouches, and electrolysis-driven pumps, which promotes a compact and inexpensive instrument design. The closed-system disposable prevents workspace amplicon contamination. The cartridge design is based on standard scalable manufacturing techniques such as injection molding. Nucleic acid amplification occurs in a two-layer pouch that enables efficient heat transfer. We have demonstrated as proof of principle the amplification and detection of Mycobacterium tuberculosis (M.tb) genomic DNA in the cartridge, using either Loop Mediated Amplification (LAMP) or the Exponential Amplification Reaction (EXPAR), both coupled to NALF detection. We envision that a refined version of this cartridge, including upstream sample preparation coupled to amplification and detection, will enable fully-automated sample-in to answer-out infectious disease diagnosis in primary care settings of low-resource countries with high disease burden. PMID:23922706

  15. Simple system for isothermal DNA amplification coupled to lateral flow detection.

    PubMed

    Roskos, Kristina; Hickerson, Anna I; Lu, Hsiang-Wei; Ferguson, Tanya M; Shinde, Deepali N; Klaue, Yvonne; Niemz, Angelika

    2013-01-01

    Infectious disease diagnosis in point-of-care settings can be greatly improved through integrated, automated nucleic acid testing devices. We have developed an early prototype for a low-cost system which executes isothermal DNA amplification coupled to nucleic acid lateral flow (NALF) detection in a mesofluidic cartridge attached to a portable instrument. Fluid handling inside the cartridge is facilitated through one-way passive valves, flexible pouches, and electrolysis-driven pumps, which promotes a compact and inexpensive instrument design. The closed-system disposable prevents workspace amplicon contamination. The cartridge design is based on standard scalable manufacturing techniques such as injection molding. Nucleic acid amplification occurs in a two-layer pouch that enables efficient heat transfer. We have demonstrated as proof of principle the amplification and detection of Mycobacterium tuberculosis (M.tb) genomic DNA in the cartridge, using either Loop Mediated Amplification (LAMP) or the Exponential Amplification Reaction (EXPAR), both coupled to NALF detection. We envision that a refined version of this cartridge, including upstream sample preparation coupled to amplification and detection, will enable fully-automated sample-in to answer-out infectious disease diagnosis in primary care settings of low-resource countries with high disease burden. PMID:23922706

  16. Electrochemical genosensor for the rapid detection of GMO using loop-mediated isothermal amplification.

    PubMed

    Ahmed, Minhaz Uddin; Saito, Masato; Hossain, M Mosharraf; Rao, S Ramachandara; Furui, Satoshi; Hino, Akihiro; Takamura, Yuzuru; Takagi, Masahiro; Tamiya, Eiichi

    2009-05-01

    In this study, we are reporting for the first time an efficient, accurate and inexpensive rapid detection system which employs the integration of isothermal amplification and subsequent analysis of unpurified amplicons by an electrochemical system. In our experiments, loop-mediated isothermal amplification (LAMP) with its higher efficiency than PCR was performed at a constant temperature (65 degrees C). Amplification products were combined with a redox active molecule Hoechst 33258 [H33258, 2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi(1H-benzimidazole)] and analyzed by a DNA stick (DS) which is integrated with a disposable electrochemical printed (DEP) chip using linear sweep voltammetry (LSV). The DNA minor groove binding of the H33258 molecule causes a significant drop in the peak current intensity of the H33258 oxidation. The phenomenon of DNA binding induced by H33258, in addition to changes in the anodic current peak, was used to detect maize CBH 351 variety (StarLink). Since laborious probe immobilization was not required, and amplification and detection were performed on a single device, our biosensor eliminates potential cross-contamination. We believe that this type of sensor will have an unprecedented impact for environmental protection. PMID:19381392

  17. Adapting Enzyme-Free DNA Circuits to the Detection of Loop-Mediated Isothermal Amplification Reactions

    PubMed Central

    Li, Bingling; Chen, Xi; Ellington, Andrew D.

    2012-01-01

    Loop-mediated isothermal amplification of DNA (LAMP) is a powerful isothermal nucleic acid amplification technique that can accumulate ~109 copies from less than 10 copies of input template within an hour or two. Unfortunately, while the amplification reactions are extremely powerful, the quantitative detection of LAMP products is still analytically difficult. In this article, in order to both improve the specificity of LAMP detection and to make direct readout of LAMP amplification simpler and much more reliable, we have developed a non-enzymatic nucleic acid circuit (catalyzed hairpin assembly, CHA) that can both amplify and integrate the specific sequence signals present in LAMP amplicons. Through a hairpin acceptor, one of the four loop products amplified from the LAMP is transduced to an active catalyst ssDNA which can in turn trigger a CHA reaction. After CHA detection, even less than 10 molecules/μL model templates (M13mp18) can produce significant signal, and both non-specific template and parasitic amplicons cannot bring interference at all. More importantly, to further enhance the specificity, we have designed a dual-CHA circuit that only gave positive responses in presence of two LAMP loops. The AND-GATE detector will act as a simultaneous, specific readout of the LAMP product, rather than of competing and parasitic amplicons. PMID:22947054

  18. [Visual Detection of Human Coronavirus NL63 by Reverse Transcription Loop-Mediated Isothermal Amplification].

    PubMed

    Geng, Heyuan; Wang, Shengqiang; Xie, Xiaoqian; Xiao, Yu; Zhang, Ting; Tan, Wenjie; Su, Chuan

    2016-01-01

    A simple and sensitive assay for rapid detection of human coronavirus NL63 (HCoV-NL63) was developed by colorimetic reverse transcription loop-mediated isothermal amplification (RT-LAMP). The method employed six specially designed primers that recognized eight distinct regions of the HCoV-NL63 nucleocapsid protein gene for amplification of target sequences under isothermal conditions at 63 degrees C for 1 h Amplification of RT-LAMP was monitored by addition of calcein before amplification. A positive reaction was confirmed by change from light-brown to yellow-green under visual detection. Specificity of the RT-LAMP assay was validated by cross-reaction with different human coronaviruses, norovirus, influenza A virus, and influenza B virus. Sensitivity was evaluated by serial dilution of HCoV-NL63 RNA from 1.6 x 10(9) to 1.6 x 10(1) per reaction. The RT-LAMP assay could achieve 1,600 RNA copies per reaction with high specificity. Hence, our colorimetric RT-LAMP assay could be used for rapid detection of human coronavirus NL63. PMID:27295884

  19. Rapid and Sensitive Isothermal Detection of Nucleic-acid Sequence by Multiple Cross Displacement Amplification

    PubMed Central

    Wang, Yi; Wang, Yan; Ma, Ai-Jing; Li, Dong-Xun; Luo, Li-Juan; Liu, Dong-Xin; Jin, Dong; Liu, Kai; Ye, Chang-Yun

    2015-01-01

    We have devised a novel amplification strategy based on isothermal strand-displacement polymerization reaction, which was termed multiple cross displacement amplification (MCDA). The approach employed a set of ten specially designed primers spanning ten distinct regions of target sequence and was preceded at a constant temperature (61–65 °C). At the assay temperature, the double-stranded DNAs were at dynamic reaction environment of primer-template hybrid, thus the high concentration of primers annealed to the template strands without a denaturing step to initiate the synthesis. For the subsequent isothermal amplification step, a series of primer binding and extension events yielded several single-stranded DNAs and single-stranded single stem-loop DNA structures. Then, these DNA products enabled the strand-displacement reaction to enter into the exponential amplification. Three mainstream methods, including colorimetric indicators, agarose gel electrophoresis and real-time turbidity, were selected for monitoring the MCDA reaction. Moreover, the practical application of the MCDA assay was successfully evaluated by detecting the target pathogen nucleic acid in pork samples, which offered advantages on quick results, modest equipment requirements, easiness in operation, and high specificity and sensitivity. Here we expounded the basic MCDA mechanism and also provided details on an alternative (Single-MCDA assay, S-MCDA) to MCDA technique. PMID:26154567

  20. Loop-mediated isothermal amplification (LAMP) for the rapid detection of Mycoplasma genitalium.

    PubMed

    Edwards, Thomas; Burke, Patricia; Smalley, Helen B; Gillies, Liz; Longhurst, Denise; Vipond, Barry; Hobbs, Glyn

    2015-09-01

    Mycoplasma genitalium is a sexually transmissible, pathogenic bacterium and a significant cause of nongonococcal urethritis in both men and women. Due to the difficulty of the culture of M. genitalium from clinical samples, the laboratory diagnosis of M. genitalium infection is almost exclusively carried out using nucleic acid amplification tests. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technology, utilising a set of 4 primers specific to 6 distinct regions of the target DNA sequence, in order to amplify target DNA in a highly specific and rapid manner. A LAMP assay was designed to the pdhD gene of M. genitalium, and the limit of detection of the assay was determined as 10 fg of M. genitalium genomic DNA, equating to ~16 copies of the M. genitalium genome, which was equally sensitive as a gold standard 16S rRNA polymerase chain reaction assay. PMID:26072150

  1. Rapid and sensitive detection of Banana bunchy top virus by loop-mediated isothermal amplification.

    PubMed

    Peng, Jun; Zhang, Junfang; Xia, Zihao; Li, Yongqiang; Huang, Junsheng; Fan, Zaifeng

    2012-11-01

    A sensitive loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of Banana bunchy top virus (BBTV) infection. The reaction was performed in a single tube at 63°C for 90 min, with an improved closed-tube detection system by adding the SYBR Green I dye to the inside of the tube lid prior to amplification. The detection limit of the LAMP assay was approximately 1 pg/μl plasmid DNA when mixed with extracted DNA from healthy banana plant, and no cross-reaction with other banana-infected pathogens was observed. Real-time turbidimetry was used to monitor the amplification result in the tubes, and it was shown that this LAMP assay was about 100-fold more sensitive than PCR. The results demonstrated that this LAMP method should be useful for both banana disease monitoring and mass propagation of virus-free banana plantlets. PMID:22771738

  2. The Development of a Loop-Mediated Isothermal Amplification Method (LAMP) for Echinococcus granulosis Coprodetection

    PubMed Central

    Salant, Harold; Abbasi, Ibrahim; Hamburger, Joseph

    2012-01-01

    We have previously developed a polymerase chain reaction (PCR) assay for detection of Echinococcus granulosus infection, which proved very sensitive and specific for identification of infected dogs. We have now developed a loop-mediated isothermal amplification (LAMP) assay, which amplifies the same genomic repeated sequences of E. granulosus for coprodetection. This assay enabled detection of a single egg in fecal samples and showed high species specificity for E. granulosus with no cross-amplification of DNA from closely related helminths, including Echinococcus multilocularis. Because the method does not require thermocycling for DNA amplification, or electrophoresis for amplicon detection, it can potentially be used for premortem identification of E. granulosus-infected dogs to enable large-scale surveys in endemic countries where highly specialized equipment to undertake PCR analysis is rare. PMID:22987649

  3. Tuberculosis Biomarker Extraction and Isothermal Amplification in an Integrated Diagnostic Device

    PubMed Central

    Creecy, Amy; Russ, Patricia K.; Solinas, Francesca; Wright, David W.; Haselton, Frederick R.

    2015-01-01

    In this study, we integrated magnetic bead-based sample preparation and isothermal loop mediated amplification (LAMP) of TB in a single tube. Surrogate sputum samples produced by the Program for Appropriate Technology in Health containing inactivated TB bacteria were used to test the diagnostic. In order to test the sample preparation method, samples were lysed, and DNA was manually extracted and eluted into water in the tube. In a thermal cycler, LAMP amplified TB DNA from 103 TB cells/mL of sputum at 53.5 ± 3.3 minutes, 104 cells/mL at 46.3 ± 2.2 minutes, and 105 cells/mL at 41.6 ± 1.9 minutes. Negative control samples did not amplify. Next, sample preparation was combined with in-tubing isothermal LAMP amplification by replacing the water elution chamber with a LAMP reaction chamber. In this intermediate configuration, LAMP amplified 103 cells/mL at 74 ± 10 minutes, 104 cells/mL at 60 ± 9 minutes, and 105 TB cells/mL of sputum at 54 ± 9 minutes. Two of three negative controls did not amplify; one amplified at 100 minutes. In the semi-automated system, DNA was eluted directly into an isothermal reaction solution containing the faster OptiGene DNA polymerase. The low surrogate sputum concentration, 103 TB cells/mL, amplified at 52.8 ± 3.3 minutes, 104 cells/mL at 45.4 ± 11.3 minutes, and 105 cells/mL at 31.8 ± 2.9 minutes. TB negative samples amplified at 66.4 ± 7.4 minutes. This study demonstrated the feasibility of a single tube design for integrating sample preparation and isothermal amplification, which with further development could be useful for point-of-care applications, particularly in a low-resource setting. PMID:26132307

  4. GMO detection in food and feed through screening by visual loop-mediated isothermal amplification assays.

    PubMed

    Wang, Cong; Li, Rong; Quan, Sheng; Shen, Ping; Zhang, Dabing; Shi, Jianxin; Yang, Litao

    2015-06-01

    Isothermal DNA/RNA amplification techniques are the primary methodology for developing on-spot rapid nucleic acid amplification assays, and the loop-mediated isothermal amplification (LAMP) technique has been developed and applied in the detection of foodborne pathogens, plant/animal viruses, and genetically modified (GM) food/feed contents. In this study, one set of LAMP assays targeting on eight frequently used universal elements, marker genes, and exogenous target genes, such as CaMV35S promoter, FMV35S promoter, NOS, bar, cry1Ac, CP4 epsps, pat, and NptII, were developed for visual screening of GM contents in plant-derived food samples with high efficiency and accuracy. For these eight LAMP assays, their specificity was evaluated by testing commercial GM plant events and their limits of detection were also determined, which are 10 haploid genome equivalents (HGE) for FMV35S promoter, cry1Ac, and pat assays, as well as five HGE for CaMV35S promoter, bar, NOS terminator, CP4 epsps, and NptII assays. The screening applicability of these LAMP assays was further validated successfully using practical canola, soybean, and maize samples. The results suggested that the established visual LAMP assays are applicable and cost-effective for GM screening in plant-derived food samples. PMID:25822163

  5. Application of Isothermal Amplification Techniques for Identification of Madurella mycetomatis, the Prevalent Agent of Human Mycetoma

    PubMed Central

    Ahmed, Sarah A.; van de Sande, Wendy W. J.; Desnos-Ollivier, Marie; Fahal, Ahmed H.; Mhmoud, Najwa A.

    2015-01-01

    Appropriate diagnosis and treatment of eumycetoma may vary significantly depending on the causative agent. To date, the most common fungus causing mycetoma worldwide is Madurella mycetomatis. This species fails to express any recognizable morphological characteristics, and reliable identification can therefore only be achieved with the application of molecular techniques. Recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) are proposed as alternatives to phenotypic methods. Species-specific primers were developed to target the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region of M. mycetomatis. Both isothermal amplification techniques showed high specificity and sufficient sensitivity to amplify fungal DNA and proved to be appropriate for detection of M. mycetomatis. Diagnostic performance of the techniques was assessed in comparison to conventional PCR using biopsy specimens from eumycetoma patients. RPA is reliable and easy to operate and has the potential to be implemented in areas where mycetoma is endemic. The techniques may be expanded to detect fungal DNA from environmental samples. PMID:26246484

  6. Simultaneous detection of RNA and DNA targets based on multiplex isothermal amplification.

    PubMed

    Dobnik, David; Morisset, Dany; Lenarčič, Rok; Ravnikar, Maja

    2014-04-01

    The detection of pathogenic microorganisms present in food, feed, plant, and other samples is important for providing safe food as well as for preventing the spread of microbes. The genome of pathogens is made of DNA or RNA, therefore a multiplex diagnostics tool would ideally be able to amplify and detect both RNA and DNA targets in parallel. With this goal we have developed an isothermal nucleic acid sequence based amplification [NASBA] implemented microarray analysis (NAIMA) procedure, suitable for the simultaneous multiplex amplification of RNA and DNA targets, coupled with the detection on ArrayTubes. The method is demonstrated to be very sensitive and specific for the detection of two economically important quarantine plant pathogens of potato, the potato spindle tuber viroid (RNA target) and Ralstonia solanacearum (DNA target). Because of its isothermal amplification and simple detection equipment, the method is also applicable for on-site analyses. NAIMA can be used in any domain where there is the need to detect RNA and DNA targets simultaneously. PMID:24625323

  7. Application of Isothermal Amplification Techniques for Identification of Madurella mycetomatis, the Prevalent Agent of Human Mycetoma.

    PubMed

    Ahmed, Sarah A; van de Sande, Wendy W J; Desnos-Ollivier, Marie; Fahal, Ahmed H; Mhmoud, Najwa A; de Hoog, G S

    2015-10-01

    Appropriate diagnosis and treatment of eumycetoma may vary significantly depending on the causative agent. To date, the most common fungus causing mycetoma worldwide is Madurella mycetomatis. This species fails to express any recognizable morphological characteristics, and reliable identification can therefore only be achieved with the application of molecular techniques. Recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) are proposed as alternatives to phenotypic methods. Species-specific primers were developed to target the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region of M. mycetomatis. Both isothermal amplification techniques showed high specificity and sufficient sensitivity to amplify fungal DNA and proved to be appropriate for detection of M. mycetomatis. Diagnostic performance of the techniques was assessed in comparison to conventional PCR using biopsy specimens from eumycetoma patients. RPA is reliable and easy to operate and has the potential to be implemented in areas where mycetoma is endemic. The techniques may be expanded to detect fungal DNA from environmental samples. PMID:26246484

  8. MALARIA DIAGNOSIS BY LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) IN THAILAND

    PubMed Central

    OCKER, Ronja; PROMPUNJAI, Yongyut; CHUTIPONGVIVATE, Salakchit; KARANIS, Panagiotis

    2016-01-01

    The loop-mediated isothermal amplification method (LAMP) is a recently developed molecular technique that amplifies nucleic acid under isothermal conditions. For malaria diagnosis, 150 blood samples from consecutive febrile malaria patients, and healthy subjects were screened in Thailand. Each sample was diagnosed by LAMP, microscopy and nested polymerase chain reaction (nPCR), using nPCR as the gold standard. Malaria LAMP was performed using Plasmodiumgenus and Plasmodium falciparum specific assays in parallel. For the genus Plasmodium, microscopy showed a sensitivity and specificity of 100%, while LAMP presented 99% of sensitivity and 93% of specificity. For P. falciparum, microscopy had a sensitivity of 95%, and LAMP of 90%, regarding the specificity; and microscopy presented 93% and LAMP 97% of specificity. The results of the genus-specific LAMP technique were highly consistent with those of nPCR and the sensitivity of P. falciparum detection was only marginally lower. PMID:27074321

  9. Visual detection of murray valley encephalitis virus by reverse transcription loop-mediated isothermal amplification.

    PubMed

    Gong, Rui; Wang, Han Hua; Qin, Hong; Guo, Xiao Ping; Ma, Xue Jun

    2015-03-01

    A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of Murray valley encephalitis virus (MVEV) infection. The reaction was performed in one step in a single tube at 63 °C for 60 min with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The detection limit of the RT-LAMP assay was 100 copies per reaction based on 10-fold dilutions of in vitro transcribed RNA derived from a synthetic MVEV DNA template. No cross-reaction was observed with other encephalitis-associated viruses. The assay was further evaluated using spiked cerebrospinal fluid sample with pseudotype virus containing the NS5 gene of MVEV. PMID:25800449

  10. Loop-mediated isothermal amplification method for a differential identification of human Taenia tapeworms.

    PubMed

    Sako, Yasuhito; Nkouawa, Agathe; Yanagida, Tetsuya; Ito, Akira

    2013-01-01

    Loop-mediated isothermal amplification (LAMP), which employs a Bst DNA polymerase with strand-displacement activity and four primers (two inner primers and two outer primers) recognizing six distinct regions on the target DNA, is a highly sensitive, specific, simple, and rapid nucleotide amplification method. Moreover, because the Bst DNA polymerase resists much DNA polymerase inhibitors present in biological specimens, the LAMP method is suitable for the detection of infectious agents from clinical material such as fecal samples. Here, we describe the LAMP method which can differentially detect and identify human Taenia tapeworms, Taenia solium, T. saginata, and T. asiatica, using DNA specimens prepared from parasite tissue and human fecal sample. PMID:24026690

  11. Microfluidic devices for nucleic acid (NA) isolation, isothermal NA amplification, and real-time detection.

    PubMed

    Mauk, Michael G; Liu, Changchun; Sadik, Mohamed; Bau, Haim H

    2015-01-01

    Molecular (nucleic acid)-based diagnostics tests have many advantages over immunoassays, particularly with regard to sensitivity and specificity. Most on-site diagnostic tests, however, are immunoassay-based because conventional nucleic acid-based tests (NATs) require extensive sample processing, trained operators, and specialized equipment. To make NATs more convenient, especially for point-of-care diagnostics and on-site testing, a simple plastic microfluidic cassette ("chip") has been developed for nucleic acid-based testing of blood, other clinical specimens, food, water, and environmental samples. The chip combines nucleic acid isolation by solid-phase extraction; isothermal enzymatic amplification such as LAMP (Loop-mediated AMPlification), NASBA (Nucleic Acid Sequence Based Amplification), and RPA (Recombinase Polymerase Amplification); and real-time optical detection of DNA or RNA analytes. The microfluidic cassette incorporates an embedded nucleic acid binding membrane in the amplification reaction chamber. Target nucleic acids extracted from a lysate are captured on the membrane and amplified at a constant incubation temperature. The amplification product, labeled with a fluorophore reporter, is excited with a LED light source and monitored in situ in real time with a photodiode or a CCD detector (such as available in a smartphone). For blood analysis, a companion filtration device that separates plasma from whole blood to provide cell-free samples for virus and bacterial lysis and nucleic acid testing in the microfluidic chip has also been developed. For HIV virus detection in blood, the microfluidic NAT chip achieves a sensitivity and specificity that are nearly comparable to conventional benchtop protocols using spin columns and thermal cyclers. PMID:25626529

  12. Comparison of fluorescent intercalating dyes for quantitative loop-mediated isothermal amplification (qLAMP).

    PubMed

    Oscorbin, Igor P; Belousova, Ekaterina A; Zakabunin, Aleksandr I; Boyarskikh, Ulyana A; Filipenko, Maksim L

    2016-01-01

    Real-time or quantitative loop-mediated isothermal amplification (qLAMP) is a promising technique for the accurate detection of pathogens in organisms and the environment. Here we present a comparative study of the performance of six fluorescent intercalating dyes-SYTO-9, SYTO-13, SYTO-82, SYBR Green I, SYBR Gold, EvaGreen-in three different qLAMP model systems. SYTO-9 and SYTO-82, which had the best results, were used for additional enzyme and template titration studies. SYTO-82 demonstrated the best combination of time-to-threshold (Tt) and signal-to-noise ratio (SNR). PMID:27401670

  13. Loop-mediated Isothermal Amplification Assay to Rapidly Detect Wheat Streak Mosaic Virus in Quarantined Plants

    PubMed Central

    Lee, Siwon; Kim, Jin-Ho; Choi, Ji-Young; Jang, Won-Cheoul

    2015-01-01

    We developed a loop-mediated isothermal amplification (LAMP) method to rapidly diagnose Wheat streak mosaic virus (WSMV) during quarantine inspections of imported wheat, corn, oats, and millet. The LAMP method was developed as a plant quarantine inspection method for the first time, and its simplicity, quickness, specificity and sensitivity were verified compared to current reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR quarantine methods. We were able to quickly screen for WSMV at quarantine sites with many test samples; thus, this method is expected to contribute to plant quarantine inspections. PMID:26674930

  14. Loop-mediated Isothermal Amplification Assay to Rapidly Detect Wheat Streak Mosaic Virus in Quarantined Plants.

    PubMed

    Lee, Siwon; Kim, Jin-Ho; Choi, Ji-Young; Jang, Won-Cheoul

    2015-12-01

    We developed a loop-mediated isothermal amplification (LAMP) method to rapidly diagnose Wheat streak mosaic virus (WSMV) during quarantine inspections of imported wheat, corn, oats, and millet. The LAMP method was developed as a plant quarantine inspection method for the first time, and its simplicity, quickness, specificity and sensitivity were verified compared to current reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR quarantine methods. We were able to quickly screen for WSMV at quarantine sites with many test samples; thus, this method is expected to contribute to plant quarantine inspections. PMID:26674930

  15. Detection and Characterization of Viral Species/Subspecies Using Isothermal Recombinase Polymerase Amplification (RPA) Assays.

    PubMed

    Glais, Laurent; Jacquot, Emmanuel

    2015-01-01

    Numerous molecular-based detection protocols include an amplification step of the targeted nucleic acids. This step is important to reach the expected sensitive detection of pathogens in diagnostic procedures. Amplifications of nucleic acid sequences are generally performed, in the presence of appropriate primers, using thermocyclers. However, the time requested to amplify molecular targets and the cost of the thermocycler machines could impair the use of these methods in routine diagnostics. Recombinase polymerase amplification (RPA) technique allows rapid (short-term incubation of sample and primers in an enzymatic mixture) and simple (isothermal) amplification of molecular targets. RPA protocol requires only basic molecular steps such as extraction procedures and agarose gel electrophoresis. Thus, RPA can be considered as an interesting alternative to standard molecular-based diagnostic tools. In this paper, the complete procedures to set up an RPA assay, applied to detection of RNA (Potato virus Y, Potyvirus) and DNA (Wheat dwarf virus, Mastrevirus) viruses, are described. The proposed procedure allows developing species- or subspecies-specific detection assay. PMID:25981257

  16. Real-Time Fluorescence Loop Mediated Isothermal Amplification for the Detection of Acinetobacter baumannii

    PubMed Central

    Wang, Qinqin; Zhou, Yanbin; Li, Shaoli; Zhuo, Chao; Xu, Siqi; Huang, Lixia; Yang, Ling; Liao, Kang

    2013-01-01

    Background Detection of Acinetobacter baumannii has been relying primarily on bacterial culture that often fails to return useful results in time. Although DNA-based assays are more sensitive than bacterial culture in detecting the pathogen, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. In addition, these molecular tools require expensive laboratory instruments. Therefore, establishing molecular tools for field use require simpler molecular platforms. The loop-mediated isothermal amplification method is relatively simple and can be improved for better use in a routine clinical bacteriology laboratory. A simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in the same platform has been developed in recent years. This method is referred to as real-time loop-mediated isothermal amplification. In this study, we attempted to utilize this method for rapid detection of A. baumannii. Methodology and Significant Findings Species-specific primers were designed to test the utility of this method. Clinical samples of A. baumannii were used to determine the sensitivity and specificity of this system compared to bacterial culture and a polymerase chain reaction method. All positive samples isolated from sputum were confirmed to be the species of Acinetobacter by 16S rRNA gene sequencing. The RealAmp method was found to be simpler and allowed real-time detection of DNA amplification, and could distinguish A. baumannii from Acinetobacter calcoaceticus and Acinetobacter genomic species 3. DNA was extracted by simple boiling method. Compared to bacterial culture, the sensitivity and specificity of RealAmp in detecting A. baumannii was 98.9% and 75.0%, respectively. Conclusion The RealAmp assay only requires a single unit, and the assay positivity can be verified by visual inspection. Therefore, this assay has

  17. Rapid and sensitive detection of Taura syndrome virus by reverse transcription loop-mediated isothermal amplification.

    PubMed

    Kiatpathomchai, Wansika; Jareonram, Wansadaj; Jitrapakdee, Sarawut; Flegel, T W

    2007-12-01

    Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, sensitivity and rapidity, which can be applied for disease diagnosis in shrimp aquaculture. The method is performed under isothermal conditions with a set of four specially designed primers that recognize six distinct sequences of the target. In this study, using the RT-LAMP method, a protocol for detecting Taura syndrome virus which is a causative agent of Penaeus vannamei was developed. Time and temperature conditions for detection of TSV were optimized for 60min at 63 degrees C. The nucleic acids of other shrimp pathogens (yellow head virus; YHV and white spot syndrome; WSSV) were not amplified by this RT-LAMP system. The detection of TSV using RT-LAMP was 10 times more sensitive than the RT-PCR but less sensitive than nested RT-PCR. However this system was more convenient, rapid, and does not require sophisticated PCR machine. PMID:17643501

  18. Development of a rapid cyprinid herpesvirus 2 detection method by loop-mediated isothermal amplification.

    PubMed

    Liang, L-G; Xie, J; Luo, D

    2014-10-01

    Cyprinid herpesvirus 2 (CyHV2) is a pathogen that causes severe disease and high mortality in goldfish and Prussian carp. We developed a six primer loop-mediated isothermal amplification (LAMP) assay targeting the intercapsomeric triplex protein gene. CyHV-2 DNA was 10-fold serially diluted (10(8)-10(0) copies μl(-1)) and was used as the template to determine primer sensitivity. LAMP assays were performed with DNA templates from other pathogens to determine specificity. The LAMP assay had an unequivocal detection limit of 10 copies μl(-1), which was 100 times lower than that of the polymerase chain reaction. Other pathogen strains were not amplified by the LAMP primers, indicating good specificity. SYBR Green I was added to visually detect the amplification products. Assay applicability was evaluated in 120 samples of Carassius auratus gibelio, and a positive rate of 92·5% was obtained. In conclusion, a conventional LAMP assay has high convenience, rapidity, sensitivity and specificity for detecting CyHV-2 in infected aquatic organisms. Significance and impact of the study: Herpesviral haematopoietic necrosis, caused by cyprinid herpesvirus 2 (CyHV-2), is a severe disease of goldfish and Prussian carp associated with high mortality. We developed a loop-mediated isothermal amplification (LAMP) assay to detect CyHV-2 at relatively low plasmid DNA copy levels. The results show that the LAMP assay has a number of advantages (simple, sensitive, rapid and specific) over the conventional polymerase chain reaction and can be applied in the laboratory and field. Particularly, the method is highly applicable to facilitate surveillance and early diagnosis of CyHV-2. PMID:24935791

  19. Rapid detection of mud crab dicistrovirus-1 using loop-mediated isothermal amplification.

    PubMed

    Guo, Zhixun; Zhang, Di; Ma, Hongling; Su, Youlu; Feng, Juan; Xu, Liwen

    2014-11-01

    Mud crab dicistrovirus-1 (MCDV-1) was isolated from the mud crab (Scylla paramamosain), resulting in mass mortality and widespread economic loss in China. In this study, a detection method for MCDV-1 using loop-mediated isothermal amplification was developed. Two pairs of primers targeting the VP2 gene were designed. These primers were the outer primers F3 and B3, and the inner primers FIP and BIP. Optimal amplification was carried out using 0.2 μmol/L F3/B3, 1.6 μmol/L FIP/BIP, 6 mmol/L Mg(2+), 0.8 mmol/L dNTPs, and 0.8 mol/L betaine, and completed in 1h at 62°C. The products demonstrated a ladder pattern on agarose gel electrophoresis and could also be detected visually according to turbidity, or by adding SYBR Green I and observing a color change from orange to green. The proposed method could specifically amplify MCDV-1 gene fragments. Sensitivity assay revealed that six copies of the viral genome could be detected by this method, which was 1000-fold more sensitive than that of conventional PCR using constructed plasmid as amplification template. At clinical sample level, sensitivity of LAMP was 100-fold higher than that of conventional PCR. PMID:25172047

  20. Detection of the food allergen celery via loop-mediated isothermal amplification technique.

    PubMed

    Zahradnik, Celine; Martzy, Roland; Mach, Robert L; Krska, Rudolf; Farnleitner, Andreas H; Brunner, Kurt

    2014-11-01

    Since 2005, celery and celery products have to be labeled according to Directive 2003/89/EC due to their allergenic potential. In order to provide a DNA-based, rapid and simple detection method suitable for high-throughput analysis, a loop-mediated isothermal amplification (LAMP) assay for the detection of celery (Apium graveolens) was developed. The assay was tested for specificity for celery since closely related species also hold food relevance. The limit of detection (LOD) for spiked food samples was found to be as low as 7.8 mg of dry celery powder per kilogram. An evaluation of different amplification and detection platforms was performed to show reliable detection independent from the instrument used for amplification (thermal cycler or heating block) and detection mechanisms (real-time fluorescence detection, agarose gel electrophoresis or nucleic acid staining). The analysis of 10 commercial food samples representing diverse and complex food matrices, and a false-negative rate of 0% for approximately 24 target copies or 0.08 ng celery DNA for three selected food matrices show that LAMP has the potential to be used as an alternative strategy for the detection of allergenic celery. The performance of the developed LAMP assay turned out to be equal or superior to the best available PCR assay for the detection of celery in food products. PMID:24880868

  1. Recombinase-based isothermal amplification of nucleic acids with self-avoiding molecular recognition systems (SAMRS).

    PubMed

    Sharma, Nidhi; Hoshika, Shuichi; Hutter, Daniel; Bradley, Kevin M; Benner, Steven A

    2014-10-13

    Recombinase polymerase amplification (RPA) is an isothermal method to amplify nucleic acid sequences without the temperature cycling that classical PCR uses. Instead of using heat to denature the DNA duplex, RPA uses recombination enzymes to swap single-stranded primers into the duplex DNA product; these are then extended using a strand-displacing polymerase to complete the cycle. Because RPA runs at low temperatures, it never forces the system to recreate base-pairs following Watson-Crick rules, and therefore it produces undesired products that impede the amplification of the desired product, complicating downstream analysis. Herein, we show that most of these undesired side products can be avoided if the primers contain components of a self-avoiding molecular recognition system (SAMRS). Given the precision that is necessary in the recombination systems for them to function biologically, it is surprising that they accept SAMRS. SAMRS-RPA is expected to be a powerful tool within the range of amplification techniques available to scientists. PMID:25209570

  2. Detection of Fusarium graminearum DNA using a loop-mediated isothermal amplification (LAMP) assay.

    PubMed

    Niessen, Ludwig; Vogel, Rudi F

    2010-06-15

    Loop-mediated isothermal amplification (LAMP) of DNA is a simple, cost effective, and rapid method for the specific detection of genomic DNA using a set of six oligonucleotide primers with eight binding sites hybridizing specifically to different regions of a target gene, and a thermophilic DNA polymerase from Geobacillus stearothermophilus for DNA amplification. The method has been applied in various assays for the diagnosis of bacterial and viral infections of humans and animals, sexing of bovine and swine embryos, and in the detection of bacteria from environmental samples. Only recently, first applications for fungal organisms were published. During the current study a LAMP assay was developed for the specific detection of Fusarium graminearum, the major causative agent of Fusarium head blight of small cereals and producer of the mycotoxins deoxynivalenol, nivalenol, and zearalenone. The assay was based on the gaoA gene (galactose oxidase) of the fungus. Amplification of DNA during the reaction was indirectly detected in situ by using calcein fluorescence as a marker without the necessity of time-consuming electrophoretic analysis. The assay was optimized for rapidness, specificity, and sensitivity and was shown to detect the presence of less than 2pg of purified target DNA per reaction within 30 min. Within 132 fungal species tested, exclusively DNA isolated from cultures of F. graminearum (lineages 1-9) resulted in a fluorescent signal after amplification with the LAMP assay. The method was demonstrated to be useful in the analysis of fungal cultures by direct analysis of surface scrapings from agar plate cultures, direct testing of single infected barley grains, and detection of F. graminearum in total genomic DNA isolated from bulk samples of ground wheat grains. Results obtained indicate that LAMP offers an interesting new assay format for the rapid and specific DNA-based detection and identification of agriculturally important toxigenic fungi in pure

  3. NAIL: Nucleic Acid detection using Isotachophoresis and Loop-mediated isothermal amplification.

    PubMed

    Borysiak, Mark D; Kimura, Kevin W; Posner, Jonathan D

    2015-04-01

    Nucleic acid amplification tests are the gold standard for many infectious disease diagnoses due to high sensitivity and specificity, rapid operation, and low limits of detection. Despite the advantages of nucleic acid amplification tests, they currently offer limited point-of-care (POC) utility due to the need for complex instruments and laborious sample preparation. We report the development of the Nucleic Acid Isotachophoresis LAMP (NAIL) diagnostic device. NAIL uses isotachophoresis (ITP) and loop-mediated isothermal amplification (LAMP) to extract and amplify nucleic acids from complex matrices in less than one hour inside of an integrated chip. ITP is an electrokinetic separation technique that uses an electric field and two buffers to extract and purify nucleic acids in a single step. LAMP amplifies nucleic acids at constant temperature and produces large amounts of DNA that can be easily detected. A mobile phone images the amplification results to eliminate the need for laser fluorescent detection. The device requires minimal user intervention because capillary valves and heated air chambers act as passive valves and pumps for automated fluid actuation. In this paper, we describe NAIL device design and operation, and demonstrate the extraction and detection of pathogenic E. coli O157:H7 cells from whole milk samples. We use the Clinical and Laboratory Standards Institute (CLSI) limit of detection (LoD) definitions that take into account the variance from both positive and negative samples to determine the diagnostic LoD. According to the CLSI definition, the NAIL device has a limit of detection (LoD) of 1000 CFU mL(-1) for E. coli cells artificially inoculated into whole milk, which is two orders of magnitude improvement to standard tube-LAMP reactions with diluted milk samples and comparable to lab-based methods. The NAIL device potentially offers significant reductions in the complexity and cost of traditional nucleic acid diagnostics for POC applications

  4. Strand Invasion Based Amplification (SIBA®): a novel isothermal DNA amplification technology demonstrating high specificity and sensitivity for a single molecule of target analyte.

    PubMed

    Hoser, Mark J; Mansukoski, Hannu K; Morrical, Scott W; Eboigbodin, Kevin E

    2014-01-01

    Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR) in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA). SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO) into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2'-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella. PMID:25419812

  5. Development of an in situ loop-mediated isothermal amplification technique for chromosomal localization of DNA sequences

    NASA Astrophysics Data System (ADS)

    Meng, Qinglei; Wang, Shi; Zhang, Lingling; Huang, Xiaoting; Bao, Zhenmin

    2013-01-01

    In situ loop-mediated isothermal amplification (in situ LAMP) combines in situ hybridization and loop-mediated isothermal amplification (LAMP) techniques for chromosomal localization of DNA sequences. In situ LAMP is a method that is generally more specific and sensitive than conventional techniques such as fluorescence in situ hybridization (FISH), primed in situ labeling (PRINS), and cycling primed in situ labeling (C-PRINS). Here, we describe the development and application of in situ LAMP to identify the chromosomal localization of DNA sequences. To benchmark this technique, we successfully applied this technique to localize the major ribosomal RNA gene on the chromosomes of the Zhikong scallop ( Chlamys farreri).

  6. Rapid Detection of Shrimp White Spot Syndrome Virus by Real Time, Isothermal Recombinase Polymerase Amplification Assay

    PubMed Central

    Xia, Xiaoming; Yu, Yongxin; Weidmann, Manfred; Pan, Yingjie; Yan, Shuling; Wang, Yongjie

    2014-01-01

    White spot syndrome virus (WSSV) causes large economic losses to the shrimp aquaculture industry, and thus far there are no efficient therapeutic treatments available against this lethal virus. In this study, we present the development of a novel real time isothermal recombinase polymerase amplification (RPA) assay for WSSV detection on a small ESEQuant Tube Scanner device. The RPA sensitivity, specificity and rapidity were evaluated by using a plasmid standard as well as viral and shrimp genomic DNAs. Compared with qPCR, the RPA assay revealed more satisfactory performance. It reached a detection limit up to 10 molecules in 95% of cases as determined by probit analysis of 8 independent experiments within 6.41±0.17 min at 39°C. Consequently, this rapid RPA method has great application potential for field use or point of care diagnostics. PMID:25121957

  7. Development of loop-mediated isothermal amplification methods for detecting Taylorella equigenitalis and Taylorella asinigenitalis

    PubMed Central

    KINOSHITA, Yuta; NIWA, Hidekazu; KATAYAMA, Yoshinari; HARIU, Kazuhisa

    2015-01-01

    ABSTRACT Taylorella equigenitalis is a causative bacterium of contagious equine metritis (CEM), and Taylorella asinigenitalis is species belonging to genus Taylorella. The authors developed two loop-mediated isothermal amplification (LAMP) methods, Te-LAMP and Ta-LAMP, for detecting T. equigenitalis and T. asinigenitalis, respectively. Using experimentally spiked samples, Te-LAMP was as sensitive as a published semi-nested PCR method, and Ta-LAMP was more sensitive than conventional PCR. Multiplex LAMP worked well without nonspecific reactions, and the analytical sensitivities of multiplex LAMP in the spiked samples were almost equivalent to those of Te-LAMP and Ta-LAMP. Therefore, the LAMP methods are considered useful tools to detect T. equigenitalis and/or T. asinigenitalis, and preventive measures will be rapidly implemented if the occurrence of CEM is confirmed by the LAMP methods. PMID:25829868

  8. Loop-mediated isothermal amplification (LAMP) method for detection of genetically modified maize T25.

    PubMed

    Xu, Junyi; Zheng, Qiuyue; Yu, Ling; Liu, Ran; Zhao, Xin; Wang, Gang; Wang, Qinghua; Cao, Jijuan

    2013-11-01

    The loop-mediated isothermal amplification (LAMP) assay indicates a potential and valuable means for genetically modified organism (GMO) detection especially for its rapidity, simplicity, and low cost. We developed and evaluated the specificity and sensitivity of the LAMP method for rapid detection of the genetically modified (GM) maize T25. A set of six specific primers was successfully designed to recognize six distinct sequences on the target gene, including a pair of inner primers, a pair of outer primers, and a pair of loop primers. The optimum reaction temperature and time were verified to be 65°C and 45 min, respectively. The detection limit of this LAMP assay was 5 g kg(-1) GMO component. Comparative experiments showed that the LAMP assay was a simple, rapid, accurate, and specific method for detecting the GM maize T25. PMID:24804053

  9. Loop-mediated isothermal amplification (LAMP)-based method for rapid mushroom species identification.

    PubMed

    Vaagt, Franziska; Haase, Ilka; Fischer, Markus

    2013-02-27

    Toxic mushroom species, such as the death cap ( Amanita phalloides ), are responsible for most mushroom poisonings. In the present work, novel loop-mediated isothermal amplification (LAMP) assays were used for the differentiation of even closely related edible and toxic mushroom species. The applicability of these methods was tested by cross-reaction studies and analysis of spiked mushroom samples (raw and fried material). Contaminations at the level of 2% (w/w) could be detected in different mushroom blends. Three detection methods were used: agarose gel analysis, fluorimetric real-time detection, and visual detection by lateral flow dipsticks (LFD). The LAMP assay combined with LFD detection allows the identification of A. phalloides in about 2 h (including DNA extraction) at a very low level of technical equipment (micropestle, water bath, and mobile centrifuge), which makes this technique perfectly suited for on-site applications. PMID:23350919

  10. A simple identification method of saliva by detecting Streptococcus salivarius using loop-mediated isothermal amplification.

    PubMed

    Nakanishi, Hiroaki; Ohmori, Takeshi; Hara, Masaaki; Takada, Aya; Shojo, Hideki; Adachi, Noboru; Saito, Kazuyuki

    2011-01-01

    We previously reported that detection of Streptococcus salivarius is feasible for proving the presence of saliva in a forensic sample. Here, a simple and rapid method for the detection of S. salivarius in forensic samples was developed that uses loop-mediated isothermal amplification (LAMP). The LAMP primer set was designed using S. salivarius-specific sequences of glucosyltransferase K. To simplify the procedure, the sample was prepared by boiling and mutanolysin treatment only, and the entire analytical process was completed within 2.5 h. The cut-off value was set at 0.1 absorbance units, measured at 660 nm, upon termination of the reaction. S. salivarius was identified in all saliva samples, but was not detected in other body fluids or on the skin surface. Using this method, S. salivarius was successfully detected in various mock forensic samples. We therefore suggest that this approach is useful for the identification of saliva in forensic practice. PMID:21198609

  11. Detection of acute toxoplasmosis in pigs using loop-mediated isothermal amplification and quantitative PCR.

    PubMed

    Wang, Yanhua; Wang, Guangxiang; Zhang, Delin; Yin, Hong; Wang, Meng

    2013-10-01

    A loop-mediated isothermal amplification (LAMP) assay allows rapid diagnosis of Toxoplasma gondii infection. In the present study, the LAMP assay was evaluated using blood from both naturally and experimentally infected pigs. The sensitivity of the LAMP assay was compared with that of Q-PCR. Both assays detected T. gondii in the blood of experimentally infected pigs, with 100% agreement. In infected blood samples, the parasite was detected as early as 2 days post-infection and reached a peak in 3-5 days. In 216 field serum samples, the detection rates of LAMP and Q-PCR assays were 6.9% and 7.8%, respectively. This result indicates that the sensitivity of the LAMP assay was slightly lower than that of the Q-PCR assay. However, the LAMP may be an attractive diagnostic method in conditions where sophisticated and expensive equipment is unavailable. This assay could be a powerful supplement to current diagnostic methods. PMID:24327785

  12. Development of a loop-mediated isothermal amplification assay for rapid detection of Burkholderia mallei.

    PubMed

    Mirzai, S; Safi, S; Mossavari, N; Afshar, D; Bolourchian, M

    2016-01-01

    The present study was conducted to establish a Loop-mediated isothermal amplification (LAMP) technique for the rapid detection of B. mallei the etiologic agent of glanders, a highly contagious disease of equines. A set of six specific primers targeting integrase gene cluster were designed for the LAMP test. The reaction was optimized using different temperatures and time intervals. The specificity of the assay was evaluated using DNA from B.pseudomallei and Pseudomonas aeruginosa. The LAMP products were analyzed both visually and under UV light after electrophoresis. The optimized conditions were found to be at 63ºC for 60 min. The assay showed high specificity and sensitivity. It was concluded that the established LAMP assay is a rapid, sensitive and practical tool for detection of B. mallei and early diagnosis of glanders. PMID:27609471

  13. Development of Loop-Mediated Isothermal Amplification for Detection of Leifsonia xyli subsp. xyli in Sugarcane

    PubMed Central

    Liu, Jing; Guo, Jinlong; Chen, Rukai; Grisham, Michael Paul

    2013-01-01

    Ratoon stunt, caused by the xylem-limited coryneform bacterium Leifsonia xyli subsp. xyli (Lxx), is a deep bacteriosis and prevalent in most of sugarcane-producing countries. Based on loop-mediated isothermal amplification (LAMP), we developed a method for detecting Lxx. The major advantages of the LAMP method are visual judgment by color and time saving with only 60 min for identification of Lxx and without the need for costly PCR apparatus and gel scanner. In the present study, positive and negative samples detected by the LAMP method were clearly distinguishable. When total DNA extracted from internode juice was used as the template, the sensitivity of LAMP was 10 times higher than that of the conventional PCR detection. The LAMP assay is a highly specific, rapid, and sensitive method for the diagnosis of ratoon stunt caused by Lxx in sugarcane. This is the first report of LAMP-based assay for the detection of Lxx in sugarcane. PMID:23710444

  14. A simple colorimetric DNA detection by target-induced hybridization chain reaction for isothermal signal amplification.

    PubMed

    Ma, Cuiping; Wang, Wenshuo; Mulchandani, Ashok; Shi, Chao

    2014-07-15

    A novel DNA detection method is presented based on a gold nanoparticle (AuNP) colorimetric assay and hybridization chain reaction (HCR). In this method, target DNA hybridized with probe DNA modified on AuNP, and triggered HCR. The resulting HCR products with a large number of negative charges significantly enhanced the stability of AuNPs, inhibiting aggregation of AuNPs at an elevated salt concentration. The approach was highly sensitive and selective. Using this enzyme-free and isothermal signal amplification method, we were able to detect target DNA at concentrations as low as 0.5 nM with the naked eye. Our method also has great potential for detecting other analytes, such as metal ions, proteins, and small molecules, if the target analytes could make HCR products attach to AuNPs. PMID:24780220

  15. Development of a loop-mediated isothermal amplification assay for detection of Trichomonas vaginalis.

    PubMed

    Reyes, John Carlo B; Solon, Juan Antonio A; Rivera, Windell L

    2014-07-01

    A loop-mediated isothermal amplification (LAMP) assay targeting the 2-kbp repeated DNA species-specific sequence was developed for detection of Trichomonas vaginalis, the causative agent of trichomoniasis. The analytical sensitivity and specificity of the LAMP assay were evaluated using pooled genital swab and urine specimens, respectively, spiked with T. vaginalis trophozoites. Genital secretion and urine did not inhibit the detection of the parasite. The sensitivity of the LAMP was 10-1000 times higher than the PCR performed. The detection limit of LAMP was 1 trichomonad for both spiked genital swab and urine specimens. Also, LAMP did not exhibit cross-reactivity with closely-related trichomonads, Trichomonas tenax and Pentatrichomonas hominis, and other enteric and urogenital microorganisms, Entamoeba histolytica, Candida albicans, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. This is the first report of a LAMP assay for the detection of T. vaginalis and has prospective application for rapid diagnosis and control of trichomoniasis. PMID:24792836

  16. Loop-Mediated Isothermal Amplification Assay for Identification of Five Human Plasmodium Species in Malaysia

    PubMed Central

    Lau, Yee-Ling; Lai, Meng-Yee; Fong, Mun-Yik; Jelip, Jenarun; Mahmud, Rohela

    2016-01-01

    The lack of rapid, affordable, and accurate diagnostic tests represents the primary hurdle affecting malaria surveillance in resource- and expertise-limited areas. Loop-mediated isothermal amplification (LAMP) is a sensitive, rapid, and cheap diagnostic method. Five species-specific LAMP assays were developed based on 18S rRNA gene. Sensitivity and specificity of LAMP results were calculated as compared with microscopic examination and nested polymerase chain reaction. LAMP reactions were highly sensitive with the detection limit of one copy for Plasmodium vivax, Plasmodium falciparum, and Plasmodium malariae and 10 copies for Plasmodium knowlesi and Plasmodium ovale. LAMP positively detected all human malaria species in all positive samples (N = 134; sensitivity = 100%) within 35 minutes. All negative samples were not amplified by LAMP (N = 67; specificity = 100%). LAMP successfully detected two samples with very low parasitemia. LAMP may offer a rapid, simple, and reliable test for the diagnosis of malaria in areas where malaria is prevalent. PMID:26598573

  17. Loop-Mediated Isothermal Amplification Assay for Identification of Five Human Plasmodium Species in Malaysia.

    PubMed

    Lau, Yee-Ling; Lai, Meng-Yee; Fong, Mun-Yik; Jelip, Jenarun; Mahmud, Rohela

    2016-02-01

    The lack of rapid, affordable, and accurate diagnostic tests represents the primary hurdle affecting malaria surveillance in resource- and expertise-limited areas. Loop-mediated isothermal amplification (LAMP) is a sensitive, rapid, and cheap diagnostic method. Five species-specific LAMP assays were developed based on 18S rRNA gene. Sensitivity and specificity of LAMP results were calculated as compared with microscopic examination and nested polymerase chain reaction. LAMP reactions were highly sensitive with the detection limit of one copy for Plasmodium vivax, Plasmodium falciparum, and Plasmodium malariae and 10 copies for Plasmodium knowlesi and Plasmodium ovale. LAMP positively detected all human malaria species in all positive samples (N = 134; sensitivity = 100%) within 35 minutes. All negative samples were not amplified by LAMP (N = 67; specificity = 100%). LAMP successfully detected two samples with very low parasitemia. LAMP may offer a rapid, simple, and reliable test for the diagnosis of malaria in areas where malaria is prevalent. PMID:26598573

  18. Rapid detection of shrimp white spot syndrome virus by real time, isothermal recombinase polymerase amplification assay.

    PubMed

    Xia, Xiaoming; Yu, Yongxin; Weidmann, Manfred; Pan, Yingjie; Yan, Shuling; Wang, Yongjie

    2014-01-01

    White spot syndrome virus (WSSV) causes large economic losses to the shrimp aquaculture industry, and thus far there are no efficient therapeutic treatments available against this lethal virus. In this study, we present the development of a novel real time isothermal recombinase polymerase amplification (RPA) assay for WSSV detection on a small ESEQuant Tube Scanner device. The RPA sensitivity, specificity and rapidity were evaluated by using a plasmid standard as well as viral and shrimp genomic DNAs. Compared with qPCR, the RPA assay revealed more satisfactory performance. It reached a detection limit up to 10 molecules in 95% of cases as determined by probit analysis of 8 independent experiments within 6.41 ± 0.17 min at 39 °C. Consequently, this rapid RPA method has great application potential for field use or point of care diagnostics. PMID:25121957

  19. Development of reverse transcription loop mediated isothermal amplification assay for rapid detection of bluetongue viruses.

    PubMed

    Mohandas, Sreekala S; Muthuchelvan, Dhanavelu; Pandey, Awadh Bihari; Biswas, Sanchay Kumar; Chand, Karam; Venkatesan, Gnanavel; Choudhary, Dheeraj; Ramakrishnan, Muthannan Andavar; Mondal, Bimalendu

    2015-09-15

    A single-step reverse transcription loop mediated isothermal amplification (RT-LAMP) assay targeting NS1 - a highly conserved gene among BTV serotypes was optimized and validated with seven serotypes: BTV-1, BTV-2, BTV-9, BTV-10, BTV-16, BTV-21 and BTV-23. The relative sensitivity of the assay was 0.3 TCID50 and no cross reactivity could be observed with foot and mouth disease, peste-des-petits-ruminants, goatpox, sheeppox and orf viruses. The established assay was also assessed by screening of clinical samples and the result is comparable with conventional RT-PCR. The RT-LAMP assay described here could be an additional tool to the existing assays for diagnosis/surveillance of BTV. PMID:26073661

  20. Loop-mediated isothermal amplification (LAMP) method for detection of genetically modified maize T25

    PubMed Central

    Xu, Junyi; Zheng, Qiuyue; Yu, Ling; Liu, Ran; Zhao, Xin; Wang, Gang; Wang, Qinghua; Cao, Jijuan

    2013-01-01

    The loop-mediated isothermal amplification (LAMP) assay indicates a potential and valuable means for genetically modified organism (GMO) detection especially for its rapidity, simplicity, and low cost. We developed and evaluated the specificity and sensitivity of the LAMP method for rapid detection of the genetically modified (GM) maize T25. A set of six specific primers was successfully designed to recognize six distinct sequences on the target gene, including a pair of inner primers, a pair of outer primers, and a pair of loop primers. The optimum reaction temperature and time were verified to be 65°C and 45 min, respectively. The detection limit of this LAMP assay was 5 g kg−1 GMO component. Comparative experiments showed that the LAMP assay was a simple, rapid, accurate, and specific method for detecting the GM maize T25. PMID:24804053

  1. Use of Malachite Green-Loop Mediated Isothermal Amplification for Detection of Plasmodium spp. Parasites

    PubMed Central

    Lucchi, Naomi W.; Ljolje, Dragan; Silva-Flannery, Luciana; Udhayakumar, Venkatachalam

    2016-01-01

    Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and rapid diagnostic tests. Isothermal nucleic acid amplification assays such as the loop-mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG) dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63°C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1–8 parasites/μL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed. PMID:26967908

  2. Isothermal target and probe amplification assay for the real-time rapid detection of Staphylococcus aureus.

    PubMed

    Shin, Hyewon; Kim, Minhwan; Yoon, Eunju; Kang, Gyoungwon; Kim, Seungyu; Song, Aelee; Kim, Jeongsoon

    2015-04-01

    Staphylococcus aureus, the species most commonly associated with staphylococcal food poisoning, is one of the most prevalent causes of foodborne disease in Korea and other parts of the world, with much damage inflicted to the health of individuals and economic losses estimated at $120 million. To reduce food poisoning outbreaks by implementing prevention methods, rapid detection of S. aureus in foods is essential. Various types of detection methods for S. aureus are available. Although each method has advantages and disadvantages, high levels of sensitivity and specificity are key aspects of a robust detection method. Here, we describe a novel real-time isothermal target and probe amplification (iTPA) method that allows the rapid and simultaneous amplification of target DNA (the S. aureus nuc gene) and a fluorescence resonance energy transfer-based signal probe under isothermal conditions at 61 °C or detection of S. aureus in real time. The assay was able to specifically detect all 91 S. aureus strains tested without nonspecific detection of 51 non-S. aureus strains. The real-time iTPA assay detected S. aureus at an initial level of 10(1) CFU in overnight cultures of preenriched food samples (kiwi dressing, soybean milk, and custard cream). The advantage of this detection system is that it does not require a thermal cycler, reducing the cost of the real-time PCR and its footprint. Combined with a miniaturized fluorescence detector, this system can be developed into a simplified quantitative hand-held real-time device, which is often required. The iTPA assay was highly reliable and therefore may be used as a rapid and sensitive means of identifying S. aureus in foods. PMID:25836397

  3. Use of Malachite Green-Loop Mediated Isothermal Amplification for Detection of Plasmodium spp. Parasites.

    PubMed

    Lucchi, Naomi W; Ljolje, Dragan; Silva-Flannery, Luciana; Udhayakumar, Venkatachalam

    2016-01-01

    Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and rapid diagnostic tests. Isothermal nucleic acid amplification assays such as the loop-mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG) dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63°C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1-8 parasites/μL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed. PMID:26967908

  4. Real-time isothermal detection of Shiga toxin-producing Escherichia coli using recombinase polymerase amplification.

    PubMed

    Murinda, Shelton E; Ibekwe, A Mark; Zulkaffly, Syaizul; Cruz, Andrew; Park, Stanley; Razak, Nur; Paudzai, Farah Md; Ab Samad, Liana; Baquir, Khairul; Muthaiyah, Kokilah; Santiago, Brenna; Rusli, Amirul; Balkcom, Sean

    2014-07-01

    Shiga toxin-producing Escherichia coli (STEC) are a major family of foodborne pathogens of public health, zoonotic, and economic significance in the United States and worldwide. To date, there are no published reports on use of recombinase polymerase amplification (RPA) for STEC detection. The primary goal of this study was to assess the potential application of RPA in detection of STEC. This study focused on designing and evaluating RPA primers and fluorescent probes for isothermal (39°C) detection of STEC. Compatible sets of candidate primers and probes were designed for detection of Shiga toxin 1 and 2 (Stx1 and 2), respectively. The sets were evaluated for specificity and sensitivity against STEC (n=12) of various stx genotypes (stx1/stx2, stx1, or stx2, respectively), including non-Stx-producing E. coli (n=28) and other genera (n=7). The primers and probes that were designed targeted amplification of the subunit A moiety of stx1 and stx2. The assay detected STEC in real time (within 5-10 min at 39°C) with high sensitivity (93.5% vs. 90%; stx1 vs. stx2), specificity (99.1% vs. 100%; stx1 vs. stx2), and predictive value (97.9% for both stx1 vs. stx2). Limits of detection of ∼ 5-50 colony-forming units/mL were achieved in serially diluted cultures grown in brain heart infusion broth. This study successfully demonstrated for the first time that RPA can be used for isothermal real-time detection of STEC. PMID:24749488

  5. Sensitive detection of transcription factors by isothermal exponential amplification-based colorimetric assay.

    PubMed

    Zhang, Yan; Hu, Juan; Zhang, Chun-Yang

    2012-11-01

    Transcription factors regulate gene expression by binding to specific DNA sequences within the regulatory regions of genes and have become potential targets in clinical diagnosis and drug development. However, traditional approaches for the detection of transcription factors are usually laborious and time-consuming with a low sensitivity. Here, we develop an isothermal exponential amplification reaction (EXPAR)-based colorimetric assay for simple and sensitive detection of transcription factor NF-κB p50. In this assay, the presence of NF-κB p50 is converted to the reporter oligonucleotides through protein-DNA interaction, exonuclease III digestion, and isothermal exponential amplification. The subsequent sandwich hybridization of the reporter oligonucleotides with the gold nanoparticle (AuNP)-labeled DNA probes generates a red-to-purple color change, allowing the visual detection of NF-κB p50 with the naked eye. Notably, this method converts the detection of transcription factors to the detection of DNA without the requirement of DNA marker-linked antibodies in the case of immuno-PCR and can sensitively measure NF-κB p50 with a detection limit of 3.8 pM, which has improved by as much as 4 orders of magnitude as compared with the conventional AuNP-based colorimetric assay and the label-free luminescence assay and up to 4 orders of magnitude as compared with fluorescence resonance energy transfer (FRET)-based assay as well. Importantly, this method can be used to measure TNF-α-induced endogenous NF-κB p50 in HeLa cell nuclear extracts and might be further applied for the detection of various DNA-binding proteins and aptamer-binding molecules. PMID:23050558

  6. Loop-Mediated Isothermal Amplification (LAMP) Method for Rapid Detection of Trypanosoma brucei rhodesiense

    PubMed Central

    Njiru, Zablon Kithinji; Mikosza, Andrew Stanislaw John; Armstrong, Tanya; Enyaru, John Charles; Ndung'u, Joseph Mathu; Thompson, Andrew Richard Christopher

    2008-01-01

    Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique that rapidly amplifies target DNA under isothermal conditions. In the present study, a LAMP test was designed from the serum resistance-associated (SRA) gene of Trypanosoma brucei rhodesiense, the cause of the acute form of African sleeping sickness, and used to detect parasite DNA from processed and heat-treated infected blood samples. The SRA gene is specific to T. b. rhodesiense and has been shown to confer resistance to lysis by normal human serum. The assay was performed at 62°C for 1 h, using six primers that recognised eight targets. The template was varying concentrations of trypanosome DNA and supernatant from heat-treated infected blood samples. The resulting amplicons were detected using SYTO-9 fluorescence dye in a real-time thermocycler, visual observation after the addition of SYBR Green I, and gel electrophoresis. DNA amplification was detected within 35 min. The SRA LAMP test had an unequivocal detection limit of one pg of purified DNA (equivalent to 10 trypanosomes/ml) and 0.1 pg (1 trypanosome/ml) using heat-treated buffy coat, while the detection limit for conventional SRA PCR was ∼1,000 trypanosomes/ml. The expected LAMP amplicon was confirmed through restriction enzyme RsaI digestion, identical melt curves, and sequence analysis. The reproducibility of the SRA LAMP assay using water bath and heat-processed template, and the ease in results readout show great potential for the diagnosis of T. b. rhodesiense in endemic regions. PMID:18253475

  7. Molecular-based isothermal tests for field diagnosis of malaria and their potential contribution to malaria elimination.

    PubMed

    Oriero, Eniyou C; Jacobs, Jan; Van Geertruyden, Jean-Pierre; Nwakanma, Davis; D'Alessandro, Umberto

    2015-01-01

    In countries where malaria transmission has decreased substantially, thanks to the scale-up of control interventions, malaria elimination may be feasible. Nevertheless, this goal requires new strategies such as the active detection and treatment of infected individuals. As the detection threshold for the currently used diagnostic methods is 100 parasites/μL, most low-density, asymptomatic infections able to maintain transmission cannot be detected. Identifying them by molecular methods such as PCR is a possible option but the field deployment of these tests is problematic. Isothermal amplification of nucleic acids (at a constant temperature) offers the opportunity of addressing some of the challenges related to the field deployment of molecular diagnostic methods. One of the novel isothermal amplification methods for which a substantial amount of work has been done is the loop-mediated isothermal amplification (LAMP) assay. The present review describes LAMP and several other isothermal nucleic acid amplification methods, such as thermophilic helicase-dependent amplification, strand displacement amplification, recombinase polymerase amplification and nucleic acid sequence-based amplification, and explores their potential use as high-throughput, field-based molecular tests for malaria diagnosis. PMID:25223973

  8. Endpoint Visual Detection of Three Genetically Modified Rice Events by Loop-Mediated Isothermal Amplification

    PubMed Central

    Chen, Xiaoyun; Wang, Xiaofu; Jin, Nuo; Zhou, Yu; Huang, Sainan; Miao, Qingmei; Zhu, Qing; Xu, Junfeng

    2012-01-01

    Genetically modified (GM) rice KMD1, TT51-1, and KF6 are three of the most well known transgenic Bt rice lines in China. A rapid and sensitive molecular assay for risk assessment of GM rice is needed. Polymerase chain reaction (PCR), currently the most common method for detecting genetically modified organisms, requires temperature cycling and relatively complex procedures. Here we developed a visual and rapid loop-mediated isothermal amplification (LAMP) method to amplify three GM rice event-specific junction sequences. Target DNA was amplified and visualized by two indicators (SYBR green or hydroxy naphthol blue [HNB]) within 60 min at an isothermal temperature of 63 °C. Different kinds of plants were selected to ensure the specificity of detection and the results of the non-target samples were negative, indicating that the primer sets for the three GM rice varieties had good levels of specificity. The sensitivity of LAMP, with detection limits at low concentration levels (0.01%–0.005% GM), was 10- to 100-fold greater than that of conventional PCR. Additionally, the LAMP assay coupled with an indicator (SYBR green or HNB) facilitated analysis. These findings revealed that the rapid detection method was suitable as a simple field-based test to determine the status of GM crops. PMID:23203072

  9. Polymerase Spiral Reaction (PSR): A novel isothermal nucleic acid amplification method

    PubMed Central

    Liu, Wei; Dong, Derong; Yang, Zhan; Zou, Dayang; Chen, Zeliang; Yuan, Jing; Huang, Liuyu

    2015-01-01

    In this study, we report a novel isothermal nucleic acid amplification method only requires one pair of primers and one enzyme, termed Polymerase Spiral Reaction (PSR) with high specificity, efficiency, and rapidity under isothermal condition. The recombinant plasmid of blaNDM-1 was imported to Escherichia coli BL21, and selected as the microbial target. PSR method employs a Bst DNA polymerase and a pair of primers designed targeting the blaNDM-1 gene sequence. The forward and reverse Tab primer sequences are reverse to each other at their 5’ end (Nr and N), whereas their 3’ end sequences are complementary to their respective target nucleic acid sequences. The PSR method was performed at a constant temperature 61 °C–65 °C, yielding a complicated spiral structure. PSR assay was monitored continuously in a real-time turbidimeter instrument or visually detected with the aid of a fluorescent dye (SYBR Greenı), and could be finished within 1 h with a high accumulation of 109 copies of the target and a fine sensitivity of 6 CFU per reaction. Clinical evaluation was also conducted using PSR, showing high specificity of this method. The PSR technique provides a convenient and cost-effective alternative for clinical screening, on-site diagnosis and primary quarantine purposes. PMID:26220251

  10. Rapid detection of infectious laryngotracheitis virus isolates by loop-mediated isothermal amplification.

    PubMed

    Xie, Qing-mei; Ji, Jun; Pickens, Tristan Tyler; Du, Li-qin; Cao, Yong-chang; Li, Hong-mei; Wang, Lin-guo; Ma, Jing-yun; Bi, Ying-zuo

    2010-04-01

    The objective of this study was to develop and evaluate a loop-mediated isothermal amplification (LAMP) method to detect infectious laryngotracheitis virus (ILTV) from commercial broiler and layer flocks in southern China. A set of six specific primers was designed to recognize six distinct genomic sequences of thymidine kinase (TK) from ILTV. The entire assay duration was recorded at 40 min under isothermal condition at 63.5 degrees C. The amplified products were analyzed by electrophoresis and visual judgment by the SYBR Green I dyeing. LAMP assay was 10-fold more sensitive than the routine PCR assay, with a detection limit of 46 copies per reaction. In detecting ILTV, the LAMP assay detected all 5 strains previously isolated, did not cross-react with other avian pathogens, and obtained a 100% sensitivity in 43 positive clinical samples with reference to virus isolation. Therefore, the LAMP assay may be a good alternative method for specific diagnosis of ILTV infection in primary care facilities, and in less well-equipped laboratories. PMID:20100518

  11. Reverse transcription loop-mediated isothermal amplification assay for detecting tomato chlorosis virus.

    PubMed

    Zhao, Li-ming; Li, Gang; Gao, Ying; Zhu, You-rong; Liu, Jin; Zhu, Xiao-ping

    2015-03-01

    A betaine-free reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and optimised for detecting tomato chlorosis virus (ToCV), one of the most important viruses that infect tomato crops worldwide. A set of four specific primers was designed against the RNA-dependent RNA polymerase (RdRp) gene. The betaine-free RT-LAMP procedure could be completed within 40 min under isothermal conditions at 60 °C without a thermal cycler, and no cross-reactivity was seen with other tomato viral pathogens. Sensitivity analysis showed that RT-LAMP could detect viral dilutions up to 2.0×10(-7)ng, which is 100-times more sensitive than reverse transcription-polymerase chain reaction (RT-PCR). In addition, naked-eye observation after staining in-tube RT-LAMP products with SYBR Green I facilitated detection of ToCV by avoiding the requirement for ethidium staining following gel electrophoresis. These results suggest that ToCV RT-LAMP is a rapid, sensitive, and affordable diagnostic tool that is more suitable than RT-PCR for the detection and surveillance of ToCV in field samples. PMID:25486081

  12. Rapid detection of European orthobunyaviruses by reverse transcription loop-mediated isothermal amplification assays.

    PubMed

    Camp, Jeremy V; Nowotny, Norbert

    2016-10-01

    The development of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assays are described herein for the detection of two orthobunyaviruses (Bunyaviridae), which represent the two main serogroups found in mosquitoes in Central Europe. The RT-LAMP assays were optimized for the detection of Ťahyňa virus (a California encephalitis group virus found in Aedes sp or Ochlerotatus sp mosquitoes) and Batai virus (also called Čalovo virus, a Bunyamwera group virus found in Anopheles maculipennis s.l. mosquitoes) nucleic acid using endemic European virus isolates. The sensitivity of the RT-LAMP assays was determined to be comparable to that of conventional tests, with a limit of detection<0.1 pfu per reaction. The assays can be performed in 60min under isothermal conditions using very simple equipment. Furthermore, it was possible to proceed with the assays without nucleic acid extraction, albeit at a 100-fold loss of sensitivity. The RT-LAMP assays are a sensitive, cost-efficient method for both arbovirus surveillance as well as diagnostic laboratories to detect the presence of these endemic orthobunyaviruses. PMID:27491341

  13. Polymerase Spiral Reaction (PSR): A novel isothermal nucleic acid amplification method.

    PubMed

    Liu, Wei; Dong, Derong; Yang, Zhan; Zou, Dayang; Chen, Zeliang; Yuan, Jing; Huang, Liuyu

    2015-01-01

    In this study, we report a novel isothermal nucleic acid amplification method only requires one pair of primers and one enzyme, termed Polymerase Spiral Reaction (PSR) with high specificity, efficiency, and rapidity under isothermal condition. The recombinant plasmid of blaNDM-1 was imported to Escherichia coli BL21, and selected as the microbial target. PSR method employs a Bst DNA polymerase and a pair of primers designed targeting the blaNDM-1 gene sequence. The forward and reverse Tab primer sequences are reverse to each other at their 5' end (Nr and N), whereas their 3' end sequences are complementary to their respective target nucleic acid sequences. The PSR method was performed at a constant temperature 61 °C-65 °C, yielding a complicated spiral structure. PSR assay was monitored continuously in a real-time turbidimeter instrument or visually detected with the aid of a fluorescent dye (SYBR Greenı), and could be finished within 1 h with a high accumulation of 10(9) copies of the target and a fine sensitivity of 6 CFU per reaction. Clinical evaluation was also conducted using PSR, showing high specificity of this method. The PSR technique provides a convenient and cost-effective alternative for clinical screening, on-site diagnosis and primary quarantine purposes. PMID:26220251

  14. Real-time loop-mediated isothermal DNA amplification in compact disc micro-reactors.

    PubMed

    Santiago-Felipe, Sara; Tortajada-Genaro, Luis A; Carrascosa, Javier; Puchades, Rosa; Maquieira, Ángel

    2016-05-15

    An integrated device composed of micro-reactors embedded onto compact discs is proposed for real-time targeted DNA determination. The method principle is based on in-disc loop-mediated isothermal amplification (iD-LAMP) and quantitative optical read-out by a disc drive. In the presence of a target, the turbidimetric or colorimetric properties of reaction solution change, and the transmitted intensity of the disc drive laser modifies according to reaction yield. Monitoring real-time curves allowed the quantitative determination of DNA template amounts. The best amplification/detection results were obtained with micro-reactors (2mm diameter and 1.1mm in depth) drilled on a digital video disc (DVD) and detection based on the colorimetric mode. As proof-of-concept, the assay was applied to detect pathogenic bacteria Salmonella spp. and to identify bovine meat in food samples. Ninety-six samples were simultaneously analysed in 15 min, with high selectivity and sensitivity (5 CFU/mL and 10 µg/g for bacteria and meat, respectively). The in-disc results were comparable to those obtained by conventional LAMP or qPCR approaches. The developed device allows low sample and reagent consumption (3 µL of reaction), portability, ease-of-use, and rapid low-cost high-throughput analyses. PMID:26716424

  15. Detection of New Bunyavirus RNA by Reverse Transcription–Loop-Mediated Isothermal Amplification

    PubMed Central

    Huang, Xue-Yong; Hu, Xiao-Ning; Ma, Hong; Du, Yan-Hua; Ma, Hong-Xia; Kang, Kai; You, Ai-Guo; Wang, Hai-Feng; Zhang, Li; Chen, Hao-Min

    2014-01-01

    Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging and epidemic infectious disease in central and northeast China. It is caused by New Bunyavirus and carries an average 12% case fatality rate. Early and rapid detection is critical for prevention and control of New Bunyavirus infection, since no vaccine or antiviral drugs are currently available, and prevention requires careful attention to control of the suspected tick vector. In this study, a simple and sensitive reverse transcription–loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid detection of New Bunyavirus. The detection limit of the RT-LAMP assay was approximately 103 50% tissue culture infective doses/ml of New Bunyavirus in culture supernatants, and no cross-reactive amplification of other viruses known to cause similar clinical manifestations was observed. The assay was further evaluated using 138 specimens from clinically suspected SFTS and 40 laboratory-proven hantavirus infection with fever and renal syndrome patients, and the assay exhibited 97% agreement compared to real-time RT-PCR and conventional RT-PCR. Using real-time RT-PCR as the diagnostic gold standard, RT-LAMP was 99% sensitive and 100% specific. The RT-LAMP assay could become a useful alternative in clinical diagnosis of SFTS caused by New Bunyavirus, especially in resource-limited hospitals or rural clinics of China. PMID:24478484

  16. Reverse transcription loop-mediated isothermal amplification assay for rapid detection of Bovine Rotavirus

    PubMed Central

    2012-01-01

    Background Bovine rotavirus (BRV) infection is common in young calves. This viral infection causes acute diarrhea leading to death. Rapid identification of infected calves is essential to control BRV successfully. Therefore development of simple, highly specific, and sensitive detection method for BRV is needed. Results A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and optimized for rapid detection of BRV. Specific primer sets were designed to target the sequences of the VP6 gene of the neonatal calf diarrhea virus (NCDV) strain of BRV. The RT-LAMP assay was performed in a water bath for 60 minutes at 63°C, and the amplification products were visualized either directly or under ultraviolet light. This BRV specific RT-LAMP assay could detect 3.32 copies of subtype A BRV. No cross-reactions were detected with other bovine pathogens. The ability of RT-LAMP to detect bovine rotavirus was further evaluated with 88 bovine rectal swab samples. Twenty-nine of these samples were found to be positive for BRV using RT-LAMP. The BRV-specific-RT-LAMP results were also confirmed by real-time RT-PCR assay. Conclusions The bovine rotavirus-specific RT-LAMP assay was highly sensitive and holds promise as a prompt and simple diagnostic method for the detection of group A bovine rotavirus infection in young calves. PMID:22894568

  17. Development and application of loop-mediated isothermal amplification (LAMP) for detection of Plasmopara viticola

    PubMed Central

    Kong, Xiangjiu; Qin, Wentao; Huang, Xiaoqing; Kong, Fanfang; Schoen, Cor D.; Feng, Jie; Wang, Zhongyue; Zhang, Hao

    2016-01-01

    A rapid LAMP (loop-mediated isothermal amplification) detection method was developed on the basis of the ITS sequence of P. viticola, the major causal agent of grape downy mildew. Among the 38 fungal and oomycete species tested, DNA isolated exclusively from P. viticola resulted in a specific product after LAMP amplification. This assay had high sensitivity and was able to detect the presence of less than 33 fg of genomic DNA per 25-μL reaction within 30 min. The infected leaves may produce sporangia that serve as a secondary inoculum. The developed LAMP assay is efficient for estimating the latent infection of grape leaves by P. viticola. When combined with the rapid and simple DNA extraction method, this assay’s total detection time is shortened to approximately one hour; therefore it is suitable for on-site detection of latent infection in the field. The sporangia levels in the air are strongly associated with disease severity. The LAMP method was also demonstrated to be able to estimate the level of sporangia released in the air in a certain period. This assay should make disease forecasting more accurate and rapid and should be helpful in decision-making regarding the control of grape downy mildew. PMID:27363943

  18. A loop-mediated isothermal amplification assay for the visual detection of duck circovirus

    PubMed Central

    2014-01-01

    Background Duck circovirus (DuCV) infection in farmed ducks is associated with growth problems or retardation syndromes. Rapid identification of DuCV infected ducks is essential to control DuCV effectively. Therefore, this study aims to develop of an assay for DuCV to be highly specific, sensitive, and simple without any specialized equipment. Methods A set of six specific primers was designed to target the sequences of the Rep gene of DuCV, and A loop-mediated isothermal amplification (LAMP) assay were developed and the reaction conditions were optimized for rapid detection of DuCV. Results The LAMP assay reaction was conducted in a 62°C water bath condition for 50 min. Then the amplification products were visualized directly for color changes. This LAMP assay is highly sensitive and able to detect twenty copies of DuCV DNA. The specificity of this LAMP assay was supported by no cross-reaction with other duck pathogens. Conclusion This LAMP method for DuCV is highly specific and sensitive and can be used as a rapid and direct diagnostic assay for testing clinical samples. PMID:24775810

  19. Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for Rapid Detection of Ehrlichia ruminantium

    PubMed Central

    2010-01-01

    Background The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater, a potential zoonotic disease of ruminants transmitted by ticks of the genus Amblyomma. The disease is distributed in nearly all of sub-Saharan Africa and some islands of the Caribbean, from where it threatens the American mainland. This report describes the development of two different loop-mediated isothermal amplification (LAMP) assays for sensitive and specific detection of E. ruminantium. Results Two sets of LAMP primers were designed from the pCS20 and sodB genes. The detection limits for each assay were 10 copies for pCS20 and 5 copies for sodB, which is at least 10 times higher than that of the conventional pCS20 PCR assay. DNA amplification was completed within 60 min. The assays detected 16 different isolates of E. ruminantium from geographically distinct countries as well as two attenuated vaccine isolates. No cross-reaction was observed with genetically related Rickettsiales, including zoonotic Ehrlichia species from the USA. LAMP detected more positive samples than conventional PCR but less than real-time PCR, when tested with field samples collected in sub-Saharan countries. Conclusions Due to its simplicity and specificity, LAMP has the potential for use in resource-poor settings and also for active screening of E. ruminantium in both heartwater-endemic areas and regions that are at risk of contracting the disease. PMID:21087521

  20. A Simple Isothermal DNA Amplification Method to Screen Black Flies for Onchocerca volvulus Infection

    PubMed Central

    Alhassan, Andy; Makepeace, Benjamin L.; LaCourse, Elwyn James; Osei-Atweneboana, Mike Y.; Carlow, Clotilde K. S.

    2014-01-01

    Onchocerciasis is a debilitating neglected tropical disease caused by infection with the filarial parasite Onchocerca volvulus. Adult worms live in subcutaneous tissues and produce large numbers of microfilariae that migrate to the skin and eyes. The disease is spread by black flies of the genus Simulium following ingestion of microfilariae that develop into infective stage larvae in the insect. Currently, transmission is monitored by capture and dissection of black flies and microscopic examination of parasites, or using the polymerase chain reaction to determine the presence of parasite DNA in pools of black flies. In this study we identified a new DNA biomarker, encoding O. volvulus glutathione S-transferase 1a (OvGST1a), to detect O. volvulus infection in vector black flies. We developed an OvGST1a-based loop-mediated isothermal amplification (LAMP) assay where amplification of specific target DNA is detectable using turbidity or by a hydroxy naphthol blue color change. The results indicated that the assay is sensitive and rapid, capable of detecting DNA equivalent to less than one microfilaria within 60 minutes. The test is highly specific for the human parasite, as no cross-reaction was detected using DNA from the closely related and sympatric cattle parasite Onchocerca ochengi. The test has the potential to be developed further as a field tool for use in the surveillance of transmission before and after implementation of mass drug administration programs for onchocerciasis. PMID:25299656

  1. A simple isothermal DNA amplification method to screen black flies for Onchocerca volvulus infection.

    PubMed

    Alhassan, Andy; Makepeace, Benjamin L; LaCourse, Elwyn James; Osei-Atweneboana, Mike Y; Carlow, Clotilde K S

    2014-01-01

    Onchocerciasis is a debilitating neglected tropical disease caused by infection with the filarial parasite Onchocerca volvulus. Adult worms live in subcutaneous tissues and produce large numbers of microfilariae that migrate to the skin and eyes. The disease is spread by black flies of the genus Simulium following ingestion of microfilariae that develop into infective stage larvae in the insect. Currently, transmission is monitored by capture and dissection of black flies and microscopic examination of parasites, or using the polymerase chain reaction to determine the presence of parasite DNA in pools of black flies. In this study we identified a new DNA biomarker, encoding O. volvulus glutathione S-transferase 1a (OvGST1a), to detect O. volvulus infection in vector black flies. We developed an OvGST1a-based loop-mediated isothermal amplification (LAMP) assay where amplification of specific target DNA is detectable using turbidity or by a hydroxy naphthol blue color change. The results indicated that the assay is sensitive and rapid, capable of detecting DNA equivalent to less than one microfilaria within 60 minutes. The test is highly specific for the human parasite, as no cross-reaction was detected using DNA from the closely related and sympatric cattle parasite Onchocerca ochengi. The test has the potential to be developed further as a field tool for use in the surveillance of transmission before and after implementation of mass drug administration programs for onchocerciasis. PMID:25299656

  2. Detection of Bar Transgenic Sugarcane with a Rapid and Visual Loop-Mediated Isothermal Amplification Assay.

    PubMed

    Zhou, Dinggang; Wang, Chunfeng; Li, Zhu; Chen, Yun; Gao, Shiwu; Guo, Jinlong; Lu, Wenying; Su, Yachun; Xu, Liping; Que, Youxiong

    2016-01-01

    Genetic engineering offers an attractive alternative in sugarcane breeding for increasing cane and sugar yields as well as disease and insect resistance. Bar transgenic sugarcane employing the herbicide tolerance is a useful agronomical trait in weed control. In this study, a loop-mediated isothermal amplification (LAMP) assay for rapid detection of the bar gene in transgenic sugarcane has been developed and evaluated. A set of six primers was designed for LAMP-based amplification of the bar gene. The LAMP reaction conditions were optimized as follows: 5.25 mM of Mg(2+), 6:1 ratio of inner vs. outer primer, and 6.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. The detection limit of the recombinant plasmid 1Ac0229 was as low as 10 copies in the developed LAMP, which was 10-fold higher sensitive than that of conventional PCR. In 100 putative transgenic lines, the bar gene was detected in 100/100 cases (100%) by LAMP and 97/100 cases (97%) by conventional PCR, respectively. In conclusion, the developed LAMP assay is visual, rapid, sensitive, reliable, and cost-effective for detection of the bar specific transgenic sugarcane. PMID:27014303

  3. Detection of Bar Transgenic Sugarcane with a Rapid and Visual Loop-Mediated Isothermal Amplification Assay

    PubMed Central

    Zhou, Dinggang; Wang, Chunfeng; Li, Zhu; Chen, Yun; Gao, Shiwu; Guo, Jinlong; Lu, Wenying; Su, Yachun; Xu, Liping; Que, Youxiong

    2016-01-01

    Genetic engineering offers an attractive alternative in sugarcane breeding for increasing cane and sugar yields as well as disease and insect resistance. Bar transgenic sugarcane employing the herbicide tolerance is a useful agronomical trait in weed control. In this study, a loop-mediated isothermal amplification (LAMP) assay for rapid detection of the bar gene in transgenic sugarcane has been developed and evaluated. A set of six primers was designed for LAMP-based amplification of the bar gene. The LAMP reaction conditions were optimized as follows: 5.25 mM of Mg2+, 6:1 ratio of inner vs. outer primer, and 6.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. The detection limit of the recombinant plasmid 1Ac0229 was as low as 10 copies in the developed LAMP, which was 10-fold higher sensitive than that of conventional PCR. In 100 putative transgenic lines, the bar gene was detected in 100/100 cases (100%) by LAMP and 97/100 cases (97%) by conventional PCR, respectively. In conclusion, the developed LAMP assay is visual, rapid, sensitive, reliable, and cost-effective for detection of the bar specific transgenic sugarcane. PMID:27014303

  4. Development of loop-mediated isothermal amplification (LAMP) for detection of Babesia gibsoni infection in dogs.

    PubMed

    Mandal, Mrityunjay; Banerjee, Partha S; Kumar, Saroj; Ram, Hira; Garg, Rajat; Pawde, Abhijit M

    2015-04-15

    Diagnosis of canine babesiosis, caused by Babesia gibsoni is difficult, especially in chronically infected dogs. A loop mediated isothermal amplification (LAMP) assay was developed and standardized by using four oligonucleotide primers targeting the hypervariable region of 18S rRNA gene (GenBank Acc. no. KC461261). The primers specifically amplified B. gibsoni DNA, while no amplification was detected with DNA from non-infected dogs as well as from dogs infected with Babesia canis vogeli, Hepatozoon canis, Ehrlichia canis and Trypanosoma evansi. The assay could detect 1.35 × 10(-7) parasitaemia and 10(-4) dilution of recombinant plasmid, equivalent to 12 pg of target DNA. All the samples were tested by nested PCR as well as LAMP assay. LAMP was found to be 10 times more sensitive than nested PCR targeting the same gene. Out of 75 suspected field samples, collected from different parts of the country, LAMP could detect B. gibsoni in 43 samples, while nested PCR and microscopy could detect 37 and 23 samples, respectively. High sensitivity, specificity and rapidity of LAMP assay may be exploited for screening large number of samples in a field setting. PMID:25749021

  5. Sensitive colorimetric detection of Listeria monocytogenes based on isothermal gene amplification and unmodified gold nanoparticles.

    PubMed

    Fu, Zhongyu; Zhou, Xiaoming; Xing, Da

    2013-12-15

    Listeria monocytogenes (L. monocytogenes), one of most problematic food-borne bacteria, is mainly transmitted through the food chain and may cause listeriosis. Therefore, the development of rapid and sensitive L. monocytogenes detection technique has become an urgent task. In this study, we proposed a method using hyperbranching rolling circle amplification (HRCA) combined with gold nanoparticle (GNP) based colorimetric strategy to offer an isothermal, highly sensitive and specific assay for the detection of L. monocytogenes. First, a linear padlock probe targeting a specific sequence in the hly gene was designed and followed with a ligation by Taq DNA ligase. After ligation, further amplification by HRCA with a thiolated primer and an unlabeled primer is performed. The resulting thiolated HRCA products were then captured onto GNP surface and made GNP more salt-tolerant. Detection of the bacteria can be achieved by a facilitated GNP based colorimetric testing using naked eyes. Through this approach, as low as 100 aM synthetic hly gene targets and about 75 copies of L. monocytogenes can be detected. The specificity is evaluated by distinguishing target L. monocytogenes from other bacteria. The artificial contaminated food samples were also detected for its potential applications in real food detection. This method described here is ideal for bacteria detection due to its simplicity and high sensitivity. PMID:23948710

  6. The use of loop-mediated isothermal amplification improves Toxoplasma gondii detection in wildlife.

    PubMed

    Trisciuoglio, Anna; Zanet, Stefania; Marello, Giuseppina; Chiesa, Francesco; Nucera, Daniele M; Bergallo, Massimiliano; Gennero, Maria S; Ferroglio, Ezio

    2015-11-01

    Toxoplasma gondii is among the most widespread parasites worldwide. Wildlife is recognized as an important reservoir and source of infection of T. gondii. The goal of the present work was to assess the performance of loop-mediated isothermal amplification (LAMP) as a diagnostic tool for T. gondii infection in the skeletal muscle and central nervous system (CNS) of free-ranging ungulates and carnivores. Fifty-seven wild animals were tested for the presence of T. gondii DNA by polymerase chain reaction (PCR) and LAMP. The use of LAMP amplification improved sensitivity in T. gondii molecular detection compared with conventional PCR on skeletal muscle (χ(2) = 5.8, P < 0.05), having a lower minimum detection limit (0.1 tachyzoite) than PCR (1 tachyzoite). No significant differences existed between the detection capacities of both assays when performed on CNS. LAMP is a valid tool to improve the diagnosis of T. gondii infection in wild game meat. The technique provides a sensitive yet specific method that can be applicable to both field surveys and large-scale testing of wildlife samples. PMID:26462761

  7. Detection of Clostridium perfringens alpha toxin gene in lambs by loop mediated isothermal amplification

    PubMed Central

    Radhika, B.; Kumar, N. Vinod; Sreenivasulu, D.

    2016-01-01

    Aim: The loop mediated isothermal amplification (LAMP) was standardized for rapid detection of Clostridium perfringens. Materials and Methods: A total of 120 fecal samples were collected from enterotoxemia suspected lambs were used for screening of C. perfringens cpa gene by LAMP. The specificity of the LAMP amplified products was tested by digesting with restriction enzyme XmnI for alpha toxin gene. Results: Out of 120 samples screened 112 (93.3%) samples were positive by both LAMP and polymerase chain reaction (PCR) for detection of cpa gene which indicated the equal sensitivity of both the tests. The enzyme produced single cut in 162 base pair amplified product of alpha toxin gene at 81 base pair resulting in a single band in gel electrophoresis. Conclusion: Both LAMP and PCR for detection of cpa gene indicated the equal sensitivity of both the tests. Standardization of LAMP reaction for amplification of epsilon and beta toxin genes will help to identify the C. perfringens toxin types from the clinical samples. The test could be a suitable alternative to the PCR in detection of toxin types without the help of sophisticated machinery like thermal cycler. Considering its simplicity in operation and high sensitivity, there is the potential use of this technique in clinical diagnosis and surveillance of infectious diseases. PMID:27051186

  8. An accurate assay for HCV based on real-time fluorescence detection of isothermal RNA amplification.

    PubMed

    Wu, Xuping; Wang, Jianfang; Song, Jinyun; Li, Jiayan; Yang, Yongfeng

    2016-09-01

    Hepatitis C virus (HCV) is one of the common reasons of liver fibrosis and hepatocellular carcinoma (HCC). Early, rapid and accurate HCV RNA detection is important to prevent and control liver disease. A simultaneous amplification and testing (SAT) assay, which is based on isothermal amplification of RNA and real-time fluorescence detection, was designed to optimize routine HCV RNA detection. In this study, HCV RNA and an internal control (IC) were amplified and analyzed simultaneously by SAT assay and detection of fluorescence using routine real-time PCR equipment. The assay detected as few as 10 copies of HCV RNA transcripts. We tested 705 serum samples with SAT, among which 96.4% (680/705) showed consistent results compared with routine real-time PCR. About 92% (23/25) discordant samples were confirmed to be same results as SAT-HCV by using a second real-time PCR. The sensitivity and specificity of SAT-HCV assay were 99.6% (461/463) and 100% (242/242), respectively. In conclusion, the SAT assay is an accurate test with a high specificity and sensitivity which may increase the detection rate of HCV. It is therefore a promising tool to diagnose HCV infection. PMID:27283884

  9. New closed tube loop mediated isothermal amplification assay for prevention of product cross-contamination

    PubMed Central

    Karthik, K.; Rathore, Rajesh; Thomas, Prasad; Arun, T.R.; Viswas, K.N.; Dhama, Kuldeep; Agarwal, R.K.

    2014-01-01

    Loop mediated isothermal amplification (LAMP) assay, a promising diagnostic test, has been developed for detection of different pathogens of human as well as animals. Various positive points support its use as a field level test but the major problem is product cross contamination leading to false positive results. Different methods were adopted by various researchers to control this false positive amplification due to cross contamination but all have their own advantages and disadvantages. A new closed tube LAMP assay based on agar dye capsule was developed in the present study and this technique has some advantages over the other closed tube technique.•Agar at the concentration of 1.5% was used to sandwich SYBR green dye I with the aid of intradermal syringe. This agar dye capsule was placed over the LAMP reaction mixture before it was amplified.•To eliminate the hazardous nature of Ultra Violet (UV) light during result visualization of LAMP products, the present study demonstrates the use of Light Emitting Diode (LED) lights for result visualization.•LAMP was carried out for Brucella species detection using this modified techniques yielding good results without any cross contamination and LED showed similar fluorescence compared to UV. PMID:26150945

  10. Specific diagnosis of Opisthorchis viverrini using loop-mediated isothermal amplification (LAMP) targeting parasite microsatellites.

    PubMed

    Arimatsu, Yuji; Kaewkes, Sasithorn; Laha, Thewarach; Sripa, Banchob

    2015-01-01

    Opisthorchis viverrini and other food-borne trematode infections are major health problems in Thailand, the Lao People's Democratic Republic, Vietnam and Cambodia. Differential diagnosis of O. viverrini based on the microscopic observation of parasite eggs is difficult in areas where Clonorchis sinensis and minute intestinal flukes coexist. Recently, loop-mediated isothermal amplification (LAMP) has been widely used for detection and identification of trematode for its simple method that is useful in low-resource or field settings. We have reported ITS1-LAMP assay to detect O. viverrini infection from human feces. The sensitivity and specificity of the test was 100% and 61.5%. The sensitivity of the test appeared to be higher than microscopic egg examination; however non-specific amplification from other parasites could not be ruled out. We therefore targeted microsatellites of O. viverrini that is a species specific sequence. By using hydroxyl naphthol blue (HNB)-LAMP, O. viverrini microsatellite 6 (OVMS6) could specifically amplify DNA from O. viverrini genome, but not other parasites such as C. sinensis, Opisthorchis felineus, Centrocestus caninus, Haplorchis taichui, Fasciola gigantica and Haplorchoodes sp. The detection limit of the test is 1 ng genomic DNA, which was 1000 times lower than the ITS1-LAMP, but targeting microstellites showed more specific detection of O. viverrini. In addition, the colorimetric LAMP assay was simple and effective; this makes it potentially applicable for point-of-care diagnosis. PMID:25268466

  11. A Novel Isothermal Assay of Borrelia burgdorferi by Recombinase Polymerase Amplification with Lateral Flow Detection

    PubMed Central

    Liu, Wei; Liu, Hui-Xin; Zhang, Lin; Hou, Xue-Xia; Wan, Kang-Lin; Hao, Qin

    2016-01-01

    A novel isothermal detection for recombinase polymerase amplification with lateral flow (LF-RPA) was established for Borrelia burgdorferi (B. burgdorferi) detection in this study. This assay with high sensitivity and specificity can get a visible result without any additional equipment in 30 min. We designed a pair of primers according to recA gene of B. burgdorferi strains and a methodology evaluation was performed. The results showed that the RPA assay based on the recA gene was successfully applied in B. burgdorferi detection, and its specific amplification was only achieved from the genomic DNA of B. burgdorferi. The detection limit of the new assay was about 25 copies of the B. burgdorferi genomic DNA. Twenty Lyme borreliosis patients’ serum samples were detected by LF-RPA assay, real-time qPCR and nested-PCR. Results showed the LF-RPA assay is more effective than nested-PCR for its shorter reaction time and considerably higher detection rate. This method is of great value in clinical rapid detection for Lyme borreliosis. Using the RPA assay might be a megatrend for DNA detection in clinics and endemic regions. PMID:27527151

  12. A Novel Isothermal Assay of Borrelia burgdorferi by Recombinase Polymerase Amplification with Lateral Flow Detection.

    PubMed

    Liu, Wei; Liu, Hui-Xin; Zhang, Lin; Hou, Xue-Xia; Wan, Kang-Lin; Hao, Qin

    2016-01-01

    A novel isothermal detection for recombinase polymerase amplification with lateral flow (LF-RPA) was established for Borrelia burgdorferi (B. burgdorferi) detection in this study. This assay with high sensitivity and specificity can get a visible result without any additional equipment in 30 min. We designed a pair of primers according to recA gene of B. burgdorferi strains and a methodology evaluation was performed. The results showed that the RPA assay based on the recA gene was successfully applied in B. burgdorferi detection, and its specific amplification was only achieved from the genomic DNA of B. burgdorferi. The detection limit of the new assay was about 25 copies of the B. burgdorferi genomic DNA. Twenty Lyme borreliosis patients' serum samples were detected by LF-RPA assay, real-time qPCR and nested-PCR. Results showed the LF-RPA assay is more effective than nested-PCR for its shorter reaction time and considerably higher detection rate. This method is of great value in clinical rapid detection for Lyme borreliosis. Using the RPA assay might be a megatrend for DNA detection in clinics and endemic regions. PMID:27527151

  13. Development and application of loop-mediated isothermal amplification (LAMP) for detection of Plasmopara viticola.

    PubMed

    Kong, Xiangjiu; Qin, Wentao; Huang, Xiaoqing; Kong, Fanfang; Schoen, Cor D; Feng, Jie; Wang, Zhongyue; Zhang, Hao

    2016-01-01

    A rapid LAMP (loop-mediated isothermal amplification) detection method was developed on the basis of the ITS sequence of P. viticola, the major causal agent of grape downy mildew. Among the 38 fungal and oomycete species tested, DNA isolated exclusively from P. viticola resulted in a specific product after LAMP amplification. This assay had high sensitivity and was able to detect the presence of less than 33 fg of genomic DNA per 25-μL reaction within 30 min. The infected leaves may produce sporangia that serve as a secondary inoculum. The developed LAMP assay is efficient for estimating the latent infection of grape leaves by P. viticola. When combined with the rapid and simple DNA extraction method, this assay's total detection time is shortened to approximately one hour; therefore it is suitable for on-site detection of latent infection in the field. The sporangia levels in the air are strongly associated with disease severity. The LAMP method was also demonstrated to be able to estimate the level of sporangia released in the air in a certain period. This assay should make disease forecasting more accurate and rapid and should be helpful in decision-making regarding the control of grape downy mildew. PMID:27363943

  14. Rapid detection of Opisthorchis viverrini copro-DNA using loop-mediated isothermal amplification (LAMP).

    PubMed

    Arimatsu, Yuji; Kaewkes, Sasithorn; Laha, Thewarach; Hong, Sung-Jong; Sripa, Banchob

    2012-03-01

    Opisthorchis viverrini and other foodborne trematode infections are major health problem in Thailand, the Lao People's Democratic Republic, Vietnam and Cambodia. Differential diagnosis of O. viverrini based on the microscopic observation of parasite eggs is difficult in areas where Clonorchis sinensis and minute intestinal flukes coexist. We therefore established a rapid, sensitive and specific method for detecting O. viverrini infection from the stool samples using the loop-mediated isothermal amplification (LAMP) method. A total of five primers from seven regions were designed to target the internal transcribed spacer 1 (ITS1) in ribosomal DNA for specific amplification. Hydroxy naphthol blue (HNB) was more effective to detect the LAMP product compared to the Real-time LAMP and turbidity assay for its simple and distinct detection. The LAMP assay specifically amplified O. viverrini ITS1 but not C. sinensis and minute intestinal flukes with the limit of detection around 10(-3)ng DNA/μL. The sensitivity of the LAMP was 100% compared to egg positive samples. While all microscopically positive samples were positive by LAMP, additionally 5 of 13 (38.5%) microscopically negative samples were also LAMP positive. The technique has great potential for differential diagnosis in endemic areas with mixed O. viverrini and intestinal fluke infections. As it is an easy and simple method, the LAMP is potentially applicable for point-of-care diagnosis. PMID:21871581

  15. Sensitive Detection of Xanthomonas oryzae Pathovars oryzae and oryzicola by Loop-Mediated Isothermal Amplification

    PubMed Central

    Lang, Jillian M.; Langlois, Paul; Nguyen, Marian Hanna R.; Triplett, Lindsay R.; Purdie, Laura; Holton, Timothy A.; Djikeng, Appolinaire; Vera Cruz, Casiana M.; Verdier, Valérie

    2014-01-01

    Molecular diagnostics for crop diseases can enhance food security by enabling the rapid identification of threatening pathogens and providing critical information for the deployment of disease management strategies. Loop-mediated isothermal amplification (LAMP) is a PCR-based tool that allows the rapid, highly specific amplification of target DNA sequences at a single temperature and is thus ideal for field-level diagnosis of plant diseases. We developed primers highly specific for two globally important rice pathogens, Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight (BB) disease, and X. oryzae pv. oryzicola, the causal agent of bacterial leaf streak disease (BLS), for use in reliable, sensitive LAMP assays. In addition to pathovar distinction, two assays that differentiate X. oryzae pv. oryzae by African or Asian lineage were developed. Using these LAMP primer sets, the presence of each pathogen was detected from DNA and bacterial cells, as well as leaf and seed samples. Thresholds of detection for all assays were consistently 104 to 105 CFU ml−1, while genomic DNA thresholds were between 1 pg and 10 fg. Use of the unique sequences combined with the LAMP assay provides a sensitive, accurate, rapid, simple, and inexpensive protocol to detect both BB and BLS pathogens. PMID:24837384

  16. Sensitive detection of Xanthomonas oryzae Pathovars oryzae and oryzicola by loop-mediated isothermal amplification.

    PubMed

    Lang, Jillian M; Langlois, Paul; Nguyen, Marian Hanna R; Triplett, Lindsay R; Purdie, Laura; Holton, Timothy A; Djikeng, Appolinaire; Vera Cruz, Casiana M; Verdier, Valérie; Leach, Jan E

    2014-08-01

    Molecular diagnostics for crop diseases can enhance food security by enabling the rapid identification of threatening pathogens and providing critical information for the deployment of disease management strategies. Loop-mediated isothermal amplification (LAMP) is a PCR-based tool that allows the rapid, highly specific amplification of target DNA sequences at a single temperature and is thus ideal for field-level diagnosis of plant diseases. We developed primers highly specific for two globally important rice pathogens, Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight (BB) disease, and X. oryzae pv. oryzicola, the causal agent of bacterial leaf streak disease (BLS), for use in reliable, sensitive LAMP assays. In addition to pathovar distinction, two assays that differentiate X. oryzae pv. oryzae by African or Asian lineage were developed. Using these LAMP primer sets, the presence of each pathogen was detected from DNA and bacterial cells, as well as leaf and seed samples. Thresholds of detection for all assays were consistently 10(4) to 10(5) CFU ml(-1), while genomic DNA thresholds were between 1 pg and 10 fg. Use of the unique sequences combined with the LAMP assay provides a sensitive, accurate, rapid, simple, and inexpensive protocol to detect both BB and BLS pathogens. PMID:24837384

  17. Development of Rapid Isothermal Amplification Assays for Detection of Phytophthora spp. in Plant Tissue.

    PubMed

    Miles, Timothy D; Martin, Frank N; Coffey, Michael D

    2015-02-01

    Several isothermal amplification techniques recently have been developed that are tolerant of inhibitors present in many plant extracts, which can reduce the need for obtaining purified DNA for running diagnostic assays. One such commercially available technique that has similarities with real-time polymerase chain reaction (PCR) for designing primers and a labeled probe is recombinase polymerase amplification (RPA). This technology was used to develop two simple and rapid approaches for detection of Phytophthora spp.: one genus-specific assay multiplexed with a plant internal control and the other species-specific assays for Phytophthora ramorum and P. kernoviae. All assays were tested for sensitivity (ranging from 3 ng to 1 fg of DNA) and specificity using DNA extracted from more than 136 Phytophthora taxa, 21 Pythium spp., 1 Phytopythium sp., and a wide range of plant species. The lower limit of linear detection using purified DNA was 200 to 300 fg of DNA in all pathogen RPA assays. Six different extraction buffers were tested for use during plant tissue maceration and the assays were validated in the field by collecting 222 symptomatic plant samples from over 50 different hosts. Only 56 samples were culture positive for Phytophthora spp. whereas 91 were positive using the Phytophthora genus-specific RPA test and a TaqMan real-time PCR assay. A technique for the generation of sequencing templates from positive RPA amplifications to confirm species identification was also developed. These RPA assays have added benefits over traditional technologies because they are rapid (results can be obtained in as little as 15 min), do not require DNA extraction or extensive training to complete, use less expensive portable equipment than PCR-based assays, and are significantly more specific than current immunologically based methods. This should provide a rapid, field-deployable capability for pathogen detection that will facilitate point-of-sample collection processing

  18. Loop-mediated isothermal amplification for detection of the tomato and potato late blight pathogen, Phytophthora infestans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aims: To design and validate a colorimetric loop-mediated isothermal amplification assay for rapid detection of P. infestans DNA. Methods and Results: Two sets of LAMP primers were designed and evaluated for their sensitivity and specificity for P. infestans. ITSII primers targeted a portion of the ...

  19. Identification of the MEAM1 cryptic species of Bemisia tabaci (Hemiptera: Aleyrodidae) by loop-mediated isothermal amplification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are two major invasive cryptic species within the Bemisia tabaci cryptic species complex in Florida, called MEAM1 or biotype B, and MED or biotype Q. We used loop-mediated isothermal amplification of DNA to detect these groups. Primer sets developed in house and those previously published wer...

  20. Current state and future perspectives of loop-mediated isothermal amplification (LAMP)-based diagnosis of filamentous fungi and yeasts.

    PubMed

    Niessen, Ludwig

    2015-01-01

    Loop-mediated isothermal amplification is a rather novel method of enzymatic deoxyribonucleic acid amplification which can be applied for the diagnosis of viruses, bacteria, and fungi. Although firmly established in viral and bacterial diagnosis, the technology has only recently been applied to a noteworthy number of species in the filamentous fungi and yeasts. The current review gives an overview of the literature so far published on the topic by discussing the different groups of fungal organisms to which the method has been applied. Moreover, the method is described in detail as well as the different possibilities available for signal detection and quantification and sample preparation. Future perspective of loop-mediated isothermal amplification-based assays is discussed in the light of applicability for fungal diagnostics. PMID:25492418

  1. Isothermal loop-mediated amplification (lamp) for diagnosis of contagious bovine pleuro-pneumonia

    PubMed Central

    2013-01-01

    Background Contagious Bovine Pleuropneumonia (CBPP) is the most important chronic pulmonary disease of cattle on the African continent causing severe economic losses. The disease, caused by infection with Mycoplasma mycoides subsp. mycoides is transmitted by animal contact and develops slowly into a chronic form preventing an early clinical diagnosis. Because available vaccines confer a low protection rate and short-lived immunity, the rapid diagnosis of infected animals combined with traditional curbing measures is seen as the best way to control the disease. While traditional labour-intensive bacteriological methods for the detection of M. mycoides subsp. mycoides have been replaced by molecular genetic techniques in the last two decades, these latter approaches require well-equipped laboratories and specialized personnel for the diagnosis. This is a handicap in areas where CBPP is endemic and early diagnosis is essential. Results We present a rapid, sensitive and specific diagnostic tool for M. mycoides subsp. mycoides detection based on isothermal loop-mediated amplification (LAMP) that is applicable to field conditions. The primer set developed is highly specific and sensitive enough to diagnose clinical cases without prior cultivation of the organism. The LAMP assay detects M. mycoides subsp. mycoides DNA directly from crude samples of pulmonary/pleural fluids and serum/plasma within an hour using a simple dilution protocol. A photometric detection of LAMP products allows the real-time visualisation of the amplification curve and the application of a melting curve/re-association analysis presents a means of quality assurance based on the predetermined strand-inherent temperature profile supporting the diagnosis. Conclusion The CBPP LAMP developed in a robust kit format can be run on a battery-driven mobile device to rapidly detect M. mycoides subsp. mycoides infections from clinical or post mortem samples. The stringent innate quality control allows a

  2. Integrated centrifugal reverse transcriptase loop-mediated isothermal amplification microdevice for influenza A virus detection.

    PubMed

    Jung, Jae Hwan; Park, Byung Hyun; Oh, Seung Jun; Choi, Goro; Seo, Tae Seok

    2015-06-15

    An integrated reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) microdevice which consists of microbead-assisted RNA purification and RT-LAMP with real-time monitoring by a miniaturized optical detector was demonstrated. The integrated RT-LAMP microdevice includes four reservoirs for a viral RNA sample (purified influenza A viral RNA or lysates), a washing solution (70% ethanol), an elution solution (RNase-free water), and an RT-LAMP cocktail, and two chambers (a waste chamber and an RT-LAMP reaction chamber). The separate reservoirs for a washing solution, an elution solution, and an RT-LAMP cocktail were designed with capillary valves for stable storage. Three influenza A virus strains (A/H1N1, A/H3N2, and A/H5N1) were used for RNA templates, and RT-LAMP primer sets were designed to detect hemagglutinin (HA) and conserved M gene. Sequential sample flow to the microbeads for RNA purification was achieved by centrifugal force with optimization of capillary valves and a siphon channel. Furthermore, the purified RNA solution was successfully isolated from the waste solution by changing the rotational direction, and combined with the RT-LAMP cocktail in the RT-LAMP reaction chamber for target gene amplification. Total process from the sample injection to the result was completed in 47 min. Influenza A H1N1 virus was confirmed on the integrated RT-LAMP microdevice even with 10 copies of viral RNAs, which revealed 10-fold higher sensitivity than that of a conventional RT-PCR. Subtyping and specificity test of influenza A H1N1 viral lysates were also performed and clinical samples were successfully genotyped to confirm influenza A virus on our proposed integrated microdevice. PMID:25569879

  3. Meat Species Identification using Loop-mediated Isothermal Amplification Assay Targeting Species-specific Mitochondrial DNA

    PubMed Central

    2014-01-01

    Meat source fraud and adulteration scandals have led to consumer demands for accurate meat identification methods. Nucleotide amplification assays have been proposed as an alternative method to protein-based assays for meat identification. In this study, we designed Loop-mediated isothermal amplification (LAMP) assays targeting species-specific mitochondrial DNA to identify and discriminate eight meat species; cattle, pig, horse, goat, sheep, chicken, duck, and turkey. The LAMP primer sets were designed and the target genes were discriminated according to their unique annealing temperature generated by annealing curve analysis. Their unique annealing temperatures were found to be 85.56±0.07℃ for cattle, 84.96±0.08℃ for pig, and 85.99±0.05℃ for horse in the BSE-LAMP set (Bos taurus, Sus scrofa domesticus and Equus caballus); 84.91±0.11℃ for goat and 83.90±0.11℃ for sheep in the CO-LAMP set (Capra hircus and Ovis aries); and 86.31±0.23℃ for chicken, 88.66±0.12℃ for duck, and 84.49±0.08℃ for turkey in the GAM-LAMP set (Gallus gallus, Anas platyrhynchos and Meleagris gallopavo). No cross-reactivity was observed in each set. The limits of detection (LODs) of the LAMP assays in raw and cooked meat were determined from 10 pg/μL to 100 fg/μL levels, and LODs in raw and cooked meat admixtures were determined from 0.01% to 0.0001% levels. The assays were performed within 30 min and showed greater sensitivity than that of the PCR assays. These novel LAMP assays provide a simple, rapid, accurate, and sensitive technology for discrimination of eight meat species. PMID:26761677

  4. Bovine embryo sex determination by multiplex loop-mediated isothermal amplification.

    PubMed

    Khamlor, Trisadee; Pongpiachan, Petai; Parnpai, Rangsun; Punyawai, Kanchana; Sangsritavong, Siwat; Chokesajjawatee, Nipa

    2015-03-15

    In cattle, the ability to determine the sex of embryos before embryo transfer is beneficial for increasing the number of animals with the desired sex. This study therefore developed a new modification of loop-mediated isothermal amplification in a multiplex format (multiplex LAMP) for highly efficient bovine embryo sexing. Two chromosomal regions, one specific for males (Y chromosome, S4 region) and the other common to both males and females (1.715 satellite DNA), were amplified in the same reaction tube. Each target was amplified by specifically designed inner primers, outer primers, and loop primers, where one of the S4 loop primers was labeled with the fluorescent dye 6-carboxyl-X-rhodamine (emitting a red color), whereas both satellite loop primers were labeled with the fluorescent dye fluorescein isothiocyanate (emitting a green color). After amplification at 63 °C for 1 hour, the amplified products were precipitated by a small volume of cationic polymer predispensed inside the reaction tube cap. Green precipitate indicated the presence of only control DNA without the Y chromosome, whereas orange precipitate indicated the presence of both target DNAs, enabling interpretation as female and male, respectively. Accuracy of the multiplex LAMP assay was evaluated using 46 bovine embryos with known sex (25 male and 21 female) generated by somatic cell nuclear transfer and confirmed by multiplex polymerase chain reaction. The multiplex LAMP showed 100% accuracy in identifying the actual sex of the embryos and provides a fast, simple, and cost-effective tool for bovine embryo sexing. PMID:25542460

  5. Visual Detection of Enterohemorrhagic Escherichia coli O157:H7 Using Loop-Mediated Isothermal Amplification

    PubMed Central

    Ranjbar, Reza; Erfanmanesh, Maryam; Afshar, Davoud; Mohammadi, Mohsen; Ghaderi, Omar; Haghnazari, Ali

    2016-01-01

    Introduction Escherichia coli O157:H7, an important foodborne pathogen, can cause serious renal damage, which can also lead to mortality. Since a rapid and sensitive method is needed to identify this pathogenic agent, we evaluated Loop-Mediated Isothermal Amplification Assay (LAMP) to detect Escherichia coli O157:H7. Methods We used six primers that specifically identified the rfbE gene. To examine the sensitivity of the method, different dilutions were subjected to the LAMP reaction. Other bacterial strains also were investigated to determine the specificity of the test. The turbidity of the amplified products was assayed by visual detection. The amplified products were detected by addition of SYBR Green II to the reaction tubes. Results Amplification products were observed as a ladder-like pattern on the agarose gel. A white turbidity emerged in the positive tubes. Under UV light, the positive samples were green, whereas the negative samples were orange. The detection limit of the LAMP was 78 pg/tube, and this indicated that it was 100 times more sensitive than PCR for the detection of EHEC. No LAMP products were detected when template DNA of non-EHEC strains were used, suggesting high specificity of the LAMP assay. Conclusion The results indicated that the LAMP assay is a valuable diagnostic assay to identify EHEC O157:H7. In addition, the simplicity, sensitivity, specificity, and rapidity of this assay make it a useful method to diagnose pathogens in primary labs without any need for expensive equipment or specialized techniques. PMID:27504175

  6. A Reverse Transcription Loop-Mediated Isothermal Amplification Assay Optimized to Detect Multiple HIV Subtypes

    PubMed Central

    Ocwieja, Karen E.; Sherrill-Mix, Scott; Liu, Changchun; Song, Jinzhao; Bau, Haim; Bushman, Frederic D.

    2015-01-01

    Diagnostic methods for detecting and quantifying HIV RNA have been improving, but efficient methods for point-of-care analysis are still needed, particularly for applications in resource-limited settings. Detection based on reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is particularly useful for this, because when combined with fluorescence-based DNA detection, RT-LAMP can be implemented with minimal equipment and expense. Assays have been developed to detect HIV RNA with RT-LAMP, but existing methods detect only a limited subset of HIV subtypes. Here we report a bioinformatic study to develop optimized primers, followed by empirical testing of 44 new primer designs. One primer set (ACeIN-26), targeting the HIV integrase coding region, consistently detected subtypes A, B, C, D, and G. The assay was sensitive to at least 5000 copies per reaction for subtypes A, B, C, D, and G, with Z-factors of above 0.69 (detection of the minor subtype F was found to be unreliable). There are already rapid and efficient assays available for detecting HIV infection in a binary yes/no format, but the rapid RT-LAMP assay described here has additional uses, including 1) tracking response to medication by comparing longitudinal values for a subject, 2) detecting of infection in neonates unimpeded by the presence of maternal antibody, and 3) detecting infection prior to seroconversion. PMID:25675344

  7. A real-time loop-mediated isothermal amplification assay for rapid detection of Shigella species.

    PubMed

    Liew, P S; Teh, C S J; Lau, Y L; Thong, K L

    2014-12-01

    Shigellosis is a foodborne illness caused by the genus Shigella and is an important global health issue. The development of effective techniques for rapid detection of this pathogen is essential for breaking the chain of transmission. Therefore, we have developed a novel loop-mediated isothermal amplification (LAMP) assay targeting the invasion plasmid antigen H (ipaH) gene to rapidly detect Shigella species. This assay could be performed in 90 min at an optimal temperature of 64ºC, with endpoint results visualized directly. Notably, the method was found to be more sensitive than conventional PCR. Indeed, the detection limit for the LAMP assay on pure bacterial cultures was 5.9 x 10(5) CFU/ml, while PCR displayed a limit of 5.9 x 10(7) CFU/ml. In spiked lettuce samples, the sensitivity of the LAMP assay was 3.6 x 10(4) CFU/g, whereas PCR was 3.6 x 10(5) CFU/g. Overall, the assay accurately identified 32 Shigella spp. with one enteroinvasive Escherichia coli displaying positive reaction while the remaining 32 non-Shigella strains tested were negative. PMID:25776596

  8. Reverse transcription loop-mediated isothermal amplification assay for rapid detection of Papaya ringspot virus.

    PubMed

    Shen, Wentao; Tuo, Decai; Yan, Pu; Yang, Yong; Li, Xiaoying; Zhou, Peng

    2014-08-01

    Papaya ringspot virus (PRSV) and Papaya leaf distortion mosaic virus (PLDMV), which causes disease symptoms similar to PRSV, threaten commercial production of both non-transgenic-papaya and PRSV-resistant transgenic papaya in China. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to detect PLDMV was developed previously. In this study, the development of another RT-LAMP assay to distinguish among transgenic, PRSV-infected and PLDMV-infected papaya by detection of PRSV is reported. A set of four RT-LAMP primers was designed based on the highly conserved region of the P3 gene of PRSV. The RT-LAMP method was specific and sensitive in detecting PRSV, with a detection limit of 1.15×10(-6)μg of total RNA per reaction. Indeed, the reaction was 10 times more sensitive than one-step RT-PCR. Field application of the RT-LAMP assay demonstrated that samples positive for PRSV were detected only in non-transgenic papaya, whereas samples positive for PLDMV were detected only in commercialized PRSV-resistant transgenic papaya. This suggests that PRSV remains the major limiting factor for non-transgenic-papaya production, and the emergence of PLDMV threatens the commercial transgenic cultivar in China. However, this study, combined with the earlier development of an RT-LAMP assay for PLDMV, will provide a rapid, sensitive and cost-effective diagnostic power to distinguish virus infections in papaya. PMID:24769198

  9. Detection of equine rotavirus by reverse transcription loop-mediated isothermal amplification (RT-LAMP).

    PubMed

    Nemoto, Manabu; Imagawa, Hiroshi; Tsujimura, Koji; Yamanaka, Takashi; Kondo, Takashi; Matsumura, Tomio

    2010-06-01

    Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was applied to detection of equine rotavirus. Because equine rotavirus of the single P genotype, P[12], is predominant in the equine population worldwide, an RT-LAMP primer set was designed to target the genotype P[12] sequence and thus detect equine rotavirus. The detection limit of the RT-LAMP assay was 10(3) copies of viral RNA, whereas that of semi-nested RT-PCR for genotype P[12] was 10(5) copies. The RT-LAMP assay specifically amplified genotype P[12] but did not amplify the other P genotype strains. The RT-LAMP assay did not amplify any pathogens related to equine intestinal disorder other than rotavirus. Using 96 diarrheal stools, the RT-LAMP assay detected equine rotavirus in 58 samples, whereas semi-nested RT-PCR only detected equine rotavirus in 25 samples. The RT-LAMP assay did not detect equine rotavirus with fecal samples collected from nine healthy foals. These results indicate that the RT-LAMP assay is specific for equine rotavirus and more sensitive than semi-nested RT-PCR. Because it is easy to manipulate without the need for a thermal cycler or gel electrophoresis, the RT-LAMP assay should be applicable to diagnosis of equine rotavirus infections in diagnostic laboratories. PMID:20160420

  10. Development and evaluation of loop-mediated isothermal amplification assay for rapid detection of Capripoxvirus

    PubMed Central

    Batra, Kanisht; Kumar, Aman; Kumar, Vinay; Nanda, Trilok; Maan, Narender S; Maan, Sushila

    2015-01-01

    Aim: The present study was undertaken to develop a nucleic acid-based diagnostic assay loop-mediated isothermal amplification assay (LAMP) targeting highly conserved genomic regions of Capripoxvirus (CaPVs) and its comparative evaluation with real-time polymerase chain reaction (PCR). Material and Methods: Lyophilized vaccine strain of sheeppox virus (SPPV) was used for optimization of LAMP assay. The LAMP assay was designed using envelope immunogenic protein (P32) coding gene targeting highly conserved genomic regions of CaPV responsible for causing sheep pox, goat pox, and lumpy skin disease in sheep, goat and cattle respectively. Serial tenfold dilution of SPPV recombinant plasmid DNA was used for a calculating limit of detection. Analytical sensitivity and specificity were performed. Results: The test described is quick (30 min), sensitive and specific for detection of CaPVs. The described assay did not show any cross-reactivity to other related viruses that cause apparently similar clinical signs. It was found to be ten times more sensitive than conventional PCR however, 100 times less sensitive than quantitative PCR (qPCR). LAMP assay results were monitored by color change method using picogreen dye and agarose gel electrophoresis. Conclusion: LAMP assay can be a very good alternative for CaPV detection to other molecular techniques requiring sophisticated equipments. PMID:27047031

  11. Rapid, sensitive, and specific detection of Clostridium tetani by loop-mediated isothermal amplification assay.

    PubMed

    Jiang, Dongneng; Pu, Xiaoyun; Wu, Jiehong; Li, Meng; Liu, Ping

    2013-01-01

    Tetanus is a specific infectious disease, which is often associated with catastrophic events such as earthquakes, traumas, and war wounds. The obligate anaerobe Clostridium tetani is the pathogen that causes tetanus. Once the infection of tetanus progresses to an advanced stage within the wounds of limbs, the rates of amputation and mortality increase manifold. Therefore, it is necessary to devise a rapid and sensitive point-of-care detection method for C. tetani so as to ensure an early diagnosis and clinical treatment of tetanus. In this study, we developed a detection method for C. tetani using loop-mediated isothermal amplification (LAMP) assay, wherein the C. tetani tetanus toxin gene was used as the target gene. The method was highly specific and sensitive, with a detection limit of 10 colony forming units (CFU)/ml, and allowed quantitative analysis. While detecting C. tetani in clinical samples, it was found that the LAMP results completely agreed with those of the traditional API 20A anaerobic bacteria identification test. As compared with the traditional API test and PCR assay, LAMP detection of C. tetani is simple and rapid, and the results can be identified through naked-eye observation. Therefore, it is an ideal and rapid point-of-care testing method for tetanus. PMID:23314360

  12. Loop-Mediated Isothermal Amplification for Detection of Staphylococcus aureus in Dairy Cow Suffering from Mastitis

    PubMed Central

    Tie, Zhang; Chunguang, Wang; Xiaoyuan, Wei; Xinghua, Zhao; Xiuhui, Zhong

    2012-01-01

    To develop a rapid detection method of Staphylococcus aureus using loop-mediated isothermal amplification (LAMP), four specific primers were designed according to six distinct sequences of the nuc gene. In addition, the specificity and sensitivity of LAMP were verified and compared with those of PCR. Results showed that the LAMP reaction was completed within 45 min at 62.5°C, and ladder bands were appeared in LAMP products analyzed by gel electrophoresis. After adding 1x SYBR Green l, the positive reaction tube showed green color and the negative reaction tube remained orange, indicating that the LAMP has high specificity. The minimal detectable concentration of LAMP was 1 × 102 CFU/mL and that of PCR was 1 × 104 CFU/mL, indicating that the LAMP was 100 times more sensitive than the PCR. The LAMP method for detection of Staphylococcus aureus has many advantages, such as simple operation, high sensitivity, high specificity, and rapid analysis. Therefore, this method is more suitable for the rapid on-site detection of Staphylococcus aureus. PMID:23251078

  13. Identification of pork in meat products using real-time loop-mediated isothermal amplification

    PubMed Central

    Yang, Lixia; Fu, Shujun; Peng, Xinkai; Li, Le; Song, Taoping; Li, Lin

    2014-01-01

    In this study, a one-step, real-time, loop-mediated isothermal amplification (RealAmp) assay was developed, for the highly specific detection of pork DNA. For the assay, the mtDNA of cytochrome b (cytb) gene was amplified at 63 °C using SYBR Green I for 45 min with a Real-Time Polymerase Chain Reaction (PCR) System that measured the fluorescent signal at one-minute intervals. As little as 1 pg of template DNA could be detected, without any cross-reactivity with non-target species. Meat mixtures, heat-treated at 100 °C for 15 min, prepared by mixing pork meat with beef at different ratios (0.01%–10%) were tested, and the RealAmp assays allowed the detection of as little as 0.01% pork in the meat mixtures. Thus, this work showed that RealAmp could be used for specific identification and sensitive quantification of meat species, even for heat-treated meat products. PMID:26019573

  14. Simple detection of Pythium irregulare using loop-mediated isothermal amplification assay.

    PubMed

    Feng, Wenzhuo; Ishiguro, Yasushi; Hotta, Keisuke; Watanabe, Hideki; Suga, Haruhisa; Kageyama, Koji

    2015-11-01

    Pythium irregulare is an important soil-borne pathogen that causes seed, stem and root rot, and seedling damping-off in various crops. Here, we have developed a rapid and reliable approach for detecting the pathogen using loop-mediated isothermal amplification (LAMP) in combination with primers designed from the sequences of the P. irregulare ribosomal DNA internal transcribed spacer region. The specificity of the primers for P. irregulare was tested using 50 isolates of 40 Pythium species, 11 Phytophthora isolates and 8 isolates of 7 other soil-borne pathogens. The assay showed that the limit of sensitivity of the LAMP method was 100 fg of pure DNA, a similar level to that of a polymerase chain reaction. LAMP detected P. irregulare from the supernatant after mixing culture medium (template DNA source) with distilled water. Similarly, positive results were obtained using a 'Plant-LAMP' method applied to a suspension rotted roots in water. A 'Bait-LAMP' method using the supernatant of autoclaved perilla seeds incubated in a soil/water mixture for 1 week at 25°C successfully detected P. irregulare from the soil. The LAMP assay described in this study is therefore a simple and effective way for practical detection of P. irregulare. PMID:26394643

  15. Centrifugal loop-mediated isothermal amplification microdevice for rapid, multiplex and colorimetric foodborne pathogen detection.

    PubMed

    Oh, Seung Jun; Park, Byung Hyun; Jung, Jae Hwan; Choi, Goro; Lee, Doh C; Kim, Do Hyun; Seo, Tae Seok

    2016-01-15

    We present a centrifugal microfluidic device which enables multiplex foodborne pathogen identification by loop-mediated isothermal amplification (LAMP) and colorimetric detection using Eriochrome Black T (EBT). Five identical structures were designed in the centrifugal microfluidic system to perform the genetic analysis of 25 pathogen samples in a high-throughput manner. The sequential loading and aliquoting of the LAMP cocktail, the primer mixtures, and the DNA sample solutions were accomplished by the optimized zigzag-shaped microchannels and RPM control. We targeted three kinds of pathogenic bacteria (Escherichia coli O157:H7, Salmonella typhimurium and Vibrio parahaemolyticus) and detected the amplicons of the LAMP reaction by the EBT-mediated colorimetric method. For the limit-of-detection (LOD) test, we carried out the LAMP reaction on a chip with serially diluted DNA templates of E. coli O157:H7, and could observe the color change with 380 copies. The used primer sets in the LAMP reaction were specific only to the genomic DNA of E. coli O157:H7, enabling the on-chip selective, sensitive, and high-throughput pathogen identification with the naked eyes. The entire process was completed in 60min. Since the proposed microsystem does not require any bulky and expensive instrumentation for end-point detection, our microdevice would be adequate for point-of-care (POC) testing with high simplicity and high speed, providing an advanced genetic analysis microsystem for foodborne pathogen detection. PMID:26322592

  16. Rapid detection of ermB gene in Clostridium difficile by loop-mediated isothermal amplification.

    PubMed

    Lin, Minyi; Liu, Wei; Wang, Pu; Tan, Jiasheng; Zhou, Youlian; Wu, Peiqun; Zhang, Ting; Yuan, Jing; Chen, Ye

    2015-08-01

    Macrolide-lincosamide-streptogramin B resistance in Clostridium difficile is mostly due to the ermB resistance determinant. Here, we describe a sensitive and rapid molecular method to detect ermB in C. difficile to contribute to the wider epidemiological study. Five sets of loop-mediated isothermal amplification (LAMP) primers were designed and optimized for rapid detection of ermB. The specificity and sensitivity of the primers for ermB were detected, and the ermB LAMP assay was compared to conventional PCR with 80 clinical isolates of C. difficile. Real-time monitoring of turbidity and chromogenic reaction were used to determine negative and positive results. A total of 26 pathogenic bacterial strains of different species were found to be negative for ermB, which indicated the high specificity of the primers. ermB was detected in 78.8 % (63/80) of the clinical isolates by both LAMP and conventional PCR. The detection limit of LAMP was 36.1  pg DNA μl- 1 and its sensitivity was 10-fold greater than that of conventional PCR. This study is the first report regarding the development and application of the LAMP assay for detection of the ermB gene in C. difficile strains. The developed LAMP method is sensitive, specific and provides a user-friendly visual approach for the rapid detection of ermB-bearing C. difficile. PMID:26272634

  17. The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii.

    PubMed

    Chen, Hua-Wei; Ching, Wei-Mei

    2016-01-01

    Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 °C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic. PMID:27167168

  18. Rapid detection of Mycoplasma synoviae by loop-mediated isothermal amplification.

    PubMed

    Kursa, Olimpia; Woźniakowski, Grzegorz; Tomczyk, Grzegorz; Sawicka, Anna; Minta, Zenon

    2015-03-01

    Mycoplasma synoviae (MS) remains a serious concern in production of poultry and affects world production of chickens and turkeys. Loop-mediated isothermal amplification (LAMP) of DNA has been recently used for the identification of different economically important avian pathogens. The aim of this study was to develop LAMP for simple and inexpensive detection of MS strains in poultry using specifically designed primers targeting hemagglutin A (vlh) gene. The assay was conducted in a water bath for 1 h at 63 °C. The results were visualized after addition of SYBR Green(®) fluorescent dye. LAMP was specific exclusively for MS without cross-reactivity with other Mycoplasma species. The sensitivity of LAMP was determined as 10(-1) CFU/ml and was 1,000 times higher than MS-specific polymerase chain reaction. LAMP assay was conducted on 18 MS field strains to ensure its reliability and usefulness. This is the first report on LAMP development and application for the rapid detection of MS isolated from chickens. This simple method may be applied by diagnostic laboratories without access to expensive equipment. PMID:25413672

  19. Detection methods for milk pathogenic bacteria by loop-mediated isothermal amplification.

    PubMed

    Yang, Wentao; Song, Xiaoning; Wang, Jingxin; Li, Zhen; Ji, Mingjiang; Li, Yufeng

    2014-12-01

    Milk is a common food, which is consumed all over the world. It is an important source of calcium. Meanwhile, it provides abundant protein, minerals and vitamins. However, pathogenic bacteria which exist in milk not only causes nutrition loss, but also produces toxins which may cause diarrhea, food poisoning, and even death. In order to control the microbial level of raw milk and eliminate the contamination of materials, this assay applied loop-mediated isothermal amplification to explore a new way to detect enterotoxigenic Escherichia coli (ETEC) in raw milk. The best reaction condition in detecting ETEC from raw milk was confirmed to be: 0.016 μM each of forward outer primer (primer F3) and backward outer primer (primer B3), 0.128 μM each of forward inner primer (primer FIP) and backward inner primer (primer BIP), 0.45 μM deoxy-ribonucleoside triphosphate (dNTPs), 2IU Bst DNA polymerase large fragment and template DNA were incubated at 63°C for 60 min. LAMP was proved to be specific, rapid and sensitive in detecting pathogenic bacteria which exist in milk. PMID:25641177

  20. Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Cannabis sativa.

    PubMed

    Kitamura, Masashi; Aragane, Masako; Nakamura, Kou; Watanabe, Kazuhito; Sasaki, Yohei

    2016-07-01

    In many parts of the world, the possession and cultivation of Cannabis sativa L. are restricted by law. As chemical or morphological analyses cannot identify the plant in some cases, a simple yet accurate DNA-based method for identifying C. sativa is desired. We have developed a loop-mediated isothermal amplification (LAMP) assay for the rapid identification of C. sativa. By optimizing the conditions for the LAMP reaction that targets a highly conserved region of tetrahydrocannabinolic acid (THCA) synthase gene, C. sativa was identified within 50 min at 60-66°C. The detection limit was the same as or higher than that of conventional PCR. The LAMP assay detected all 21 specimens of C. sativa, showing high specificity. Using a simple protocol, the identification of C. sativa could be accomplished within 90 min from sample treatment to detection without use of special equipment. A rapid, sensitive, highly specific, and convenient method for detecting and identifying C. sativa has been developed and is applicable to forensic investigations and industrial quality control. PMID:27118244

  1. Development of Lyophilized Loop-Mediated Isothermal Amplification Reagents for the Detection of Leptospira.

    PubMed

    Chen, Hua-Wei; Weissenberger, Giulia; Ching, Wei-Mei

    2016-05-01

    Leptospirosis is considered to be the most widespread zoonosis. This worldwide emerging infectious disease is caused by the pathogenic species belonging to the genus Leptospira. Polymerase chain reaction (PCR)-based diagnostic assays have been developed for detecting Leptospira DNA in cell cultures and clinical samples. Because PCR requires specialized equipment and extensive end-user training, it is not suitable for routine work in resource-limited areas. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of Leptospira in patient, animal and environmental samples using lyophilized reagents at a single temperature of around 63°C with a heating block. The sensitivity of this LAMP assay is very similar to the PCR method. The amplified DNA products can be visualized with the naked eyes using hydroxy naphthol blue or detected by the fluorescence signal of SYBR green dye in the reaction when an ultraviolet lamp or compact fluorescence tube scanner is used. This LAMP assay is simple, rapid, and can be performed with a water bath or heating block. The lyophilized LAMP reagents are stable for 3 months when stored at 4°C and 1 month when stored at 25°C, respectively. It is ideal for resource-limited settings where leptospirosis is endemic. PMID:27168577

  2. Development of double loop-mediated isothermal amplification to detect Listeria monocytogenes in food.

    PubMed

    Wu, Rina; Liu, Xiang; Guo, Bangcheng; Chen, Fusheng; Wang, Xiaohong

    2014-12-01

    In this study, a double loop-mediated isothermal amplification (dLAMP) based on two target genes hlyA and iap was developed for the rapid detection of Listeria monocytogenes in food. The results revealed that the detection time and temperature of our dLAMP assay for L. monocytogenes were 15 min and 63 °C respectively, with a sensitivity of 10 fg DNA of L. monocytogenes per tube. While normal LAMP (nLAMP) of hlyA or iap was 100 fg DNA of L. monocytogenes per tube for 45 min and 63 °C. Furthermore, mineral oil and GoldViewII nucleic acid stain were chosen as the basic materials to develop a simple visualized identification of the positive samples. A total of 450 food samples were tested for L. monocytogenes using the dLAMP protocol developed in this study. The results showed that the accuracy of the dLAMP and the "gold standard" culture-biotechnical method were 100 % identical, suggesting that the modified dLAMP assay would provide a potential for detection of L. monocytogenes in food products. PMID:25086581

  3. Development of a loop-mediated isothermal amplification method for rapid detection of pigeon circovirus.

    PubMed

    Tsai, Shinn Shyong; Chang, Yeng Ling; Huang, Yen Li; Liu, Hung Jen; Ke, Guan Ming; Chiou, Chwei Jang; Hsieh, Yao Ching; Chang, Tsung Chou; Cheng, Li Ting; Chuang, Kuo Pin

    2014-05-01

    There are no effective antiviral treatments for pigeon circovirus (PiCV); thus, rapid diagnosis is critical for effective control of the disease caused by this virus. The recent development of a novel LAMP technique that amplifies nucleic acids rapidly with high specificity and sensitivity under isothermal conditions has overcome some of the deficiencies of nucleic-acid-based diagnostic tests. We established a LAMP method for rapid detection of PiCV using two pairs of primers that were designed from PiCV and compared its sensitivity and specificity with that of PCR. Amplification by LAMP was optimal at 63 °C for 60 min. The detection limit was nearly 0.5 pg of PiCV DNA, making it ten times more sensitive than PCR. There was no cross-reaction with porcine circovirus type 2 (PCV2), pigeon Trichomonas gallinae, or pigeon herpesvirus (PHV) under the same conditions. The assay also successfully detected the pathogen DNA in the tissues of infected pigeons. This is the first report indicating that LAMP is a valuable, rapid method of detecting PiCV with high sensitivity and specificity. PMID:24193953

  4. Loop-Mediated Isothermal Amplification Targeting Actin DNA of Trichomonas vaginalis.

    PubMed

    Goo, Youn-Kyoung; Shin, Won-Sik; Yang, Hye-Won; Joo, So-Young; Song, Su-Min; Ryu, Jae-Sook; Kong, Hyun-Hee; Lee, Won-Ki; Chung, Dong-Il; Hong, Yeonchul

    2016-06-01

    Trichomoniasis caused by Trichomonas vaginalis is a common sexually transmitted disease. Its association with several health problems, including preterm birth, pelvic inflammatory disease, cervical cancer, and transmission of human immunodeficiency virus, emphasizes the importance of improved access to early and accurate detection of T. vaginalis. In this study, a rapid and efficient loop-mediated isothermal amplification-based method for the detection of T. vaginalis was developed and validated, using vaginal swab specimens from subjects suspected to have trichomoniasis. The LAMP assay targeting the actin gene was highly sensitive with detection limits of 1 trichomonad and 1 pg of T. vaginalis DNA per reaction, and specifically amplified the target gene only from T. vaginalis. Validation of this assay showed that it had the highest sensitivity and better agreement with PCR (used as the gold standard) compared to microscopy and multiplex PCR. This study showed that the LAMP assay, targeting the actin gene, could be used to diagnose early infections of T. vaginalis. Thus, we have provided an alternative molecular diagnostic tool and a point-of-care test that may help to prevent trichomoniasis transmission and associated complications. PMID:27417089

  5. Loop-Mediated Isothermal Amplification Targeting Actin DNA of Trichomonas vaginalis

    PubMed Central

    Goo, Youn-Kyoung; Shin, Won-Sik; Yang, Hye-Won; Joo, So-Young; Song, Su-Min; Ryu, Jae-Sook; Kong, Hyun-Hee; Lee, Won-Ki; Chung, Dong-Il; Hong, Yeonchul

    2016-01-01

    Trichomoniasis caused by Trichomonas vaginalis is a common sexually transmitted disease. Its association with several health problems, including preterm birth, pelvic inflammatory disease, cervical cancer, and transmission of human immunodeficiency virus, emphasizes the importance of improved access to early and accurate detection of T. vaginalis. In this study, a rapid and efficient loop-mediated isothermal amplification-based method for the detection of T. vaginalis was developed and validated, using vaginal swab specimens from subjects suspected to have trichomoniasis. The LAMP assay targeting the actin gene was highly sensitive with detection limits of 1 trichomonad and 1 pg of T. vaginalis DNA per reaction, and specifically amplified the target gene only from T. vaginalis. Validation of this assay showed that it had the highest sensitivity and better agreement with PCR (used as the gold standard) compared to microscopy and multiplex PCR. This study showed that the LAMP assay, targeting the actin gene, could be used to diagnose early infections of T. vaginalis. Thus, we have provided an alternative molecular diagnostic tool and a point-of-care test that may help to prevent trichomoniasis transmission and associated complications. PMID:27417089

  6. Detection of the Quarantine Species Thrips palmi by Loop-Mediated Isothermal Amplification

    PubMed Central

    Przybylska, Arnika; Fiedler, Żaneta; Kucharczyk, Halina; Obrępalska-Stęplowska, Aleksandra

    2015-01-01

    Thrips palmi (from the order Thysanoptera) is a serious insect pest of various crops, including vegetables, fruits and ornamental plants, causing significant economic losses. Its presence constitutes a double threat; not only does T. palmi feed on the plants, it is also a vector for several plant viruses. T. palmi originated in Asia, but has spread to North and Central America, Africa, Oceania and the Caribbean in recent decades. This species has been sporadically noted in Europe and is under quarantine regulation in the European Union. For non-specialists its larval stages are indistinguishable morphologically from another widespread and serious insect pest Frankliniella occidentalis (a non-quarantine species in the European Union) as well as other frequently occurring thrips. In this study, we have developed a loop-mediated isothermal amplification protocol to amplify rDNA regions of T. palmi. The results were consistent whether isolated DNA or crushed insects were used as template, indicating that the DNA isolation step could be omitted. The described method is species-specific and sensitive and provides a rapid diagnostic tool to detect T. palmi in the field. PMID:25793743

  7. Development of a loop-mediated isothermal amplification method for detection of Perkinsus spp. in mollusks.

    PubMed

    Feng, Chunyan; Wang, Caixia; Lin, Xiangmei; Zhang, Yongning; Lv, Jizhou; Deng, Junhua; Yuan, Xiangfen; Mei, Lin; Wu, Shaoqiang

    2013-05-27

    Perkinsus is a genus of unicellular protozoan parasite responsible for mass mortality of several commercially valuable mollusks. Surveillance and inspection of its epidemiology in the field calls for convenient and rapid detection methods. Here, a loop-mediated isothermal amplification (LAMP) assay was developed to detect the presence of Perkinsus spp. in mollusks. Specific LAMP primers were designed targeting the conserved internal transcribed spacer 2 (ITS-2) region of the rRNA gene of Perkinsus spp. Using ITS-2 recombinant plasmid as a template, we optimized the LAMP reaction system and conditions and then evaluated the analytical sensitivity and specificity of the assay. The LAMP assay was validated using clam samples collected from coastal areas in eastern China and oysters imported to China and compared with the traditional Ray's fluid thioglycollate culture method (RFTM). Our results showed that the LAMP detection method for Perkinsus was successful. The detection limit was 10 copies of plasmid DNA. Compared to the RFTM assay, the LAMP detection method was more sensitive (56 versus 52 positive out of 60 samples). P. olseni and P. marinus from infected hosts were successfully detected by this method. The LAMP method is rapid, sensitive, and specific for Perkinsus spp. detection, and could be used to screen for perkinsosis both on farms and at ports. PMID:23709467

  8. Multicenter Clinical Evaluation of the Novel Alere i Strep A Isothermal Nucleic Acid Amplification Test

    PubMed Central

    Russo, Michael E.; Jaggi, Preeti; Kline, Jennifer; Gluckman, William; Parekh, Amisha

    2015-01-01

    Rapid detection of group A beta-hemolytic streptococcus (GAS) is used routinely to help diagnose and treat pharyngitis. However, available rapid antigen detection tests for GAS have relatively low sensitivity, and backup testing is recommended in children. Newer assays are more sensitive yet require excessive time for practical point-of-care use as well as laboratory personnel. The Alere i strep A test is an isothermal nucleic acid amplification test designed to offer highly sensitive results at the point of care within 8 min when performed by nonlaboratory personnel. The performance of the Alere i strep A test was evaluated in a multicenter prospective trial in a Clinical Laboratory Improvement Amendments (CLIA)-waived setting in comparison to bacterial culture in 481 children and adults. Compared to culture, the Aleri i strep A test had 96.0% sensitivity and 94.6% specificity. Discrepant results were adjudicated by PCR and found the Alere i strep A test to have 98.7% sensitivity and 98.5% specificity. Overall, the Alere i strep A test could provide a one-step, rapid, point-of-care testing method for GAS pharyngitis and obviate backup testing on negative results. PMID:25972418

  9. Development and evaluation of probe based real time loop mediated isothermal amplification for Salmonella: A new tool for DNA quantification.

    PubMed

    Mashooq, Mohmad; Kumar, Deepak; Niranjan, Ankush Kiran; Agarwal, Rajesh Kumar; Rathore, Rajesh

    2016-07-01

    A one step, single tube, accelerated probe based real time loop mediated isothermal amplification (RT LAMP) assay was developed for detecting the invasion gene (InvA) of Salmonella. The probe based RT LAMP is a novel method of gene amplification that amplifies nucleic acid with high specificity and rapidity under isothermal conditions with a set of six primers. The whole procedure is very simple and rapid, and amplification can be obtained in 20min. Detection of gene amplification was accomplished by amplification curve, turbidity and addition of DNA binding dye at the end of the reaction results in colour difference and can be visualized under normal day light and in UV. The sensitivity of developed assay was found 10 fold higher than taqman based qPCR. The specificity of the RT LAMP assay was validated by the absence of any cross reaction with other members of enterobacteriaceae family and other gram negative bacteria. These results indicate that the probe based RT LAMP assay is extremely rapid, cost effective, highly specific and sensitivity and has potential usefulness for rapid Salmonella surveillance. PMID:27130353

  10. Sensitive and rapid detection of Giardia lamblia infection in pet dogs using loop-mediated isothermal amplification.

    PubMed

    Li, Jie; Wang, Peiyuan; Zhang, Aiguo; Zhang, Ping; Alsarakibi, Muhamd; Li, Guoqing

    2013-04-01

    Giardia lamblia is recognized as one of the most prevalent parasites in dogs. The present study aimed to establish a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of G. lamblia from dogs. The fecal samples were collected and prepared for microscopic analysis, and then the genomic DNA was extracted directly from purified cysts. The concentration of DNA samples of G. lamblia were diluted by 10-fold serially ranging from 10(-1) to 10(-5) ng/µl for LAMP and PCR assays. The LAMP assay allows the amplification to be finished within 60 min under isothermal conditions of 63℃ by employing 6 oligonucleotide primers designed based on G. lamblia elongation factor 1 alpha (EF1α) gene sequence. Our tests showed that the specific amplification products were obtained only with G. lamblia, while no amplification products were detected with DNA of other related protozoans. Sensitivity evaluation indicated that the LAMP assay was sensitive 10 times more than PCR. It is concluded that LAMP is a rapid, highly sensitive and specific DNA amplification technique for detection of G. lamblia, which has implications for effective control and prevention of giardiasis. PMID:23710094

  11. G-quadruplex based two-stage isothermal exponential amplification reaction for label-free DNA colorimetric detection.

    PubMed

    Nie, Ji; Zhang, De-Wen; Tie, Cai; Zhou, Ying-Lin; Zhang, Xin-Xiang

    2014-06-15

    A novel G-quadruplex based two-stage isothermal exponential amplification reaction (GQ-EXPAR) was developed for label-free DNA colorimetric detection in this work. The exponential amplified trigger DNA in the first stage can convert into G-quadruplex sequence EAD2 by a linear amplification circuit in the second stage. Created EAD2 can form G-quadruplex/hemin DNAzyme to act as a direct signal readout element. The GQ-EXPAR combines the exponential amplification of DNA sequence and the peroxidase-mimicking DNAzyme induced signal amplification, which achieves tandem dual-amplification. Taking advantages of isothermal incubation, this label-free homogeneous assay obviates the need of thermal cycling . As no complex synthesis or extra downstream operation is needed, the whole easy handling procedure can be finished in no more than 1h. This assay allows the sensing of the model DNA with the limit of detection to be 2.5pM. Moreover, it demonstrates good discrimination of mismatched sequences. The strategy has also been successfully implemented to sensitively detect Tay-Sachs genetic disorder mutant. PMID:24508547

  12. Detection of foot-and-mouth disease virus RNA by reverse transcription loop-mediated isothermal amplification.

    PubMed

    Chen, Hao-tai; Zhang, Jie; Liu, Yong-sheng; Liu, Xiang-tao

    2011-01-01

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for foot-and-mouth disease virus (FMDV) RNA. The amplification was able to finish in 45 min under isothermal condition at 64°C by employing a set of four primers targeting FMDV 2B. The assay showed higher sensitivity than RT-PCR. No cross reactivity was observed from other RNA viruses including classical swine fever virus, swine vesicular disease, porcine reproductive and respiratory syndrome virus, Japanese encephalitis virus. Furthermore, the assay correctly detected 84 FMDV positive samples but not 65 FMDV negative specimens. The result indicated the potential usefulness of the technique as a simple and rapid procedure for the detection of FMDV infection. PMID:22070774

  13. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid diagnosis of chilli veinal mottle virus.

    PubMed

    Banerjee, Amrita; Roy, Somnath; Sharma, Susheel Kumar; Dutta, Sudip Kumar; Chandra, Satish; Ngachan, S V

    2016-07-01

    Chilli veinal mottle virus (ChiVMV) causes significant economic loss to chilli cultivation in northeastern India, as well as in eastern Asia. In this study, we have developed a single-tube one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid, sensitive and specific diagnosis of ChiVMV. Amplification could be visualized after adding SYBR Green I (1000×) dye within 60 min under isothermal conditions at 63 °C, with a set of four primers designed based on the large nuclear inclusion protein (NIb) domain of ChiVMV (isolate KC-ML1). The RT-LAMP method was 100 times more sensitive than one-step reverse transcription polymerase chain reaction (RT-PCR), with a detection limit of 0.0001 ng of total RNA per reaction. PMID:27063408

  14. Detection of BCR-ABL Fusion mRNA Using Reverse Transcriptase Loop-mediated Isothermal Amplification

    SciTech Connect

    Dugan, L C; Hall, S; Kohlgruber, A; Urbin, S; Torres, C; Wilson, P

    2011-12-08

    RT-PCR is commonly used for the detection of Bcr-Abl fusion transcripts in patients diagnosed with chronic myelogenous leukemia, CML. Two fusion transcripts predominate in CML, Br-Abl e13a2 and e14a2. They have developed reverse transcriptase isothermal loop-mediated amplification (RT-LAMP) assays to detect these two fusion transcripts along with the normal Bcr transcript.

  15. Development of a loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of Arcanobacterium pluranimalium.

    PubMed

    Abdulmawjood, A; Wickhorst, J; Sammra, O; Lämmler, C; Foster, G; Wragg, P N; Prenger-Berninghoff, E; Klein, G

    2015-12-01

    In the present study 28 Arcanobacterium pluranimalium strains isolated from various origins could successfully be identified with a newly designed loop-mediated isothermal amplification (LAMP) assay based on gene pla encoding pluranimaliumlysin. No comparable reaction could be observed with control strains representing five species of genus Arcanobacterium and five species of genus Trueperella. The presented pla LAMP assay might allow a reliable and low cost identification of this bacterial pathogen also in laboratories with less specified equipment. PMID:26093093

  16. Loop‑mediated Isothermal Amplification assay (LAMP) based detection of Pasteurella multocida in cases of haemorrhagic septicaemia and fowl cholera.

    PubMed

    Bhimani, Mayurkumar; Bhanderi, Bharat; Roy, Ashish

    2015-01-01

    Twenty two isolates of Pasteurella multocida were obtained from different tissues of dead birds and animals (cattle, buffalo, sheep, and goat) suspected of fowl cholera and haemorrhagic septicaemia. The isolates were confirmed as P. multocida by various biochemical tests and PM PCR. An attempt was made to standardize Loop mediated isothermal amplification (LAMP) using newly designed primer sequences of KMT1 gene. Loop mediated isothermal amplification was conducted using 6 sets of primers at 65°C for 30 minutes and the result was confirmed by visual observation using SYBR green fluorescence dye as marker of positive reaction under UV transilluminator. On electrophoretic analysis of the products on 2% agarose gel, a ladder like pattern was observed, which suggested a positive amplification, whereas no amplification was observed in negative controls. Additionally, product of positive reaction yielded a green fluorescence following addition of SYBR green under UV transilluminator. It was observed that LAMP is a more sensitive test than polymerase chain reaction (PCR), as the former could detect DNA to lower limit of 22.8 pg/µl, while the latter could detect DNA to lower limit of 2.28 ng/ µl, thus LAMP could detect 100 times lesser concentration of DNA in comparison to PCR. Loop mediated isothermal amplification is a rather newer molecular technique, which can be used for rapid detection of infectious agent at field level and which does not require sophisticated instrument, i.e. thermal cycler. Furthermore, unlike the conventional PCR technique, LAMP requires lesser time to perform and result can be read visually. PMID:26129662

  17. Rapid detection of Streptococcus uberis in raw milk by loop-mediated isothermal amplification.

    PubMed

    Cornelissen, J B W J; De Greeff, A; Heuvelink, A E; Swarts, M; Smith, H E; Van der Wal, F J

    2016-06-01

    A loop-mediated isothermal amplification (LAMP) method to detect Streptococcus uberis in raw milk was developed and evaluated. Three genes (sodA, pauA, cpn60) were assessed for their suitability as targets in LAMP. The analytical sensitivity was 120, 120, and 12 fg per assay for the sodA, pauA, and cpn60 assays, respectively, with a detectable signal within 8 min for the highest concentration (12ng/assay) and ~60 min for the lowest concentrations. The LAMP assays correctly identified 7 Strep. uberis strains among a set of 83 mastitis pathogens. To enable DNA isolation from raw milk, a new method was used in which a pretreatment with a cocktail of lysing enzymes was performed before an established procedure. This method resulted in an analytical sensitivity of 48 cfu/assay for the sodA LAMP assay using raw milk spiked with Strep. uberis, corresponding to 2.4×10(4) cfu/mL milk. For raw milk samples from cows experimentally infected with Strep. uberis, results of enumeration were largely reflected by results of LAMP. Evaluation of the sodA LAMP assay with 100 raw milk field samples, of which 50 were Strep. uberis culture-negative and 50 Strep. uberis culture-positive, showed that the assay had a diagnostic sensitivity of 96.0% and a diagnostic specificity of 96.0%. In conclusion, the described LAMP assay may offer a simple alternative for convenient and sensitive detection of S. uberis in raw milk, provided a compatible rapid DNA isolation procedure is available. PMID:27060835

  18. Loop-mediated isothermal amplification for rapid and semiquantitative detection of Loa loa infection.

    PubMed

    Drame, Papa M; Fink, Doran L; Kamgno, Joseph; Herrick, Jesica A; Nutman, Thomas B

    2014-06-01

    Rapid and accurate tests are currently needed to identify individuals with high levels of Loa loa microfilaria (mf), so that these individuals may be excluded from mass ivermectin administration campaigns against onchocerciasis and lymphatic filariasis being conducted in areas where Onchocerca volvulus, Wuchereria bancrofti, and L. loa are coendemic. To address this need, colorimetric loop-mediated isothermal amplification (LAMP) assays targeting the L. loa-specific gene sequences LLMF72 and LLMF342 were developed for the detection and quantification of L. loa microfilaremia. Both LAMP assays were highly specific (100%) for L. loa infection compared to the absence of infection or infection with related filarial pathogens. The LLMF72-based LAMP assay showed greater analytic sensitivity (limit of detection, 0.1 pg/ml of genomic DNA [gDNA] and/or 5 mf/ml) than the LLMF342-based LAMP assay (10 pg/ml of gDNA and/or 50 mf/ml), and its analytic sensitivity was similar to that of LLMF72-based quantitative PCR (qPCR). A high level of correlation was observed between microfilaria counts as determined by LLMF72-based qPCR and time to positivity by the LAMP assay, and performance measures of sensitivity, specificity, and positive and negative predictive values were similar for both assays when applied to field-collected clinical samples. By simply varying the run time, the LAMP assay was able to accurately distinguish individuals at risk for serious adverse events (SAEs) after exposure to ivermectin, using thresholds of >5,000 mf/ml and >30,000 mf/ml as indicators of increasing levels of risk. In summary, LLMF72 LAMP represents a new molecular diagnostic tool that is readily applicable as a point-of-care method for L. loa microfilarial detection and quantification in resource-limited countries where L. loa infection is endemic. PMID:24696020

  19. Visual detection of goose haemorrhagic polyomavirus in geese and ducks by loop-mediated isothermal amplification.

    PubMed

    Woźniakowski, Grzegorz; Tarasiuk, Karolina

    2015-01-01

    Goose haemorrhagic polyomavirus (GHPV) is an aetiological agent of haemorrhagic nephritis and enteritis of geese occurring in geese (Anser anser). GHPV may also infect Muscovy ducks (Carina mochata) and mule ducks. Early detection of GHPV is important to isolate the infected birds from the rest of the flock thus limiting infection transmission. The current diagnosis of haemorrhagic nephritis and enteritis of geese is based on virus isolation, histopathological examination, haemagglutination inhibition assay, ELISA and polymerase chain reaction (PCR). Recently, real-time PCR assay was developed which considerably improved detection of GHPV. In spite of many advantages, these methods are still time-consuming and inaccessible for laboratories with limited access to ELISA plate readers or PCR thermocyclers. The aim of our study was to develop loop-mediated isothermal amplification (LAMP) that may be conducted in a water bath. Two pairs of specific primers complementary to VP1 gene of GHPV were designed. The results of GHPV LAMP were recorded under ultraviolet light. Our study showed LAMP was able to specifically amplify VP1 fragment of a GHPV without cross-reactivity with other pathogens of geese and ducks. LAMP detected as little as 1.5 pg of DNA extracted from a GHPV standard strain (150 pg/µl). The optimized LAMP was used to examine 18 field specimens collected from dead and clinically diseased geese and ducks aged from 1 to 12 weeks. The positive signal for GHPV was detected in three out of 18 (16.6%) specimens. These results were reproducible and consistent with those of four real-time PCR. To the best of our knowledge this is the first report on LAMP application for the GHPV detection. PMID:25959267

  20. An isothermal amplification reactor with an integrated isolation membrane for point-of-care detection of infectious diseases

    PubMed Central

    Liu, Changchun; Geva, Eran; Mauk, Michael; Qiu, Xianbo; Abrams, William R.; Malamud, Daniel; Curtis, Kelly; Owen, S. Michele; Bau, Haim H.

    2015-01-01

    A simple, point of care, inexpensive, disposable cassette for the detection of nucleic acids extracted from pathogens was designed, constructed, and tested. The cassette utilizes a single reaction chamber for isothermal amplification of nucleic acids. The chamber is equipped with an integrated, flow-through, Flinders Technology Associates (Whatman FTA®) membrane for the isolation, concentration, and purification of DNA and/or RNA. The nucleic acids captured by the membrane are used directly as templates for amplification without elution, thus simplifying the cassette’s flow control. The FTA membrane also serves another critical role—enabling the removal of inhibitors that dramatically reduce detection sensitivity. Thermal control is provided with a thin film heater external to the cassette. The amplification process was monitored in real time with a portable, compact fluorescent reader. The utility of the integrated, single-chamber cassette was demonstrated by detecting the presence of HIV-1 in oral fluids. The HIV RNA was reverse transcribed and subjected to loop-mediated, isothermal amplification (LAMP). A detection limit of less than 10 HIV particles was demonstrated. The cassette is particularly suitable for resource poor regions, where funds and trained personnel are in short supply. The cassette can be readily modified to detect nucleic acids associated with other pathogens borne in saliva, urine, and other body fluids as well as in water and food. PMID:21455542

  1. Mismatch extension of DNA polymerases and high-accuracy single nucleotide polymorphism diagnostics by gold nanoparticle-improved isothermal amplification.

    PubMed

    Chen, Feng; Zhao, Yue; Fan, Chunhai; Zhao, Yongxi

    2015-09-01

    Sequence mismatches may induce nonspecific extension reaction, causing false results for SNP diagnostics. Herein, we systematically investigated the impact of various 3'-terminal mismatches on isothermal amplification catalyzed by representative DNA polymerases. Despite their diverse efficiencies depending on types of mismatch and kinds of DNA polymerase, all 12 kinds of single 3'-terminal mismatches induced the extension reaction. Generally, only several mismatches (primer-template, C-C, G-A, A-G, and A-A) present an observable inhibitory effect on the amplification reaction, whereas other mismatches trigger amplified signals as high as those of Watson-Crick pairs. The related mechanism was deeply discussed, and a primer-design guideline for specific SNP analysis was summarized. Furthermore, we found that the addition of appropriate gold nanoparticles (AuNPs) can significantly inhibit mismatch extension and enhance the amplification specificity. Also the high-accuracy SNP analysis of human blood genomic DNA has been demonstrated by AuNPs-improved isothermal amplification, the result of which was verified by sequencing (the gold standard method for SNP assay). Collectively, this work provides mechanistic insight into mismatch behavior and achieves accurate SNP diagnostics, holding great potential for the application in molecular diagnostics and personalized medicine. PMID:26249366

  2. Nanocrystal-based electrochemiluminescence sensor for cell detection with Au nanoparticles and isothermal circular double-assisted signal amplification.

    PubMed

    Dai, Pan-Pan; Li, Jin-Yi; Yu, Tao; Xu, Jing-Juan; Chen, Hong-Yuan

    2015-08-15

    Here we have developed a sensitive cancer cell amplified detection method which combined Au NPs enhanced electrochemiluminescence (ECL) of CdS nanocrystals (NCs) film, with isothermal circular amplification reaction of polymerase. In DNA circular amplification detection system, hairpin DNA beacon/Au NPs composite modified CdS NCs film was used as an ECL emitter. Messenger DNA is hybridized with the aptamer modified on magnetic beads (MBs) to form MB-Au bioconjugates. In the presence of HL-60 cell, the aptamer would conjugate with the glycoprotein at cell surface and messenger DNA sequence would be released. The released messenger DNA sequence was then introduced into the cycle amplification system to trigger circular polymerizations. This assay allows us to determine the released messenger DNA equivalent to 10 cells and exhibits a significant specificity for HL-60 cells. PMID:25966387

  3. Development of primer sets for loop-mediated isothermal amplification that enables rapid and specific detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three ...

  4. [Detection of the Zaire Subtype of the Ebola Virus by Isothermal Multiple Self-matching Initiated Amplification].

    PubMed

    Li, Xinna; Nie, Kai; Wang, Ji; Zhang, Dan; Guan, Li; Liu, Jun; Ke, Yuehua; Zhou, Hangyu; Ma, Xuejun

    2016-01-01

    Given the Ebola outbreak in West Africa and the risks of spread to other regions, a rapid, sensitive and simple method for the detection of the Ebola virus (EBOV) is of great significance for the prevention and control of Ebola. We developed a simple colorimetric isothermal multiple self-matching initiated amplification (IMSA) for rapid detection of the Zaire subtype of the Ebola virus (EBOV-Z). This method employed six primers that recognized seven sites of the EBOV-Z nucleoprotein gene for amplification of nucleic acids under isothermal conditions at 63 degrees C for 1 h. Amplification products were detected through visual inspection of color change by pre-addition of hydroxyl naphthol blue dye. Relative sensitivity was validated by detection of serial tenfold dilutions of virus-like particles containing the partial EBOV-Z nucleoprotein gene and mock clinical sample. Specificity of IMSA was validated by detection of the plasma of 30 healthy volunteers, the dengue virus, and Japanese encephalitis virus. IMSA had comparable sensitivity to Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and cross-reaction with human plasma or other viruses was not observed. Reverse transcription-isothermal multiple self-matching initiated amplification (RT-IMSA) was also evaluated and compared in parallel with the commercial RT-qPCR kit for detection of EBOV-suspected samples of human blood in Sierra Leone. Sensitivity and specificity of the RT-IMSA was 91.4% and 100%, respectively. These data suggest that RT-IMSA is a valuable tool for the detection of the EBOV with the distinct advantages of simplicity and low cost compared with RT-qPCR. PMID:27295876

  5. Reading Out Single-Molecule Digital RNA and DNA Isothermal Amplification in Nanoliter Volumes with Unmodified Camera Phones.

    PubMed

    Rodriguez-Manzano, Jesus; Karymov, Mikhail A; Begolo, Stefano; Selck, David A; Zhukov, Dmitriy V; Jue, Erik; Ismagilov, Rustem F

    2016-03-22

    Digital single-molecule technologies are expanding diagnostic capabilities, enabling the ultrasensitive quantification of targets, such as viral load in HIV and hepatitis C infections, by directly counting single molecules. Replacing fluorescent readout with a robust visual readout that can be captured by any unmodified cell phone camera will facilitate the global distribution of diagnostic tests, including in limited-resource settings where the need is greatest. This paper describes a methodology for developing a visual readout system for digital single-molecule amplification of RNA and DNA by (i) selecting colorimetric amplification-indicator dyes that are compatible with the spectral sensitivity of standard mobile phones, and (ii) identifying an optimal ratiometric image-process for a selected dye to achieve a readout that is robust to lighting conditions and camera hardware and provides unambiguous quantitative results, even for colorblind users. We also include an analysis of the limitations of this methodology, and provide a microfluidic approach that can be applied to expand dynamic range and improve reaction performance, allowing ultrasensitive, quantitative measurements at volumes as low as 5 nL. We validate this methodology using SlipChip-based digital single-molecule isothermal amplification with λDNA as a model and hepatitis C viral RNA as a clinically relevant target. The innovative combination of isothermal amplification chemistry in the presence of a judiciously chosen indicator dye and ratiometric image processing with SlipChip technology allowed the sequence-specific visual readout of single nucleic acid molecules in nanoliter volumes with an unmodified cell phone camera. When paired with devices that integrate sample preparation and nucleic acid amplification, this hardware-agnostic approach will increase the affordability and the distribution of quantitative diagnostic and environmental tests. PMID:26900709

  6. Using the ubiquitous pH meter combined with a loop mediated isothermal amplification method for facile and sensitive detection of Nosema bombycis genomic DNA PTP1.

    PubMed

    Xie, Shunbi; Yuan, Yali; Song, Yue; Zhuo, Ying; Li, Tian; Chai, Yaqin; Yuan, Ruo

    2014-12-28

    Here we show an amplification-coupled detection method for directly measuring released hydrogen ions during the loop mediated isothermal amplification (LAMP) procedure by using a pH meter. The genomic DNA of Nosema bombycis (N. bombycis) was amplified and detected by employing this LAMP-pH meter platform for the first time. PMID:25381873

  7. Improved loop-mediated isothermal amplification for HLA-DRB1 genotyping using RecA and a restriction enzyme for enhanced amplification specificity.

    PubMed

    Mitsunaga, Shigeki; Shimizu, Sayoko; Okudaira, Yuko; Oka, Akira; Tanaka, Masafumi; Kimura, Minoru; Kulski, Jerzy K; Inoue, Ituro; Inoko, Hidetoshi

    2013-06-01

    Our aim was to test and develop the use of loop-mediated isothermal amplification (LAMP) for HLA-DRB1 genotyping. Initially, we found that the conventional LAMP protocols produced non-specific and variable amplification results depending on the sample DNA conditions. Experiments with different concentrations of DNase in the reaction mixture with and without T4 DNA ligase-treated samples suggested that the strand displacement activity of DNA polymerase in LAMP, at least in part, started from randomly existing nicks because T4 DNA ligase treatment of sample DNA resulted in no amplification. Such non-specific amplification due to the randomly existing nicks was improved specifically by the addition of RecA of Escherichia coli and a restriction enzyme, for example, PvuII, to the reaction mixture. We applied the modified LAMP (mLAMP) (1) to detect specific HLA-DRB1 alleles by using only specific primers for amplification or (2) for genotyping in multiple samples with a multi-probe typing system. In the latter case, HLA-DRB1 genotyping was developed by combining the mLAMP with amplicon capture using polymorphic region-specific probes fixed onto the bottom of the wells of a 96-well plate and the captured amplicons visualized as a black spot at the bottom of the well. The multi-probe human leukocyte antigen (HLA) typing method and the specific HLA allele detection method could be applied for point-of-care testing due to no requirement for specific and expensive instruments. PMID:23474534

  8. Rapid and sensitive identification of the herbal tea ingredient Taraxacum formosanum using loop-mediated isothermal amplification.

    PubMed

    Lai, Guan-Hua; Chao, Jung; Lin, Ming-Kuem; Chang, Wen-Te; Peng, Wen-Huang; Sun, Fang-Chun; Lee, Meng-Shiunn; Lee, Meng-Shiou

    2015-01-01

    Taraxacum formosanum (TF) is a medicinal plant used as an important component of health drinks in Taiwan. In this study, a rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) assay for authenticating TF was established. A set of four specific LAMP primers was designed based on the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA (nrDNA) of TF. LAMP amplicons were successfully amplified and detected when purified genomic DNA of TF was added in the LAMP reaction under isothermal condition (65 °C) within 45 min. These specific LAMP primers have high specificity and can accurately discriminate Taraxacum formosanum from other adulterant plants; 1 pg of genomic DNA was determined to be the detection limit of the LAMP assay. In conclusion, using this novel approach, TF and its misused plant samples obtained from herbal tea markets were easily identified and discriminated by LAMP assay for quality control. PMID:25584616

  9. Rapid and Sensitive Identification of the Herbal Tea Ingredient Taraxacum formosanum Using Loop-Mediated Isothermal Amplification

    PubMed Central

    Lai, Guan-Hua; Chao, Jung; Lin, Ming-Kuem; Chang, Wen-Te; Peng, Wen-Huang; Sun, Fang-Chun; Lee, Meng-Shiunn; Lee, Meng-Shiou

    2015-01-01

    Taraxacum formosanum (TF) is a medicinal plant used as an important component of health drinks in Taiwan. In this study, a rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) assay for authenticating TF was established. A set of four specific LAMP primers was designed based on the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA (nrDNA) of TF. LAMP amplicons were successfully amplified and detected when purified genomic DNA of TF was added in the LAMP reaction under isothermal condition (65 °C) within 45 min. These specific LAMP primers have high specificity and can accurately discriminate Taraxacum formosanum from other adulterant plants; 1 pg of genomic DNA was determined to be the detection limit of the LAMP assay. In conclusion, using this novel approach, TF and its misused plant samples obtained from herbal tea markets were easily identified and discriminated by LAMP assay for quality control. PMID:25584616

  10. Detection of cucumber mosaic virus isolates from banana by one-step reverse transcription loop-mediated isothermal amplification.

    PubMed

    Peng, Jun; Shi, Minjing; Xia, Zihao; Huang, Junsheng; Fan, Zaifeng

    2012-11-01

    Cucumber mosaic virus (CMV) is one of the most devastating threats to the banana industry. A single-tube, one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid detection of CMV-infected banana and plantain (Musa spp.). The reaction was performed in a single tube at 63 °C for 90 min using a real-time turbidimeter, with an improved closed-tube visual detection system in which fluorescent dye was added to the inside of the lid prior to amplification. This RT-LAMP assay is an alternative method for the rapid detection of CMV in banana plants and tissue culture materials. PMID:22782136

  11. Loop mediated isothermal amplification of 5.8S rDNA for specific detection of Tritrichomonas foetus.

    PubMed

    Oyhenart, Jorge; Martínez, Florencia; Ramírez, Rosana; Fort, Marcelo; Breccia, Javier D

    2013-03-31

    Tritrichomonas foetus is the causative agent of bovine trichomonosis, a sexually transmitted disease leading to infertility and abortion. A test based on loop mediated isothermal amplification (LAMP) targeting the 5.8S rDNA subunit was designed for the specific identification of T. foetus. The LAMP assay was validated using 28 T. foetus and 35 non-T. foetus trichomonads strains. It did not exhibit cross-reaction with closely related parasites commonly found in smegma cultures like Tetratrichomonas spp. and Pentatrichomonas hominis. Bovine smegma did not show interferences for the detection of the parasite and, the sensitivity of the method (4×10(3) CFU/mL, approximately 10 cells/reaction) was slightly higher than that found for PCR amplification with TFR3 and TFR4 primers. The LAMP approach has potential applications for diagnosis and control of T. foetus and, practical use for low skill operators in rural areas. PMID:23265811

  12. Application of a loop-mediated isothermal amplification (LAMP) assay for molecular identification of Trueperella pyogenes isolated from various origins.

    PubMed

    Abdulmawjood, A; Wickhorst, J; Hashim, O; Sammra, O; Hassan, A A; Alssahen, M; Lämmler, C; Prenger-Berninghoff, E; Klein, G

    2016-08-01

    In the present study 28 Trueperella pyogenes strains isolated from various origins could successfully be identified with a newly designed loop-mediated isothermal amplification (LAMP) assay based on gene cpn60 encoding chaperonin. No cross reaction could be observed with control strains representing four species of genus Trueperella and seven species of closely related genus Arcanobacterium. The present cpn60 LAMP assay might allow a reliable and low cost identification of T. pyogenes also in laboratories with less specified equipment. PMID:27242007

  13. Visual detection of H3 subtype avian influenza viruses by reverse transcription loop-mediated isothermal amplification assay

    PubMed Central

    2011-01-01

    Background Recent epidemiological investigation of different HA subtypes of avian influenza viruses (AIVs) shows that the H3 subtype is the most predominant among low pathogenic AIVs (LPAIVs), and the seasonal variations in isolation of H3 subtype AIVs are consistent with that of human H3 subtype influenza viruses. Consequently, the development of a rapid, simple, sensitive detection method for H3 subtype AIVs is required. The loop-mediated isothermal amplification (LAMP) assay is a simple, rapid, sensitive and cost-effective nucleic acid amplification method that does not require any specialized equipment. Results A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect the H3 subtype AIVs visually. Specific primer sets target the sequences of the hemagglutinin (HA) gene of H3 subtype AIVs were designed, and assay reaction conditions were optimized. The established assay was performed in a water bath for 50 minutes, and the amplification result was visualized directly as well as under ultraviolet (UV) light reflections. The detection limit of the RT-LAMP assay was 0.1pg total RNA of virus, which was one hundred-fold higher than that of RT-PCR. The results on specificity indicated that the assay had no cross-reactions with other subtype AIVs or avian respiratory pathogens. Furthermore, a total of 176 clinical samples collected from birds at the various live-bird markets (LBMs) were subjected to the H3-subtype-specific RT-LAMP (H3-RT-LAMP). Thirty-eight H3 subtype AIVs were identified from the 176 clinical samples that were consistent with that of virus isolation. Conclusions The newly developed H3-RT-LAMP assay is simple, sensitive, rapid and can identify H3 subtype AIVs visually. Consequently, it will be a very useful screening assay for the surveillance of H3 subtype AIVs in underequipped laboratories as well as in field conditions. PMID:21729297

  14. Detection of Puccinia kuehnii Causing Sugarcane Orange Rust with a Loop-Mediated Isothermal Amplification-Based Assay.

    PubMed

    Chandra, Amaresh; Keizerweerd, Amber T; Grisham, Michael P

    2016-03-01

    Puccinia kuehnii is a fungal pathogen that causes orange rust in sugarcane, which is now prevalent in many countries. At the early stage of disease, it is almost indistinguishable from brown rust, which is caused by Puccinia melanocephala. Although several PCR assays are available to detect these diseases, the loop-mediated isothermal amplification (LAMP)-based assay has been reported to be more economical and easier to perform. Under isothermal conditions, DNA is amplified with high specificity and rapidity. Moreover, visual judgment of color change without further post-amplification processing makes the method convenient. The present study was undertaken to detect P. kuehnii genomic DNA using four primers corresponding to a unique DNA sequence of P. kuehnii. The LAMP assay was found to be optimal when 8 mM MgSO4 was used and the reaction was incubated at 63 °C for 90 min. Positive samples showed a color change from orange to green upon SYBR Green I dye addition. Specificity of the LAMP test was checked with DNA of P. melanocephala, which showed no reaction. Sensitivity of the LAMP method was observed to be the same as real-time PCR at 0.1 ng, thus providing a rapid and more affordable option for early disease detection. PMID:26837389

  15. One-step reverse transcription loop-mediated isothermal amplification for the rapid detection of cucumber green mottle mosaic virus.

    PubMed

    Li, Jin-yu; Wei, Qi-wei; Liu, Yong; Tan, Xin-qiu; Zhang, Wen-na; Wu, Jian-yan; Charimbu, Miriam Karwitha; Hu, Bai-shi; Cheng, Zhao-bang; Yu, Cui; Tao, Xiao-rong

    2013-11-01

    Cucumber green mottle mosaic virus (CGMMV) has caused serious damage to Cucurbitaceae crops worldwide. The virus is considered one of the most serious Cucurbitaceae quarantine causes in many countries. In this study, a highly efficient and practical one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of CGMMV. The total RNA or crude RNA extracted from watermelon plants or seeds could be detected easily by this RT-LAMP assay. The RT-LAMP assay was conducted in isothermal (63°C) conditions within 1h. The amplified products of CGMMV could be detected as ladder-like bands using agarose gel electrophoresis or visualized in-tube under UV light with the addition of a fluorescent dye. The RT-LAMP amplification was specific to CGMMV, as no cross-reaction was observed with other viruses. The RT-LAMP assay was 100-fold more sensitive than that of reverse-transcription polymerase chain reaction (RT-PCR). This is the first report of the application of the RT-LAMP assay to detect CGMMV. The sensitive, specific and rapid RT-LAMP assay developed in this study can be applied widely in laboratories, the field and quarantine surveillance of CGMMV. PMID:23933076

  16. The isothermal amplification detection of double-stranded DNA based on a double-stranded fluorescence probe.

    PubMed

    Shi, Chao; Shang, Fanjin; Pan, Mei; Liu, Sen; Ma, Cuiping

    2016-06-15

    Here we have developed a novel method of isothermal amplification detection of double-stranded DNA (dsDNA) based on double-stranded fluorescence probe (ds-probe). Target dsDNA repeatedly generated single-stranded DNA (ssDNA) with polymerase and nicking enzyme. The ds-probe as a primer hybridized with ssDNA and extended to its 5'-end. The displaced ssDNA served as a new detection target to initiate above-described reaction. Meanwhile, the extended ds-probe could dynamically dissociate from ssDNA and self-hybridize, converting into a turn-back structure to initiate another amplification reaction. In particular, the ds-probe played a key role in the entire experimental process, which not only was as a primer but also produced the fluorescent signal by an extension and displacement reaction. Our method could detect the pBluescript II KS(+) plasmid with a detection limit of 2.3 amol, and it was also verified to exhibit a high specificity, even one-base mismatch. Overall, it was a true isothermal dsDNA detection strategy with a strongly anti-jamming capacity and one-pot, only requiring one ds-probe, which greatly reduced the cost and the probability of contamination. With its advantages, the approach of dsDNA detection will offer a promising tool in the field of point-of-care testing (POCT). PMID:26803414

  17. Rapid Point-of-Care Isothermal Amplification Assay for the Detection of Malaria without Nucleic Acid Purification

    PubMed Central

    Modak, Sayli S.; Barber, Cheryl A.; Geva, Eran; Abrams, William R.; Malamud, Daniel; Ongagna, Yhombi Serge Yvon

    2016-01-01

    Malaria remains one of the most prevalent infectious diseases and results in significant mortality. Isothermal amplification (loop-mediated isothermal amplification) is used to detect malarial DNA at levels of ~1 parasite/µL blood in ≤30 minutes without the isolation of parasite nucleic acid from subject’s blood or saliva. The technique targets the mitochondrial cytochrome oxidase subunit 1 gene and is capable of distinguishing Plasmodium falciparum from Plasmodium vivax. Malarial diagnosis by the gold standard microscopic examination of blood smears is generally carried out only after moderate-to-severe symptoms appear. Rapid diagnostic antigen tests are available but generally require infection levels in the range of 200–2,000 parasites/µL for a positive diagnosis and cannot distinguish if the disease has been cleared due to the persistence of circulating antigen. This study describes a rapid and simple molecular assay to detect malarial genes directly from whole blood or saliva without DNA isolation. PMID:26819557

  18. Rapid and sensitive detection of Citrus Bacterial Canker by loop-mediated isothermal amplification combined with simple visual evaluation methods

    PubMed Central

    2010-01-01

    Background Citrus Bacterial Canker (CBC) is a major, highly contagious disease of citrus plants present in many countries in Asia, Africa and America, but not in the Mediterranean area. There are three types of Citrus Bacterial Canker, named A, B, and C that have different genotypes and posses variation in host range within citrus species. The causative agent for type A CBC is Xanthomonas citri subsp. citri, while Xanthomonas fuscans subsp. aurantifolii, strain B causes type B CBC and Xanthomonas fuscans subsp. aurantifolii strain C causes CBC type C. The early and accurate identification of those bacteria is essential for the protection of the citrus industry. Detection methods based on bacterial isolation, antibodies or polymerase chain reaction (PCR) have been developed previously; however, these approaches may be time consuming, laborious and, in the case of PCR, it requires expensive laboratory equipment. Loop-mediated isothermal amplification (LAMP), which is a novel isothermal DNA amplification technique, is sensitive, specific, fast and requires no specialized laboratory equipment. Results A loop-mediated isothermal amplification assay for the diagnosis of Citrus Bacterial Canker (CBC-LAMP) was developed and evaluated. DNA samples were obtained from infected plants or cultured bacteria. A typical ladder-like pattern on gel electrophoresis was observed in all positive samples in contrast to the negative controls. In addition, amplification products were detected by visual inspection using SYBRGreen and using a lateral flow dipstick, eliminating the need for gel electrophoresis. The sensitivity and specificity of the assay were evaluated in different conditions and using several sample sources which included purified DNA, bacterium culture and infected plant tissue. The sensitivity of the CBC-LAMP was 10 fg of pure Xcc DNA, 5 CFU in culture samples and 18 CFU in samples of infected plant tissue. No cross reaction was observed with DNA of other phytopathogenic

  19. Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification.

    PubMed

    Itonaga, Masahiro; Matsuzaki, Ibu; Warigaya, Kenji; Tamura, Takaaki; Shimizu, Yuki; Fujimoto, Masakazu; Kojima, Fumiyoshi; Ichinose, Masao; Murata, Shin-Ichi

    2016-01-01

    Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid-locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP), for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD) of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V) of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation. PMID:26999437

  20. Rapid detection of newly isolated Tembusu-related Flavivirus by reverse-transcription loop-mediated isothermal amplification assay

    PubMed Central

    2011-01-01

    Background From April 2010 to January 2011, a severe new viral disease had devastated most duck-farming regions in China. This disease affected not only laying ducks but also meat ducks, causing huge economic losses for the poultry industry. The objective of this study is to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of the new virus related to Tembusu-related Flavivirus. Results The RT-LAMP assay is very simple and rapid, and the amplification can be completed within 50 min under isothermal conditions at 63°C by a set of 6 primers targeting the E gene based on the sequences analysis of the newly isolated viruses and other closely related Flavivirus.The monitoring of gene amplification can also be visualized by using SYBR green I fluorescent dye. In addition, the RT-LAMP assay for newly isolated Tembusu-related Flavivirus showed higher sensitivity with an RNA detection-limit of 2 copies/μL compared with 190 copies/μL of the conventional RT-PCR method. The specificity was identified without cross reaction to other common avian pathogens. By screening a panel of clinical samples this method was more feasible in clinical settings and there was higher positive coincidence rate than conventional RT-PCR and virus isolation. Conclusion The RT-LAMP assay for newly isolated Tembusu-related Flavivirus is a valuable tool for the rapid and real-time detection not only in well-equipped laboratories but also in general conditions. PMID:22185513

  1. Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification

    PubMed Central

    Warigaya, Kenji; Tamura, Takaaki; Shimizu, Yuki; Fujimoto, Masakazu; Kojima, Fumiyoshi; Ichinose, Masao; Murata, Shin-ichi

    2016-01-01

    Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid—locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP), for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD) of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V) of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation. PMID:26999437

  2. GMO detection using a bioluminescent real time reporter (BART) of loop mediated isothermal amplification (LAMP) suitable for field use

    PubMed Central

    2012-01-01

    Background There is an increasing need for quantitative technologies suitable for molecular detection in a variety of settings for applications including food traceability and monitoring of genetically modified (GM) crops and their products through the food processing chain. Conventional molecular diagnostics utilising real-time polymerase chain reaction (RT-PCR) and fluorescence-based determination of amplification require temperature cycling and relatively complex optics. In contrast, isothermal amplification coupled to a bioluminescent output produced in real-time (BART) occurs at a constant temperature and only requires a simple light detection and integration device. Results Loop mediated isothermal amplification (LAMP) shows robustness to sample-derived inhibitors. Here we show the applicability of coupled LAMP and BART reactions (LAMP-BART) for determination of genetically modified (GM) maize target DNA at low levels of contamination (0.1-5.0% GM) using certified reference material, and compare this to RT-PCR. Results show that conventional DNA extraction methods developed for PCR may not be optimal for LAMP-BART quantification. Additionally, we demonstrate that LAMP is more tolerant to plant sample-derived inhibitors, and show this can be exploited to develop rapid extraction techniques suitable for simple field-based qualitative tests for GM status determination. We also assess the effect of total DNA assay load on LAMP-BART quantitation. Conclusions LAMP-BART is an effective and sensitive technique for GM detection with significant potential for quantification even at low levels of contamination and in samples derived from crops such as maize with a large genome size. The resilience of LAMP-BART to acidic polysaccharides makes it well suited to rapid sample preparation techniques and hence to both high throughput laboratory settings and to portable GM detection applications. The impact of the plant sample matrix and genome loading within a reaction must

  3. Point of care nucleic acid detection of viable pathogenic bacteria with isothermal RNA amplification based paper biosensor

    NASA Astrophysics Data System (ADS)

    Liu, Hongxing; Xing, Da; Zhou, Xiaoming

    2014-09-01

    Food-borne pathogens such as Listeria monocytogenes have been recognized as a major cause of human infections worldwide, leading to substantial health problems. Food-borne pathogen identification needs to be simpler, cheaper and more reliable than the current traditional methods. Here, we have constructed a low-cost paper biosensor for the detection of viable pathogenic bacteria with the naked eye. In this study, an effective isothermal amplification method was used to amplify the hlyA mRNA gene, a specific RNA marker in Listeria monocytogenes. The amplification products were applied to the paper biosensor to perform a visual test, in which endpoint detection was performed using sandwich hybridization assays. When the RNA products migrated along the paper biosensor by capillary action, the gold nanoparticles accumulated at the designated Test line and Control line. Under optimized experimental conditions, as little as 0.5 pg/μL genomic RNA from Listeria monocytogenes could be detected. The whole assay process, including RNA extraction, amplification, and visualization, can be completed within several hours. The developed method is suitable for point-of-care applications to detect food-borne pathogens, as it can effectively overcome the false-positive results caused by amplifying nonviable Listeria monocytogenes.

  4. Loop-mediated isothermal amplification (LAMP) based detection of Colletotrichum falcatum causing red rot in sugarcane

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Red rot, caused by Colletotrichum falcatum, is a destructive disease prevalent in most sugarcane-producing countries. Disease-free sugarcane planting materials are essential as the pathogen spreads primarily through infected setts. The present study was undertaken to develop loop-mediated isothermal...

  5. Real-time isothermal detection of Shiga toxin-producing Escherichia coli using recombinase polymerase amplification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin (Stx) producing E. coli (STEC) are a major family of foodborne pathogens of immense public health, zoonotic and economic significance in the US and worldwide. To date, there are no published reports on use of recombinase polymerase amplification (RPA) for STEC detection. The primary goal...

  6. Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens.

    PubMed

    Kersting, Sebastian; Rausch, Valentina; Bier, Frank F; von Nickisch-Rosenegk, Markus

    2014-01-01

    We report on the development of an on-chip RPA (recombinase polymerase amplification) with simultaneous multiplex isothermal amplification and detection on a solid surface. The isothermal RPA was applied to amplify specific target sequences from the pathogens Neisseria gonorrhoeae, Salmonella enterica and methicillin-resistant Staphylococcus aureus (MRSA) using genomic DNA. Additionally, a positive plasmid control was established as an internal control. The four targets were amplified simultaneously in a quadruplex reaction. The amplicon is labeled during on-chip RPA by reverse oligonucleotide primers coupled to a fluorophore. Both amplification and spatially resolved signal generation take place on immobilized forward primers bount to expoxy-silanized glass surfaces in a pump-driven hybridization chamber. The combination of microarray technology and sensitive isothermal nucleic acid amplification at 38 °C allows for a multiparameter analysis on a rather small area. The on-chip RPA was characterized in terms of reaction time, sensitivity and inhibitory conditions. A successful enzymatic reaction is completed in <20 min and results in detection limits of 10 colony-forming units for methicillin-resistant Staphylococcus aureus and Salmonella enterica and 100 colony-forming units for Neisseria gonorrhoeae. The results show this method to be useful with respect to point-of-care testing and to enable simplified and miniaturized nucleic acid-based diagnostics. FigureThe combination of multiplex isothermal nucleic acid amplification with RPA and spatially-resolved signal generation on specific immobilized oligonucleotides. PMID:25253912

  7. Determination of ABO blood group genotypes using the real-time loop-mediated isothermal amplification method

    PubMed Central

    ZHANG, CHAO; ZHU, JUANLI; YANG, JIANGCUN; WAN, YINSHENG; MA, TING; CUI, YALI

    2015-01-01

    ABO genotyping is commonly used in several situations, including blood transfusion, personal identification and disease detection. The present study developed a novel method for ABO genotyping, using loop-mediated isothermal amplification (LAMP). This method allows the simultaneous determination of six ABO genotypes under 40 min at a constant temperature of 62°C. The genotypes of 101 blood samples were determined to be AA (n=6), AO (n=38), BB (n=12), BO (n =29), AB (n=8) and OO (n=8) by the LAMP assay. The results were compared with the phenotypes determined by serological assay and the genotypes determined by direct sequencing, and no discrepancies were observed. This novel and rapid method, with good accuracy and reasonably cost effective, provides a supplement to routine serological ABO typing and may also be useful in other point-of-care testing. PMID:26238310

  8. A Sensitive, Colorimetric, High-Throughput Loop-Mediated Isothermal Amplification Assay for the Detection of Plasmodium knowlesi.

    PubMed

    Britton, Sumudu; Cheng, Qin; Grigg, Matthew J; William, Timothy; Anstey, Nicholas M; McCarthy, James S

    2016-07-01

    The simian parasite Plasmodium knowlesi is now the commonest cause of malaria in Malaysia and can rapidly cause severe and fatal malaria. However, microscopic misdiagnosis of Plasmodium species is common, rapid antigen detection tests remain insufficiently sensitive and confirmation of P. knowlesi requires polymerase chain reaction (PCR). Thus available point-of-care diagnostic tests are inadequate. This study reports the development of a simple, sensitive, colorimetric, high-throughput loop-mediated isothermal amplification assay (HtLAMP) diagnostic test using novel primers for the detection of P. knowlesi. This assay is able to detect 0.2 parasites/μL, and compared with PCR has a sensitivity of 96% for the detection of P. knowlesi, making it a potentially field-applicable point-of-care diagnostic tool. PMID:27162264

  9. A rapid and precise diagnostic method for detecting the Pinewood nematode Bursaphelenchus xylophilus by loop-mediated isothermal amplification.

    PubMed

    Kikuchi, Taisei; Aikawa, Takuya; Oeda, Yuka; Karim, Nurul; Kanzaki, Natsumi

    2009-12-01

    ABSTRACT Bursaphelenchus xylophilus is the causal agent of pine wilt disease, which is a major forest disease in Japan, Korea, China, Taiwan, and Portugal. A diagnostic method which is rapid, precise, and simple could greatly help the proper management of this disease. Here, we present a novel detection method using loop-mediated isothermal amplification (LAMP) targeting the internal transcribed spacer region of ribosomal DNA of the nematode. Specificity of the primers and LAMP was confirmed using DNA from various nematode species related to B. xylophilus. Our experimental results suggest that LAMP can detect B. xylophilus faster and with higher sensitivity than the traditional diagnostic method. Moreover, because it does not require expensive equipment or specialized techniques, this LAMP-based diagnostic method has the potential to be used under field conditions. PMID:19900002

  10. Use of the loop-mediated isothermal amplification method for identification of PCR ribotype 027 Clostridium difficile.

    PubMed

    Kato, Haru; Arakawa, Yoshichika

    2011-08-01

    The loop-mediated isothermal amplification (LAMP) assay detecting the slpA gene of slpA sequence type gc8 (slpA-gc8) was established for the identification of a hypervirulent Clostridium difficile strain, PCR ribotype 027. Of 107 isolates examined, 27 belonging to PCR ribotype 027 were all positive for the LAMP assay. The remaining 80 isolates were typed into 47 different PCR ribotypes other than type 027, and were negative for the LAMP assay with the exception of two isolates. The sensitivity and specificity of the LAMP method for identification of PCR ribotype 027 were 100 % and 98 %, respectively. The LAMP assay detecting slpA-gc8 is a reliable tool for the identification of PCR ribotype 027 C. difficile. This simple and rapid method will contribute to detection of the hypervirulent strain. PMID:21459906

  11. Evaluation of the rapid loop-mediated isothermal amplification assay Illumigene for diagnosis of Clostridium difficile in an outbreak situation.

    PubMed

    Norén, Torbjörn; Unemo, Magnus; Magnusson, Cecilia; Eiserman, Maud; Matussek, Andreas

    2014-02-01

    An outbreak of Clostridium difficile infection (CDI) at Höglandet Hospital Eksjö in southern Sweden in 2011 was mainly due to a multidrug-resistant PCR ribotype 046 (30% of all samples). Diagnostics used routinely was the Vidas CDAB assay, but to control the outbreak the rapid loop-mediated isothermal amplification (LAMP) assay Illumigene was introduced and both techniques were compared to Toxigenic culture (TC) prospectively. The LAMP assay had a superior sensitivity, that is, 98% compared to 79% for the Vidas CDAB assay. Most importantly, the mean turn-around-time from collecting sample to result was reduced from 59 h to 2 h enabling early isolation of patients and effective hygiene precautions. This may potentially decrease the morbidity and nosocomial transmissions of C. difficile. PMID:23758095

  12. Reverse transcription loop-mediated isothermal amplification for the rapid detection of infectious bronchitis virus in infected chicken tissues.

    PubMed

    Chen, Hao-tai; Zhang, Jie; Ma, Yan-ping; Ma, Li-Na; Ding, Yao-zhong; Liu, Xiang-tao; Cai, Xue-peng; Ma, Li-qing; Zhang, Yong-guang; Liu, Yong-sheng

    2010-04-01

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the nucleocapsid phosphoprotein gene of infectious bronchitis virus (IBV) was developed. The detection limits for the IBV RT-LAMP assay were 10(1) 50% egg infection dose (EID(50)) per 50 microl of titrated viruses and no cross-reaction of IBV RT-LAMP was found when tested with other viruses including Newcastle disease virus (NDV), avian reovirus (ARV), and infectious laryngotrachietis virus (ILTV) due to their mismatch with IBV RT-LAMP primers. A total of 187 clinical tissues samples (88 blood, 62 kidney and 37 lung) were evaluated and compared to conventional RT-PCR. The sensitivity of RT-LAMP and RT-PCR assays for detecting IBV RNA in clinical specimens was 99.5% and 98.4%, respectively. These findings showed that the RT-LAMP assay has potential usefulness for rapid and sensitive diagnosis in outbreak of IBV. PMID:19835950

  13. Rapid detection of Prunus necrotic ringspot virus using magnetic nanoparticle-assisted reverse transcription loop-mediated isothermal amplification.

    PubMed

    Zong, Xiaojuan; Wang, Wenwen; Wei, Hairong; Wang, Jiawei; Chen, Xin; Xu, Li; Zhu, Dongzi; Tan, Yue; Liu, Qingzhong

    2014-11-01

    Prunus necrotic ringspot virus (PNRSV) has seriously reduced the yield of Prunus species worldwide. In this study, a highly efficient and specific two-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed to detect PNRSV. Total RNA was extracted from sweet cherry leaf samples using a commercial kit based on a magnetic nanoparticle technique. Transcripts were used as the templates for the assay. The results of this assay can be detected using agarose gel electrophoresis or by assessing in-tube fluorescence after adding SYBR Green I. The assay is highly specific for PNRSV, and it is more sensitive than reverse-transcription polymerase chain reaction (RT-PCR). Restriction enzyme digestion verified further the reliability of this RT-LAMP assay. To our knowledge, this is the first report of the application of RT-LAMP to PNRSV detection in Prunus species. PMID:25110116

  14. Development of a loop-mediated isothermal amplification assay for rapid detection of Trichosporon asahii in experimental and clinical samples.

    PubMed

    Zhou, Jianfeng; Liao, Yong; Li, Haitao; Lu, Xuelian; Han, Xiufeng; Tian, Yanli; Chen, Shanshan; Yang, Rongya

    2015-01-01

    Invasive trichosporonosis is a deep mycosis found mainly in immunocompromised hosts, and the major pathogen is Trichosporon asahii. We detected the species-specific intergenic spacers (IGS) of rRNA gene of T. asahii using a loop-mediated isothermal amplification (LAMP) assay in 15 isolates with 3 different visualization methods, including SYBR green detection, gel electrophoresis, and turbidimetric methods. The LAMP assay displayed superior rapidity to other traditional methods in the detection time; that is, only 1 h was needed for detection and identification of the pathogen DNA. Furthermore, the detection limit of the LAMP assay was more sensitive than the PCR assay. We also successfully detect the presence of T. asahii in samples from experimentally infected mice and samples from patients with invasive trichosporonosis caused by T. asahii, suggesting that this method may become useful in clinical applications in the near future. PMID:25692144

  15. Development of loop-mediated isothermal amplification method for detecting Wuchereria bancrofti DNA in human blood and vector mosquitoes.

    PubMed

    Takagi, Hidekazu; Itoh, Makoto; Kasai, Shinji; Yahathugoda, Thishan C; Weerasooriya, Mirani V; Kimura, Eisaku

    2011-12-01

    We have developed loop-mediated isothermal amplification (LAMP) method to detect Wuchereria bancrofti DNA. The sensitivity and specificity of LAMP method were equivalent to those of PCR method which detects SspI repeat sequence in W. bancrofti genomic DNA: both methods detected one thousandth of W. bancrofti DNA from one microfilaria (Mf), and did not cross-react with DNAs of Brugia malayi, B. pahangi, Dirofilaria immitis, human and Culex quinquefasciatus. We also examined the sensitivity of LAMP using the mimic samples of patient's blood or blood-fed mosquitoes containing one W. bancrofti Mf per sample. The LAMP method was able to detect W. bancrofti DNA in 1000 μl of blood or in a pool of 60 mosquitoes, indicating its usefulness in detecting/monitoring W. bancrofti infection in humans and vector mosquitoes in endemic areas. PMID:21930238

  16. Point-of-care multiplexed assays of nucleic acids using microcapillary-based loop-mediated isothermal amplification.

    PubMed

    Zhang, Yi; Zhang, Lu; Sun, Jiashu; Liu, Yulei; Ma, Xingjie; Cui, Shangjin; Ma, Liying; Xi, Jianzhong Jeff; Jiang, Xingyu

    2014-07-15

    This report demonstrates a straightforward, robust, multiplexed and point-of-care microcapillary-based loop-mediated isothermal amplification (cLAMP) for assaying nucleic acids. This assay integrates capillaries (glass or plastic) to introduce and house sample/reagents, segments of water droplets to prevent contamination, pocket warmers to provide heat, and a hand-held flashlight for a visual readout of the fluorescent signal. The cLAMP system allows the simultaneous detection of two RNA targets of human immunodeficiency virus (HIV) from multiple plasma samples, and achieves a high sensitivity of two copies of standard plasmid. As few nucleic acid detection methods can be wholly independent of external power supply and equipment, our cLAMP holds great promise for point-of-care applications in resource-poor settings. PMID:24937125

  17. Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Trichosporon asahii in Experimental and Clinical Samples

    PubMed Central

    Zhou, Jianfeng; Liao, Yong; Li, Haitao; Lu, Xuelian; Han, Xiufeng; Tian, Yanli; Chen, Shanshan; Yang, Rongya

    2015-01-01

    Invasive trichosporonosis is a deep mycosis found mainly in immunocompromised hosts, and the major pathogen is Trichosporon asahii. We detected the species-specific intergenic spacers (IGS) of rRNA gene of T. asahii using a loop-mediated isothermal amplification (LAMP) assay in 15 isolates with 3 different visualization methods, including SYBR green detection, gel electrophoresis, and turbidimetric methods. The LAMP assay displayed superior rapidity to other traditional methods in the detection time; that is, only 1 h was needed for detection and identification of the pathogen DNA. Furthermore, the detection limit of the LAMP assay was more sensitive than the PCR assay. We also successfully detect the presence of T. asahii in samples from experimentally infected mice and samples from patients with invasive trichosporonosis caused by T. asahii, suggesting that this method may become useful in clinical applications in the near future. PMID:25692144

  18. Rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) by real-time, isothermal recombinase polymerase amplification assay.

    PubMed

    Xia, Xiaoming; Yu, Yongxin; Hu, Linghao; Weidmann, Manfred; Pan, Yingjie; Yan, Shuling; Wang, Yongjie

    2015-04-01

    Infectious hypodermal and hematopoietic necrosis virus (IHHNV) causes mortality or runt deformity syndrome in penaeid shrimps and is responsible for significant economic losses in the shrimp aquaculture industry. Here, we describe a novel real-time isothermal recombinase polymerase amplification (RPA) assay developed for IHHNV detection. Using IHHNV plasmid standards and DNA samples from a variety of organisms, we evaluated the ability of the IHHNV-RPA assay to detect IHHNV based on analysis of its sensitivity, specificity, rapidity, and reproducibility. Probit analysis of eight independent experimental replicates indicated satisfactory performance of the RPA assay, which is sufficiently sensitive to detect as few as 4 copies of the IHHNV genome within 7 min at 39 °C with 95 % reliability. Therefore, this rapid RPA method has great potential for applications, either in field use or as a point of care diagnostic technique. PMID:25655264

  19. Comparison of Conventional PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Arcobacter Species

    PubMed Central

    Wang, Xiaoyu; Seo, Dong Joo; Lee, Min Hwa

    2014-01-01

    This study aimed to develop a loop-mediated isothermal amplification (LAMP) method for the rapid detection of Arcobacter species. Specific primers targeting the 23S ribosomal RNA gene were used to detect Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii. The specificity of the LAMP primer set was assessed using DNA samples from a panel of Arcobacter and Campylobacter species, and the sensitivity was determined using serial dilutions of Arcobacter species cultures. LAMP showed a 10- to 1,000-fold-higher sensitivity than multiplex PCR, with a detection limit of 2 to 20 CFU per reaction in vitro. Whereas multiplex PCR showed cross-reactivity with Campylobacter species, the LAMP method developed in this study was more sensitive and reliable than conventional PCR or multiplex PCR for the detection of Arcobacter species. PMID:24478488

  20. Development of a loop-mediated isothermal amplification method for rapid detection of streptococcal pyrogenic exotoxin B.

    PubMed

    Cao, Cuiming; Zhang, Fang; Ji, Mingyu; Pei, Fengyan; Fan, Xiujie; Shen, Hong; Wang, Qingxi; Yang, Weihua; Wang, Yunshan

    2016-07-01

    We developed a visual loop-mediated isothermal amplification (LAMP) technique to detect the streptococcal pyrogenic exotoxin B (speB) gene. Fifteen strains (from American Type Culture Collection or clinical isolates) were used to determine the specificity and sensitivity of the LAMP assay. Clinical samples were collected from 132 patients with suspected Streptococcus pyogenes (S. pyogenes) infection to verify the feasibility of the LAMP assay for detection of the speB gene. By using a set of five primers (a pair of outer primers, a pair of inner primers and one loop primer) targeting the speB gene, the amplification reaction was rapidly performed in a regular water bath under isothermal conditions at 63 °C for approximately 60 min. Only the two S. pyogenes strains showed positive results which were easily observed with the naked eye, and the other strains showed negative results. The detection limit of the LAMP assay was 0.01 ng/μl of template, showing higher sensitivity than conventional PCR (with a detection limit of 1.0 ng/μl). The detection rate of the speB gene in clinical samples was 71.21% and was consistent with the PCR results. The rapid detection of the speB gene by the LAMP assay is highly specific and sensitive, is simple to perform and cost-effective, and is expected to be a new reliable method for the rapid diagnosis of S. pyogenes infection, that is particularly suitable for rural or community hospitals in developing countries. PMID:27045360

  1. Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of yellow head virus in shrimp.

    PubMed

    Mekata, Tohru; Sudhakaran, Raja; Kono, Tomoya; U-taynapun, Kittichon; Supamattaya, Kidchakan; Suzuki, Yoshihiro; Sakai, Masahiro; Itami, Toshiaki

    2009-12-01

    A real-time reverse transcription loop-mediated isothermal amplification (real-time RT-LAMP) method was applied for detecting the replicase polyprotein-encoding gene of yellow head virus (YHV) in shrimp, Penaeus monodon. It is a novel, gene-specific assay that amplifies nucleic acid with high specificity, sensitivity and rapidity under isothermal conditions using a set of six specially designed primers that recognize eight distinct sequences of the target gene. This method works with even low copies of DNA and is based on magnesium pyrophosphate turbidity detection by an inexpensive photometer for quantitative analysis. A user-friendly protocol was developed with optimal conditions standardized at 63 degrees C for 60 min. With this protocol, the assay sensitivity was 10 times higher than the widely used YHV nested RT-PCR system. Cross-reactivity analysis using other shrimp virus DNA/cDNA and YHV-negative shrimp demonstrated high specificity of the assay. The real-time RT-LAMP method was performed also for an internal control gene, EF-1alpha, to compare with the expressions of the YHV gene in different organs of infected shrimp, and the resulting standard curves showed high correlation coefficient values. These results suggest that this assay is applicable widely as a new quantitative detection method in the pursuit of YHV-free shrimp culture. PMID:19646483

  2. Loop-mediated isothermal amplification: rapid visual and real-time methods for detection of genetically modified crops.

    PubMed

    Randhawa, Gurinder Jit; Singh, Monika; Morisset, Dany; Sood, Payal; Zel, Jana

    2013-11-27

    A rapid, reliable, and sensitive loop-mediated isothermal amplification (LAMP) system was developed for screening of genetically modified organisms (GMOs). The optimized LAMP assays using designed primers target commonly employed promoters, i.e., Cauliflower Mosaic Virus 35S (P-35S) and Figwort Mosaic Virus promoter (P-FMV), and marker genes, i.e., aminoglycoside 3'-adenyltransferase (aadA), neomycin phosphotransferase II (nptII), and β-glucuronidase (uidA). The specificity and performance of the end-point and real-time LAMP assays were confirmed using eight genetically modified (GM) cotton events on four detection systems, employing two chemistries. LAMP assays on the isothermal real-time system were found to be most sensitive, detecting up to four target copies, within 35 min. The LAMP assays herein presented using alternate detection systems can be effectively utilized for rapid and cost-effective screening of the GM status of a sample, irrespective of the crop species or GM trait. These assays coupled with a fast and simple DNA extraction method may further facilitate on-site GMO screening. PMID:24188249

  3. Analysis of a Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) for yellow fever diagnostic.

    PubMed

    Nunes, Marcio R T; Vianez, João Lídio; Nunes, Keley N B; da Silva, Sandro Patroca; Lima, Clayton P S; Guzman, Hilda; Martins, Lívia C; Carvalho, Valéria L; Tesh, Robert B; Vasconcelos, Pedro F C

    2015-12-15

    Yellow Fever virus (YFV) is an important human pathogen in tropical areas of Africa and South America. Although an efficient vaccine is available and has been used since the early 1940s, sylvatic YFV transmission still occurs in forested areas where anthropogenic actions are present, such as mineral extraction, rearing livestock and agriculture, and ecological tourism. In this context, two distinct techniques based on the RT-PCR derived method have been previously developed, however both methods are expensive due to the use of thermo cyclers and labeled probes. We developed isothermal genome amplification, which is a rapid, sensitive, specific and low cost molecular approach for YFV genome detection. This assay used a set of degenerate primers designed for the NS1 gene and was able to amplify, within 30 min in isothermal conditions, the YFV 17D vaccine strain derived from an African wild prototype strain (Asibi), as well as field strains from Brazil, other endemic countries from South and Central America, and the Caribbean. The generic RT-LAMP assay could be helpful for YFV surveillance in field and rapid response during outbreaks in endemic areas. PMID:26459206

  4. Rapid visual detection of phytase gene in genetically modified maize using loop-mediated isothermal amplification method.

    PubMed

    Huang, Xin; Chen, Lili; Xu, Jiangmin; Ji, Hai-Feng; Zhu, Shuifang; Chen, Hongjun

    2014-08-01

    Transgenic maize plant expressing high phytase activity has been reported and approved by Chinese government in 2009. Here, we report a highly specific loop-mediated isothermal amplification (LAMP) method to detect the phytase gene in the GMO maize. The LAMP reaction takes less than 20min and the amplification is visible without gel electrophoresis. The detection sensitivity of the LAMP method is about 30 copies of phytase genomic DNA, which is 33.3 times greater than the conventional PCR method with gel electrophoresis. The quantitative detection results showed that the LAMP method has a good linear correlation between the DNA copy number and the associated Tt values over a large dynamic range of template concentration from 6×10(1) to 6×10(7) copies, with a quantification limit of 60 copies. Therefore, the LAMP method is visual, faster, and more sensitive, and does not need special equipment compared to traditional PCR technique, which is very useful for field tests and fast screening of GMO feeds. PMID:24629956

  5. Visual detection of Ebola virus using reverse transcription loop-mediated isothermal amplification combined with nucleic acid strip detection.

    PubMed

    Xu, Changping; Wang, Hualei; Jin, Hongli; Feng, Na; Zheng, Xuexing; Cao, Zengguo; Li, Ling; Wang, Jianzhong; Yan, Feihu; Wang, Lina; Chi, Hang; Gai, Weiwei; Wang, Chong; Zhao, Yongkun; Feng, Yan; Wang, Tiecheng; Gao, Yuwei; Lu, Yiyu; Yang, Songtao; Xia, Xianzhu

    2016-05-01

    Ebola virus (species Zaire ebolavirus) (EBOV) is highly virulent in humans. The largest recorded outbreak of Ebola hemorrhagic fever in West Africa to date was caused by EBOV. Therefore, it is necessary to develop a detection method for this virus that can be easily distributed and implemented. In the current study, we developed a visual assay that can detect EBOV-associated nucleic acids. This assay combines reverse transcription loop-mediated isothermal amplification and nucleic acid strip detection (RT-LAMP-NAD). Nucleic acid amplification can be achieved in a one-step process at a constant temperature (58 °C, 35 min), and the amplified products can be visualized within 2-5 min using a nucleic acid strip detection device. The assay is capable of detecting 30 copies of artificial EBOV glycoprotein (GP) RNA and RNA encoding EBOV GP from 10(2) TCID50 recombinant viral particles per ml with high specificity. Overall, the RT-LAMP-NAD method is simple and has high sensitivity and specificity; therefore, it is especially suitable for the rapid detection of EBOV in African regions. PMID:26831931

  6. Development and evaluation of a novel and rapid detection assay for Botrytis cinerea based on loop-mediated isothermal amplification.

    PubMed

    Duan, Ya-Bing; Ge, Chang-Yan; Zhang, Xiao-Ke; Wang, Jian-Xin; Zhou, Ming-Guo

    2014-01-01

    Botrytis cinerea is a devastating plant pathogen that causes grey mould disease. In this study, we developed a visual detection method of B. cinerea based on the Bcos5 sequence using loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue dye (HNB). The LAMP reaction was optimal at 63 °C for 45 min. When HNB was added prior to amplification, samples with B. cinerea DNA developed a characteristic sky blue color after the reaction but those without DNA or with DNA of other plant pathogenic fungi did not. Results of HNB staining method were reconfirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for B. cinerea was 10(-3) ng µL(-1) of genomic DNA per reaction, which was 10-fold more sensitive than conventional PCR (10(-2) ng µL(-1)). Detection of the LAMP assay for inoculum of B. cinerea was possible in the inoculated tomato and strawberry petals. In the 191 diseased samples, 180 (94.2%) were confirmed as positive by LAMP, 172 (90.1%) positive by the tissue separation, while 147 (77.0%) positive by PCR. Because the LAMP assay performed well in aspects of sensitivity, specificity, repeatability, reliability, and visibility, it is suitable for rapid detection of B. cinerea in infected plant materials prior to storage and during transportation, such as cut flowers, fruits and vegetables. PMID:25329402

  7. A Loop-Mediated Isothermal Amplification Assay and Sample Preparation Procedure for Sensitive Detection of Xanthomonas fragariae in Strawberry

    PubMed Central

    Wang, Hehe; Turechek, William W.

    2016-01-01

    Xanthomonas fragariae is a bacterium that causes angular leaf spot of strawberry. Asymptomatic infection is common and contributes to the difficulties in disease management. The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay as an efficient method for detection of asymptomatic infections of X. fragariae. In addition, a new method of sample preparation was developed that allows sampling of a larger amount of plant tissue, hence increasing the detection rate in real-life samples. The sample preparation procedure includes an overnight incubation of strawberry tissues in phosphate-buffered saline (PBS), followed by a quick sample concentration and a boiling step to extract DNA for amplification. The detection limit of the LAMP assay was approximately 2×103 CFU/mL for pure bacteria culture and 300 CFU/mL for bacteria spiked strawberry leaf and petiole samples. LAMP provided a 2–3 fold lower detection limit than the standard qPCR assay but was faster, and more user-friendly. The LAMP assay should serve as a rapid, sensitive and cost-effective tool for detecting asymptomatic infections of X. fragariae in strawberry nursery stock and contribute to improved disease management. PMID:26766068

  8. Development of a rapid assay to detect the jellyfish Cyanea nozakii using a loop-mediated isothermal amplification method.

    PubMed

    Liu, Zhongyuan; Dong, Zhijun; Liu, Dongyan

    2016-07-01

    Blooms of the harmful jellyfish Cyanea nozakii, which are a severe nuisance to fisheries and tourisms, frequently occur in the northern East China Sea, Yellow Sea, and Bohai Sea. To provide early warning of this species, a simple and effective molecular method for identifying C. nozakii was developed using the loop-mediated isothermal amplification method (LAMP). The LAMP assay is highly specific and uses a set of four primers that target six different regions on the mitochondrial cytochrome c oxidase subunit I (COI) gene of C. nozakii. The amplification conditions, including the dNTP and betaine concentrations, the inner primer to outer primer concentration ratio, reaction time and temperature, were optimized. The LAMP assay amplified DNA extracted from tissue samples of C. nozakii but did not amplify DNA from other common scyphozoans and hydrozoans collected in the same region. In addition, the LAMP assay was more sensitive than conventional PCR. Therefore, the established LAMP assay is a sensitive, specific, fast, and easily performed method for detection of C. nozakii at different stages in their life cycle. PMID:25774948

  9. Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection.

    PubMed

    Nzelu, Chukwunonso O; Gomez, Eduardo A; Cáceres, Abraham G; Sakurai, Tatsuya; Martini-Robles, Luiggi; Uezato, Hiroshi; Mimori, Tatsuyuki; Katakura, Ken; Hashiguchi, Yoshihisa; Kato, Hirotomo

    2014-04-01

    Entomological monitoring of Leishmania infection in leishmaniasis endemic areas offers epidemiologic advantages for predicting the risk and expansion of the disease, as well as evaluation of the effectiveness of control programs. In this study, we developed a highly sensitive loop-mediated isothermal amplification (LAMP) method for the mass screening of sand flies for Leishmania infection based on the 18S rRNA gene. The LAMP technique could detect 0.01 parasites, which was more sensitive than classical PCR. The method was robust and could amplify the target DNA within 1h from a crude sand fly template without DNA purification. Amplicon detection could be accomplished by the newly developed colorimetric malachite green (MG)--mediated naked eye visualization. Pre-addition of MG to the LAMP reaction solution did not inhibit amplification efficiency. The field applicability of the colorimetric MG-based LAMP assay was demonstrated with 397 field-caught samples from the endemic areas of Ecuador and eight positive sand flies were detected. The robustness, superior sensitivity, and ability to produce better visual discriminatory reaction products than existing LAMP fluorescence and turbidity assays indicated the field potential usefulness of this new method for surveillance and epidemiological studies of leishmaniasis in developing countries. PMID:24388795

  10. Loop-Mediated Isothermal Amplification Procedure (LAMP) for Detection of the Potato Zebra Chip Pathogen "Candidatus Liberibacter solanacearum".

    PubMed

    Ravindran, Aravind; Lévy, Julien; Pierson, Elizabeth; Gross, Dennis C

    2015-01-01

    An efficient loop-mediated isothermal amplification procedure (LAMP) for the detection of "Candidatus Liberibacter solanacearum" (Lso), the bacterial causal agent of potato zebra chip (ZC) disease, is described in this chapter. Similar to the polymerase chain reaction (PCR), the LAMP employs a bacterial polymerase to amplify specific DNA sequences. However, the method differs from conventional PCR in that it uses six primers specific to the target region to generate a loop structure and autocycling strand displacement rather than thermocycling for sequence amplification. Moreover, unlike PCR that requires agarose gel electrophoresis for resolution, the positive LAMP results can be visualized directly as a precipitate within the reaction tubes. The 16S rDNA gene of "Ca. Liberibacter solanacearum" was used as the target for the design of the six LAMP primers. The LAMP technique is a reliable, rapid, and cost-effective method of detecting the "Ca. Liberibacter solanacearum" pathogen in the potato/tomato psyllid, Bactericera cockerelli, and in field-grown potato plants and tubers. PMID:25981248

  11. Development of a loop-mediated isothermal amplification assay for the detection of Streptococcus agalactiae in bovine milk.

    PubMed

    Bosward, Katrina L; House, John K; Deveridge, Amber; Mathews, Karen; Sheehy, Paul A

    2016-03-01

    Streptococcus agalactiae is a well-characterized bovine mastitis pathogen that is known to be highly contagious and capable of spreading rapidly in affected dairy herds. Loop-mediated isothermal amplification (LAMP) is a novel molecular diagnostic method that has the capability to provide rapid, cost-effective screening for pathogens to support on-farm disease control and eradication programs. In the current study, a LAMP test was developed to detect S. agalactiae in milk. The assay was validated on a bank of existing clinical mastitis milk samples that had previously been identified as S. agalactiae positive via traditional microbiological culture techniques and PCR. The LAMP assay was conducted on bacterial colonies and DNA extracted from milk in tube- and plate-based formats using multiple detection platforms. The 1-h assay conducted at 64 °C exhibited repeatability (coefficient of variation) of 2.07% (tube) and 8.3% (plate), sensitivity to ~20 pg of extracted DNA/reaction, and specificity against a panel of known bacterial mastitis pathogens. Of the 109 known S. agalactiae isolates assessed by LAMP directly from bacterial cells in culture, 108 were identified as positive, in accordance with PCR analysis. The LAMP analysis from the corresponding milk samples indicated that 104 of these milks exhibited a positive amplification curve. Although exhibiting some limitations, this assay provides an opportunity for rapid screening of milk samples to facilitate on-farm management of this pathogen. PMID:26778303

  12. Optimal terminal sequences for continuous or serial isothermal amplification of dsRNA with norovirus RNA replicase.

    PubMed

    Arai, Hidenao; Nishigaki, Koichi; Nemoto, Naoto; Suzuki, Miho; Husimi, Yuzuru

    2014-01-01

    The norovirus RNA replicase (NV3D(pol), 56 kDa, single chain monomeric protein) can amplify double-stranded (ds) RNA isothermally. It will play an alternative role in the in vitro evolution against traditional Qβ RNA replicase, which cannot amplify dsRNA and consists of four subunits, three of which are borrowed from host E.coli. In order to identify the optimal 3'-terminal sequence of the RNA template for NV3D(pol), an in vitro selection using the serial transfer was performed for a random library having the 3'-terminal sequence of ---UUUUUUNNNN-3'. The population landscape on the 4-dimensional sequence space of the 17(th) round of transfer gave a main peak around ---CAAC-3'. In the preceding studies on the batch amplification reaction starting from a single-stranded RNA, a template with 3'-terminal C-stretch was amplified effectively. It was confirmed that in the batch amplification the ---CCC-3' was much more effective than the ---CAAC-3', but in the serial transfer condition in which the ----CAAC-3' was sustained stably, the ---CCC-3' was washed out. Based on these results we proposed the existence of the "shuttle mode" replication of dsRNA. We also proposed the optimal terminal sequences of RNA for in vitro evolution with NV3D(pol). PMID:27493494

  13. Optimization of loop-mediated isothermal amplification (LAMP) assays for the detection of Leishmania DNA in human blood samples.

    PubMed

    Abbasi, Ibrahim; Kirstein, Oscar D; Hailu, Asrat; Warburg, Alon

    2016-10-01

    Visceral leishmaniasis (VL), one of the most important neglected tropical diseases, is caused by Leishmania donovani eukaryotic protozoan parasite of the genus Leishmania, the disease is prevalent mainly in the Indian sub-continent, East Africa and Brazil. VL can be diagnosed by PCR amplifying ITS1 and/or kDNA genes. The current study involved the optimization of Loop-mediated isothermal amplification (LAMP) for the detection of Leishmania DNA in human blood or tissue samples. Three LAMP systems were developed; in two of those the primers were designed based on shared regions of the ITS1 gene among different Leishmania species, while the primers for the third LAMP system were derived from a newly identified repeated region in the Leishmania genome. The LAMP tests were shown to be sufficiently sensitive to detect 0.1pg of DNA from most Leishmania species. The green nucleic acid stain SYTO16, was used here for the first time to allow real-time monitoring of LAMP amplification. The advantage of real time-LAMP using SYTO 16 over end-point LAMP product detection is discussed. The efficacy of the real time-LAMP tests for detecting Leishmania DNA in dried blood samples from volunteers living in endemic areas, was compared with that of qRT-kDNA PCR. PMID:27288706

  14. Development and Evaluation of a Novel and Rapid Detection Assay for Botrytis cinerea Based on Loop-Mediated Isothermal Amplification

    PubMed Central

    Duan, Ya-Bing; Ge, Chang-Yan; Zhang, Xiao-Ke; Wang, Jian-Xin; Zhou, Ming-Guo

    2014-01-01

    Botrytis cinerea is a devastating plant pathogen that causes grey mould disease. In this study, we developed a visual detection method of B. cinerea based on the Bcos5 sequence using loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue dye (HNB). The LAMP reaction was optimal at 63°C for 45 min. When HNB was added prior to amplification, samples with B. cinerea DNA developed a characteristic sky blue color after the reaction but those without DNA or with DNA of other plant pathogenic fungi did not. Results of HNB staining method were reconfirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for B. cinerea was 10−3 ng µL−1 of genomic DNA per reaction, which was 10-fold more sensitive than conventional PCR (10−2 ng µL−1). Detection of the LAMP assay for inoculum of B. cinerea was possible in the inoculated tomato and strawberry petals. In the 191 diseased samples, 180 (94.2%) were confirmed as positive by LAMP, 172 (90.1%) positive by the tissue separation, while 147 (77.0%) positive by PCR. Because the LAMP assay performed well in aspects of sensitivity, specificity, repeatability, reliability, and visibility, it is suitable for rapid detection of B. cinerea in infected plant materials prior to storage and during transportation, such as cut flowers, fruits and vegetables. PMID:25329402

  15. Operational Feasibility of Using Loop-Mediated Isothermal Amplification for Diagnosis of Pulmonary Tuberculosis in Microscopy Centers of Developing Countries▿

    PubMed Central

    Boehme, Catharina C.; Nabeta, Pamela; Henostroza, German; Raqib, Rubhana; Rahim, Zeaur; Gerhardt, Martina; Sanga, Erica; Hoelscher, Michael; Notomi, Tsugunori; Hase, Tetsu; Perkins, Mark D.

    2007-01-01

    The characteristics of loop-mediated isothermal amplification (LAMP) make it a promising platform for the molecular detection of tuberculosis (TB) in developing countries. Here, we report on the first clinical evaluation of LAMP for the detection of pulmonary TB in microscopy centers in Peru, Bangladesh, and Tanzania to determine its operational applicability in such settings. A prototype LAMP assay with simplified manual DNA extraction was evaluated for accuracy and ease of use. The sensitivity of LAMP in smear- and culture-positive sputum specimens was 97.7% (173/177 specimens; 95% confidence interval [CI], 95.5 to 99.9%), and the sensitivity in smear-negative, culture-positive specimens was 48.8% (21/43 specimens; CI, 33.9 to 63.7%). The specificity in culture-negative samples was 99% (500/505 specimens; CI, 98.1 to 99.9%). The average hands-on time for testing six samples and two controls was 54 min, similar to that of sputum smear microscopy. The optimal amplification time was 40 min. No indeterminate results were reported, and the interreader variability was 0.4%. Despite the use of a single room without biosafety cabinets for all procedures, no DNA contamination was observed. The assay was robust, with high end-point stability and low rates of test failure. Technicians with no prior molecular experience easily performed the assay after 1 week of training, and opportunities for further simplification of the assay were identified. PMID:17392443

  16. Optimal terminal sequences for continuous or serial isothermal amplification of dsRNA with norovirus RNA replicase

    PubMed Central

    Arai, Hidenao; Nishigaki, Koichi; Nemoto, Naoto; Suzuki, Miho; Husimi, Yuzuru

    2014-01-01

    The norovirus RNA replicase (NV3Dpol, 56 kDa, single chain monomeric protein) can amplify double-stranded (ds) RNA isothermally. It will play an alternative role in the in vitro evolution against traditional Qβ RNA replicase, which cannot amplify dsRNA and consists of four subunits, three of which are borrowed from host E.coli. In order to identify the optimal 3′-terminal sequence of the RNA template for NV3Dpol, an in vitro selection using the serial transfer was performed for a random library having the 3′-terminal sequence of ---UUUUUUNNNN-3′. The population landscape on the 4-dimensional sequence space of the 17th round of transfer gave a main peak around ---CAAC-3′. In the preceding studies on the batch amplification reaction starting from a single-stranded RNA, a template with 3′-terminal C-stretch was amplified effectively. It was confirmed that in the batch amplification the ---CCC-3′ was much more effective than the ---CAAC-3′, but in the serial transfer condition in which the ----CAAC-3′ was sustained stably, the ---CCC-3′ was washed out. Based on these results we proposed the existence of the “shuttle mode” replication of dsRNA. We also proposed the optimal terminal sequences of RNA for in vitro evolution with NV3Dpol. PMID:27493494

  17. Shewanella putrefaciens in cultured tilapia detected by a new calcein-loop-mediated isothermal amplification (Ca-LAMP) method.

    PubMed

    Suebsing, Rungkarn; Kampeera, Jantana; Sirithammajak, Sarawut; Pradeep, Padmaja Jayaprasad; Jitrakorn, Sarocha; Arunrut, Narong; Sangsuriya, Pakkakul; Saksmerprome, Vanvimon; Senapin, Saengchan; Withyachumnarnkul, Boonsirm; Kiatpathomchai, Wansika

    2015-12-01

    Shewanella putrefaciens is being increasingly isolated from a wide variety of sources and is pathogenic to many marine and freshwater fish. For better control of this pathogen, there is a need for the development of simple and inexpensive but highly specific, sensitive, and rapid detection methods suitable for application in field laboratories. Our colorogenic loop-mediated isothermal amplification (LAMP) assay combined with calcein (Ca-LAMP) for unaided visual confirmation of LAMP amplicons is a simple method for fish pathogen detection in cultured tilapia. Here, we describe the detection of S. putrefaciens using the same platform. As before, the method gave positive results (orange to green color change) in 45 min at 63°C with sensitivity 100 times higher than that of a conventional PCR assay, with no cross-amplification of other known fish bacterial pathogens tested. Using the assay with 389 samples of gonads, fertilized eggs, and fry of farmed Nile and red tilapia Oreochromis spp., 35% of samples were positive for S. putrefaciens. The highest prevalence was found in samples of gonads (55%) and fertilized eggs (55%) from adult breeding stocks, indicating that S. putrefaciens could be passed on easily to fry used for stocking production ponds. Tissue tropism assays revealed that the spleen showed the highest colonization by S. putrefaciens in naturally infected tilapia and that it would be the most suitable organ for screening and monitoring fish stocks for presence of the bacteria. PMID:26648105

  18. Evaluation of Loop-Mediated Isothermal Amplification Assay for the Detection of Pneumocystis jirovecii in Immunocompromised Patients

    PubMed Central

    Singh, Preeti; Singh, Sundeep; Mirdha, Bijay Ranjan; Guleria, Randeep; Agarwal, Sanjay Kumar; Mohan, Anant

    2015-01-01

    Pneumocystis pneumonia (PCP) is one of the common opportunistic infection among HIV and non-HIV immunocompromised patients. The lack of a rapid and specific diagnostic test necessitates a more reliable laboratory diagnostic test for PCP. In the present study, the loop-mediated isothermal amplification (LAMP) assay was evaluated for the detection of Pneumocystis jirovecii. 185 clinical respiratory samples, including both BALF and IS, were subjected to GMS staining, nested PCR, and LAMP assay. Of 185 respiratory samples, 12/185 (6.5%), 41/185 (22.2%), and 49/185 (26.5%) samples were positive by GMS staining, nested PCR, and LAMP assay, respectively. As compared to nested PCR, additional 8 samples were positive by LAMP assay and found to be statistically significant (p < 0.05) with the detection limit of 1 pg. Thus, the LAMP assay may serve as a better diagnostic tool for the detection of P. jirovecii with high sensitivity and specificity, less turn-around time, operational simplicity, single-step amplification, and immediate visual detection. PMID:26664746

  19. Real-time isothermal RNA amplification of toxic marine microalgae using preserved reagents on an integrated microfluidic platform.

    PubMed

    Tsaloglou, Maria-Nefeli; Laouenan, Florian; Loukas, Christos-Moritz; Monsalve, Lisandro Gabriel; Thanner, Christine; Morgan, Hywel; Ruano-López, Jesus M; Mowlem, Matthew C

    2013-01-21

    Quantitation of specific RNA sequences is a useful technique in marine biology that can elucidate cell abundance, speciation and viability, especially for early detection of harmful algal blooms. We are thus developing an integrated microfluidic system for cell concentration and lysis, RNA extraction/purification and quantitative RNA detection for environmental applications. The portable system is based on a microfluidic cartridge, or "lab-card", using a low-cost injection moulded device, with a laminated lid. Here we present real-time isothermal RNA amplification using reagent master-mixes preserved on-chip in a gel at 4 °C for up to eight months. We demonstrate quantitation by reference to an internal control in a competitive assay with 500 cell equivalents of the toxic microalga Karenia brevis. Annealing of primers, amplification at 41 °C and real-time fluorescence detection of the internal control and target using sequence-specific molecular beacons were all performed on-chip. PMID:23171958

  20. Detection of shrimp Taura syndrome virus by loop-mediated isothermal amplification using a designed portable multi-channel turbidimeter.

    PubMed

    Sappat, Assawapong; Jaroenram, Wansadaj; Puthawibool, Teeranart; Lomas, Tanom; Tuantranont, Adisorn; Kiatpathomchai, Wansika

    2011-08-01

    In this study, a portable turbidimetric end-point detection method was devised and tested for the detection of Taura syndrome virus (TSV) using spectroscopic measurement of a loop-mediated isothermal amplification (LAMP) by-product: magnesium pyrophosphate (Mg(2)P(2)O(7)). The device incorporated a heating block that maintained an optimal temperature of 63°C for the duration of the RT-LAMP reaction. Turbidity of the RT-LAMP by-product was measured when light from a light-emitting diode (LED) passed through the tube to reach a light dependent resistance (LDR) detector. Results revealed that turbidity measurement of the RT-LAMP reactions using this device provided the same detection sensitivity as the agarose gel electrophoresis detection of RT-LAMP and nested RT-PCR (IQ2000™) products. Cross reactions with other shrimp viruses were not found, indicating that the RT-LAMP-turbidity measurement was highly specific to TSV. The combination of 10 min for rapid RNA preparation with 30 min for RT-LAMP amplification followed by turbidity measurement resulted in a total assay time of less than 1h compared to 4-8h for the nested RT-PCR method. RT-LAMP plus turbidity measurement constitutes a platform for the development of more rapid and user-friendly detection of TSV in the field. PMID:21619895

  1. [Development of rapid detection of infectious hypodermal and hematopoietic necrosis virus by loop-mediated isothermal amplification].

    PubMed

    He, Lin; Xu, Hai-Sheng; Wang, Mei-Zhen; Rong, Hua-Nan

    2010-11-01

    Loop-mediated isothermal amplification (LAMP) assay is a novel method of gene amplification with high specificity, sensitivity and rapidity, which can be applied for disease diagnosis in shrimp aquaculture. The method is performed under isothermal conditions with a set of four specially designed primers that recognize six distinct sequences of the target. In the present study, according to the conservative regions of non-structural protein gene NS1, a set of four specific primers were designed, and a rapid detection of IHHNV was established by LAMP assay. The parameters of reaction time and temperature were optimized, and its specificity and sensitivity were assessed. The reactions were carried out at 60 degrees C, 62 degrees C, 63 degrees C, 64 degrees C, 65 degrees C, 66 degrees C, 67 degrees C, 68 degrees C for different time (0 min; 15 min; 30 min; 45 min; 60 min; 75 min). A plasmid pMDIHHNV carrying target sequence of LAMP detection was constructed. Ten-fold serially diluted pMDIHHNV (10(7)-10(0)copies/microL) was used as template for LAMP assay to investigate the detection limit. To determine the specificity, LAMP assays were carried out with DNA templates from other pathogens (White spot syndrome virus; WSSV, Taura Syndrome Virus; TSV, Aeromonas. hydrophila, V. alginolyticus, Vibrio. parahaemolytious, Escherichia. coli). The results showed the optimized LAMP assay for the rapid detection of IHHNV was performed at 65 degrees C for 60 min. The LAMP assay had an unequivocal detection limit of 100 copies/microL, and it was 1,000 times lower than that of PCR. The nucleic acids of other pathogens were not amplified by this LAMP system with the specific primers, which showed a good specificity. The resulting amplicons were detected using visual observation after the addition of SYBR Green I and gel electrophoresis. We investigated the efficacy of UNG (uracil-N-glycosylase) and dUTP in avoiding carry-over contamination in the LAMP assay procedure and explored its

  2. Development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of Sugarcane mosaic virus and Sorghum mosaic virus in sugarcane

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV) in sugarcane. Six sets of four primers corresponding to the conserved coat protein gene were designed for each virus and their succ...

  3. Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for Clostridium difficile Detection Challenges Cytotoxin B Cell Test and Culture as Gold Standard▿

    PubMed Central

    Norén, Torbjörn; Alriksson, Ingegärd; Andersson, Josefin; Åkerlund, Thomas; Unemo, Magnus

    2011-01-01

    Compared to the composite gold standard cytotoxin B assay and toxigenic culture, the loop-mediated isothermal amplification (LAMP) test for Clostridium difficile had a sensitivity and specificity of 98%, positive predictive value of 92%, and negative predictive value of >99%. A one-hour turnaround time for the LAMP test provides rapid diagnosis and cost savings. PMID:21106782

  4. Rapid and sensitive loop-mediated isothermal amplification test for Clostridium difficile detection challenges cytotoxin B cell test and culture as gold standard.

    PubMed

    Norén, Torbjörn; Alriksson, Ingegärd; Andersson, Josefin; Akerlund, Thomas; Unemo, Magnus

    2011-02-01

    Compared to the composite gold standard cytotoxin B assay and toxigenic culture, the loop-mediated isothermal amplification (LAMP) test for Clostridium difficile had a sensitivity and specificity of 98%, positive predictive value of 92%, and negative predictive value of >99%. A one-hour turnaround time for the LAMP test provides rapid diagnosis and cost savings. PMID:21106782

  5. Development of reverse transcription loop-mediated isothermal amplification assay for rapid detection of an emerging potyvirus: tomato necrotic stunt virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tomato necrotic stunt virus (ToNStV) is an emerging potyvirus that causes severe stunting to the infected tomato plants. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for a sensitive detection of ToNStV. The sensitivity of RT-LAMP was comparable to th...

  6. Development of a quantitative loop-mediated isothermal amplification (qLAMP) assay for the detection of Magnaporthe oryzae airborne inoculum in turf ecosystems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Grey Leaf Spot (GLS) is a detrimental disease of perennial ryegrass caused by a host-specialized form of Magnaporthe oryzae (Mot). In order to improve turf management, a quantitative loop-mediated isothermal amplification (LAMP) assay coupled with a simple spore trap is being developed to monitor GL...

  7. Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39 °C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in ...

  8. Protein detection through different platforms of immuno-loop-mediated isothermal amplification

    PubMed Central

    2013-01-01

    Different immunoassay-based methods have been devised to detect protein targets. These methods have some challenges that make them inefficient for assaying ultra-low-amounted proteins. ELISA, iPCR, iRCA, and iNASBA are the common immunoassay-based methods of protein detection, each of which has specific and common technical challenges making it necessary to introduce a novel method in order to avoid their problems for detection of target proteins. Here we propose a new method nominated as ‘immuno-loop-mediated isothermal amplification’ or ‘iLAMP’. This new method is free from the problems of the previous methods and has significant advantages over them. In this paper we also offer various configurations in order to improve the applicability of this method in real-world sample analyses. Important potential applications of this method are stated as well. PMID:24237767

  9. Tip-enhanced fluorescence with radially polarized illumination for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA

    NASA Astrophysics Data System (ADS)

    Wei, Shih-Chung; Chuang, Tsung-Liang; Wang, Da-Shin; Lu, Hui-Hsin; Gu, Frank X.; Sung, Kung-Bin; Lin, Chii-Wann

    2015-02-01

    A tip nanobiosensor for monitoring DNA replication was presented. The effects of excitation power and polarization on tip-enhanced fluorescence (TEF) were assessed with the tip immersed in fluorescein isothiocyanate solution first. The photon count rose on average fivefold with radially polarized illumination at 50 mW. We then used polymerase-functionalized tips for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA. The amplicon-SYBR Green I complex was detected and compared to real-time loop-mediated isothermal amplification. The signals of the reaction using 4 and 0.004 ng/μl templates were detected 10 and 30 min earlier, respectively. The results showed the potential of TEF in developing a nanobiosensor for real-time DNA amplification.

  10. Successful diagnosis of tuberculous lymphadenitis by loop-mediated isothermal amplification of cutaneous samples from an ulcerated surface lesion: a case report

    PubMed Central

    2014-01-01

    Introduction Tuberculous lymphadenitis is the most frequent form of extrapulmonary tuberculous. Although nucleic acid amplification assays such as polymerase chain reaction have recently become mainstream techniques for diagnosing tuberculous lymphadenitis, they are still not routinely performed in developing countries because of their high costs and complicated procedures. Case presentation We describe a case of tuberculous lymphadenitis in a 79-year-old Japanese man who had been on continuous hemodialysis for end-stage renal disease. We employed loop-mediated isothermal amplification and the procedure for ultrarapid extraction to develop a fast and easy-to-perform procedure for diagnosing tuberculous lymphadenitis. Conclusions The commercially available loop-mediated isothermal amplification assay kit and a rapid purification procedure enabled us to identify and amplify a Mycobacterium tuberculosis–specific gene within just 1.5 hours. PMID:25030753

  11. Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay.

    PubMed

    Yao, Xiefeng; Li, Pingfang; Xu, Jinghua; Zhang, Man; Ren, Runsheng; Liu, Guang; Yang, Xingping

    2016-01-01

    Didymella bryoniae is a pathogenic fungus that causes gummy stem blight (GSB) in Cucurbitaceae crops (e.g., cantaloupe, muskmelon, cucumber, and watermelon). GSB produces lesions on the stems and leaves, and can also be spread by seeds. Here, we developed a rapid, visual, and sensitive loop-mediated amplification (LAMP) assay for D. bryoniae detection based on sequence-characterized amplified regions (GenBank accession nos GQ872461 and GQ872462) common to the two random amplification of polymorphic DNA group genotypes (RGI and RGII) of D. bryoniae; ideal conditions for detection were optimized for completion in 45 min at 63°C. The sensitivity and specificity of the LAMP assay were further analyzed in comparison with those of a conventional polymerase chain reaction (PCR). The sensitivity of the LAMP assay was 1000-fold higher than that of conventional PCR with a detection limit of 0.1 fg μL(-1) of targeted DNA. The LAMP assay could be accomplished in about 45 min, with the results visible to the naked eye. The assay showed high specificity in discriminating all D. bryoniae isolates from seven other fungal pathogens that occur in Cucurbitaceae crops. The LAMP assay also detected D. bryoniae infection in young muskmelon leaves with suspected early symptoms of GSB disease. Hence, the technique has great potential for developing rapid and sensitive visual detection methods for the D. bryoniae pathogen in crops and seeds. This method has potential application in early prediction of disease and reducing the risk of epidemics. PMID:27625648

  12. A Simple and Rapid Identification Method for Mycobacterium bovis BCG with Loop-Mediated Isothermal Amplification

    PubMed Central

    Kouzaki, Yuji; Maeda, Takuya; Sasaki, Hiroaki; Tamura, Shinsuke; Hamamoto, Takaaki; Yuki, Atsushi; Sato, Akinori; Miyahira, Yasushi; Kawana, Akihiko

    2015-01-01

    Bacillus Calmette-Guérin (BCG) is widely used as a live attenuated vaccine against Mycobacterium tuberculosis and is an agent for standard prophylaxis against the recurrence of bladder cancer. Unfortunately, it can cause severe infectious diseases, especially in immunocompromised patients, and the ability to immediately distinguish BCG from other M. tuberculosis complexes is therefore important. In this study, we developed a simple and easy-to-perform identification procedure using loop-mediated amplification (LAMP) to detect deletions within the region of difference, which is deleted specifically in all M. bovis BCG strains. Reactions were performed at 64°C for 30 min and successful targeted gene amplifications were detected by real-time turbidity using a turbidimeter and visual inspection of color change. The assay had an equivalent detection limit of 1.0 pg of genomic DNA using a turbidimeter whereas it was 10 pg with visual inspection, and it showed specificity against 49 strains of 44 pathogens, including M. tuberculosis complex. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. We employed the Procedure for Ultra Rapid Extraction (PURE) kit to isolate mycobacterial DNA and found that the highest sensitivity limit with a minimum total cell count of mycobacterium (including DNA purification with PURE) was up to 1 × 103 cells/reaction, based on color changes under natural light with FDA reagents. The detection limit of this procedure when applied to artificial serum, urine, cerebrospinal fluid, and bronchoalveolar lavage fluid samples was also about 1 × 103 cells/reaction. Therefore, this substitute method using conventional culture or clinical specimens followed by LAMP combined with PURE could be a powerful tool to enable the rapid identification of M. bovis BCG as point-of-care testing. It is suitable for practical use not only in resource-limited situations, but also in any clinical situation

  13. Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay

    PubMed Central

    Yao, Xiefeng; Li, Pingfang; Xu, Jinghua; Zhang, Man; Ren, Runsheng; Liu, Guang; Yang, Xingping

    2016-01-01

    Didymella bryoniae is a pathogenic fungus that causes gummy stem blight (GSB) in Cucurbitaceae crops (e.g., cantaloupe, muskmelon, cucumber, and watermelon). GSB produces lesions on the stems and leaves, and can also be spread by seeds. Here, we developed a rapid, visual, and sensitive loop-mediated amplification (LAMP) assay for D. bryoniae detection based on sequence-characterized amplified regions (GenBank accession nos GQ872461 and GQ872462) common to the two random amplification of polymorphic DNA group genotypes (RGI and RGII) of D. bryoniae; ideal conditions for detection were optimized for completion in 45 min at 63°C. The sensitivity and specificity of the LAMP assay were further analyzed in comparison with those of a conventional polymerase chain reaction (PCR). The sensitivity of the LAMP assay was 1000-fold higher than that of conventional PCR with a detection limit of 0.1 fg μL-1 of targeted DNA. The LAMP assay could be accomplished in about 45 min, with the results visible to the naked eye. The assay showed high specificity in discriminating all D. bryoniae isolates from seven other fungal pathogens that occur in Cucurbitaceae crops. The LAMP assay also detected D. bryoniae infection in young muskmelon leaves with suspected early symptoms of GSB disease. Hence, the technique has great potential for developing rapid and sensitive visual detection methods for the D. bryoniae pathogen in crops and seeds. This method has potential application in early prediction of disease and reducing the risk of epidemics. PMID:27625648

  14. Real-time, label-free isothermal solid-phase amplification/detection (ISAD) device for rapid detection of genetic alteration in cancers.

    PubMed

    Shin, Yong; Perera, Agampodi Promoda; Kim, Kyung Woo; Park, Mi Kyoung

    2013-06-01

    Here, we first present an isothermal solid-phase amplification/detection (ISAD) technique for the detection of single-point mutations that can be performed without labelling in real-time by utilizing both silicon microring-based solid-phase amplification and isothermal recombinase polymerase amplification (RPA). The ISAD technique was performed on a silicon microring device with a plastic chamber containing 10 μL of the reaction mixture, and characterized with an assay for the detection of the HRAS (Harvey RAS) gene single-point mutation. For the solid-phase amplification, the primer of the gene was directly attached to the surface of the device via an amine modification reaction. The amplified DNA was detected, without a label, by measuring the optical wavelength shift of the silicon microring resonator during the reaction. We demonstrated that the sensitivity of the ISAD technique was 100-times higher than that of RPA and conventional PCR methods. Moreover, this technique can be used to distinguish a single-point mutation of the HRAS gene via target amplification. This novel DNA amplification/detection technique will be useful for the detection of sequence alterations such as mutations and single-nucleotide polymorphisms as DNA biomarkers in human diseases. PMID:23609609

  15. Sensitive and rapid detection of genetic modified soybean (Roundup Ready) by loop-mediated isothermal amplification.

    PubMed

    Liu, Mei; Luo, Yan; Tao, Ran; He, Ru; Jiang, Keyong; Wang, Baojie; Wang, Lei

    2009-11-01

    Using the LAMP method, a highly specific and sensitive detection system for genetically modified soybean (Roundup Ready) was designed. In this detection system, a set of four primers was designed by targeting the exogenous 35S epsps gene. Target DNA was amplified and visualized on agarose gel within 45 min under isothermal conditions at 65 degrees C. Without gel electrophoresis, the LAMP amplicon was visualized directly in the reaction tube by the addition of SYBR Green I for naked-eye inspection. The detection sensitivity of LAMP was 10-fold higher than the nested PCR established in our laboratory. Moreover, the LAMP method was much quicker, taking only 70 min, as compared with 300 min for nested PCR to complete the analysis of the GM soybean. Compared with traditional PCR approaches, the LAMP procedure is faster and more sensitive, and there is no need for a special PCR machine or electrophoresis equipment. Hence, this method can be a very useful tool for GMO detection and is particularly convenient for fast screening. PMID:19897926

  16. On-Chip Isothermal Nucleic Acid Amplification on Flow-Based Chemiluminescence Microarray Analysis Platform for the Detection of Viruses and Bacteria.

    PubMed

    Kunze, A; Dilcher, M; Abd El Wahed, A; Hufert, F; Niessner, R; Seidel, M

    2016-01-01

    This work presents an on-chip isothermal nucleic acid amplification test (iNAAT) for the multiplex amplification and detection of viral and bacterial DNA by a flow-based chemiluminescence microarray. In a principle study, on-chip recombinase polymerase amplification (RPA) on defined spots of a DNA microarray was used to spatially separate the amplification reaction of DNA from two viruses (Human adenovirus 41, Phi X 174) and the bacterium Enterococcus faecalis, which are relevant for water hygiene. By establishing the developed assay on the microarray analysis platform MCR 3, the automation of isothermal multiplex-amplification (39 °C, 40 min) and subsequent detection by chemiluminescence imaging was realized. Within 48 min, the microbes could be identified by the spot position on the microarray while the generated chemiluminescence signal correlated with the amount of applied microbe DNA. The limit of detection (LOD) determined for HAdV 41, Phi X 174, and E. faecalis was 35 GU/μL, 1 GU/μL, and 5 × 10(3) GU/μL (genomic units), which is comparable to the sensitivity reported for qPCR analysis, respectively. Moreover the simultaneous amplification and detection of DNA from all three microbes was possible. The presented assay shows that complex enzymatic reactions like an isothermal amplification can be performed in an easy-to-use experimental setup. Furthermore, iNAATs can be potent candidates for multipathogen detection in clinical, food, or environmental samples in routine or field monitoring approaches. PMID:26624222

  17. Rapid, sensitive detection of Vibrio anguillarum using loop-mediated isothermal amplification

    NASA Astrophysics Data System (ADS)

    Gao, Hongwei; Li, Fuhua; Zhang, Xiaojun; Wang, Bing; Xiang, Jianhai

    2010-01-01

    Vibrio anguillarum is an important bacterial pathogen of aquatic organisms and a significant problem in aquatic farming. The rapid detection and identification of V. anguillarum, and other pathogens that infect marine organisms, is crucial to effective disease management. In this study, we developed a loop-mediated amplification (LAMP) assay to detect V. anguillarum in an hour in a single tube without the need for thermal cycling. Conserved regions of the metalloproteinase ( empA) gene of V. anguillarum served as the targets for primer design. A fragment of the empA gene was amplified at 65°C in the presence of the primer mixture and Bst DNA polymerase. In the optimized LAMP assay, 6.7 pg of V. anguillarum DNA could be detected. Six strains of V. anguillarum and 17 strains of non- V. anguillarum bacteria were used in this study to evaluate the species specificity of the primers. The six V. anguillarum strains gave a positive result in the LAMP assay. This method was also validated in V. anguillarum-infected fish. This LAMP method is more sensitive than PCR in the detection of V. anguillarum and shows good species specificity. The LAMP assay is therefore an effective method for the quick detection of V. anguillarum both in the laboratory and in the field.

  18. Target-triggered triple isothermal cascade amplification strategy for ultrasensitive microRNA-21 detection at sub-attomole level.

    PubMed

    Cheng, Fang-Fang; Jiang, Nan; Li, Xiaoyan; Zhang, Li; Hu, Lihui; Chen, Xiaojun; Jiang, Li-Ping; Abdel-Halim, E S; Zhu, Jun-Jie

    2016-11-15

    MicroRNA-21 (miR-21) is a promising diagnostic biomarker for breast cancer screening and disease progression, thus the method for the sensitive and selective detection of miR-21 is vital to its clinical diagnosis. Herein, we develop a novel method to quantify miR-21 levels as low as attomolar sensitivity by a target-triggered triple isothermal cascade amplification (3TICA) strategy. An ingenious unimolecular DNA template with three functional parts has been designed: 5'-fragment as the miR-21 recognition unit, middle fragment as the miR-21 analogue amplification unit, and 3'-fragment as the 8-17 DNAzyme production unit. Triggered by miR-21 and accompanied by polymerase-nicking enzyme cascade, new miR-21 analogues autonomously generated for the successive re-triggering and cleavage process. Simultaneously, the 8-17 DNAzyme-contained sequence could be exponentially released and activated for the second cyclic cleavage toward a specific ribonucleotide (rA)-contained substrate, inducing a remarkably amplified generation of HRP-mimicking DNAzyme in the presence of hemin. Finally, the amperometric technique was used to record the catalytic reduction current of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. The increase in the steady-state current was proportional with the increase of the miR-21 concentration from 1 aM to 100 pM. An ultra-low detection limit of 0.5 aM with an excellent selectivity for even discriminating differences between 1-base mismatched target and miR-21 was achieved. This simple and cost-effective 3TICA strategy is promising for the detection of any short oligonucleotides, simply by altering the target recognition unit in the template sequence. PMID:27311114

  19. Development of Reverse Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of Batai Virus in Cattle and Mosquitoes.

    PubMed

    Liu, Hao; Li, Xin Tong; Hu, Bo; Zhang, Lei; Xue, Xiang-Hong; Lv, Shuang; Lu, Rong-Guang; Shi, Ning; Yan, Xi-Jun

    2016-06-01

    Batai virus (BATV) is an arthropod-borne single-stranded RNA virus belonging to the genus Orthobunyavirus of the family Bunyaviridae that is primarily transmitted by mosquitoes. Methods for detecting BATV are currently limited to serological surveillance, virus isolation, and conventional reverse transcription-polymerase chain reaction (RT-PCR) assay. In this study, we sought to develop a BATV detection assay that needs no specialized equipment and is highly specific, sensitive, and simple. We first developed and optimized a reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of BATV that uses two pairs of primers to amplify a conserved region of the BATV M gene. The optimal reaction conditions for this RT-LAMP BATV detection assay were 40 min at 65°C. The amplification products could be visualized directly for color changes. This RT-LAMP method has a detection limit of 2.86 copies/μL and a sensitivity that was approximately 10- and 100-fold greater than real-time and conventional RT-PCR, respectively. RT-LAMP for BATV detection showed no cross-reactivity with other viruses and its sensitivity was validated with cattle blood and mosquito specimens. Our results suggest that this RT-LAMP method was simpler and faster than conventional RT-PCR or real-time RT-PCR. Moreover, RT-LAMP represents a potential tool to test for BATV in clinical and mosquito samples, especially in rural areas of China. This method also shows promise as a diagnostic tool due to its rapid and sensitive detection without the need for sophisticated equipment or complicated protocols. PMID:27027481

  20. Label-free fluorescence strategy for sensitive microRNA detection based on isothermal exponential amplification and graphene oxide.

    PubMed

    Li, Wei; Hou, Ting; Wu, Min; Li, Feng

    2016-02-01

    MicroRNAs (miRNAs) play an important role in many biological processes, and have been regarded as potential targets and biomarkers in cancer diagnosis and therapy. Also, to meet the big challenge imposed by the characteristics of miRNAs, such as small size and vulnerability to enzymatic digestion, it is of great importance to develop accurate, sensitive and simple miRNA assays. Herein, we developed a label-free fluorescence strategy for sensitive miRNA detection by combining isothermal exponential amplification and the unique features of SYBR Green I (SG) and graphene oxide (GO), in which SG gives significantly enhanced fluorescence upon intercalation into double-stranded DNAs (dsDNAs), and GO selectively adsorbs miRNA, single-stranded DNA and SG, to protect miRNA from enzymatic digestion, and to quench the fluorescence of the adsorbed SG. In the presence of the target miRNA, the ingeniously designed hairpin probe (HP) is unfolded and the subsequent polymerization and strand displacement reaction takes place to initiate the target recycling process. The newly formed dsDNAs are then recognized and cleaved by the nicking enzyme, generating new DNA triggers with the same sequence as the target miRNA, which hybridize with intact HPs to initiate new extension reactions. As a result, the circular exponential amplification for target miRNA is achieved and large amount of dsDNAs are formed to generate significantly enhanced fluorescence upon the intercalation of SG. Thus sensitive and selective fluorescence miRNA detection is realized, and the detection limit of 3 fM is obtained. Besides, this method exhibits additional advantages of simplicity and low cost, since expensive and tedious labeling process is avoided. Therefore, the as-proposed label-free fluorescence strategy has great potential in the applications in miRNA-related clinical practices and biochemical researches. PMID:26653431

  1. Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick

    PubMed Central

    2014-01-01

    Background Citrus Huanglongbing (HLB) is the most devastating bacterial citrus disease worldwide. Three Candidatus Liberibacter species are associated with different forms of the disease: Candidatus Liberibacter asiaticus, Candidatus Liberibacter americanus and Candidatus Liberibacter africanus. Amongst them, Candidatus Liberibacter asiaticus is the most widespread and economically important. These Gram-negative bacterial plant pathogens are phloem-limited and vectored by citrus psyllids. The current management strategy of HLB is based on early and accurate detection of Candidatus Liberibacter asiaticus in both citrus plants and vector insects. Nowadays, real time PCR is the method of choice for this task, mainly because of its sensitivity and reliability. However, this methodology has several drawbacks, namely high equipment costs, the need for highly trained personnel, the time required to conduct the whole process, and the difficulty in carrying out the detection reactions in field conditions. Results A recent DNA amplification technique known as Loop Mediated Isothermal Amplification (LAMP) was adapted for the detection of Candidatus Liberibacter asiaticus. This methodology was combined with a Lateral Flow Dipstick (LFD) device for visual detection of the resulting amplicons, eliminating the need for gel electrophoresis. The assay was highly specific for the targeted bacterium. No cross-reaction was observed with DNA from any of the other phytopathogenic bacteria or fungi assayed. By serially diluting purified DNA from an infected plant, the sensitivity of the assay was found to be 10 picograms. This sensitivity level was proven to be similar to the values obtained running a real time PCR in parallel. This methodology was able to detect Candidatus Liberibacter asiaticus from different kinds of samples including infected citrus plants and psyllids. Conclusions Our results indicate that the methodology here reported constitutes a step forward in the development

  2. Development of a highly sensitive loop-mediated isothermal amplification (LAMP) method for the detection of Loa loa.

    PubMed

    Fernández-Soto, Pedro; Mvoulouga, Prosper Obolo; Akue, Jean Paul; Abán, Julio López; Santiago, Belén Vicente; Sánchez, Miguel Cordero; Muro, Antonio

    2014-01-01

    The filarial parasite Loa loa, the causative agent of loiasis, is endemic in Central and Western Africa infecting 3-13 million people. L. loa has been associated with fatal encephalopathic reactions in high Loa-infected individuals receiving ivermectin during mass drug administration programs for the control of onchocerciasis and lymphatic filariasis. In endemic areas, the only diagnostic method routinely used is the microscopic examination of mid-day blood samples by thick blood film. Improved methods for detection of L. loa are needed in endemic regions with limited resources, where delayed diagnosis results in high mortality. We have investigated the use of a loop-mediated isothermal amplification (LAMP) assay to facilitate rapid, inexpensive, molecular diagnosis of loiasis. Primers for LAMP were designed from a species-specific repetitive DNA sequence from L. loa retrieved from GenBank. Genomic DNA of a L. loa adult worm was used to optimize the LAMP conditions using a thermocycler or a conventional heating block. Amplification of DNA in the LAMP mixture was visually inspected for turbidity as well as addition of fluorescent dye. LAMP specificity was evaluated using DNA from other parasites; sensitivity was evaluated using DNA from L. loa 10-fold serially diluted. Simulated human blood samples spiked with DNA from L. loa were also tested for sensitivity. Upon addition of fluorescent dye, all positive reactions turned green while the negative controls remained orange under ambient light. After electrophoresis on agarose gels, a ladder of multiple bands of different sizes could be observed in positive samples. The detection limit of the assay was found to be as little as 0.5 ag of L. loa genomic DNA when using a heating block. We have designed, for the first time, a highly sensitive LAMP assay for the detection of L. loa which is potentially adaptable for field diagnosis and disease surveillance in loiasis-endemic areas. PMID:24722638

  3. Detection of Middle East respiratory syndrome coronavirus using reverse transcription loop-mediated isothermal amplification (RT-LAMP)

    PubMed Central

    2014-01-01

    Background The first documented case of Middle East Respiratory Syndrome coronavirus (MERS-CoV) occurred in 2012, and outbreaks have continued ever since, mainly in Saudi Arabia. MERS-CoV is primarily diagnosed using a real-time RT-PCR assay, with at least two different genomic targets required for a positive diagnosis according to the case definition of The World Health Organization (WHO) as of 3 July 2013. Therefore, it is urgently necessary to develop as many specific genetic diagnostic methods as possible to allow stable diagnosis of MERS-CoV infections. Methods Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) is a genetic diagnostic method used widely for the detection of viral pathogens, which requires only a single temperature for amplification, and can be completed in less than 1 h. This study developed a novel RT-LAMP assay for detecting MERS-CoV using primer sets targeting a conserved nucleocapsid protein region. Results The RT-LAMP assay was capable of detecting as few as 3.4 copies of MERS-CoV RNA, and was highly specific, with no cross-reaction to other respiratory viruses. Pilot experiments to detect MERS-CoV from medium containing pharyngeal swabs inoculated with pre-titrated viruses were also performed. The RT-LAMP assay exhibited sensitivity similar to that of MERS-CoV real-time RT-PCR. Conclusions These results suggest that the RT-LAMP assay described here is a useful tool for the diagnosis and epidemiologic surveillance of human MERS-CoV infections. PMID:25103205

  4. Development of mitochondrial loop-mediated isothermal amplification for detection of the small liver fluke Opisthorchis viverrini (Opisthorchiidae; Trematoda; Platyhelminthes).

    PubMed

    Le, Thanh Hoa; Nguyen, Nga Thi Bich; Truong, Nam Hai; De, Nguyen Van

    2012-04-01

    Mitochondrial DNA sequences offer major advantages over the more usual nuclear targets for loop-mediated isothermal amplification approaches (mito-LAMP) because multiple copies occur in every cell. Four LAMP primers [F3, FIP(F1c+F2), BIP(B1c+B2), and B3] were designed based on the mitochondrial nad1 sequence of Opisthorchis viverrini and used for a highly specific assay (mito-OvLAMP) to distinguish DNA of O. viverrini from that of another opisthorchiid (Clonorchis sinensis) and other trematodes (Haplorchis pumilio, Haplorchis taichui, Fasciola hepatica, and Fasciola gigantica). Conventional PCR was applied using F3/B3 primer pairs to verify the specificity of the primers for O. viverrini DNA templates. All LAMP-positive samples could be detected with the naked eye in sunlight, by gel electrophoresis (stained with ethidium bromide), and by addition of SYBR green I to the product in sunlight or under UV light. Only DNA from O. viverrini yielded amplification products by LAMP (and by PCR verification), and the LAMP limit of detection was as little as 100 fg (10(-4) ng DNA), indicating that this assay is 10 to 100 times more sensitive than PCR. Field testing was done using representative egg and metacercarial samples collected from localities where the fluke is endemic. With the advantages of simplicity, rapidity, sensitivity, and cost effectiveness, mito-OvLAMP is a good tool for molecular detection and epidemiology studies in regions or countries where O. viverrini is endemic, which can lead to more effective control of opisthorchiasis and trematodiasis. PMID:22322346

  5. The application of loop-mediated isothermal amplification (LAMP) in food testing for bacterial pathogens and fungal contaminants.

    PubMed

    Niessen, Ludwig; Luo, Jie; Denschlag, Carla; Vogel, Rudi F

    2013-12-01

    Bacterial pathogens and toxicants, parasites as well as mycotoxin producing fungi are the major biotic factors influencing the safety of food. Moreover, viral infections and prions may be present as quasi biotic challenging factors. A vast array of culture dependent analytical methods and protocols for food safety testing has been developed during the past decades. Presently, protocols involving molecular biological techniques such as PCR-based nucleic acid amplification and hybridization have become available for many of the known pathogens with their major advantages being rapidness, high sensitivity and specificity. However, this type of assays is still quite labor- and cost intensive and mostly cannot be operated directly in the field. Recently, loop-mediated isothermal amplification (LAMP) of DNA has emerged as an alternative to the use of PCR-based methods not only in food safety testing but also in a wide array of application. Its advantages over PCR-based techniques are even shorter reaction time, no need for specific equipment, high sensitivity and specificity as well as comparably low susceptibility to inhibitors present in sample materials which enables detection of the pathogens in sample materials even without time consuming sample preparation. The present article presents a critical review of the application of LAMP-based methods and their usefulness in detecting and identifying food borne bacterial pathogens and toxicants as well as mycotoxin producing food borne fungi as compared to other methods. Moreover does it elaborate on new developments in the design and automation of LAMP-based assays and their implications for the future developments of food testing. PMID:24010598

  6. Loop mediated isothermal amplification (LAMP) assay for detection of coconut root wilt disease and arecanut yellow leaf disease phytoplasma.

    PubMed

    Nair, Smita; Manimekalai, Ramaswamy; Ganga Raj, Palliyath; Hegde, Vinayaka

    2016-07-01

    The coconut root wilt disease (RWD) and the arecanut yellow leaf disease (YLD) are two major phytoplasma associated diseases affecting palms in South India. Greatly debilitating the palm health, these diseases cause substantial yield reduction and economic loss to farmers. A rapid and robust diagnostic technique is crucial in efficient disease management. We established phytoplasma 16S rDNA targeted loop mediated isothermal amplification (LAMP) and real time LAMP based diagnostics for coconut RWD and arecanut YLD. The LAMP reaction was set at 65 °C and end point detection made using hydroxynaphthol blue (HNB) and agarose gel electrophoresis. Molecular typing of LAMP products were made with restriction enzyme HpyCH4 V. Conventional PCR with LAMP external primers and sequencing of amplicons was carried out. Real time LAMP was performed on the Genei II platform (Optigene Ltd., UK). An annealing curve analysis was programmed at the end of the incubation to check the fidelity of the amplicons. The phytoplasma positive samples produced typical ladder like bands on agarose gel, showed colour change from violet to blue with HNB and produced unique annealing peak at 85 ± 0.5 °C in the real time detection. Restriction digestion produced predicted size fragments. Sequencing and BLASTN analysis confirmed that the amplification corresponded to phytoplasma 16S rRNA gene. LAMP method devised here was found to be more robust compared to conventional nested PCR and hence has potential applications in detection of phytoplasma from symptomatic palm samples and in rapid screening of healthy seedlings. PMID:27263003

  7. Rapid and simple detection of methicillin-resistance staphylococcus aureus by orfX loop-mediated isothermal amplification assay

    PubMed Central

    2014-01-01

    Background Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most prevalent pathogens responsible for nosocomial infections throughout the world. As clinical MRSA diagnosis is concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for MRSA detection. This study aimed at developing a simple loop-mediated isothermal amplification (LAMP) assay targeting on orfX for the rapid detection of methicillin-resistance Staphylococcus aureus (MRSA). Results The protocol was designed by targeting orfX, a highly conserved open reading frame in S. aureus. One hundred and sixteen reference strains, including 52 Gram-positive and 64 Gram-negative isolates, were included for evaluation and optimization of the orfX-LAMP assay. This assay had been further performed on 667 Staphylococcus (566 MRSA, 25 MSSA, 53 MRCNS and 23 MSCNS) strains and were comparatively validated by PCR assay using primers F3 and B3, with rapid template DNA processing, simple equipments (water bath) and direct result determination (both naked eye and under UV light) applied. The indispensability of each primer had been confirmed, and the optimal amplification was obtained under 65°C for 45 min. The 25 μl reactant was found to be the most cost-efficient volume, and the detection limit was determined to be 10 DNA copies and 10 CFU/reaction. High specificity was observed when orfX-LAMP assay was subjected to 116 reference strains. For application, 557 (98.4%, 557/566) and 519 (91.7%, 519/566) tested strains had been detected positive by LAMP and PCR assays. The detection rate, positive predictive value (PPV) and negative predictive value (NPV) of orfX-LAMP were 98.4%, 100% and 92.7% respectively. Conclusions The established orfX-LAMP assay had been demonstrated to be a valid and rapid detection method on MRSA. PMID:24456841

  8. Electrochemical biosensor for microRNA detection based on poly(U) polymerase mediated isothermal signal amplification.

    PubMed

    Zhou, Yunlei; Yin, Huanshun; Li, Jie; Li, Bingchen; Li, Xue; Ai, Shiyun; Zhang, Xiansheng

    2016-05-15

    MicroRNAs play crucial role in post-transcriptional regulation for gene expression in animals, plants, and viruses. For the better understanding of microRNA and its functions, it is very important to develop effectively analytical method for microRNA detection. Herein, a novel electrochemical biosensor was fabricated for sensitive and selective detection of microRNA based on poly(U) polymerase mediated isothermal signal amplification, where poly(U) polymerase can catalyze the template independent addition of UMP from UTP to the 3' end of RNA. Using this activity, the target microRNA can be successfully labeled with biotin conjugated UMPs at its 3'-end using biotin conjugated UTP (biotin-UTP) as donor. Then, the avidin conjugated alkaline phosphatase can be further captured to the 3'-end of the target microRNA based on the specific interaction between biotin and avidin. Finally, under the catalytic activity of alkaline phosphatase, the substrate of p-nitrophenyl phosphate disodium salt hexahydrate can be hydrolyzed to produce 4-nitrophenol. According to the relationship between the electrochemical signal of p-nitrophenol and the concentration of microRNA-319a, the content of microRNA-319a can be detected. This signal amplification method is simple and sensitive. The developed method can detect as low as 1.7 fM microRNA and produce precise and accurate linear dynamic range from 10 to 1000 fM. The fabricated biosensor was further applied to detect the expression level change of microRNA-319a in rice seedlings after incubation with five kinds of different phytohormones. PMID:26700579

  9. Detection of Mycoplasma pneumoniae by loop-mediated isothermal amplification (LAMP) assay and serology in pediatric community-acquired pneumonia.

    PubMed

    Gotoh, Kensei; Nishimura, Naoko; Ohshima, Yasunori; Arakawa, Yasuko; Hosono, Haruki; Yamamoto, Yasuto; Iwata, Yasushi; Nakane, Kazumasa; Funahashi, Keiji; Ozaki, Takao

    2012-10-01

    Rapid diagnosis of Mycoplasma pneumoniae pneumonia is required for treatment with effective antimicrobial agents without delay; however, this capacity has not yet been established in clinical practice. Recently, a novel nucleic acid amplification method termed loop-mediated isothermal amplification (LAMP) has been used to rapidly diagnose various infectious diseases. In this study, we prospectively evaluated the efficacy of the LAMP assay to rapidly diagnose M. pneumoniae pneumonia in clinical practice. Three hundred sixty-eight children (median age, 3.8 years; range, 0.1-14.3 years) admitted to our hospital between April 2009 and March 2010 for community-acquired pneumonia were enrolled in this study. We obtained throat swabs on admission to detect M. pneumoniae DNA and paired serum samples on admission and at discharge to assay M. pneumoniae antibody titers. M. pneumoniae pneumonia was diagnosed by either a positive LAMP assay or a fourfold or greater increase in antibody titer. Overall, 46 children (12.5% of the patients with pneumonia) were diagnosed with M. pneumoniae pneumonia; of these, 27 (58.7%) were aged less than 6 years. Of the aforementioned 46 children, 38 (82.6%) and 37 (80.4%) were identified by LAMP and serology, respectively. When the results of serology were taken as the standard, the sensitivity and specificity and positive and negative predictive values of the LAMP assay were 78.4%, 97.3%, 76.3%, and 97.6%, respectively. We concluded the LAMP assay may be useful for rapid diagnosis of M. pneumoniae pneumonia. PMID:22370920

  10. Detection of shrimp infectious myonecrosis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick.

    PubMed

    Puthawibool, Teeranart; Senapin, Saengchan; Kiatpathomchai, Wansika; Flegel, Timothy W

    2009-03-01

    Infectious myonecrosis virus (IMNV) has caused a slowly progressive disease with cumulative mortalities of up to 70% or more in cultured Penaeus (Litopenaeus) vannamei in Northeast Brazil and Indonesia. Rapid detection of viruses by loop-mediated isothermal amplification (LAMP) of genomic material with high specificity and sensitivity can be applied for diagnosis, monitoring and control of diseases in shrimp aquaculture. Using an IMNV template, successful detection was achieved after a 60-min RT-LAMP reaction using biotin-labeled primers followed by 5min hybridization with an FITC-labeled DNA probe and 5min assay using a chromatographic lateral flow dipstick (LFD). Thus, the combined system of RT-LAMP and LFD required a total assay interval of less than 75min, excluding the RNA extraction time. The sensitivity of detection was comparable to that of other commonly used methods for nested RT-PCR detection of IMNV. In addition to reducing amplicon detection time when compared to electrophoresis, LFD confirmed amplicon identity by hybridization and eliminated the need to handle carcinogenic ethidium bromide. The RT-LAMP-LFD method gave negative test results with nucleic acid extracts from normal shrimp and from shrimp infected with other viruses including infectious hypodermal hematopoietic necrosis virus (IHHNV), monodon baculovirus (MBV), a hepatopancreatic parvovirus from P. monodon (PmDNV), white spot syndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus (TSV), Macrobrachium rosenbergii nodavirus (MrNV) and gill associated virus (GAV). PMID:19022295

  11. Loop-mediated isothermal amplification of specific endoglucanase gene sequence for detection of the bacterial wilt pathogen Ralstonia solanacearum.

    PubMed

    Lenarčič, Rok; Morisset, Dany; Pirc, Manca; Llop, Pablo; Ravnikar, Maja; Dreo, Tanja

    2014-01-01

    The increased globalization of crops production and processing industries also promotes the side-effects of more rapid and efficient spread of plant pathogens. To prevent the associated economic losses, and particularly those related to bacterial diseases where their management relies on removal of the infected material from production, simple, easy-to-perform, rapid and cost-effective tests are needed. Loop-mediated isothermal amplification (LAMP) assays that target 16S rRNA, fliC and egl genes were compared and evaluated as on-site applications. The assay with the best performance was that targeted to the egl gene, which shows high analytical specificity for diverse strains of the betaproteobacterium Ralstonia solanacearum, including its non-European and non-race 3 biovar 2 strains. The additional melting curve analysis provides confirmation of the test results. According to our extensive assessment, the egl LAMP assay requires minimum sample preparation (a few minutes of boiling) for the identification of pure cultures and ooze from symptomatic material, and it can also be used in a high-throughput format in the laboratory. This provides sensitive and reliable detection of R. solanacearum strains of different phylotypes. PMID:24763488

  12. Efficient and Specific Detection of Salmonella in Food Samples Using a stn-Based Loop-Mediated Isothermal Amplification Method

    PubMed Central

    Srisawat, Mevaree; Panbangred, Watanalai

    2015-01-01

    The Salmonella enterotoxin (stn) gene exhibits high homology among S. enterica serovars and S. bongori. A set of 6 specific primers targeting the stn gene were designed for detection of Salmonella spp. using the loop-mediated isothermal amplification (LAMP) method. The primers amplified target sequences in all 102 strains of 87 serovars of Salmonella tested and no products were detected in 57 non-Salmonella strains. The detection limit in pure cultures was 5 fg DNA/reaction when amplified at 65°C for 25 min. The LAMP assay could detect Salmonella in artificially contaminated food samples as low as 220 cells/g of food without a preenrichment step. However, the sensitivity was increased 100-fold (~2 cells/g) following 5 hr preenrichment at 35°C. The LAMP technique, with a preenrichment step for 5 and 16 hr, was shown to give 100% specificity with food samples compared to the reference culture method in which 67 out of 90 food samples gave positive results. Different food matrixes did not interfere with LAMP detection which employed a simple boiling method for DNA template preparation. The results indicate that the LAMP method, targeting the stn gene, has great potential for detection of Salmonella in food samples with both high specificity and high sensitivity. PMID:26543859

  13. Loop Mediated Isothermal Amplification for Detection of Trypanosoma brucei gambiense in Urine and Saliva Samples in Nonhuman Primate Model

    PubMed Central

    Ngotho, Maina; Kagira, John Maina; Gachie, Beatrice Muthoni; Karanja, Simon Muturi; Waema, Maxwell Wambua; Maranga, Dawn Nyawira; Maina, Naomi Wangari

    2015-01-01

    Human African trypanosomiasis (HAT) is a vector-borne parasitic zoonotic disease. The disease caused by Trypanosoma brucei gambiense is the most prevalent in Africa. Early diagnosis is hampered by lack of sensitive diagnostic techniques. This study explored the potential of loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) in the detection of T. b. gambiense infection in a vervet monkey HAT model. Six vervet monkeys were experimentally infected with T. b. gambiense IL3253 and monitored for 180 days after infection. Parasitaemia was scored daily. Blood, cerebrospinal fluid (CSF), saliva, and urine samples were collected weekly. PCR and LAMP were performed on serum, CSF, saliva, and urine samples. The detection by LAMP was significantly higher than that of parasitological methods and PCR in all the samples. The performance of LAMP varied between the samples and was better in serum followed by saliva and then urine samples. In the saliva samples, LAMP had 100% detection between 21 and 77 dpi, whereas in urine the detection it was slightly lower, but there was over 80% detection between 28 and 91 dpi. However, LAMP could not detect trypanosomes in either saliva or urine after 140 and 126 dpi, respectively. The findings of this study emphasize the importance of LAMP in diagnosis of HAT using saliva and urine samples. PMID:26504841

  14. Tracing Phosphate Ions Generated during Loop-Mediated Isothermal Amplification for Electrochemical Detection of Nosema bombycis Genomic DNA PTP1.

    PubMed

    Xie, Shunbi; Yuan, Yali; Chai, Yaqin; Yuan, Ruo

    2015-10-20

    Traditionally, amplified DNA detection in a loop-mediated isothermal amplification (LAMP) was carried out in a complicated gel electrophoresis or with expensive fluorescence-based methods. Here, instead of direct detection that relies on amplified DNA, the indirect detection based on tracing phosphate ions (Pi) generated during LAMP by using an electrochemical method has been proposed for sensitive nucleic acid detection. Pyrophosphate (PPi) as the byproduct of nucleic acid polymerization reaction in LAMP was hydrolyzed into Pi by the preaddition of thermostable inorganic pyrophosphatase (PPase). Thus, the total amount of Pi in the LAMP-amplified sample was proportional to the amount of starting DNA templates. The obtained Pi could then react with acidic molybdate to form the molybdophosphate precipitates on the electrode surface, which serve as redox mediators to give a readily measurable electrochemical signal. The practicality of this strategy has been further demonstrated by employing it for sensitive and accurate quantification of Nosema bombycis genomic DNA PTP1. The electrochemical method allowed the quantitative analysis for target genomic DNA with a detection limit of 17 fg/μL. Thus, we suppose that the novel method proposed in this work with superior sensitivity and specificity, as well as the simple feature, can be easily established for quantitative analysis of many other kinds of nucleic acids in the assistance of LAMP. PMID:26412581

  15. Rapid detection of aflatoxin producing fungi in food by real-time quantitative loop-mediated isothermal amplification.

    PubMed

    Luo, Jie; Vogel, Rudi F; Niessen, Ludwig

    2014-12-01

    Aflatoxins represent a serious risk for human and animal health. They are mainly produced by Aspergillus flavus and Aspergillus parasiticus but also by Aspergillus nomius. Three species specific turbidimeter based real-time LAMP (loop-mediated isothermal amplification) assays were developed to quantify the three species individually in conidial solutions and to define contamination levels in samples of shelled Brazil nuts, maize, and peanuts. Standard curves relating spore numbers to time to threshold (Tt) values were set up for each of the species. Assays had detection limits of 10, 100 and 100 conidia per reaction of A. flavus, A. parasiticus, and A. nomius, respectively. Analysis of contaminated sample materials revealed that the A. nomius specific real-time LAMP assay detected a minimum of 10 conidia/g in Brazil nuts while assays specific for A. flavus and A. parasiticus had detection limits of 10(2) conidia/g and 10(5) conidia/g, respectively in peanut samples as well as 10(4) conidia/g and 10(4) conidia/g, respectively in samples of maize. The real-time LAMP assays developed here appear to be promising tools for the prediction of potential aflatoxigenic risk at an early stage and in all critical control points of the food and feed production chain. PMID:25084656

  16. Development of loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of ostrich meat.

    PubMed

    Abdulmawjood, Amir; Grabowski, Nils; Fohler, Svenja; Kittler, Sophie; Nagengast, Helga; Klein, Guenter

    2014-01-01

    Animal species identification is one of the primary duties of official food control. Since ostrich meat is difficult to be differentiated macroscopically from beef, therefore new analytical methods are needed. To enforce labeling regulations for the authentication of ostrich meat, it might be of importance to develop and evaluate a rapid and reliable assay. In the present study, a loop-mediated isothermal amplification (LAMP) assay based on the cytochrome b gene of the mitochondrial DNA of the species Struthio camelus was developed. The LAMP assay was used in combination with a real-time fluorometer. The developed system allowed the detection of 0.01% ostrich meat products. In parallel, a direct swab method without nucleic acid extraction using the HYPLEX LPTV buffer was also evaluated. This rapid processing method allowed detection of ostrich meat without major incubation steps. In summary, the LAMP assay had excellent sensitivity and specificity for detecting ostrich meat and could provide a sampling-to-result identification-time of 15 to 20 minutes. PMID:24963709

  17. Development of phage immuno-loop-mediated isothermal amplification assays for organophosphorus pesticides in agro-products.

    PubMed

    Hua, Xiude; Yin, Wei; Shi, Haiyan; Li, Ming; Wang, Yanru; Wang, Hong; Ye, Yonghao; Kim, Hee Joo; Gee, Shirley J; Wang, Minghua; Liu, Fengquan; Hammock, Bruce D

    2014-08-19

    Two immuno-loop-mediated isothermal amplification assays (iLAMP) were developed by using a phage-borne peptide that was isolated from a cyclic eight-peptide phage library. One assay was used to screen eight organophosphorus (OP) pesticides with limits of detection (LOD) between 2 and 128 ng mL(-1). The iLAMP consisted of the competitive immuno-reaction coupled to the LAMP reaction for detection. This method provides positive results in the visual color of violet, while a negative response results in a sky blue color; therefore, the iLAMP allows one to rapidly detect analytes in yes or no fashion. We validated the iLAMP by detecting parathion-methyl, parathion, and fenitrothion in Chinese cabbage, apple, and greengrocery, and the detection results were consistent with the enzyme-linked immunosorbent assay (ELISA). In conclusion, the iLAMP is a simple, rapid, sensitive, and economical method for detecting OP pesticide residues in agro-products with no instrumental requirement. PMID:25135320

  18. Rapid detection of contagious ecthyma by loop-mediated isothermal amplification and epidemiology in Jilin Province China

    PubMed Central

    WANG, Kai; SHAO, Hongze; PEI, Zhihua; HU, Guixue

    2015-01-01

    The aim of this experiment was to develop a loop-mediated isothermal amplification (LAMP) assay and to research the recent epidemiology of contagious ecthyma in Jilin Province, China, using the assay. A LAMP assay targeting a highly conserved region of the F1L gene was developed to detect contagious ecthyma virus (CEV). Three hundred and sixty-five cases from 64 flocks in 9 different areas of Jilin Province, China, from 2011 to 2014 were tested using the LAMP assay. The results showed that the sensitivity of the LAMP assay was 100 copies of the standard plasmid, which is 100-fold higher than the sensitivity of PCR. No cross-reactivity was observed with capripoxvirus, fowlpox virus, foot-and-mouth disease virus serotype O, foot-and-mouth disease virus serotype Asia I and bluetongue virus. The average positive rate was 19.73% (72/365), and the positive rate was highest in lambs aged 1–6 months. Our results demonstrated that CEV infection was very widespread in the flocks of Jilin Province and that the LAMP assay allows for easy, rapid, accurate and sensitive detection of CEV infection. PMID:26346652

  19. Application of novel loop-mediated isothermal amplification (LAMP) for rapid authentication of the herbal tea ingredient Hedyotis diffusa Willd.

    PubMed

    Li, Ming; Wong, Yuk-Lau; Jiang, Li-Li; Wong, Ka-Lok; Wong, Yuen-Ting; Lau, Clara Bik-San; Shaw, Pang-Chui

    2013-12-01

    Hedyotis diffusa Willd. (Baihuasheshecao) is an ingredient of herbal teas commonly consumed in the Orient and tropical Asia for cancer treatment and health maintenance. In the market, this ingredient is frequently adulterated by the related species Hedyotis corymbosa (L.) Lam. The objective of this study is to develop a novel loop-mediated isothermal amplification (LAMP) technique to differentiate H. diffusa from its adulterant H. corymbosa. A set of four internal control primers (F3, FIP, BIP and B3) were designed based on six loci in the internal transcribed spacer (ITS) for LAMP of both H. diffusa and H. corymbosa. Two specific primers (S_F3 and S_FIP) were designed for specific LAMP detection of H. diffusa only. Our data showed that LAMP was successful for both H. diffusa and H. corymbosa in internal control. In contrast, only H. diffusa was detected in specific LAMP using the specific primers S_F3 and S_FIP. This study showed that LAMP was useful to differentiate H. diffusa from its adulterant H. corymbosa. This study is significant for the verification of the authenticity for better quality control of this common herbal tea ingredient. The strategy of including an internal control assures the quality of the concerned DNA region for LAMP. PMID:23870990

  20. Development of a loop-mediated isothermal amplification method for the rapid diagnosis of Ascochyta rabiei L. in chickpeas

    PubMed Central

    Chen, Xiaolu; Ma, Lijuan; Qiang, Song; Ma, Deying

    2016-01-01

    Ascochyta blight (AB) is a devastating fungal disease of chickpeas that has spread to nearly all of the chickpea cultivating regions of the world. The rapid diagnosis of Ascochyta rabiei L. (A. rabiei), the pathogen that causes AB, plays an important role in A. rabiei epidemic tracking and AB management. In this study, a group of loop-mediated isothermal amplification (LAMP) primers was designed to detect A. rabiei in chickpea plants and seeds via a LAMP method and a conventional PCR method based on an internal transcribed spacer (ITS) sequence analysis of A. rabiei. Compared with the conventional PCR method, the LAMP method not only exhibited greater sensitivity and specificity in the detection of A. rabiei but also used simpler equipment and required less operational time. The minimum detectable concentration of the A. rabiei genomic DNA solution with the LAMP method was 6.01 × 10−6 ng/μl, which was 100 times lower than that of the conventional PCR method with the same outer primers. The greatest advantage of the LAMP method is that results can be observed via the visualization of color changes in SYBR Green I dye with the naked eye, and it does not require expensive instruments, also with less time consumption. PMID:27161564

  1. Rapid detection of nusG and fadA in Fusobacterium nucleatum by loop-mediated isothermal amplification.

    PubMed

    Huang, Simo; Yang, Zhan; Zou, Dayang; Dong, Derong; Liu, Anheng; Liu, Wei; Huang, Liuyu

    2016-08-01

    Fusobacterium nucleatum is associated with various human diseases such as periodontal disease and colorectal cancer (CRC); thus, F. nucleatum detection might serve as a novel diagnostic tool. Here, we describe the development of a sensitive and rapid molecular method for detecting two F. nucleatum genes: the highly conserved nusG and fadA, which encode a critical host colonization factor. Loop-mediated isothermal amplification (LAMP) primer sets for the rapid detection of nusG and fadA were designed and optimized. The nusG primers yielded consistent negative results for 20 non-F. nucleatum bacterial strains, confirming the high specificity of the primers. LAMP reaction primer sensitivity was determined, and its detection rate in comparison to conventional PCR was assessed using 57 clinical stool samples. The LAMP detection limit for nusG and fadA was 22.5 and 0.225 pg µl-1, respectively, indicating that the sensitivity of this method was 10-fold higher than that of conventional PCR. These results suggest that the LAMP technique is able to effectively identify F. nucleatum via nusG as well as detect its virulence factor. To the best of our knowledge, this study is the first to report the application of LAMP for the detection of nusG and fadA in F. nucleatum. The LAMP method constitutes a sensitive and specific visual assay for the rapid detection of the pathogen F. nucleatum. PMID:27339262

  2. Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

    PubMed Central

    Barkway, Christopher P.; Pocock, Rebecca L.; Vrba, Vladimir; Blake, Damer P.

    2015-01-01

    Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm’s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable. PMID:25741643

  3. Automated Detection of Toxigenic Clostridium difficile in Clinical Samples: Isothermal tcdB Amplification Coupled to Array-Based Detection

    PubMed Central

    Pasko, Chris; Groves, Benjamin; Ager, Edward; Corpuz, Maylene; Frech, Georges; Munns, Denton; Smith, Wendy; Warcup, Ashley; Denys, Gerald; Ledeboer, Nathan A.; Lindsey, Wes; Owen, Charles; Rea, Larry; Jenison, Robert

    2012-01-01

    Clostridium difficile can carry a genetically variable pathogenicity locus (PaLoc), which encodes clostridial toxins A and B. In hospitals and in the community at large, this organism is increasingly identified as a pathogen. To develop a diagnostic test that combines the strengths of immunoassays (cost) and DNA amplification assays (sensitivity/specificity), we targeted a genetically stable PaLoc region, amplifying tcdB sequences and detecting them by hybridization capture. The assay employs a hot-start isothermal method coupled to a multiplexed chip-based readout, creating a manual assay that detects toxigenic C. difficile with high sensitivity and specificity within 1 h. Assay automation on an electromechanical instrument produced an analytical sensitivity of 10 CFU (95% probability of detection) of C. difficile in fecal samples, along with discrimination against other enteric bacteria. To verify automated assay function, 130 patient samples were tested: 31/32 positive samples (97% sensitive; 95% confidence interval [CI], 82 to 99%) and 98/98 negative samples (100% specific; 95% CI, 95 to 100%) were scored correctly. Large-scale clinical studies are now planned to determine clinical sensitivity and specificity. PMID:22675134

  4. Loop-mediated isothermal amplification (LAMP) assays for rapid detection and differentiation of Nosema apis and N. ceranae in honeybees.

    PubMed

    Ptaszyńska, Aneta A; Borsuk, Grzegorz; Woźniakowski, Grzegorz; Gnat, Sebastian; Małek, Wanda

    2014-08-01

    Nosemosis is a contagious disease of honeybees (Apis mellifera) manifested by increased winter mortality, poor spring build-up and even the total extinction of infected bee colonies. In this paper, loop-mediated isothermal amplifications (LAMP) were used for the first time to identify and differentiate N. apis and N. ceranae, the causative agents of nosemosis. LAMP assays were performed at a constant temperature of 60 °C using two sets of six species-specific primers, recognising eight distinct fragments of 16S rDNA gene and GspSSD polymerase with strand displacement activity. The optimal time for LAMP and its Nosema species sensitivity and specificity were assessed. LAMP only required 30 min for robust identification of the amplicons. Ten-fold serial dilutions of total DNA isolated from bees infected with microsporidia were used to determine the detection limit of N. apis and N. ceranae DNAs by LAMP and standard PCR assays. LAMP appeared to be 10(3) -fold more sensitive than a standard PCR in detecting N. apis and N. ceranae. LAMP methods developed by us are highly Nosema species specific and allow to identify and differentiate N. apis and N. ceranae. PMID:24975021

  5. Rapid Detection of the Marek's Disease Viral Genome in Chicken Feathers by Loop-Mediated Isothermal Amplification

    PubMed Central

    Baskaran, Subasty; Gopal, Dhinakar Raj; Devarajan, Jeyanthi; Kathaperumal, Kumanan

    2012-01-01

    A loop-mediated isothermal amplification (LAMP) method for the rapid detection of serotype 1 Marek's disease virus (MDV) was developed. The method used a set of three pairs of primers to amplify the MEQ gene for detecting serotype 1 MDV. The MDV LAMP method did not cross-react with serotype 2 and serotype 3, nor did the LAMP primers have binding sites for the common avian DNA viruses (reticuloendotheliosis virus, chicken anemia virus, subgroup J of the avian leukosis virus). Additionally, the assay could detect up to 10 copies of the MEQ gene in the MD viral genome, and it had 10 times higher sensitivity than the traditional PCR methods. The LAMP master mix was stable for 90 days at −20°C. Furthermore, the efficiency of LAMP for detection of serotype 1 MDV in clinical samples was comparable to those of PCR and viral isolation. The LAMP procedure is simple and does not rely on any special equipment. The detection of serotype 1 MDV by LAMP will be useful for detecting and controlling oncogenic Marek's disease. PMID:22170920

  6. Loop-mediated isothermal amplification (LAMP) assays for detection of Theileria parva infections targeting the PIM and p150 genes.

    PubMed

    Thekisoe, Oriel M M; Rambritch, Natasha E; Nakao, Ryo; Bazie, Raoul S; Mbati, Peter; Namangala, Boniface; Malele, Imna; Skilton, Robert A; Jongejan, Frans; Sugimoto, Chihiro; Kawazu, Shin-Ichiro; Inoue, Noboru

    2010-01-01

    We have developed two loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria parva, the causative agent of East Coast fever (ECF), an economically important cattle disease in eastern, central and southern Africa. These assays target the polymorphic immunodominant molecule (PIM) and p150 LAMP genes. The primer set for each gene target consists of six primers, and each set recognises eight distinct regions on the target gene to give highly specific detection of T. parva. The detection limit of each primer set is 1fg, which is equivalent to one copy of the PIM and p150 T. parva genes. These PIM and p150 LAMP primer sets amplify DNA of T. parva isolates from cattle and buffalo from different countries including Kenya, South Africa, Tanzania, Rwanda, Uganda and Burundi, indicating their ability to detect T. parva from different countries. With the advantages of simplicity, rapidity and cost effectiveness, these LAMP assays are good candidates for molecular epidemiology studies and for monitoring control programs in ECF-endemic, resource poor countries. PMID:19654009

  7. Accuracy of loop-mediated isothermal amplification for the diagnosis of Clostridium difficile infection: a systematic review.

    PubMed

    Lloyd, Aaron; Pasupuleti, Vinay; Thota, Priyaleela; Pant, Chaitanya; Rolston, David D K; Hernandez, Adrian V; Benites-Zapata, Vicente A; Fraser, Thomas G; Donskey, Curtis J; Deshpande, Abhishek

    2015-05-01

    Loop-mediated isothermal DNA amplification (LAMP) is currently used as standalone diagnostic test for C. difficile infection (CDI). We assessed the diagnostic accuracy of LAMP for the diagnosis of CDI. We searched 5 databases to identify studies that compared LAMP with culture cytotoxicity neutralization assay or anaerobic toxigenic culture (TC) of C. difficile. We used the random-effects model to calculate pooled sensitivities, specificities, diagnostic odds ratios, and their 95% confidence intervals (CIs). The search of the databases yielded 16 studies (6979 samples) that met inclusion criteria. When TC was used as the gold standard (6572 samples), bivariate analysis yielded a mean sensitivity of 0.95 (95% CI, 0.93-0.97; I(2)=67.4) and a mean specificity of 0.99 (95% CI, 0.96-1.00; I(2)=97.0). LAMP is a useful diagnostic tool with high sensitivity and specificity for detecting CDI. The results should, however, be interpreted only in the presence of clinical suspicion and symptoms of CDI. PMID:25752201

  8. Fluorescence detection of telomerase activity in cancer cell extracts based on autonomous exonuclease III-assisted isothermal cycling signal amplification.

    PubMed

    Ding, Caifeng; Li, Xiaoqian; Wang, Wei; Chen, Yaoyao

    2016-09-15

    Based on the extension reaction of a telomerase substrate (TS) primer in the presence of the telomerase, strand-displacement process to perform more stable longer duplex chain, and stepwise hydrolysis of mononucleotides from the blunt or the recessed 3'-hydroxyl termini of duplex DNA in the presence of Exonuclease III (Exo III), an amplified fluorescence detection of telomerase activity in the cancer cells was described in this manuscript. A fluorescence probe DNA, a quencher DNA, and a TS primer were mixed to construct a three-chain DNA structure and a two-chain DNA structure because the amount of the TS primer was less than the other two DNA. In the presence of the telomerase, the quencher DNA was replaced from the probe DNA and the telomerase activity could be determined with the fluorescence enhancement. The telomerase activity in HeLa extracts equivalent to 6-2000 cells was detected by this method. Moreover, the strategy was further proved by using telomerase extracted from Romas cells. With the multiple rounds of isothermal strand displacement and the hydrolysis process, constituted consecutive of signal amplification for the novel detection paradigm that allowed measuring of telomerase activity in crude cancer cell extracts confirmed the reliability and practicality of the protocol, which reveal this platform holds great promise in the biochemical assay for the telomerase activity in early diagnosis for cancers. PMID:27108253

  9. Evaluation of loop-mediated isothermal amplification suitable for molecular monitoring of schistosome-infected snails in field laboratories.

    PubMed

    Hamburger, Joseph; Abbasi, Ibrahim; Kariuki, Curtis; Wanjala, Atsabina; Mzungu, Elton; Mungai, Peter; Muchiri, Eric; King, Charles H

    2013-02-01

    We previously described loop-mediated isothermal amplification (LAMP) for detection of Schistosoma haematobium and S. mansoni DNA in infected snails. In the present study, we adapted the LAMP assay for application in field laboratories in schistosomiasis-endemic areas. Isolation of DNA was simplified by blotting snail tissue (extracted in NaOH/sodium dodecyl sulfate) onto treated membranes, which enabled preservation at ambient temperatures. A ready-mix of LAMP reagents, suitable for shipment at ambient temperature and storage in minimal refrigeration, was used. Local survey teams without experience in molecular biology acquired operational expertise with this test within a few hours. Fifty-four field-caught snails were tested locally by LAMP and 59 were tested at similar conditions in Jerusalem. The LAMP results were consistent with those of a polymerase chain reaction; only four samples showed false-negative results. Results indicate that LAMP assays are suitable for detection of S. haematobium and S. mansoni in low-technology parasitology laboratories in which schistosomiasis elimination activities are undertaken. PMID:23208875

  10. Graphene oxide based fluorescence resonance energy transfer and loop-mediated isothermal amplification for white spot syndrome virus detection.

    PubMed

    Waiwijit, U; Phokaratkul, D; Kampeera, J; Lomas, T; Wisitsoraat, A; Kiatpathomchai, W; Tuantranont, A

    2015-10-20

    Graphene oxide (GO) is attractived for biological or medical applications due to its unique electrical, physical, optical and biological properties. In particular, GO can adsorb DNA via π-π stacking or non-covalent interactions, leading to fluorescence quenching phenomenon applicable for bio-molecular detection. In this work, a new method for white spot syndrome virus (WSSV)-DNA detection is developed based on loop-mediated isothermal amplification (LAMP) combined with fluorescence resonance energy transfer (FRET) between GO and fluorescein isothiocyanate-labeled probe (FITC-probe). The fluorescence quenching efficiency of FITC-probe was found to increase with increasing GO concentration and reached 98.7% at a GO concentration of 50 μg/ml. The fluorescence intensity of FITC-probe was recovered after hybridization with WSSV LAMP product with an optimal hybridization time of 10 min and increased accordingly with increasing amount of LAMP products. The detection limit was estimated to be as low as 10 copies of WSSV plasmid DNA or 0.6 fg of the total DNA extracted from shrimp infected with WSSV. In addition, no cross reaction was observed with other common shrimp viral pathogens. Therefore, the GO-FRET-LAMP technique is promising for fast, sensitive and specific detection of DNAs. PMID:26277651

  11. Rapid detection of measles virus using reverse transcription loop-mediated isothermal amplification coupled with a disposable lateral flow device.

    PubMed

    Xu, Changping; Feng, Yan; Chen, Yin; Gao, Jian; Lu, Yiyu

    2016-06-01

    The measles virus (MeV) causes a highly contagious disease and efforts to reduce its spread are critical. A reverse transcription loop-mediated isothermal amplification assay coupled with a disposable lateral flow device (RT-LAMP-LFD) was developed for the rapid detection of MeV. The assay was performed in 40 min at an optimal temperature of 58 °C, with endpoint results visualized directly. A probe that was complementary to the RT-LAMP amplicon was designed to enhance assay specificity. Detection limit of the assay was 8.8 copies/μL synthetic RNA, which equals the sensitivity of real-time RT-PCR. Clinical specimens were used to validate the RT-LAMP-LFD in provincial Center for Disease Control and Prevention (CDC) (n = 245) and six municipal CDCs (n = 249). The results obtained using RT-LAMP-LFD and real-time RT-PCR were highly concordant. The RT-LAMP-LFD is rapid, stable, and does not require expensive equipment, which can be used for routine MeV monitoring in CDC laboratories. PMID:27117517

  12. Development of multiplex loop-mediated isothermal amplification assays to detect medically important yeasts in dairy products.

    PubMed

    Kasahara, Kohei; Ishikawa, Hiroshi; Sato, Sumie; Shimakawa, Yasuhisa; Watanabe, Koichi

    2014-08-01

    Rapid detection of yeast contamination is important in the food industry. We have developed loop-mediated isothermal amplification (LAMP) assays to detect the emerging opportunistic pathogenic yeasts: Candida albicans, Candida glabrata, Candida tropicalis, the Candida parapsilosis group, Trichosporon asahii, and Trichosporon mucoides. These yeasts may cause deep-seated candidiasis or trichosporonosis. Four LAMP primer sets specific for Candida were designed to target the internal transcribed spacer 2 (ITS2) region between the 5.8S and 26S rRNA genes, and two LAMP primer sets specific for Trichosporon were designed to target the intergenic spacer 1 (IGS1) region between the 26S and 5S rRNA genes. The LAMP assays could detect these yeasts in a range between 10(0) and 10(3)  cells mL(-1) in a contaminated dairy product within 1 h. We also developed multiplex LAMP assays to detect these Candida or Trichosporon species in a single reaction. Multiplex LAMP assays can detect contamination if at least one of the target species is present; they are more time- and cost-efficient than conventional methods and could detect target yeasts with sensitivity close to that of the LAMP assays. Multiplex LAMP assays established in this study can be used as a primary screening method for yeast contamination in food products. PMID:24965944

  13. Development and application of loop-mediated isothermal amplification for detecting the highly benzimidazole-resistant isolates in Sclerotinia sclerotiorum.

    PubMed

    Duan, Ya Bing; Yang, Ying; Wang, Jian Xin; Liu, Cong Chao; He, Ling Ling; Zhou, Ming Guo

    2015-01-01

    Resistance of benzimidazole fungicides is related to the point mutation of the β-tubulin gene in Sclerotinia sclerotiorum. The point mutation at codon 198 (GAG → GCG, E198A) occurs in more than 90% of field resistant populations in China. Traditional detection methods of benzimidazole-resistant mutants of S. sclerotiorum are time-consuming, tedious and inefficient. To establish a suitable and rapid detection of benzimidazole-resistant mutants of S. sclerotiorum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). Eight sets of LAMP primers were designed and four sets were optimized to specially distinguish benzimidazole-resistant mutants of S. sclerotiorum. With the optimal LAMP primers, the concentration of LAMP components was optimized and the reaction conditions were set as 60-64 °C for 60 min. This method had a good specificity, sensitivity, stability and repeatability. In the 1491 sclerotia, 614 (41.18%) were positive by LAMP, and 629 (42.19%) positive by MIC. Therefore, the LAMP assay is more feasible to detect benzimidazole-resistant mutants of S. sclerotiorum than traditional detection methods. PMID:26606972

  14. A Novel Loop-Mediated Isothermal Amplification Assay for Serogroup Identification of Neisseria meningitidis in Cerebrospinal Fluid

    PubMed Central

    Lee, DoKyung; Kim, Eun Jin; Kilgore, Paul E.; Takahashi, Hideyuki; Ohnishi, Makoto; Tomono, Jun; Miyamoto, Shigehiko; Omagari, Daisuke; Kim, Dong Wook; Seki, Mitsuko

    2016-01-01

    We have developed a novel Neisseria meningitidis serogroup-specific loop-mediated isothermal amplification (LAMP) assay for six of the most common meningococcal serogroups (A, B, C, W, X, and Y). The assay was evaluated using a set of 31 meningococcal LAMP assay positive cerebrospinal fluid (CSF) specimens from 1574 children with suspected meningitis identified in prospective surveillance between 1998 and 2002 in Vietnam, China, and Korea. Primer specificity was validated using 15 N. meningitidis strains (including serogroups A, B, C, E, W, X, Y, and Z) and 19 non-N. meningitidis species. The N. meningitidis serogroup LAMP detected down to ten copies and 100 colony-forming units per reaction. Twenty-nine CSF had N. meningitidis serogroup identified by LAMP compared with two CSF in which N. meningitidis serogroup was identified by culture and multi-locus sequence typing. This is the first report of a serogroup-specific identification assay for N. meningitidis using the LAMP method. Our results suggest that this assay will be a rapid, sensitive, and uniquely serogroup-specific assay with potential for application in clinical laboratories and public health surveillance systems. PMID:26793181

  15. Rapid detection of contagious ecthyma by loop-mediated isothermal amplification and epidemiology in Jilin Province China.

    PubMed

    Wang, Kai; Shao, Hongze; Pei, Zhihua; Hu, Guixue

    2016-01-01

    The aim of this experiment was to develop a loop-mediated isothermal amplification (LAMP) assay and to research the recent epidemiology of contagious ecthyma in Jilin Province, China, using the assay. A LAMP assay targeting a highly conserved region of the F1L gene was developed to detect contagious ecthyma virus (CEV). Three hundred and sixty-five cases from 64 flocks in 9 different areas of Jilin Province, China, from 2011 to 2014 were tested using the LAMP assay. The results showed that the sensitivity of the LAMP assay was 100 copies of the standard plasmid, which is 100-fold higher than the sensitivity of PCR. No cross-reactivity was observed with capripoxvirus, fowlpox virus, foot-and-mouth disease virus serotype O, foot-and-mouth disease virus serotype Asia I and bluetongue virus. The average positive rate was 19.73% (72/365), and the positive rate was highest in lambs aged 1-6 months. Our results demonstrated that CEV infection was very widespread in the flocks of Jilin Province and that the LAMP assay allows for easy, rapid, accurate and sensitive detection of CEV infection. PMID:26346652

  16. A portable automatic endpoint detection system for amplicons of loop mediated isothermal amplification on microfluidic compact disk platform.

    PubMed

    Uddin, Shah Mukim; Ibrahim, Fatimah; Sayad, Abkar Ahmed; Thiha, Aung; Pei, Koh Xiu; Mohktar, Mas S; Hashim, Uda; Cho, Jongman; Thong, Kwai Lin

    2015-01-01

    In recent years, many improvements have been made in foodborne pathogen detection methods to reduce the impact of food contamination. Several rapid methods have been developed with biosensor devices to improve the way of performing pathogen detection. This paper presents an automated endpoint detection system for amplicons generated by loop mediated isothermal amplification (LAMP) on a microfluidic compact disk platform. The developed detection system utilizes a monochromatic ultraviolet (UV) emitter for excitation of fluorescent labeled LAMP amplicons and a color sensor to detect the emitted florescence from target. Then it processes the sensor output and displays the detection results on liquid crystal display (LCD). The sensitivity test has been performed with detection limit up to 2.5 × 10(-3) ng/µL with different DNA concentrations of Salmonella bacteria. This system allows a rapid and automatic endpoint detection which could lead to the development of a point-of-care diagnosis device for foodborne pathogens detection in a resource-limited environment. PMID:25751077

  17. A Portable Automatic Endpoint Detection System for Amplicons of Loop Mediated Isothermal Amplification on Microfluidic Compact Disk Platform

    PubMed Central

    Uddin, Shah Mukim; Ibrahim, Fatimah; Sayad, Abkar Ahmed; Thiha, Aung; Pei, Koh Xiu; Mohktar, Mas S.; Hashim, Uda; Cho, Jongman; Thong, Kwai Lin

    2015-01-01

    In recent years, many improvements have been made in foodborne pathogen detection methods to reduce the impact of food contamination. Several rapid methods have been developed with biosensor devices to improve the way of performing pathogen detection. This paper presents an automated endpoint detection system for amplicons generated by loop mediated isothermal amplification (LAMP) on a microfluidic compact disk platform. The developed detection system utilizes a monochromatic ultraviolet (UV) emitter for excitation of fluorescent labeled LAMP amplicons and a color sensor to detect the emitted florescence from target. Then it processes the sensor output and displays the detection results on liquid crystal display (LCD). The sensitivity test has been performed with detection limit up to 2.5 × 10−3 ng/µL with different DNA concentrations of Salmonella bacteria. This system allows a rapid and automatic endpoint detection which could lead to the development of a point-of-care diagnosis device for foodborne pathogens detection in a resource-limited environment. PMID:25751077

  18. Meningococcal carriage rates in healthy individuals in Japan determined using Loop-Mediated Isothermal Amplification and oral throat wash specimens.

    PubMed

    Takahashi, Hideyuki; Haga, Masae; Sunagawa, Tomimasa; Saitoh, Takehito; Kitahara, Takeru; Matsumoto, Sohkichi; Ohnishi, Makoto

    2016-07-01

    The detailed epidemiology of meningococcal diseases in Japan has yet to be determined and, moreover, the healthy carriage rate is also unknown. In this study, to obtain insight into the carriage rate of Neisseria meningitidis in healthy individuals in Japan, we developed a new method to detect the N. meningitidis-specific ctrB gene, one of the genes encoding enzymes for capsule synthesis, by Loop-Mediated Isothermal Amplification (LAMP) and examined the meningococcal carriage rate by using self-collected oral throat wash specimens from 836 students at a university. Examination by LAMP showed that 7 out of 836 samples were positive for N. meningitidis DNA, and the results were also verified by the nested PCR method for the meningococcus specific ggt gene. The N. meningitidis carriage rate in healthy individuals was estimated to be 0.84%. Moreover, we further confirmed by the nested-PCR-based serogroup typing method that 5 of the positive samples belonged to serogroup Y, 1 belonged to group B and 1 was unidentifiable. Considering the epidemiology for meningococcal diseases in Japan, the carriage rate and the serogroup profile seem to be consistent with low incidence of meningococcal diseases and serogroup distribution of clinical meningococcal isolates in Japan, respectively. PMID:26895673

  19. Loop-mediated isothermal amplification (LAMP) assay for the rapid detection of the sexually-transmitted parasite, Trichomonas vaginalis.

    PubMed

    Adao, Davin Edric V; Rivera, Windell L

    2016-01-01

    A loop-mediated isothermal amplification (LAMP) assay was developed to detect the sexually-transmitted parasite, Trichomonas vaginalis in vaginal swabs. The presence of T. vaginalis was detected from 121 female sex workers attending a social hygiene clinic in Balibago, Angeles City, Pampanga, Philippines using culture, polymerase chain reaction (PCR), and the developed LAMP assay. The high analytical sensitivity of LAMP detected a higher prevalence of T. vaginalis (42.06%) compared to culture (8.26%) and PCR (7.44%). Additionally, this assay did not cross-react with DNAs of other trichomonads that can infect humans such as Trichomonas tenax and Pentatrichomonas hominis as well as the pathogens, Candida albicans and Staphylococcus aureus. The LAMP assay developed had a limit of detection (0.036 ng/μl) lower than that of PCR using the primers TvK3 and TvK7 (0.36 ng/μl). Prevalence of T. vaginalis in female sex workers in this area of the Philippines may be higher than previously estimated. Discordant results of PCR and LAMP may be due to different reactions to different kinds of inhibitors in the vaginal swabs. PMID:27262954

  20. Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for Rapid Detection of Aspergillus fumigatus.

    PubMed

    Tang, Qing; Tian, Shuguang; Yu, Nong; Zhang, Xi; Jia, Xiaodong; Zhai, Hongyan; Sun, Qun; Han, Li

    2016-04-01

    Aspergillus fumigatusis a conditional pathogen and the major cause of life-threatening invasive aspergillosis (IA) in immunocompromised patients. The early and rapid detection ofA. fumigatusinfection is still a major challenge. In this study, the new member of the fungal annexin family, annexin C4, was chosen as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, specific, and sensitive detection ofA. fumigatus The evaluation of the specificity of the LAMP assay that was developed showed that no false-positive results were observed for the 22 non-A. fumigatusstrains, including 5 species of theAspergillusgenus. Its detection limit was approximately 10 copies per reaction in reference plasmids, with higher sensitivity than that of real-time quantitative PCR (qPCR) at 10(2)copies for the same target. Clinical samples from a total of 69 patients with probable IA (n =14) and possible IA (n= 55) were subjected to the LAMP assay, and positive results were found for the 14 patients with probable IA (100%) and 34 patients with possible IA (61.82%). When detection using the LAMP assay was compared with that using qPCR in the 69 clinical samples, the LAMP assay demonstrated a sensitivity of 89.19% and the concordance rate for the two methods was 72.46%. Accordingly, we report that a valuable LAMP assay for the rapid, specific, and simple detection ofA. fumigatusin clinical testing has been developed. PMID:26791368

  1. Establishment and application of a loop-mediated isothermal amplification (LAMP) system for detection of cry1Ac transgenic sugarcane

    PubMed Central

    Zhou, Dinggang; Guo, Jinlong; Xu, Liping; Gao, Shiwu; Lin, Qingliang; Wu, Qibin; Wu, Luguang; Que, Youxiong

    2014-01-01

    To meet the demand for detection of foreign genes in genetically modified (GM) sugarcane necessary for regulation of gene technology, an efficient method with high specificity and rapidity was developed for the cry1Ac gene, based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed using the sequence of cry1Ac along with optimized reaction conditions: 5.25 mM of Mg2+, 4:1 ratio of inner primer to outer primer, 2.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. Three post-LAMP detection methods (precipitation, calcein (0.60 mM) with Mn2+ (0.05 mM) complex and SYBR Green I visualization), were shown to be effective. The sensitivity of the LAMP method was tenfold higher than that of conventional PCR when using templates of the recombinant cry1Ac plasmid or genomic DNA from cry1Ac transgenic sugarcane plants. More importantly, this system allowed detection of the foreign gene on-site when screening GM sugarcane without complex and expensive instruments, using the naked eye. This method can not only provide technological support for detection of cry1Ac, but can also further facilitate the use of this detection technique for other transgenes in GM sugarcane. PMID:24810230

  2. Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid and Sensitive Identification of Ostrich Meat

    PubMed Central

    Abdulmawjood, Amir; Grabowski, Nils; Fohler, Svenja; Kittler, Sophie; Nagengast, Helga; Klein, Guenter

    2014-01-01

    Animal species identification is one of the primary duties of official food control. Since ostrich meat is difficult to be differentiated macroscopically from beef, therefore new analytical methods are needed. To enforce labeling regulations for the authentication of ostrich meat, it might be of importance to develop and evaluate a rapid and reliable assay. In the present study, a loop-mediated isothermal amplification (LAMP) assay based on the cytochrome b gene of the mitochondrial DNA of the species Struthio camelus was developed. The LAMP assay was used in combination with a real-time fluorometer. The developed system allowed the detection of 0.01% ostrich meat products. In parallel, a direct swab method without nucleic acid extraction using the HYPLEX LPTV buffer was also evaluated. This rapid processing method allowed detection of ostrich meat without major incubation steps. In summary, the LAMP assay had excellent sensitivity and specificity for detecting ostrich meat and could provide a sampling-to-result identification-time of 15 to 20 minutes. PMID:24963709

  3. Rapid and Sensitive Detection of Plesiomonas shigelloides by Loop-Mediated Isothermal Amplification of the hugA Gene

    PubMed Central

    Meng, Shuang; Xu, Jianguo; Xiong, Yanwen; Ye, Changyun

    2012-01-01

    Plesiomonas shigelloides is one of the causative agents of human gastroenteritis, with increasing number of reports describing such infections in recent years. In this study, the hugA gene was chosen as the target to design loop-mediated isothermal amplification (LAMP) assays for the rapid, specific, and sensitive detection of P. shigelloides. The performance of the assay with reference plasmids and spiked human stools as samples was evaluated and compared with those of quantitative PCR (qPCR). No false-positive results were observed for the 32 non-P. shigelloides strains used to evaluate assay specificity. The limit of detection for P. shigelloides was approximately 20 copies per reaction in reference plasmids and 5×103 CFU per gram in spiked human stool, which were more sensitive than the results of qPCR. When applied in human stool samples spiked with 2 low levels of P. shigelloides, the LAMP assays achieved accurate detection after 6-h enrichment. In conclusion, the LAMP assay developed in this study is a valuable method for rapid, cost-effective, and simple detection of P. shigelloides in basic clinical and field laboratories in the rural areas of China. PMID:23077478

  4. Development and application of loop-mediated isothermal amplification for detecting the highly benzimidazole-resistant isolates in Sclerotinia sclerotiorum

    PubMed Central

    Duan, Ya Bing; Yang, Ying; Wang, Jian Xin; Liu, Cong Chao; He, Ling Ling; Zhou, Ming Guo

    2015-01-01

    Resistance of benzimidazole fungicides is related to the point mutation of the β-tubulin gene in Sclerotinia sclerotiorum. The point mutation at codon 198 (GAG → GCG, E198A) occurs in more than 90% of field resistant populations in China. Traditional detection methods of benzimidazole-resistant mutants of S. sclerotiorum are time-consuming, tedious and inefficient. To establish a suitable and rapid detection of benzimidazole-resistant mutants of S. sclerotiorum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). Eight sets of LAMP primers were designed and four sets were optimized to specially distinguish benzimidazole-resistant mutants of S. sclerotiorum. With the optimal LAMP primers, the concentration of LAMP components was optimized and the reaction conditions were set as 60–64 °C for 60 min. This method had a good specificity, sensitivity, stability and repeatability. In the 1491 sclerotia, 614 (41.18%) were positive by LAMP, and 629 (42.19%) positive by MIC. Therefore, the LAMP assay is more feasible to detect benzimidazole-resistant mutants of S. sclerotiorum than traditional detection methods. PMID:26606972

  5. Rapid and sensitive detection of Little cherry virus 2 using isothermal reverse transcription-recombinase polymerase amplification.

    PubMed

    Mekuria, Tefera A; Zhang, Shulu; Eastwell, Kenneth C

    2014-09-01

    Little cherry virus 2 (LChV2) (genus Ampelovirus) is the primary causal agent of little cherry disease (LCD) in sweet cherry (Prunus avium) in North America and other parts of the world. This mealybug-transmitted virus does not induce significant foliar symptoms in most sweet cherry cultivars, but does cause virus-infected trees to yield unevenly ripened small fruits with poor flavor. Most fruits from infected trees are unmarketable. In the present study, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) technique was developed using LChV2 coat protein specific primers and probe. Detection of terminally labeled amplicons was achieved with a high affinity lateral flow strip. The RT-RPA is confirmed to be simple, fast, and specific. In comparison, although it retains the sensitivity of RT-PCR, it is a more cost-effective procedure. RT-RPA will be a very useful tool for detecting LChV2 from crude extracts in any growth stage of sweet cherry from field samples. PMID:24797461

  6. Rapid and Sensitive Detection of RNA Viruses Based on Reverse Transcription Loop-Mediated Isothermal Amplification, Magnetic Nanoparticles, and Chemiluminescence.

    PubMed

    Wang, Jiuhai; Lu, Peng; Yan, Jieni; Zhang, Yufan; Huang, Lanye; Ali, Zeeshan; Li, Zhiyang; He, Nongyue

    2016-04-01

    RNA viruses, particularly, the highly pathogenic avian influenza (HPAI) virus, pose serious health concerns, and cause huge economic losses worldwide. Diagnostic tools for the early detection of these deadly RNA viruses are urgently needed to implement treatment and disease control strategies. Conventional reverse transcription polymerase chain reaction (RT-PCR)-based chemiluminescent (RT-PCR-CL) detection is frequently used for the diagnosis of viral infections. However, the requirements for expensive PCR machines and longer thermocycling times are significant drawbacks. In this study, we propose a method based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with chemiluminescence (CL) to detect H7N9 virus. The proposed method does not require any expensive instruments, and processing time is remarkably shortened compared to that of RT-PCR-CL. Since several factors including RT-LAMP temperature, probe concentration, hybridization temperature, and hybridization duration might affect the CL signal, each of these parameters was investigated and optimized. One thousand copies/mL of H7N9 RNA were detectable using the optimized RT-LAMP-CL method. The detection time was significantly reduced by using RT-LAMP, in comparison with conventional RT-PCR-CL. This technique holds great promise for viral detection and diagnosis, especially with regard to avian influenza virus. PMID:27301197

  7. Rapid detection of duck hepatitis A virus genotype C using reverse transcription loop-mediated isothermal amplification.

    PubMed

    Li, Chuanfeng; Chen, Zongyan; Meng, Chunchun; Liu, Guangqing

    2014-02-01

    A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was used and optimized to develop a rapid and sensitive detection system for duck hepatitis A virus genotype C (DHAV-C) RNA. A set of four specific primers was designed against highly conserved sequences located within the 3D gene from DHAV (strain GX1201). Under optimal reaction conditions, the sensitivity of DHAV-C-specific RT-LAMP was 100-fold higher than that of reverse transcriptase-polymerase chain reaction (RT-PCR), with a detection limit of 0.3pg (6.59×10(4) copies) per reaction. No cross-reactivity was observed from the samples of other duck viruses, which is in good accordance with RT-PCR. Furthermore, a positive reaction can be visually inspected by observing turbidity or color change after the addition of SYBR green I dye. The DHAV-C-specific RT-LAMP assay was applied to the samples and compared with RT-PCR. The positive-sample ratios were 26.7% (12 of 45) by RT-LAMP and 20% (9 of 45) by RT-PCR. Therefore, the newly developed RT-LAMP assay is a rapid, specific, sensitive, and cost-effective method of DHAV-C detection. This assay has potential applications in both clinical diagnosis and field surveillance of DHAV-C infection. PMID:24291148

  8. A Novel Loop-Mediated Isothermal Amplification Assay for Serogroup Identification of Neisseria meningitidis in Cerebrospinal Fluid.

    PubMed

    Lee, DoKyung; Kim, Eun Jin; Kilgore, Paul E; Takahashi, Hideyuki; Ohnishi, Makoto; Tomono, Jun; Miyamoto, Shigehiko; Omagari, Daisuke; Kim, Dong Wook; Seki, Mitsuko

    2015-01-01

    We have developed a novel Neisseria meningitidis serogroup-specific loop-mediated isothermal amplification (LAMP) assay for six of the most common meningococcal serogroups (A, B, C, W, X, and Y). The assay was evaluated using a set of 31 meningococcal LAMP assay positive cerebrospinal fluid (CSF) specimens from 1574 children with suspected meningitis identified in prospective surveillance between 1998 and 2002 in Vietnam, China, and Korea. Primer specificity was validated using 15 N. meningitidis strains (including serogroups A, B, C, E, W, X, Y, and Z) and 19 non-N. meningitidis species. The N. meningitidis serogroup LAMP detected down to ten copies and 100 colony-forming units per reaction. Twenty-nine CSF had N. meningitidis serogroup identified by LAMP compared with two CSF in which N. meningitidis serogroup was identified by culture and multi-locus sequence typing. This is the first report of a serogroup-specific identification assay for N. meningitidis using the LAMP method. Our results suggest that this assay will be a rapid, sensitive, and uniquely serogroup-specific assay with potential for application in clinical laboratories and public health surveillance systems. PMID:26793181

  9. Loop-mediated isothermal amplification (LAMP) for rapid detection of Renibacterium salmoninarum, the causative agent of bacterial kidney disease.

    PubMed

    Saleh, Mona; Soliman, Hatem; El-Matbouli, Mansour

    2008-08-27

    A loop-mediated isothermal amplification (LAMP) assay was developed for rapid, specific and sensitive detection of Renibacterium salmoninarum in 1 h without thermal cycling. A fragment of R. salmoninarum p57 gene was amplified at 63 degrees C in the presence of Bst polymerase and a specially designed primer mixture. The specificity of the BKD-LAMP assay was demonstrated by the absence of any cross reaction with other bacterial strains, followed by restriction digestion of the amplified products. Detections of BKD-LAMP amplicons by visual inspection, agrose gel electrophoresis, and real-time monitoring using a turbidimeter were equivalently sensitive. The BKD-LAMP assay has the sensitivity of the nested PCR method, and 10 times the sensitivity of one-round PCR assay. The lower detection limit of BKD-LAMP and nested PCR is 1 pg genomic R. salmoninarum DNA, compared to 10 pg genomic R. salmoninarum DNA for one-round PCR assay. In comparison to other available diagnostic methods, the BKD-LAMP assay is rapid, simple, sensitive, specific, and cost effective with a high potential for field application. PMID:18924379

  10. Development of a loop-mediated isothermal amplification method for the rapid diagnosis of Ascochyta rabiei L. in chickpeas.

    PubMed

    Chen, Xiaolu; Ma, Lijuan; Qiang, Song; Ma, Deying

    2016-01-01

    Ascochyta blight (AB) is a devastating fungal disease of chickpeas that has spread to nearly all of the chickpea cultivating regions of the world. The rapid diagnosis of Ascochyta rabiei L. (A. rabiei), the pathogen that causes AB, plays an important role in A. rabiei epidemic tracking and AB management. In this study, a group of loop-mediated isothermal amplification (LAMP) primers was designed to detect A. rabiei in chickpea plants and seeds via a LAMP method and a conventional PCR method based on an internal transcribed spacer (ITS) sequence analysis of A. rabiei. Compared with the conventional PCR method, the LAMP method not only exhibited greater sensitivity and specificity in the detection of A. rabiei but also used simpler equipment and required less operational time. The minimum detectable concentration of the A. rabiei genomic DNA solution with the LAMP method was 6.01 × 10(-6 )ng/μl, which was 100 times lower than that of the conventional PCR method with the same outer primers. The greatest advantage of the LAMP method is that results can be observed via the visualization of color changes in SYBR Green I dye with the naked eye, and it does not require expensive instruments, also with less time consumption. PMID:27161564

  11. Rapid genome detection of Schmallenberg virus and bovine viral diarrhea virus by use of isothermal amplification methods and high-speed real-time reverse transcriptase PCR.

    PubMed

    Aebischer, Andrea; Wernike, Kerstin; Hoffmann, Bernd; Beer, Martin

    2014-06-01

    Over the past few years, there has been an increasing demand for rapid and simple diagnostic tools that can be applied outside centralized laboratories by using transportable devices. In veterinary medicine, such mobile test systems would circumvent barriers associated with the transportation of samples and significantly reduce the time to diagnose important infectious animal diseases. Among a wide range of available technologies, high-speed real-time reverse transcriptase quantitative PCR (RT-qPCR) and the two isothermal amplification techniques loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) represent three promising candidates for integration into mobile pen-side tests. The aim of this study was to investigate the performance of these amplification strategies and to evaluate their suitability for field application. In order to enable a valid comparison, novel pathogen-specific assays have been developed for the detection of Schmallenberg virus and bovine viral diarrhea virus. The newly developed assays were evaluated in comparison with established standard RT-qPCR using samples from experimentally or field-infected animals. Even though all assays allowed detection of the target virus in less than 30 min, major differences were revealed concerning sensitivity, specificity, robustness, testing time, and complexity of assay design. These findings indicated that the success of an assay will depend on the integrated amplification technology. Therefore, the application-specific pros and cons of each method that were identified during this study provide very valuable insights for future development and optimization of pen-side tests. PMID:24648561

  12. Development of reverse transcription loop-mediated isothermal amplification assay as a simple detection method of Chrysanthemum stem necrosis virus in chrysanthemum and tomato.

    PubMed

    Suzuki, Ryoji; Fukuta, Shiro; Matsumoto, Yuho; Hasegawa, Toru; Kojima, Hiroko; Hotta, Makiko; Miyake, Noriyuki

    2016-10-01

    For a simple and rapid detection of Chrysanthemum stem necrosis virus (CSNV) from chrysanthemum and tomato, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed. A primer set designed to the genome sequences of CSNV worked most efficiently at 63°C and could detect CSNV RNA within 12min by fluorescence monitoring using an isothermal DNA amplification and fluorescence detection device. The result of a specificity test using seven other viruses and one viroid-infectable chrysanthemum or tomato showed that the assay could amplify CSNV specifically, and a sensitivity comparison showed that the RT-LAMP assay was as sensitive as the reverse transcriptase polymerase chain reaction. The RT-LAMP assay using crude RNA, extracted simply, could detect CSNV. Overall, the RT-LAMP assay was found to be a simple, specific, convenient, and time-saving method for CSNV detection. PMID:27400833

  13. Loop-mediated isothermal amplification for diagnosis of 18 World Organization for Animal Health (OIE) notifiable viral diseases of ruminants, swine and poultry.

    PubMed

    Mansour, Shimaa M G; Ali, Haytham; Chase, Christopher C L; Cepica, Arnost

    2015-12-01

    Loop-mediated isothermal amplification (LAMP) is a simple, powerful state-of-the-art gene amplification technique used for the rapid diagnosis and early detection of microbial diseases. Many LAMP assays have been developed and validated for important epizootic diseases of livestock. We review the LAMP assays that have been developed for the detection of 18 viruses deemed notifiable of ruminants, swine and poultry by the World Organization for Animal Health (OIE). LAMP provides a fast (the assay often takes less than an hour), low cost, highly sensitive, highly specific and less laborious alternative to detect infectious disease agents. The LAMP procedure can be completed under isothermal conditions so thermocyclers are not needed. The ease of use of the LAMP assay allows adaptability to field conditions and works well in developing countries with resource-limited laboratories. However, this technology is still underutilized in the field of veterinary diagnostics despite its huge capabilities. PMID:25900363

  14. A lab-on-a-chip system with integrated sample preparation and loop-mediated isothermal amplification for rapid and quantitative detection of Salmonella spp. in food samples.

    PubMed

    Sun, Yi; Quyen, Than Linh; Hung, Tran Quang; Chin, Wai Hoe; Wolff, Anders; Bang, Dang Duong

    2015-04-21

    Foodborne disease is a major public health threat worldwide. Salmonellosis, an infectious disease caused by Salmonella spp., is one of the most common foodborne diseases. Isolation and identification of Salmonella by conventional bacterial culture or molecular-based methods are time consuming and usually take a few hours to days to complete. In response to the demand for rapid on line or on site detection of pathogens, in this study, we describe for the first time an eight-chamber lab-on-a-chip (LOC) system with integrated magnetic bead-based sample preparation and loop-mediated isothermal amplification (LAMP) for rapid and quantitative detection of Salmonella spp. in food samples. The whole diagnostic procedures including DNA isolation, isothermal amplification, and real-time detection were accomplished in a single chamber. Up to eight samples could be handled simultaneously and the system was capable to detect Salmonella at concentration of 50 cells per test within 40 min. The simple design, together with high level of integration, isothermal amplification, and quantitative analysis of multiple samples in short time, will greatly enhance the practical applicability of the LOC system for rapid on-site screening of Salmonella for applications in food safety control, environmental surveillance, and clinical diagnostics. PMID:25715949

  15. An integrated direct loop-mediated isothermal amplification microdevice incorporated with an immunochromatographic strip for bacteria detection in human whole blood and milk without a sample preparation step.

    PubMed

    Lee, Dohwan; Kim, Yong Tae; Lee, Jee Won; Kim, Do Hyun; Seo, Tae Seok

    2016-05-15

    We have developed an integrated direct loop-mediated isothermal amplification (Direct LAMP) microdevice incorporated with an immunochromatographic strip (ICS) to identify bacteria contaminated in real samples. The Direct LAMP is a novel isothermal DNA amplification technique which does not require thermal cycling steps as well as any sample preparation steps such as cell lysis and DNA extraction for amplifying specific target genes. In addition, the resultant amplicons were colorimetrically detected on the ICS, thereby enabling the entire genetic analysis process to be simplified. The two functional units (Direct LAMP and ICS) were integrated on a single device without use of the tedious and complicated microvalve and tubing systems. The utilization of a slidable plate allows us to manipulate the fluidic control in the microchannels manually and the sequential operation of the Direct LAMP and ICS detection could be performed by switching the slidable plate to each functional unit. Thus, the combination of the direct isothermal amplification without any sample preparation and thermal cycling steps, the ICS based amplicon detection by naked eyes, and the slidable plate to eliminate the microvalves in the integrated microdevice would be an ideal platform for point-of-care DNA diaganotics. On the integrated Direct LAMP-ICS microdevice, we could analyze Staphylococcus aureus (S. aureus) and Escherichia coli O157:H7 (E. coli O157:H7) contaminated in human whole blood or milk at a single-cell level within 1h. PMID:26710344

  16. Loop-mediated isothermal amplification assay for 16S rRNA methylase genes in Gram-negative bacteria.

    PubMed

    Nagasawa, Mitsuaki; Kaku, Mitsuo; Kamachi, Kazunari; Shibayama, Keigo; Arakawa, Yoshichika; Yamaguchi, Keizo; Ishii, Yoshikazu

    2014-10-01

    Using the loop-mediated isothermal amplification (LAMP) method, we developed a rapid assay for detection of 16S rRNA methylase genes (rmtA, rmtB, and armA), and investigated 16S rRNA methylase-producing strains among clinical isolates. Primer Explorer V3 software was used to design the LAMP primers. LAMP primers were prepared for each gene, including two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB). Detection was performed with the Loopamp DNA amplification kit. For all three genes (rmtA, rmtB, and armA), 10(2) copies/tube could be detected with a reaction time of 60 min. When nine bacterial species (65 strains saved in National Institute of Infectious Diseases) were tested, which had been confirmed to possess rmtA, rmtB, or armA by PCR and DNA sequencing, the genes were detected correctly in these bacteria with no false negative or false positive results. Among 8447 clinical isolates isolated at 36 medical institutions, the LAMP method was conducted for 191 strains that were resistant to aminoglycosides based on the results of antimicrobial susceptibility tests. Eight strains were found to produce 16S rRNA methylase (0.09%), with rmtB being identified in three strains (0.06%) of 4929 isolates of Enterobacteriaceae, rmtA in three strains (0.10%) of 3284 isolates of Pseudomonas aeruginosa, and armA in two strains (0.85%) of 234 isolates of Acinetobacter spp. At present, the incidence of strains possessing 16S rRNA methylase genes is very low in Japan. However, when Gram-negative bacteria showing high resistance to aminoglycosides are isolated by clinical laboratories, it seems very important to investigate the status of 16S rRNA methylase gene-harboring bacilli and monitor their trends among Japanese clinical settings. PMID:25179393

  17. Surveillance for Western Equine Encephalitis, St. Louis Encephalitis, and West Nile Viruses Using Reverse Transcription Loop-Mediated Isothermal Amplification.

    PubMed

    Wheeler, Sarah S; Ball, Cameron S; Langevin, Stanley A; Fang, Ying; Coffey, Lark L; Meagher, Robert J

    2016-01-01

    Collection of mosquitoes and testing for vector-borne viruses is a key surveillance activity that directly influences the vector control efforts of public health agencies, including determining when and where to apply insecticides. Vector control districts in California routinely monitor for three human pathogenic viruses including West Nile virus (WNV), Western equine encephalitis virus (WEEV), and St. Louis encephalitis virus (SLEV). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) offers highly sensitive and specific detection of these three viruses in a single multiplex reaction, but this technique requires costly, specialized equipment that is generally only available in centralized public health laboratories. We report the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect WNV, WEEV, and SLEV RNA extracted from pooled mosquito samples collected in California, including novel primer sets for specific detection of WEEV and SLEV, targeting the nonstructural protein 4 (nsP4) gene of WEEV and the 3' untranslated region (3'-UTR) of SLEV. Our WEEV and SLEV RT-LAMP primers allowed detection of <0.1 PFU/reaction of their respective targets in <30 minutes, and exhibited high specificity without cross reactivity when tested against a panel of alphaviruses and flaviviruses. Furthermore, the SLEV primers do not cross-react with WNV, despite both viruses being closely related members of the Japanese encephalitis virus complex. The SLEV and WEEV primers can also be combined in a single RT-LAMP reaction, with discrimination between amplicons by melt curve analysis. Although RT-qPCR is approximately one order of magnitude more sensitive than RT-LAMP for all three targets, the RT-LAMP technique is less instrumentally intensive than RT-qPCR and provides a more cost-effective method of vector-borne virus surveillance. PMID:26807734

  18. Evaluation of loop-mediated isothermal amplification for the rapid, reliable, and robust detection of Salmonella in produce.

    PubMed

    Yang, Qianru; Wang, Fei; Jones, Kelly L; Meng, Jianghong; Prinyawiwatkul, Witoon; Ge, Beilei

    2015-04-01

    Rapid, reliable, and robust detection of Salmonella in produce remains a challenge. In this study, loop-mediated isothermal amplification (LAMP) was comprehensively evaluated against real-time quantitative PCR (qPCR) for detecting diverse Salmonella serovars in various produce items (cantaloupe, pepper, and several varieties of lettuce, sprouts, and tomato). To mimic real-world contamination events, produce samples were surface-inoculated with low concentrations (1.1-2.9 CFU/25 g) of individual Salmonella strains representing ten serovars and tested after aging at 4 °C for 48 h. Four DNA extraction methods were also compared using produce enrichment broths. False-positive or false-negative results were not observed among 178 strains (151 Salmonella and 27 non-Salmonella) used to evaluate assay specificity. The detection limits for LAMP were 1.8-4 CFU per reaction in pure culture and 10(4)-10(6) CFU per 25 g (i.e., 10(2)-10(4) CFU per g) in produce without enrichment, comparable to those obtained by qPCR. After 6-8 h of enrichment, both LAMP and qPCR consistently detected these low concentrations of Salmonella of diverse serovars in all produce items except sprouts. The PrepMan Ultra sample preparation reagent yielded the best results among the four DNA extraction methods. Upon further validation, LAMP may be a valuable tool for routine Salmonella testing in produce. The difficulty of detecting Salmonella in sprouts, whether using LAMP or qPCR, warrants further study. PMID:25475319

  19. Surveillance for Western equine encephalitis St. Louis encephalitis and West Nile viruses using reverse transcription loop-mediated isothermal amplification

    DOE PAGESBeta

    Meagher, Robert J.; Ball, Cameron Scott; Langevin, Stanley A.; Fang, Ying; Wheeler, Sarah S.; Coffey, Lark L.

    2016-01-25

    In this study, collection of mosquitoes and testing for vector-borne viruses is a key surveillance activity that directly influences the vector control efforts of public health agencies, including determining when and where to apply insecticides. Vector control districts in California routinely monitor for three human pathogenic viruses including West Nile virus (WNV), Western equine encephalitis virus (WEEV), and St. Louis encephalitis virus (SLEV). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) offers highly sensitive and specific detection of these three viruses in a single multiplex reaction, but this technique requires costly, specialized equipment that is generally only available in centralized publicmore » health laboratories. We report the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect WNV, WEEV, and SLEV RNA extracted from pooled mosquito samples collected in California, including novel primer sets for specific detection of WEEV and SLEV, targeting the nonstructural protein 4 (nsP4) gene of WEEV and the 3’ untranslated region (3’-UTR) of SLEV. Our WEEV and SLEV RT-LAMP primers allowed detection of <0.1 PFU/reaction of their respective targets in <30 minutes, and exhibited high specificity without cross reactivity when tested against a panel of alphaviruses and flaviviruses. Furthermore, the SLEV primers do not cross-react with WNV, despite both viruses being closely related members of the Japanese encephalitis virus complex. The SLEV and WEEV primers can also be combined in a single RT-LAMP reaction, with discrimination between amplicons by melt curve analysis. Although RT-qPCR is approximately one order of magnitude more sensitive than RT-LAMP for all three targets, the RT-LAMP technique is less instrumentally intensive than RT-qPCR and provides a more cost-effective method of vector-borne virus surveillance.« less

  20. Performance of reversed transcription loop-mediated isothermal amplification technique detecting EV71: a systematic review with meta-analysis.

    PubMed

    Lei, Xiaoying; Wen, Hongling; Zhao, Li; Yu, Xuejie

    2014-04-01

    Human enterovirus 71 (EV71) is the major etiological agent of hand, foot and mouth disease (HFMD), which is a common infectious disease in young children. Studies in the past have shown that reversed transcription loop-mediated isothermal amplification (RT-LAMP) was a rapid approach for the detection of EV71 in HFMD. This meta-analysis study is to evaluate the diagnostic role of RT-LAMP in detecting EV71 infection. A comprehensive literature research of PubMed, Embase, Wan Fang Data, and Chinese National Knowledge Infrastructure databases was conducted on articles aiming at the diagnostic performance of RT-LAMP in EV71 detection published before February 10, 2014. Data from selected studies were pooled to yield the summary sensitivity, specificity, positive and negative likelihood ratio (PLR, NLR), diagnostic odds ratio (DOR), and receiver operating characteristic (SROC) curve by using STATA VERSION 12.0 software. Ten studies including a total of 907 clinical samples were of high quality in this meta-analysis. Overall, the pooled sensitivity, specificity, PLR, NLR, DOR, and the area under the SROC curve was 0.99 (0.97, 1.00), 0.97 (0.94, 1.00), 5.90 (95% CI: 3.90-8.94), 0.20 (95% CI: 0.14-0.29), and 1.00 (95% CI: 0.99-1.00), respectively. The univariate analysis of potential variables showed some changes in the diagnostic performance, but none of the differences reached statistical significance. Despite inter-study variability, the test performance of RT-LAMP was consistent with real-time RT-PCR in detecting EV71. This meta-analysis suggests that RT-LAMP is a useful diagnostic tool with high sensitivity and specificity for detecting EV71. PMID:24815384

  1. Loop-Mediated Isothermal Amplification Assays for Detecting Shiga Toxin-Producing Escherichia coli in Ground Beef and Human Stools

    PubMed Central

    Wang, Fei; Jiang, Lin

    2012-01-01

    Shiga toxin-producing Escherichia coli (STEC), encompassing E. coli O157 and non-O157 STEC, is a significant cause of food-borne illnesses and deaths in the United States and worldwide. Shiga toxins (encoded by stx) and intimin (encoded by eae) are important virulence factors for STEC strains linked to severe human illnesses such as hemorrhagic colitis and hemolytic-uremic syndrome. In this study, the stx1, stx2, and eae genes were chosen as targets to design loop-mediated isothermal amplification (LAMP) assays for the rapid, specific, sensitive, and quantitative detection of STEC strains. The assay performances in pure culture and spiked ground beef and human stools were evaluated and compared with those of quantitative PCR (qPCR). No false-positive or false-negative results were observed among 90 bacterial strains used to evaluate assay specificity. The limits of detection for seven STEC strains of various serogroups (O26, O45, O103, O111, O121, O145, and O157) were approximately 1 to 20 CFU/reaction in pure culture and 103 to 104 CFU/g in spiked ground beef, which were comparable to the results of qPCR. Standard curves generated suggested good linear relationships between STEC cell numbers and LAMP turbidity signals. When applied in ground beef samples spiked with two low levels (1 to 2 and 10 to 20 CFU/25 g) of STEC cultures, the LAMP assays achieved accurate detection after 6 to 8 h enrichment. The assays also consistently detected STEC in human stool specimens spiked with 103 or 104 CFU/0.5 g stool after 4 h enrichment, while qPCR required 4 to 6 h. In conclusion, the LAMP assays developed in this study may facilitate rapid and reliable identification of STEC contaminations in high-risk food commodities and also facilitate prompt diagnosis of STEC infections in clinical laboratories. PMID:22031701

  2. Genome Filtering for New DNA Biomarkers of Loa loa Infection Suitable for Loop-Mediated Isothermal Amplification

    PubMed Central

    Poole, Catherine B.; Ettwiller, Laurence; Tanner, Nathan A.; Evans, Thomas C.; Wanji, Samuel; Carlow, Clotilde K. S.

    2015-01-01

    Loa loa infections have emerged as a serious public health problem in patients co-infected with Onchocerca volvulus or Wuchereria bancrofti because of severe adverse neurological reactions after treatment with ivermectin. Accurate diagnostic tests are needed for careful mapping in regions where mass drug administration is underway. Loop-mediated isothermal amplification (LAMP) has become a widely adopted screening method because of its operational simplicity, rapidity and versatility of visual detection readout options. Here, we present a multi-step bioinformatic pipeline to generate diagnostic candidates suitable for LAMP and experimentally validate this approach using one of the identified candidates to develop a species-specific LAMP assay for L. loa. The pipeline identified ~140 new L. loa specific DNA repeat families as putative biomarkers of infection. The consensus sequence of one family, repeat family 4 (RF4), was compiled from ~ 350 sequences dispersed throughout the L. loa genome and maps to a L. loa-specific region of the long terminal repeats found at the boundaries of Bel/Pao retrotransposons. PCR and LAMP primer sets targeting RF4 specifically amplified L. loa but not W. bancrofti, O. volvulus, Brugia malayi, human or mosquito DNA. RF4 LAMP detects the DNA equivalent of one microfilaria (100 pg) in 25–30 minutes and as little as 0.060 pg of L. loa DNA (~1/1600th of a microfilaria) purified from spiked blood samples in approximately 50 minutes. In summary, we have successfully employed a bioinformatic approach to mine the L. loa genome for species-specific repeat families that can serve as new DNA biomarkers for LAMP. The RF4 LAMP assay shows promise as a field tool for the implementation and management of mass drug administration programs and warrants further testing on clinical samples as the next stage in development towards this goal. PMID:26414073

  3. Determination of the prevalence of trypanosome species in cattle from Monduli district, northern Tanzania, by loop mediated isothermal amplification.

    PubMed

    Haji, Isihaka Juma; Sugimoto, Chihiro; Kajino, Kiichi; Malele, Imna; Simukoko, Humphrey; Chitambo, Harrison; Namangala, Boniface

    2015-08-01

    Bovine African trypanosomosis (BAT) remains one of the major vector-borne diseases with serious impediment to cattle production and economic advancement in sub-Saharan Africa. The present study evaluated the performance of the trypanosome-species-specific loop-mediated isothermal amplification (LAMP), using parasite DNA obtained from 295 indigenous Tanzanian short horn Zebu (TSHZ) and Boran crosses in Monduli district within northern Tanzania, against routine microscopy on Giemsa-stained blood films. Compared to parasitological data in which the prevalence of BAT was estimated at 2.4% (95% CI 0.7-4.1%), LAMP increased the prevalence to 27.8% (95% CI 22.3-32.5%), of which 11.9% (95% CI 8.2-15.6%) were monolytic infections with Trypanosoma vivax, while 13.6% (95% CI 9.7-17.5%) were coinfections of either T. vivax and Trypanosoma brucei subspecies or T. vivax and Trypanosoma congolense, respectively. Among the T. brucei subspecies detected, 0.7% (95% CI 0-1.7%) were human-infective Trypanosoma brucei rhodesiense. Our study is in concordance with previous reports and suggests that LAMP is a potential tool for routine diagnosis of trypanosomes in domestic animals in BAT endemic regions. According to LAMP, T. vivax seems to be the predominant trypanosome species circulating among the indigenous Monduli cattle. Importantly, the detection of T. b. rhodesiense in cattle in such wildlife-domestic-animal-human-interface areas poses a risk of contracting human African trypanosomiasis (HAT) by local communities and tourists. Continuous trypanosome surveillances in domestic animals, humans, and tsetse flies using sensitive and specific tests such as LAMP are recommended. PMID:25953023

  4. Visual Detection of Brucella spp. in Spiked Bovine Semen Using Loop-Mediated Isothermal Amplification (LAMP) Assay.

    PubMed

    Prusty, Bikash R; Chaudhuri, Pallab; Chaturvedi, V K; Saini, Mohini; Mishra, B P; Gupta, Praveen K

    2016-06-01

    Several pathogens including Brucella spp. are shed in semen of infected bulls and can be transmitted to cows through contaminated semen during artificial insemination. The present study reports omp2a and bcsp31 gene based loop-mediated isothermal amplification (LAMP) assays for detection of Brucella genomic DNA in semen from infected bulls. The positive results could be interpreted visually by change in colour of reaction mixture containing hydroxyl naphthol blue (HNB) dye from violet to sky blue. LAMP assays based on omp2a and bcsp31 could detect as little as 10 and 100 fg of B. abortus S19 genomic DNA, respectively. Sensitivity of omp2a and bcsp31 LAMP assays for direct detection of organisms in bovine semen was 2.28 × 10(1) CFU and 2.28 × 10(2) CFU of B. abortus S19 in spiked bovine semen, respectively. The omp2a LAMP assay was found equally sensitive to TaqMan probe based real-time PCR and 100 times more sensitive than conventional PCR in identifying Brucella in spiked semen. The diagnostic applicability of the omp2a LAMP assay was evaluated with seventy-nine bovine semen samples and results were re-evaluated through TaqMan probe based real-time PCR and conventional PCR. Taken together, the omp2a LAMP assay is easy to perform, rapid and sensitive in diagnosis of Brucella spp. in bovine semen. PMID:27570305

  5. Genome Filtering for New DNA Biomarkers of Loa loa Infection Suitable for Loop-Mediated Isothermal Amplification.

    PubMed

    Poole, Catherine B; Ettwiller, Laurence; Tanner, Nathan A; Evans, Thomas C; Wanji, Samuel; Carlow, Clotilde K S

    2015-01-01

    Loa loa infections have emerged as a serious public health problem in patients co-infected with Onchocerca volvulus or Wuchereria bancrofti because of severe adverse neurological reactions after treatment with ivermectin. Accurate diagnostic tests are needed for careful mapping in regions where mass drug administration is underway. Loop-mediated isothermal amplification (LAMP) has become a widely adopted screening method because of its operational simplicity, rapidity and versatility of visual detection readout options. Here, we present a multi-step bioinformatic pipeline to generate diagnostic candidates suitable for LAMP and experimentally validate this approach using one of the identified candidates to develop a species-specific LAMP assay for L. loa. The pipeline identified ~140 new L. loa specific DNA repeat families as putative biomarkers of infection. The consensus sequence of one family, repeat family 4 (RF4), was compiled from ~ 350 sequences dispersed throughout the L. loa genome and maps to a L. loa-specific region of the long terminal repeats found at the boundaries of Bel/Pao retrotransposons. PCR and LAMP primer sets targeting RF4 specifically amplified L. loa but not W. bancrofti, O. volvulus, Brugia malayi, human or mosquito DNA. RF4 LAMP detects the DNA equivalent of one microfilaria (100 pg) in 25-30 minutes and as little as 0.060 pg of L. loa DNA (~1/1600th of a microfilaria) purified from spiked blood samples in approximately 50 minutes. In summary, we have successfully employed a bioinformatic approach to mine the L. loa genome for species-specific repeat families that can serve as new DNA biomarkers for LAMP. The RF4 LAMP assay shows promise as a field tool for the implementation and management of mass drug administration programs and warrants further testing on clinical samples as the next stage in development towards this goal. PMID:26414073

  6. Rapid and simple identification of Beijing genotype strain of Mycobacterium tuberculosis using a loop-mediated isothermal amplification assay.

    PubMed

    Nagai, Yuhki; Iwade, Yoshito; Nakano, Manabu; Akachi, Shigehiro; Kobayashi, Takashi; Nishinaka, Takamichi

    2016-07-01

    Beijing genotype strains of Mycobacterium tuberculosis are geographically widespread and pose a notorious public health problem, these strains causing outbreaks of multidrug-resistant tuberculosis (TB); some studies have reported an association with drug resistance. Because the prevalence of Beijing strain has a substantial impact on TB control programs, the availability of a rapid and reliable method for detecting these strains is important for epidemiological monitoring of their circulation. The main methods currently used to identify Beijing genotype strains are IS6110 DNA fingerprinting, spoligotyping and PCR to detect specific deletions such as region of difference (RD)207. More recently, multiplex PCR assay using a Beijing-specific single nucleotide polymorphism (SNP) has been developed for detecting Beijing lineage strains. However, these methods are time-consuming and technically demanding. In the present study, a loop-mediated isothermal amplification (LAMP) assay that allows specific identification of Beijing genotype strain was developed. This Beijing genotype strain-identifying LAMP assay was performed 214 clinical isolates and the results compared with those of conventional PCR that targeted RD207 and Rv0679c-targreting multiplex PCR for Beijing lineage identification. LAMP assay showed 100% sensitivity and specificity compared with RD207-PCR. Furthermore, the sensitivity and specificity were 99.3% and 100%, respectively, compared with Rv0679c-multiplex PCR. This LAMP assay could be used routinely in local laboratories to monitor the prevalence of the Beijing genotype strain and thereby used to help control the spread of these potentially highly virulent and drug resistant strains. PMID:27213686

  7. Development of loop-mediated isothermal amplification to detect Streptococcus suis and its application to retail pork meat in Japan.

    PubMed

    Arai, Sakura; Tohya, Mari; Yamada, Ryoko; Osawa, Ro; Nomoto, Ryohei; Kawamura, Yoshiaki; Sekizaki, Tsutomu

    2015-09-01

    We here developed a novel loop-mediated isothermal amplification (LAMP) method to detect Streptococcus suis in raw pork meat. This method, designated LAMPSS, targeted the recombination/repair protein (recN) gene of S. suis and detected all serotypes of S. suis, except those taxonomically removed from authentic S. suis, i.e., serotypes 20, 22, 26, 32, 33, and 34. The specificity of LAMPSS was confirmed and its detection limit was 5.4cfu/reaction. Among the 966 raw pork meat samples examined, including sliced pork, minced pork, and the liver, tongue, heart, and small intestine, 255 samples tested positive with LAMPSS. The rate of contamination was higher in the organs than in pork. No significant difference was observed in the total bacterial count between LAMPSS-positive and -negative samples. The number of shops that provided LAMPSS-positive pork was slightly higher in those that sold swine organs and pork than in those that sold only pork, suggesting that cross contamination occurred from the organs to pork. Among the 255 which tested positive for LAMPSS, only 47 samples tested positive for the previously described LAMP specific for S. suis serotype 2. Two isolates of S. suis serotype 2, belonging to sequence type 28, which is potentially hazardous to humans, as well as those of some other serotypes were obtained from 19 out of 47 samples by combining LAMP with a replica plating method. These results suggest that LAMPSS will be a useful tool for the surveillance of raw pork meat in the retail market. PMID:26043307

  8. Development of a loop-mediated isothermal amplification method for rapid detection of porcine boca-like virus.

    PubMed

    Li, Bin; Ma, Jun-jie; Xiao, Shao-bo; Zhang, Xue-han; Wen, Li-bin; Mao, Li; Ni, Yan-xiu; Guo, Rong-li; Zhou, Jun-ming; Lv, Li-xin; He, Kong-wang

    2012-02-01

    The porcine boca-like virus (Pbo-likeV) was recently discovered in Swedish pigs with post-weaning multisystemic wasting syndrome (PMWS). In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for rapid, specific and sensitive detection of Pbo-likeV. A set of four primers specific for six regions of Pbo-likeV VP1/2 genes was designed with the online software. The reaction temperature and time were optimized to 65 °C and 60 min, respectively. LAMP products were detected by agarose gel electrophoresis or by visual inspection of a color change due to addition of fluorescent dye. The developed method was highly specific for detection of Pbo-likeV, and no cross-reaction was observed with other swine viruses, such as porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV) and classic swine fever virus (CSFV) found commonly in China. The lower detection limit of the LAMP assay was approximately 10 copies per reaction, and it was 100 times more sensitive than that of conventional PCR. Furthermore, the efficiency of LAMP for detection Pbo-likeV in clinical samples was comparable to PCR and sequencing. These results showed that the LAMP assay is a simple, rapid, sensitive and specific technique for detection of Pbo-likeV, and the procedure of LAMP does not rely on any special equipment. It has capacity for the detection of Pbo-likeV both in the laboratory and on farms. PMID:22172971

  9. Surveillance for Western Equine Encephalitis, St. Louis Encephalitis, and West Nile Viruses Using Reverse Transcription Loop-Mediated Isothermal Amplification

    PubMed Central

    Wheeler, Sarah S.; Ball, Cameron S.; Langevin, Stanley A.; Fang, Ying; Coffey, Lark L.; Meagher, Robert J.

    2016-01-01

    Collection of mosquitoes and testing for vector-borne viruses is a key surveillance activity that directly influences the vector control efforts of public health agencies, including determining when and where to apply insecticides. Vector control districts in California routinely monitor for three human pathogenic viruses including West Nile virus (WNV), Western equine encephalitis virus (WEEV), and St. Louis encephalitis virus (SLEV). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) offers highly sensitive and specific detection of these three viruses in a single multiplex reaction, but this technique requires costly, specialized equipment that is generally only available in centralized public health laboratories. We report the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect WNV, WEEV, and SLEV RNA extracted from pooled mosquito samples collected in California, including novel primer sets for specific detection of WEEV and SLEV, targeting the nonstructural protein 4 (nsP4) gene of WEEV and the 3’ untranslated region (3’-UTR) of SLEV. Our WEEV and SLEV RT-LAMP primers allowed detection of <0.1 PFU/reaction of their respective targets in <30 minutes, and exhibited high specificity without cross reactivity when tested against a panel of alphaviruses and flaviviruses. Furthermore, the SLEV primers do not cross-react with WNV, despite both viruses being closely related members of the Japanese encephalitis virus complex. The SLEV and WEEV primers can also be combined in a single RT-LAMP reaction, with discrimination between amplicons by melt curve analysis. Although RT-qPCR is approximately one order of magnitude more sensitive than RT-LAMP for all three targets, the RT-LAMP technique is less instrumentally intensive than RT-qPCR and provides a more cost-effective method of vector-borne virus surveillance. PMID:26807734

  10. A Pilot Study of Quantitative Loop-mediated Isothermal Amplification-guided Target Therapies for Hospital-acquired Pneumonia

    PubMed Central

    Wang, Fang; Li, Ran; Shang, Ying; Wang, Can; Wang, Guo-Qing; Zhou, De-Xun; Yang, Dong-Hong; Xi, Wen; Wang, Ke-Qiang; Bao, Jing; Kang, Yu; Gao, Zhan-Cheng

    2016-01-01

    Background: It is important to achieve the definitive pathogen identification in hospital-acquired pneumonia (HAP), but the traditional culture results always delay the target antibiotic therapy. We assessed the method called quantitative loop-mediated isothermal amplification (qLAMP) as a new implement for steering of the antibiotic decision-making in HAP. Methods: Totally, 76 respiratory tract aspiration samples were prospectively collected from 60 HAP patients. DNA was isolated from these samples. Specific DNA fragments for identifying 11 pneumonia-related bacteria were amplified by qLAMP assay. Culture results of these patients were compared with the qLAMP results. Clinical data and treatment strategies were analyzed to evaluate the effects of qLAMP results on clinical data. McNemar test and Fisher's exact test were used for statistical analysis. Results: The detection of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Stenotrophomonas maltophilia, Streptococcus pneumonia, and Acinetobacter baumannii by qLAMP was consistent with sputum culture (P > 0.05). The qLAMP results of 4 samples for Haemophilus influenzae, Legionella pneumophila, or Mycoplasma pneumonia (MP) were inconsistent with culture results; however, clinical data revealed that the qLAMP results were all reliable except 1 MP positive sample due to the lack of specific species identified in the final diagnosis. The improvement of clinical condition was more significant (P < 0.001) in patients with pathogen target-driven therapy based on qLAMP results than those with empirical therapy. Conclusion: qLAMP is a more promising method for detection of pathogens in an early, rapid, sensitive, and specific manner than culture. PMID:26830989

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

    PubMed Central

    2014-01-01

    Background Malaria that is caused by Plasmodium vivax is the most widely distributed human malaria. Its recent resurgence in many parts of the world, including the Republic of Korea (ROK), emphasizes the importance of improved access to the early and accurate detection of P. vivax to reduce disease burden. In this study, a rapid and efficient loop-mediated isothermal amplification (LAMP)-based method was developed and validated using blood samples from malaria-suspected patients. Method A LAMP assay targeting the α-tubulin gene for the detection of P. vivax was developed with six primers that recognize different regions of the target gene. The diagnostic performance of the α-tubulin LAMP assay was compared to three other tests: microscopic examinations, rapid diagnostic tests (RDTs), and nested polymerase chain reactions (PCRs) using 177 whole blood specimens obtained from ROK military personnel from May to December 2011. Results The α-tubulin LAMP assay was highly sensitive with a detection limit of 100 copies of P. vivax α-tubulin gene per reaction within 50 min. It specifically amplified the target gene only from P. vivax. Validation of the α-tubulin LAMP assay showed that the assay had the highest sensitivity (P < 0.001 versus microscopy; P = 0.0023 versus RDT) when nested PCR was used as the gold standard and better agreement (concordance: 94.9%, kappa value: 0.865) with nested PCR than RDT and microscopy. A Receiver Operation Characteristics analysis showed that the diagnostic accuracy of the α-tubulin LAMP assay for vivax malaria was higher (Area Under Curve = 0.908) than RDT and microscopy. Conclusion This study showed that the P. vivax α-tubulin LAMP assay, which can be used to diagnose early infections of vivax malaria, is an alternative molecular diagnostic tool and a point-of-care test that may help to prevent transmission in endemic areas. PMID:24981710

  12. Loop-Mediated Isothermal Amplification (LAMP) assay for the identification of Echinococcus multilocularis infections in canine definitive hosts

    PubMed Central

    2014-01-01

    Background Alveolar echinococcosis, caused by the metacestode larval stage of Echinococcus multilocularis, is a zoonosis of public health significance and is highly prevalent in northwest China. To effectively monitor its transmission, we developed a new rapid and cheap diagnostic assay, based on loop-mediated isothermal amplification (LAMP), to identify canine definitive hosts infected with E. multilocularis. Methods The primers used in the LAMP assay were based on the mitochondrial nad5 gene of E. multilocularis and were designed using Primer Explorer V4 software. The developed LAMP assay was compared with a conventional PCR assay, using DNA extracted from the feces of dogs experimentally infected with E. multilocularis, on 189 dog fecal samples collected from three E. multilocularis-endemic regions in Qinghai province, the People’s Republic of China, and 30 negative control copro-samples from dogs from an area in Gansu province that had been subjected to an intensive de-worming program. Light microscopy was also used to examine the experimentally obtained and field collected dog copro-samples for the presence of E. multilocularis eggs. Results The E. multilocularis-positivity rates obtained for the field-collected fecal samples were 16.4% and 5.3% by the LAMP and PCR assays, respectively, and all samples obtained from the control dogs were negative. The LAMP assay was able to detect E. multilocularis DNA in the feces of experimentally infected dogs at 12 days post-infection, whereas the PCR assay was positive on the 17th day and eggs were first detectable by light microscopy at day 44 post-challenge. Conclusion The earlier specific detection of an E. multilocularis infection in dog copro-samples indicates that the LAMP assay we developed is a realistic alternative method for the field surveillance of canines in echinococcosis-endemic areas. PMID:24886279

  13. Mixed-Dye-Based Label-Free and Sensitive Dual Fluorescence for the Product Detection of Nucleic Acid Isothermal Multiple-Self-Matching-Initiated Amplification.

    PubMed

    Ding, Xiong; Wu, Wenshuai; Zhu, Qiangyuan; Zhang, Tao; Jin, Wei; Mu, Ying

    2015-10-20

    Visual detections based on fluorescence and the color changes under natural light are two promising product detections for isothermal nucleic acid amplifications (INAAs) such as the isothermal multiple-self-matching-initiated amplification (IMSA) as point-of-care testing techniques. However, the currently used approaches have shortcomings in application. For the former, fluorescence changes recognized by naked eye may be indistinguishable because of single fluorescence emitted and strong background noise, which requires empirical preset of cutoff intensity values. For the latter, visual detection sensitivity under natural light is not comparable to that based on fluorescence. Herein, hydroxyl naphthol blue (HNB) and SYBR Green I (SG) were coupled to acquire a label-free dual fluorescence for the visual product detection of IMSA. The mixed-dye-loaded off-chip (tube-based) and on-chip (microfluidic chip-based) IMSAs for the detection of hepatitis B virus were conducted. The results demonstrated that this dual fluorescence could realize distinguishable fluorescent color changes to improve visual detection sensitivity and avoid the preset of cutoff values. Moreover, the mixed dye is stable when kept at room temperature and compatible with the IMSA's reagents without a contamination-prone step of opening tubes after amplification. Also, this coupled dye inherits the advantages of achieving color changes under natural light from HNB and real-time detection from SG. In conclusion, the mixed-dye-based dual fluorescence has a potential in the point-of-care testing application for realizing off-chip and on-chip product detection of IMSA, loop-mediated isothermal amplification (LAMP), or other INAAs. PMID:26383158

  14. Development of loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Penicillium nordicum in dry-cured meat products.

    PubMed

    Ferrara, M; Perrone, G; Gallo, A; Epifani, F; Visconti, A; Susca, A

    2015-06-01

    The need of powerful diagnostic tools for rapid, simple, and cost-effective detection of food-borne fungi has become very important in the area of food safety. Currently, several isothermal nucleic acid amplification methods have been developed as an alternative to PCR-based analyses. Loop-mediated isothermal amplification (LAMP) is one of these innovative methods; it requires neither gel electrophoresis to separate and visualize the products nor expensive laboratory equipment and it has been applied already for detection of pathogenic organisms. In the current study, we developed a LAMP assay for the specific detection of Penicillium nordicum, the major causative agent of ochratoxin A contamination in protein-rich food, especially dry-cured meat products. The assay was based on targeting otapksPN gene, a key gene in the biosynthesis of ochratoxin A (OTA) in P. nordicum. Amplification of DNA during the reaction was detected directly in-tube by color transition of hydroxynaphthol blue from violet to sky blue, visible to the naked eye, avoiding further post amplification analyses. Only DNAs isolated from several P. nordicum strains led to positive results and no amplification was observed from non-target OTA and non OTA-producing strains. The assay was able to detect down to 100 fg of purified targeted genomic DNA or 10(2) conidia/reaction within 60 min. The LAMP assay for detection and identification of P. nordicum was combined with a rapid DNA extraction method set up on serially diluted conidia, providing an alternative rapid, specific and sensitive DNA-based method suitable for application directly "on-site", notably in key steps of dry-cured meat production. PMID:25771218

  15. Loop Mediated Isothermal Amplification (LAMP) Accurately Detects Malaria DNA from Filter Paper Blood Samples of Low Density Parasitaemias

    PubMed Central

    González, Iveth J.; Polley, Spencer D.; Bell, David; Shakely, Delér; Msellem, Mwinyi I.; Björkman, Anders; Mårtensson, Andreas

    2014-01-01

    Background Loop mediated isothermal amplification (LAMP) provides an opportunity for improved, field-friendly detection of malaria infections in endemic areas. However data on the diagnostic accuracy of LAMP for active case detection, particularly low-density parasitaemias, are lacking. We therefore evaluated the performance of a new LAMP kit compared with PCR using DNA from filter paper blood spots. Methods and Findings Samples from 865 fever patients and 465 asymptomatic individuals collected in Zanzibar were analysed for Pan (all species) and Pf (P. falciparum) DNA with the Loopamp MALARIA Pan/Pf kit. Samples were amplified at 65°C for 40 minutes in a real-time turbidimeter and results were compared with nested PCR. Samples with discordant results between LAMP and nested PCR were analysed with real-time PCR. The real-time PCR corrected nested PCR result was defined as gold standard. Among the 117 (13.5%) PCR detected P. falciparum infections from fever patients (mean parasite density 7491/µL, range 6–782,400) 115, 115 and 111 were positive by Pan-LAMP, Pf-LAMP and nested PCR, respectively. The sensitivities were 98.3% (95%CI 94–99.8) for both Pan and Pf-LAMP. Among the 54 (11.6%) PCR positive samples from asymptomatic individuals (mean parasite density 10/µL, range 0–4972) Pf-LAMP had a sensitivity of 92.7% (95%CI 80.1–98.5) for detection of the 41 P. falciparum infections. Pan-LAMP had sensitivities of 97% (95%CI 84.2–99.9) and 76.9% (95%CI 46.2–95) for detection of P. falciparum and P. malariae, respectively. The specificities for both Pan and Pf-LAMP were 100% (95%CI 99.1–100) in both study groups. Conclusion Both components of the Loopamp MALARIA Pan/Pf detection kit revealed high diagnostic accuracy for parasite detection among fever patients and importantly also among asymptomatic individuals of low parasite densities from minute blood volumes preserved on filter paper. These data support LAMPs potential role for improved detection of low

  16. Diagnostic accuracy of loop-mediated isothermal amplification in detection of Clostridium difficile in stool samples: a meta-analysis

    PubMed Central

    Wei, Chen; Yang-Ming, Li; Shan, Luo; Yi-Ming, Zhong

    2015-01-01

    Introduction Clostridium difficile infection (CDI) remains a diagnostic challenge for clinicians. More recently, loop-mediated isothermal amplification (LAMP) has become readily available for the diagnosis of CDI, and many studies have investigated the usefulness of LAMP for rapid and accurate diagnosis of CDI. However, the overall diagnostic accuracy of LAMP for CDI remains unclear. In this meta-analysis, our aim was to establish the overall diagnostic accuracy of LAMP in detection of Clostridium difficile (CD) in stool samples. Material and methods A search was done in PubMed, MEDLINE, EMBASE and Cochrane Library databases up to February 2014 to identify published studies that evaluated the diagnostic role of LAMP for CD. Methodological quality was assessed according to the quality assessment for studies of diagnostic accuracy (QUADAS) instrument. The sensitivities (SEN), specificities (SPE), positive likelihood ratio (PLR), negative likelihood ratio (NLR) and diagnostic odds ratio (DOR) were pooled statistically using random effects models. Statistical analysis was performed by employing Meta-Disc 1.4 software. Summary receiver operating characteristic (SROC) curves were used to summarize overall test performance. Funnel plots were used to test the potential publication bias. Result A total of 9 studies met inclusion criteria for the present meta-analysis. The pooled SEN and SPE for diagnosing CD were 0.93 (95% CI: 0.91–0.95) and 0.98 (95% CI: 0.98–0.99), respectively. The PLR was 47.72 (95% CI: 15.10–150.82), NLR was 0.07 (95% CI: 0.04–0.14) and DOR was 745.19 (95% CI: 229.30−2421.72). The area under the ROC was 0.98. Meta-regression indicated that the total number of samples was a source of heterogeneity for LAMP in detection of CD. The funnel plots suggested no publication bias. Conclusions The LAMP meets the minimum desirable characteristics of a diagnostic test of SEN, SPE and other measures of accuracy in the diagnosis of CD, and it is suitable

  17. Loop-Mediated Isothermal Amplification for Laboratory Confirmation of Buruli Ulcer Disease—Towards a Point-of-Care Test

    PubMed Central

    Beissner, Marcus; Phillips, Richard Odame; Battke, Florian; Bauer, Malkin; Badziklou, Kossi; Sarfo, Fred Stephen; Maman, Issaka; Rhomberg, Agata; Piten, Ebekalisai; Frimpong, Michael; Huber, Kristina Lydia; Symank, Dominik; Jansson, Moritz; Wiedemann, Franz Xaver; Banla Kere, Abiba; Herbinger, Karl-Heinz; Löscher, Thomas; Bretzel, Gisela

    2015-01-01

    Background As the major burden of Buruli ulcer disease (BUD) occurs in remote rural areas, development of point-of-care (POC) tests is considered a research priority to bring diagnostic services closer to the patients. Loop-mediated isothermal amplification (LAMP), a simple, robust and cost-effective technology, has been selected as a promising POC test candidate. Three BUD-specific LAMP assays are available to date, but various technical challenges still hamper decentralized application. To overcome the requirement of cold-chains for transport and storage of reagents, the aim of this study was to establish a dry-reagent-based LAMP assay (DRB-LAMP) employing lyophilized reagents. Methodology/Principal Findings Following the design of an IS2404 based conventional LAMP (cLAMP) assay suitable to apply lyophilized reagents, a lyophylization protocol for the DRB-LAMP format was developed. Clinical performance of cLAMP was validated through testing of 140 clinical samples from 91 suspected BUD cases by routine assays, i.e. IS2404 dry-reagent-based (DRB) PCR, conventional IS2404 PCR (cPCR), IS2404 qPCR, compared to cLAMP. Whereas qPCR rendered an additional 10% of confirmed cases and samples respectively, case confirmation and positivity rates of DRB-PCR or cPCR (64.84% and 56.43%; 100% concordant results in both assays) and cLAMP (62.64% and 52.86%) were comparable and there was no significant difference between the sensitivity of the assays (DRB PCR and cPCR, 86.76%; cLAMP, 83.82%). Likewise, sensitivity of cLAMP (95.83%) and DRB-LAMP (91.67%) were comparable as determined on a set of 24 samples tested positive in all routine assays. Conclusions/Significance Both LAMP formats constitute equivalent alternatives to conventional PCR techniques. Provided the envisaged availability of field friendly DNA extraction formats, both assays are suitable for decentralized laboratory confirmation of BUD, whereby DRB-LAMP scores with the additional advantage of not requiring cold

  18. Rapid, Simple and Sensitive Detection of Q Fever by Loop-Mediated Isothermal Amplification of the htpAB Gene

    PubMed Central

    Pan, Lei; Zhang, Lijuan

    2013-01-01

    Background Q fever is the most widespread zoonosis, and domestic animals are the most common sources of transmission. It is not only difficult to distinguish from other febrile diseases because of the lack of specific clinical manifestations in humans, but it is also difficult to identify the disease in C. burnetii-carrying animals because of the lack of identifiable features. Conventional serodiagnosis requires sera from the acute and convalescent stages of infection, which are unavailable at early diagnosis. Nested PCR and real-time PCR require equipment. In this study, we developed a Loop-Mediated Isothermal Amplification (LAMP) assay to identify C. burnetii rapidly and sensitively. Methods A universal LAMP primer set was designed to detect the repeated sequence IS1111a of the htpAB gene of C. burnetii using PrimerExplorer V4 software. The sensitivity of the LAMP assay was evaluated using known quantities of recombined reference plasmids containing the targeted genes. The specificity of the developed LAMP assay was determined using 26 members of order Rickettsiae and 18 other common pathogens. The utility of the LAMP assay was further compared with real time PCR by the examination 24 blood samples including 6 confirmed and 18 probable Q fever cases, which diagnosed by IFA serological assessment and real time PCR. In addition, 126 animal samples from 4 provinces including 97 goats, 7 cattle, 18 horses, 3 marmots and 1 deer were compared by these two methods. Results The limits of detection of the LAMP assay for the htpAB gene were 1 copy per reaction. The specificity of the LAMP assay was 100%, and no cross-reaction was observed among the bacteria used in the study. The positive rate of unknown febrile patients was 33.3%(95%CI 30.2%–36.4%) for the LAMP assay and 8.3%(95%CI 7.4%–9.2%) for the real time PCR(P<0.05). Similarly, the total positive rate of animals was 7.9%(95%CI 7.1%–8.7%) for the LAMP assay and 0.8%(95%CI 0.7%–0.9%)for the real time PCR(P<0

  19. [Investigation on Toxoplasma gondii by polymerase chain reaction and loop-mediated isothermal amplification in water samples from Giresun, Turkey].

    PubMed

    Demirel, Elif; Kolören, Zeynep; Karaman, Ulkü; Ayaz, Emine

    2014-10-01

    Toxoplasmosis is generally asymptomatic in immunocompetent subjects, however serious manifestations of the disease may develop in immunocompromised patients and in pregnant women. The mean Toxoplasma gondii seroprevalence which is approximately 40% in Turkish population, indicates the high risk for the development of acute toxoplasmosis in those cases. One of the transmission ways of T.gondii is the consumption of contaminated water. The aim of this study was to detect the presence of T.gondii in the environmental and drinking water samples by using standard polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) methods. A total of 96 water samples, of them 76 were environmental water samples collected from creeks in Giresun province center and its various districts (Piraziz, Bulancak, Keşap, Espiye) and 20 from drinking water samples, were included in the study. The samples were precipitated by the aluminium sulfate method and DNAs were isolated from the pellets formed with the sucrose gradient method. PCR and LAMP were applied to the isolated DNAs, by the use of primers F3 and B3 specific to B1 gene of the parasite. In our study, T.gondii was detected in none of the drinking water samples by PCR and LAMP, however T.gondii DNAs were positive in 13.2% (10/76) of the environmental water samples. Both of the methods yielded the same results in those samples. The stations that were positive for T.gondii were Aksu creek in Giresun province center; Gelivera creek in Espiye; Yolağzı, Keşap, Keşap Login Bridge creeks in Keşap; Bulancak, Karadere and İncivez creeks in Bulancak; Piraziz and Çayırağzı creeks in Piraziz counties. Resistance of T.gondii to chlorination and the inadequacy of the filtration processes, create a serious threat among people in contact with rivers, sea and drinking waters particularly in areas with high humidity. As far as the national literature was considered, no report about the water-borne T

  20. Development and application of loop-mediated isothermal amplification assays based on ITS-1 for rapid detection of Toxoplasma gondii in pork.

    PubMed

    Zhuo, Xunhui; Huang, Bin; Luo, Jiaqing; Yu, Haijie; Yan, Baolong; Yang, Yi; Du, Aifang

    2015-03-15

    The loop-mediated isothermal amplification (LAMP) assay is a novel method that rapidly amplifies DNA with high specificity and sensitivity under isothermal conditions. In this study, we established a LAMP assay with six primers targeting a highly conserved region of Toxoplasma gondii ITS-1 sequence. The amplification protocol completes within 30 min under isothermal condition in a 65°C water bath while specificity tests confirmed no cross-reactivity with DNA templates of Neospora caninum, Eimeria tenella, Cryptosporidium parvum, Listeria monocytogenes and Streptococcus suis. The detection limit of the LAMP assay was 0.9 fg T. gondii genomic DNA, a sensitivity that was 10-fold higher than that of a conventional PCR assay. Both LAMP assay and conventional PCR were applied to detect T. gondii genomic DNA in 118 diaphragm samples obtained from pig farms in Zhejiang Province, China. Our results showed that the LAMP assay is more sensitive than conventional PCR (13.56% and 9.32%). The LAMP assay established in this study provides a simple, specific, sensitive and rapid method of T. gondii genomic DNA detection, hence is expected to plays an important role in the monitoring of T. gondii contamination in various food products. PMID:25624074

  1. Real-Time Sequence-Validated Loop-Mediated Isothermal Amplification Assays for Detection of Middle East Respiratory Syndrome Coronavirus (MERS-CoV)

    PubMed Central

    Bhadra, Sanchita; Jiang, Yu Sherry; Kumar, Mia R.; Johnson, Reed F.; Hensley, Lisa E.; Ellington, Andrew D.

    2015-01-01

    The Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF)1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD) for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU) (5 to 50 PFU/ml) of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens. PMID:25856093

  2. Loop mediated isothermal amplification assay using hydroxy naphthol blue, conventional polymerase chain reaction and real-time PCR in the diagnosis of intraocular tuberculosis.

    PubMed

    Balne, P K; Basu, S; Rath, S; Barik, M R; Sharma, S

    2015-01-01

    This study is a comparative evaluation (Chi-square test) of a closed tube loop mediated isothermal amplification assay using hydroxy naphthol blue dye (HNB-LAMP), real-time polymerase chain reaction (PCR) and conventional PCR in the diagnosis of intraocular tuberculosis. Considering clinical presentation as the gold standard in 33 patients, the sensitivity of HNB-LAMP assay (75.8%) was higher (not significant, P value 0.2) than conventional PCR (57.6%) and lower than real-time PCR (90.9%). Specificity was 100% by all three methods. No amplification was observed in negative controls (n = 20) by all three methods. The cost of the HNB-LAMP assay was Rs. 500.00 and it does not require thermocycler, therefore, it can be used as an alternative to conventional PCR in resource-poor settings. PMID:26470966

  3. Rapid Detection of Subtype H10N8 Influenza Virus by One-Step Reverse Transcription–Loop-Mediated Isothermal Amplification Methods

    PubMed Central

    Bao, Hongmei; Feng, Xiaoxiao; Ma, Yong; Shi, Jianzhong; Zhao, Yuhui; Gu, Linlin

    2015-01-01

    We developed hemagglutinin- and neuraminidase-specific one-step reverse transcription–loop-mediated isothermal amplification assays for detecting the H10N8 virus. The detection limit of the assays was 10 copies of H10N8 virus, and the assays did not amplify nonspecific RNA. The assays can detect H10N8 virus from chicken samples with high sensitivity and specificity, and they can serve as an effective tool for detecting and monitoring H10N8 virus in live poultry markets. PMID:26378283

  4. Development and comparative evaluation of loop mediated isothermal amplification (LAMP) assay for simple visual detection of orf virus in sheep and goats.

    PubMed

    Venkatesan, G; Bhanuprakash, V; Balamurugan, V

    2015-06-01

    A loop-mediated isothermal amplification (LAMP) assay targeting DNA Pol gene was optimized and evaluated for the rapid detection of orf virus in clinical samples. The LAMP assay was found to be specific and sensitive. The detection rate of LAMP (89.3%) was better than PCR (67.9%) and comparable to real-time PCR (91.1%) in clinical samples by gel electrophoresis and visual detection methods. This LAMP assay is simple and does not rely upon any special equipment and could be employed in clinical diagnosis and epidemiological survey of orf infection. PMID:25828693

  5. Development and evaluation of loop-mediated isothermal amplification (LAMP) for the rapid diagnosis of Penicillium marneffei in archived tissue samples.

    PubMed

    Sun, Jiufeng; Li, Xiqing; Zeng, Hanxiang; Xie, Zhi; Lu, Changming; Xi, Liyan; de Hoog, Gert S

    2010-04-01

    Penicillium marneffei is the etiologic agent of a severe systemic disease in immunocompromised hosts in Southeast Asia. In the present study, a novel method, known as loop-mediated isothermal amplification (LAMP), is described for the rapid and specific detection of the species, using a primer set derived from the internal transcribed spacer (ITS) region of the rRNA gene. Amplification products can be detected macroscopically by visual inspection in vials using SYBR Green I as well as by electrophoresis on agarose gel. The LAMP assay resulted in specific amplification of P. marneffei ITS using pure cultures after a 1-h reaction at 65 degrees C in a water bath; no cross-reactivity with other fungi including other biverticillate penicillia was observed. The detectable DNA limit was two copies. In addition, specific amplification was achieved using paraffin wax-embedded tissue samples from patients with penicilliosis marneffei and tissue samples from bamboo rats. The method provides a powerful tool for rapid diagnostics in the clinical lab, and has potential for use in ecological studies. PMID:20113352

  6. Multiplex, Rapid, and Sensitive Isothermal Detection of Nucleic-Acid Sequence by Endonuclease Restriction-Mediated Real-Time Multiple Cross Displacement Amplification.

    PubMed

    Wang, Yi; Wang, Yan; Zhang, Lu; Liu, Dongxin; Luo, Lijuan; Li, Hua; Cao, Xiaolong; Liu, Kai; Xu, Jianguo; Ye, Changyun

    2016-01-01

    We have devised a novel isothermal amplification technology, termed endonuclease restriction-mediated real-time multiple cross displacement amplification (ET-MCDA), which facilitated multiplex, rapid, specific and sensitive detection of nucleic-acid sequences at a constant temperature. The ET-MCDA integrated multiple cross displacement amplification strategy, restriction endonuclease cleavage and real-time fluorescence detection technique. In the ET-MCDA system, the functional cross primer E-CP1 or E-CP2 was constructed by adding a short sequence at the 5' end of CP1 or CP2, respectively, and the new E-CP1 or E-CP2 primer was labeled at the 5' end with a fluorophore and in the middle with a dark quencher. The restriction endonuclease Nb.BsrDI specifically recognized the short sequence and digested the newly synthesized double-stranded terminal sequences (5' end short sequences and their complementary sequences), which released the quenching, resulting on a gain of fluorescence signal. Thus, the ET-MCDA allowed real-time detection of single or multiple targets in only a single reaction, and the positive results were observed in as short as 12 min, detecting down to 3.125 fg of genomic DNA per tube. Moreover, the analytical specificity and the practical application of the ET-MCDA were also successfully evaluated in this study. Here, we provided the details on the novel ET-MCDA technique and expounded the basic ET-MCDA amplification mechanism. PMID:27242766

  7. Rapid authentication of the precious herb saffron by loop-mediated isothermal amplification (LAMP) based on internal transcribed spacer 2 (ITS2) sequence

    PubMed Central

    Zhao, Mingming; Shi, Yuhua; Wu, Lan; Guo, Licheng; Liu, Wei; Xiong, Chao; Yan, Song; Sun, Wei; Chen, Shilin

    2016-01-01

    Saffron is one of the most expensive species of Chinese herbs and has been subjected to various types of adulteration because of its high price and limited production. The present study introduces a loop-mediated isothermal amplification (LAMP) technique for the differentiation of saffron from its adulterants. This novel technique is sensitive, efficient and simple. Six specific LAMP primers were designed on the basis of the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA of Crocus sativus. All LAMP amplifications were performed successfully, and visual detection occurred within 60 min at isothermal conditions of 65 °C. The results indicated that the LAMP primers are accurate and highly specific for the discrimination of saffron from its adulterants. In particular, 10 fg of genomic DNA was determined to be the limit for template accuracy of LAMP in saffron. Thus, the proposed novel, simple, and sensitive LAMP assay is well suited for immediate on-site discrimination of herbal materials. Based on the study, a practical standard operating procedure (SOP) for utilizing the LAMP protocol for herbal authentication is provided. PMID:27146605

  8. Rapid authentication of the precious herb saffron by loop-mediated isothermal amplification (LAMP) based on internal transcribed spacer 2 (ITS2) sequence.

    PubMed

    Zhao, Mingming; Shi, Yuhua; Wu, Lan; Guo, Licheng; Liu, Wei; Xiong, Chao; Yan, Song; Sun, Wei; Chen, Shilin

    2016-01-01

    Saffron is one of the most expensive species of Chinese herbs and has been subjected to various types of adulteration because of its high price and limited production. The present study introduces a loop-mediated isothermal amplification (LAMP) technique for the differentiation of saffron from its adulterants. This novel technique is sensitive, efficient and simple. Six specific LAMP primers were designed on the basis of the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA of Crocus sativus. All LAMP amplifications were performed successfully, and visual detection occurred within 60 min at isothermal conditions of 65 °C. The results indicated that the LAMP primers are accurate and highly specific for the discrimination of saffron from its adulterants. In particular, 10 fg of genomic DNA was determined to be the limit for template accuracy of LAMP in saffron. Thus, the proposed novel, simple, and sensitive LAMP assay is well suited for immediate on-site discrimination of herbal materials. Based on the study, a practical standard operating procedure (SOP) for utilizing the LAMP protocol for herbal authentication is provided. PMID:27146605

  9. Establishment and application of a real-time loop-mediated isothermal amplification system for the detection of CYP2C19 polymorphisms.

    PubMed

    Zhang, Chao; Yao, Yao; Zhu, Juan-Li; Zhang, Si-Nong; Zhang, Shan-Shan; Wei, Hua; Hui, Wen-Li; Cui, Ya-Li

    2016-01-01

    Single-nucleotide polymorphisms (SNPs) represent the most widespread type of genetic variation (approximately 90%) in the human genome, and the demand to overcome such variation has received more attention now than ever before. The capacity to rapidly assess SNPs that correlate with disease predisposition, drug efficacy and drug toxicity is a key step for the development of personalized medicine. In this work, a rapid one-step SNP detection method, real-time loop-mediated isothermal amplification (RT-LAMP), was first applied for CYP2C19 polymorphisms testing. The optimized method was established with specifically designed primers for target amplification by real-time detection in approximately 30 min under isothermal conditions. RT-LAMP amplified few copies of template to produce significant amounts of product and quantitatively detected human DNA with compatible specificity and sensitivity. The success in the establishment of this RT-LAMP protocol for CYP2C19 polymorphism testing is significant for the extension of this technique for the detection of other SNPs, which will further facilitate the development of personalized medicine. PMID:27246657

  10. A rapid and sensitive loop-mediated isothermal amplification procedure (LAMP) for Mycoplasma hyopneumoniae detection based on the p36 gene.

    PubMed

    Liu, M J; Du, G M; Bai, F F; Wu, Y Z; Xiong, Q Y; Feng, Z X; Li, B; Shao, G Q

    2015-01-01

    The aim of this study was to establish a method for sensitive and rapid diagnosis of Mycoplasma hyopneumoniae in clinical specimens. To this effect, we employed three sets of primers specifically designed for amplification of nucleic acids under isothermal conditions. After optimization of reaction conditions, M. hyopneumoniae could be successfully detected at 63°C in 45 min through use of the loop-mediated isothermal amplification (LAMP) assay. A positive reaction was identified visually as white precipitate and confirmed by gel electrophoresis. The detection limit for this assay was 10 copies/μL, as observed by electrophoretic analysis. The accuracy of the LAMP reaction was confirmed by restriction endonuclease digestion as well as by direct sequencing of the amplified product. This method can specifically detect M. hyopneumoniae; other species with high homology and other bacterial and virus strains gave negative results. To test the utility of this procedure, the LAMP assay was applied to 40 clinical samples collected from swine lung tissues experimentally challenged with M. hyopneumoniae isolates, and compared to the results from a real-time polymerase chain reaction (PCR) assay. A concordance of 100% was observed between the two assays. In conclusion, the results from our study demonstrated that the LAMP assay provided a rapid reaction and was inexpensive to perform, with no need of complex instruments or systems such as Geneamp PCR. The LAMP assay may therefore be applied in routine diagnosis in the clinical laboratory and for in-field detection of M. hyopneumoniae infection. PMID:25966242

  11. Application of loop-mediated isothermal amplification assay for the sensitive and rapid diagnosis of visceral leishmaniasis and post-kala-azar dermal leishmaniasis.

    PubMed

    Verma, Sandeep; Avishek, Kumar; Sharma, Vanila; Negi, Narendra Singh; Ramesh, Venkatesh; Salotra, Poonam

    2013-04-01

    Loop-mediated isothermal amplification (LAMP) is at the forefront in the search for innovative diagnostics for rapid and specific amplification of target DNA under isothermal conditions. We have applied LAMP assay using SYBR Green for clear-cut naked eye detection of Leishmania (Leishmania) donovani in 200 clinical samples of visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL). The assay was positive in 53/55 VL blood samples (sensitivity, 96.4%; 95% confidence interval [CI], 87.7-99%), 15/15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI, 79.6-100%), 60/62 PKDL tissue biopsy samples (sensitivity, 96.8%; 95% CI, 88.9-99.1%), and 1/68 control samples (specificity, 98.5%; 95% CI, 92.1-99.7%). The assay was specific for L. (L.) donovani, the causative species for VL and negative for L. (L.) infantum, L. (L.) tropica, and L. (L.) major. This is the first comprehensive clinical study demonstrating the applicability of the LAMP assay for a rapid and reliable molecular diagnosis of VL and PKDL. PMID:23433714

  12. One simple DNA extraction device and its combination with modified visual loop-mediated isothermal amplification for rapid on-field detection of genetically modified organisms.

    PubMed

    Zhang, Miao; Liu, Yinan; Chen, Lili; Quan, Sheng; Jiang, Shimeng; Zhang, Dabing; Yang, Litao

    2013-01-01

    Quickness, simplicity, and effectiveness are the three major criteria for establishing a good molecular diagnosis method in many fields. Herein we report a novel detection system for genetically modified organisms (GMOs), which can be utilized to perform both on-field quick screening and routine laboratory diagnosis. In this system, a newly designed inexpensive DNA extraction device was used in combination with a modified visual loop-mediated isothermal amplification (vLAMP) assay. The main parts of the DNA extraction device included a silica gel membrane filtration column and a modified syringe. The DNA extraction device could be easily operated without using other laboratory instruments, making it applicable to an on-field GMO test. High-quality genomic DNA (gDNA) suitable for polymerase chain reaction (PCR) and isothermal amplification could be quickly isolated from plant tissues using this device within 15 min. In the modified vLAMP assay, a microcrystalline wax encapsulated detection bead containing SYBR green fluorescent dye was introduced to avoid dye inhibition and cross-contaminations from post-LAMP operation. The system was successfully applied and validated in screening and identification of GM rice, soybean, and maize samples collected from both field testing and the Grain Inspection, Packers, and Stockyards Administration (GIPSA) proficiency test program, which demonstrated that it was well-adapted to both on-field testing and/or routine laboratory analysis of GMOs. PMID:23181490

  13. Establishment and application of a real-time loop-mediated isothermal amplification system for the detection of CYP2C19 polymorphisms

    PubMed Central

    Zhang, Chao; Yao, Yao; Zhu, Juan-Li; Zhang, Si-Nong; Zhang, Shan-Shan; Wei, Hua; Hui, Wen-Li; Cui, Ya-Li

    2016-01-01

    Single-nucleotide polymorphisms (SNPs) represent the most widespread type of genetic variation (approximately 90%) in the human genome, and the demand to overcome such variation has received more attention now than ever before. The capacity to rapidly assess SNPs that correlate with disease predisposition, drug efficacy and drug toxicity is a key step for the development of personalized medicine. In this work, a rapid one-step SNP detection method, real-time loop-mediated isothermal amplification (RT-LAMP), was first applied for CYP2C19 polymorphisms testing. The optimized method was established with specifically designed primers for target amplification by real-time detection in approximately 30 min under isothermal conditions. RT-LAMP amplified few copies of template to produce significant amounts of product and quantitatively detected human DNA with compatible specificity and sensitivity. The success in the establishment of this RT-LAMP protocol for CYP2C19 polymorphism testing is significant for the extension of this technique for the detection of other SNPs, which will further facilitate the development of personalized medicine. PMID:27246657

  14. 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.

    PubMed

    Khorosheva, Eugenia M; Karymov, Mikhail A; Selck, David A; Ismagilov, Rustem F

    2016-01-29

    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. PMID:26358811

  15. 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

    PubMed Central

    Khorosheva, Eugenia M.; Karymov, Mikhail A.; Selck, David A.; Ismagilov, Rustem F.

    2016-01-01

    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. PMID:26358811

  16. Asymmetric exponential amplification reaction on a toehold/biotin featured template: an ultrasensitive and specific strategy for isothermal microRNAs analysis

    PubMed Central

    Chen, Jun; Zhou, Xueqing; Ma, Yingjun; Lin, Xiulian; Dai, Zong; Zou, Xiaoyong

    2016-01-01

    The sensitive and specific analysis of microRNAs (miRNAs) without using a thermal cycler instrument is significant and would greatly facilitate biological research and disease diagnostics. Although exponential amplification reaction (EXPAR) is the most attractive strategy for the isothermal analysis of miRNAs, its intrinsic limitations of detection efficiency and inevitable non-specific amplification critically restrict its use in analytical sensitivity and specificity. Here, we present a novel asymmetric EXPAR based on a new biotin/toehold featured template. A biotin tag was used to reduce the melting temperature of the primer/template duplex at the 5′ terminus of the template, and a toehold exchange structure acted as a filter to suppress the non-specific trigger of EXPAR. The asymmetric EXPAR exhibited great improvements in amplification efficiency and specificity as well as a dramatic extension of dynamic range. The limit of detection for the let-7a analysis was decreased to 6.02 copies (0.01 zmol), and the dynamic range was extended to 10 orders of magnitude. The strategy enabled the sensitive and accurate analysis of let-7a miRNA in human cancer tissues with clearly better precision than both standard EXPAR and RT-qPCR. Asymmetric EXPAR is expected to have an important impact on the development of simple and rapid molecular diagnostic applications for short oligonucleotides. PMID:27257058

  17. Development of a loop-mediated isothermal amplification targeting a gene within the pyruvate dehydrogenase complex, the pdhA gene, for rapid detection of Mycoplasma gallisepticum.

    PubMed

    Zhang, Fanqing; Bao, Shijun; Yu, Shengqing; Cheng, Jinghua; Tan, Lei; Qiu, Xvsheng; Song, Cuiping; Dai, Yabin; Fei, Rongmei; Ding, Chan

    2015-05-01

    Mycoplasma gallisepticum infections impose a significant economic burden on the poultry industry. In the current study, a loop-mediated isothermal amplification (LAMP) assay was developed and optimized to detect M. gallisepticum based on a gene within the pyruvate dehydrogenase complex, the pdhA gene, which codes for the major subunit (E1α) in the complex. The reaction conditions were optimized, and the specificity was confirmed by successful amplification of several M. gallisepticum strains, while no amplification was detected with 20 other major bacterial and viral pathogens of poultry. Additionally, the LAMP assay achieved 10-fold higher sensitivity than an existing polymerase chain reaction (PCR) method. The LAMP assay was applied to swab samples collected from poultry farms and compared with PCR. The positive detection rate was 20.2% (37/183) by LAMP and 13.1% (24/183) by PCR. The LAMP assay could provide a cost-effective, quick, and sensitive method for the detection of M. gallisepticum. PMID:26038479

  18. Visual Detection of West Nile Virus Using Reverse Transcription Loop-Mediated Isothermal Amplification Combined with a Vertical Flow Visualization Strip

    PubMed Central

    Cao, Zengguo; Wang, Hualei; Wang, Lina; Li, Ling; Jin, Hongli; Xu, Changping; Feng, Na; Wang, Jianzhong; Li, Qian; Zhao, Yongkun; Wang, Tiecheng; Gao, Yuwei; Lu, Yiyu; Yang, Songtao; Xia, Xianzhu

    2016-01-01

    West Nile virus (WNV) causes a severe zoonosis, which can lead to a large number of casualties and considerable economic losses. A rapid and accurate identification method for WNV for use in field laboratories is urgently needed. Here, a method utilizing reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip (RT-LAMP-VF) was developed to detect the envelope (E) gene of WNV. The RT-LAMP-VF assay could detect 102 copies/μl of an WNV RNA standard using a 40 min amplification reaction followed by a 2 min incubation of the amplification product on the visualization strip, and no cross-reaction with other closely related members of the Flavivirus genus was observed. The assay was further evaluated using cells and mouse brain tissues infected with a recombinant rabies virus expressing the E protein of WNV. The assay produced sensitivities of 101.5 TCID50/ml and 101.33 TCID50/ml for detection of the recombinant virus in the cells and brain tissues, respectively. Overall, the RT-LAMP-VF assay developed in this study is rapid, simple and effective, and it is therefore suitable for clinical application in the field. PMID:27148234

  19. A sensitive loop-mediated isothermal amplification (LAMP) method for detection of Renibacterium salmoninarum, causative agent of bacterial kidney disease in salmonids.

    PubMed

    Gahlawat, S K; Ellis, A E; Collet, B

    2009-06-01

    Loop-mediated isothermal amplification (LAMP) is a novel technique for nucleic acid amplification with high specificity, sensitivity and rapidity and does not require expensive equipment or reagents. In the present study, we developed and evaluated a LAMP method for the rapid detection of Renibacterium salmoninarum causing the bacterial kidney disease in salmonids. This method was more sensitive than quantitative real-time polymerase chain reaction (qPCR). Using DNA template extracted from cultured R. salmoninarum, the LAMP method gave an amplification signal from template diluted to 10(-8) while the limit of detection of qPCR was10(-7). The LAMP method was also highly specific and did not amplify DNA purified from five other Gram-positive and -negative bacterial fish pathogens. The method also worked well using extracts of macrophages infected with R. salmoninarum and kidney material from rainbow trout, which were positive for R. salmoninarum by qPCR and crude R. salmoninarum culture. There was some evidence for inhibitors of the LAMP reaction in the kidney samples, which was overcome by diluting the sample. PMID:19538642

  20. Quenching of Unincorporated Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright, Single-Step, Closed-Tube, and Multiplexed Detection of RNA Viruses.

    PubMed

    Ball, Cameron S; Light, Yooli K; Koh, Chung-Yan; Wheeler, Sarah S; Coffey, Lark L; Meagher, Robert J

    2016-04-01

    Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read "quasar"), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA. PMID:26980448

  1. Development and Application of a Loop-Mediated Isothermal Amplification (LAMP) Approach for the Rapid Detection of Dirofilaria repens from Biological Samples

    PubMed Central

    Raele, Donato Antonio; Pugliese, Nicola; Galante, Domenico; Latorre, Laura Maria; Cafiero, Maria Assunta

    2016-01-01

    Dirofilariasis by Dirofilaria repens is an important mosquito vector borne parasitosis, and the dog represents the natural host and reservoir of the parasite. This filarial nematode can also induce disease in humans, and in the last decades an increasing number of cases have been being reported. The present study describes the first loop mediated isothermal amplification (LAMP) assay to detect D. repens DNA in blood and mosquitoes. Two versions of the technique have been developed and described: in the first, the amplification is followed point by point through a real time PCR instrument (ReT-LAMP); in the second, the amplification is visualized by checking UV fluorescence of the reaction mixture after addition of propidium iodide (PI-LAMP). The two variants use the same set of 4 primers targeting the D. repens cytochrome oxidase subunit I (COI) gene. To assess the specificity of the method, reactions were carried out by using DNA from the major zoonotic parasites of the family of Onchocercidae, and no amplification was observed. The lower limit of detection of the ReT-LAMP assay was 0.15 fg/μl (corresponding to about 50 copy of COI gene per μl). Results suggest that the described assay is specific, and its sensitivity is higher than the conventional PCR based on the same gene. It is also provide a rapid and cost-effective molecular detection of D. repens, mainly when PI-LAMP is applied, and it should be performed in areas where this emerging parasitosis is endemic. PMID:27341205

  2. Multiplex, Rapid, and Sensitive Isothermal Detection of Nucleic-Acid Sequence by Endonuclease Restriction-Mediated Real-Time Multiple Cross Displacement Amplification

    PubMed Central

    Wang, Yi; Wang, Yan; Zhang, Lu; Liu, Dongxin; Luo, Lijuan; Li, Hua; Cao, Xiaolong; Liu, Kai; Xu, Jianguo; Ye, Changyun

    2016-01-01

    We have devised a novel isothermal amplification technology, termed endonuclease restriction-mediated real-time multiple cross displacement amplification (ET-MCDA), which facilitated multiplex, rapid, specific and sensitive detection of nucleic-acid sequences at a constant temperature. The ET-MCDA integrated multiple cross displacement amplification strategy, restriction endonuclease cleavage and real-time fluorescence detection technique. In the ET-MCDA system, the functional cross primer E-CP1 or E-CP2 was constructed by adding a short sequence at the 5′ end of CP1 or CP2, respectively, and the new E-CP1 or E-CP2 primer was labeled at the 5′ end with a fluorophore and in the middle with a dark quencher. The restriction endonuclease Nb.BsrDI specifically recognized the short sequence and digested the newly synthesized double-stranded terminal sequences (5′ end short sequences and their complementary sequences), which released the quenching, resulting on a gain of fluorescence signal. Thus, the ET-MCDA allowed real-time detection of single or multiple targets in only a single reaction, and the positive results were observed in as short as 12 min, detecting down to 3.125 fg of genomic DNA per tube. Moreover, the analytical specificity and the practical application of the ET-MCDA were also successfully evaluated in this study. Here, we provided the details on the novel ET-MCDA technique and expounded the basic ET-MCDA amplification mechanism. PMID:27242766

  3. Detection of Early and Single Infections of Schistosoma japonicum in the Intermediate Host Snail, Oncomelania hupensis, by PCR and Loop-Mediated Isothermal Amplification (LAMP) Assay

    PubMed Central

    Kumagai, Takashi; Furushima-Shimogawara, Rieko; Ohmae, Hiroshi; Wang, Tian-Ping; Lu, Shaohong; Chen, Rui; Wen, Liyong; Ohta, Nobuo

    2010-01-01

    Polymerase chain reaction (PCR) with the specific primer set amplifying 28S ribosomal DNA (rDNA) of Schistosoma japonicum was able to detect genomic DNA of S. japonicum, but not S. mansoni, at 100 fg. This procedure enabled us to detect the DNA from a single miracidium and a snail infected with one miracidium at just 1 day after infection. We compared these results with those from loop-mediated isothermal amplification (LAMP) targeting 28S rDNA and found similar results. The LAMP could amplify the specific DNA from a group of 100 normal snails mixed with one infected snail A PCR screening of infected snails from endemic regions in Anhui Province revealed schistosomal DNA even in snails found negative by microscopy. PCR and LAMP show promise for monitoring the early infection rate in snails, and they may be useful for predicting the risk of infection in the endemic places. PMID:20810818

  4. Detection of Coconut cadang-cadang viroid (CCCVd) in oil palm by reverse transcription loop-mediated isothermal amplification (RT-LAMP).

    PubMed

    Thanarajoo, Sathis Sri; Kong, Lih Ling; Kadir, Jugah; Lau, Wei Hongi; Vadamalai, Ganesan

    2014-06-01

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) detected Coconut cadang-cadang viroid (CCCVd) within 60 min at 60 °C in total nucleic acid extracted from oil palm leaves infected with CCCVd. Positive reactions showed colour change from orange to green in the reaction mix after the addition of fluorescent reagent, and a laddering pattern band on 2% agarose gel electrophoresis. Conventional RT-PCR with LAMP primers produced amplicons with a sequence identical to the 297-nt CCCVd oil palm variant with the primers being specific for CCCVd and not for other viroids such as PSTVd and CEVd. RT-LAMP was found to be rapid and specific for detecting oil palm CCCVd. PMID:24631346

  5. Comparison of a TaqMan real-time polymerase chain reaction assay with a loop-mediated isothermal amplification assay for detection of Gallid herpesvirus 1.

    PubMed

    Ou, Shan-Chia; Giambrone, Joseph J; Macklin, Kenneth S

    2012-01-01

    A TaqMan real-time polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) assay were developed to detect Gallid herpesvirus 1 (GaHV-1, formerly Infectious laryngotracheitis virus). The standard curve of real-time PCR was established, and the sensitivity reached 10 copies/μl. In the current study, the conversion between viral titer and GaHV-1 genomic copy number was constructed. Six primers for LAMP assay amplified target gene at 65°C within 45 min, and the detection limit was 60 copies/μl. The 6 primers were highly specific, sensitive, and reproducible for detection of GaHV-1. Although the sensitivity of LAMP was lower than that of real-time PCR, LAMP was faster, less expensive, and did not require a thermocycler. The LAMP assay would be a viable alternative assay in diagnostic laboratories that do not employ real-time PCR technology. PMID:22362944

  6. Loop-mediated isothermal amplification (RT-LAMP): a new approach for the detection of foot-and-mouth disease virus and its sero-types in Pakistan.

    PubMed

    Farooq, U; Latif, A; Irshad, H; Ullah, A; Zahur, A B; Naeem, K; Khan, S U H; Ahmed, Z; Rodriguez, L L; Smoliga, G

    2015-01-01

    Successful disease management requires a rapid and sensitive diagnosis method that can recognize early infection even before the manifestation of its clinical signs. The only available field diagnostic tests for foot-and-mouth disease (FMD) are lateral flow devices, commonly known as chromatographic strips. Low sensitivity and inability to detect FMD virus (FMDV) at the serotype level are limitations of lateral flow devices. Therefore, a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) was standardized using universal and sero-type specific genes in a single tube. This test does not require sophisticated equipment and can detect FMDV at serotype level in about 60 min. In addition, the sensitivity and specificity of this test is comparable to conventional reverse transcriptase PCR and real time PCR (rRT-PCR). PMID:27175198

  7. Development of a quadruplex loop-mediated isothermal amplification assay for field detection of four Vibrio species associated with fish disease.

    PubMed

    Zhou, Shun; Gao, Zhi-Xin; Zhang, Min; Liu, Dan-Yang; Zhao, Xin-Peng; Liu, Yong

    2016-01-01

    A quadruplex loop-mediated isothermal amplification (LAMP) method was developed to detect four Vibrio species, including Vibrio ichthyoenteri, Vibrio parahaemolyticus, Vibrio scophthalmi, and Vibrio vulnificus, simultaneously. Four sets of species-specific primers were designed with different restriction sites contained in the inner primers. The quadruplex LAMP method could distinguish four Vibrio species via the subsequent restriction enzyme analysis. The sensitivity of the quadruplex LAMP method were 10(2)-10(3) times higher than the sensitivity of conventional PCR. V. scophthalmi, V. vulnificus, V. parahaemolyticus and V. ichthyoenteri could be detected in the different tissues of the infected fish by the quadruplex LAMP method simply and conveniently through using SYBR Green I to facilitate visual inspection of the LAMP products. The method we developed in this study could be a simple and convenient diagnostic tool for field detection of Vibrio infection in fish. PMID:27468405

  8. Development of a loop-mediated isothermal amplification assay for rapid detection of Nocardia salmonicida, the causative agent of nocardiosis in fish.

    PubMed

    Xia, Liqun; Zhang, Honglian; Lu, Yishan; Cai, Jia; Wang, Bei; Jian, Jichang

    2015-03-01

    Nocardia salmonicida is one of the main pathogens of fish nocardiosis. The purpose of this study was to build a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of N. salmonicida. A set of four primers were designed from the 16S-23S rRNA intergenic spacer region of N. salmonicida, and conditions for LAMP were optimized as incubating all the reagents for 60 min at 64°C. LAMP products were judged with agar gel electrophoresis as well as with the naked eye after the addition of SYBR Green I. Results showed the sensitivity of the LAMP assay was 1.68 × 10(3) CFU/ml (16.8 CFU per reaction) and 10-fold higher than that of PCR. The LAMP method was also effectively applied to detect N. salmonicida in diseased fish samples, and it may potentially facilitate the surveillance and early diagnosis of fish nocardiosis. PMID:25262681

  9. Loop-mediated isothermal amplification (RT-LAMP): a new approach for the detection of foot-and-mouth disease virus and its sero-types in Pakistan

    PubMed Central

    Farooq, U; Latif, A; Irshad, H; Ullah, A; Zahur, A. B; Naeem, K; Khan, S. U. H; Ahmed, Z; Rodriguez, L. L; Smoliga, G

    2015-01-01

    Successful disease management requires a rapid and sensitive diagnosis method that can recognize early infection even before the manifestation of its clinical signs. The only available field diagnostic tests for foot-and-mouth disease (FMD) are lateral flow devices, commonly known as chromatographic strips. Low sensitivity and inability to detect FMD virus (FMDV) at the serotype level are limitations of lateral flow devices. Therefore, a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) was standardized using universal and sero-type specific genes in a single tube. This test does not require sophisticated equipment and can detect FMDV at serotype level in about 60 min. In addition, the sensitivity and specificity of this test is comparable to conventional reverse transcriptase PCR and real time PCR (rRT-PCR). PMID:27175198

  10. Development of a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of common genetically modified organisms (GMOs).

    PubMed

    Feng, Jiawang; Tang, Shiming; Liu, Lideng; Kuang, Xiaoshan; Wang, Xiaoyu; Hu, Songnan; You, Shuzhu

    2015-03-01

    Here, we developed a loop-mediated isothermal amplification (LAMP) assay for 11 common transgenic target DNA in GMOs. Six sets of LAMP primer candidates for each target were designed and their specificity, sensitivity, and reproductivity were evaluated. With the optimized LAMP primers, this LAMP assay was simply run within 45-60 min to detect all these targets in GMOs tested. The sensitivity, specificity, and reproductivity of the LAMP assay were further analyzed in comparison with those of Real-Time PCR. In consistent with real-time PCR, detection of 0.5% GMOs in equivalent background DNA was possible using this LAMP assay for all targets. In comparison with real-time PCR, the LAMP assay showed the same results with simple instruments. Hence, the LAMP assay developed can provide a rapid and simple approach for routine screening as well as specific events detection of many GMOs. PMID:25582179

  11. Giardia and Cryptosporidium spp. dissemination during wastewater treatment and comparative detection via immunofluorescence assay (IFA), nested polymerase chain reaction (nested PCR) and loop mediated isothermal amplification (LAMP).

    PubMed

    Gallas-Lindemann, Carmen; Sotiriadou, Isaia; Plutzer, Judit; Noack, Michael J; Mahmoudi, Mohammad Reza; Karanis, Panagiotis

    2016-06-01

    Environmental water samples from the Lower Rhine area in Germany were investigated via immunofluorescence assays (IFAs), nested polymerase chain reaction (nested PCR) and loop-mediated isothermal amplification (LAMP) to detect the presence of Giardia spp. (n=185) and Cryptosporidium spp. (n=227). The samples were concentrated through filtration or flocculation, and oocysts were purified via centrifugation through a sucrose density gradient. For all samples, IFA was performed first, followed by DNA extraction for the nested PCR and LAMP assays. Giardia cysts were detected in 105 samples (56.8%) by IFA, 62 samples (33.5%) by nested PCR and 79 samples (42.7%) by LAMP. Cryptosporidium spp. were detected in 69 samples (30.4%) by IFA, 95 samples (41.9%) by nested PCR and 99 samples (43.6%) by LAMP. According to these results, the three detection methods are complementary for monitoring Giardia and Cryptosporidium in environmental waters. PMID:26880717

  12. Development of Primer Sets for Loop-Mediated Isothermal Amplification that Enables Rapid and Specific Detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae

    PubMed Central

    Wang, Deguo; Liu, Yanhong

    2015-01-01

    Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three pathogens are not available. With the 16S rRNA gene and 16S-23S rRNA intergenic spacers as targets, four sets of LAMP primers were designed for identification and differentiation of S. dysgalactiae, S. uberis and S. agalactiae. The detection limit of all four LAMP primer sets were 0.1 pg DNA template per reaction, the LAMP method with 16S rRNA gene and 16S-23S rRNA intergenic spacers as the targets can differentiate the three pathogens, which is potentially useful in epidemiological studies. PMID:26016433

  13. Development of Primer Sets for Loop-Mediated Isothermal Amplification that Enables Rapid and Specific Detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae.

    PubMed

    Wang, Deguo; Liu, Yanhong

    2015-06-01

    Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three pathogens are not available. With the 16S rRNA gene and 16S-23S rRNA intergenic spacers as targets, four sets of LAMP primers were designed for identification and differentiation of S. dysgalactiae, S. uberis and S. agalactiae. The detection limit of all four LAMP primer sets were 0.1 pg DNA template per reaction, the LAMP method with 16S rRNA gene and 16S-23S rRNA intergenic spacers as the targets can differentiate the three pathogens, which is potentially useful in epidemiological studies. PMID:26016433

  14. Loop-Mediated Isothermal Amplification (LAMP) for Rapid Detection and Quantification of Dehalococcoides Biomarker Genes in Commercial Reductive Dechlorinating Cultures KB-1 and SDC-9

    PubMed Central

    Kanitkar, Yogendra H.; Stedtfeld, Robert D.; Steffan, Robert J.; Hashsham, Syed A.

    2016-01-01

    Real-time quantitative PCR (qPCR) protocols specific to the reductive dehalogenase (RDase) genes vcrA, bvcA, and tceA are commonly used to quantify Dehalococcoides spp. in groundwater from chlorinated solvent-contaminated sites. In this study, loop-mediated isothermal amplification (LAMP) was developed as an alternative approach for the quantification of these genes. LAMP does not require a real-time thermal cycler (i.e., amplification is isothermal), allowing the method to be performed using less-expensive and potentially field-deployable detection devices. Six LAMP primers were designed for each of three RDase genes (vcrA, bvcA, and tceA) using Primer Explorer V4. The LAMP assays were compared to conventional qPCR approaches using plasmid standards, two commercially available bioaugmentation cultures, KB-1 and SDC-9 (both contain Dehalococcoides species). DNA was extracted over a growth cycle from KB-1 and SDC-9 cultures amended with trichloroethene and vinyl chloride, respectively. All three genes were quantified for KB-1, whereas only vcrA was quantified for SDC-9. A comparison of LAMP and qPCR using standard plasmids indicated that quantification results were similar over a large range of gene concentrations. In addition, the quantitative increase in gene concentrations over one growth cycle of KB-1 and SDC-9 using LAMP was comparable to that of qPCR. The developed LAMP assays for vcrA and tceA genes were validated by comparing quantification on the Gene-Z handheld platform and a real-time thermal cycler using DNA isolated from eight groundwater samples obtained from an SDC-9-bioaugmented site (Tulsa, OK). These assays will be particularly useful at sites subject to bioaugmentation with these two commonly used Dehalococcoides species-containing cultures. PMID:26746711

  15. High sensitivity, loop-mediated isothermal amplification combined with colorimetric gold-nanoparticle probes for visual detection of high risk human papillomavirus genotypes 16 and 18.

    PubMed

    Kumvongpin, Ratchanida; Jearanaikool, Patcharee; Wilailuckana, Chotechana; Sae-Ung, Nattaya; Prasongdee, Prinya; Daduang, Sakda; Wongsena, Metee; Boonsiri, Patcharee; Kiatpathomchai, Wansika; Swangvaree, Sukumarn Sanersak; Sandee, Alisa; Daduang, Jureerut

    2016-08-01

    High-risk human papillomavirus (HR-HPV) causes cervical cancer. HPV16 and HPV18 are the most prevalent strains of the virus reported in women worldwide. Loop-mediated isothermal amplification (LAMP) is an alternative method for DNA detection under isothermal conditions. However, it results in a turbid amplified product which is not easily detected by the naked eye. This study aimed to develop an improved technique by using gold nanoparticles (AuNPs) attached to a single-stranded DNA probe for the detection of HPV16 and HPV18. Detection of the LAMP product by AuNP color change was compared with detection by visual turbidity. The optimal conditions for this new LAMP-AuNP assay were an incubation time of 20min and a temperature of 65°C. After LAMP amplification was complete, its products were hybridized with the AuNP probe for 5min and then detected by the addition of magnesium salt. The color changed from red to blue as a result of aggregation of the AuNP probe under high ionic strength conditions produced by the addition of the salt. The sensitivity of the LAMP-AuNP assay was greater than the LAMP turbidity assay by up to 10-fold for both HPV genotypes. The LAMP-AuNP assay showed higher sensitivity and ease of visualization than did the LAMP turbidity for the detection of HPV16 and HPV18. Additionally, AuNP-HPV16 and AuNP-HPV18 probes were stable for over 1year. The combination of LAMP and the AuNP-probe colorimetric assay offers a simple, rapid and highly sensitive alternative diagnostic tool for the detection of HPV16 and HPV18 in district hospitals or field studies. PMID:27086727

  16. Detection of spring viraemia of carp virus (SVCV) by loop-mediated isothermal amplification (LAMP) in koi carp, Cyprinus carpio L

    USGS Publications Warehouse

    Shivappa, R.B.; Savan, R.; Kono, T.; Sakai, M.; Emmenegger, E.; Kurath, G.; Levine, J.F.

    2008-01-01

    Spring viraemia of carp virus (SVCV) is a rhabdovirus associated with systemic illness and mortality in cyprinids. Several diagnostic tests are available for detection of SVCV. However, most of these tests are time consuming and are not well adapted for field-based diagnostics. In this study, a diagnostic tool for SVCV detection based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been developed. Based on the nucleotide sequence of the glycoprotein (G) gene of SVCV North Carolina (NC) isolate, four sets (each set containing two outer and two inner) of primers were designed. Temperature and time conditions were optimized to 65 ??C and 60 min, respectively, for LAMP and RT-LAMP using one primer set. In vitro specificity was evaluated using four different strains of fish rhabdoviruses and RT-LAMP was found to be specific to SVCV. Serial dilutions of SVCV NC isolate was used to evaluate the in vitro sensitivity of RT-LAMP. Sensitivity of the assays was similar to RT-PCR and detected SVCV even at the lowest dilution of 10 1 TCID50 mL-1. The ability of RT-LAMP to detect SVCV from infected carp was also tested and the assay detected SVCV from all infected fish. The isothermal temperature requirements, high specificity and sensitivity, and short incubation time of the RT-LAMP assay make it an excellent choice as a field diagnostic test for SVCV. ?? 2008 The Authors.

  17. Development of reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a diagnostic tool of Toxoplasma gondii in pork.

    PubMed

    Qu, Daofeng; Zhou, Huaiyu; Han, Jianzhong; Tao, Siyue; Zheng, Bailing; Chi, Na; Su, Chunlei; Du, Aifang

    2013-02-18

    A fast, sensitive and specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of Toxoplasma gondii (T. gondii) in pork was developed. In this study, we used a conserved sequence of 18s rRNA of Toxoplasma gondii to design primers for RT-LAMP test. The amplication was able to finish in 60 min under isothermal condition at 63°C by employing a set of six primers. The assay showed higher sensitivity than RT-PCR using T. gondii RNA as template. The RT-LAMP assay was also assessed for specificity and was found to precisely discriminate between positive and negative test samples. Furthermore, the assay correctly detected T. gondii from contaminated pork, and had the detect limit of 1 tachyzoite in 1g pork. This is the first report of a study which applied the RT-LAMP method to detect T. gondii from pork. As RT-LAMP requires very basic instruments and the results can be obtained by visual observation, this technique provides a simple and reliable tool for inspecting food which are T. gondii-contaminated. PMID:23146414

  18. Rapid Identification of Officinal Akebiae Caulis and Its Toxic Adulterant Aristolochiae Manshuriensis Caulis (Aristolochia manshuriensis) by Loop-Mediated Isothermal Amplification

    PubMed Central

    Wu, Lan; Wang, Bo; Zhao, Mingming; Liu, Wei; Zhang, Peng; Shi, Yuhua; Xiong, Chao; Wang, Ping; Sun, Wei; Chen, Shilin

    2016-01-01

    Mu-tong (Akebiae Caulis) is a traditional Chinese medicine commonly used as a diuretic and antiphlogistic. A common adulterant of Mu-tong is Guan-mu-tong (Aristolochiae Manshuriensis Caulis), which is derived from the stem of Aristolochia manshuriensis Komarov, and contains carcinogenic aristolochic acids. We used an alternative technique, loop-mediated isothermal amplification (LAMP), to differentiate Mu-tong from Guan-mu-tong because LAMP is quick, highly sensitive, and specific. We designed a set of four common primers (G-F3, G-B3, G-FIP, and G-BIP) and a loop primer (G-LB) for LAMP based on the internal transcribed spacer 2 sequence of Ar. manshuriensis. We successfully amplified the LAMP assays and visual detection occurred within 60 min at isothermal conditions of 65°C. The LAMP reaction exhibited a tenfold increase in detection (4.22 pg/μl DNA) over conventional polymerase chain reaction demonstrating that LAMP is a useful technique to detect Guan-mu-tong. We conclude that the LAMP technique is a potentially valuable safety control method for simple and efficient discrimination of Mu-tong from its adulterant Guan-mu-tong. PMID:27379153

  19. Rapid Identification of Officinal Akebiae Caulis and Its Toxic Adulterant Aristolochiae Manshuriensis Caulis (Aristolochia manshuriensis) by Loop-Mediated Isothermal Amplification.

    PubMed

    Wu, Lan; Wang, Bo; Zhao, Mingming; Liu, Wei; Zhang, Peng; Shi, Yuhua; Xiong, Chao; Wang, Ping; Sun, Wei; Chen, Shilin

    2016-01-01

    Mu-tong (Akebiae Caulis) is a traditional Chinese medicine commonly used as a diuretic and antiphlogistic. A common adulterant of Mu-tong is Guan-mu-tong (Aristolochiae Manshuriensis Caulis), which is derived from the stem of Aristolochia manshuriensis Komarov, and contains carcinogenic aristolochic acids. We used an alternative technique, loop-mediated isothermal amplification (LAMP), to differentiate Mu-tong from Guan-mu-tong because LAMP is quick, highly sensitive, and specific. We designed a set of four common primers (G-F3, G-B3, G-FIP, and G-BIP) and a loop primer (G-LB) for LAMP based on the internal transcribed spacer 2 sequence of Ar. manshuriensis. We successfully amplified the LAMP assays and visual detection occurred within 60 min at isothermal conditions of 65°C. The LAMP reaction exhibited a tenfold increase in detection (4.22 pg/μl DNA) over conventional polymerase chain reaction demonstrating that LAMP is a useful technique to detect Guan-mu-tong. We conclude that the LAMP technique is a potentially valuable safety control method for simple and efficient discrimination of Mu-tong from its adulterant Guan-mu-tong. PMID:27379153

  20. Development of a rapid, simple method for detecting Naegleria fowleri visually in water samples by loop-mediated isothermal amplification (LAMP).

    PubMed

    Mahittikorn, Aongart; Mori, Hirotake; Popruk, Supaluk; Roobthaisong, Amonrattana; Sutthikornchai, Chantira; Koompapong, Khuanchai; Siri, Sukhontha; Sukthana, Yaowalark; Nacapunchai, Duangporn

    2015-01-01

    Naegleria fowleri is the causative agent of the fatal disease primary amebic meningoencephalitis. Detection of N. fowleri using conventional culture and biochemical-based assays is time-consuming and laborious, while molecular techniques, such as PCR, require laboratory skills and expensive equipment. We developed and evaluated a novel loop-mediated isothermal amplification (LAMP) assay targeting the virulence-related gene for N. fowleri. Time to results is about 90 min and amplification products were easily detected visually using hydroxy naphthol blue. The LAMP was highly specific after testing against related microorganisms and able to detect one trophozoite, as determined with spiked water and cerebrospinal fluid samples. The assay was then evaluated with a set of 80 water samples collected during the flooding crisis in Thailand in 2011, and 30 natural water samples from border areas of northern, eastern, western, and southern Thailand. N. fowleri was detected in 13 and 10 samples using LAMP and PCR, respectively, with a Kappa coefficient of 0.855. To the best of our knowledge, this is the first report of a LAMP assay for N. fowleri. Due to its simplicity, speed, and high sensitivity, the LAMP method described here might be useful for quickly detecting and diagnosing N. fowleri in water and clinical samples, particularly in resource-poor settings. PMID:25822175

  1. Development of a Rapid, Simple Method for Detecting Naegleria fowleri Visually in Water Samples by Loop-Mediated Isothermal Amplification (LAMP)

    PubMed Central

    Mahittikorn, Aongart; Mori, Hirotake; Popruk, Supaluk; Roobthaisong, Amonrattana; Sutthikornchai, Chantira; Koompapong, Khuanchai; Siri, Sukhontha; Sukthana, Yaowalark; Nacapunchai, Duangporn

    2015-01-01

    Naegleria fowleri is the causative agent of the fatal disease primary amebic meningoencephalitis. Detection of N. fowleri using conventional culture and biochemical-based assays is time-consuming and laborious, while molecular techniques, such as PCR, require laboratory skills and expensive equipment. We developed and evaluated a novel loop-mediated isothermal amplification (LAMP) assay targeting the virulence-related gene for N. fowleri. Time to results is about 90 min and amplification products were easily detected visually using hydroxy naphthol blue. The LAMP was highly specific after testing against related microorganisms and able to detect one trophozoite, as determined with spiked water and cerebrospinal fluid samples. The assay was then evaluated with a set of 80 water samples collected during the flooding crisis in Thailand in 2011, and 30 natural water samples from border areas of northern, eastern, western, and southern Thailand. N. fowleri was detected in 13 and 10 samples using LAMP and PCR, respectively, with a Kappa coefficient of 0.855. To the best of our knowledge, this is the first report of a LAMP assay for N. fowleri. Due to its simplicity, speed, and high sensitivity, the LAMP method described here might be useful for quickly detecting and diagnosing N. fowleri in water and clinical samples, particularly in resource-poor settings. PMID:25822175

  2. Erwinia amylovora loop-mediated isothermal amplification (LAMP) assay for rapid pathogen detection and on-site diagnosis of fire blight.

    PubMed

    Bühlmann, Andreas; Pothier, Joël F; Rezzonico, Fabio; Smits, Theo H M; Andreou, Michael; Boonham, Neil; Duffy, Brion; Frey, Jürg E

    2013-03-01

    Several molecular methods have been developed for the detection of Erwinia amylovora, the causal agent of fire blight in pear and apple, but none are truly applicable for on-site use in the field. We developed a fast, reliable and field applicable detection method using a novel target on the E. amylovora chromosome that we identified by applying a comparative genomic pipeline. The target coding sequences (CDSs) are both uniquely specific for and all-inclusive of E. amylovora genotypes. This avoids potential false negatives that can occur with most commonly used methods based on amplification of plasmid gene targets, which can vary among strains. Loop-mediated isothermal AMPlification (LAMP) with OptiGene Genie II chemistry and instrumentation proved to be an exceptionally rapid (under 15 min) and robust method for detecting E. amylovora in orchards, as well as simple to use in the plant diagnostic laboratory. Comparative validation results using plant samples from inoculated greenhouse trials and from natural field infections (of regional and temporal diverse origin) showed that our LAMP had an equivalent or greater performance regarding sensitivity, specificity, speed and simplicity than real-time PCR (TaqMan), other LAMP assays, immunoassays and plating, demonstrating its utility for routine testing. PMID:23275135

  3. Development and application of loop-mediated isothermal amplification for detection of the F167Y mutation of carbendazim-resistant isolates in Fusarium graminearum.

    PubMed

    Duan, Yabing; Zhang, Xiaoke; Ge, Changyan; Wang, Yong; Cao, Junhong; Jia, Xiaojing; Wang, Jianxin; Zhou, Mingguo

    2014-01-01

    Resistance of Fusarium graminearum to carbendazim is caused by point mutations in the β2-tubulin gene. The point mutation at codon 167 (TTT → TAT, F167Y) occurs in more than 90% of field resistant isolates in China. To establish a suitable method for rapid detection of the F167Y mutation in F. graminearum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed and optimized to specially distinguish the F167Y mutation genotype. The LAMP reaction was optimal at 63 °C for 60 min. When hydroxynaphthol blue dye (HNB) was added prior to amplification, samples with DNA of the F167Y mutation developed a characteristic sky blue color after the reaction but those without DNA or with different DNA did not. Results of HNB staining method were reconfirmed by gel electrophoresis. The developed LAMP had good specificity, stability and repeatability and was suitable for monitoring carbendazim-resistance populations of F. graminearum in agricultural production. PMID:25403277

  4. Ultrasensitive electrochemical detection of avian influenza A (H7N9) virus DNA based on isothermal exponential amplification coupled with hybridization chain reaction of DNAzyme nanowires.

    PubMed

    Yu, Yanyan; Chen, Zuanguang; Jian, Wensi; Sun, Duanping; Zhang, Beibei; Li, Xinchun; Yao, Meicun

    2015-02-15

    In this work, a simple and label-free electrochemical biosensor with duel amplification strategy was developed for DNA detection based on isothermal exponential amplification (EXPAR) coupled with hybridization chain reaction (HCR) of DNAzymes nanowires. Through rational design, neither the primer nor the DNAzymes containing molecular beacons (MBs) could react with the duplex probe which were fixed on the electrode surface. Once challenged with target, the duplex probe cleaved and triggered the EXPAR mediated target recycle and regeneration circles as well as the HCR process. As a result, a greater amount of targets were generated to cleave the duplex probes. Subsequently, the nanowires consisting of the G-quadruplex units were self-assembled through hybridization with the strand fixed on the electrode surface. In the presence of hemin, the resulting catalytic G-quadruplex-hemin HRP-mimicking DNAzymes were formed. Electrochemical signals can be obtained by measuring the increase in reduction current of oxidized 3.3',5.5'-tetramethylbenzidine sulfate (TMB), which was generated by DNAzyme in the presence of H2O2. This method exhibited ultrahigh sensitivity towards avian influenza A (H7N9) virus DNA sequence with detection limits of 9.4 fM and a detection range of 4 orders of magnitude. The biosensor was also capable of discriminating single-nucleotide difference among concomitant DNA sequences and performed well in spiked cell lysates. PMID:25310490

  5. A method for simultaneous detection and identification of Brazilian dog- and vampire bat-related rabies virus by reverse transcription loop-mediated isothermal amplification assay.

    PubMed

    Saitou, Yasumasa; Kobayashi, Yuki; Hirano, Shinji; Mochizuki, Nobuyuki; Itou, Takuya; Ito, Fumio H; Sakai, Takeo

    2010-09-01

    At present, the sporadic occurrence of human rabies in Brazil can be attributed primarily to dog- and vampire bat-related rabies viruses. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was employed as a simultaneous detection method for both rabies field variants within 60 min. Vampire bat-related rabies viruses could be distinguished from dog variants by digesting amplicons of the RT-LAMP reaction using the restriction enzyme AlwI. Amplification and digestion could both be completed within 120 min after RNA extraction. In addition, the RT-LAMP assay also detected rabies virus in isolates from Brazilian frugivorous bats and Ugandan dog, bovine and goat samples. In contrast, there were false negative results from several Brazilian insectivorous bats and all of Chinese dog, pig, and bovine samples using the RT-LAMP assay. This study showed that the RT-LAMP assay is effective for the rapid detection of rabies virus isolates from the primary reservoir in Brazil. Further improvements are necessary so that the RT-LAMP assay can be employed for the universal detection of genetic variants of rabies virus in the field. PMID:20403387

  6. Invading stacking primer: A trigger for high-efficiency isothermal amplification reaction with superior selectivity for detecting microRNA variants.

    PubMed

    Liu, Weipeng; Zhu, Minjun; Liu, Hongxing; Wei, Jitao; Zhou, Xiaoming; Xing, Da

    2016-07-15

    Searching for a strategy to enhance the efficiency of nucleic acid amplification and achieve exquisite discrimination of nucleic acids at the single-base level for biological detection has become an exciting research direction in recent years. Here, we have developed a simple and universal primer design strategy which produces a fascinating effect on isothermal strand displacement amplification (iSDA). We refer to the resultant primer as "invading stacking primer (IS-Primer)" which is based on contiguous stacking hybridization and toehold-mediated exchange reaction and function by merely changing the hybridization location of the primer. Using the IS-Primer, the sensitivity in detecting the target miR-21 is improved approximately five fold compared with the traditional iSDA reaction. It was further demonstrated that the IS-Primer acts as an invading strand to initiate branch migration which can increase the efficiency of the untwisting of the hairpin probe. This effect is equivalent to reducing the free energy of the stem, and the technique shows superior selectivity for single-base mismatches. By demonstrating the enhanced effect of the IS-Primer in the iSDA reaction, this work may provide a potentially new avenue for developing more sensitive and selective nucleic acids assays. PMID:26985583

  7. Detection of Cucurbit chlorotic yellows virus from Bemisia tabaci captured on sticky traps using reverse transcription loop-mediated isothermal amplification (RT-LAMP) and simple template preparation.

    PubMed

    Okuda, Mitsuru; Okuda, Shiori; Iwai, Hisashi

    2015-09-01

    Cucurbit chlorotic yellows virus (CCYV) of the genus Crinivirus within the family Closteroviridae is an emerging infectious agent of cucurbits leading to severe disease and significant economic losses. Effective detection and identification methods for this virus are urgently required. In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect CCYV from its vector Bemisia tabaci. LAMP primer sets to detect CCYV were evaluated for their sensitivity and specificity, and a primer set designed from the HSP70h gene with corresponding loop primers were selected. The RT-LAMP assay was applied to detect CCYV from viruliferous B. tabaci trapped on sticky traps. A simple extraction procedure using RNAsecure™ was developed for template preparation. CCYV was detected in all of the B. tabaci 0, 1, 7 and 14 days after they were trapped. Although the rise of turbidity was delayed in reactions using RNA from B. tabaci trapped for 7 and 14 days compared with those from 0 and 1 day, the DNA amplification was sufficient to detect CCYV in all of the samples. These findings therefore present a simple template preparation method and an effective RT-LAMP assay, which can be easily and rapidly performed to monitor CCYV-viruliferous B. tabaci in the field. PMID:25912723

  8. Development of a Reverse Transcription Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Subtype H7N9 Avian Influenza Virus

    PubMed Central

    Bao, Hongmei; Zhao, Yuhui; Wang, Yunhe; Xu, Xiaolong; Shi, Jianzhong; Zeng, Xianying; Wang, Xiurong; Chen, Hualan

    2014-01-01

    A novel influenza A (H7N9) virus has emerged in China. To rapidly detect this virus from clinical samples, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of the H7N9 virus. The minimum detection limit of the RT-LAMP assay was 0.01 PFU H7N9 virus, making this method 100-fold more sensitive to the detection of the H7N9 virus than conventional RT-PCR. The H7N9 virus RT-LAMP assays can efficiently detect different sources of H7N9 influenza virus RNA (from chickens, pigeons, the environment, and humans). No cross-reactive amplification with the RNA of other subtype influenza viruses or of other avian respiratory viruses was observed. The assays can effectively detect H7N9 influenza virus RNA in drinking water, soil, cloacal swab, and tracheal swab samples that were collected from live poultry markets, as well as human H7N9 virus, in less than 30 min. These results suggest that the H7N9 virus RT-LAMP assays were efficient, practical, and rapid diagnostic methods for the epidemiological surveillance and diagnosis of influenza A (H7N9) virus from different resource samples. PMID:24689044

  9. Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay.

    PubMed

    Nkouawa, Agathe; Sako, Yasuhito; Okamoto, Munehiro; Ito, Akira

    2016-06-01

    For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical. PMID:27044566

  10. Development and application of loop-mediated isothermal amplification for detection of the F167Y mutation of carbendazim-resistant isolates in Fusarium graminearum

    PubMed Central

    Duan, Yabing; Zhang, Xiaoke; Ge, Changyan; Wang, Yong; Cao, Junhong; Jia, Xiaojing; Wang, Jianxin; Zhou, Mingguo

    2014-01-01

    Resistance of Fusarium graminearum to carbendazim is caused by point mutations in the β2-tubulin gene. The point mutation at codon 167 (TTT → TAT, F167Y) occurs in more than 90% of field resistant isolates in China. To establish a suitable method for rapid detection of the F167Y mutation in F. graminearum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed and optimized to specially distinguish the F167Y mutation genotype. The LAMP reaction was optimal at 63°C for 60 min. When hydroxynaphthol blue dye (HNB) was added prior to amplification, samples with DNA of the F167Y mutation developed a characteristic sky blue color after the reaction but those without DNA or with different DNA did not. Results of HNB staining method were reconfirmed by gel electrophoresis. The developed LAMP had good specificity, stability and repeatability and was suitable for monitoring carbendazim-resistance populations of F. graminearum in agricultural production. PMID:25403277

  11. A rapid molecular diagnosis of cutaneous leishmaniasis by colorimetric malachite green-loop-mediated isothermal amplification (LAMP) combined with an FTA card as a direct sampling tool.

    PubMed

    Nzelu, Chukwunonso O; Cáceres, Abraham G; Guerrero-Quincho, Silvia; Tineo-Villafuerte, Edwin; Rodriquez-Delfin, Luis; Mimori, Tatsuyuki; Uezato, Hiroshi; Katakura, Ken; Gomez, Eduardo A; Guevara, Angel G; Hashiguchi, Yoshihisa; Kato, Hirotomo

    2016-01-01

    Leishmaniasis remains one of the world's most neglected diseases, and early detection of the infectious agent, especially in developing countries, will require a simple and rapid test. In this study, we established a quick, one-step, single-tube, highly sensitive loop-mediated isothermal amplification (LAMP) assay for rapid detection of Leishmania DNA from tissue materials spotted on an FTA card. An FTA-LAMP with pre-added malachite green was performed at 64°C for 60min using a heating block and/or water bath and DNA amplification was detected immediately after incubation. The LAMP assay had high detection sensitivity down to a level of 0.01 parasites per μl. The field- and clinic-applicability of the colorimetric FTA-LAMP assay was demonstrated with 122 clinical samples collected from patients suspected of having cutaneous leishmaniasis in Peru, from which 71 positives were detected. The LAMP assay in combination with an FTA card described here is rapid and sensitive, as well as simple to perform, and has great potential usefulness for diagnosis and surveillance of leishmaniasis in endemic areas. PMID:26516109

  12. Loop-mediated isothermal amplification of vanA gene enables a rapid and naked-eye detection of vancomycin-resistant enterococci infection.

    PubMed

    Kim, Hye Jin; Kim, Yu Jin; Yong, Dong Eun; Lee, Kyungwon; Park, Jeon Han; Lee, Jae Myun; Yoon, Sang Sun

    2014-09-01

    Vancomycin-resistant enterococci (VRE) are one of the leading causes of nosocomial infection at intensive care unit (ICU). A rapid and sensitive detection of VRE infection is in high demand for timely and suitable antibiotic treatment. Here, we optimized a distinct DNA-based diagnostic technique, loop-mediated isothermal amplification (LAMP) for a rapid detection of the presence of vanA gene, a critical component of the gene cluster required for vancomycin resistance. Amplification efficiency was optimal at 62°C and with 2mM MgSO4. The detection limit of the DNA template was 80pg and LAMP amplicons were detected within 40min; thereby suggesting a potential applicability of LAMP as a sensitive and urgent diagnostic method. Furthermore, positive LAMP reaction was directly detected with the naked-eye by monitoring the formation of a white precipitate or the color change induced by hydroxy naphthol blue (HNB) dye. Finally, 56 clinical isolates were successfully tested for the presence of vanA gene by LAMP, which was determined to be more sensitive than PCR. Together, our results clearly demonstrate the usefulness of LAMP for the diagnosis of VRE infection. PMID:24925601

  13. Rapid diagnostic detection of plum pox virus in Prunus plants by isothermal AmplifyRP(®) using reverse transcription-recombinase polymerase amplification.

    PubMed

    Zhang, Shulu; Ravelonandro, Michel; Russell, Paul; McOwen, Nathan; Briard, Pascal; Bohannon, Seven; Vrient, Albert

    2014-10-01

    Plum pox virus (PPV) causes the most destructive viral disease known as plum pox or Sharka disease in stone fruit trees. As an important regulated pathogen, detection of PPV is thus of critical importance to quarantine and eradication of the spreading disease. In this study, the innovative development of two AmplifyRP(®) tests is reported for a rapid isothermal detection of PPV using reverse transcription-recombinase polymerase amplification. In an AmplifyRP(®) test, all specific recombination and amplification reactions occur at a constant temperature without thermal cycling and the test results are either recorded in real-time with a portable fluorescence reader or displayed using a lateral flow strip contained inside an amplicon detection chamber. The major improvement of this assay is that the entire test from sample preparation to result can be completed in as little as 20min and can be performed easily both in laboratories and in the field. The results from this study demonstrated the ability of the AmplifyRP(®) technique to detect all nine PPV strains (An, C, CR, D, EA, M, Rec, T, or W). Among the economic benefits to pathogen surveys is the higher sensitivity of the AmplifyRP(®) to detect PPV when compared to the conventional ELISA and ImmunoStrip(®) assays. This is the first report describing the use of such an innovative technique to detect rapidly plant viruses affecting perennial crops. PMID:25010790

  14. Comparative study of sensitivity, linearity, and resistance to inhibition of digital and nondigital polymerase chain reaction and loop mediated isothermal amplification assays for quantification of human cytomegalovirus.

    PubMed

    Nixon, Gavin; Garson, Jeremy A; Grant, Paul; Nastouli, Eleni; Foy, Carole A; Huggett, Jim F

    2014-05-01

    Performing nucleic acid amplification techniques (NAATs) in digital format using limiting dilution provides potential advantages that have recently been demonstrated with digital polymerase chain reaction (dPCR). Key benefits that have been claimed are the ability to quantify nucleic acids without the need of an external calibrator and a greater resistance to inhibitors than real-time quantitative PCR (qPCR). In this study, we evaluated the performance of four NAATs, qPCR, dPCR, real-time quantitative loop mediated isothermal amplification (qLAMP), and digital LAMP (dLAMP), for the detection and quantification of human cytomegalovirus (hCMV). We used various DNA templates and inhibitors to compare the performance of these methods using a conventional real-time thermocycler platform (Bio-Rad CFX96) and a chip based digital platform (Fluidigm Biomark 12.765 Digital Array). dPCR performed well and demonstrated greater resistance to inhibitors than the other methods although this resistance did not apply equally to all inhibitors tested. dLAMP was found to be less sensitive than dPCR, but its quantitative performance was better than qLAMP, the latter being unable to quantify below 1000 copies. dLAMP was also more resistant to inhibitors than qLAMP. Unlike qPCR, both digital methods were able to quantify viral genomes without requiring a calibrator; however, neither can currently compete with the large reaction volumes, and thus the greater absolute sensitivity, of qPCR. With the introduction of digital instrumentation that will enable larger reaction volumes, digital amplification methods such as those evaluated in this study could potentially offer a robust alternative to qPCR for nucleic acid quantification. PMID:24684191

  15. Feasibility and Operational Performance of Tuberculosis Detection by Loop-Mediated Isothermal Amplification Platform in Decentralized Settings: Results from a Multicenter Study.

    PubMed

    Gray, Christen M; Katamba, Achilles; Narang, Pratibha; Giraldo, Jorge; Zamudio, Carlos; Joloba, Moses; Narang, Rahul; Paramasivan, C N; Hillemann, Doris; Nabeta, Pamela; Amisano, Danielle; Alland, David; Cobelens, Frank; Boehme, Catharina C

    2016-08-01

    Currently available nucleic acid amplification platforms for tuberculosis (TB) detection are not designed to be simple or inexpensive enough to implement in decentralized settings in countries with a high burden of disease. The loop-mediated isothermal amplification platform (LAMP) may change this. We conducted a study in adults with symptoms suggestive of TB in India, Uganda, and Peru to establish the feasibility of using TB-LAMP (Eiken Chemical Co.) in microscopy laboratories compared with using smear microscopy against a reference standard of solid and liquid cultures. Operational characteristics were evaluated as well. A total of 1,777 participants met the eligibility criteria and were included for analysis. Overall, TB-LAMP sensitivities among culture-positive samples were 97.2% (243/250; 95% confidence interval [CI], 94.3% to 98.2%) and 62.0% (88/142; 95% CI, 53.5% to 70.0%) for smear-positive and smear-negative TB, respectively, but varied widely by country and operator. Specificities ranged from 94.5% (446/472; 95% CI, 92.0% to 96.4%) to 98.0% (350/357; 95% CI, 96.0% to 99.2%) by country. A root cause analysis identified high temperatures, high humidity, and/or low reaction volumes as possible causes for false-positive results, as they may result in nonspecific amplification. The study was repeated in India with training focused on vulnerable steps and an updated protocol; 580 participants were included for analysis. Specificity in the repeat trial was 96.6% (515/533; 95% CI, 94.7% to 97.9%). To achieve acceptable performance of LAMP at the microscopy center level, significant training and infrastructure requirements are necessary. PMID:27194691

  16. Improved Detection Limit in Rapid Detection of Human Enterovirus 71 and Coxsackievirus A16 by a Novel Reverse Transcription–Isothermal Multiple-Self-Matching-Initiated Amplification Assay

    PubMed Central

    Ding, Xiong; Nie, Kai; Shi, Lei; Zhang, Yong; Guan, Li; Zhang, Dan

    2014-01-01

    Rapid detection of human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) is important in the early phase of hand-foot-and-mouth disease (HFMD). In this study, we developed and evaluated a novel reverse transcription–isothermal multiple-self-matching-initiated amplification (RT-IMSA) assay for the rapid detection of EV71 and CVA16 by use of reverse transcriptase, together with a strand displacement DNA polymerase. Real-time RT-IMSA assays using a turbidimeter and visual RT-IMSA assays to detect EV71 and CVA16 were established and completed in 1 h, and the reported corresponding real-time reverse transcription–loop-mediated isothermal amplification (RT-LAMP) assays targeting the same regions of the VP1 gene were adopted as parallel tests. Through testing VP1 RNAs transcribed in vitro, the real-time RT-IMSA assays exhibited better linearity of quantification, with R2 values of 0.952 (for EV71) and 0.967 (for CVA16), than the real-time RT-LAMP assays, which had R2 values of 0.803 (for EV71) and 0.904 (for CVA16). Additionally, the detection limits of the real-time RT-IMSA assays (approximately 937 for EV71 and 67 for CVA16 copies/reaction) were higher than those of real-time RT-LAMP assays (approximately 3,266 for EV71 and 430 for CVA16 copies/reaction), and similar results were observed in the visual RT-IMSA assays. The new approaches also possess high specificities for the corresponding targets, with no cross-reactivity observed. In clinical assessment, compared to commercial reverse transcription-quantitative PCR (qRT-PCR) kits, the diagnostic sensitivities of the real-time RT-IMSA assays (96.4% for EV71 and 94.6% for CVA16) were higher than those of the real-time RT-LAMP assays (91.1% for EV71 and 90.8% for CVA16). The visual RT-IMSA assays also exhibited the same results. In conclusion, this proof-of-concept study suggests that the novel RT-IMSA assay is superior to the RT-LAMP assay in terms of detection limit and has the potential to rapidly detect EV71

  17. Development and Application of Loop-Mediated Isothermal Amplification Assays for Rapid Visual Detection of cry2Ab and cry3A Genes in Genetically-Modified Crops

    PubMed Central

    Li, Feiwu; Yan, Wei; Long, Likun; Qi, Xing; Li, Congcong; Zhang, Shihong

    2014-01-01

    The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs) containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP) method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM) crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field. PMID:25167136

  18. Development and application of loop-mediated isothermal amplification assays for rapid visual detection of cry2Ab and cry3A genes in genetically-modified crops.

    PubMed

    Li, Feiwu; Yan, Wei; Long, Likun; Qi, Xing; Li, Congcong; Zhang, Shihong

    2014-01-01

    The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs) containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP) method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM) crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field. PMID:25167136

  19. Rapid and Sensitive Detection of Shigella spp. and Salmonella spp. by Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification Technique.

    PubMed

    Wang, Yi; Wang, Yan; Luo, Lijuan; Liu, Dongxin; Luo, Xia; Xu, Yanmei; Hu, Shoukui; Niu, Lina; Xu, Jianguo; Ye, Changyun

    2015-01-01

    Shigella and Salmonella are frequently isolated from various food samples and can cause human gastroenteritis. Here, a novel multiple endonuclease restriction real-time loop-mediated isothermal amplification technology (MERT-LAMP) were successfully established and validated for simultaneous detection of Shigella strains and Salmonella strains in only a single reaction. Two sets of MERT-LAMP primers for 2 kinds of pathogens were designed from ipaH gene of Shigella spp. and invA gene of Salmonella spp., respectively. Under the constant condition at 63°C, the positive results were yielded in as short as 12 min with the genomic DNA extracted from the 19 Shigella strains and 14 Salmonella strains, and the target pathogens present in a sample could be simultaneously identified based on distinct fluorescence curves in real-time format. Accordingly, the multiplex detection assay significantly reduced effort, materials and reagents used, and amplification and differentiation were conducted at the same time, obviating the use of postdetection procedures. The analytical sensitivity of MERT-LAMP was found to be 62.5 and 125 fg DNA/reaction with genomic templates of Shigella strains and Salmonella strains, which was consist with normal LAMP assay, and at least 10- and 100-fold more sensitive than that of qPCR and conventional PCR approaches. The limit of detection of MERT-LAMP for Shigella strains and Salmonella strains detection in artificially contaminated milk samples was 5.8 and 6.4 CFU per vessel. In conclusion, the MERT-LAMP methodology described here demonstrated a potential and valuable means for simultaneous screening of Shigella and Salmonella in a wide variety of samples. PMID:26697000

  20. Rapid and Sensitive Detection of Shigella spp. and Salmonella spp. by Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification Technique

    PubMed Central

    Wang, Yi; Wang, Yan; Luo, Lijuan; Liu, Dongxin; Luo, Xia; Xu, Yanmei; Hu, Shoukui; Niu, Lina; Xu, Jianguo; Ye, Changyun

    2015-01-01

    Shigella and Salmonella are frequently isolated from various food samples and can cause human gastroenteritis. Here, a novel multiple endonuclease restriction real-time loop-mediated isothermal amplification technology (MERT-LAMP) were successfully established and validated for simultaneous detection of Shigella strains and Salmonella strains in only a single reaction. Two sets of MERT-LAMP primers for 2 kinds of pathogens were designed from ipaH gene of Shigella spp. and invA gene of Salmonella spp., respectively. Under the constant condition at 63°C, the positive results were yielded in as short as 12 min with the genomic DNA extracted from the 19 Shigella strains and 14 Salmonella strains, and the target pathogens present in a sample could be simultaneously identified based on distinct fluorescence curves in real-time format. Accordingly, the multiplex detection assay significantly reduced effort, materials and reagents used, and amplification and differentiation were conducted at the same time, obviating the use of postdetection procedures. The analytical sensitivity of MERT-LAMP was found to be 62.5 and 125 fg DNA/reaction with genomic templates of Shigella strains and Salmonella strains, which was consist with normal LAMP assay, and at least 10- and 100-fold more sensitive than that of qPCR and conventional PCR approaches. The limit of detection of MERT-LAMP for Shigella strains and Salmonella strains detection in artificially contaminated milk samples was 5.8 and 6.4 CFU per vessel. In conclusion, the MERT-LAMP methodology described here demonstrated a potential and valuable means for simultaneous screening of Shigella and Salmonella in a wide variety of samples. PMID:26697000

  1. Rapid and simple method for detecting the toxin B gene of Clostridium difficile in stool specimens by loop-mediated isothermal amplification.

    PubMed

    Kato, Haru; Yokoyama, Toshiyuki; Kato, Hideaki; Arakawa, Yoshichika

    2005-12-01

    We applied the loop-mediated isothermal amplification (LAMP) assay to the detection of the toxin B gene (tcdB) of Clostridium difficile for identification of toxin B (TcdB)-positive C. difficile strains and detection of tcdB in stool specimens. tcdB was detected in all toxin A (TcdA)-positive, TcdB-positive (A(+)B(+)) and TcdA-negative, TcdB-positive (A(-)B(+)) C. difficile strains but not from TcdA-negative, TcdB-negative strains. Of the 74 stool specimens examined, A(+)B(+) or A(-)B(+) C. difficile was recovered from 39 specimens, of which 38 specimens were LAMP positive and one was negative. Amplification was obtained in 10 specimens that were culture negative, indicating that LAMP is highly sensitive. The LAMP assay was applied to detection of tcdB in DNA extracted by a simple boiling method from 47 of those 74 specimens, which were cultured overnight in cooked-meat medium (CMM). Twenty-two of 24 culture-positive specimens were positive for LAMP on DNA from the culture in CMM. Four specimens were culture negative but positive by LAMP on DNA from CMM cultures. The LAMP assay is a reliable tool for identification of TcdB-positive C. difficile as well as for direct detection of tcdB in stool specimens with high sensitivity. Detection of tcdB by LAMP from overnight cultures in CMM could be an alternative method of diagnostic testing at clinical laboratories without special apparatus. PMID:16333105

  2. Smartphone-Imaged HIV-1 Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) on a Chip from Whole Blood

    PubMed Central

    Damhorst, Gregory L.; Duarte-Guevara, Carlos; Chen, Weili; Ghonge, Tanmay; Cunningham, Brian T.; Bashir, Rashid

    2015-01-01

    Viral load measurements are an essential tool for the long-term clinical care of hum an immunodeficiency virus (HIV)-positive individuals. The gold standards in viral load instrumentation, however, are still too limited by their size, cost, and sophisticated operation for these measurements to be ubiquitous in remote settings with poor healthcare infrastructure, including parts of the world that are disproportionately affected by HIV infection. The challenge of developing a point-of-care platform capable of making viral load more accessible has been frequently approached but no solution has yet emerged that meets the practical requirements of low cost, portability, and ease-of-use. In this paper, we perform reverse-transcription loop-mediated isothermal amplification (RT-LAMP) on minimally processed HIV-spiked whole blood samples with a microfluidic and silicon microchip platform, and perform fluorescence measurements with a consumer smartphone. Our integrated assay shows amplification from as few as three viruses in a ~ 60 nL RT-LAMP droplet, corresponding to a whole blood concentration of 670 viruses per µL of whole blood. The technology contains greater power in a digital RT-LAMP approach that could be scaled up for the determination of viral load from a finger prick of blood in the clinical care of HIV-positive individuals. We demonstrate that all aspects of this viral load approach, from a drop of blood to imaging the RT-LAMP reaction, are compatible with lab-on-a-chip components and mobile instrumentation. PMID:26705482

  3. A Loop-Mediated Isothermal Amplification (LAMP) Assay for Early Detection of Schistosoma mansoni in Stool Samples: A Diagnostic Approach in a Murine Model

    PubMed Central

    Fernández-Soto, Pedro; Gandasegui Arahuetes, Javier; Sánchez Hernández, Alicia; López Abán, Julio; Vicente Santiago, Belén; Muro, Antonio

    2014-01-01

    Background Human schistosomiasis, mainly due to Schistosoma mansoni species, is one of the most prevalent parasitic diseases worldwide. To overcome the drawbacks of classical parasitological and serological methods in detecting S. mansoni infections, especially in acute stage of the disease, development of cost-effective, simple and rapid molecular methods is still needed for the diagnosis of schistosomiasis. A promising approach is the loop-mediated isothermal amplification (LAMP) technology. Compared to PCR-based assays, LAMP has the advantages of reaction simplicity, rapidity, specificity, cost-effectiveness and higher amplification efficiency. Additionally, as results can be inspected by the naked eye, the technique has great potential for use in low-income countries. Methodology/Principal findings A sequence corresponding to a mitochondrial S. mansoni minisatellite DNA region was selected as a target for designing a LAMP-based method to detect S. mansoni DNA in stool samples. We used a S. mansoni murine model to obtain well defined stool and sera samples from infected mice with S. mansoni cercariae. Samples were taken weekly from week 0 to 8 post-infection and the Kato-Katz and ELISA techniques were used for monitoring the infection. Primer set designed were tested using a commercial reaction mixture for LAMP assay and an in house mixture to compare results. Specificity of LAMP was tested using 16 DNA samples from different parasites, including several Schistosoma species, and no cross-reactions were found. The detection limit of our LAMP assay (SmMIT-LAMP) was 1 fg of S. mansoni DNA. When testing stool samples from infected mice the SmMIT-LAMP detected S. mansoni DNA as soon as 1 week post-infection. Conclusions/Significance We have developed, for the first time, a cost-effective, easy to perform, specific and sensitive LAMP assay for early detection of S. mansoni in stool samples. The method is potentially and readily adaptable for field diagnosis and

  4. Hyd5 gene-based detection of the major gushing-inducing Fusarium spp. in a loop-mediated isothermal amplification (LAMP) assay.

    PubMed

    Denschlag, Carla; Vogel, Rudi F; Niessen, Ludwig

    2012-06-01

    Fusarium graminearum and the closely related F. culmorum were found to be associated with over foaming of bottled beer (gushing) when contaminated brewing malt is used. The presence of highly surface active hydrophobins produced by these fungi upon growth on wheat or barley in the field or during malting may affect bubble formation and stability in gushing beers and other carbonated beverages. Aiming for a method for the rapid and user friendly analysis of unmalted and malted cereals during quality control in the brewing industry, a loop-mediated isothermal amplification (LAMP) assay for the detection of Fusarium spp. capable of producing the gushing inducing hydrophobin Hyd5p was set up. A set of primers was designed towards a 221 bp region within the hyd5 gene of F. culmorum. The LAMP product was verified by sequencing a 150 bp portion. Testing specificity with purified DNA from 99 different fungal species as well as barley and wheat showed that DNA synthesis only occurred during LAMP when DNA of the closely related species F. graminearum, F. culmorum, F. cerealis and F. lunulosporum were used as template. In-tube indirect detection of DNA amplification was applied using manganese-quenched calcein as fluorescence indicator for pyrophosphate produced during DNA synthesis. The assay had a detection limit of 0.74 pg of purified target DNA which corresponds 20 copy numbers per reaction within 30 minutes using a simple heating block. Analysis of Fusarium infected cereals revealed that the assay was able to detect F. graminearum at a level of 0.5% of infected grains in uninfected barley by analysis of surface washings without further sample preparation. Results show that the hyd5 based LAMP assay can be a rapid, useful and sensitive tool for quality control in the brewing and malting industry. PMID:22554927

  5. Loop-mediated isothermal amplification method for diagnosing Pneumocystis pneumonia in HIV-uninfected immunocompromised patients with pulmonary infiltrates.

    PubMed

    Nakashima, Kei; Aoshima, Masahiro; Ohkuni, Yoshihiro; Hoshino, Eri; Hashimoto, Kohei; Otsuka, Yoshihito

    2014-12-01

    Loop-mediated isothermal amplification (LAMP) is becoming an established nucleic acid amplification method offering rapid, accurate, and cost-effective diagnosis of infectious diseases. We retrospectively evaluated 78 consecutive HIV-uninfected patients who underwent LAMP method for diagnosing Pneumocystis pneumonia (PCP). Diagnosis of PCP was made by the detection of Pneumocystis jirovecii (P. jirovecii) with positive LAMP or conventional staining (CS) (Grocott methenamine silver staining or Diff-Quick™) on the basis of compatible clinical symptoms and radiologic findings. Additionally, we reviewed HIV-uninfected immunocompromised patients who underwent subcontract PCR as a historical control. LAMP was positive in 10 (90.9%) of 11 positive-CS patients. Among 13 negative-CS patients with positive LAMP, 11 (84.6%) had PCP, and the remaining 2 were categorized as having P. jirovecii colonization. LDH levels in negative-CS PCP were higher than in positive-CS PCP (p = 0.026). (1 → 3)-β-D-glucan levels in negative-CS PCP were lower than in positive-CS PCP (p = 0.011). The interval from symptom onset to diagnosis as PCP in LAMP group (3.45 ± 1.77 days; n = 22) was shorter than in subcontract PCR group (6.90 ± 2.28 days; n = 10; p < 0.001). As for patients without PCP, duration of unnecessary PCP treatment in LAMP group (2; 2-3 days; n = 10) was shorter than in subcontract PCR group (7; 7-12.25 days; n = 6; p = 0.003). LAMP showed higher sensitivity (95.4%) and positive predictive value (91.3%) than subcontract PCR did. Pneumocystis LAMP method is a sensitive and cost-effective diagnostic method and is easy to administer in general hospitals. In-house LAMP method would realize early diagnosis of PCP, resulting in improving PCP prognosis and reducing unnecessary PCP-specific treatment. PMID:25187511

  6. Ag(I)-coordinated hairpin DNA for homogenous electronic monitoring of hepatitis C virus accompanying isothermal cycling signal amplification strategy.

    PubMed

    Lu, Minghua; Xu, Linfang; Zhang, Xiaona; Xiao, Rui; Wang, Youmei

    2015-11-15

    This work designs a new homogenous electronic monitoring platform for sensitive detection of hepatitis C virus (HCV) on an immobilization-free Ag(I)-assisted hairpin DNA through the cytosine-Ag(+)-cytosine coordination chemistry. The assay consists of target-induced Ag(+) dissociation from hairpin DNA and an isothermal circular strand-displacement polymerization (ICSDP) reaction. Upon target analyte introduction, HCV DNA initially hybridizes with hairpin DNA to disrupt the Ag(I)-coordinated hairpin probe and releases the coordinated Ag(+) ion, then the newly formed DNA duplex induces the ICSDP reaction with the aid of primer and polymerase, and then the displaced target DNA retriggers Ag(I)-coordinated hairpin DNA with target recycling, thereby resulting in formation of numerous free Ag(+) ions in the detection cell. The released Ag(+) ions can be readily captured by the negatively charged screen-printed carbon electrode, and subsequent anodic-stripping voltammetric detection of the captured Ag(+) ions are conducted to form the anodic current for the production of the electrochemical signal within the applied potential. Under optimal conditions, the ICSDP-based homogenous sensing system can be utilized for the detection of HCV DNA at a concentration as low as 2.3 pM. Intra- and inter-assay coefficients of variation with identical batches are below 9.5% and 10.5%, respectively. The analysis in 5 clinical serum specimens shows good accordance between results obtained by the developed method and commercial Cobas® Amplicor HCV Test Analyzer. PMID:26071691

  7. Loop-mediated isothermal amplification assay targeting the blaCTX-M9 gene for detection of extended spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae.

    PubMed

    Thirapanmethee, Krit; Pothisamutyothin, Kanokporn; Nathisuwan, Surakit; Chomnawang, Mullika T; Wiwat, Chanpen

    2014-12-01

    Extended-spectrum β-lactamases (ESBLs) produced by Enterobacteriaceae are one of the resistance mechanisms to most β-lactam antibiotics. ESBLs are currently a major problem in both hospitals and community settings worldwide. Rapid and reliable means of detecting ESBL-producing bacteria is necessary for identification, prevention and treatment. Loop-mediated isothermal amplification (LAMP) is a technique that rapidly amplifies DNA with high specificity and sensitivity under isothermal conditions. This study was aimed to develop a convenient, accurate and inexpensive method for detecting ESBL-producing bacteria by a LAMP technique. ESBLs-producing Escherichia coli and Klebsiella pneumoniae were isolated from a tertiary hospital in Bangkok, Thailand and reconfirmed by double-disk synergy test. A set of four specific oligonucleotide primers of LAMP for detection of bla(CTX-M9) gene was designed based on bla(CTX-M9) from E. coli (GenBank Accession No. AJ416345). The LAMP reaction was amplified under isothermal temperature at 63°C for 60 min. Ladder-like patterns of band sizes from 226 bp of the bla(CTX-M9) DNA target was observed. The LAMP product was further analyzed by restriction digestion with MboI and TaqI endonucleases. The fragments generated were approximately 168, 177 and 250 bp in size for MboI digestion and 165, 193, 229, 281 and 314 bp for TaqI digestion, which is in agreement with the predicted sizes. The sensitivity of the LAMP technique to bla(CTX-M9) was greater than that of the PCR method by at least 10,000-fold. These results showed that the LAMP primers specifically amplified only the bla(CTX-M9) gene. Moreover, the presence of LAMP amplicon was simply determined by adding SYBR Green I in the reaction. In conclusion, this technique for detection of ESBLs is convenient, reliable and easy to perform routinely in hospitals or laboratory units in developing countries. PMID:25284314

  8. Sensitive Visual Detection of AHPND Bacteria Using Loop-Mediated Isothermal Amplification Combined with DNA-Functionalized Gold Nanoparticles as Probes

    PubMed Central

    Arunrut, Narong; Kampeera, Jantana; Sirithammajak, Sarawut; Sanguanrut, Piyachat; Proespraiwong, Porranee; Suebsing, Rungkarn; Kiatpathomchai, Wansika

    2016-01-01

    Acute hepatopancreatic necrosis disease (AHPND) is a component cause of early mortality syndrome (EMS) of shrimp. In 2013, the causative agent was found to be unique isolates of Vibrio parahaemolyticus (VPAHPND) that contained a 69 kbp plasmid (pAP1) carrying binary Pir-like toxin genes PirvpA and PirvpB. In Thailand, AHPND was first recognized in 2012, prior to knowledge of the causative agent, and it subsequently led to a precipitous drop in shrimp production. After VPAHPND was characterized, a major focus of the AHPND control strategy was to monitor broodstock shrimp and post larvae for freedom from VPAHPND by nucleic acid amplification methods, most of which required use of expensive and sophisticated equipment not readily available in a shrimp farm setting. Here, we describe a simpler but equally sensitive approach for detection of VPAHPND based on loop-mediated isothermal amplification (LAMP) combined with unaided visual reading of positive amplification products using a DNA-functionalized, ssDNA-labled nanogold probe (AuNP). The target for the special set of six LAMP primers used was the VPAHPND PirvpA gene. The LAMP reaction was carried out at 65°C for 45 min followed by addition of the red AuNP solution and further incubation at 65°C for 5 min, allowing any PirvpA gene amplicons present to hybridize with the probe. Hybridization protected the AuNP against aggregation, so that the solution color remained red upon subsequent salt addition (positive test result) while unprotected AuNP aggregated and underwent a color change from red to blue and eventually precipitated (negative result). The total assay time was approximately 50 min. The detection limit (100 CFU) was comparable to that of other commonly-used methods for nested PCR detection of VPAHPND and 100-times more sensitive than 1-step PCR detection methods (104 CFU) that used amplicon detection by electrophoresis or spectrophotometry. There was no cross reaction with DNA templates derived from non

  9. Rapid and sensitive detection of porcine epidemic diarrhea virus by reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip.

    PubMed

    Gou, Hongchao; Deng, Jieru; Wang, Jiaying; Pei, Jingjing; Liu, Wenjun; Zhao, Mingqiu; Chen, Jinding

    2015-02-01

    Porcine epidemic diarrhea virus (PEDV) is an important pathogen that causes vomiting, diarrhea, and dehydration, leading to serious damage to the swine industry worldwide. The establishment of effective diagnostic methods is imperative. However, traditional methods are often unsuitable. In this study, reverse transcription loop-mediated isothermal amplification (RT-LAMP) was combined with a vertical flow (VF) nucleic acid detection strip to detect PEDV. Parameters that affect the RT-LAMP reaction were optimized. The RT-LAMP-VF assay that we established was performed at 62 °C for 40 min, and then directly evaluated on the VF visualization strip cassette. The method demonstrated high specificity for PEDV. The detection limit was 10 pg of ribonucleic acid, consistent with RT-PCR, RT-LAMP detected products on agarose gels and by direct calcein fluorescence. Application of this method to clinical samples yielded a positivity rate that was comparable to that obtained for RT-PCR. This technique saves time and is efficient, and is thus expected to be useful for the diagnosis of PEDV infection in the field. PMID:25444939

  10. The detection of T-Nos, a genetic element present in GMOs, by cross-priming isothermal amplification with real-time fluorescence.

    PubMed

    Zhang, Fang; Wang, Liu; Fan, Kai; Wu, Jian; Ying, Yibin

    2014-05-01

    An isothermal cross-priming amplification (CPA) assay for Agrobacterium tumefaciens nopaline synthase terminator (T-Nos) was established and investigated in this work. A set of six specific primers, recognizing eight distinct regions on the T-Nos sequence, was designed. The CPA assay was performed at a constant temperature, 63 °C, and detected by real-time fluorescence. The results indicated that real-time fluorescent CPA had high specificity, and the limit of detection was 1.06 × 10(3) copies of rice genomic DNA, which could be detected in 40 min. Comparison of real-time fluorescent CPA and conventional polymerase chain reaction (PCR) was also performed. Results revealed that real-time fluorescent CPA had a comparable sensitivity to conventional real-time PCR and had taken a shorter time. In addition, different contents of genetically modified (GM)-contaminated rice seed powder samples were detected for practical application. The result showed real-time fluorescent CPA could detect 0.5 % GM-contaminated samples at least, and the whole reaction could be finished in 35 min. Real-time fluorescent CPA is sensitive enough to monitor labeling systems and provides an attractive method for the detection of GMO. PMID:24748469

  11. Development and evaluation of a loop-mediated isothermal amplification assay combined with enrichment culture for rapid detection of very low numbers of Vibrio parahaemolyticus in seafood samples.

    PubMed

    Di, Huiling; Ye, Lei; Neogi, Sucharit Basu; Meng, Hecheng; Yan, He; Yamasaki, Shinji; Shi, Lei

    2015-01-01

    The aim of this study was to develop and evaluate a rapid and effective method to detect Vibrio parahaemolyticus, a leading pathogen causing seafood-borne gastroenteritis. A newly designed loop-mediated isothermal amplification (LAMP) assay including a short enrichment period was optimized. This assay correctly detected all the target strains (n=61) but none of the non-target strains (n=34). Very low numbers of V. parahaemolyticus (2 colony forming unit (CFU) per gram of seafood) could be detected within 3 h and the minimum time of the whole assay was only 5 h. Comparative screening of various seafood samples (n=70) indicated that the LAMP assay is superior to polymerase chain reaction (PCR) and conventional culture methods because it is more rapid and less complex. This highly sensitive LAMP assay can be applicable as the method of choice in large-scale and rapid screening of seafood and environmental samples to detect V. parahaemolyticus strains. PMID:25744462

  12. Field Evaluation of Malaria Microscopy, Rapid Malaria Tests and Loop-Mediated Isothermal Amplification in a Rural Hospital in South Western Ethiopia

    PubMed Central

    Prieto-Pérez, Laura; Martin-Martin, Ines; Berzosa, Pedro; González, Vicenta; Tisiano, Gebre; Balcha, Seble; Ramos, José Manuel; Górgolas, Miguel

    2015-01-01

    Background In up to one third of the hospitals in some rural areas of Africa, laboratory services in malaria diagnosis are limited to microscopy by thin film, as no capability to perform thick film exists (gold standard in terms of sensitivity for malaria diagnosis). A new rapid molecular malaria diagnostic test called Loop-mediated isothermal DNA amplification (LAMP) has been recently validated in clinical trials showing exceptional sensitivity and specificity features. It could be a reliable diagnostic tool to be implemented without special equipment or training. Objective The objective of this proof of concept study was to confirm the feasibility of using LAMP technique for diagnosis of malaria in a rural Ethiopian hospital with limited resources. Methodology/Principal Findings This study was carried out in Gambo General Hospital, West Arsi Province (Ethiopia), from November 1st to December 31st 2013. A total of 162 patients with a non-focal febrile syndrome were investigated. The diagnostic capability (sensitivity, specificity, positive predictive and negative predictive values) of rapid malaria tests and microscopy by thin film was evaluated in comparison with LAMP. Eleven (6.79%) out of the 162 patients with fever and suspected malaria, tested positive for LAMP, 3 (1.85%) for rapid malaria tests and none of the eleven cases was detected by thin film microscopy. Conclusions/Significance LAMP can be performed in basic rural laboratories without the need for specialized infrastructure and it may set a reliable tool for malaria control to detect a low level parasitemia. PMID:26555068

  13. Development and Diagnostic Evaluation of Loop-Mediated Isothermal Amplification Using a New Gene Target for Rapid Detection of Helicobacter pylori

    PubMed Central

    Bakhtiari, Somaye; Alvandi, Amirhooshang; Pajavand, Hamid; Navabi, Jafar; Najafi, Farid; Abiri, Ramin

    2016-01-01

    Background Helicobacter pylori cause chronic gastritis and subsequent diseases like gastric and duodenal ulcers and gastric adenocarcinoma. Current methods for detecting H. pylori have several disadvantages and it is of utmost importance to develop a simple, quick, accurate, and cost-effective diagnostic test. Objectives The aim of this study was to set up and evaluate a diagnostic value of loop- mediated isothermal amplification (LAMP) for detecting H. pylori. Patients and Methods The analytical sensitivity values (limit of detection) of LAMP and polymerase chain reaction (PCR) were determined using serial dilutions of H. pylori DNA. Analytical specificity of the methods using new designed primers targeted ureC gene was also determined. Results The detection limits of the LAMP and PCR assay were similar and were 10 fg of pure DNA of H. pylori, which is equal to 6 copy numbers of H. pylori genome. Analytical specificity of the tests was 100% because the tests were positive only with H. pylori DNA. Conclusions The analytical sensitivity of LAMP and PCR methods, using the designed primers, was 8 times more than any other reported methods. The designed methods are specific and sensitive for detection of H. pylori in different clinical and environmental samples. PMID:27540449

  14. Development of a Loop-Mediated Isothermal Amplification Assay for Rapid and Specific Identification of ACT Producing Alternaria alternata, the Agent of Brown Spot Disease in Tangerine.

    PubMed

    Moghimi, Hamid; Moradi, Amir; Hamedi, Javad; Basiri, Mina

    2016-03-01

    Rapid, accurate, and easy identification of pathogenic agents has always been important in medicine, veterinary, and agriculture. The brown spot infection is among the most common diseases in tangerine caused by Alternaria alternata. Due to the existence of seven various pathotypes of A. alternata species, it is challenging and time consuming to detect a pathotype responsible for citrus brown spot. In this study, we were seeking a rapid and specific approach to identify the tangerine pathotype within the A. alternata-pathogenic species, using the loop-mediated isothermal amplification (LAMP) method and actts2 gene as a marker molecule. Nine pathogenic samples were obtained from the region of Ramsar, Iran, and certified as A. alternata-pathogenic isolates. Specific primers were designed for regions coding for Alternaria citri toxin (ACT), and the PCR and LAMP reactions were performed. Our data showed that the primers designed for the tangerine pathotype of A. alternata were specific, and in both reactions, positive results were only observed in desired pathotypes. In the other pathotypes of this species as well as other standard fungal samples as negative controls, no positive result was observed. Therefore, our results suggest the possibility to detect the tangerine-specific A. alternata pathotype from other related species with a high accuracy and in early stages of the disease. PMID:26638210

  15. Development of a Loop Mediated Isothermal Amplification (LAMP) - Surface Enhanced Raman spectroscopy (SERS) Assay for the Detection of Salmonella Enterica Serotype Enteritidis

    PubMed Central

    Draz, Mohamed Shehata; Lu, Xiaonan

    2016-01-01

    As a major foodborne pathogen, Salmonella enterica serotype Enteritidis is increasingly rising as a global health concern. Here, we developed an integrated assay that combines loop mediated isothermal amplification (LAMP) and surface enhanced Raman spectroscopy (SERS) for DNA detection of S. Enteritidis using specifically designed Raman active Au-nanoprobes. The target DNA was amplified by LAMP and then labeled with Au-nanoprobes comprised of gold nanoparticle-modified with specific cy5/DNA probes to allow the detection by SERS. The sensitivity of the developed LAMP-SERS detection assay (66 CFU/mL) was ~100-fold higher than the conventional polymerase chain reaction (PCR) method. Significantly, this technique allowed highly specific detection of the target DNA of S. Enteritidis and could differentiate it from the DNA of closely related bacterial species or non-specific contamination, making it more accurate and reliable than the standard LAMP technique. The applicability of detection of S. Enteritidis in milk samples using LAMP-SERS assay was validated as well. In sum, the developed LAMP-SERS assay is highly specific and sensitive, and has the potential to be applied for rapid detection of different foodborne pathogens and other microbial contaminants. PMID:26941845

  16. Establishment and application of a loop-mediated isothermal amplification method for simple, specific, sensitive and rapid detection of Toxoplasma gondii.

    PubMed

    Cao, Lili; Cheng, Ronghua; Yao, Lin; Yuan, Shuxian; Yao, Xinhua

    2014-01-01

    The Loop-mediated isothermal amplification (LAMP) method amplifies DNA with high simply, specificity, sensitivity and rapidity. In this study, A LAMP assay with 6 primers targeting a highly conserved region of the GRA1 gene was developed to diagnose Toxoplasma gondii. The reaction time of the LAMP assay was shortened to 30 min after optimizing the reaction system. The LAMP assay was found to be highly specific and stable. The detection limit of the LAMP assay was 10 copies, the same as that of the conventional PCR. We used the LAMP assay to develop a real-time fluorogenic protocol to quantitate T. gondii DNA and generated a log-linear regression plot by plotting the time-to-threshold values against genomic equivalent copies. Furthermore, the LAMP assay was applied to detect T. gondii DNA in 423 blood samples and 380 lymph node samples from 10 pig farms, and positive results were obtained for 7.8% and 8.2% of samples, respectively. The results showed that the LAMP method is slightly more sensitive than conventional PCR (6.1% and 7.6%). Positive samples obtained from 6 pig farms. The LAMP assay established in this study resulted in simple, specific, sensitive and rapid detection of T. gondii DNA and is expected to play an important role in clinical detection of T. gondii. PMID:23965849

  17. Development of a Loop Mediated Isothermal Amplification (LAMP) - Surface Enhanced Raman spectroscopy (SERS) Assay for the Detection of Salmonella Enterica Serotype Enteritidis.

    PubMed

    Draz, Mohamed Shehata; Lu, Xiaonan

    2016-01-01

    As a major foodborne pathogen, Salmonella enterica serotype Enteritidis is increasingly rising as a global health concern. Here, we developed an integrated assay that combines loop mediated isothermal amplification (LAMP) and surface enhanced Raman spectroscopy (SERS) for DNA detection of S. Enteritidis using specifically designed Raman active Au-nanoprobes. The target DNA was amplified by LAMP and then labeled with Au-nanoprobes comprised of gold nanoparticle-modified with specific cy5/DNA probes to allow the detection by SERS. The sensitivity of the developed LAMP-SERS detection assay (66 CFU/mL) was ~100-fold higher than the conventional polymerase chain reaction (PCR) method. Significantly, this technique allowed highly specific detection of the target DNA of S. Enteritidis and could differentiate it from the DNA of closely related bacterial species or non-specific contamination, making it more accurate and reliable than the standard LAMP technique. The applicability of detection of S. Enteritidis in milk samples using LAMP-SERS assay was validated as well. In sum, the developed LAMP-SERS assay is highly specific and sensitive, and has the potential to be applied for rapid detection of different foodborne pathogens and other microbial contaminants. PMID:26941845

  18. Development of a loop-mediated isothermal amplification (LAMP) for the detection of F5 fimbriae gene in enterotoxigenic Escherichia coli (ETEC).

    PubMed

    Jiang, Kuiyu; Zhu, Ying; Liu, Wenxin; Feng, Yufei; He, Lili; Guan, Weikun; Hu, Wenxia; Shi, Dongfang

    2012-11-01

    The objective of this study was to establish a loop-mediated isothermal amplification (LAMP) method for the detection of F5 fimbriae gene in Enterotoxigenic Escherichia coli. A set of four primers were designed based on the conservative sequence of coding F5 fimbriae. Temperature and time condition, specificity test, and sensitivity test were performed with the DNA of Escherichia coli (F5+). The results showed that the optimal reaction condition for LAMP was achieved at 61 °C for 45 min in a water bath. Ladder-like products were produced with those F5-positive samples by LAMP, while no product was generated with other negative samples. The assay of LAMP had a detection limit equivalent to 72 cfu/tube, which was more sensitive than PCR (7.2 × 10(2) cfu/tube). The agreement rate between LAMP and PCR was 100 % in detecting simulation samples. Thus, the LAMP assay may be a new method for rapid detection of F5 fimbriae gene of ETEC. PMID:22890294

  19. Investigation of false positives associated with loop-mediated isothermal amplification assays for detection of Toxoplasma gondii in archived tissue samples of captive felids.

    PubMed

    Suleman, Essa; Mtshali, Moses Sibusiso; Lane, Emily

    2016-09-01

    Toxoplasma gondii is a ubiquitous protozoan parasite that infects humans and many different animals, including felids. Many molecular and serologic tests have been developed for detection of T. gondii in a wide range of hosts. Loop-mediated isothermal amplification (LAMP) is a field-friendly technique that lacks the practical drawbacks of other molecular and serologic tests, and LAMP assays have been successfully developed for detection of T. gondii in fresh tissue samples. In the current study, both a previously published and a de-novo designed LAMP assay were compared to a quantitative real-time (q)PCR assay, for the detection of T. gondii in archived formalin-fixed, paraffin-embedded (FFPE) tissue samples from captive wildlife. The LAMP assays produced conflicting results, generating both false positives and false negatives. Furthermore, the LAMP assays were unable to positively identify samples with low levels of parasites as determined by qPCR and histopathology. Therefore, these LAMP assays may not be the most suitable assays for detection of T. gondii in archived FFPE and frozen tissue samples. PMID:27449130

  20. A novel and rapid diagnostic method for discriminating between feces of sika deer and Japanese serow by loop-mediated isothermal amplification.

    PubMed

    Aikawa, T; Horino, S; Ichihara, Y

    2015-08-01

    Severe damages to natural vegetation, agriculture, and forestry caused by overpopulation of sika deer (Cervus nippon) have markedly increased in Japan in recent years. To devise a population management plan of sika deer, information on the distribution and population size of the animal in each region is indispensable. An easy and effective method to obtain this information is to count the fecal pellets in the field. However, the habitat of sika deer in Japan overlaps that of Japanese serow (Capricornis crispus). Additionally, it is difficult to discriminate between the feces of both animals. Here, we present a rapid and precise diagnostic method for discriminating between the feces of sika deer and Japanese serow using loop-mediated isothermal amplification (LAMP) targeting cytochrome b gene in the mitochondrial DNA. Our results showed that the LAMP can discriminate between the feces of sika deer and Japanese serow, and the method is simpler and more sensitive than the conventional molecular diagnostic method. Since LAMP method does not require special skills for molecular biology techniques, even the field researchers who have never done a molecular experiment can easily carry out the protocol. In addition, the entire protocol, from DNA extraction from fecal pellet to identification of species, takes only about 75 min and does not require expensive equipment. Hence, this diagnostic method is simple, fast, and accessible to anyone. As such, the method can be a useful tool to estimate distribution and population size of sika deer. PMID:26084704

  1. A highly specific and sensitive loop-mediated isothermal amplification method for the detection of Escherichia coli O157:H7.

    PubMed

    Ravan, Hadi; Amandadi, Mojdeh; Sanadgol, Nima

    2016-02-01

    E. coli O157:H7 is one of the most important foodborne pathogen that causes some human illnesses such as bloody diarrhea, hemolytic-uremic syndrome, and kidney failure. We developed a loop-mediated isothermal amplification (LAMP) assay with six special primers that target a highly specific 299-bp region of the Z3276 gene for the detection of E. coli O157:H7. Among 117 bacterial strains tested in this study, positive results were only obtained from E. coli O157:H7 strains. The sensitivity level of the Z3276-LAMP assay was determined to be 5 CFU/reaction tube in pure bacterial culture. Moreover, the LAMP assay was successfully applied to artificially contaminated ground beef with a sensitivity level of 10(3) CFU/mL without pre-enrichment and 10 CFU/mL after a 4-h pre-enrichment. In conclusion, the present LAMP assay would be a useful and powerful tool for the rapid, sensitive, and specific diagnosis of E. coli O157:H7 strains in resource limited laboratories. PMID:26724736

  2. Development and application of a loop-mediated isothermal amplification assay for rapid identification of aflatoxigenic molds and their detection in food samples.

    PubMed

    Luo, Jie; Vogel, Rudi F; Niessen, Ludwig

    2012-10-15

    Aflatoxins are the most thoroughly studied mycotoxins. They are produced by several members of the genus Aspergillus in section Flavi with Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nomius being frequently isolated from contaminated food sources. In this work, we describe the development and evaluation of loop-mediated isothermal amplification (LAMP) assays for rapid detection of the three species in separate analyses. The acl1-gene of A. flavus and amy1-genes of A. nomius and A. parasiticus were used as target genes. The detection limits were 2.4, 7.6 and 20pg of pure DNA/reaction for A. flavus, A. nomius and A. parasiticus, respectively. For specificity testing, DNA extracted from mycelia of representative strains of 39 Aspergillus species, 23 Penicillium species, 75 Fusarium species and 37 other fungal species was used as a template for the specific LAMP primer sets developed for the three target species. The LAMP assay was combined with a DNA extraction method for the analysis of pure fungal cultures as well as artificially contaminated Brazil nuts, peanuts and green coffee beans. It is suggested that the developed LAMP assay is a promising tool in the prediction of a potential aflatoxin risk in food and food raw materials and may therefore be suitable for high throughput analysis in the food industry. PMID:23107500

  3. Colorimetric Method of Loop-Mediated Isothermal Amplification with the Pre-Addition of Calcein for Detecting Flavobacterium columnare and its Assessment in Tilapia Farms.

    PubMed

    Suebsing, Rungkarn; Kampeera, Jantana; Sirithammajak, Sarawut; Withyachumnarnkul, Boonsirm; Turner, Warren; Kiatpathomchai, Wansika

    2015-03-01

    Flavobacterium columnare, the causative agent of columnaris disease in fish, affects many economically important freshwater fish species. A colorimetric method of loop-mediated isothermal amplification with the pre-addition of calcein (LAMP-calcein) was developed and used to detect the presence of F. columnare in farmed tilapia (Nile Tilapia Oreochromis niloticus and red tilapia [Nile Tilapia × Mozambique Tilapia O. mossambicus]) and rearing water. The detection method, based on a change in color from orange to green, could be performed within 45 min at 63°C. The method was highly specific, as it had no cross-detections with 14 other bacterial species, including other fish pathogens and two Flavobacterium species. The method has a minimum detection limit of 2.2 × 10(2) F. columnare CFU; thus, it is about 10 times more sensitive than conventional PCR. With this method, F. columnare was detected in gonad, gill, and blood samples from apparently healthy tilapia broodstock as well as in samples of fertilized eggs, newly hatched fry, and rearing water. The bacteria isolated from the blood were further characterized biochemically and found to be phenotypically identical to F. columnare. The amplified products from the LAMP-calcein method had 97% homology with the DNA sequence of F. columnare. PMID:25584663

  4. Evaluation of loop-mediated isothermal amplification method (LAMP) for pathogenic Leptospira spp. detection with leptospires isolation and real-time PCR.

    PubMed

    Suwancharoen, Duangjai; Sittiwicheanwong, Busara; Wiratsudakul, Anuwat

    2016-09-01

    Leptospirosis has been one of the worldwide zoonotic diseases caused by pathogenic Leptospira spp. Many molecular techniques have consecutively been developed to detect such pathogen including loop-mediated isothermal amplification method (LAMP). The objectives of this study were to evaluate the diagnostic accuracy of LAMP assay and real-time PCR using bacterial culture as the gold standard and to assess the agreement among these three tests using Cohen's kappa statistics. In total, 533 urine samples were collected from 266 beef and 267 dairy cattle reared in central region of Thailand. Sensitivity and specificity of LAMP were 96.8% (95% CI 81.5-99.8) and 97.0% (95% CI 94.9-98.2), respectively. The accuracy of LAMP (97.0%) was significantly higher than that of real-time PCR (91.9%) at 95% CI. With Cohen's kappa statistics, culture method and LAMP were substantially agreed with each other (77.4%), whereas real-time PCR only moderately agreed with culture (47.7%) and LAMP (45.3%), respectively. Consequently, LAMP was more effective than real-time PCR in detecting Leptospira spp. in the urine of cattle. Besides, LAMP had less cost and was simpler than real-time PCR. Thus, LAMP was an excellent alternative for routine surveillance of leptospirosis in cattle. PMID:27150208

  5. Development and application of a rapid and visual loop-mediated isothermal amplification for the detection of Sporisorium scitamineum in sugarcane.

    PubMed

    Su, Yachun; Yang, Yuting; Peng, Qiong; Zhou, Dinggang; Chen, Yun; Wang, Zhuqing; Xu, Liping; Que, Youxiong

    2016-01-01

    Smut is a fungal disease with widespread prevalence in sugarcane planting areas. Early detection and proper identification of Sporisorium scitamineum are essential in smut management practices. In the present study, four specific primers targeting the core effector Pep1 gene of S. scitamineum were designed. Optimal concentrations of Mg(2+), primer and Bst DNA polymerase, the three important components of the loop-mediated isothermal amplification (LAMP) reaction system, were screened using a single factor experiment method and the L16(4(5)) orthogonal experimental design. Hence, a LAMP system suitable for detection of S. scitamineum was established. High specificity of the LAMP method was confirmed by the assay of S. scitamineum, Fusarium moniliforme, Pestalotia ginkgo, Helminthospcrium sacchari, Fusarium oxysporum and endophytes of Yacheng05-179 and ROC22. The sensitivity of the LAMP method was equal to that of the conventional PCR targeting Pep1 gene and was 100 times higher than that of the conventional PCR assay targeting bE gene in S. scitamineum. The results suggest that this novel LAMP system has strong specificity and high sensitivity. This method not only provides technological support for the epidemic monitoring of sugarcane smut, but also provides a good case for development of similar detection technology for other plant pathogens. PMID:27035751

  6. Loop-mediated isothermal amplification assay for detection of Histomonas meleagridis infection in chickens targeting the 18S rRNA sequences.

    PubMed

    Xu, Jinjun; Qu, Chanbao; Tao, Jianping

    2014-01-01

    Histomonas meleagridis is the causative agent of histomonosis, a disease of gallinaceous fowl characterized by necrotic typhlitis, hepatitis, and high mortality. To develop a rapid and sensitive method for specific detection of H. meleagridis, an assay based on loop-mediated isothermal amplification (LAMP) targeting the 18S rRNA gene was established. The detection limit of the LAMP assay was 10 copies for standard plasmids containing an 18S rRNA gene fragment, which was superior to that of a classical PCR method. Specificity tests revealed that there was no cross-reaction with other protozoa such as Trichomonas gallinae, Blastocytis sp, Tetratrichomonas gallinarum, Plasmodium gallinaceum, Toxoplasma gondii, Eimeria tenella, Leucocytozoon caulleryi and Leucocytozoon sabrazesi. The assay was evaluated for its diagnostic utility using liver and caeca samples collected from suspected field cases, the detection rate was 100 and 97.92%, respectively. These results indicate that the LAMP assay may be a useful tool for rapid detection and identification of H. meleagridis in poultry. PMID:24320623

  7. Establishment of reverse transcription loop-mediated isothermal amplification for rapid detection and differentiation of canine distemper virus infected and vaccinated animals.

    PubMed

    Liu, Da-Fei; Liu, Chun-Guo; Tian, Jin; Jiang, Yi-Tong; Zhang, Xiao-Zhan; Chai, Hong-Liang; Yang, Tian-Kuo; Yin, Xiu-Chen; Zhang, Hong-Ying; Liu, Ming; Hua, Yu-Ping; Qu, Lian-Dong

    2015-06-01

    Although widespread vaccination against canine distemper virus (CDV) has been conducted for many decades, several canine distemper outbreaks in vaccinated animals have been reported frequently. In order to detect and differentiate the wild-type and vaccine strains of the CDV from the vaccinated animals, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed. A set of four primers-two internal and two external-were designed to target the H gene for the specific detection of wild-type CDV variants. The CDV-H RT-LAMP assay rapidly amplified the target gene, within 60 min, using a water bath held at a constant temperature of 65°C. The assay was 100-fold more sensitive than conventional RT-PCR, with a detection limit of 10(-1)TCID50ml(-1). The system showed a preference for wild-type CDV, and exhibited less sensitivity to canine parvovirus, canine adenovirus type 1 and type 2, canine coronavirus, and canine parainfluenza virus. The assay was validated using 102 clinical samples obtained from vaccinated dog farms, and the results were comparable to a multiplex nested RT-PCR assay. The specific CDV-H RT-LAMP assay provides a simple, rapid, and sensitive tool for the detection of canines infected with wild-type CDV from canines vaccinated with attenuated vaccine. PMID:25769803

  8. Development and application of a rapid and visual loop-mediated isothermal amplification for the detection of Sporisorium scitamineum in sugarcane

    PubMed Central

    Su, Yachun; Yang, Yuting; Peng, Qiong; Zhou, Dinggang; Chen, Yun; Wang, Zhuqing; Xu, Liping; Que, Youxiong

    2016-01-01

    Smut is a fungal disease with widespread prevalence in sugarcane planting areas. Early detection and proper identification of Sporisorium scitamineum are essential in smut management practices. In the present study, four specific primers targeting the core effector Pep1 gene of S. scitamineum were designed. Optimal concentrations of Mg2+, primer and Bst DNA polymerase, the three important components of the loop-mediated isothermal amplification (LAMP) reaction system, were screened using a single factor experiment method and the L16(45) orthogonal experimental design. Hence, a LAMP system suitable for detection of S. scitamineum was established. High specificity of the LAMP method was confirmed by the assay of S. scitamineum, Fusarium moniliforme, Pestalotia ginkgo, Helminthospcrium sacchari, Fusarium oxysporum and endophytes of Yacheng05-179 and ROC22. The sensitivity of the LAMP method was equal to that of the conventional PCR targeting Pep1 gene and was 100 times higher than that of the conventional PCR assay targeting bE gene in S. scitamineum. The results suggest that this novel LAMP system has strong specificity and high sensitivity. This method not only provides technological support for the epidemic monitoring of sugarcane smut, but also provides a good case for development of similar detection technology for other plant pathogens. PMID:27035751

  9. [Laboratory-based evaluation of loop-mediated isothermal amplification (LAMP) to detect Cryptosporidium oocyst and Giardia lamblia cyst in stool specimens].

    PubMed

    Nago, Tamami T; Tokashiki, Yoshino T; Kisanuki, Kyoko; Nakasone, Isamu; Yamane, Nobuhisa

    2010-08-01

    To establish an alternative and more sensitive test method to detect oocyst of Cryptosporidium parvum and cyst of Giardia lamblia in clinical stool specimens, loop-mediated isothermal amplification (LAMP) was evaluated. Minimum cell concentrations at which LAMP assay could detect C. parvum oocyst and G. lamblia cyst were determined as 6.25 x 10(-1) and 3.12 x 10(-1) cells/assay when the stool specimens were spiked with the respective parasites. The results indicated 400 times higher sensitivities or more when compared to the microscopic readings. Twenty and nineteen diarrhea stool specimens spiked with C. parvum oocyst or G. lamblia cyst were assayed by LAMP. The results indicated that 14 (70%) and 16 (84%) samples successfully resulted in positive readings. But the remaining 6 and 3 samples were read as negative probably due to residual stool color. However, further dilutions of DNA extraction samples and addition of bovine serum albumin to LAMP reaction mixture showed positive effects on the occurrence of false-negative readings. With these results, we can conclude that the LAMP assay provides us an accurate and highly sensitive test method to detect C. parvum oocyst and cyst of G. lamblia, in place of labor-intensive and experience-dependent microscopic examination, in clinical laboratories. PMID:20860168

  10. Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification.

    PubMed

    Hammond, Rosemarie W; Zhang, Shulu

    2016-10-01

    A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39°C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in infected leaf and seed tissues. The performance of the AmplifyRP(®) Acceler8™ RT-RPA diagnostic assay, utilizing a lateral flow strip contained within an amplicon detection chamber, was evaluated and the results were compared with a standard RT-PCR assay. The AmplifyRP(®) Acceler8™ assay was specific for TCDVd in leaf and seed tissues, its sensitivity was comparable to conventional RT-PCR in leaf tissues, and it does not require extensive sample purification, specialized equipment, or technical expertise. This is the first report utilizing an RT-RPA assay to detect viroids and the assay can be used both in the laboratory and in the field for TCDVd detection. PMID:27427473

  11. Identification of human DNA in forensic evidence by loop-mediated isothermal amplification combined with a colorimetric gold nanoparticle hybridization probe.

    PubMed

    Watthanapanpituck, Khanistha; Kiatpathomchai, Wansika; Chu, Eric; Panvisavas, Nathinee

    2014-11-01

    A DNA test based on loop-mediated isothermal amplification (LAMP) and colorimetric gold nanoparticle (AuNP) hybridization probe to detect the presence of human DNA in forensic evidence was developed. The LAMP primer set targeted eight regions of the human cytochrome b, and its specificity was verified against the DNA of 11 animal species, which included animals closely related to humans, such as chimpanzee and orangutan. By using the AuNP probe, sequence-specific LAMP product could be detected and the test result could be visualized through the change in color. The limit of detection was demonstrated with reproducibility to be as low as 718 fg of genomic DNA, which is equivalent to approximately 100 plasmid DNA copies containing the cytochrome b DNA target region. A simple DNA extraction method for the commonly found forensic biological samples was also devised to streamline the test process. This LAMP-AuNP human DNA test showed to be a robust, specific, and cost-effective tool for the forensic identification of human specimens without requiring sophisticated laboratory instruments. PMID:24827529

  12. The Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Salmonella enterica serovar Typhi

    PubMed Central

    Fan, Fenxia; Du, Pengcheng; Kan, Biao; Yan, Meiying

    2015-01-01

    Typhoid fever remains a public health threat in many countries. A positive result in traditional culture is a gold-standard for typhoid diagnosis, but this method is time consuming and not sensitive enough for detection of samples containing a low copy number of the target organism. The availability of the loop-mediated isothermal amplification (LAMP) assay, which offers high speed and simplicity in detection of specific targets, has vastly improved the diagnosis of numerous infectious diseases. However, little research efforts have been made on utilizing this approach for diagnosis of Salmonella enterica serovar Typhi by targeting a single and specific gene. In this study, a LAMP assay for rapid detection of S. Typhi based on a novel marker gene, termed STY2879-LAMP, was established and evaluated with real-time PCR (RT-PCR). The specificity tests showed that STY2879 could be amplified in all S. Typhi strains isolated in different years and regions in China, whereas no amplification was observable in non-typhoidal strains covering 34 Salmonella serotypes and other pathogens causing febrile illness. The detection limit of STY2879-LAMP for S. Typhi was 15 copies/reaction in reference plasmids, 200 CFU/g with simple heat-treatment of DNA extracted from simulated stool samples and 20 CFU/ml with DNA extracted from simulated blood samples, which was 10 fold more sensitive than the parallel RT-PCR control experiment. Furthermore, the sensitivity of STY2879-LAMP and RT-PCR combining the traditional culture enrichment method for simulated stool and blood spiked with lower S. Typhi count during the 10 h enrichment time was also determined. In comparison with LAMP, the positive reaction time for RT-PCR required additional 2-3 h enrichment time for either simulated stool or blood specimens. Therefore, STY2879-LAMP is of practical value in the clinical settings and has a good potential for application in developing regions due to its easy-to-use protocol. PMID:25910059

  13. Evaluation of a loop-mediated isothermal amplification suite for the rapid, reliable, and robust detection of Shiga toxin-producing Escherichia coli in produce.

    PubMed

    Wang, Fei; Yang, Qianru; Qu, Yinzhi; Meng, Jianghong; Ge, Beilei

    2014-04-01

    Shiga toxin-producing Escherichia coli (STEC) strains are a leading cause of produce-associated outbreaks in the United States. Rapid, reliable, and robust detection methods are needed to better ensure produce safety. We recently developed a loop-mediated isothermal amplification (LAMP) suite for STEC detection. In this study, the STEC LAMP suite was comprehensively evaluated against real-time quantitative PCR (qPCR) using a large panel of bacterial strains (n = 156) and various produce items (several varieties of lettuce, spinach, and sprouts). To simulate real-world contamination events, produce samples were surface inoculated with a low level (1.2 to 1.8 CFU/25 g) of individual STEC strains belonging to seven serogroups (O26, O45, O103, O111, O121, O145, and O157) and held at 4°C for 48 h before testing. Six DNA extraction methods were also compared using produce enrichment broths. All STEC targets and their subtypes were accurately detected by the LAMP suite. The detection limits were 1 to 20 cells per reaction in pure culture and 10(5) to 10(6) CFU per 25 g (i.e., 10(3) to 10(4) CFU per g) in produce, except for strains harboring the stx2c, eae-β, and eae-θ subtypes. After 6 to 8 h of enrichment, the LAMP suite achieved accurate detection of low levels of STEC strains of various stx2 and eae subtypes in lettuce and spinach varieties but not in sprouts. A similar trend of detection was observed for qPCR. The PrepMan Ultra sample preparation reagent yielded the best results among the six DNA extraction methods. This research provided a rapid, reliable, and robust method for detecting STEC in produce during routine sampling and testing. The challenge with sprouts detection by both LAMP and qPCR calls for special attention to further analysis. PMID:24509927

  14. Evaluation of a Loop-Mediated Isothermal Amplification Suite for the Rapid, Reliable, and Robust Detection of Shiga Toxin-Producing Escherichia coli in Produce

    PubMed Central

    Wang, Fei; Yang, Qianru; Qu, Yinzhi; Meng, Jianghong

    2014-01-01

    Shiga toxin-producing Escherichia coli (STEC) strains are a leading cause of produce-associated outbreaks in the United States. Rapid, reliable, and robust detection methods are needed to better ensure produce safety. We recently developed a loop-mediated isothermal amplification (LAMP) suite for STEC detection. In this study, the STEC LAMP suite was comprehensively evaluated against real-time quantitative PCR (qPCR) using a large panel of bacterial strains (n = 156) and various produce items (several varieties of lettuce, spinach, and sprouts). To simulate real-world contamination events, produce samples were surface inoculated with a low level (1.2 to 1.8 CFU/25 g) of individual STEC strains belonging to seven serogroups (O26, O45, O103, O111, O121, O145, and O157) and held at 4°C for 48 h before testing. Six DNA extraction methods were also compared using produce enrichment broths. All STEC targets and their subtypes were accurately detected by the LAMP suite. The detection limits were 1 to 20 cells per reaction in pure culture and 105 to 106 CFU per 25 g (i.e., 103 to 104 CFU per g) in produce, except for strains harboring the stx2c, eae-β, and eae-θ subtypes. After 6 to 8 h of enrichment, the LAMP suite achieved accurate detection of low levels of STEC strains of various stx2 and eae subtypes in lettuce and spinach varieties but not in sprouts. A similar trend of detection was observed for qPCR. The PrepMan Ultra sample preparation reagent yielded the best results among the six DNA extraction methods. This research provided a rapid, reliable, and robust method for detecting STEC in produce during routine sampling and testing. The challenge with sprouts detection by both LAMP and qPCR calls for special attention to further analysis. PMID:24509927

  15. Development and application of a rapid detection system for human papillomavirus and Herpes simplex virus-2 by loop-mediated isothermal amplification assay.

    PubMed

    Yang, Jin-Fang; Zhao, Chang-Zhen; Lu, Ke-Xin

    2016-08-01

    Human papillomavirus (HPV) infection is an important factor that causes cervical cancer and non-melanoma skin cancer (NMSC), while HSV-2 plays an important role when HR-HPV triggers the cancer. Thus, a quick and convenient assay in the detection of HPV and HSV-2in the screening of HPV and HSV-2 infection is required. Two respective HPV and HSV-2 detection methods were established based on loop-mediated isothermal amplification (LAMP) assay. Specific outer primers, inner primers, and loop primers were designed according to the conserved domains of HPV and HSV-2 genomes, respectively, while degenerate primers were used for HPV assay. After optimizing the reaction conditions, the results were observed by LAMP Tubidimeter real-time LA-320. Standard plasmids HPV-L-P and HSV-2-L-P were cloned and used in sensitivity tests of HPV LAMP and HSV-2 LAMP, respectively. Fifty samples of actinic keratosis (AK), 20 samples of squamous cell carcinoma (SCC), 50 samples of basal cell carcinoma (BCC) and 20 samples of seborrheic keratosis (SK) were detected by HPV assay. Seventy three clinical samples of vaginitis, chronic cervicitis, cervical intraepithelial neoplasias and cervical cancer level positive were detected with HPV and HSV-2 assays. The reaction conditions of two assays were the same with a reaction temperature of 63 °C and a reaction time of 45 min. The sensitivity of HPV LAMP assay was 10 copies/μL, while that of the HSV-2 LAMP assay was 100 copies/μL. No cross-reactivity was observed. The HPV positive rates of AK, SCC, BCC and SK samples were 80% (40/50), 75% (15/20), 44% (22/50) and 21% (15/72), respectively. As an economic and quick diagnostic tool, LAMP assay is conducive to the extensive screening of HPV and HSV-2 and has huge potential to be promoted in resource-limited hospitals. PMID:27287497

  16. Loop-Mediated Isothermal Amplification for Rickettsia typhi (the Causal Agent of Murine Typhus): Problems with Diagnosis at the Limit of Detection

    PubMed Central

    Dittrich, Sabine; Castonguay-Vanier, Josée; Moore, Catrin E.; Thongyoo, Narongchai; Newton, Paul N.

    2014-01-01

    Murine typhus is a flea-borne disease of worldwide distribution caused by Rickettsia typhi. Although treatment with tetracycline antibiotics is effective, treatment is often misguided or delayed due to diagnostic difficulties. As the gold standard immunofluorescence assay is imperfect, we aimed to develop and evaluate a loop-mediated isothermal amplification (LAMP) assay. LAMP assays have the potential to fulfill the WHO ASSURED criteria (affordable, sensitive, specific, user friendly, robust and rapid, equipment free, deliverable to those who need them) for diagnostic methodologies, as they can detect pathogen-derived nucleic acid with low technical expenditure. The LAMP assay was developed using samples of bacterial isolates (n = 41), buffy coat specimens from R. typhi PCR-positive Lao patients (n = 42), and diverse negative controls (n = 47). The method was then evaluated prospectively using consecutive patients with suspected scrub typhus or murine typhus (n = 266). The limit of detection was ∼40 DNA copies/LAMP reaction, with an analytical sensitivity of <10 DNA copies/reaction based on isolate dilutions. Despite these low cutoffs, the clinical sensitivity was disappointing, with 48% (95% confidence interval [95% CI], 32.5 to 62.7%) (specificity, 100% [95% CI, 100 to 100%]) in the developmental phase and 33% (95% CI, 9.2 to 56.8%) (specificity, 98.5% [95% CI, 97.0% to 100%]) in the prospective study. This low diagnostic accuracy was attributed to low patient R. typhi bacterial loads (median, 210 DNA copies/ml blood; interquartile range, 130 to 500). PCR-positive but LAMP-negative samples demonstrated significantly lower bacterial loads than LAMP-positive samples. Our findings highlight the diagnostic challenges for diseases with low pathogen burdens and emphasize the need to integrate pathogen biology with improved template production for assay development strategies. PMID:24371248

  17. A new surveillance and response tool: risk map of infected Oncomelania hupensis detected by Loop-mediated isothermal amplification (LAMP) from pooled samples.

    PubMed

    Tong, Qun-Bo; Chen, Rui; Zhang, Yi; Yang, Guo-Jing; Kumagai, Takashi; Furushima-Shimogawara, Rieko; Lou, Di; Yang, Kun; Wen, Li-Yong; Lu, Shao-Hong; Ohta, Nobuo; Zhou, Xiao-Nong

    2015-01-01

    Although schistosomiasis remains a serious health problem worldwide, significant achievements in schistosomiasis control has been made in the People's Republic of China. The disease has been eliminated in five out of 12 endemic provinces, and the prevalence in remaining endemic areas is very low and is heading toward elimination. A rapid and sensitive method for monitoring the distribution of infected Oncomelania hupensis is urgently required. We applied a loop-mediated isothermal amplification (LAMP) assay targeting 28S rDNA for the rapid and effective detection of Schistosoma japonicum DNA in infected and prepatent infected O. hupensis snails. The detection limit of the LAMP method was 100 fg of S. japonicum genomic DNA. To promote the application of the approach in the field, the LAMP assay was used to detect infection in pooled samples of field-collected snails. In the pooled sample detection, snails were collected from 28 endemic areas, and 50 snails from each area were pooled based on the maximum pool size estimation, crushed together and DNA was extracted from each pooled sample as template for the LAMP assay. Based on the formula for detection from pooled samples, the proportion of positive pooled samples and the positive proportion of O. hupensis detected by LAMP of Xima village reached 66.67% and 1.33%, while those of Heini, Hongjia, Yangjiang and Huangshan villages were 33.33% and 0.67%, and those of Tuanzhou and Suliao villages were 16.67% and 0.33%, respectively. The remaining 21 monitoring field sites gave negative results. A risk map for the transmission of schistosomiasis was constructed using ArcMap, based on the positive proportion of O. hupensis infected with S. japonicum, as detected by the LAMP assay, which will form a guide for surveillance and response strategies in high risk areas. PMID:24495631

  18. Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira.

    PubMed

    Nurul Najian, A B; Engku Nur Syafirah, E A R; Ismail, Nabilah; Mohamed, Maizan; Yean, Chan Yean

    2016-01-15

    In recent years extensive numbers of molecular diagnostic methods have been developed to meet the need of point-of-care devices. Efforts have been made towards producing rapid, simple and inexpensive DNA tests, especially in the diagnostics field. We report on the development of a label-based lateral flow dipstick for the rapid and simple detection of multiplex loop-mediated isothermal amplification (m-LAMP) amplicons. A label-based m-LAMP lateral flow dipstick assay was developed for the simultaneous detection of target DNA template and a LAMP internal control. This biosensor operates through a label based system, in which probe-hybridization and the additional incubation step are eliminated. We demonstrated this m-LAMP assay by detecting pathogenic Leptospira, which causes the re-emerging disease Leptospirosis. The lateral flow dipstick was developed to detect of three targets, the LAMP target amplicon, the LAMP internal control amplicon and a chromatography control. Three lines appeared on the dipstick, indicating positive results for all representative pathogenic Leptospira species, whereas two lines appeared, indicating negative results, for other bacterial species. The specificity of this biosensor assay was 100% when it was tested with 13 representative pathogenic Leptospira species, 2 intermediate Leptospira species, 1 non-pathogenic Leptospira species and 28 other bacteria species. This study found that this DNA biosensor was able to detect DNA at concentrations as low as 3.95 × 10(-1) genomic equivalent ml(-1). An integrated m-LAMP and label-based lateral flow dipstick was successfully developed, promising simple and rapid visual detection in clinical diagnostics and serving as a point-of-care device. PMID:26709307

  19. Exploration of fluorescence-based real-time loop-mediated isothermal amplification (LAMP) assay for detection of Isospora suis oocysts.

    PubMed

    Huang, Cuiqin; Wen, Fuli; Yue, Liangping; Chen, Renfeng; Zhou, Wei; Hu, Lingying; Chen, Meizhen; Wang, Shoukun

    2016-06-01

    Isospora suis is an intestinal protozoan parasite in pigs. The 2-3 weeks old piglets are most often infected by I. suis because their immune system is not fully developed. The infection exhibits clinical features such as diarrhea and dehydration and seriously affects the economic interests of farmers. The traditional method of identifying I. suis relies on the detection of fecal oocysts, which depends heavily on the accumulation of experience. Thus, missed detection, and false alarms often occur during detection. With the development of molecular-based detection methods, development of a simple, convenient and more sensitive method for the detection of I. suis is an urgent need. In this study, based on the 18S rRNA gene sequence, a fluorescence -based real-time loop-mediated isothermal amplification (LAMP) assay was established for the detection of I. suis. The results showed that the assay is highly specific and sensitive, with a detection limit of 2.74 × 10(2) copies/μL recombinant plasmid of I. suis, corresponding to 1 fg/μL plasmid when converted to DNA concentration. The sensitivity is about 100 times higher than conventional PCR. Additionally, DNA extracted from a certain number of oocysts was used for detection, and it showed that the LAMP assay had a detection limit of 5 oocysts, lower than that of 13 oocysts of conventional PCR. The established LAMP assay overcomes the shortage of the traditional microscopy-based method, and provides a valuable way for molecular detection of I. suis. PMID:26965400

  20. Reverse transcription loop-mediated isothermal amplification assays for rapid identification of eastern and western strains of bluetongue virus in India.

    PubMed

    Maan, S; Maan, N S; Batra, K; Kumar, A; Gupta, A; Rao, Panduranga P; Hemadri, Divakar; Reddy, Yella Narasimha; Guimera, M; Belaganahalli, M N; Mertens, P P C

    2016-08-01

    Bluetongue virus (BTV) infects all ruminants, including cattle, goats and camelids, causing bluetongue disease (BT) that is often severe in naïve deer and sheep. Reverse-transcription-loop-mediated-isothermal-amplification (RT-LAMP) assays were developed to detect eastern or western topotype of BTV strains circulating in India. Each assay uses four primers recognizing six distinct sequences of BTV genome-segment 1 (Seg-1). The eastern (e)RT-LAMP and western (w)RT-LAMP assay detected BTV RNA in all positive isolates that were tested (n=52, including Indian BTV-1, -2, -3, -5, -9, -10, -16, -21 -23, and -24 strains) with high specificity and efficiency. The analytical sensitivity of the RT-LAMP assays is comparable to real-time RT-PCR, but higher than conventional RT-PCR. The accelerated eRT-LAMP and wRT-LAMP assays generated detectable levels of amplified DNA, down to 0.216 fg of BTV RNA template or 108 fg of BTV RNA template within 60-90min respectively. The assays gave negative results with RNA from foot-and-mouth-disease virus (FMDV), peste des petits ruminants virus (PPRV), or DNA from Capripox viruses and Orf virus (n=10), all of which can cause clinical signs similar to BT. Both RT-LAMP assays did not show any cross-reaction among themselves. The assays are rapid, easy to perform, could be adapted as a 'penside' test making them suitable for 'front-line' diagnosis, helping to identify and contain field outbreaks of BTV. PMID:27054888

  1. Rapid and Sensitive Detection of Bartonella bacilliformis in Experimentally Infected Sand Flies by Loop-Mediated Isothermal Amplification (LAMP) of the Pap31 Gene

    PubMed Central

    Angkasekwinai, Nasikarn; Atkins, Erin H.; Johnson, Richard N.; Grieco, John P.; Ching, Wei Mei; Chao, Chien Chung

    2014-01-01

    Background Carrion' disease, caused by Bartonella bacilliformis, remains truly neglected due to its focal geographical nature. A wide spectrum of clinical manifestations, including asymptomatic bacteremia, and lack of a sensitive diagnostic test can potentially lead to a spread of the disease into non-endemic regions where competent sand fly vectors may be present. A reliable test capable of detecting B. bacilliformis is urgently needed. Our objective is to develop a loop-mediated isothermal amplification (LAMP) assay targeting the pap31 gene to detect B. bacilliformis. Methods and Findings The sensitivity of the LAMP was evaluated in comparison to qPCR using plasmid DNA containing the target gene and genomic DNA in the absence and presence of human or sand fly DNA. The detection limit of LAMP was 1 to 10 copies/µL, depending on the sample metrics. No cross-reaction was observed when testing against a panel of various closely related bacteria. The utility of the LAMP was further compared to qPCR by the examination of 74 Lutzomyia longipalpis sand flies artificially fed on blood spiked with B. bacilliformis and harvested at days (D) 1, 3, 5, 7 and 9 post feeding. Only 86% of sand flies at D1 and 63% of flies at D3 were positive by qPCR. LAMP was able to detect B. bacilliformis in all those flies confirmed positive by qPCR. However, none of the flies after D3 were positive by either LAMP or qPCR. In addition to demonstrating the sensitivity of the LAMP assay, these results suggest that B. bacilliformis cannot propagate in artificially fed L. longipalpis. Conclusions The LAMP assay is as sensitive as qPCR for the detection of B. bacilliformis and could be useful to support diagnosis of patients in low-resource settings and also to identify B. bacilliformis in the sand fly vector. PMID:25522230

  2. Development and evaluation of a loop-mediated isothermal amplification assay for detection of Ehrlichia canis DNA in naturally infected dogs using the p30 gene.

    PubMed

    Pinhanelli, V C; Costa, P N M; Silva, G; Aguiar, D M; Silva, C M L; Fachin, A L; Marins, M

    2015-01-01

    Canine monocytic ehrlichiosis (CME) is a common tick-borne disease caused by the rickettsial bacterium Ehrlichia canis (Rickettsiales: Anaplasmataceae). In view of the different stages and variable clinical signs of CME, which can overlap with those of other infections, a conclusive diagnosis can more readily be obtained by combining clinical and hematological evaluations with molecular diagnostic methods. In this study, a loop-mediated isothermal amplification (LAMP) assay targeting the p30 gene of E. canis was developed. The assay was developed using DNA extracted from E. canis-infected cultures of the macrophage cell line DH82 and samples from dogs testing positive for E. canis DNA by PCR. The LAMP assay was compared to a p30-based PCR assay, using DNA extracted from EDTA-anticoagulated blood samples of 137 dogs from an endemic region in Brazil. The LAMP assay was sensitive enough to detect a single copy of the target gene, and identified 74 (54.0%) E. canis DNA-positive samples, while the p30 PCR assay detected 50 positive samples (36.5%) among the field samples. Agreement between the two assays was observed in 42 positive and 55 negative samples. However, 32 positive samples that were not detected by the PCR assay were identified by the LAMP assay, while eight samples identified as E. canis-positive by PCR showed negative results in LAMP. The developed E. canis LAMP assay showed the potential to maximize the use of nucleic acid tests in a veterinary clinical laboratory, and to improve the diagnosis of CME. PMID:26782434

  3. Most-probable-number loop-mediated isothermal amplification-based procedure enhanced with K antigen-specific immunomagnetic separation for quantifying tdh(+) Vibrio parahaemolyticus in molluscan Shellfish.

    PubMed

    Tanaka, Natsuko; Iwade, Yoshito; Yamazaki, Wataru; Gondaira, Fumio; Vuddhakul, Varaporn; Nakaguchi, Yoshitsugu; Nishibuchi, Mitsuaki

    2014-07-01

    Although thermostable direct hemolysin-producing (tdh(+)) Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis, the enumeration of tdh(+) V. parahaemolyticus remains challenging due to its low densities in the environment. In this study, we developed a most-probable-number (MPN)-based procedure designated A-IS(1)-LAMP, in which an immunomagnetic separation (IMS) technique targeting as many as 69 established K antigens and a loop-mediated isothermal amplification (LAMP) assay targeting the thermostable direct hemolysin (tdh) gene were applied in an MPN format. Our IMS employed PickPen, an eight-channel intrasolution magnetic particle separation device, which enabled a straightforward microtiter plate-based IMS procedure (designated as PickPen-IMS). The ability of the procedure to quantify a wide range of tdh(+) V. parahaemolyticus levels was evaluated by testing shellfish samples in Japan and southern Thailand, where shellfish products are known to contain relatively low and high levels of total V. parahaemolyticus, respectively. The Japanese and Thai shellfish samples showed, respectively, relatively low (< 3 to 11 MPN/10 g) and considerably higher (930 to 110,000 MPN/10 g) levels of tdh(+) V. parahaemolyticus, raising concern about the safety of Thai shellfish products sold to domestic consumers at local morning markets. LAMP showed similar or higher performance than conventional PCR in the detection and quantification of a wide range of tdh(+) V. parahaemolyticus levels in shellfish products. Whereas a positive effect of PickPen-IMS was not observed in MPN determination, PickPen-IMS was able to concentrate tdh(+) V. parahaemolyticus 32-fold on average from the Japanese shellfish samples at an individual tube level, suggesting a possibility of using PickPen-IMS as an optional tool for specific shellfish samples. The A-IS(1)-LAMP procedure can be used by any health authority in the world to measure the tdh(+) V. parahaemolyticus levels in

  4. Detection of Magnaporthe oryzae chrysovirus 1 in Japan and establishment of a rapid, sensitive and direct diagnostic method based on reverse transcription loop-mediated isothermal amplification.

    PubMed

    Komatsu, Ken; Urayama, Syun-Ichi; Katoh, Yu; Fuji, Shin-Ichi; Hase, Shu; Fukuhara, Toshiyuki; Arie, Tsutomu; Teraoka, Tohru; Moriyama, Hiromitsu

    2016-02-01

    Magnaporthe oryzae chrysovirus 1 (MoCV1) is a mycovirus with a dsRNA genome that infects the rice blast fungus Magnaporthe oryzae and impairs its growth. To date, MoCV1 has only been found in Vietnamese isolates of M. oryzae, and the distribution of this virus in M. oryzae isolates from other parts of the world remains unknown. In this study, using a one-step reverse transcription PCR (RT-PCR) assay, we detected a MoCV1-related virus in M. oryzae in Japan (named MoCV1-AK) whose sequence shares considerable similarity with that of the MoCV1 Vietnamese isolate. To establish a system for a comprehensive survey of MoCV1 infection in the field, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for direct detection of the virus. The sensitivity of the RT-LAMP assay was at least as high as that of the one-step RT-PCR assay. In addition, we detected MoCV1-AK in M. oryzae-infected oatmeal agar plates and lesions on rice leaves using the RT-LAMP assay without dsRNA extraction, by simple sampling with a toothpick. Preliminary screening of MoCV1 in Japanese M. oryzae isolates indicated that MoCV1 is currently distributed in rice fields in Japan. Our results provide a first example of the application of RT-LAMP for the detection of mycoviruses, which will accelerate surveys for mycovirus infection. PMID:26547578

  5. An aptamer-based bio-barcode assay with isothermal recombinase polymerase amplification for cytochrome-c detection and anti-cancer drug screening.

    PubMed

    Loo, Jacky F C; Lau, P M; Ho, H P; Kong, S K

    2013-10-15

    Based on a recently reported ultra-sensitive bio-barcode (BBC) assay, we have developed an aptamer-based bio-barcode (ABC) alternative to detect a cell death marker cytochrome-c (Cyto-c) and its subsequent application to screen anti-cancer drugs. Aptamer is a short single-stranded DNA selected from a synthetic DNA library by virtue of its high binding affinity and specificity to its target based on its unique 3D structure from the nucleotide sequence after folding. In the BBC assay, an antigen (Ag) in analytes is captured by a micro-magnetic particle (MMP) coated with capturing antibodies (Abs). Gold nanoparticles (NPs) with another recognition Ab against the same target and hundreds of identical DNA molecules of known sequence are subsequently added to allow the formation of sandwich structures ([MMP-Ab1]-Ag-[Ab2-NP-DNA]). After isolating the sandwiches by a magnetic field, the DNAs hybridized to their complementary DNAs covalently bound on the NPs are released from the sandwiches after heating. Acting as an Ag identification tag, these bio-barcode DNAs with known DNA sequence are then amplified by polymerase chain reaction (PCR) and detected by fluorescence. In our ABC assay, we employed a Cyto-c-specific aptamer to substitute both the recognition Ab and barcode DNAs on the NPs in the BBC assay; and a novel isothermal recombinase polymerase amplification for the time-consuming PCR. The detection limit of our ABC assay for the Cyto-c was found to be 10 ng/mL and this new assay can be completed within 3h. Several potential anti-cancer drugs have been tested in vitro for their efficacy to kill liver cancer with or without multi-drug resistance. PMID:24054573

  6. The Sensitivity and Specificity of Loop-Mediated Isothermal Amplification (LAMP) Assay for Tuberculosis Diagnosis in Adults with Chronic Cough in Malawi

    PubMed Central

    Nliwasa, Marriott; MacPherson, Peter; Chisala, Palesa; Kamdolozi, Mercy; Khundi, McEwen; Kaswaswa, Kruger; Mwapasa, Mphatso; Msefula, Chisomo; Sohn, Hojoon; Flach, Clare; Corbett, Elizabeth L.

    2016-01-01

    Background Current tuberculosis diagnostics lack sensitivity, and are expensive. Highly accurate, rapid and cheaper diagnostic tests are required for point of care use in low resource settings with high HIV prevalence. Objective To investigate the sensitivity and specificity, and cost of loop-mediated isothermal amplification (LAMP) assay for tuberculosis diagnosis in adults with chronic cough compared to Xpert® MTB/RIF, fluorescence smear microscopy. Methods Between October 2013 and March 2014, consecutive adults at a primary care clinic were screened for cough, offered HIV testing and assessed for tuberculosis using LAMP, Xpert® MTB/RIF and fluorescence smear microscopy. Sensitivity and specificity (with culture as reference standard), and costs were estimated. Results Of 273 adults recruited, 44.3% (121/273) were HIV-positive and 19.4% (53/273) had bacteriogically confirmed tuberculosis. The sensitivity of LAMP compared to culture was 65.0% (95% CI: 48.3% to 79.4%) with 100% (95% CI: 98.0% to 100%) specificity. The sensitivity of Xpert® MTB/RIF (77.5%, 95% CI: 61.5% to 89.2%) was similar to that of LAMP, p = 0.132. The sensitivity of concentrated fluorescence smear microscopy with routine double reading (87.5%, 95% CI: 73.2% to 95.8%) was higher than that of LAMP, p = 0.020. All three tests had high specificity. The lowest cost per test of LAMP was at batch size of 14 samples (US$ 9.98); this was lower than Xpert® MTB/RIF (US$ 13.38) but higher than fluorescence smear microscopy (US$ 0.65). Conclusion The sensitivity of LAMP was similar to Xpert® MTB/RIF but lower than fluorescence smear microscopy; all three tests had high specificity. These findings support the Malawi policy that recommends a combination of fluorescence smear microscopy and Xpert® MTB/RIF prioritised for people living with HIV, already found to be smear-negative, or being considered for retreatment of tuberculosis. PMID:27171380

  7. Development and Evaluation of Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay Coupled with a Portable Device for Rapid Diagnosis of Ebola Virus Disease in Guinea

    PubMed Central

    Kurosaki, Yohei; Magassouba, N’Faly; Oloniniyi, Olamide K.; Cherif, Mahamoud S.; Sakabe, Saori; Takada, Ayato; Hirayama, Kenji; Yasuda, Jiro

    2016-01-01

    Given the current absence of specific drugs or vaccines for Ebola virus disease (EVD), rapid, sensitive, and reliable diagnostic methods are required to stem the transmission chain of the disease. We have developed a rapid detection assay for Zaire ebolavirus based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and coupled with a novel portable isothermal amplification and detection platform. The RT-LAMP assay is based on primer sets that target the untranscribed trailer region or nucleoprotein coding region of the viral RNA. The test could specifically detect viral RNAs of Central and West African Ebola virus strains within 15 minutes with no cross-reactivity to other hemorrhagic fever viruses and arboviruses, which cause febrile disease. The assay was evaluated using a total of 100 clinical specimens (serum, n = 44; oral swab, n = 56) collected from suspected EVD cases in Guinea. The specificity of this diagnostic test was 100% for both primer sets, while the sensitivity was 100% and 97.9% for the trailer and nucleoprotein primer sets, respectively, compared with a reference standard RT-PCR test. These observations suggest that our diagnostic assay is useful for identifying EVD cases, especially in the field or in settings with insufficient infrastructure. PMID:26900929

  8. Evaluation of a loop-mediated isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Channel catfish Ictalurus punctatus infected with Edwardsiella ictaluri results in $40 - 50 million annual losses in profits to catfish producers. Early detection of this pathogen is necessary for disease control and reduction of economic loss. In this communication, the loop-mediated isothermal a...

  9. Development of a Loop-Mediated Isothermal Amplification Method for Detecting Streptococcus equi subsp. zooepidemicus and Analysis of Its Use with Three Simple Methods of Extracting DNA from Equine Respiratory Tract Specimens

    PubMed Central

    KINOSHITA, Yuta; NIWA, Hidekazu; KATAYAMA, Yoshinari

    2014-01-01

    ABSTRACT Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a dominant pathogenic bacterium in equine pneumonia. We developed a specific loop-mediated isothermal amplification (LAMP) method, which targets the gene encoding sorbitol-6-phosphate 2-dehydrogenase (sorD), for detecting S. zooepidemicus and examined the clinical efficacies of its use in combination with each of 3 DNA extraction methods easily used by veterinary practitioners, namely the Loopamp PURE DNA Extraction Kit, InstaGene Matrix and a conventional boiling method. The LAMP method plus the Loopamp PURE DNA Extraction Kit gave higher rates of positivity than the other combinations in both clinical and spiked samples containing clinically significant concentrations (>1 × 104 CFU/ml) of S. zooepidemicus. PMID:24871644

  10. Increased Robustness of Single-Molecule Counting with Microfluidics, Digital Isothermal Amplification, and a Mobile Phone versus Real-Time Kinetic Measurements

    PubMed Central

    Selck, David A.; Karymov, Mikhail A.; Sun, Bing; Ismagilov, Rustem F.

    2013-01-01

    Quantitative bioanalytical measurements are commonly performed in a kinetic format, and are known to not be robust to perturbation that affects the kinetics itself, or the measurement of kinetics. We hypothesized that the same measurements performed in a “digital” (single-molecule) format would show increased robustness to such perturbations. Here, we investigated the robustness of an amplification reaction (reverse-transcription loop-mediated amplification, RT-LAMP) in the context of fluctuations in temperature and time when this reaction is used for quantitative measurements of HIV-1 RNA molecules under limited-resource settings (LRS). The digital format that counts molecules using dRT-LAMP chemistry detected a two-fold change in concentration of HIV-1 RNA despite a 6 °C temperature variation (p-value = 6.7×10−7), whereas the traditional kinetic (real-time) format did not (p-value = 0.25). Digital analysis was also robust to a 20-minute change in reaction time, to poor imaging conditions obtained with a consumer cell-phone camera, and to automated cloud-based processing of these images (R2 = 0.9997 vs. true counts over a 100-fold dynamic range). Fluorescent output of multiplexed PCR amplification could also be imaged with the cell phone camera using flash as the excitation source. Many nonlinear amplification schemes based on organic, inorganic, and biochemical reactions have been developed but their robustness is not well understood. This work implies that these chemistries may be significantly more robust in the digital, rather than kinetic, format. It also calls for theoretical studies to predict robustness of these chemistries, and more generally to design robust reaction architectures. The SlipChip that we used here and other digital microfluidic technologies already exist to enable testing of these predictions. Such work may lead to identification or creation of robust amplification chemistries that enable rapid and precise quantitative molecular

  11. Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. cubense tropical race 4 in soil.

    PubMed

    Zhang, Xin; Zhang, He; Pu, Jinji; Qi, Yanxiang; Yu, Qunfang; Xie, Yixian; Peng, Jun

    2013-01-01

    Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 10(3) spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China. PMID:24376590

  12. Development of a Real-Time Fluorescence Loop-Mediated Isothermal Amplification Assay for Rapid and Quantitative Detection of Fusarium oxysporum f. sp. cubense Tropical Race 4 In Soil

    PubMed Central

    Pu, Jinji; Qi, Yanxiang; Yu, Qunfang; Xie, Yixian; Peng, Jun

    2013-01-01

    Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 103 spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China. PMID:24376590

  13. Application of ethidium bromide monoazide for quantification of viable and dead cells of Salmonella enterica by real-time loop-mediated isothermal amplification.

    PubMed

    Wu, Guo Ping; Chen, Su Hua; Levin, Robert E

    2015-10-01

    A rapid and efficient method for quantification and discrimination of Salmonella enterica ser. Enteritidis between viable and dead cells killed by heat was developed using ethidium bromide monoazide (EMA) in combination with a real-time loop amplified (Rti-LAMP) DNA assay. The use of 8.0 μg/ml or less of EMA did not inhibit DNA amplification in Rti-LAMP assays derived from viable cells. However, 8.0 μg/ml of EMA notably inhibited DNA amplification and significantly increased the Tp values with dead cells. When the DNA from 2000 viable CFU was subjected to EMA-Rti-LAMP the resulting Tp value was 13 min. In contrast, the DNA from 2000 CFU completely heat destroyed CFU still yielded a Tp value, which was greatly increased to 33.1 min. When the DNA from viable plus heat killed CFU at a ratio of 7:1993 was subjected to EMA-Rti-LAMP, the resulting Tp value was 19.3 min, which was statistically identical (P<0.05) to the Tp value of 19.9 min. obtained with the DNA from only 7 viable CFU. These results indicate that even though 2000 dead cells yielded a Tp value of 33.1 min., low numbers of viable cells in the presence of much higher numbers of dead cells still yielded a linear plot for enumerating viable CFU from Tp values. In addition, propidium monoazide (PMA) was found to be ineffective in distinguishing between low numbers of viable and heat killed cells of S. enterica. PMID:26187777

  14. Rapid and sensitive detection of Babesia bovis and Babesia bigemina by loop-mediated isothermal amplification combined with a lateral flow dipstick.

    PubMed

    Yang, Yimin; Li, Qun; Wang, Suhua; Chen, Xueqiu; Du, Aifang

    2016-03-30

    Babesia spp. are apicomplexan protozoan parasites of the red blood cells of mammals and are transmitted by ticks. Bovine babesiosis mainly caused by Babesia bovis and Babesia bigemina occurs worldwide, which is a great threat to animal health. Microscopy examination is a gold standard for the diagnosis of babesiosis. However, its sensitivity is too low. This study was conducted to establish a simple, efficient and fast LAMP-LFP method used for early diagnosis of animal babesiosis. LAMP was developed with a set of four primers targeting and amplifying six distinct regions of cytochrome b gene of Babesia spp. under isothermal conditions. Afterwards, a chromatographic lateral-flow dipstick (LFD) was used to detect LAMP products that were labeled with FITC at the 5' end, avoiding gel electrophoresis. The LAMP-LFD method was very specific, yielding no positive results with DNA templates of Theileria sergenti, Thenileria ovis, Theileria equi and Toxoplasma gondii. The LAMP-LFP was highly sensitive and could detect 0.85 fg B. bigemina DNA and 0.14 fg B. bovis DNA, 100-fold higher than a conventional PCR assay. This method could be adapted for quick and accurate diagnosis of bovine babesiosis in the fields in case the whole blood could be directly used, especially for identifying carrier animals with very low parasitaemia. PMID:26921043

  15. Preliminary Validation of Direct Detection of Foot-And-Mouth Disease Virus within Clinical Samples Using Reverse Transcription Loop-Mediated Isothermal Amplification Coupled with a Simple Lateral Flow Device for Detection

    PubMed Central

    Waters, Ryan A.; Fowler, Veronica L.; Armson, Bryony; Nelson, Noel; Gloster, John; Paton, David J.; King, Donald P.

    2014-01-01

    Rapid, field-based diagnostic assays are desirable tools for the control of foot-and-mouth disease (FMD). Current approaches involve either; 1) Detection of FMD virus (FMDV) with immuochromatographic antigen lateral flow devices (LFD), which have relatively low analytical sensitivity, or 2) portable RT-qPCR that has high analytical sensitivity but is expensive. Loop-mediated isothermal amplification (LAMP) may provide a platform upon which to develop field based assays without these drawbacks. The objective of this study was to modify an FMDV-specific reverse transcription–LAMP (RT-LAMP) assay to enable detection of dual-labelled LAMP products with an LFD, and to evaluate simple sample processing protocols without nucleic acid extraction. The limit of detection of this assay was demonstrated to be equivalent to that of a laboratory based real-time RT-qPCR assay and to have a 10,000 fold higher analytical sensitivity than the FMDV-specific antigen LFD currently used in the field. Importantly, this study demonstrated that FMDV RNA could be detected from epithelial suspensions without the need for prior RNA extraction, utilising a rudimentary heat source for amplification. Once optimised, this RT-LAMP-LFD protocol was able to detect multiple serotypes from field epithelial samples, in addition to detecting FMDV in the air surrounding infected cattle, pigs and sheep, including pre-clinical detection. This study describes the development and evaluation of an assay format, which may be used as a future basis for rapid and low cost detection of FMDV. In addition it provides providing “proof of concept” for the future use of LAMP assays to tackle other challenging diagnostic scenarios encompassing veterinary and human health. PMID:25165973

  16. Comparison of real-time PCR, reverse transcriptase real-time PCR, loop-mediated isothermal amplification, and the FDA conventional microbiological method for the detection of Salmonella spp. in produce.

    PubMed

    Zhang, Guodong; Brown, Eric W; González-Escalona, Narjol

    2011-09-01

    Contamination of foods, especially produce, with Salmonella spp. is a major concern for public health. Several methods are available for the detection of Salmonella in produce, but their relative efficiency for detecting Salmonella in commonly consumed vegetables, often associated with outbreaks of food poisoning, needs to be confirmed. In this study, the effectiveness of three molecular methods for detection of Salmonella in six produce matrices was evaluated and compared to the FDA microbiological detection method. Samples of cilantro (coriander leaves), lettuce, parsley, spinach, tomato, and jalapeno pepper were inoculated with Salmonella serovars at two different levels (10(5) and <10(1) CFU/25 g of produce). The inoculated produce was assayed by the FDA Salmonella culture method (Bacteriological Analytical Manual) and by three molecular methods: quantitative real-time PCR (qPCR), quantitative reverse transcriptase real-time PCR (RT-qPCR), and loop-mediated isothermal amplification (LAMP). Comparable results were obtained by these four methods, which all detected as little as 2 CFU of Salmonella cells/25 g of produce. All control samples (not inoculated) were negative by the four methods. RT-qPCR detects only live Salmonella cells, obviating the danger of false-positive results from nonviable cells. False negatives (inhibition of either qPCR or RT-qPCR) were avoided by the use of either a DNA or an RNA amplification internal control (IAC). Compared to the conventional culture method, the qPCR, RT-qPCR, and LAMP assays allowed faster and equally accurate detection of Salmonella spp. in six high-risk produce commodities. PMID:21803916

  17. Comparison of Real-Time PCR, Reverse Transcriptase Real-Time PCR, Loop-Mediated Isothermal Amplification, and the FDA Conventional Microbiological Method for the Detection of Salmonella spp. in Produce ▿ †

    PubMed Central

    Zhang, Guodong; Brown, Eric W.; González-Escalona, Narjol

    2011-01-01

    Contamination of foods, especially produce, with Salmonella spp. is a major concern for public health. Several methods are available for the detection of Salmonella in produce, but their relative efficiency for detecting Salmonella in commonly consumed vegetables, often associated with outbreaks of food poisoning, needs to be confirmed. In this study, the effectiveness of three molecular methods for detection of Salmonella in six produce matrices was evaluated and compared to the FDA microbiological detection method. Samples of cilantro (coriander leaves), lettuce, parsley, spinach, tomato, and jalapeno pepper were inoculated with Salmonella serovars at two different levels (105 and <101 CFU/25 g of produce). The inoculated produce was assayed by the FDA Salmonella culture method (Bacteriological Analytical Manual) and by three molecular methods: quantitative real-time PCR (qPCR), quantitative reverse transcriptase real-time PCR (RT-qPCR), and loop-mediated isothermal amplification (LAMP). Comparable results were obtained by these four methods, which all detected as little as 2 CFU of Salmonella cells/25 g of produce. All control samples (not inoculated) were negative by the four methods. RT-qPCR detects only live Salmonella cells, obviating the danger of false-positive results from nonviable cells. False negatives (inhibition of either qPCR or RT-qPCR) were avoided by the use of either a DNA or an RNA amplification internal control (IAC). Compared to the conventional culture method, the qPCR, RT-qPCR, and LAMP assays allowed faster and equally accurate detection of Salmonella spp. in six high-risk produce commodities. PMID:21803916

  18. Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Leishmania infantum in canine leishmaniasis based on cysteine protease B genes.

    PubMed

    Chaouch, Melek; Mhadhbi, Moez; Adams, Emily R; Schoone, Gerard J; Limam, Sassi; Gharbi, Zyneb; Darghouth, Mohamed Aziz; Guizani, Ikram; BenAbderrazak, Souha

    2013-11-15

    We developed a Leishmania infantum specific LAMP assay that was carried out using a set of, six primers targeting the cysteine protease B multi copy gene of L. infantum. Our result shows that we, successfully detect the L. infantum DNA and that amplification is specific as no cross reaction was seen, with L. major, L. tropica, L. turanica, L. aethiopica, L. tarentolae, L. gerbilii, Trypanosoma cruzi or, human genomic DNA. When compared to conventional cpb based PCR, the sensitivity of LAMP assay, was higher with a detection limit of 50 fg/μl of genomic L. infantum parasite DNA. Accurate and rapid, diagnosis of canine leishmaniasis (CanL) is an important issue that allows early treatment and, prevents transmission. Our developed LAMP assay was used to evaluate occurrences of Leishmania infantum in seventy five (75) dogs from the field. Blood samples were used to perform LAMP assay, classical PCR, IFAT and microscopy that was used as gold standard. The IFAT in addition to, microscopy, are the basic techniques used for CanL diagnosis at the School of Veterinary Medicine, where we obtained our samples. Compared to molecular methods, the serology (IFAT) test shows the, best sensitivity (88.57%) with, however, a much lower specificity (52.5%) due to a relatively high, number of false-positive results (22 animals). The PCR assay shows a low sensitivity (37.14%) and, specificity around (82.5%). Our LAMP assay shows a suitable sensitivity (54%) and a good specificity, (80%), with however, positive (70%) and negative (66%) predictive values. Furthermore, the best, positive likelihood ratio (LR+) was obtained by LAMP assay (2.7). This technique presents the highest, kappa value (with a fair agreement of 0.34). Moreover, the relative stability of the reagents indicates, that LAMP may be a good alternative to a conventional PCR, especially under field conditions. Finally in, a brief cost evaluation, the LAMP assay compares favorably with other molecular diagnostic tests. This

  19. Rapid and Specific Detection of Escherichia coli Serogroups O26, O45, O103, O111, O121, O145, and O157 in Ground Beef, Beef Trim, and Produce by Loop-Mediated Isothermal Amplification

    PubMed Central

    Wang, Fei; Jiang, Lin; Yang, Qianru; Prinyawiwatkul, Witoon

    2012-01-01

    Escherichia coli O157 and six additional serogroups of Shiga toxin-producing E. coli (STEC) (O26, O45, O103, O111, O121, and O145) account for the majority of STEC infections in the United States. In this study, O serogroup-specific genes (wzx or wzy) were used to design loop-mediated isothermal amplification (LAMP) assays for the rapid and specific detection of these leading STEC serogroups. The assays were evaluated in pure culture and spiked food samples (ground beef, beef trim, lettuce, and spinach) and compared with real-time quantitative PCR (qPCR). No false-positive or false-negative results were observed among 120 bacterial strains used to evaluate assay specificity. The limits of detection of various STEC strains belonging to these target serogroups were approximately 1 to 20 CFU/reaction mixture in pure culture and 103 to 104 CFU/g in spiked food samples, which were comparable to those of qPCR. Standard curves generated suggested good linear relationships between STEC cell numbers and LAMP turbidity signals. In various beef and produce samples spiked with two low levels (1 to 2 and 10 to 20 CFU/25 g) of respective STEC strains, the LAMP assays consistently achieved accurate detection after 6 to 8 h of enrichment. In conclusion, these newly developed LAMP assays may facilitate rapid and reliable detection of the seven major STEC serogroups in ground beef, beef trim, and produce during routine sample testing. PMID:22327594

  20. Isothermal Calorimeter

    NASA Technical Reports Server (NTRS)

    Rowlette, John J.

    1990-01-01

    Pressure-feedback signal indicates rate of heating. Improved isothermal calorimeter measures rate of heating in object under test. Called "isothermal" because chamber holding object and its environment maintained at or near constant temperature to minimize spurious tranfers of heat introducing errors into measurements. When item under test generates heat, rate of boiling and pressure in inner chamber increase. Servo-valve opens wider to maintain preset differential pressure. Valve-control voltage used as measure of rate of heating.

  1. Evaluation of commercial kit based on loop-mediated isothermal amplification for rapid detection of low levels of uninjured and injured Salmonella on duck meat, bean sprouts, and fishballs in Singapore.

    PubMed

    Lim, Hazel Sin Yue; Zheng, Qianwang; Miks-Krajnik, Marta; Turner, Matthew; Yuk, Hyun-Gyun

    2015-06-01

    The objective of this study was to evaluate performance of the commercial kit based on loop-mediated isothermal amplification (LAMP) in comparison with the International Organization for Standardization method for detecting uninjured and sublethally injured Salmonella cells artificially inoculated at levels of 10(0) and 10(1) CFU/25 g on raw duck wing, raw mung bean sprouts, and processed fishballs. Injured cells were prepared by a heat treatment for duck wings and fishball samples and a chlorine treatment for bean sprout samples. Additionally, a validation study was performed on naturally contaminated food samples sold in Singapore. A total of 110 samples of each commodity were analyzed in this study. Regardless of inoculum levels, the detection by the commercial LAMP kit showed 100% sensitivity and specificity for both inoculated and uninoculated samples compared with the International Organization for Standardization method, with the exception of bean sprout samples. Only 20% of bean sprout samples inoculated with 10(0) CFU/25 g injured Salmonella cells were positive by using the commercial LAMP-based kit. However, all negative samples became positive following a secondary enrichment in Rappaport-Vassiliadis medium with soy broth or after concentration by centrifugation. These results suggest that secondary enrichment or centrifugation should be considered as an additional step to increase the sensitivity of the commercial LAMP-based kit with low numbers of injured target cells in samples with high background microflora (such as mung bean sprouts). The validation study also showed that the commercial LAMP-based kit provided 91% sensitivity and 95% specificity for naturally contaminated samples. Thus, this study demonstrates that the commercial LAMP-based kit might be a cost-effective method, as this system could provide rapid, accurate detection of both uninjured and injured Salmonella cells on raw duck wings, raw mung bean sprouts, and processed fishballs in

  2. Application of loop-mediated isothermal amplification assays for direct identification of pure cultures of Aspergillus flavus, A. nomius, and A. caelatus and for their rapid detection in shelled Brazil nuts.

    PubMed

    Luo, Jie; Taniwaki, Marta H; Iamanaka, Beatriz T; Vogel, Rudi F; Niessen, Ludwig

    2014-02-17

    Brazil nuts have a high nutritional content and are a very important trade commodity for some Latin American countries. Aflatoxins are carcinogenic fungal secondary metabolites. In Brazil nuts they are produced predominantly by Aspergillus (A.) nomius and A. flavus. In the present study we applied and evaluated two sets of primers previously published for the specific detection of the two species using loop-mediated isothermal amplification (LAMP) technology. Moreover, a primer set specific for A. caelatus as a frequently occurring non-aflatoxigenic member of Aspergillus section Flavi in Brazil nuts was newly developed. LAMP assays were combined with a simplified DNA release method and used for rapid identification of pure cultures and rapid detection of A. nomius and A. flavus from samples of shelled Brazil nuts. An analysis of pure cultures of 68 isolates representing the major Aspergillus species occurring on Brazil nuts showed that the three LAMP assays had individual accuracies of 61.5%, 84.4%, and 93.3% for A. flavus, A. nomius, and A. caelatus, respectively when morphological identification was used as a reference. The detection limits for conidia added directly to the individual LAMP reactions were found to be 10⁵ conidia per reaction with the primer set ID9 for A. nomius and 10⁴ conidia per reaction with the primer set ID58 for A. flavus. Sensitivity was increased to 10¹ and 10² conidia per reaction for A. nomius and A. flavus, respectively, when sample preparation included a spore disruption step. The results of LAMP assays obtained during the analysis of 32 Brazil nut samples from different regions of Brazil and from different steps in the production process of the commodity were compared with results obtained from mycological analysis and aflatoxin analysis of corresponding samples. Compared with mycological analysis of the samples, the Negative Predictive Values of LAMP assays were 42.1% and 12.5% while the Positive Predictive Values were 61

  3. Comparison of conventional RT-PCR, reverse-transcription loop-mediated isothermal amplification, and SYBR green I-based real-time RT-PCR in the rapid detection of bovine viral diarrhea virus nucleotide in contaminated commercial bovine sera batches.

    PubMed

    Zhang, Shu-Qin; Tan, Bin; Li, Peng; Wang, Feng-Xue; Guo, Li; Yang, Yong; Sun, Na; Zhu, Hong-Wei; Wen, Yong-Jun; Cheng, Shi-Peng

    2014-10-01

    Bovine viral diarrhea virus (BVDV) can contaminate biological products produced in bovine or porcine cells or manufactured using bovine sera. A rapid, specific, sensitive, and practical method of detecting BVDV in bio-products is needed. The purpose of this study was to compare three assays with respect to their ability to accurately detect BVDV in biological samples, namely reverse-transcription loop-mediated isothermal amplification (RT-LAMP), SYBR green I-based real-time RT-PCR, and conventional RT-PCR. All assays detected BVDV nucleotide and differentiated between BVDV-free and -contaminated bovine sera successfully. In addition, the results were specific to BVDV: the amplification of samples containing the closely related classical swine fever virus or other pathogenic bovine viruses yielded negative results. The lowest detection threshold, 10(1) copies, was displayed by the SYBR green I-based real-time RT-PCR and RT-LAMP assay. This assay was also the most effective in the detection of BVDV contamination in a set of commercially available bovine sera. The field conditions suggest that RT-LAMP is specific and sensitive to detecting BVDV in biological samples and may be used for quality control of biomaterials. PMID:25019170

  4. Electrical and Electrochemical Monitoring of Nucleic Acid Amplification

    PubMed Central

    Goda, Tatsuro; Tabata, Miyuki; Miyahara, Yuji

    2015-01-01

    Nucleic acid amplification is a gold standard technique for analyzing a tiny amount of nucleotides in molecular biology, clinical diagnostics, food safety, and environmental testing. Electrical and electrochemical monitoring of the amplification process draws attention over conventional optical methods because of the amenability toward point-of-care applications as there is a growing demand for nucleic acid sensing in situations outside the laboratory. A number of electrical and electrochemical techniques coupled with various amplification methods including isothermal amplification have been reported in the last 10 years. In this review, we highlight recent developments in the electrical and electrochemical monitoring of nucleic acid amplification. PMID:25798440

  5. Isothermal Gaseous Detonation Model

    NASA Astrophysics Data System (ADS)

    Prokhorov, E. S.

    2015-05-01

    We propose an isothermal gaseous detonation model taking into account the initial pressure of the explosive mixture that permits describing in a simplified form both the self-sustaining and the supercompressed and undercompressed detonation regimes. The exactness of this model has been estimated on the basis of a comparative analysis with the results of equilibrium calculations of the gas-dynamic parameters at the front of detonation waves.

  6. Isothermal separation processes

    NASA Technical Reports Server (NTRS)

    England, C.

    1982-01-01

    The isothermal processes of membrane separation, supercritical extraction and chromatography were examined using availability analysis. The general approach was to derive equations that identified where energy is consumed in these processes and how they compare with conventional separation methods. These separation methods are characterized by pure work inputs, chiefly in the form of a pressure drop which supplies the required energy. Equations were derived for the energy requirement in terms of regular solution theory. This approach is believed to accurately predict the work of separation in terms of the heat of solution and the entropy of mixing. It can form the basis of a convenient calculation method for optimizing membrane and solvent properties for particular applications. Calculations were made on the energy requirements for a membrane process separating air into its components.

  7. Miniaturized nucleic acid amplification systems for rapid and point-of-care diagnostics: a review.

    PubMed

    Ahmad, Farhan; Hashsham, Syed A

    2012-07-01

    Point-of-care (POC) genetic diagnostics critically depends on miniaturization and integration of sample processing, nucleic acid amplification, and detection systems. Polymerase chain reaction (PCR) assays have extensively applied for the diagnosis of genetic markers of disease. Microfluidic chips for microPCR with different materials and designs have been reported. Temperature cycling systems with varying thermal masses and conductivities, thermal cycling times, flow-rates, and cross-sectional areas, have also been developed to reduce the nucleic acid amplification time. Similarly, isothermal amplification techniques (e.g., loop-mediated isothermal amplification or LAMP), which are still are emerging, have a better potential as an alternative to PCR for POC diagnostics. Isothermal amplification techniques have: (i) moderate incubation temperature leading to simplified heating and low power consumption, (ii) yield high amount of amplification products, which can be detected either visually or by simple detectors, (iii) allow direct genetic amplification from bacterial cells due to the superior tolerance to substances that typically inhibit PCR, (iv) have high specificity, and sensitivity, and (v) result in rapid detection often within 10-20 min. The aim of this review is to provide a better understanding of the advantages and limitations of microPCR and microLAMP systems for rapid and POC diagnostics. PMID:22704369

  8. NASBA: A detection and amplification system uniquely suited for RNA

    SciTech Connect

    Sooknanan, R.; Malek, L.T.

    1995-06-01

    The invention of PCR (polymerase chain reaction) has revolutionized our ability to amplify and manipulate a nucleic acid sequence in vitro. The commercial rewards of this revolution have driven the development of other nuclei acid amplification and detection methodologies. This has created an alphabet soup of technologies that use different amplification methods, including NASBA (nucleic acid sequence-based amplification), LCR (ligase chain reaction), SDA (strand displacement amplification), QBR (Q-beta replicase), CPR (cycling probe reaction), and bDNA (branched DNA). Despite the differences in their processes, these amplification systems can be separated into two broad categories based on how they achieve their goal: sequence-based amplification systems, such as PCR, NASBA, and SDA, amplify a target nucleic acid sequence. Signal-based amplification systems, such as LCR, QBR, CPR and bDNA, amplify or alter a signal from a detection reaction that is target-dependent. While the various methods have relative strengths and weaknesses, only NASBA offers the unique ability to homogeneously amplify an RNA analyte in the presence of homologous genomic DNA under isothermal conditions. Since the detection of RNA sequences almost invariably measures biological activity, it is an excellent prognostic indicator of activities as diverse as virus production, gene expression, and cell viability. The isothermal nature of the reaction makes NASBA especially suitable for large-scale manual screening. These features extend NASBA`s application range from research to commercial diagnostic applications. Field test kits are presently under development for human diagnostics as well as the burgeoning fields of food and environmental diagnostic testing. These developments suggest future integration of NASBA into robotic workstations for high-throughput screening as well. 17 refs., 1 tab.

  9. Early amplification options.

    PubMed

    Gabbard, Sandra Abbott; Schryer, Jennifer

    2003-01-01

    Children with permanent hearing loss have been remediated with hearing amplification devices for decades. The influx of young infants identified with hearing loss through successful newborn hearing screening programs has established a need for amplification resources for infants within the first six months of life. For the approximately two of every 1000 infants born who are identified with bilateral hearing loss [Mehl and Thomson, 1998, Pediatrics 101, p. e4], the use of amplification is commonly the first step in treating the sequella of their loss. The use of hearing aids, combined with early intervention, has been shown to significantly improve the speech and language skills of young children with hearing loss [Yoshinaga-Itano, 2000, Seminars in Hearing 21, p. 309]. Speech and language delays have contributed to compromised academic performance of school aged children with hearing loss [Johnson et al., 1997, Educational Audiology Handbook, Singular Publishing, San Diego]. Most hard-of-hearing and deaf children use hearing aids and other assistive listening devices every day throughout their lifetime and the life expectancy of a hearing aid is only five to eight years. The current challenge for pediatric audiologists is selecting and evaluating the available amplification to provide the best options for children and their families. Amplification technology has seen an explosion in growth the past few years and the options continue to expand rapidly. This article examines currently available amplification technology and reviews the selection criteria that may be used for infants and young children. Issues such as style, type, amplification features, signal processing strategies, and verification and validation tools are also discussed. PMID:14648816

  10. Quantum Feedback Amplification

    NASA Astrophysics Data System (ADS)

    Yamamoto, Naoki

    2016-04-01

    Quantum amplification is essential for various quantum technologies such as communication and weak-signal detection. However, its practical use is still limited due to inevitable device fragility that brings about distortion in the output signal or state. This paper presents a general theory that solves this critical issue. The key idea is simple and easy to implement: just a passive feedback of the amplifier's auxiliary mode, which is usually thrown away. In fact, this scheme makes the controlled amplifier significantly robust, and furthermore it realizes the minimum-noise amplification even under realistic imperfections. Hence, the presented theory enables the quantum amplification to be implemented at a practical level. Also, a nondegenerate parametric amplifier subjected to a special detuning is proposed to show that, additionally, it has a broadband nature.

  11. On soliton amplification

    NASA Technical Reports Server (NTRS)

    Leibovich, S.; Randall, J. D.

    1979-01-01

    The paper considers a modified Korteweg-de Vries equation that permits wave amplification or damping. A 'terminal similarity' solution is identified for large times in amplified systems. Numerical results are given which confirm that the terminal similarity solution is a valid local approximation for mu t sufficiently large and positive, even though the approximation is not uniformly valid in space.

  12. Questioning cochlear amplification

    NASA Astrophysics Data System (ADS)

    van der Heijden, Marcel; Versteegh, Corstiaen P. C.

    2015-12-01

    Thirty years ago it was hypothesized that motile processes inject mechanical energy into cochlear traveling waves. This mechanical amplification, alternatively described as negative damping, is invoked to explain both the sensitivity and the nonlinear compression of cochlear responses. There is a recent trend to present cochlear amplification as an established fact, even though the evidence is at most circumstantial and several thorny problems have remained unresolved. We analyze several of these issues, and present new basilar membrane recordings that allowed us to quantify cochlear energy flow. Specifically, we address the following questions: (1) Does auditory sensitivity require narrowband amplification? (2) Has the "RC problem" (lowpass filtering of outer hair cell receptor potential) been resolved? (3) Can OHC motility improve auditory sensitivity? (4) Is there a net power gain between neighboring locations on the basilar membrane? The analyses indicate that mechanical amplification in the cochlea is neither necessary nor useful, and that realizing it by known forms of motility would reduce sensitivity rather than enhance it. Finally, our experimental data show that the peaking of the traveling wave is realized by focusing the acoustic energy rather than amplifying it. (Abbreviations. BM: basilar membrane; CF: characteristic frequency; IHC: inner hair cell; ME: middle ear; MT; mechanotransducer; OHC: outer hair cell; SPL: sound pressure level.)

  13. Isothermal-Gas-Transfer Program

    NASA Technical Reports Server (NTRS)

    Levine, Don I.

    1989-01-01

    Isothermal Gas Transfer program (GASXFER) solves variety of problems in which gas or gas mixture transferred between two containers. Special features of program include ease of entering data and ease of obtaining output. Program displays, prints, or graphs complete pressure history of each gas as function of time. Written in Lotus Symphony macrolanguage.

  14. Scaling in Supersonic Isothermal Turbulence

    NASA Astrophysics Data System (ADS)

    Kritsuk, A. G.; Wagner, R.; Norman, M. L.

    2015-10-01

    An exact relation for third-order structure functions in isothermal compressible turbulence (Galtier & Banerjee 2011) is verified using numerical data from a simulation at Mach 6. The analysis supports a Kolmogorov-like cascade phenomenology and yields a reduced version of the relation for high Mach number turbulence.

  15. Isothermal Dendritic Growth Experiment Video

    NASA Technical Reports Server (NTRS)

    1997-01-01

    This video, captured during the Isothermal Dendritic Growth Experiment (IDGE) flown on STS-87 as a part of the fourth United States Microgravity payload, shows the growth of a dendrite, and the surface solidification that occurred on the front and back windows of the growth chamber. Dendrites are tiny, tree like structures that form as metals solidify.

  16. Gravitomagnetic amplification in cosmology

    SciTech Connect

    Tsagas, Christos G.

    2010-02-15

    Magnetic fields interact with gravitational waves in various ways. We consider the coupling between the Weyl and the Maxwell fields in cosmology and study the effects of the former on the latter. The approach is fully analytical and the results are gauge invariant. We show that the nature and the outcome of the gravitomagnetic interaction depends on the electric properties of the cosmic medium. When the conductivity is high, gravitational waves reduce the standard (adiabatic) decay rate of the B field, leading to its superadiabatic amplification. In poorly conductive environments, on the other hand, Weyl-curvature distortions can result into the resonant amplification of large-scale cosmological magnetic fields. Driven by the gravitational waves, these B fields oscillate with an amplitude that is found to diverge when the wavelengths of the two sources coincide. We present technical and physical aspects of the gravitomagnetic interaction and discuss its potential implications.

  17. Light amplification using semiconductors

    SciTech Connect

    Dupuis, R.D.

    1987-06-01

    During the summer of 1953, John von Neumann discussed his ideas concerning light amplification using semiconductors with Edward Teller. In September of that year, von Neumann sent a manuscript containing his ideas and calculations on this subject to Teller for his comments. To the best of our knowledge, von Neumann did not take time to work further on these ideas, and the manuscript remained unpublished. These previously unpublished writings of John von Neumann on the subject of light amplification in semiconductors are printed as a service to the laser community. While von Neumann's original manuscript and his letter to Teller are available to anyone who visits the Library of Congress, it is much more convenient to have this paper appear in an archival journal.

  18. Voltage Amplification using Plasma

    SciTech Connect

    Farias, E. E.; Cavalcanti, G. H.; Santiago, M. A. M.

    2006-12-04

    The purpose of this work is to present experimental results about voltage amplification using plasma produced by a simple neon lamp, series connected with a signal generator and discrete circuit elements. The main advantage of employing plasma as an amplifier is due to its ability to drive larger power and potentially to operate in a larger frequency range compared with traditional amplifiers. Our results show that both, the voltage gain and the frequency range where the gain is bigger than one, are related to the plasma density which may be adjusted by a proper control of electrical discharge conditions. The plasma produced into the neon lamp exhibits a diode characteristic that is the principal responsible by the nonlinear plasma response. The amplification occurs when the plasma shows a negative conductance. In this regime the lamp works as an active amplifier and voltage gain higher than 18 was obtained.

  19. Controlled Microwave Heating Accelerates Rolling Circle Amplification

    PubMed Central

    Yoshimura, Takeo; Suzuki, Takamasa; Mineki, Shigeru; Ohuchi, Shokichi

    2015-01-01

    Rolling circle amplification (RCA) generates single-stranded DNAs or RNA, and the diverse applications of this isothermal technique range from the sensitive detection of nucleic acids to analysis of single nucleotide polymorphisms. Microwave chemistry is widely applied to increase reaction rate as well as product yield and purity. The objectives of the present research were to apply microwave heating to RCA and indicate factors that contribute to the microwave selective heating effect. The microwave reaction temperature was strictly controlled using a microwave applicator optimized for enzymatic-scale reactions. Here, we showed that microwave-assisted RCA reactions catalyzed by either of the four thermostable DNA polymerases were accelerated over 4-folds compared with conventional RCA. Furthermore, the temperatures of the individual buffer components were specifically influenced by microwave heating. We concluded that microwave heating accelerated isothermal RCA of DNA because of the differential heating mechanisms of microwaves on the temperatures of reaction components, although the overall reaction temperatures were the same. PMID:26348227

  20. Understand rotating isothermal collapses yet

    SciTech Connect

    Tohline, J.E.

    1985-01-01

    A scalar virial equation is used to describe the dynamic properties of equilibrium gas clouds, taking into account the relative effects of surface pressure, rotation, self gravity and internal isothermal pressure. Details concerning the internal structure of the clouds are ignored in order to obtain a globalized analytical expression. The obtained solution to the equation is found to agree with the surface-pressure-dominated model of Stahler (1983), and the rotation-dominated model of Hayashi, Narita, and Miyama (1982). On the basis of the analytical expression of virial equilibrium in the clouds, some of the limiting properties of isothermal clouds are described, and a realistic starting model for cloud collapse is proposed. 18 references.

  1. IDGE: Isothermal Dendritic Growth Experiment

    NASA Technical Reports Server (NTRS)

    1994-01-01

    The Isothermal Dendritic Growth Experiment (IDGE) flew on STS-62 to study the microscopic, tree-like structures (dendrites) that form within metals as they solidify from molten materials. The size, shape, and orientation of these dendrites affect the strength and usefulness of metals. Data from this experiment will be used to test and improve the mathematical models that support the industrial production of metals.

  2. Coherent white light amplification

    DOEpatents

    Jovanovic, Igor; Barty, Christopher P.

    2004-05-25

    A system for coherent simultaneous amplification of a broad spectral range of light that includes an optical parametric amplifier and a source of a seed pulse is described. A first angular dispersive element is operatively connected to the source of a seed pulse. A first imaging telescope is operatively connected to the first angular dispersive element and operatively connected to the optical parametric amplifier. A source of a pump pulse is operatively connected to the optical parametric amplifier. A second imaging telescope is operatively connected to the optical parametric amplifier and a second angular dispersive element is operatively connected to the second imaging telescope.

  3. Isothermal compressors for process gases

    SciTech Connect

    Wiederuh, E.; Meinhart, D. )

    1992-09-01

    This paper reports on isothermal compressors which are more efficient for all gases. The study of several representative gases considered stage efficiencies, pressure ratios and pressure losses of the intercoolers. Generally there are two ways to reduce power consumption of a gas compression process: minimize losses of the compressor or improve the thermodynamics of the process. But there are some new ways to reduce losses of turbocompressors. Losses of the impeller labyrinth seals and the balance piston labyrinth seal can be reduced by optimizing the labyrinth geometry and minimizing labyrinth clearances. Therefore, conventional labyrinth seals are still being studied and will be improved.

  4. [Recombinase Polymerase Amplification and its Applications in Parasite Detection].

    PubMed

    ZHENG, Wen-bin; WU, Yao-dong; MA, Jian-gang; ZHU, Xing-quan; ZHOU, Dong-hui

    2015-10-01

    Recombinase polymerase amplification (RPA) is a recently -developed isothermal nucleic-acid-amplification technology that is based on the nucleic acid replication mechanism in T4 bacteriophage. With this technique, nucleic-acid templates can be amplified to measurable levels within 20 min at 37-42 °C. The. RPA process has high sensitivity and specificity, and is simple to operate, thus nucleic acids can be detected rapidly in non-laboratory conditions. Since its development in 2006, the RPA technique has been applied in agriculture, food safety, medicine, transgene detection, etc. In this review, we will give an overview on the research progress of RPA and its application in parasite detection. PMID:26931046

  5. Evidence of high-elevation amplification versus Arctic amplification

    NASA Astrophysics Data System (ADS)

    Wang, Qixiang; Fan, Xiaohui; Wang, Mengben

    2016-01-01

    Elevation-dependent warming in high-elevation regions and Arctic amplification are of tremendous interest to many scientists who are engaged in studies in climate change. Here, using annual mean temperatures from 2781 global stations for the 1961-2010 period, we find that the warming for the world’s high-elevation stations (>500 m above sea level) is clearly stronger than their low-elevation counterparts; and the high-elevation amplification consists of not only an altitudinal amplification but also a latitudinal amplification. The warming for the high-elevation stations is linearly proportional to the temperature lapse rates along altitudinal and latitudinal gradients, as a result of the functional shape of Stefan-Boltzmann law in both vertical and latitudinal directions. In contrast, neither altitudinal amplification nor latitudinal amplification is found within the Arctic region despite its greater warming than lower latitudes. Further analysis shows that the Arctic amplification is an integrated part of the latitudinal amplification trend for the low-elevation stations (≤500 m above sea level) across the entire low- to high-latitude Northern Hemisphere, also a result of the mathematical shape of Stefan-Boltzmann law but only in latitudinal direction.

  6. Evidence of high-elevation amplification versus Arctic amplification

    PubMed Central

    Wang, Qixiang; Fan, Xiaohui; Wang, Mengben

    2016-01-01

    Elevation-dependent warming in high-elevation regions and Arctic amplification are of tremendous interest to many scientists who are engaged in studies in climate change. Here, using annual mean temperatures from 2781 global stations for the 1961–2010 period, we find that the warming for the world’s high-elevation stations (>500 m above sea level) is clearly stronger than their low-elevation counterparts; and the high-elevation amplification consists of not only an altitudinal amplification but also a latitudinal amplification. The warming for the high-elevation stations is linearly proportional to the temperature lapse rates along altitudinal and latitudinal gradients, as a result of the functional shape of Stefan-Boltzmann law in both vertical and latitudinal directions. In contrast, neither altitudinal amplification nor latitudinal amplification is found within the Arctic region despite its greater warming than lower latitudes. Further analysis shows that the Arctic amplification is an integrated part of the latitudinal amplification trend for the low-elevation stations (≤500 m above sea level) across the entire low- to high-latitude Northern Hemisphere, also a result of the mathematical shape of Stefan-Boltzmann law but only in latitudinal direction. PMID:26753547

  7. Evidence of high-elevation amplification versus Arctic amplification.

    PubMed

    Wang, Qixiang; Fan, Xiaohui; Wang, Mengben

    2016-01-01

    Elevation-dependent warming in high-elevation regions and Arctic amplification are of tremendous interest to many scientists who are engaged in studies in climate change. Here, using annual mean temperatures from 2781 global stations for the 1961-2010 period, we find that the warming for the world's high-elevation stations (>500 m above sea level) is clearly stronger than their low-elevation counterparts; and the high-elevation amplification consists of not only an altitudinal amplification but also a latitudinal amplification. The warming for the high-elevation stations is linearly proportional to the temperature lapse rates along altitudinal and latitudinal gradients, as a result of the functional shape of Stefan-Boltzmann law in both vertical and latitudinal directions. In contrast, neither altitudinal amplification nor latitudinal amplification is found within the Arctic region despite its greater warming than lower latitudes. Further analysis shows that the Arctic amplification is an integrated part of the latitudinal amplification trend for the low-elevation stations (≤500 m above sea level) across the entire low- to high-latitude Northern Hemisphere, also a result of the mathematical shape of Stefan-Boltzmann law but only in latitudinal direction. PMID:26753547

  8. [Desorption isotherms in amaranth flours].

    PubMed

    Alvarado, J D; Toaza, E; Coloma, G

    1990-09-01

    In milled seeds amaranth (Amaranthus hybridus) samples locally known as "ataco or sangoracha" and harvested in two consecutive years, the vapor pressure at four temperatures (15 degrees, 20 degrees, 25 degrees, 30 degrees C) was determined in samples obtained for drying in oven at different times, within a range between 60 g water/100 g dry matter for fresh seeds to 10 g water/100 g dry matter or below, as measured in a Brabender equipment. Vapor pressure of distilled water was also determined for water activity calculation. The isotherms for each temperature are presented. Application of the G.A.B. model (Guggenheim-Anderson-De Boer) allowed to establish the water content of greater stability within a range of 9.9 to 7.6 g/100 g. The third degree polynomial equations presented, adjust satisfactorily with the experimental data, and can be used to calculate equilibrium moisture content from 0.15 to 0.95 water activity values. Water desorption isotherms of foods are important to determine the equilibrium relationship between the moisture content of foods and the water activity, information which permits to establish likely physical, chemical or biological changes. It is considered that the results obtained are useful in drying, milling or storing amaranth flour. PMID:2134142

  9. Modular development of a prototype point of care molecular diagnostic platform for sexually transmitted infections.

    PubMed

    Branavan, Manoharanehru; Mackay, Ruth E; Craw, Pascal; Naveenathayalan, Angel; Ahern, Jeremy C; Sivanesan, Tulasi; Hudson, Chris; Stead, Thomas; Kremer, Jessica; Garg, Neha; Baker, Mark; Sadiq, Syed T; Balachandran, Wamadeva

    2016-08-01

    This paper presents the design of a modular point of care test platform that integrates a proprietary sample collection device directly with a microfluidic cartridge. Cell lysis, within the cartridge, is conducted using a chemical method and nucleic acid purification is done on an activated cellulose membrane. The microfluidic device incorporates passive mixing of the lysis-binding buffers and sample using a serpentine channel. Results have shown extraction efficiencies for this new membrane of 69% and 57% compared to the commercial Qiagen extraction method of 85% and 59.4% for 0.1ng/µL and 100ng/µL salmon sperm DNA respectively spiked in phosphate buffered solution. Extraction experiments using the serpentine passive mixer cartridges incorporating lysis and nucleic acid purification showed extraction efficiency around 80% of the commercial Qiagen kit. Isothermal amplification was conducted using thermophillic helicase dependant amplification and recombinase polymerase amplification. A low cost benchtop real-time isothermal amplification platform has been developed capable of running six amplifications simultaneously. Results show that the platform is capable of detecting 1.32×10(6) of sample DNA through thermophillic helicase dependant amplification and 1×10(5) copy numbers Chlamydia trachomatis genomic DNA within 10min through recombinase polymerase nucleic acid amplification tests. PMID:27238759

  10. Gene amplification during myogenic differentiation

    PubMed Central

    Fischer, Ulrike; Ludwig, Nicole; Raslan, Abdulrahman; Meier, Carola; Meese, Eckart

    2016-01-01

    Gene amplifications are mostly an attribute of tumor cells and drug resistant cells. Recently, we provided evidence for gene amplifications during differentiation of human and mouse neural progenitor cells. Here, we report gene amplifications in differentiating mouse myoblasts (C2C12 cells) covering a period of 7 days including pre-fusion, fusion and post-fusion stages. After differentiation induction we found an increase in copy numbers of CDK4 gene at day 3, of NUP133 at days 4 and 7, and of MYO18B at day 4. The amplification process was accompanied by gamma-H2AX foci that are indicative of double stand breaks. Amplifications during the differentiating process were also found in primary human myoblasts with the gene CDK4 and NUP133 amplified both in human and mouse myoblasts. Amplifications of NUP133 and CDK4 were also identified in vivo on mouse transversal cryosections at stage E11.5. In the course of myoblast differentiation, we found amplifications in cytoplasm indicative of removal of amplified sequences from the nucleus. The data provide further evidence that amplification is a fundamental mechanism contributing to the differentiation process in mammalians. PMID:26760505

  11. Electricity-Free Amplification and Detection for Molecular Point-of-Care Diagnosis of HIV-1

    PubMed Central

    Singleton, Jered; Osborn, Jennifer L.; Lillis, Lorraine; Hawkins, Kenneth; Guelig, Dylan; Price, Will; Johns, Rachel; Ebels, Kelly; Boyle, David; Weigl, Bernhard; LaBarre, Paul

    2014-01-01

    In resource-limited settings, the lack of decentralized molecular diagnostic testing and sparse access to centralized medical facilities can present a critical barrier to timely diagnosis, treatment, and subsequent control and elimination of infectious diseases. Isothermal nucleic acid amplification methods, including reverse transcription loop-mediated isothermal amplification (RT-LAMP), are well-suited for decentralized point-of-care molecular testing in minimal infrastructure laboratories since they significantly reduce the complexity of equipment and power requirements. Despite reduced complexity, however, there is still a need for a constant heat source to enable isothermal nucleic acid amplification. This requirement poses significant challenges for laboratories in developing countries where electricity is often unreliable or unavailable. To address this need, we previously developed a low-cost, electricity-free heater using an exothermic reaction thermally coupled with a phase change material. This heater achieved acceptable performance, but exhibited considerable variability. Furthermore, as an enabling technology, the heater was an incomplete diagnostic solution. Here we describe a more precise, affordable, and robust heater design with thermal standard deviation <0.5°C at operating temperature, a cost of approximately US$.06 per test for heater reaction materials, and an ambient temperature operating range from 16°C to 30°C. We also pair the heater with nucleic acid lateral flow (NALF)-detection for a visual readout. To further illustrate the utility of the electricity-free heater and NALF-detection platform, we demonstrate sensitive and repeatable detection of HIV-1 with a ß-actin positive internal amplification control from processed sample to result in less than 80 minutes. Together, these elements are building blocks for an electricity-free platform capable of isothermal amplification and detection of a variety of pathogens. PMID:25426953

  12. Isothermal titration calorimetry of RNA.