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Sample records for helicase-dependent isothermal amplification

  1. Comparison of isothermal helicase-dependent amplification and PCR for the detection of Mycobacterium tuberculosis by an electrochemical genomagnetic assay.

    PubMed

    Barreda-García, Susana; Miranda-Castro, Rebeca; de-Los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo J; Lobo-Castañón, M Jesús

    2016-12-01

    Methods for the early and sensitive detection of pathogenic bacteria suited to low-resource settings could impact diagnosis and management of diseases. Helicase-dependent isothermal amplification (HDA) is an ideal tool for this purpose, especially when combined with a sequence-specific detection method able to improve the selectivity of the assay. The implementation of this approach requires that its analytical performance is shown to be comparable with the gold standard method, polymerase chain reaction (PCR). In this study, we optimize and compare the asymmetric amplification of an 84-base-long DNA sequence specific for Mycobacterium tuberculosis by PCR and HDA, using an electrochemical genomagnetic assay for hybridization-based detection of the obtained single-stranded amplicons. The results indicate the generalizability of the magnetic platform with electrochemical detection for quantifying amplification products without previous purification. Moreover, we demonstrate that under optimal conditions the same gene can be amplified by either PCR or HDA, allowing the detection of as low as 30 copies of the target gene sequence with acceptable reproducibility. Both assays have been applied to the detection of M. tuberculosis in sputum, urine, and pleural fluid samples with comparable results. Simplicity and isothermal nature of HDA offer great potential for the development of point-of-care devices. Graphical Abstract Comparative evaluation of isothermal helicase-dependent amplification and PCR for electrochemical detection of Mycobacterium tuberculosis.

  2. Attomolar quantitation of Mycobacterium tuberculosis by asymmetric helicase-dependent isothermal DNA-amplification and electrochemical detection.

    PubMed

    Barreda-García, Susana; González-Álvarez, María José; de-los-Santos-Álvarez, Noemí; Palacios-Gutiérrez, Juan José; Miranda-Ordieres, Arturo J; Lobo-Castañón, María Jesús

    2015-06-15

    A highly sensitive and robust method for the quantification of specific DNA sequences based on coupling asymmetric helicase-dependent DNA amplification to electrochemical detection is described. This method relies on the entrapment of the amplified ssDNA sequences on magnetic beads followed by a post-amplification hybridization assay to provide an added degree of specificity. As a proof-of-concept a 84-bases long sequence specific of Mycobacterium tuberculosis is amplified at 65°C, providing 3×10(6) amplification after 90 min. Using this system 0.5 aM, corresponding to 15 copies of the target gene in 50 µL of sample, can be successfully detected and reliably quantified under isothermal conditions in less than 4h. The assay has been applied to the detection of M. tuberculosis in sputum, pleural fluid and urine samples. Besides this application, the proposed assays is a powerful and general tool for molecular diagnostic that can be applied to the detection of other specific DNA sequences, taking full advantage of the plethora of genomic information now available.

  3. 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.

  4. 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.

  5. Isothermal Multiple Displacement Amplification

    PubMed Central

    Luthra, Rajyalakshmi; Medeiros, L. Jeffrey

    2004-01-01

    Isothermal multiple strand displacement amplification (IMDA) of the whole human genome is a promising method for procuring abundant DNA from valuable and often limited clinical specimens. However, whether DNA generated by this method is of high quality and a faithful replication of the DNA in the original specimen, allowing for subsequent molecular diagnostic testing, requires verification. In this study, we evaluated the suitability of IMDA-generated DNA (IMDA-DNA) for detecting antigen receptor gene rearrangements, chromosomal translocations, and gene mutations using Southern blot analysis, polymerase chain reaction (PCR) methods, or sequencing methods in 28 lymphoma and leukemia clinical specimens. Molecular testing before and after whole genome amplification of these specimens using the IMDA technique showed concordance in 27 of 28 (96%) specimens. Analysis of IMDA-DNA by Southern blot analysis detected restriction fragments >12 kilobases long. No amplification bias was observed at all loci tested demonstrating that this method can be useful in generating large amounts of unbiased, high molecular weight DNA from limited clinical specimens. PMID:15269301

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

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

    SciTech Connect

    Torres, C.

    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.

  12. 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.

  13. Loop-mediated isothermal amplification of single pollen grains.

    PubMed

    Bektaş, Ali; Chapela, Ignacio

    2014-08-01

    The polymerase chain reaction (PCR) has been a reliable and fruitful method for many applications in ecology. Nevertheless, unavoidable technical and instrumental requirements of PCR have limited its widespread application in field situations. The recent development of isothermal DNA amplification methods provides an alternative to PCR, which circumvents key limitations of PCR for direct amplification in the field. Being able to analyze DNA in the pollen cloud of an ecosystem would provide very useful ecological information, yet would require a field-enabled, high-throughput method for this potential to be realized. Here, we demonstrate the applicability of the loop-mediated DNA amplification method (LAMP), an isothermal DNA amplification technique, to be used in pollen analysis. We demonstrate that LAMP can provide a reliable method to identify species from the pollen cloud, and that it can amplify successfully with sensitivity down to single pollen grains, thus opening the possibility of field-based, high-throughput analysis.

  14. Integration of isothermal amplification methods in microfluidic devices: Recent advances.

    PubMed

    Giuffrida, Maria Chiara; Spoto, Giuseppe

    2017-04-15

    The integration of nucleic acids detection assays in microfluidic devices represents a highly promising approach for the development of convenient, cheap and efficient diagnostic tools for clinical, food safety and environmental monitoring applications. Such tools are expected to operate at the point-of-care and in resource-limited settings. The amplification of the target nucleic acid sequence represents a key step for the development of sensitive detection protocols. The integration in microfluidic devices of the most popular technology for nucleic acids amplifications, polymerase chain reaction (PCR), is significantly limited by the thermal cycling needed to obtain the target sequence amplification. This review provides an overview of recent advances in integration of isothermal amplification methods in microfluidic devices. Isothermal methods, that operate at constant temperature, have emerged as promising alternative to PCR and greatly simplify the implementation of amplification methods in point-of-care diagnostic devices and devices to be used in resource-limited settings. Possibilities offered by isothermal methods for digital droplet amplification are discussed.

  15. 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

  16. 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.

  17. 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

  18. 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...

  19. Single primer-triggered isothermal amplification for double-stranded DNA detection.

    PubMed

    Ma, Cuiping; Han, Dianang; Deng, Meilian; Wang, Jingfei; Shi, Chao

    2015-01-11

    Here we have devised a new generation of isothermal double-stranded DNA (dsDNA) detection method, termed single primer-triggered isothermal amplification (SAMP). It is very simple only requiring one primer and a few copies of dsDNA in less than an hour are detectable with multiple signal amplification steps.

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

    PubMed

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

    2015-10-20

    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.

  1. 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. PMID:27600235

  2. A fluorescence biosensor for VEGF detection based on DNA assembly structure switching and isothermal amplification.

    PubMed

    Li, Junlong; Sun, Kexin; Chen, Zhongping; Shi, Jifei; Zhou, Dandan; Xie, Guoming

    2017-03-15

    Vascular endothelial growth factor (VEGF) is an important biomarker in cancer angiogenesis. Here, we develop a aptasensor method for VEGF detection based on DNA assembly structure switching and isothermal amplification. The design employs a DNA assembly made of a isothermal amplification template, a aptamer, a primer and a protector chain. The DNA assembly is unable to undergo isothermal amplification in the absence of target. The presence of the target, however, triggers a structure switching event that causes hybridization of primer with template to facilitate isothermal amplification. Whereafter, toehold-mediated DNA strand displacement reaction between the generated (single-stranded DNA) ssDNA and fluorescent/quencher labeled probe are performed. Then, the increase in fluorescence provides an analytical signal. This strategy opens up a sensitive, selective and simple sensing platform for detection of VEGF. The system was also implemented to analyze the VEGF in human serum samples with satisfactory results.

  3. Self-primed isothermal amplification for genomic DNA detection of human papillomavirus.

    PubMed

    Lu, Wei; Yuan, Qingpan; Yang, Zhiliu; Yao, Bo

    2017-04-15

    Rolling circle amplification (RCA) is an isothermal amplification technique with high efficiency and perfect accuracy for nucleic acids detection. However, RCA technique suffers the limitation to detect short DNA or RNA molecules. For long nucleic acid molecules, enzymatic restriction as well as heat denaturation process is usually required, which makes the amplification not effective and strictly isothermal. In this article, a simple and efficient one-pot self-primed isothermal amplification (SIA) was developed for detection of genomic DNA directly based on the combination of nicking endonuclease assisted strand displacement amplification (SDA) and exponential RCA. In virtue of numerous nicking sites on the genome, a pre-amplification of the whole genome was performed through SDA with the specific cleaving of nicking endonuclease. Meanwhile, the single strand DNA with HPV target sequence generated from SDA could hybrid with the circle probe as a primer and trigger the exponential RCA as a result of the existence of nicking endonuclease. As the reaction temperature and enzyme were the same, the amplification could be operated in one pot. The reaction solution after amplification was added on the electrode for hybridization with the sulfydryl probe to achieve the electrochemical signal. Based on the isothermal amplification, genotyping of HPV 11, 16, 18 and the detection of HPV 18 in Hela cell line were attempted with satisfied results. This approach should be a promising tool for pathogene detection in clinical diagnostics and research.

  4. 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.

  5. 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...

  6. Detection of Aleutian disease virus by loop-mediated isothermal amplification.

    PubMed

    Zhang, Zhuo; Wang, Bin; Hu, Shouping; Zhang, Jiaoer; He, Xijun; Zheng, Shimin

    2015-09-01

    In this study, a loop-mediated isothermal amplification (LAMP) assay was developed and optimized for the detection of Aleutian disease virus (ADV) in minks. The amplification could be completed within 45 min under isothermal condition by employing a set of six ADV genome-specific primers. The amplification results could be visualized directly with the naked eye by using fluorescent dye. Comparative experiments showed that the LAMP assay is superior to conventional polymerase chain reaction for the detection of both experimental and field samples. Results of current study indicated that the LAMP assay is a rapid and reliable technique for routine diagnosis of ADV infection in minks.

  7. Design of a New Type of Compact Chemical Heater for Isothermal Nucleic Acid Amplification

    PubMed Central

    Shah, Kamal G.; Guelig, Dylan; Diesburg, Steven; Buser, Joshua; Burton, Robert; LaBarre, Paul; Richards-Kortum, Rebecca; Weigl, Bernhard

    2015-01-01

    Previous chemical heater designs for isothermal nucleic acid amplification have been based on solid-liquid phase transition, but using this approach, developers have identified design challenges en route to developing a low-cost, disposable device. Here, we demonstrate the feasibility of a new heater configuration suitable for isothermal amplification in which one reactant of an exothermic reaction is a liquid-gas phase-change material, thereby eliminating the need for a separate phase-change compartment. This design offers potentially enhanced performance and energy density compared to other chemical and electric heaters. PMID:26430883

  8. 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

  9. Evaluation of a commercial loop-mediated isothermal amplification assay for diagnosis of Bordetella pertussis infection.

    PubMed

    Kamachi, Kazunari; Moriuchi, Takumi; Hiramatsu, Yukihiro; Otsuka, Nao; Shibayama, Keigo

    2017-02-01

    We evaluated a commercial loop-mediated isothermal amplification (LAMP) assay kit for Bordetella pertussis detection. The LAMP primers were designed to target the ptxP1 allele of the pertussis toxin promoter, but the assay could detect B. pertussis ptxP3 and ptxP8 strains in addition to ptxP1 strains, with high analytical sensitivity.

  10. 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...

  11. 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 ...

  12. Comprehensive evaluation of molecular enhancers of the isothermal exponential amplification reaction

    PubMed Central

    Mok, Ellie; Wee, Eugene; Wang, Yuling; Trau, Matt

    2016-01-01

    The exponential amplification reaction (EXPAR) is an emerging isothermal nucleic acid amplification method with high potential for molecular diagnostics due to its isothermal nature and high amplification efficiency. However, the use of EXPAR is limited by the high levels of non-specific amplification. Hence, methods that can improve the specificity of EXPAR are desired to facilitate its widespread adoption in practice. Herein, we proposed a strategy to improve EXPAR performance by using molecular enhancers. Eight small molecules were investigated, including ethylene glycol, propylene glycol, betaine, dimethyl sulfoxide (DMSO), trehalose, tetramethylammonium chloride (TMAC), bovine serum albumin (BSA) and single-stranded binding (SSB) proteins. A combination of kinetic and end-point analysis was adopted to investigate how these molecules affected EXPAR performance. Trehalose, TMAC, BSA and SSB proteins were found to have positive effects on EXPAR with trehalose being able to increase the efficiency of EXPAR. In contrast, TMAC, BSA and SSB proteins were shown to increase the specificity of EXPAR. We applied our findings to demonstrate the combination of trehalose and TMAC could simultaneously improve both the efficiency and specificity of an EXPAR-based miRNA detection method. The information provided in this study may serve as a reference to benefit the wider isothermal amplification community. PMID:27910874

  13. 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

  14. Ultrasmall volume molecular isothermal amplification in microfluidic chip with advanced surface processing

    NASA Astrophysics Data System (ADS)

    Huang, Guoliang; Ma, Li; Yang, Xiaoyong; Yang, Xu

    2011-01-01

    In this paper, we developed a metal micro-fluidic chip with advanced surface processing for ultra-small volume molecular isothermal amplification. This method takes advantages of the nucleic acid amplification with good stability and consistency, high sensitivity about 31 genomic DNA copies and bacteria specific gene identification. Based on the advanced surface processing, the bioreaction assays of nucleic acid amplification was dropped about 392nl in volume. A high numerical aperture confocal optical detection system was advanced to sensitively monitor the DNA amplification with low noise and high power collecting fluorescence near to the optical diffraction limit. A speedy nucleic acid isothermal amplification was performed in the ultra-small volume microfluidic chip, where the time at the inflexions of second derivative to DNA exponential amplified curves was brought forward and the sensitivity was improved about 65 folds to that of in current 25μl Ep-tube amplified reaction, which indicates a promising clinic molecular diagnostics in the droplet amplification.

  15. 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.

  16. A novel loop-mediated isothermal amplification approach for sex identification of Columbidae birds.

    PubMed

    Chan, K-W; Liu, P-C; Yang, W-C; Kuo, J; Chang, C L-T; Wang, C-Y

    2012-10-01

    Because it is difficult to differentiate male and female Columbidae birds (e.g., Columba livia) on the basis of morphology, detection of DNA fragments associated with Chromobox-Helicase-DNA binding genes or female-specific genes have been widely used. The objective was to establish a loop-mediated isothermal amplification system involving the 18S ribosomal RNA gene and a female-specific gene for sex identification of Columba livia birds. Unlike polymerase chain reaction (PCR), random amplification polymorphic DNA-PCR and amplified fragment length polymorphism-PCR, target DNA was amplified under isothermal conditions (the entire process was completed in <60 min). By modulating various parameters involved in amplification, e.g., concentrations of MgSO(4), betaine, Bst polymerase, and deoxynucleotide triphosphates, as well as the relative ratio of outer/inner primers and temperatures, optimal conditions for both targets were established that had equal detection limits (62.5 ng). To simplify sex determination, direct observations of the presence of white precipitate (derived from magnesium pyrophosphates) were used for positive samples, which was compared with the whitish ring which formed in a negative sample after addition of CuSO(4). This approach was a rapid alternative to electrophoresis or turbidimetry. DNA extracted from the blood and feathers of various birds were tested using loop-mediated isothermal amplification; results were consistent with a standard PCR. Thus, the assay was a simple, accurate, fast, and economical alternative suitable for veterinary practice.

  17. 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.

  18. Isothermal strand displacement amplification (iSDA): a rapid and sensitive method of nucleic acid amplification for point-of-care diagnosis.

    PubMed

    Toley, Bhushan J; Covelli, Isabela; Belousov, Yevgeniy; Ramachandran, Sujatha; Kline, Enos; Scarr, Noah; Vermeulen, Nic; Mahoney, Walt; Lutz, Barry R; Yager, Paul

    2015-11-21

    We present a method of rapid isothermal amplification of DNA without initial heat denaturation of the template, and methods and probes for (a) real-time fluorescence detection and (b) lateral flow detection of amplicons. Isothermal strand displacement amplification (iSDA) can achieve >10(9)-fold amplification of the target sequence in <20 minutes at 49 °C, which makes it one of the fastest existing isothermal DNA amplification methods. iSDA initiates at sites where DNA base pairs spontaneously open or transiently convert into Hoogsteen pairs, i.e. "breathe", and proceeds to exponential amplification by repeated nicking, extension, and displacement of single strands. We demonstrate successful iSDA amplification and lateral flow detection of 10 copies of a Staphylococcus aureus gene, NO.-inducible l-lactate dehydrogenase (ldh1) (Richardson, Libby, and Fang, Science, 2008, 319, 1672-1676), in a clean sample and 50 copies in the presence of high concentrations of genomic DNA and mucins in <30 minutes. We also present a simple kinetic model of iSDA that incorporates competition between target and primer-dimer amplification. This is the first model that quantitates the effects of primer-dimer products in isothermal amplification reactions. Finally, we demonstrate the multiplexing capability of iSDA by the simultaneous amplification of the target gene and an engineered internal control sequence. The speed, sensitivity, and specificity of iSDA make it a powerful method for point-of-care molecular diagnosis.

  19. Label-Free Isothermal Amplification Assay for Specific and Highly Sensitive Colorimetric miRNA Detection

    PubMed Central

    2016-01-01

    We describe a new method for the detection of miRNA in biological samples. This technology is based on the isothermal nicking enzyme amplification reaction and subsequent hybridization of the amplification product with gold nanoparticles and magnetic microparticles (barcode system) to achieve naked-eye colorimetric detection. This platform was used to detect a specific miRNA (miRNA-10b) associated with breast cancer, and attomolar sensitivity was demonstrated. The assay was validated in cell culture lysates from breast cancer cells and in serum from a mouse model of breast cancer. PMID:27713932

  20. Simple and specific detection of Bordetella holmesii by using a loop-mediated isothermal amplification assay.

    PubMed

    Otsuka, Nao; Yoshino, Shuji; Kawano, Kimiko; Toyoizumi-Ajisaka, Hiromi; Shibayama, Keigo; Kamachi, Kazunari

    2012-07-01

    A loop-mediated isothermal amplification (LAMP) assay for simple detection of Bordetella holmesii was developed. This assay discriminates between B. holmesii and other Bordetella species and successfully detect B. holmesii DNA in nasopharyngeal swab samples from subjects with suspected pertussis. The LAMP assay results were in complete agreement with the results of previously published real-time PCR assay, indicating that the former is a powerful tool for the accurate diagnosis and surveillance of B. holmesii.

  1. 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

  2. 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

  3. Simultaneous multiple target detection in real-time loop-mediated isothermal amplification.

    PubMed

    Tanner, Nathan A; Zhang, Yinhua; Evans, Thomas C

    2012-08-01

    Loop-mediated isothermal amplification (LAMP) is a rapid and reliable sequence-specific isothermal nucleic acid amplification technique. To date, all reported real-time detection methods for LAMP have been restricted to single targets, limiting the utility of this technique. Here, we adapted standard LAMP primers to contain a quencher-fluorophore duplex region that upon strand separation results in a gain of fluorescent signal. This approach permitted the real-time detection of 1-4 target sequences in a single LAMP reaction tube utilizing a standard real-time fluorimeter. The methodology was highly reproducible and sensitive, detecting below 100 copies of human genomic DNA. It was also robust, with a 7-order of magnitude dynamic range of detectable targets. Furthermore, using a new strand-displacing DNA polymerase or its warm-start version, Bst 2.0 or Bst 2.0 WarmStart DNA polymerases, resulted in 50% faster amplification signals than wild-type Bst DNA polymerase, large fragment in this new multiplex LAMP procedure. The coupling of this new multiplex technique with next generation isothermal DNA polymerases should increase the utility of the LAMP method for molecular diagnostics.

  4. 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.

  5. 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.

  6. [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.

  7. Direct loop mediated isothermal amplification on filters for quantification of Dehalobacter in groundwater.

    PubMed

    Stedtfeld, Robert D; Stedtfeld, Tiffany M; Samhan, Farag; Kanitkar, Yogendra H; Hatzinger, Paul B; Cupples, Alison M; Hashsham, Syed A

    2016-12-01

    Nucleic acid amplification of biomarkers is increasingly used to monitor microbial activity and assess remedial performance in contaminated aquifers. Previous studies described the use of filtration, elution, and direct isothermal amplification (i.e. no DNA extraction and purification) as a field-able means to quantify Dehalococcoides spp. in groundwater. This study expands previous work with direct loop mediated isothermal amplification (LAMP) for the detection and quantification of Dehalobacter spp. in groundwater. Experiments tested amplification of DNA with and without crude lysis and varying concentrations of humic acid. Three separate field-able methods of biomass concentration with eight aquifer samples were also tested, comparing direct LAMP with traditional DNA extraction and quantitative PCR (qPCR). A new technique was developed where filters were amplified directly within disposable Gene-Z chips. The direct filter amplification (DFA) method eliminated an elution step and provided a detection limit of 10(2)Dehalobacter cells per 100mL. LAMP with crudely lysed Dehalobacter had a negligible effect on threshold time and sensitivity compared to lysed samples. The LAMP assay was more resilient than traditional qPCR to humic acid in sample, amplifying with up to 100mg per L of humic acid per reaction compared to 1mg per L for qPCR. Of the tested field-able concentrations methods, DFA had the lowest coefficient of variation among Dehalobacter spiked groundwater samples and lowest threshold time indicating high capture efficiency and low inhibition. While demonstrated with Dehalobacter, the DFA method can potentially be used for a number of applications requiring field-able, rapid (<60min) and highly sensitive quantification of microorganisms in environmental water samples.

  8. A general solution for opening double-stranded DNA for isothermal amplification

    PubMed Central

    Chen, Gangyi; Dong, Juan; Yuan, Yi; Li, Na; Huang, Xin; Cui, Xin; Tang, Zhuo

    2016-01-01

    Nucleic acid amplification is the core technology of molecular biology and genetic engineering. Various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). However, most of these methods can only detect single stranded nucleic acid. Herein, we put forward a simple solution for opening double-stranded DNA for isothermal detection methods. The strategy employs recombination protein from E. coli (RecA) to form nucleoprotein complex with single-stranded DNA, which could scan double-stranded template for homologous sites. Then, the nucleoprotein can invade the double-stranded template to form heteroduplex in the presence of ATP, resulting in the strand exchange. The ATP regeneration system could be eliminated by using high concentration of ATP, and the 3′-OH terminal of the invasion strand can be recognized by other DNA modifying enzymes such as DNA polymerase or DNA ligase. Moreover, dATP was found to be a better cofactor for RecA, which make the system more compatible to DNA polymerase. The method described here is a general solution to open dsDNA, serving as a platform to develop more isothermal nucleic acids detection methods for real DNA samples based on it. PMID:27687498

  9. 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.

  10. 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

  11. Thermal stability of quadruplex primers for highly versatile isothermal DNA amplification.

    PubMed

    Mathias, Jordan; Okyere, Robert; Lomidze, Levan; Gvarjaladze, David; Musier-Forsyth, Karin; Kankia, Besik

    2014-01-01

    Quadruplex priming amplification (QPA) allows isothermal amplification of nucleic acids with improved yield and simplified detection. This assay is based on a DNA quadruplex, GGGTGGGTGGGTGGG (G3T), which in the presence of specific cations possesses unusually high thermal stability. QPA employs truncated G3T sequences as primers, which upon polymerase elongation, self-dissociate from the binding site and allow the next round of priming without thermal unfolding of amplicons. The rate of amplification strongly depends on the thermal stability of the primer/primer binding site (PBS) complex and to date QPA has been demonstrated to work over a narrow temperature range. To expand the capabilities of QPA, in the present study, we studied the fold and thermodynamic properties of the wild-type G3T and variants containing sequence modifications or extensions at the 5'-end. Circular dichroism studies demonstrate that the substitution of thymidines by other nucleotides or GC addition at the 5'-end does not change the parallel fold of G3T. Thermal unfolding experiments revealed that purine bases incorporated at loop positions and 5'-end dinucleotide extension significantly destabilize the quadruplex, while loop pyrimidines have almost no effect. Overall, the results of these studies suggest that linear isothermal QPA can be performed over a wide temperature range to accommodate both thermophilic and mesophilic DNA polymerases.

  12. Detecting a novel Eriocheir sinensis reovirus by reverse transcription loop-mediated isothermal amplification assay.

    PubMed

    Ma, Y; Dai, T; Serwadda, A; Shen, H

    2016-11-01

    The novel Eriocheir sinensis reovirus (EsRV) is a pathogen that causes severe disease and high mortality rates in cultivated crabs. Here, we established a highly sensitive and specific rapid reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay that was cheaper and more suitable for field applications in crab aquaculture than those of traditional reverse transcription-polymerase chain reaction (RT-PCR) analysis. The amplification was completed within 45 min under isothermal conditions at 65°C. The RT-LAMP test for EsRV had a detection limit of 15 pg, and sensitivity was 100 times greater than that of conventional RT-PCR. The LAMP primers for EsRV were not amplified by other pathogen strains, indicating good specificity. In addition to detection by electrophoresis, RT-LAMP results were detectable by visual observations of reaction tube turbidity, and calcein was added to visually detect the amplification products. These results indicate that this highly convenient, rapid and sensitive RT-LAMP assay can be used to detect EsRV-infected aquatic organisms.

  13. Rapid detection of IHNV by molecular padlock recognition and surface-associated isothermal amplification

    NASA Astrophysics Data System (ADS)

    McCarthy, Erik L.; Egeler, Teressa J.; Bickerstaff, Lee E.; Pereira da Cunha, Mauricio; Millard, Paul J.

    2005-11-01

    RNA sequences derived from infectious hematopoeitic necrosis virus (IHNV) could be detected using a combination of surface-associated molecular padlock DNA probes (MPP) and rolling circle amplification (RCA) in microcapillary tubes. DNA oligonucleotides with base sequences identical to RNA obtained from IHNV were recognized by MPP. Circularized MPP were then captured on the inner surface of glass microcapillary tubes by immobilized DNA oligonucleotide primers. Extension of the immobilized primers by isothermal RCA gave rise to DNA concatamers, which were in turn bound by the fluorescent reporter SYBR Green II nucleic acid stain, and measured by microfluorimetry. Surface-associated molecular padlock technology, combined with isothermal RCA, exhibited high selectivity and sensitivity without thermal cycling. This technology is applicable to direct RNA and DNA detection, permitting detection of a variety of viral or bacterial pathogens.

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

    PubMed

    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.

  15. Fluorescence aptameric sensor for isothermal circular strand-displacement polymerization amplification detection of adenosine triphosphate.

    PubMed

    Song, Weiling; Zhang, Qiao; Xie, Xuxu; Zhang, Shusheng

    2014-11-15

    In this work, isothermal circular strand-displacement polymerization amplification assay is developed for highly specific and sensitive detection of adenosine triphosphate (ATP). The amplification process consists of circular common target molecule-displacement polymerization (CCDP) and circular nucleic acid strand-displacement polymerization (CNDP). In the presence of ATP, the complementary strand was released from the aptamer by the target recognition of ATP, and catalyzed the subsequent cycle reaction. With the polymerase and primer, the displaced target triggers the process of CCDP. With the involvement of nicking endonuclease, the released complementary strand triggers the CNDP. Combined CCDP with CNDP, the exponentially produced fluorescence probes are obtained, achieving a detection limit of ATP as low as 2.6 × 10(-10)M. Moreover, the proposed strategy exhibits an excellent specificity and is successfully applied in real sample assay which demonstrates potential application in practical samples.

  16. IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION

    PubMed Central

    Boese, B. J.; Corbino, K.; Breaker, R. R.

    2017-01-01

    We sought to create new cellulose-binding RNA aptamers for use as modular components in the engineering of complex functional nucleic acids. We designed our in vitro selection strategy to incorporate self-sustained sequence replication (3SR), which is an isothermal nucleic acid amplification protocol that allows for the rapid amplification of RNAs with little manipulation. The best performing aptamer representative was chosen for reselection and further optimization. The aptamer exhibits robust affinity for cellulose in both the powdered and paper form, but did not show any significant affinity for closely related polysaccharides. The minimal cellulose-binding RNA aptamer also can be grafted onto other RNAs to permit the isolation of RNAs from complex biochemical mixtures via cellulose affinity chromatography. This was demonstrated by fusing the aptamer to a glmS ribozyme sequence, and selectively eluting ribozyme cleavage products from cellulose using the glucosamine 6-phosphate to activate glmS ribozyme function. PMID:18696364

  17. Rapid and Sensitive Detection of Lymphocystis Disease Virus Genotype VII by Loop-Mediated Isothermal Amplification.

    PubMed

    Valverde, Estefanía J; Cano, Irene; Castro, Dolores; Paley, Richard K; Borrego, Juan J

    2017-03-01

    Lymphocystis disease virus (LCDV) infections have been described in gilthead seabream (Sparus aurata L.) and Senegalese sole (Solea senegalensis, Kaup), two of the most important marine fish species in the Mediterranean aquaculture. In this study, a rapid, specific, and sensitive detection method for LCDV genotype VII based on loop-mediated isothermal amplification (LAMP) was developed. The LAMP assay, performed using an apparatus with real-time amplification monitoring, was able to specifically detect LCDV genotype VII from clinically positive samples in less than 12 min. In addition, the assay allowed the detection of LCDV in all asymptomatic carrier fish analysed, identified by qPCR, showing an analytical sensitivity of ten copies of viral DNA per reaction. The LCDV LAMP assay has proven to be a promising diagnostic method that can be used easily in fish farms to detect the presence and spread of this iridovirus.

  18. Rapid and sensitive detection of Lophophora williamsii by loop-mediated isothermal amplification.

    PubMed

    Sasaki, Yohei; Fujimoto, Tsuguto; Aragane, Masako; Yasuda, Ichiro; Nagumo, Seiji

    2009-05-01

    We have developed a convenient method for the detection of Lophophora williamsii using loop-mediated isothermal amplification (LAMP). We designed six species-specific primers for L. williamsii, including two loop primers. This L. williamsii-specific primer set was used for LAMP of total DNA extracted from L. williamsii and from L. diffusa. Real-time monitoring of LAMP was achieved by measuring turbidity due to the formation of magnesium pyrophosphate. Amplification occurred in samples mixed with total DNA from L. williamsii, but not in those mixed with total DNA from L. diffusa. We could also visually observe L. williamsii by adding fluorescent detection reagent to the reaction tube and exposing it to UV light. This new method amplified L. williamsii selectively and is expected to be applicable to the detection of peyote.

  19. Loop-Mediated Isothermal Amplification (LAMP) for Detection of Phytoplasmas in the Field.

    PubMed

    Dickinson, Matt

    2015-01-01

    Loop-mediated isothermal amplification (LAMP) is a nucleic acid-based detection method with many applications. This chapter details its use for detection of phytoplasma diseases, combining a rapid 2-min DNA extraction method with real-time LAMP product detection such that the entire procedure can be undertaken and completed in the field within 40 min. Furthermore, the assays include an anneal curve validation step to guard against false positives and an assay for host plant DNA to guard against false negatives.

  20. 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.

  1. Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis

    PubMed Central

    2014-01-01

    Background Nucleic acid amplification is the most sensitive and specific method to detect Plasmodium falciparum. However the polymerase chain reaction remains laboratory-based and has to be conducted by trained personnel. Furthermore, the power dependency for the thermocycling process and the costly equipment necessary for the read-out are difficult to cover in resource-limited settings. This study aims to develop and evaluate a combination of isothermal nucleic acid amplification and simple lateral flow dipstick detection of the malaria parasite for point-of-care testing. Methods A specific fragment of the 18S rRNA gene of P. falciparum was amplified in 10 min at a constant 38°C using the isothermal recombinase polymerase amplification (RPA) method. With a unique probe system added to the reaction solution, the amplification product can be visualized on a simple lateral flow strip without further labelling. The combination of these methods was tested for sensitivity and specificity with various Plasmodium and other protozoa/bacterial strains, as well as with human DNA. Additional investigations were conducted to analyse the temperature optimum, reaction speed and robustness of this assay. Results The lateral flow RPA (LF-RPA) assay exhibited a high sensitivity and specificity. Experiments confirmed a detection limit as low as 100 fg of genomic P. falciparum DNA, corresponding to a sensitivity of approximately four parasites per reaction. All investigated P. falciparum strains (n = 77) were positively tested while all of the total 11 non-Plasmodium samples, showed a negative test result. The enzymatic reaction can be conducted under a broad range of conditions from 30-45°C with high inhibitory concentration of known PCR inhibitors. A time to result of 15 min from start of the reaction to read-out was determined. Conclusions Combining the isothermal RPA and the lateral flow detection is an approach to improve molecular diagnostic for P. falciparum in

  2. Portrait Toxigenic Clostridium difficile assay, an isothermal amplification assay detects toxigenic C. difficile in clinical stool specimens.

    PubMed

    Denys, Gerald A

    2014-01-01

    The Portrait Toxigenic Clostridium difficile assay is a rapid, qualitative assay for the detection of the tcdB gene of C. difficile in stool specimens from patients suspected of C. difficile infections, and received 510(k) clearance by the US FDA in March 2012. The Portrait Toxigenic C. difficile assay combines novel blocked-primer-mediated helicase-dependent multiplex amplification (bpHDA) technology and chip-based detection in an automated sample-to-result format. The assay requires minimal sample preparation and results are available within 90 min. In a multicenter evaluation, the Portrait Toxigenic C. difficile assay had a sensitivity of 98.2% and specificity of 92.8% compared with toxigenic culture. A comparative study between the Portrait Toxigenic C. difficile assay and three FDA-cleared molecular assays for the detection of toxigenic C. difficile exhibited a high degree of agreement (93.8-97.5%). The Portrait Toxigenic C. difficile assay provides a simple, cost-effective method with broad applicability to panel-based approaches, potentially simplifying workflow.

  3. A loop-mediated isothermal amplification method for rapid detection of NDM-1 gene.

    PubMed

    Qi, Jing; Du, Yijun; Zhu, Xiaoling; Bai, Hua; Luo, Yanbo; Liu, Yuqing

    2012-08-01

    A loop-mediated isothermal amplification (LAMP) method was developed for the rapid and sensitive detection of the emerging resistance gene New Delhi Metallo-β-lactamase-1 (NDM-1), with its specificity and sensitivity having been evaluated. Six primers, including a pair of outer primers, a pair of inner primers, and a pair of loop primers, were specially designed for recognizing eight distinct sequences on the target NDM-1 gene. The amplification reaction was performed within only 40 min under isothermal conditions at 65°C in a regular water bath. The LAMP assay showed good specificity and higher sensitivity than the conventional polymerase chain reaction (PCR), with a detection limit of 1 pg genomic DNA per tube of one NDM-1-positive reference strain. The detection result for the 345 clinical samples showed 100% consistence with the result by the PCR method, and three contaminated samples could be detected correctly by LAMP assays, while they could not be detected by PCR. The LAMP method reported here demonstrated a potential and valuable means for detection of the NDM-1 gene: easy, rapid, visual, specific, accurate, and sensitive, especially useful for on-the-spot investigation.

  4. 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.

  5. 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.

  6. Rapid detection of Brucella spp. using loop-mediated isothermal amplification (LAMP).

    PubMed

    Chen, Shouyi; Li, Xunde; Li, Juntao; Atwill, Edward R

    2013-01-01

    Brucella spp. are facultative intracellular bacteria that cause zoonotic disease of brucellosis worldwide. Livestock that are most vulnerable to brucellosis include cattle, goats, and pigs. Brucella spp. cause serious health problems to humans and animals and economic losses to the livestock industry. Traditional methods for detection of Brucella spp. take 48-72 h (Kumar et al., J Commun Dis 29:131-137, 1997; Barrouin-Melo et al., Res Vet Sci 83:340-346, 2007) that do not meet the food industry's need of rapid detection. Therefore, there is an urgent need of fast, specific, sensitive, and inexpensive method for diagnosing of Brucella spp. Loop-mediated isothermal amplification (LAMP) is a method to amplify nucleic acid at constant temperatures. Amplification can be detected by visual detection, fluorescent stain, turbidity, and electrophoresis. We targeted at the Brucella-specific gene omp25 and designed LAMP primers for detection of Brucella spp. Amplification of DNA with Bst DNA polymerase can be completed at 65 °C in 60 min. Amplified products can be detected by SYBR Green I stain and 2.0% agarose gel electrophoresis. The LAMP method is feasible for detection of Brucella spp. from blood and milk samples.

  7. 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.

  8. Rapid detection of microbial DNA by a novel isothermal genome exponential amplification reaction (GEAR) assay.

    PubMed

    Prithiviraj, Jothikumar; Hill, Vincent; Jothikumar, Narayanan

    2012-04-20

    In this study we report the development of a simple target-specific isothermal nucleic acid amplification technique, termed genome exponential amplification reaction (GEAR). Escherichia coli was selected as the microbial target to demonstrate the GEAR technique as a proof of concept. The GEAR technique uses a set of four primers; in the present study these primers targeted 5 regions on the 16S rRNA gene of E. coli. The outer forward and reverse Tab primer sequences are complementary to each other at their 5' end, whereas their 3' end sequences are complementary to their respective target nucleic acid sequences. The GEAR assay was performed at a constant temperature 60 °C and monitored continuously in a real-time PCR instrument in the presence of an intercalating dye (SYTO 9). The GEAR assay enabled amplification of as few as one colony forming units of E. coli per reaction within 30 min. We also evaluated the GEAR assay for rapid identification of bacterial colonies cultured on agar media directly in the reaction without DNA extraction. Cells from E. coli colonies were picked and added directly to GEAR assay mastermix without prior DNA extraction. DNA in the cells could be amplified, yielding positive results within 15 min.

  9. 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).

  10. 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.

  11. An improved loop-mediated isothermal amplification assay for the detection of Mycoplasma bovis

    PubMed Central

    HIGA, Yumiko; UEMURA, Ryoko; YAMAZAKI, Wataru; GOTO, Shinya; GOTO, Yoshitaka; SUEYOSHI, Masuo

    2016-01-01

    We improved a loop-mediated isothermal amplification (LAMP) assay permitting sensitive and rapid Mycoplasma bovis detection. A total of 55 bacterial strains were examined in this study, including 33 M. bovis strains, 14 non-M. bovis mycoplasmas and eight non-mycoplasma bacterial strains. M. bovis was successfully detected by the LAMP assay within 60 min without cross-reaction to any other bacteria. Furthermore, a total of 135 nasal swab samples were tested directly using our LAMP assays, the previously reported LAMP assay, conventional PCR assay without pre-culture and comparing standard culture methods. The improved LAMP assay showed sensitivity and specificity of 97.2% and 90.9%, respectively (with a kappa coefficient of 0.8231), and the sensitivity of our revised LAMP assay was increased compared to existing methods. PMID:27109067

  12. Scanning tunneling microscopy of cauliflower-like DNA nanostructures synthesised by loop-mediated isothermal amplification.

    PubMed

    Gill, P; Ranjbar, B; Saber, R

    2011-03-01

    DNA nanotechnology is a novel approach for synthesis of DNA-based nanostructures. Stem-loops, nanojunctions, sticky-ends and periodic lengths of DNA are the most essential nanostructures in DNA nanofabrications. Loop-mediated isothermal amplification (LAMP) is a powerful technology for repetitive synthesis of double-stranded and cauliflower-like DNAs. The process leads to long and repetitive sequences of DNAs, which are fabricated via loop primers. The authors demonstrate here scanning tunneling micrographs of LAMP-synthesised DNAs deposited on highly ordered pyrolytic graphite. The scans are compared with natural DNAs. Scanning tunneling microscopy (STM) images indicated the creation of periodic long DNAs, stem-looped DNAs and three-way DNA nanojunctions. It is also suggested that such nanomaterials could be promising candidates for use in DNA-based nanodevices.

  13. 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.

  14. 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.

  15. Rapid detection of Salmonella Typhi by loop-mediated isothermal amplification (LAMP) method.

    PubMed

    Abdullah, J; Saffie, N; Sjasri, F A R; Husin, A; Abdul-Rahman, Z; Ismail, A; Aziah, I; Mohamed, M

    2014-01-01

    An in-house loop-mediated isothermal amplification (LAMP) reaction was established and evaluated for sensitivity and specificity in detecting the presence of Salmonella Typhi (S. Typhi) isolates from Kelantan, Malaysia. Three sets of primers consisting of two outer and 4 inner were designed based on locus STBHUCCB_38510 of chaperone PapD of S. Typhi genes. The reaction was optimised using genomic DNA of S. Typhi ATCC7251 as the template. The products were visualised directly by colour changes of the reaction. Positive results were indicated by green fluorescence and negative by orange colour. The test was further evaluated for specificity, sensitivity and application on field samples. The results were compared with those obtained by gold standard culture method and Polymerase Chain Reaction (PCR). This method was highly specific and -10 times more sensitive in detecting S. Typhi compared to the optimised conventional polymerase chain reaction (PCR) method.

  16. 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.

  17. Loop-mediated isothermal amplification test for Trypanosoma vivax based on satellite repeat DNA.

    PubMed

    Njiru, Z K; Ouma, J O; Bateta, R; Njeru, S E; Ndungu, K; Gitonga, P K; Guya, S; Traub, R

    2011-08-25

    Trypanosoma vivax is major cause of animal trypanosomiasis and responsible for enormous economic burden in Africa and South America animal industry. T. vivax infections mostly run low parasitaemia with no apparent clinical symptoms, making diagnosis a challenge. This work reports the design and evaluation of a loop-mediated isothermal amplification (LAMP) test for detecting T. vivax DNA based on the nuclear satellite repeat sequence. The assay is rapid with results obtained within 35 min. The analytical sensitivity is ∼ 1 trypanosome/ml while that of the classical PCR tests ranged from 10 to 10(3)trypanosomes/ml. The T. vivax LAMP test reported here is simple, robust and has future potential in diagnosis of animal trypanosomiasis in the field.

  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.

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

    NASA Astrophysics Data System (ADS)

    Pourhassan-Moghaddam, Mohammad; Rahmati-Yamchi, Mohammad; Akbarzadeh, Abolfazl; Daraee, Hadis; Nejati-Koshki, Kazem; Hanifehpour, Younes; Joo, Sang Woo

    2013-11-01

    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.

  20. Real-time fluorescence loop-mediated isothermal amplification for the diagnosis of hemorrhagic enteritis virus.

    PubMed

    Liu, Xuemei; Li, Yuhao; Xu, Chenggang; Qin, Jianru; Hao, Jianyong; Feng, Min; Tan, Liqiang; Jia, Weixin; Liao, Ming; Cao, Weisheng

    2014-04-01

    Suspected cases of hemorrhagic enteritis associated with hemorrhagic enteritis virus (HEV) are becoming more frequent among yellow chickens in the Guangdong Province of China. In this study, we have developed a one-step, ecumenical, real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay for the rapid diagnosis of HEV. The RealAmp assay was performed at 63°C and reduced the assay time to 15min, using a simple and portable device, the ESE-Quant Tube Scanner. The detection limit of DNA was 1fg/μl, and the detection was specific only to HEV. We also used nested PCR to evaluate the application of the RealAmp assay. The coincidence rate of the two methods was 100%. Our data indicated that the RealAmp assay provides a sensitive, specific, and user-friendly diagnostic tool for the identification and quantification of HEV for field diagnosis and in laboratory research.

  1. Rapid detection of duck hepatitis virus type-1 by reverse transcription loop-mediated isothermal amplification.

    PubMed

    Song, Cuiping; Wan, Hongquan; Yu, Shengqing; Han, Xiangan; Qiu, Xusheng; Hu, Qinghai; Tan, Lei; Ding, Chan

    2012-06-01

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of duck hepatitis virus type-1 (DHV-1) was established. Using primers specific to the highly conserved 3D gene of DHV-1, the developed RT-LAMP assay detected the viral RNA of DHV-1 extracted from both allantoic fluid and liver samples of infected ducks. The assay is as sensitive as RT-PCR, and shows no cross-reaction with other common avian viral and bacterial pathogens. In addition to detection via ethidium bromide staining following gel electrophoresis, naked-eye observation after staining with SYBR Green I dye can be used to detect RT-LAMP products; this enables field application of this assay. The findings demonstrate that RT-LAMP can serve as a helpful tool for the detection and surveillance of DHV-1 in the poultry industry.

  2. 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.

  3. Development of loop-mediated isothermal amplification assay for detection of human coronavirus-NL63.

    PubMed

    Pyrc, Krzysztof; Milewska, Aleksandra; Potempa, Jan

    2011-07-01

    Human coronavirus NL63 was identified in 2004 in the Netherlands. Due to the high prevalence and world-wide distribution of this pathogen, it is essential to develop a sensitive and specific detection assay suitable for use in a routine diagnostic laboratory. Techniques based on PCR or real-time PCR are laborious and expensive. Detailed analysis of the HCoV-NL63 genome permitted the identification of a conserved nucleic acid sequential motif, which was sufficient for the design of a loop-mediated isothermal amplification (LAMP) assay. Evaluation of the method showed that the test is specific to HCoV-NL63 and that it does not cross-react with other respiratory viruses. The detection limit was found to be 1 copy of RNA template per reaction in cell culture supernatants and clinical specimens.

  4. Rapid and sensitive diagnosis of Acanthamoeba keratitis by loop-mediated isothermal amplification.

    PubMed

    Ge, Z; Qing, Y; Zicheng, S; Shiying, S

    2013-11-01

    A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of Acanthamoeba. The sensitivity of the LAMP assay was tested using different copies of positive DNA. The specificity of the assay was tested using DNA extracted from Acanthamoeba, Pseudomonas aeruginosa, Candida albicans, herpes simplex virus-1 and human corneal epithelial cells. Its effectiveness was evaluated and compared with culture, corneal smear examination and real-time PCR in corneal samples from mice with Acanthamoeba keratitis. We also tested three corneal samples from patients with suspected Acanthamoeba or fungal infection using LAMP. Loop-mediated isothermal amplification was confirmed to be very sensitive, with the lowest detection limit being ten copies/tube of Acanthamoeba DNA. The LAMP primers only amplified Acanthamoeba DNA. During the development of Acanthamoeba keratitis in mice, almost all of the positive rates of LAMP at each time post-infection were higher than those of culture or corneal smear examination. The total positive rate of LAMP was significantly higher than those of culture and corneal smear examination (p <0.05), whereas the sensitivities of LAMP and real-time PCR were comparable. However, the trends of positive change in these different test methods were generally similar. Of the three clinical corneal specimens, two with suspected Acanthamoeba keratitis tested positive for Acanthamoeba using LAMP along with culture or corneal smear examination, whereas the other suspected fungal keratitis tested negative. The LAMP assay is a simple, rapid, highly specific and sensitive method for the diagnosis of keratitis caused by Acanthamoeba.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. Rapid detection of rabies virus by reverse transcription loop-mediated isothermal amplification.

    PubMed

    Boldbaatar, Bazartseren; Inoue, Satoshi; Sugiura, Naoko; Noguchi, Akira; Orbina, Jun Ryan C; Demetria, Catalino; Miranda, Mary Elizabeth; Yamada, Akio

    2009-05-01

    In this study, reverse transcription loop-mediated isothermal amplification (RT-LAMP) was established which can detect 10(3) copies of viral RNA corresponding to approximately 5 fg of RNA. RT-LAMP with the Phil primer set designed according to the nucleotide sequences obtained from a Kyoto patient who contracted rabies in the Philippines was able to amplify all 16 street viral sequences derived from the Philippines. The specificity of RT-LAMP products was easily confirmed by digestion with RsaI restriction enzyme. The reaction of RT-LAMP could be completed within 1 h and could be conducted under isothermal conditions using a conventional water bath or heat blocks, indicating that RT-LAMP is ideal for the diagnosis of rabies in developing countries. Although further study is required to establish more universal RT-LAMP primers applicable to viruses from other regions or countries, the fast, easy, simple, sensitive and specific RT-LAMP method established here might be useful for rabies diagnosis and can facilitate studies of rabies epidemiology where rabies is enzootic, particularly in developing countries.

  10. Species and sex identification of Formosa landlocked salmon using loop-mediated isothermal amplification.

    PubMed

    Hsu, Te-Hua; Adiputra, Yudha T; Ohta, Hiromi; Gwo, Jin-Chywan

    2011-09-01

    Species and sex identification are among the most important parameters for conservation management. However, it is extremely difficult to perform such identification in Formosa landlocked salmon (Oncorhynchus masou formosanus). Both sexual dimorphism in landlocked dwarf form Formosa landlocked salmon and morphological difference among cherry salmon complex are minimal. We developed a simple, rapid and noninvasive method for identifying sex and species of this critically endangered species using a loop-mediated isothermal amplification (LAMP) technique. The LAMP assay showed the advantage of simple detection (evaluated by visual inspection), rapid reaction time (< 1 h), isothermal condition (less equipment required) and high efficiency (only 0.5-5 pg of DNA was required in the reaction mixture). Therefore, the method is more economical and practical than PCR. The LAMP assay can be easily performed in the field and is a valuable tool for detecting sex ratios in wild populations and identifying species in commercial imports. This is the first application of LAMP in identifying species and sex of salmonids as far as we know and clearly shows the potential application of LAMP in molecular ecology and conservation efforts.

  11. Detection of Pathogen-Specific Antibodies by Loop-Mediated Isothermal Amplification

    PubMed Central

    Burbulis, Ian E.; Yamaguchi, Kumiko; Nikolskaia, Olga V.; Prigge, Sean T.; Magez, Stefan; Bisser, Sylvie; Reller, Megan E.

    2015-01-01

    Loop-mediated isothermal amplification (LAMP) is a method for enzymatically replicating DNA that has great utility for clinical diagnosis at the point of care (POC), given its high sensitivity, specificity, speed, and technical requirements (isothermal conditions). Here, we adapted LAMP for measuring protein analytes by creating a protein-DNA fusion (referred to here as a “LAMPole”) that attaches oligonucleotides (LAMP templates) to IgG antibodies. This fusion consists of a DNA element covalently bonded to an IgG-binding polypeptide (protein L/G domain). In our platform, LAMP is expected to provide the most suitable means for amplifying LAMPoles for clinical diagnosis at the POC, while quantitative PCR is more suitable for laboratory-based quantification of antigen-specific IgG abundance. As proof of concept, we measured serological responses to a protozoan parasite by quantifying changes in solution turbidity in real time. We observed a >6-log fold difference in signal between sera from vaccinated versus control mice and in a clinical patient sample versus a control. We assert that LAMPoles will be useful for increasing the sensitivity of measuring proteins, whether it be in a clinical laboratory or in a field setting, thereby improving acute diagnosis of a variety of infections. PMID:25651920

  12. Endpoint visual detection of three genetically modified rice events by loop-mediated isothermal amplification.

    PubMed

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

    2012-11-07

    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.

  13. 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.

  14. 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

  15. 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

  16. Isothermal amplification coupled with rapid flow-through hybridisation for sensitive diagnosis of Plum pox virus.

    PubMed

    Olmos, Antonio; Bertolini, Edson; Cambra, Mariano

    2007-01-01

    A nucleic acid sequence-based amplification method coupled with rapid flow-through hybridisation (NASBA-FH) was developed for diagnosis of Plum pox virus (PPV). The sensitivity level achieved by NASBA-FH was 10 times higher than that obtained by Co-PCR and 1000 times higher than the sensitivity afforded by RT-PCR. In addition, samples from 262 stone-fruit trees collected during winter and spring seasons were analysed. These samples were tested using methods recommended by the European and Mediterranean Plant Protection Organization to detect PPV (DASI-ELISA, RT-PCR and Co-PCR) and by NASBA-FH. Winter PPV diagnostic results by ELISA and NASBA-FH coincided in 90.8%, while ELISA and PCR-based methods coincided in 91.6% and PCR-based methods with NASBA-FH agreed in 95.4%. In spring, diagnostic results were similar with all the molecular techniques, which agreed with ELISA results for 98.8% of the trees. NASBA-FH was able to detect more positive infections in winter, which were later confirmed in spring. These results indicate that NASBA-FH is a suitable molecular method for routine PPV detection in the winter and spring. This user-friendly isothermal RNA amplification coupled with a very fast flow-through hybridisation (15 min) opens up new possibilities for rapid and reliable diagnosis of a variety of pathogens.

  17. Genotyping of the CCR5 chemokine receptor by isothermal NASBA amplification and differential probe hybridization.

    PubMed

    Romano, J W; Tetali, S; Lee, E M; Shurtliff, R N; Wang, X P; Pahwa, S; Kaplan, M H; Ginocchio, C C

    1999-11-01

    The human CCR5 chemokine receptor functions as a coreceptor with CD4 for infection by macrophage-tropic isolates of human immunodeficiency virus type 1 (HIV-1). A mutated CCR5 allele which encodes a protein that does not function as a coreceptor for HIV-1 has been identified. Thus, expression of the wild-type and/or mutation allele is relevant to determining the infectability of patient peripheral blood mononuclear cells (PBMC) and affects disease progression in vivo. We developed a qualitative CCR5 genotyping assay using NASBA, an isothermal nucleic acid amplification technology. The method involves three enzymes and two oligonucleotides and targets the CCR5 mRNA, which is expressed in PBMC at a copy number higher than 2, the number of copies of DNA present encoding the gene. The single oligonucleotide set amplifies both alleles, and genotyping is achieved by separate hybridizations of wild-type- and mutation-specific probes directly to the single-stranded RNA amplification product. Assay sensitivity and specificity were demonstrated with RNAs produced in vitro from plasmid clones bearing the DNA encoding each allele. No detectable cross-reactivity between wild-type and mutation probes was found, and 50 copies of each allele were readily detectable. Analysis of patient samples found that 20% were heterozygous and 1% were homozygous for the CCR5 mutation. Thus, NASBA is a sensitive and specific means of rapidly determining CCR5 genotype and provides several technical advantages over alternative assay systems.

  18. Genotyping of the CCR5 Chemokine Receptor by Isothermal NASBA Amplification and Differential Probe Hybridization

    PubMed Central

    Romano, Joseph W.; Tetali, Surya; Lee, Eun Mi; Shurtliff, Roxanne N.; Wang, Xue Ping; Pahwa, Savita; Kaplan, Mark H.; Ginocchio, Christine C.

    1999-01-01

    The human CCR5 chemokine receptor functions as a coreceptor with CD4 for infection by macrophage-tropic isolates of human immunodeficiency virus type 1 (HIV-1). A mutated CCR5 allele which encodes a protein that does not function as a coreceptor for HIV-1 has been identified. Thus, expression of the wild-type and/or mutation allele is relevant to determining the infectibility of patient peripheral blood mononuclear cells (PBMC) and affects disease progression in vivo. We developed a qualitative CCR5 genotyping assay using NASBA, an isothermal nucleic acid amplification technology. The method involves three enzymes and two oligonucleotides and targets the CCR5 mRNA, which is expressed in PBMC at a copy number higher than 2, the number of copies of DNA present encoding the gene. The single oligonucleotide set amplifies both alleles, and genotyping is achieved by separate hybridizations of wild-type- and mutation-specific probes directly to the single-stranded RNA amplification product. Assay sensitivity and specificity were demonstrated with RNAs produced in vitro from plasmid clones bearing the DNA encoding each allele. No detectable cross-reactivity between wild-type and mutation probes was found, and 50 copies of each allele were readily detectable. Analysis of patient samples found that 20% were heterozygous and 1% were homozygous for the CCR5 mutation. Thus, NASBA is a sensitive and specific means of rapidly determining CCR5 genotype and provides several technical advantages over alternative assay systems. PMID:10548593

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. Ultrasensitive and rapid detection of β-conglutin combining aptamers and isothermal recombinase polymerase amplification.

    PubMed

    Jauset-Rubio, Miriam; Sabaté Del Río, Jonathan; Mairal, Teresa; Svobodová, Markéta; El-Shahawi, Mohammad S; Bashammakh, Abdulaziz S; Alyoubi, Abdulrahman O; O'Sullivan, Ciara K

    2017-01-01

    Lupin is increasingly being used in a variety of food products due to its nutritional, functional and nutraceutical properties. However, several examples of severe and even fatal food-associated anaphylaxis due to lupin inhalation or ingestion have been reported, resulting in the lupin subunit β-conglutin, being defined as the Lup an 1 allergen by the International Union of Immunological Societies (IUIS) in 2008. Here, we report an innovative method termed aptamer-recombinase polymerase amplification (Apta-RPA) exploiting the affinity and specificity of a DNA aptamer selected against the anaphylactic β-conglutin allergen termed β-conglutin binding aptamer II (β-CBA II), facilitating ultrasensitive detection via isothermal amplification. Combining magnetic beads as the solid phase with Apta-RPA detection, the total assay time was reduced from 210 min to just 25 min, with a limit of detection of 3.5 × 10(-11) M, demonstrating a rapid and ultrasensitive generic methodology that can be used with any aptamer. Future work will focus on further simplification of the assay to a lateral flow format. Graphical Abstract Schematic representation of the rapid and novel bead-based Apta-RPA assay.

  4. Development of a loop-mediated isothermal amplification method for detecting virulent Rhodococcus equi.

    PubMed

    Kinoshita, Yuta; Niwa, Hidekazu; Higuchi, Tohru; Katayama, Yoshinari

    2016-09-01

    Rhodococcus equi is the most important causative bacterium of severe pneumonia in foals. We report herein the development of a specific loop-mediated isothermal amplification (LAMP) assay, which targets a gene encoding vapA for detecting virulent R. equi The detection limit of the LAMP assay was 10(4) colony forming units (CFU)/mL, which was equal to 10 CFU/reaction. The clinical efficacy of the LAMP assay was compared with those of 2 published PCR-based methods: nested PCR and quantitative real-time (q)PCR. Agreements between bacterial culture, which is the gold standard for detection of R. equi, and each of the 3 molecular tests were measured by calculating a kappa coefficient. The kappa coefficients of the LAMP (0.760), nested PCR (0.583), and qPCR (0.888) indicated substantial agreement, moderate agreement, and almost perfect agreement, respectively. Although the clinical efficacy of LAMP was not the best among the 3 methods tested, LAMP could be more easily introduced into less well-equipped clinics because it does not require special equipment (such as a thermocycler) for gene amplification. Veterinary practitioners could diagnose R. equi pneumonia more quickly by using LAMP and could use the results to select an appropriate initial treatment.

  5. 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.

  6. Loop-mediated isothermal amplification (LAMP): A novel rapid detection platform for pathogens.

    PubMed

    Li, Yanmei; Fan, Penghui; Zhou, Shishui; Zhang, Li

    2017-03-18

    Foodborne bacterial infections and diseases have been considered to be a major threat for public health in the worldwide. Increased incidence of human diseases caused by foodborne pathogens have been correlated with growing world population and mobility. Loop-mediated isothermal amplification (LAMP) has been regarded as an innovative gene amplification technology and emerged as an alternative to PCR-based methodologies in both clinical laboratory and food safety testing. Nowadays, LAMP has been applied to detection and identification on pathogens from microbial diseases, as it showed significant advantage in high sensitivity, specificity and rapidity. The high sensitivity of LAMP enables detection of the pathogens in sample materials even without time consuming sample preparation. An overview of LAMP mainly containing the development history, reaction principle and its application to four kind of foodborne pathogens detection are presented in this paper. As concluded, with the advantages of rapidity, simplicity, sensitivity, specificity and robustness, LAMP is capable of applications for clinical diagnosis as well as surveillance of infection diseases. Moreover, the main purpose of this paper is to provide theoretical basis for the clinical application of LAMP technology.

  7. Loop-Mediated Isothermal Amplification and LFD Combination for Detection of Plasmodium falciparum and Plasmodium vivax.

    PubMed

    Kongkasuriyachai, Darin; Yongkiettrakul, Suganya; Kiatpathomchai, Wansika; Arunrut, Narong

    2017-01-01

    Loop-mediated isothermal amplification (LAMP) has been used to detect several pathogens including malaria parasites from field and clinical samples. In this protocol, the malaria LAMP technology is developed to differentiate between Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) species by targeting the dihydrofolate reductase thymidylate synthase (dhfr-ts) gene, a known target for the antifolate class of drugs such as Pyrimethamine. LAMP primer sets are designed and validated for species specific amplification. Additionally, specific probes help improve detection and visualization of the products when combined with lateral flow dipstick-based (LFD) detection. The protocols are further simplified to eliminate tedious sample preparation steps, such that crude lysis prepared simply by diluting few microliter (μL) of blood sample with distilled water is sufficient. The LAMP-LFD malaria dhfr-ts protocols are sensitive and can detect as little as 1 picogram (pg) of PfDNA and 1 nanogram (ng) of PvDNA, or a few microliters of crude lysate from infected blood samples (Yongkiettrakul et al., Parasitol Int 63: 777-784, 2014). These simplified steps not only reduce cost but also increase the potential for large application in the fields and clinical settings.

  8. 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

    2016-08-03

    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.

  9. 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.

  10. [Establishment of loop-mediated isothermal amplification technique for rapid detection of NDM-1 gene].

    PubMed

    Zhang, Yuanyi; Wu, Na; Zhu, Baoli; Chen, Lei; Zhu, Yuzhuo

    2011-08-01

    We established a rapid detection method of New Delhi Metallo-beta-Lactamase Gene (NDM-1) based on Loop-mediated Isothermal Amplification (LAMP). With the application of LAMP, we designed four sets of LAMP premiers, using NDM-1 gene as the target sequence, and selected the set of optimal primers. Meanwhile, we established optimal reaction systems and conditions to carry out the sensitivity and specificity experiments. The experiment results showed that the whole detection process took only one hour and could be observed visually. In the experiment of sensitivity, NDM-1 gene had a detection limit of 6 copies in each reaction. In the experiment of specificity, we detected NDM-1 gene in 4 pathogen strains (Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae), and the total DNA from intestinal microbes and the total DNA from soil microbes. We had not detected the amplification reactions. The detection method established could rapidly detect NDM-1 gene and visualize the experiment result. The method is easy to operate and has high sensitivity and specificity and thus has great application value in basic research laboratories, emergent detection and spot detection.

  11. 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

  12. An integrated portable hand-held analyser for real-time isothermal nucleic acid amplification.

    PubMed

    Smith, Matthew C; Steimle, George; Ivanov, Stan; Holly, Mark; Fries, David P

    2007-08-29

    A compact hand-held heated fluorometric instrument for performing real-time isothermal nucleic acid amplification and detection is described. The optoelectronic instrument combines a Printed Circuit Board/Micro Electro Mechanical Systems (PCB/MEMS) reaction detection/chamber containing an integrated resistive heater with attached miniature LED light source and photo-detector and a disposable glass waveguide capillary to enable a mini-fluorometer. The fluorometer is fabricated and assembled in planar geometry, rolled into a tubular format and packaged with custom control electronics to form the hand-held reactor. Positive or negative results for each reaction are displayed to the user using an LED interface. Reaction data is stored in FLASH memory for retrieval via an in-built USB connection. Operating on one disposable 3 V lithium battery >12, 60 min reactions can be performed. Maximum dimensions of the system are 150 mm (h) x 48 mm (d) x 40 mm (w), the total instrument weight (with battery) is 140 g. The system produces comparable results to laboratory instrumentation when performing a real-time nucleic acid sequence-based amplification (NASBA) reaction, and also displayed comparable precision, accuracy and resolution to laboratory-based real-time nucleic acid amplification instrumentation. A good linear response (R2 = 0.948) to fluorescein gradients ranging from 0.5 to 10 microM was also obtained from the instrument indicating that it may be utilized for other fluorometric assays. This instrument enables an inexpensive, compact approach to in-field genetic screening, providing results comparable to laboratory equipment with rapid user feedback as to the status of the reaction.

  13. 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

  14. Development of one-step Loop-Mediated Isothermal Amplification (LAMP) for the detection of norovirus in oysters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this study was to develop a simple and rapid technique for detecting human norovirus (NoV). The loop-mediated isothermal amplification (LAMP) technique was evaluated and found to be sensitive, highly specific, and useful for routine oyster testing. Reverse transcription-LAMP (RT-LAMP) pri...

  15. Development of a Loop-Mediated Isothermal Amplification Assay Targeting the mpb64 Gene for Diagnosis of Intraocular Tuberculosis

    PubMed Central

    Balne, Praveen Kumar; Barik, Manas Ranjan; Sharma, Savitri

    2013-01-01

    A loop-mediated isothermal amplification (LAMP) assay targeting the mpb64 gene for the diagnosis of intraocular tuberculosis was highly specific (100%), sensitive (85.7%), rapid, and easy to perform. The LAMP assay can be an alternative to conventional PCR for the diagnosis of ocular tuberculosis in resource-limited settings. PMID:23966513

  16. 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 ...

  17. A loop-mediated isothermal amplification assay and sample preparation procedure for sensitive detection of Xanthomonas fragariae in strawberry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Xanthomonas fragariae is a bacterium that causes angular leaf spot of strawberry. Asymptomatic infections are common and contribute to the difficulties in disease management. The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay with a bacterial enrichment proced...

  18. A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods.

    PubMed

    Nixon, Gavin J; Svenstrup, Helle F; Donald, Carol E; Carder, Caroline; Stephenson, Judith M; Morris-Jones, Stephen; Huggett, Jim F; Foy, Carole A

    2014-12-01

    Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR). There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These 'isothermal' methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT), akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP) assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

  19. Loop-mediated isothermal amplification: rapid detection of Angiostrongylus cantonensis infection in Pomacea canaliculata

    PubMed Central

    2011-01-01

    Background Angiostrongylus cantonensis is a zoonotic parasite that causes eosinophilic meningitis in humans. The most common source of infection with A. cantonensis is the consumption of raw or undercooked mollusks (e.g., snails and slugs) harbouring infectious third-stage larvae (L3). However, the parasite is difficult to identify in snails. The purpose of this study was to develop a quick, simple molecular method to survey for A. cantonensis in intermediate host snails. Findings We used a loop-mediated isothermal amplification (LAMP) assay, which was performed using Bst DNA polymerase. Reactions amplified the A. cantonensis 18S rRNA gene and demonstrated high sensitivity; as little as 1 fg of DNA was detected in the samples. Furthermore, no cross-reactivity was found with other parasites such as Toxoplasma gondii, Plasmodium falciparum, Schistosoma japonicum, Clonorchis sinensis, Paragonimus westermani and Anisakis. Pomacea canaliculata snails were exposed to A. cantonensis first-stage larvae (L1) in the laboratory, and L3 were observed in the snails thirty-five days after infection. All nine samples were positive as determined by the LAMP assay for A. cantonensis, which was identified as positive by using PCR and microscopy, this demonstrates that LAMP is sensitive and effective for diagnosis. Conclusions LAMP is an appropriate diagnostic method for the routine identification of A. cantonensis within its intermediate host snail P. canaliculata because of its simplicity, sensitivity, and specificity. It holds great promise as a useful monitoring tool for A. cantonensis in endemic regions. PMID:22023992

  20. 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

  1. Loop-mediated isothermal amplification targeting 18S ribosomal DNA for rapid detection of Acanthamoeba.

    PubMed

    Yang, Hye-Won; Lee, Yu-Ran; Inoue, Noboru; Jha, Bijay Kumar; Danne, Dinzouna-Boutamba Sylvatrie; Kim, Hong-Kyun; Lee, Junhun; Goo, Youn-Kyoung; Kong, Hyun-Hee; Chung, Dong-Il; Hong, Yeonchul

    2013-06-01

    Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner.

  2. Development of a Loop Mediated Isothermal Amplification for Diagnosis of Ascaris lumbricoides in Fecal Samples

    PubMed Central

    Eric, Agola L.; Mwangi, Ibrahim N.; Maina, Geoffrey M.; Kinuthia, Joseph M.; Mutuku, Martin W.; Mugambi, Robert M.; Mwandi, Jackson M.; Mkoji, Gerald M.

    2016-01-01

    Ascaris lumbricoides is a nematode parasite that causes the common tropical infection ascariasis in humans. It is also considered among the neglected tropical diseases. Diagnosis relies mainly on microscopy-based methods which are laborious, are limited by low sensitivity, and require high expertise. We have developed a loop mediated isothermal amplification (LAMP) for diagnosis of ascariasis in fecal samples, based on the first internal transcribed (ITS-1) spacer region of the ribosomal DNA. We used Primer Explorer V4 software to design primers. Ascaris adult and ova were obtained from naturally infected school children, whose parents/guardians gave consent for their participation in the study. Genomic DNA was extracted using alkaline lysis method and amplified by LAMP at 63°C for 45 minutes. LAMP products were visualized by naked eyes after adding SYBR Green dye and also on agarose gel. LAMP successfully and reliably detected Ascaris DNA from a single egg and in fecal samples. The assay specifically detected Ascaris DNA without amplifying DNA from ova of other parasites which commonly coexist with A. lumbricoides in feces. The developed LAMP assay has great potential for use in ascariasis diagnosis at the point of care and in low infection intensity situation that characterize control and elimination campaigns. PMID:27882242

  3. Development of a Loop Mediated Isothermal Amplification for Diagnosis of Ascaris lumbricoides in Fecal Samples.

    PubMed

    Shiraho, Esther A; Eric, Agola L; Mwangi, Ibrahim N; Maina, Geoffrey M; Kinuthia, Joseph M; Mutuku, Martin W; Mugambi, Robert M; Mwandi, Jackson M; Mkoji, Gerald M

    2016-01-01

    Ascaris lumbricoides is a nematode parasite that causes the common tropical infection ascariasis in humans. It is also considered among the neglected tropical diseases. Diagnosis relies mainly on microscopy-based methods which are laborious, are limited by low sensitivity, and require high expertise. We have developed a loop mediated isothermal amplification (LAMP) for diagnosis of ascariasis in fecal samples, based on the first internal transcribed (ITS-1) spacer region of the ribosomal DNA. We used Primer Explorer V4 software to design primers. Ascaris adult and ova were obtained from naturally infected school children, whose parents/guardians gave consent for their participation in the study. Genomic DNA was extracted using alkaline lysis method and amplified by LAMP at 63°C for 45 minutes. LAMP products were visualized by naked eyes after adding SYBR Green dye and also on agarose gel. LAMP successfully and reliably detected Ascaris DNA from a single egg and in fecal samples. The assay specifically detected Ascaris DNA without amplifying DNA from ova of other parasites which commonly coexist with A. lumbricoides in feces. The developed LAMP assay has great potential for use in ascariasis diagnosis at the point of care and in low infection intensity situation that characterize control and elimination campaigns.

  4. Detection of canine distemper virus in blood samples by reverse transcription loop-mediated isothermal amplification.

    PubMed

    Cho, H S; Park, N Y

    2005-11-01

    Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was used to detect canine distemper virus (CDV) genomic RNA. A set of four primers, two outer and two inner, were designed from CDV genomic RNA targeting the nucleocapsid protein gene. The optimal reaction time and temperature for LAMP were determined to be 60 min at 65 degrees C. The relative sensitivity and specificity of RT-LAMP was found to be 100% and 93.3%, respectively, based on 50 canine blood samples and using RT-PCR as the gold standard. The detection limit of the RT-LAMP method was 100 times lower than with RT-PCR (10-1TCID50 ml(-1) versus 10TCID50 ml(-1)). In addition to the advantage resulting from the visual detection of the end-product, the LAMP method is fast, requiring only 1 h to complete the assay. The LAMP method is a viable alternative to RT-PCR for diagnosing CDV infection in dogs. The LAMP method might be useful as an on site diagnostic assay for detecting CDV.

  5. Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Acanthamoeba

    PubMed Central

    Yang, Hye-Won; Lee, Yu-Ran; Inoue, Noboru; Jha, Bijay Kumar; Danne, Dinzouna-Boutamba Sylvatrie; Kim, Hong-Kyun; Lee, Junhun; Goo, Youn-Kyoung; Kong, Hyun-Hee; Chung, Dong-Il

    2013-01-01

    Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner. PMID:23864737

  6. 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.

  7. Detection of Papaya leaf distortion mosaic virus by reverse-transcription loop-mediated isothermal amplification.

    PubMed

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

    2014-01-01

    Papaya leaf distortion mosaic virus (PLDMV) can infect transgenic papaya resistant to a related pathogen, Papaya ringspot virus (PRSV), posing a substantial threat to papaya production in China. Current detection methods, however, are unable to be used for rapid detection in the field. Here, a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of PLDMV, using a set of four RT-LAMP primers designed based on the conserved sequence of PLDMV CP. The RT-LAMP method detected specifically PLDMV and was highly sensitive, with a detection limit of 1.32×10(-6) μg of total RNA per reaction. Indeed, the reaction was 10 times more sensitive than one-step RT-PCR, while also requiring significantly less time and equipment. The effectiveness of RT-LAMP and one-step RT-PCR in detecting the virus were compared using 90 field samples of non-transgenic papaya and 90 field samples of commercialized PRSV-resistant transgenic papaya from Hainan Island. None of the non-transgenic papaya tested positive for PLDMV using either method. In contrast, 19 of the commercialized PRSV-resistant transgenic papaya samples tested positive by RT-LAMP assay, and 6 of those tested negative by RT-PCR. Therefore, the PLDMV-specific RT-LAMP is a simple, rapid, sensitive, and cost-effective tool in the field diagnosis and control of PLDMV.

  8. Loop-Mediated Isothermal Amplification (LAMP): Emergence As an Alternative Technology for Herbal Medicine Identification

    PubMed Central

    Li, Jing-jian; Xiong, Chao; Liu, Yue; Liang, Jun-song; Zhou, Xing-wen

    2016-01-01

    Correct identification of medicinal plant ingredients is essential for their safe use and for the regulation of herbal drug supply chain. Loop-mediated isothermal amplification (LAMP) is a recently developed approach to identify herbal medicine species. This novel molecular biology technique enables timely and accurate testing, especially in settings where infrastructures to support polymerase chain reaction facilities are lacking. Studies that used this method have altered our view on the extent and complexity of herbal medicine identification. In this review, we give an introduction into LAMP analysis, covers the basic principles and important aspects in the development of LAMP analysis method. Then we presented a critical review of the application of LAMP-based methods in detecting and identifying raw medicinal plant materials and their processed products. We also provide a practical standard operating procedure (SOP) for the utilization of the LAMP protocol in herbal authentication, and consider the prospects of LAMP technology in the future developments of herbal medicine identification and the challenges associated with its application. PMID:28082999

  9. 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.

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

    PubMed

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

    2014-09-03

    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.

  11. 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.

  12. Loop-mediated isothermal amplification method for differentiation and rapid detection of Taenia species.

    PubMed

    Nkouawa, Agathe; Sako, Yasuhito; Nakao, Minoru; Nakaya, Kazuhiro; Ito, Akira

    2009-01-01

    Rapid detection and differentiation of Taenia species are required for the control and prevention of taeniasis and cysticercosis in areas where these diseases are endemic. Because of the lower sensitivity and specificity of the conventional diagnosis based on microscopical examination, molecular tools are more reliable for differential diagnosis of these diseases. In this study, we developed and evaluated a loop-mediated isothermal amplification (LAMP) assay for differential diagnosis of infections with Taenia species with cathepsin L-like cysteine peptidase (clp) and cytochrome c oxidase subunit 1 (cox1) genes. LAMP with primer sets to the cox1 gene could differentiate between three species, and LAMP with primer sets to the clp gene could differentiate Taenia solium from Taenia saginata/Taenia asiatica. Restriction enzyme digestion of the LAMP products from primer set Tsag-clp allowed the differentiation of Taenia saginata from Taenia asiatica. We demonstrated the high specificity of LAMP by testing known parasite DNA samples extracted from proglottids (n = 100) and cysticerci (n = 68). LAMP could detect one copy of the target gene or five eggs of T. asiatica and T. saginata per gram of feces, showing sensitivity similar to that of PCR methods. Furthermore, LAMP could detect parasite DNA in all taeniid egg-positive fecal samples (n = 6). Due to the rapid, simple, specific, and sensitive detection of Taenia species, the LAMP assays are valuable tools which might be easily applicable for the control and prevention of taeniasis and cysticercosis in countries where these diseases are endemic.

  13. 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.

  14. Rapid detection of BoHV-1 genomic DNA by loop-mediated isothermal amplification assay.

    PubMed

    El-Kholy, Alaa A; Abdelrahman, Khaled; Soliman, Hatem

    2014-08-01

    Bovine herpes virus-1 (BoHV-1) is a serious viral pathogen of domestic and wild cattle. Herein, we report development of a new molecular diagnostic assay for rapid and sensitive detection of BoHV-1 utilizing the loop-mediated isothermal amplification (LAMP) technique. BoHV-1-LAMP assay was optimized to amplify the target DNA by incubation the Bst-DNA polymerase enzyme with a set of specially constructed six primers, based on the gE-gene of BoHV-1 virus, at 65°C for 60min. BoHV-1-LAMP products were detected by visual inspection using SYBR Green-I stain and had a ladder-like appearance by gel electrophoresis analysis. Negative results obtained with DNA from other tested fish viruses confirmed the specificity of the assay. The analytical sensitivity of the BoHV-1-LAMP assay was 1fg of BoHV-1 DNA (dilution of 10(6)). The developed assay could successfully detect BoVH-1 DNA from clinical samples. Results of this study indicate that the developed BoHV-1-LAMP is rapid and highly sensitive assay not only for detection of BoHV-1 in clinical samples, but also for differentiation between wild-type (gE-positive) and gE-negative BoHV-1 viruses, which will improve the control programs of BoHV-1 in Egypt.

  15. Ultrasensitive detection of mucin 1 biomarker by immuno-loop-mediated isothermal amplification.

    PubMed

    Cao, Hongmei; Fang, Xueen; Li, Haipeng; Li, Hua; Kong, Jilie

    2017-03-01

    Mucin 1 (MUC-1), a glycoprotein over-expressed on most malignant epithelial cell surfaces, such as colorectal, lung, prostate, pancreatic, ovarian, and bladder carcinomas, has been confirmed as a useful biomarker for the diagnosis of early cancers. Therefore, it is very important for early cancer diagnoses to develop the sensitive and specific detection approach of MUC-1. In this paper, we report a novel technique of aptamer-based Immuno-Loop-Mediated Isothermal Amplification (Im-LAMP) for the quantitative detection of MUC-1 by MUC-1 aptamer with high affinity for MUC-1. The cycle time is linearly dependent on the logarithm (log) of the concentration of mucin 1 ranging from 1.0 pM to 1.0 aM. We call this technique Im-LAMP, which is a novel method for the detection of low-abundance proteins with high sensitivity (a low detection limit of 120 MUC-1 molecules by calculation)and specificity. Finally, this approach is also successfully applied in the analysis of human blood serum samples. It also lays the foundation for the early diagnosis of different types of low-abundance cancer biomarkers.

  16. Detection of Brucellosis in Sika Deer ( Cervus nippon ) through Loop-mediated Isothermal Amplification (LAMP).

    PubMed

    Liu, Qianhong; Wei, Jie; Sun, Qingsong; Wang, Ben; Wang, Yuting; Hu, Ying; Wu, Wenrong

    2017-03-20

    Brucellosis (Brucella bovis) in sika deer ( Cervus nippon ) can cause enormous losses to stag breeding, especially in areas in which stag breeding has become an important industry. It also poses a threat to humans because it is a zoonotic disease. Use of the loop-mediated isothermal amplification (LAMP) assay has been poorly described in the diagnosis of brucellosis in deer. We developed a LAMP assay targeting the omp25 gene sequence to detect brucellosis (Brucella bovis) in sika deer. The reaction can be completed in 60 min at 63 C and, with a detection limit of 17 pg, it was more sensitive than conventional PCR, with its detection limit of 1.7 ng. No cross-reactivity was observed with four bacteria: Escherichia coli , Salmonella enterica subsp. enterica, Clostridium pasteurianum , and Pseudomonas aeruginosa . We used 263 samples of blood to evaluate the reaction. The percentage of agreement between LAMP and PCR reached 91%; relative specificity reached 87%, and relative sensitivity reached 100%. The results indicate LAMP can be a simple and rapid diagnostic tool for detecting brucellosis in sika deer, particularly in the field, where it is essential to control brucellosis in deer with a rapid and accurate diagnosis for removal of positive animals.

  17. 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.

  18. Rapid and visual Chlamydia trachomatis detection using loop-mediated isothermal amplification and hydroxynaphthol blue.

    PubMed

    Choopara, I; Arunrut, N; Kiatpathomchai, W; Dean, D; Somboonna, N

    2017-01-01

    We developed an assay comprising crude DNA lysis by simple heat treatment coupled loop-mediated isothermal amplification with hydroxynaphthol blue for Chlamydia trachomatis detection (petty patent pending), and evaluated the developed assay for its feasibility as a one-step point-of-care detection on 284 endocervical swab specimens from clinically symptomatic C. trachomatis and healthy subjects. This assay is sensitive to 0·04 pg of ompA, specific with six primers targeting C. trachomatis ompA region, rapid (45 min total assay time), inexpensive (approx. 3 USD/reaction), does not require sophisticated instrumentation, and has comparable assay effectiveness (95% specificity, 90-100% sensitivity) to bacterial DNA isolation by a commercial kit coupled with polymerase chain reaction and gel electrophoresis (98-100% specificity, 87-100% sensitivity) based on the clinical samples test. The test result could be read by naked eye through the colour change from violet (negative) to sky blue (positive) for C. trachomatis-infected specimens. Further, this assay uses all safe chemical reagents and is hence safe to the users.

  19. Attomolar Zika virus oligonucleotide detection based on loop-mediated isothermal amplification and AC susceptometry.

    PubMed

    Tian, Bo; Qiu, Zhen; Ma, Jing; Zardán Gómez de la Torre, Teresa; Johansson, Christer; Svedlindh, Peter; Strömberg, Mattias

    2016-12-15

    Because of the serological cross-reactivity among the flaviviruses, molecular detection methods, such as reverse-transcription polymerase chain reaction (RT-PCR), play an important role in the recent Zika outbreak. However, due to the limited sensitivity, the detection window of RT-PCR for Zika viremia is only about one week after symptom onset. By combining loop-mediated isothermal amplification (LAMP) and AC susceptometry, we demonstrate a rapid and homogeneous detection system for the Zika virus oligonucleotide. Streptavidin-magnetic nanoparticles (streptavidin-MNPs) are premixed with LAMP reagents including the analyte and biotinylated primers, and their hydrodynamic volumes are dramatically increased after a successful LAMP reaction. Analyzed by a portable AC susceptometer, the changes of the hydrodynamic volume are probed as Brownian relaxation frequency shifts, which can be used to quantify the Zika virus oligonucleotide. The proposed detection system can recognize 1 aM synthetic Zika virus oligonucleotide in 20% serum with a total assay time of 27min, which can hopefully widen the detection window for Zika viremia and is therefore promising in worldwide Zika fever control.

  20. 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.

  1. Reverse transcription loop-mediated isothermal amplification for sensitive and rapid detection of Korean sacbrood virus.

    PubMed

    Yoo, Mi-Sun; Noh, Jin-Hyeong; Yoon, Byoung-Su; Reddy, Kondreddy Eswar; Kweon, Chang-Hee; Jung, Suk-Chan; Kang, Seung-Won

    2012-12-01

    Sacbrood virus (SBV) is one of the most serious honeybee viruses. The virus causes failure to pupate and death in both larvae and adult bees. Recently, the Korean sacbrood virus (KSBV) caused great losses in Korean honeybee (Apis cerana) colonies. Although KSBV shows high homology with SBV strains, it has unique motifs and causes different symptoms. Therefore, a simple, sensitive and specific method for detecting KSBV is needed urgently. In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting KSBV using total RNA extracted from honeybees (A. cerana) infected with SBV. The LAMP and the polymerase chain reaction (PCR) methods were then compared for their ability to detect KSBV in clinical samples. The virus was detected in RT-LAMP reactions containing 10(3) copies of pBX-KSBV within 30min, which was comparable to RT-PCR. In addition, the LAMP was able to distinguish between KSBV and other closely-related SBV strains, indicating a high degree of specificity. This simple and sensitive RT-LAMP assay is a useful method for the rapid diagnosis of KSBV infection in honeybees.

  2. Reverse transcription-loop-mediated isothermal amplification for the detection of rodent coronaviruses.

    PubMed

    Hanaki, Ken-Ichi; Ike, Fumio; Hatakeyama, Rika; Hirano, Norio

    2013-02-01

    Mouse hepatitis virus (MHV) is one of the most prevalent viruses detected in laboratory mouse colonies. Enterotropic strains predominate in natural infections, and molecular techniques for the detection of MHV shedding in feces are powerful enough to diagnose active infections. A reverse transcription-loop-mediated isothermal amplification (RT-LAMP) technique was developed for the detection of rodent coronaviruses within 90 min. The specificity of this technique was confirmed by its ability to detect all 17 different strains of MHV and 6 strains of rat coronaviruses as well as its failure to detect human, bovine, and porcine coronaviruses nonspecifically. The sensitivity of RT-LAMP was 3.2-fold higher than that of reverse transcription-polymerase chain reaction (RT-PCR) and 31.6-fold lower than that of nested RT-PCR. An evaluation of the diagnostic performance of RT-LAMP performed in duplicate using mouse fecal specimens showed that the sensitivity and specificity with respect to nested RT-PCR were 85.7% and 100%, respectively. RT-LAMP assays would be suitable for monitoring active MHV infection in mouse colonies.

  3. 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.

  4. Rapid detection of the Klebsiella pneumoniae carbapenemase (KPC) gene by loop-mediated isothermal amplification (LAMP).

    PubMed

    Nakano, Ryuichi; Nakano, Akiyo; Ishii, Yoshikazu; Ubagai, Tsuneyuki; Kikuchi-Ueda, Takane; Kikuchi, Hirotoshi; Tansho-Nagakawa, Shigeru; Kamoshida, Go; Mu, Xiaoqin; Ono, Yasuo

    2015-03-01

    Klebsiella pneumoniae carbapenemases (KPC), which are associated with resistance to carbapenem, have recently spread worldwide and have become a global concern. It is necessary to detect KPC-producing organisms in clinical settings to be able to control the spread of this resistance. We have developed a loop-mediated isothermal amplification (LAMP) method for rapid detection of KPC producers. LAMP primer sets were designed to recognize the homologous regions of blaKPC-2 to blaKPC-17 and could amplify blaKPC rapidly. The specificity and sensitivity of the primers in the LAMP reactions for blaKPC detection were determined. This LAMP assay was able to specifically detect KPC producers at 68 °C, and no cross-reactivity was observed for other types of β-lactamase (class A, B, C, or D) producers. The detection limit for this assay was found to be 10(0) CFU per tube, in 25 min, which was 10-fold more sensitive than a PCR assay for blaKPC detection. Then, the sensitivity of the LAMP reactions for blaKPC detection in human specimens (sputum samples, urine samples, fecal samples and blood samples) was analyzed; it was observed that the LAMP assay had almost the same sensitivity in these samples as when using purified DNA. The LAMP assay is easy to perform and rapid. It may therefore be routinely applied for detection of KPC producers in the clinical laboratory.

  5. Diagnostic potential of multi-targeted LAMP (loop-mediated isothermal amplification) for osteoarticular tuberculosis.

    PubMed

    Sharma, Kusum; Sharma, Megha; Batra, Nitya; Sharma, Aman; Dhillon, Mandeep Singh

    2017-02-01

    Delay in diagnosing osteoarticular tuberculosis (OATB) contributes significantly to morbidity by causing disfiguration and neurological sequelae. The delay caused by conventional culture and the expertise and expense involved in other nucleic acid based tests, make LAMP (loop-mediated isothermal amplification) assay a favorable middle path. We evaluated LAMP assay using IS6110 and MPB64 for rapid diagnosis of OATB by comparing with IS6110 PCR and culture. LAMP assay was performed on 140 synovial fluid and pus samples (10 culture-positive proven cases, 80 culture-negative probable cases, and 50 negative controls) using three set of primer pairs each for IS6110 and MPB64. LAMP assay, using two-target approach, had an overall sensitivity and specificity of 90% and 100% in detecting OATB. Sensitivity of IS6110 PCR, IS6110 LAMP, and MPB64 LAMP was 80%, 100%, and 100%, respectively, for confirmed cases and 72.5%, 81.75%, and 86.25%, respectively, for probable cases. Six additional cases were picked using two-target approach. LAMP assay utilizing IS6110 and MPB64 is a cost-effective technique for an early and reliable diagnosis of OATB. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:361-365, 2017.

  6. Loop-Mediated Isothermal Amplification (LAMP): Emergence As an Alternative Technology for Herbal Medicine Identification.

    PubMed

    Li, Jing-Jian; Xiong, Chao; Liu, Yue; Liang, Jun-Song; Zhou, Xing-Wen

    2016-01-01

    Correct identification of medicinal plant ingredients is essential for their safe use and for the regulation of herbal drug supply chain. Loop-mediated isothermal amplification (LAMP) is a recently developed approach to identify herbal medicine species. This novel molecular biology technique enables timely and accurate testing, especially in settings where infrastructures to support polymerase chain reaction facilities are lacking. Studies that used this method have altered our view on the extent and complexity of herbal medicine identification. In this review, we give an introduction into LAMP analysis, covers the basic principles and important aspects in the development of LAMP analysis method. Then we presented a critical review of the application of LAMP-based methods in detecting and identifying raw medicinal plant materials and their processed products. We also provide a practical standard operating procedure (SOP) for the utilization of the LAMP protocol in herbal authentication, and consider the prospects of LAMP technology in the future developments of herbal medicine identification and the challenges associated with its application.

  7. 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

  8. 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.

  9. 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

    2011-11-09

    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.

  10. Lyophilized Visually Readable Loop-Mediated Isothermal Reverse Transcriptase Nucleic Acid Amplification Test for Detection Ebola Zaire RNA.

    PubMed

    Carter, Christoph; Akrami, Kevan; Hall, Drew; Smith, Davey; Aronoff-Spencer, Eliah

    2017-02-24

    Recent viral outbreaks highlight the need for reliable, yet broadly deployable diagnostics for detection of epidemic and emerging pathogens. In this study we designed and optimized methods to visually detect viral nucleic acid by isothermal amplification and SYBR dye intercalation. We designed and tested loop-mediated isothermal amplification (LAMP) primers and lyophilized reactions to optimize the detection of Zaire Ebola Virus (ZEBOV) and further evolved the LAMP platform to allow room-temperature storage for deployment in resource limited settings. Our results demonstrated excellent sensitivity and specificity for viral nucleic acid sequences with lower limits of detection of less than 100 copies. Moreover, lyophilized reaction mixtures retained activity for prolonged periods under dry conditions at room temperature. This approach offers a way for detection of emerging viruses in resource limited settings.

  11. 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.

  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.

  13. Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay

    PubMed Central

    Liu, Wei; Huang, Simo; Liu, Ningwei; Dong, Derong; Yang, Zhan; Tang, Yue; Ma, Wen; He, Xiaoming; Ao, Da; Xu, Yaqing; Zou, Dayang; Huang, Liuyu

    2017-01-01

    This study established a constant-temperature fluorescence quantitative detection method, combining loop-mediated isothermal amplification (LAMP) with molecular beacons. The advantages of LAMP are its convenience and efficiency, as it does not require a thermocycler and results are easily visualized by the naked eye. However, a major disadvantage of current LAMP techniques is the use of indirect evaluation methods (e.g., electrophoresis, SYBR Green I dye, precipitation, hydroxynaphthol blue dye, the turbidimetric method, calcein/Mn2+ dye, and the composite probe method), which cannot distinguish between the desired products and products of nonspecific amplification, thereby leading to false positives. Use of molecular beacons avoids this problem because molecular beacons produce fluorescence signals only when binding to target DNA, thus acting as a direct indicator of amplification products. Our analyses determined the optimal conditions for molecular beacons as an evaluation tool in LAMP: beacon length of 25–45 bp, beacon concentration of 0.6–1 pmol/μL, and reaction temperature of 60–65 °C. In conclusion, we validated a novel molecular beacon loop-mediated isothermal amplification method (MB-LAMP), realizing the direct detection of LAMP product. PMID:28059137

  14. 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.

  15. Development of a loop-mediated isothermal amplification assay for rapid detection of capripoxviruses.

    PubMed

    Das, Amaresh; Babiuk, Shawn; McIntosh, Michael T

    2012-05-01

    Sheep pox (SP), goat pox (GP), and lumpy skin disease (LSD), caused by capripoxviruses (CaPVs), are economically important diseases of sheep, goats, and cattle, respectively. Here, we report the development of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of CaPVs. LAMP primers were designed to target a conserved gene encoding the poly(A) polymerase small subunit (VP39) of CaPVs. Hydroxynaphthol blue (HNB) was incorporated to monitor assay progress by color change from violet when negative to sky blue when positive, and results were verified by agarose gel electrophoresis. The LAMP assay was shown to be highly specific for CaPVs, with no apparent cross-reactivity to other related viruses (near neighbors) or viruses that cause similar clinical signs (look-a-like viruses). The performance of LAMP was compared to that of a highly sensitive quantitative real-time PCR (qPCR) assay. LAMP and qPCR exhibited similar analytical sensitivities, with limits of detection of 3 and 8 viral genome copies, respectively. Diagnostic specificity was assessed on 36 negative specimens, including swabs and EDTA blood from control sheep, goats, and cattle. Diagnostic sensitivity was assessed on 275 specimens, including EDTA blood, swabs, and tissues from experimentally infected sheep, goats, and cattle. Overall agreement on diagnostic test results between the two assays was 90 to 95% for specificity and 89 to 100% for sensitivity. The LAMP assay described in this report is simple to use, inexpensive, highly sensitive, and particularly well suited for the diagnosis of capripox in less well equipped laboratories and in rural settings where resources are limited.

  16. 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.

  17. Loop-mediated isothermal amplification for rapid detection of Chinese sacbrood virus.

    PubMed

    Ma, Mingxiao; Ma, Chen; Li, Mng; Wang, Shuquan; Yang, Song; Wang, Shude

    2011-09-01

    Chinese sacbrood virus (CSBV) has emerged as an important etiologic agent of honeybee infections and is lethal for individual bees, even causing the collapse of entire colonies. Although diagnostic methods for CSBV have been established in many clinical laboratories, application of these methods is largely restricted by the apparatus needed to carry out the reaction and by cost, therefore a simpler and less expensive diagnostic method for CSBV infection is required. In this study a simple and inexpensive system is described that is based on the loop-mediated isothermal amplification (LAMP) assay. The LAMP and the polymerase chain reaction (PCR) methods were compared for their ability to detect CSBV in 31 clinical samples, in purified CSBV-LNQY strains or to be able to discriminate between cDNA samples from other viruses. The detection limit of the LAMP method was 1pg, showing that LAMP is as sensitive as reverse transcriptase (RT)-PCR for CSBV detection. In addition, no DNA band from other related viruses samples was amplified by either method, suggesting that this LAMP assay is as specific as RT-PCR for CSBV detection. All 31 clinical samples that were LAMP assay-positive were also amplified by RT-PCR, however the LAMP assay was faster, more cost effective, and easier to perform as the target gene amplified rapidly, within 2h, and only a standard laboratory water bath or heat block was required for the reaction. The results demonstrate clearly that this LAMP-based assay is a useful tool for the rapid and sensitive diagnosis of CSBV infection of bees.

  18. Specific detection of Pectobacterium carotovorum by loop-mediated isothermal amplification.

    PubMed

    Yasuhara-Bell, Jarred; Marrero, Glorimar; De Silva, Asoka; Alvarez, Anne M

    2016-12-01

    Potatoes are an important agroeconomic crop worldwide and maceration diseases caused by pectolytic bacterial pathogens result in significant pre- and post-harvest losses. Pectobacterium carotovorum shares a common host range with other Pectobacterium spp. and other members of the Enterobacteriaceae, such as Dickeya spp. As these pathogens cannot be clearly differentiated on the basis of the symptoms they cause, improved methods of identification are critical for the determination of sources of contamination. Current standardized methods for the differentiation of pectolytic species are time consuming and require trained personnel, as they rely on traditional bacteriological practices that do not always produce conclusive results. In this growing world market, there is a need for rapid diagnostic tests that can differentiate between pectolytic pathogens, as well as separate them from non-pectolytic enteric bacteria associated with soft rots of potato. An assay has been designed previously to detect the temperate pathogen Pectobacterium atrosepticum, but there is currently no recognized rapid assay for the detection of the tropical/subtropical counterpart, Pectobacterium carotovorum. This report describes the development of a loop-mediated isothermal amplification (LAMP) assay that detects P. carotovorum with high specificity. The assay was evaluated using all known species of Pectobacterium and only showed positive reactions for P. carotovorum. This assay was also tested against 15 non-target genera of plant-associated bacteria and did not produce any false positives. The LAMP assay described here can be used as a rapid test for the differentiation of P. carotovorum from other pectolytic pathogens, and its gene target can be the basis for the development of other molecular-based detection assays.

  19. 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 ...

  20. 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.

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

    PubMed Central

    2016-01-01

    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

  2. Digital isothermal quantification of nucleic acids via simultaneous chemical initiation of recombinase polymerase amplification reactions on SlipChip.

    PubMed

    Shen, Feng; Davydova, Elena K; Du, Wenbin; Kreutz, Jason E; Piepenburg, Olaf; Ismagilov, Rustem F

    2011-05-01

    In this paper, digital quantitative detection of nucleic acids was achieved at the single-molecule level by chemical initiation of over one thousand sequence-specific, nanoliter isothermal amplification reactions in parallel. Digital polymerase chain reaction (digital PCR), a method used for quantification of nucleic acids, counts the presence or absence of amplification of individual molecules. However, it still requires temperature cycling, which is undesirable under resource-limited conditions. This makes isothermal methods for nucleic acid amplification, such as recombinase polymerase amplification (RPA), more attractive. A microfluidic digital RPA SlipChip is described here for simultaneous initiation of over one thousand nL-scale RPA reactions by adding a chemical initiator to each reaction compartment with a simple slipping step after instrument-free pipet loading. Two designs of the SlipChip, two-step slipping and one-step slipping, were validated using digital RPA. By using the digital RPA SlipChip, false-positive results from preinitiation of the RPA amplification reaction before incubation were eliminated. End point fluorescence readout was used for "yes or no" digital quantification. The performance of digital RPA in a SlipChip was validated by amplifying and counting single molecules of the target nucleic acid, methicillin-resistant Staphylococcus aureus (MRSA) genomic DNA. The digital RPA on SlipChip was also tolerant to fluctuations of the incubation temperature (37-42 °C), and its performance was comparable to digital PCR on the same SlipChip design. The digital RPA SlipChip provides a simple method to quantify nucleic acids without requiring thermal cycling or kinetic measurements, with potential applications in diagnostics and environmental monitoring under resource-limited settings. The ability to initiate thousands of chemical reactions in parallel on the nanoliter scale using solvent-resistant glass devices is likely to be useful for a broader

  3. Improvement and Evaluation of Loop-Mediated Isothermal Amplification for Rapid Detection of Toxoplasma gondii Infection in Human Blood Samples

    PubMed Central

    Sun, Xi-meng; Ji, Yong-sheng; Liu, Xian-yong; Xiang, Mei; He, Guang; Xie, Li; Suo, Jing-xia; Suo, Xun

    2017-01-01

    Loop-mediated isothermal amplification (LAMP), an attractive DNA amplification method, was developed as a valuable tool for the rapid detection of Toxoplasma gondii. In this study, species-specific LAMP primers were designed by targeting the AF146527 sequence, which was a conserved sequence of 200- to 300-fold repetitive 529 bp fragment of T.gondii. LAMP reaction system was optimized so that it could detect the minimal DNA sample such as a single tachyzoite or 10 copies of recombinant plasmid. No cross-reactivity was found when using DNA from other parasites as templates. Subsequently, a total of 200 human blood samples were directly investigated by two diagnostic methods, LAMP and conventional PCR. Fourteen of 200 (7%) samples were positive for Toxoplasma by LAMP (the primers developed in this study), whereas only 5 of 200 (2.5%) were proved positive by conventional PCR. The procedure of the LAMP assay was very simple, as the reaction would be carried out in a single tube under isothermal conditions at 64°C and the result would be read out with 1 h (as early as 35 min with loop primers). Thus, this method has the advantages of rapid amplification, simple operation, and easy detection and would be useful for rapid and reliable clinical diagnosis of acute toxoplasmosis, especially in developing countries. PMID:28056092

  4. 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.

  5. Application of a molecular beacon based real-time isothermal amplification (MBRTIA) technology for simultaneous detection of Bacillus cereus and Staphylococcus aureus.

    PubMed

    Mandappa, I M; Joglekar, Prasanna; Manonmani, H K

    2015-07-01

    A multiplex real-time isothermal amplification assay was developed using molecular beacons for the detection of Bacillus cereus and Staphylococcus aureus by targeting four important virulence genes. A correlation between targeting highly accessible DNA sequences and isothermal amplification based molecular beacon efficiency and sensitivity was demonstrated using phi(Φ)29 DNA polymerase at a constant isothermal temperature of 30 °C. It was very selective and consistently detected down to 10(1) copies of DNA. The specificity and sensitivity of this assay, when tested with pure culture were high, surpassing those of currently used PCR assays for the detection of these organisms. The molecular beacon based real-time isothermal amplification (MBRTIA) assay could be carried out entirely in 96 well plates or well strips, enabling a rapid and high-throughput detection of food borne pathogens.

  6. 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

  7. Ultrasensitive detection of low-abundance surface-marker protein using isothermal rolling circle amplification in a microfluidic nanoliter platform.

    PubMed

    Konry, Tania; Smolina, Irina; Yarmush, Joel M; Irimia, Daniel; Yarmush, Martin L

    2011-02-07

    With advances in immunology and cancer biology, there is an unmet need for increasingly sensitive systems to monitor the expression of specific cell markers for the development of new diagnostic and therapeutic tools. To address this challenge, a highly sensitive labeling method that translates antigen-antibody recognition processes into DNA detection events that can be greatly amplified via isothermal rolling circle amplification (RCA) is applied. By merging the single-molecule detection power of RCA reactions with microfluidic technology, it is demonstrated that the identification of specific protein markers can be achieved on tumor-cell surfaces in miniaturized nanoliter reaction droplets. Furthermore, this combined approach of signal amplification in a microfluidic format could extend the utility of existing methods by reducing sample and reagent consumption and enhancing the sensitivities and specificities for various applications, including early diagnosis of cancer.

  8. 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.

  9. 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.

  10. A field evaluation of an isothermal DNA amplification assay for the detection of Theileria annulata infection in cattle.

    PubMed

    Gomes, Jacinto; Santos, Marcos; Amaro, Ana; Pereira da Fonseca, Isabel; Santos-Gomes, Gabriela; Inácio, João

    2017-02-01

    A loop-mediated isothermal amplification (LAMP) assay was evaluated for the detection of Theileria annulata infection in cattle. The results were compared with a real-time PCR used for the quantification of T. annulata parasitaemia. One hundred bovine blood samples from 16 cattle farms were tested with LAMP and real-time PCR, with T. annulata DNA being detected in 66% and 67% of the samples, respectively. The results showed that the LAMP assay detects a parasitaemia as low as 0.00025%, indicating a high analytical sensitivity of LAMP for clinical diagnosis of bovine theileriosis.

  11. Evaluation of a Loop-Mediated Isothermal Amplification-Based Methodology To Detect Carbapenemase Carriage in Acinetobacter Clinical Isolates

    PubMed Central

    Vergara, Andrea; Zboromyrska, Yuliya; Mosqueda, Noraida; Morosini, María Isabel; García-Fernández, Sergio; Roca, Ignasi; Cantón, Rafael; Marco, Francesc

    2014-01-01

    Carbapenem-resistant Acinetobacter baumannii is a major source of nosocomial infections worldwide and is mainly associated with the acquisition of OXA-type carbapenemases and, to a lesser extent, metallo-β-lactamases (MBLs). In this study, 82 nonepidemiologically related Acinetobacter strains carrying different types of OXA or MBL enzymes were tested using the Eazyplex system, a loop-mediated isothermal amplification (LAMP)-based method to rapidly detect carbapenemase carriage. The presence/absence of carbapenem-hydrolyzing enzymes was correctly determined for all isolates in <30 min. PMID:25224010

  12. Concurrent reactivation of herpes simplex and varicella zoster viruses confirmed by the loop-mediated isothermal amplification assay.

    PubMed

    Kobayashi, Tsukane; Yagami, Akiko; Suzuki, Kayoko; Yoshikawa, Tetsushi; Matsunaga, Kayoko

    2014-01-01

    Concurrent reactivation of herpes simplex and varicella zoster viruses is rare. Here, we describe the case of an elderly patient with herpes labialis and herpes zoster manifesting as a right-side facial eruption with vesicles and crusting. The loop-mediated isothermal amplification (LAMP) assay demonstrated the presence of both herpes simplex virus type 1 and varicella zoster virus in swab samples taken from the face, which was confirmed by real-time PCR, suggesting concurrent reactivation of both viruses. The use of the LAMP assay in the present case indicates its usefulness in the diagnosis of atypical herpes infections.

  13. [Colorimetric detection of human influenza A H1N1 virus by reverse transcription loop mediated isothermal amplification].

    PubMed

    Nie, Kai; Wang, Da-Yan; Qin, Meng; Gao, Rong-Bao; Wang, Miao; Zou, Shu-Mei; Han, Feng; Zhao, Xiang; Li, Xi-Yan; Shu, Yue-Long; Ma, Xue-Jun

    2010-03-01

    A simple, rapid and sensitive colorimetric Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) method was established to detect human influenza A H1N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the HA gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for one and half hour. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP assay was validated by cross-reaction with different swine and human influenza virus including human seasonal influenza A /H1N1 A /H3N2, influenza B and swine A /H1N1. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of human influenza A H1N1 HA gene. The assay was further evaluated with 30 clinical specimens with suspected pandemic influenza A H1N1 virus infection in parallel with RT-PCR detection and 26 clinical specimens with seasonal influenza virus infection. Our results showed that the RT-LAMP was able to achieve a sensitivity of 60 RNA copies with high specificity, and detection rate was comparable to that of the RT-PCR with the clinical samples of pandemic influenza A H1N1 infection. The RT-LAMP reaction with HNB could also be measured at 650nm in a microplate reader for quantitative analysis. Thus, we concluded that this colorimetric RT-LAMP assay had potential for the rapid screening of the human influenza A H1N1 virus infection in National influenza monitoring network laboratories and sentinel hospitals of provincial and municipal region in China.

  14. Ultrasensitive DNA detection by cycle isothermal amplification based on nicking endonuclease and its application to logic gates.

    PubMed

    Li, Xuemei; Ding, Tianrong; Sun, Li; Mao, Changming

    2011-12-15

    In recent years, an intense interest has grown in the DNA logic gates having high potential for computation at literally the "nano-size" level. A limitation of traditional DNA logic gates is that each target strand hybridizes with only a single copy of the probe. This 1:1 hybridization radio limits the gain of the approach and thus its sensitivity. The exponential amplification of nucleic acids has become a core technology in medical diagnostics and has been widely used for the construction of DNA sensor, DNA nanomachine and DNA sequencing. It would be of great interest to develop DNA-based logic systems with exponential amplification for the output signal. In the present study, a series of three-input DNA logic gates with the cycle isothermal amplification based on nicking endonuclease (NEase) are designed. Very low concentrations of the analytes were sufficient to initiate an autocatalytic cascade, achieving a significant improvement of the detection limit, 100-fold improvement compared to the non-autocatalytic system. This was achieved by engineering a simple and flexible biological circuit designed to initiate a cascade of events to detect and amplify a specific DNA sequence. This procedure has the potential to greatly simplify the logic operation because amplification can be performed in "one-pot".

  15. Label-free detection of microRNA based on coupling multiple isothermal amplification techniques

    PubMed Central

    Zheng, Xiangjiang; Niu, Li; Wei, Di; Li, Xuemei; Zhang, Shusheng

    2016-01-01

    MicroRNA (miRNA) was a promising class of cancer biomarkers. Here we developed a label-free method for sensitive measurement of let-7d miRNA based on multiple amplification techniques. The primer will bind to the duplex strand DNA that was formed by stem-loop template and target let-7d to initiate strand displacement amplification (SDA) in tandem. The released single strand DNA will be a primer to bind the circular template to initiate rolling circle amplification (RCA). The products based on multiple amplifications will be detected by a standard fluorimeter with N-methyl mesoporphyrin IX (NMM) as the fluorescent indicator. The proposed method exhibited excellent selectivity and high sensitivity with a detection limit of as low as 1.5 × 10−13 M. Moreover, this methodology was used for the determination of biomolecules in real serum samples with satisfying results. PMID:27777399

  16. Rapid and specific detection of the thermostable direct hemolysin gene in Vibrio parahaemolyticus by loop-mediated isothermal amplification.

    PubMed

    Nemoto, Jiro; Sugawara, Chiyo; Akahane, Kenji; Hashimoto, Keiji; Kojima, Tadashi; Ikedo, Masanari; Konuma, Hirotaka; Hara-Kudo, Yukiko

    2009-04-01

    Several investigators have reported that thermostable direct hemolysin (TDH) and TDH-related hemolysin are important virulence factors of Vibrio parahaemolyticus, but it has been difficult to detect these factors rapidly in seafood and other environmental samples. A novel nucleic acid amplification method, termed the loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity and rapidity under isothermal conditions, was applied. In this study, we designed tdh gene-specific LAMP primers for detection of TDH-producing V. parahaemolyticus. The specificity of this assay was evaluated with 32 strains of TDH-producing V. parahaemolyticus, one strain of TDH-producing Grimontia hollisae, 10 strains of TDH-nonproducing V. parahaemolyticus, and 94 strains of TDH-nonproducing bacteria, and the sensitivity was high enough to detect one cell per test. Moreover, to investigate the detection of TDH-producing V. parahaemolyticus in oysters, the LAMP assay was performed with enrichment culture in alkaline peptone water of oyster samples inoculated with TDH-producing V. parahaemolyticus and TDH-nonproducing V. parahaemolyticus and V. alginolyticus after enrichment in alkaline peptone water. These results suggest that the LAMP assay targeting tdh gene has high sensitivity and specificity and is useful to detect TDH-producing V. parahaemolyticus in oyster after enrichment.

  17. A novel, sensitive and label-free loop-mediated isothermal amplification detection method for nucleic acids using luminophore dyes.

    PubMed

    Roy, Sharmili; Wei, Sim Xiao; Ying, Jean Liew Zhi; Safavieh, Mohammadali; Ahmed, Minhaz Uddin

    2016-12-15

    Electrochemiluminescence (ECL) has been widely rendered for nucleic acid testing. Here, we integrate loop-mediated isothermal amplification (LAMP) with ECL technique for DNA detection and quantification. The target LAMP DNA bound electrostatically with [Ru(bpy)3](+2) on the carbon electrode surface, and an ECL reaction was triggered by tripropylamine (TPrA) to yield luminescence. We illustrated this method as a new and highly sensitive strategy for the detection of sequence-specific DNA from different meat species at picogram levels. The proposed strategy renders the signal amplification capacities of TPrA and combines LAMP with inherently high sensitivity of the ECL technique, to facilitate the detection of low quantities of DNA. By leveraging this technique, target DNA of Sus scrofa (pork) meat was detected as low as 1pg/µL (3.43×10(-1)copies/µL). In addition, the proposed technique was applied for detection of Bacillus subtilis DNA samples and detection limit of 10pg/µL (2.2×10(3)copies/µL) was achieved. The advantages of being isothermal, sensitive and robust with ability for multiplex detection of bio-analytes makes this method a facile and appealing sensing modality in hand-held devices to be used at the point-of-care (POC).

  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. 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

  20. 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

  1. 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.

  2. Use of a novel metal indicator to judge loop-mediated isothermal amplification for detecting the 35S promoter.

    PubMed

    Wang, Xiaofu; Fu, Zhenfang; Chen, Xiaoyun; Peng, Cheng; Xu, Xiaoli; Wei, Wei; Li, Feiwu; Xu, Junfeng

    2017-02-01

    Loop-mediated isothermal amplification (LAMP) is a widely used isothermal nucleic acid amplification method. Here we developed a new closed-tube colorimetric method for judging LAMP with a novel metal indicator. First, the metal indicator, acid chrome blue K (ACBK), was evaluated in the LAMP reaction with various combinations of reaction reagents, such as reaction buffer, dNTP mixtures, primer mixtures, or Mg(2+). We found that the solution color of the LAMP reaction with ACBK changed from red to blue based on a decrease in the Mg(2+) concentration in the reaction solution. We then optimized the LAMP with ACBK method for detecting the Cauliflower Mosaic Virus 35S promoter. Further, the specificity of the new colorimetric assay using ACBK in the LAMP reaction for detecting the 35S promoter was tested with diverse transgenic events in different crops, and the sensitivity threshold of the assay was ∼50 copies for transgenic rice genomic DNA and 100 ng of 0.1 % DNA from rice, soybean, rapeseed, and maize. Finally, the applicability of the LAMP assay was successfully validated using practical maize samples. All the detection results could be easily discerned either by UV-vis spectroscopy or the naked eye. Graphical Abstract The visual detect LAMP amplification by the addition of ACBK as a signal indicator. The color of the LAMP-ACBK solution turned from red to blue as the concentration of free Mg(2+) decreases. The detection results could be easily discerned either by UV-vis spectroscopy or the naked eye.

  3. Ratiometric fluorescence transduction by hybridization after isothermal amplification for determination of zeptomole quantities of oligonucleotide biomarkers with a paper-based platform and camera-based detection.

    PubMed

    Noor, M Omair; Hrovat, David; Moazami-Goudarzi, Maryam; Espie, George S; Krull, Ulrich J

    2015-07-23

    Paper is a promising platform for the development of decentralized diagnostic assays owing to the low cost and ease of use of paper-based analytical devices (PADs). It can be challenging to detect on PADs very low concentrations of nucleic acid biomarkers of lengths as used in clinical assays. Herein we report the use of thermophilic helicase-dependent amplification (tHDA) in combination with a paper-based platform for fluorescence detection of probe-target hybridization. Paper substrates were patterned using wax printing. The cellulosic fibers were chemically derivatized with imidazole groups for the assembly of the transduction interface that consisted of immobilized quantum dot (QD)-probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as the acceptor dye in a fluorescence resonance energy transfer (FRET)-based transduction method. After probe-target hybridization, a further hybridization event with a reporter sequence brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs, triggering a FRET sensitized emission that served as an analytical signal. Ratiometric detection was evaluated using both an epifluorescence microscope and a low-cost iPad camera as detectors. Addition of the tHDA method for target amplification to produce sequences of ∼100 base length allowed for the detection of zmol quantities of nucleic acid targets using the two detection platforms. The ratiometric QD-FRET transduction method not only offered improved assay precision, but also lowered the limit of detection of the assay when compared with the non-ratiometric QD-FRET transduction method. The selectivity of the hybridization assays was demonstrated by the detection of single nucleotide polymorphism.

  4. A novel CMOS image sensor system for quantitative loop-mediated isothermal amplification assays to detect food-borne pathogens.

    PubMed

    Wang, Tiantian; Kim, Sanghyo; An, Jeong Ho

    2017-02-01

    Loop-mediated isothermal amplification (LAMP) is considered as one of the alternatives to the conventional PCR and it is an inexpensive portable diagnostic system with minimal power consumption. The present work describes the application of LAMP in real-time photon detection and quantitative analysis of nucleic acids integrated with a disposable complementary-metal-oxide semiconductor (CMOS) image sensor. This novel system works as an amplification-coupled detection platform, relying on a CMOS image sensor, with the aid of a computerized circuitry controller for the temperature and light sources. The CMOS image sensor captures the light which is passing through the sensor surface and converts into digital units using an analog-to-digital converter (ADC). This new system monitors the real-time photon variation, caused by the color changes during amplification. Escherichia coli O157 was used as a proof-of-concept target for quantitative analysis, and compared with the results for Staphylococcus aureus and Salmonella enterica to confirm the efficiency of the system. The system detected various DNA concentrations of E. coli O157 in a short time (45min), with a detection limit of 10fg/μL. The low-cost, simple, and compact design, with low power consumption, represents a significant advance in the development of a portable, sensitive, user-friendly, real-time, and quantitative analytic tools for point-of-care diagnosis.

  5. 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.

  6. [Evaluating the Stability of Loop-Mediated Isothermal Amplification Reagents at Irregular Storage Temperatures for On-Site Diagnosis].

    PubMed

    Inoshima, Yasuo; Ishiguro, Naotaka

    2015-01-01

    Temperature-stability of loop-mediated isothermal amplification (LAMP) reagents was determined for their use in on-site diagnosis, such as in farms/pastures. Bst and Csa DNA polymerases and the reagents that were stored at different temperatures (4 or 25°C) for 1, 2, or 4 days were used for the LAMP assay to detect orf virus DNA as a model. After storage at 4 and 25°C for 2 days, the enzymes and reagents were found to retain sufficient activity to carry out successful DNA amplification. Visual diagnosis was also possible with the reagents (Loopamp Fluorescent Detection Reagent or hydroxy naphthol blue, as well as DNA amplification checker, D-Quick) that were stored for 2 days at different temperatures. Although the time taken to obtain the positive/negative results were delayed, the enzymes and reagents, stored at 25°C for 4 days, were active and had the ability to efficiently amplify DNA in less than 50 min. These results indicate that LAMP assay can be successfully utilized for the diagnosis of infectious diseases under non-clinical settings such as for on-site diagnosis in farms/pastures, owing to the fact that the relevant enzymes and reagents does not require restricted temperature storage.

  7. Development of a multiplex loop-mediated isothermal amplification method for the simultaneous detection of Salmonella spp. and Vibrio parahaemolyticus

    PubMed Central

    Liu, Ningwei; Zou, Dayang; Dong, Derong; Yang, Zhan; Ao, Da; Liu, Wei; Huang, Liuyu

    2017-01-01

    Rapid detection of food-borne pathogens is important in the food industry, to monitor and prevent the spread of these pathogens through contaminated food products. We therefore established a multiplex real-time loop-mediated isothermal amplification (LAMP) assay to simultaneously detect and distinguish Salmonella spp. and Vibrio parahaemolyticus DNA in a single reaction. Two target sequences, one specific for Salmonella and the other specific for Vibrio parahaemolyticus, were amplified by specific LAMP primers in the same reaction tube. After amplification at 65 °C for 60 min, the amplified products were subjected to melting curve analysis and thus could be distinguished based on the different melting temperatures (Tm values) of the two specifically amplified products. The specificity of the multiplex LAMP assay was evaluated using 19 known bacterial strains, including one V. parahaemolyticus and seven Salmonella spp. strains. The multiplex LAMP showed 100% inclusivity and exclusivity, and a detection limit similar to that of multiplex PCR. In addition, we observed and corrected preferential amplification induced by what we call LAMP selection in the multiplex LAMP reaction. In conclusion, our assay was rapid, specific, and quantitative, making it a useful tool for the food industry. PMID:28349967

  8. Development of a loop-mediated isothermal amplification assay for rapid detection of iridovirus in the Chinese giant salamander.

    PubMed

    Meng, Yan; Zhang, Hui; Liang, Hongwei; Zeng, Lingbing; Xiao, Hanbing; Xie, Congxin

    2013-12-01

    The Chinese giant salamander (Andrias davidianus) iridovirus (GSIV) is an emerging infectious pathogen responsible for severe hemorrhagic disease and high mortality in cultured Chinese giant salamanders. A loop-mediated isothermal amplification (LAMP) assay based on the major caspid protein (MCP) gene has been developed to detect this virus. Primer pairs for the LAMP assay were designed based on the GSIV MCP gene sequence. Amplification results indicate that under optimized conditions the LAMP assay has the ability to specifically detect the virus in both diseased animals and infected epithelioma papilloma cyprinid (EPC) cells. The assay was shown to be 10-fold more sensitive than nested PCR and was able to detect concentrations of 10(-9) (approximately 0.01 pg/μL). The LAMP assay is relatively easy to perform in situ and the amplification products can be observed directly under UV light or via staining with SYBR Green I. The LAMP assay is also rapid and cost-effective. This study establishes the use of a LAMP assay for rapid detection of GSIV, which is a novel and important tool for the diagnosis of GSIV infection in laboratory or farmed Chinese giant salamanders.

  9. 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...

  10. Isothermal amplification of DNA using quadruplex primers with fluorescent pteridine base analogue 3-methyl isoxanthopterin.

    PubMed

    Gogichaishvili, Shota; Johnson, John; Gvarjaladze, David; Lomidze, Levan; Kankia, Besik

    2014-06-01

    We previously developed a method, known as quadruplex priming amplification (QPA), which greatly simplifies DNA amplification and quantification assays. QPA employs specific primers based on GGGTGGGTGGGTGGG (G3T) sequence, which upon polymerase elongation spontaneously dissociates from the target and folds into a stable quadruplex. Fluorescent nucleotide analogs, when incorporated into these primers, emit light upon quadruplex formation and permit simple, specific, and sensitive quantification without the attachment of probe molecules. Here, we studied optical [fluorescence and circular dichroism (CD)] and thermodynamic properties of the G3T sequence and variants incorporating 3-methylisoxanthopterin (3MI), a highly fluorescent nucleotide analog suitable for QPA. CD studies demonstrate that the incorporation of 3MI does not change the overall tertiary structure of G3T; however, thermal unfolding experiments revealed that it significantly destabilizes the quadruplex. Enzymatic studies revealed that Taq and Bst are practically unable to incorporate any nucleotides opposite to template 3MI. Based on this knowledge, we designed QPA assays with truncated targets that demonstrate efficient amplification around 55°C. Overall, these studies suggest that 3MI-based QPA is a useful assay for DNA amplification and detection.

  11. 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...

  12. Loop-Mediated Isothermal Amplification for Direct Detection of Mycobacterium tuberculosis Complex, M. avium, and M. intracellulare in Sputum Samples

    PubMed Central

    Iwamoto, Tomotada; Sonobe, Toshiaki; Hayashi, Kozaburo

    2003-01-01

    Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react under isothermal conditions with high specificity, efficiency, and rapidity. We used LAMP for detection of Mycobacterium tuberculosis complex, Mycobacterium avium, and Mycobacterium intracellulare directly from sputum specimens as well as for detection of culture isolates grown in a liquid medium (MGIT; Nippon Becton Dickinson Co., Ltd., Tokyo, Japan) or on a solid medium (Ogawa's medium). Species-specific primers were designed by targeting the gyrB gene, and their specificities were validated on 24 mycobacterial species and 7 nonmycobacterial species. The whole procedure is quite simple, starting with the mixing of all reagents in a single tube, followed by an isothermal reaction during which the reaction mixture is held at 63°C. The resulting amplicons are visualized by adding SYBR Green I to the reaction tube. The only equipment needed for the amplification reaction is a regular laboratory water bath or heat block that furnishes a constant temperature of 63°C. The assay had a detection limit of 5 to 50 copies of purified DNA with a 60-min incubation time. The reaction time could be shortened to 35 min for the species identification of M. tuberculosis complex, M. avium, and M. intracellulare from a solid-medium culture. Residual DNA lysates prepared for the Amplicor assay (Roche Diagnostics GmbH) from 66 sputum specimens were tested in the LAMP assay. Although the sample size used for the latter assay was small, 2.75 μl of the DNA lysates, it showed a performance comparable with that of the Amplicor assay, which required 50 μl of the lysates. This LAMP-based assay is simple, rapid, and sensitive; a result is available in 35 min for a solid-medium culture and in 60 min for a liquid-medium culture or for a sputum specimen that contains a corresponding amount of DNA available for testing. PMID:12791888

  13. 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.

  14. Detection of HbsAg and hATIII genetically modified goats (Caprahircus) by loop-mediated isothermal amplification.

    PubMed

    Tao, Chenyu; Zhang, Qingde; Zhai, Shanli; Liu, Bang

    2013-11-01

    In this study, sensitive and rapid detection systems were designed using a loop-mediated isothermal amplification (LAMP) method to detect the genetically modified goats. A set of 4 primers were designed for each exogenous nucleic acids HBsAg and hATIII. The DNA samples were first amplified with the outer and inner primers and released a single-stranded DNA,of which both ends were stem-loop structure. Then one inner primer hybridized with the loop, and initiated displacement synthesis in less than 1 h. The result could be visualized by both agarose gel electrophoresis and unaided eyes directly after adding SYBR GREEN 1. The detection limit of LAMP was ten copies of target molecules, indicating that LAMP was tenfold more sensitive than the classical PCR. Furthermore, all the samples of genetically modified goats were tested positively by LAMP, and the results demonstrated that the LAMP was a rapid and sensitive method for detecting the genetically modified organism.

  15. Reverse transcription loop-mediated isothermal amplification for rapid and sensitive detection of nervous necrosis virus in groupers.

    PubMed

    Sung, Chia-Hsuan; Lu, Jenn-Kan

    2009-08-01

    The reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is a sensitive nucleic acid diagnostic method that can amplify rapidly a target template; it can be applied for the diagnosis of viral disease in grouper aquaculture. In this study, two outer and two inner primers were designed from nervous necrosis virus (NNV) coat protein gene sequence. The reaction temperature and time for the detection of NNV were optimized at 65 degrees C for 60min. The detection limit of RT-LAMP is 10(-6) NNV-RNA from infected groupers, and more sensitive than the one-step RT-PCR and nested RT-PCR. The combination of RNA rapid extraction and RT-LAMP, the process can be completed within 2h. Thus, the RT-LMAP is a rapid, sensitive, specific and efficient method for detection of NNV in groupers.

  16. The development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of abalone herpesvirus DNA.

    PubMed

    Chen, M H; Kuo, S T; Renault, T; Chang, P H

    2014-02-01

    A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of abalone herpesvirus DNA. Two pairs of primers were designed, based on the sequence of the DNA polymerase gene of abalone herpesvirus. The reaction temperature and time were optimized to 63°C and 60min, respectively. LAMP amplicons were analyzed by 2% agarose gel electrophoresis or by visual inspection of a colour change emitted by fluorescent dye. The method developed was specific for the detection of abalone herpesvirus, without cross-reactions with other tested herpesviruses including ostreid herpesvirus 1 (OsHV-1), European eel herpesvirus, koi herpesvirus (KHV) and an avian herpesvirus. The LAMP assay was 100 folds more sensitive than a conventional PCR and 10 folds less sensitive than a SYBR Green PCR. These results indicate that the developed LAMP assay is a simple, rapid, sensitive, specific and reliable technique for the detection of abalone herpesvirus.

  17. Detection of methicillin-resistant Staphylococcus aureus directly by loop-mediated isothermal amplification and direct cefoxitin disk diffusion tests.

    PubMed

    Metwally, L; Gomaa, N; Hassan, R

    2014-05-01

    We evaluated the utility of 2 methods for detection of methicillin-resistant Staphylococcus aureus (MRSA) directly from signal-positive blood culture bottles: loop-mediated isothermal amplification (LAMP) assay, and direct cefoxitin disk diffusion (DCDD) test using a 30 μg cefoxitin disk. In parallel, standard microbiological identification and oxacillin susceptibility testing with MecA PCR was performed. Of 60 blood cultures positive for Gram-positive cocci in clusters, LAMP (via detection of the FemA and MecA genes) showed 100% sensitivity and specificity for identification of MRSA/MSSA. When coagulase-negative staphylococci were tested, sensitivity for detection of methicillin resistance was 91.7% and specificity was 100%. DCDD along with direct tube coagulase assay detected only 80.6% of MRSA/MSSA. LAMP showed higher diagnostic accuracy although DCDD was more cost-effective and did not require additional reagents or supplies.

  18. Rapid detection of hepatitis C virus RNA by a reverse transcription loop-mediated isothermal amplification assay.

    PubMed

    Wang, Qin-qin; Zhang, Jie; Hu, Jin-song; Chen, Hao-tai; Du, Li; Wu, Li-qin; Ding, Yao-zhong; Xiong, Sheng-he; Huang, Xin-cheng; Zhang, Yin-hong; Liu, Yong-sheng

    2011-10-01

    The usefulness of reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the rapid diagnosis of hepatitis C virus (HCV) RNA was evaluated. This assay showed higher sensitivities than that of nested RT-PCR, with a detection limit of 600 IU mL(-1) , and no cross-reactivity was observed with hepatitis A virus, hepatitis B virus and hepatitis E virus. Furthermore, 106 stored sera from recently diagnosed cases were retrospectively investigated with real-time RT-PCR, the nested RT-PCR, in parallel with this new assay. The general detection rates of HCV RT-LAMP, real-time PCR and the nested RT-PCR for 106 stored sera samples were 95%, 96% and 88%, respectively. This study provides the first data on the usefulness of HCV RT-LAMP in the diagnosis of HCV RNA, especially in the early clinical diagnosis of acute HCV infection.

  19. Detection methods for rice viruses by a reverse-transcription loop-mediated isothermal amplification (RT-LAMP).

    PubMed

    Sasaya, Takahide

    2015-01-01

    Developing a quick and accurate method to diagnose rice viruses in host plants and in vector insects is very important to control virus diseases of rice. A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay, one of the most promising molecular diagnostic methods, was established to detect nine viruses, including eight RNA viruses and one DNA virus, in infected rice plants and the viruliferous vector insects. The sensitivities of the assays were either higher than or similar to those of one-step RT-PCR. With a combination of rapid RNA extraction and a RT-LAMP assay, these nine viruses were detected within 2 h from infected rice plants and the viruliferous insects without expensive or unusual equipment. This RT-LAMP method for rice viruses can therefore be adopted not only for diagnosis but also to study the epidemiology and molecular pathology of rice viruses.

  20. Detection of Francisella piscicida in Atlantic cod (Gadus morhua L) by the loop-mediated isothermal amplification (LAMP) reaction.

    PubMed

    Caipang, Christopher M A; Kulkarni, Amod; Brinchmann, Monica F; Korsnes, Kjetil; Kiron, Viswanath

    2010-06-01

    The loop-mediated isothermal amplification (LAMP) reaction was evaluated for its speed and sensitivity in detecting the presence of Francisella piscicida, the causative agent of francisellosis in Atlantic cod (Gadus morhua). Four primer sets, consisting of two outer and two inner, were designed from the groEL gene of the pathogen. The LAMP reaction was optimised at 63 degrees C for 1h using bacterial genomic DNA as the template and the products were visualised under ultra-violet light and analysed by agarose gel electrophoresis. A ladder-like pattern of bands, specific for F. piscicida, was amplified from positive samples. The method was highly specific for the detection of F. piscicida and was 100 times more sensitive than conventional PCR. In addition, the LAMP assay was able to detect the pathogen in kidney and splenic samples of naturally-infected Atlantic cod.

  1. 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.

  2. Flower-like ZnO nanostructure assisted loop-mediated isothermal amplification assay for detection of Japanese encephalitis virus.

    PubMed

    Ma, Yonghua; Lu, Yan; Guan, Guiquan; Luo, Jianxun; Niu, Qingli; Liu, Junlong; Yin, Hong; Liu, Guangyuan

    2017-03-15

    In this study, we described a novel and effective flower-like ZnO nanostructure assisted Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) method to detect Japanese Encephalitis Virus (JEV). The effects of different concentrations of ZnO nanoflower on the RT-LAMP reaction were investigated. With the increase of concentration of ZnO nanoflower, RT-LAMP reaction obtained optimization, until the concentration exceeded 1.5nM, RT-LAMP reaction was inhibited. Made 1nM as optimum concentration of ZnO nanoflower, we found that optimum RT-LAMP reaction temperature and time were 60°C and 30min, respectively. The optimization might be connected with good adsorption to DNA and thermal conductivity of ZnO nanoflower, but mechanism of the RT-LAMP reaction affected by ZnO nanoflower needs to be explored further.

  3. A loop-mediated isothermal amplification assay for rapid and sensitive detection of bovine papular stomatitis virus.

    PubMed

    Kurosaki, Yohei; Okada, Sayaka; Nakamae, Sayuri; Yasuda, Jiro

    2016-12-01

    Bovine papular stomatitis virus (BPSV) causes pustular cutaneous disease in cattle worldwide. This paper describes the development of a specific loop-mediated isothermal amplification (LAMP) assay to detect BPSV which did not cross-react with other parapoxviruses. To assess analytical sensitivity of this LAMP assay, DNA was extracted from serially diluted BPSV from which the infectious titer was determined by a novel assay based on calf kidney epithelial cells. The LAMP assay had equivalent analytical sensitivity to quantitative PCR, and could detect as few as 86 copies of viral DNA per reaction. These results suggest that the assay is a specific and sensitive technique to rapidly diagnose bovine papular stomatitis in domestic animals.

  4. A loop mediated isothermal amplification (LAMP) method for detection of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Gunimaladevi, I; Kono, T; Lapatra, S E; Sakai, M

    2005-05-01

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) protocol was developed for detection of infectious hematopoietic necrosis virus (IHNV) RNA in rainbow trout (Oncorhynchus mykiss). A set of four primers, two outer and two inner primers for the RT-LAMP and the LAMP assay, were designed based on the sequence of G-protein of IHNV. Time and temperature conditions were optimized for 60 min at 63 degrees C for both RT-LAMP and LAMP protocols. The detection limit was found to be similar for both RT-LAMP and LAMP. When the sensitivity of RT-LAMP and LAMP were compared with conventional nested PCR, a10-fold higher sensitivity was seen for the LAMP protocols.

  5. 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

  6. 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.

  7. One-step reverse transcription loop-mediated isothermal amplification for the detection of Maize chlorotic mottle virus in maize.

    PubMed

    Chen, Ling; Jiao, Zhiyuan; Liu, Dongmei; Liu, Xingliang; Xia, Zihao; Deng, Congliang; Zhou, Tao; Fan, Zaifeng

    2017-02-01

    Maize chlorotic mottle virus (MCMV) is spreading in many regions worldwide, causing maize lethal necrosis when co-infected with a potyvirid. In this study, one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect MCMV in maize. A set of four specific primers was designed based on the conserved coat protein gene sequences of MCMV. The RT-LAMP could be completed within 60min under isothermal condition at 63°C. The sensitivity test showed that the RT-LAMP was about 10-fold more sensitive than RT-PCR and no cross-reactivity was detected with other viral pathogens infecting maize in China. Moreover, the results of RT-LAMP could be visually inspected by SYBR Green I staining in a closed-tube, facilitating high-throughput application of MCMV detection. This method was further verified by testing field-collected samples. These results suggested that the developed MCMV RT-LAMP technique is a rapid, efficient and sensitive method which could be used as a routine screen for MCMV infection.

  8. Loop-mediated isothermal amplification with the Procedure for Ultra Rapid Extraction kit for the diagnosis of pneumocystis pneumonia.

    PubMed

    Kawano, Shuichi; Maeda, Takuya; Suzuki, Takefumi; Abe, Tatsuhiro; Mikita, Kei; Hamakawa, Yusuke; Ono, Takeshi; Sonehara, Wataru; Miyahira, Yasushi; Kawana, Akihiko

    2015-03-01

    Loop-mediated isothermal amplification (LAMP) is an innovative molecular technique requiring only a heating device and isothermal conditions to amplify a specific target gene. The results of current microscopic diagnostic tools for pneumocystis pneumonia are not sufficiently consistent for detecting infection with a low-density of Pneumocystis jirovecii. Although polymerase chain reaction (PCR) is highly sensitive, it is not suitable for resource-limited facilities. LAMP is a potential diagnostic replacement for PCR in such settings but a critical disadvantage of DNA extraction was still remained. Therefore, we employed the Procedure for Ultra Rapid Extraction (PURE) kit, which uses a porous material, to isolate the DNA from clinical samples in a simple way in combination with previously reported LAMP procedure for diagnosing PCP. The detection limit of the PURE-LAMP method applied to artificial bronchoalveolar lavage fluid samples was 100 copies/tube, even with the use of massive blood-contaminated solutions. In addition, we concluded the diagnostic procedure within 1 h without the need for additional equipment. PURE-LAMP coupled with suitable primers for specific pathogens has good potential for diagnosing various infectious diseases.

  9. Identification of human metapneumovirus genotypes A and B from clinical specimens by reverse transcription loop-mediated isothermal amplification.

    PubMed

    Song, Qinwei; Zhu, Runan; Sun, Yu; Zhao, Linqing; Wang, Fang; Deng, Jie; Qian, Yuan

    2014-02-01

    Human metapneumovirus (hMPV) has been recognized as an important pathogen for acute respiratory infections in children worldwide and classified into genotypes A and B. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay is a rapid diagnostic method for detecting nucleic acids with a single step under isothermal conditions in less than 1h. RT-LAMP targeting the M gene of hMPV was developed for detecting and identifying hMPV genotypes A and B. The detection limit of the genotype-specific hMPV RT-LAMP assay was 10 times greater than that of conventional reverse transcription polymerase chain reaction (RT-PCR). No cross-reactivity was found with respiratory syncytial virus, parainfluenza virus 1-3, adenovirus, human bocavirus, human rhinovirus, influenza virus A and B, human coronaviruses and enteroviruses. One hundred and fifteen clinical specimens were detected for hMPV genotypes A and B with RT-LAMP, RT-PCR and real-time SYBR PCR. Kappa coefficients showed that there was a good agreement among these three methods. Compared with RT-PCR and real-time SYBR PCR, the genotype-specific RT-LAMP showed better specificity, sensitivity and is more convenient to perform with reduced turn-around time.

  10. 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.

  11. Development of a reverse-transcription loop-mediated isothermal amplification method for detection of rabbit hemorrhagic disease virus.

    PubMed

    Yuan, Dongwei; Guo, Dongchun; Liu, Jiasen; Si, Changde; Jiang, Qian; Lin, Huan; Yang, Tiankuo; Qu, Liandong

    2013-02-01

    Rabbit hemorrhagic disease virus (RHDV) causes haemagglutination and severe liver damage, with a high mortality rate. To develop a rapid and sensitive method for the surveillance of RHDV, a one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was established using a set of four primers specific for the VP60 gene segment of RHDV. The established assay was performed at 64°C for 40 min under isothermal conditions, and the results were visualized directly by electrophoresis or as fluorescent signals under ultraviolet light. The detection limit of the RT-LAMP assay was 10 copies of viral RNA per reaction, which was comparable to quantitative real-time RT-PCR, and 100-fold more sensitive than standard RT-PCR. Furthermore, seven viral RNAs of field isolates in China could be detected successfully using this assay. Overall, the newly established RT-LAMP assay indicates the potential usefulness of the technique as a simple, rapid and sensitive procedure, and can visually detect RHDV infection without the need for any specialized equipment.

  12. Rapid sex identification of papaya (Carica papaya) using multiplex loop-mediated isothermal amplification (mLAMP).

    PubMed

    Hsu, Te-Hua; Gwo, Jin-Chywan; Lin, Kuan-Hung

    2012-10-01

    Papaya (Carica papaya L.) is established as a cash crop throughout the tropical and subtropical regions due to its easy adaptation to diverse agricultural conditions, high yields, and prompt returns. The sex types of papaya plants are hermaphrodite, male, and female. Among them, hermaphroditic plants are the major type in papaya production, because the fruit has commercial advantages over that of the other sexes. Sex inheritance in papaya is determined by the M and M(h) dominant alleles in males and hermaphrodites, respectively, and a recessive m allele in females. Currently, all hermaphrodite seeds are not available due to the lethality of dominant homozygosity. Therefore, in this study, six male-hermaphrodite-specific markers were developed for a rapid sex identification using multiplex loop-mediated isothermal amplification (mLAMP) to efficiently and precisely select hermaphroditic individuals in the seedling or early growth stage. The LM1-LAMP assay consisted of two sex-LAMP reactions for amplifying two male-specific markers (T12 and Cpsm90) in one reaction, and showed several advantages in terms of a rapid reaction time (<1 h), isothermal conditions (less equipment required), a high efficiency (0.5 ng of DNA required in the reaction mixture), and an economical reaction system (5 μl in volume). The established method can be easily performed in the field by visual inspection and facilitates the selection of all hermaphroditic individuals in papaya production.

  13. Development of a pan-serotype reverse transcription loop-mediated isothermal amplification assay for the detection of dengue virus.

    PubMed

    Dauner, Allison L; Mitra, Indrani; Gilliland, Theron; Seales, Sajeewane; Pal, Subhamoy; Yang, Shih-Chun; Guevara, Carolina; Chen, Jiann-Hwa; Liu, Yung-Chuan; Kochel, Tadeusz J; Wu, Shuenn-Jue L

    2015-09-01

    During dengue outbreaks, acute diagnosis at the patient's point of need followed by appropriate supportive therapy reduces morbidity and mortality. To facilitate needed diagnosis, we developed and optimized a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay that detects all 4 serotypes of dengue virus (DENV). We used a quencher to reduce nonspecific amplification. The assay does not require expensive thermocyclers, utilizing a simple water bath to maintain the reaction at 63 °C. Results can be visualized using UV fluorescence, handheld readers, or lateral flow immunochromatographic tests. We report a sensitivity of 86.3% (95% confidence interval [CI], 72.7-94.8%) and specificity of 93.0% (95% CI, 83.0-98.1%) using a panel of clinical specimens characterized by DENV quantitative reverse transcription-polymerase chain reaction. This pan-serotype DENV RT-LAMP can be adapted to field-expedient formats where it can provide actionable diagnosis near the patient's point of need.

  14. 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

    2015-12-09

    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.

  15. 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.

  16. 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.

  17. An electrochemical biosensor for double-stranded Wnt7B gene detection based on enzymatic isothermal amplification.

    PubMed

    Li, Junlong; Chen, Zhongping; Xiang, Yu; Zhou, Lili; Wang, Ting; Zhang, Zhang; Sun, Kexin; Yin, Dan; Li, Yi; Xie, Guoming

    2016-12-15

    Wnt7B gene plays an important role in the development and progression of breast cancer, gastric cancer, esophageal cancer and pancreatic cancer. While, the natural state of DNA is double stranded, which makes it difficult to be directly detected. Here, we develop an electrochemical biosensor method for Wnt7B gene detection without the need to denature the target. This method firstly used nicking enzyme for exploiting in the double-stranded DNA (dsDNA). Then, long single-stranded DNA (ssDNA) was generated from the cutting site through polymerase extension reaction. Whereafter, the long ssDNA triggered a hairpin self-assembly recycling reaction, which gave rise to another isothermal amplification reaction. Last, short ssDNA was formed after the this amplification process, which could hybridize with the capture probe immobilized on Au electrode and result in signal variation. This method showed excellent analytical performance for dsDNA, of which the linear range was 2fM to 500pM and the detection limit was 1.6fM (S/N=3). It also showed an good results when applied to the real sample of Wnt7B gene detection.

  18. Loop Mediated Isothermal Amplification (LAMP) for Embryo Sex Determination in Pregnant Women at Eight Weeks of Pregnancy

    PubMed Central

    Almasi, Mohammad Amin; Almasi, Galavizh

    2017-01-01

    Background: In human, SRY (sex-determining region of the Y chromosome) is the major gene for the testis-determining factor which is found in normal XY males and in the rare XX males, and it is absent in normal XX females and many XY females. There are several methods which can indicate a male genotype by the presence of the amplified product of SRY gene. The aim of this study was to identify the SRY gene for embryo sex determination in human during pregnancy using loop mediated isothermal amplification (LAMP) method. Methods: A total of 15 blood samples from pregnant women at eight weeks of pregnancy were collected, and Plasma DNA was extracted. LAMP assay was performed using DNA obtained for detection of SRY gene. Furthermore, colorimetric LAMP assay for rapid and easy detection of SRY gene was developed. Results: LAMP results revealed that the positive reaction was highly specific only with samples containing XY chromosomes, while no amplification was found in samples containing XX chromosomes. A total of 15 blood samples from pregnant women were seven male embryos (46.6%) and eight female embryos (53.4%). All used visual components in the colorimetric assay could successfully make a clear distinction between positive and negative ones. Conclusion: The LAMP assay developed in this study is a valuable tool capable of monitoring the purity and detection of SRY gene for sex determination. PMID:28377900

  19. Detection of Vibrio cholerae by isothermal cross-priming amplification combined with nucleic acid detection strip analysis.

    PubMed

    Zhang, Xia; Du, Xin-Jun; Guan, Chun; Li, Ping; Zheng, Wen-Jie; Wang, Shuo

    2015-08-01

    Vibrio cholerae is a water- and food-borne human pathogen, and V. cholerae serotypes O1 and O139 have attracted attention because of their severe pathogenesis. However, non-O1, non-O139 cholera vibrios (NCVs) were also recently recognized as having virulence properties. In this study, we developed a cross-priming amplification (CPA) method for the detection of all serotypes of V. cholerae. The specificity of the CPA method was tested using a panel of 60 different bacterial strains. All of the V. cholerae strains showed positive results, and 41 other types of bacteria gave negative results. The limit of detection of the CPA method was 79.28 fg of genomic DNA, 4.2 × 10(2) CFU/ml for bacteria in pure culture, and 5.6 CFU per 25 g of sample with pre-enrichment. This method showed a higher sensitivity than the loop-mediated isothermal amplification (LAMP) method did and was more convenient to perform. These results indicate that the CPA method can be used for the rapid preliminary screening of V. cholerae.

  20. Fast detection of Southern tomato virus by one-step transcription loop-mediated isothermal amplification (RT-LAMP).

    PubMed

    Elvira-González, L; Puchades, A V; Carpino, C; Alfaro-Fernandez, A; Font-San-Ambrosio, M I; Rubio, L; Galipienso, L

    2017-03-01

    Southern tomato virus (STV) is a double stranded RNA (dsRNA) virus belonging to genus Amalgavirus (family Amalgamaviridae) which has been detected in tomato plants showing stunting, fruit discoloration and size reduction. A one-step reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of STV in total RNA or sap extracts (obtained just by grinding in buffer) from STV-infected tomato plants by using a set of three primers pairs which were designed to the sequence of the STV putative coat protein. Amplification products were visualized by gel electrophoresis or direct staining of DNA. The sensitivity of RT-LAMP was identical to that of the conventional RT-PCR and less affected by the presence of polymerase inhibitors. STV was detected by RT-LAMP in different tomato tissues, i.e. leaves, roots, fruits and seeds. Also the virus was successfully detected by RT-LAMP from sap extracts obtained from field tomato plants whereas conventional RT-PCR did not. Results of this work show that RT-LAMP is a specific, rapid and cheap procedure to detect STV and it could be implemented on field surveys and sanitation programs.

  1. 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

  2. Rapid and Sensitive Detection of Listeria ivanovii by Loop-Mediated Isothermal Amplification of the smcL Gene

    PubMed Central

    Wang, Yi; Wang, Yan; Xu, Huaqing; Dai, Hang; Meng, Shuang; Ye, Changyun

    2014-01-01

    A loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive detection of the L. ivanovii strains had been developed and evaluated in this study. Oligonucleotide primers specific for L. ivanovii species were designed corresponding to smcL gene sequences. The primers set comprise six primers targeting eight regions on the species-specific gene smcL. The LAMP assay could be completed within 1 h at 64°C in a water bath. Amplification products were directly observed by the Loopamp Fluorescent Detection Reagent (FD) or detected by agarose gel electrophoresis. Moreover, the LAMP reactions were also detected by real-time measurement of turbidity. The exclusivity of 77 non-L. ivanovii and the inclusivity of 17 L. ivanovii were both 100% in the assay. Sensitivity of the LAMP assay was 250 fg DNA and 16 CFU per reaction for detection of L. ivanovii in pure cultures and simulated human stool. The LAMP assay was 10 and 100-fold more sensitive than quantitative PCR (qPCR) and conventional PCR assays,respectively. When applied to human stool samples spiked with low level (8 CFU/0.5 g) of L. ivanovii strains, the new LAMP assay described here achieved positive detection after 6 hours enrichment. In conclusion, the new LAMP assay in this study can be used as a valuable, rapid and sensitive detection tool for the detection of L. ivanovii in field, medical and veterinary laboratories. PMID:25549337

  3. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of viral hemorrhagic septicaemia virus (VHS).

    PubMed

    Soliman, H; El-Matbouli, M

    2006-05-31

    A one step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of viral hemorrhagic septicaemia virus (VHS). A set of six primers were designed, based on the G-protein sequence of the VHS virus serotypes (He, F1, 23.75, Klapmolle and Rindsholm). The assay was optimised to amplify VHS RNA by incubation at 63 degrees C for only 1h, and required only a simple water bath or heating block to provide a constant temperature of 63 degrees C. RT-LAMP amplification products were detected by visual inspection using SYBR Green I stain and had a ladder-like appearance when electrophoresed on an agarose gel. The detection limit of the RT-LAMP assay was found to be similar to the commonly used RT-PCR method: both methods detected VHS RNA at a dilution of 10(6). The assay was evaluated using clinical samples and the results indicated the suitability and simplicity of the test as a rapid, field diagnostic tool for VHS virus.

  4. Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of lymphocystis disease virus.

    PubMed

    Li, Qiong; Yue, Zhiqin; Liu, Hong; Liang, Chengzhu; Zheng, Xiaolong; Zhao, Yuran; Chen, Xiao; Xiao, Xizhi; Chen, Changfu

    2010-02-01

    A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of lymphocystis disease virus (LCDV). A set of five specific primers, two inner and two outer primers and a loop primer, were designed on the basis of the major capsid protein gene of LCDV. The reaction time and temperatures were optimized for 60 min at 63 degrees C, respectively. LAMP amplification products were detected by a ladder-like appearance on agarose gel electrophoresis or a naked-eye inspection of a color change in the reaction tube by addition of SYBR Green I. The assay was specific for LCDV, and there was no cross-reactivity with white spot syndrome virus (WSSV) or six other Iridoviridae viruses (epizootic hematopoietic necrosis virus, EHNV; tiger frog virus, TFV; Bohle iridovirus, BIV; soft-shelled turtle iridovirus, STIV; infectious spleen and kidney necrosis virus, ISKNV; red sea bream iridovirus, RSIV). The detection limit of the LAMP assay was 15 fg, which was similar to that of real-time quantitative polymerase chain reaction (PCR) and 10-fold higher than the conventional PCR. The LAMP assay was evaluated using 109 clinical samples, and the results indicated the suitability and simplicity of the test as a rapid, field diagnostic tool for detection of LCDV. The LCDV LAMP assay has potential for early diagnosis of LCDV infection.

  5. 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.

  6. 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.

  7. One-step reverse-transcription loop-mediated isothermal amplification for detection of infectious bursal disease virus.

    PubMed

    Lee, Meng-Shiou; Lin, Yi-Chiu; Lai, Guan-Hua; Lai, Su-Yaun; Chen, Hsi-Jien; Wang, Min-Ying

    2011-04-01

    A fast, sensitive, and specific reverse-transcription (RT) loop-mediated isothermal amplification (RT-LAMP) assay was developed that involved a single tube and a 1-step reaction for detecting infectious bursal disease virus (IBDV). Four specific primers were used for amplification of the VP2 gene of IBDV. The amplified LAMP products were detected by DNA electrophoresis and by direct observation with the naked eye in the presence of SYBR Green I. The sensitivity of RT-LAMP was determined to be 0.01 fg of IBDV viral RNA. This assay for IBDV is more sensitive than the conventional RT-polymerase chain reaction assay, which has a detection limit of 1 ng. The LAMP assay was also assessed for specificity and was found to precisely discriminate between positive and negative test samples. This newly established LAMP assay, combined with RT, is a practical diagnostic tool because IBDV-infected and uninfected clinical samples collected from an experimental farm could be discriminated. Full verification of a sample's IBDV status was obtained within 40 min of extraction of the viral RNA, which could then be directly added to the RT-LAMP reaction mixture.

  8. Direct detection of various pathogens by loop-mediated isothermal amplification assays on bacterial culture and bacterial colony.

    PubMed

    Yan, Muxia; Li, Weidong; Zhou, Zhenwen; Peng, Hongxia; Luo, Ziyan; Xu, Ling

    2017-01-01

    In this work, loop-mediated isothermal amplification based detection assay using bacterial culture and bacterial colony for various common pathogens direct detection had been established, evaluated and further applied. A total of five species of common pathogens and nine detection targets (tlh, tdh and trh for V. Parahaemolyticus, rfbE, stx1 and stx2 for E. coli, oprI for P. aeruginosa, invA for Salmonella and hylA for L. monocytogenes) were performed on bacterial culture and bacterial colony LAMP. To evaluate and optimize this assay, a total of 116 standard strains were included. Then, for each detected targets, 20 random selected strains were applied. Results were determined through both visual observation of the changed color by naked eye and electrophoresis, which increased the accuracy of survey. The minimum adding quantity of each primer had been confirmed, and the optimal amplification was obtained under 65 °C for 45 min with 25 μl reaction volume. The detection limit of bacterial culture LAMP and PCR assay were determined to be 10(2) and 10(4) or 10(5) CFU/reaction, respectively. No false positive amplification was observed when subjecting the bacterial -LAMP assay to 116 reference strains. This was the first report of colony-LAMP and culture-LAMP assay, which had been demonstrated to be a fast, reliable, cost-effective and simple method on detection of various common pathogens.

  9. A new visually improved and sensitive loop mediated isothermal amplification (LAMP) for diagnosis of symptomatic falciparum malaria.

    PubMed

    Mohon, Abu Naser; Elahi, Rubayet; Khan, Wasif A; Haque, Rashidul; Sullivan, David J; Alam, Mohammad Shafiul

    2014-06-01

    Molecular diagnosis of malaria by nucleotide amplification requires sophisticated and expensive instruments, typically found only in well-established laboratories. Loop-mediated isothermal amplification (LAMP) has provided a new platform for an easily adaptable molecular technique for molecular diagnosis of malaria without the use of expensive instruments. A new primer set has been designed targeting the 18S rRNA gene for the detection of Plasmodium falciparum in whole blood samples. The efficacy of LAMP using the new primer set was assessed in this study in comparison to that of a previously described set of LAMP primers as well as with microscopy and real-time PCR as reference methods for detecting P. falciparum. Pre-addition of hydroxy napthol blue (HNB) in the LAMP reaction caused a distinct color change, thereby improving the visual detection system. The new LAMP assay was found to be 99.1% sensitive compared to microscopy and 98.1% when compared to real-time PCR. Meanwhile, its specificity was 99% and 100% in contrast to microscopy and real-time PCR, respectively. Moreover, the LAMP method was in very good agreement with microscopy and real-time PCR (0.94 and 0.98, respectively). This new LAMP method can detect at least 5parasites/μL of infected blood within 35min, while the other LAMP method tested in this study, could detect a minimum of 100parasites/μL of human blood after 60min of amplification. Thus, the new method is sensitive and specific, can be carried out in a very short time, and can substitute PCR in healthcare clinics and standard laboratories.

  10. Development of an isothermal recombinase polymerase amplification assay for rapid detection of pseudorabies virus.

    PubMed

    Yang, Yang; Qin, Xiaodong; Zhang, Wei; Li, Zhiyong; Zhang, Shuaijun; Li, Yanmin; Zhang, Zhidong

    2017-03-22

    Recombinase polymerase amplification assays using real-time fluorescent detection (real-time RPA assay) and lateral flow dipstick (RPA LFD assay) were developed targeting the gD gene of pseudorabies virus (PRV). Both assays were performed at 39 °C within 20 min. The sensitivity of the real-time RPA assay and the RPA LFD assay was 100 copies per reaction and 160 copies per reaction, respectively. Both assays did not detect DNAs from other virus or PRV negative samples. Therefore, the developed RPA assays provide a rapid, simple, sensitive and specific alternative tool for detection of PRV.

  11. 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...

  12. 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 ...

  13. 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...

  14. 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...

  15. 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.

  16. 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

  17. 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

  18. 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

    2013-06-07

    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.

  19. Development and evaluation of loop-mediated isothermal amplification assay for detection of Crimean Congo hemorrhagic fever virus in Sudan.

    PubMed

    Osman, Hana A M; Eltom, Kamal H; Musa, Nasreen O; Bilal, Nasreldin M; Elbashir, Mustafa I; Aradaib, Imadeldin E

    2013-06-01

    Crimean-Congo hemorrhagic fever (CCHF) virus (CCHFV) activity has been detected in Kordufan region of the Sudan in 2008 with high case-fatality rates in villages and rural hospitals in the region. Therefore, in the present study, a reverse transcription (RT) loop-mediated isothermal amplification (RT-LAMP) assay was developed and compared to nested RT-PCR for rapid detection of CCHFV targeting the small (S) RNA segment. A set of RT-LAMP primers, designed from a highly conserved region of the S segment of the viral genome, was employed to identify all the Sudanese CCHFV strains. The sensitivity studies indicated that the RT-LAMP detected 10fg of CCHFV RNA as determined by naked eye turbidity read out, which is more likely the way it would be read in a resource-poor setting. This level of sensitivity is good enough to detect most acute cases. Using agarose gel electrophoresis, the RT-LAMP assay detected as little as 0.1fg of viral RNA (equivalent to 50 viral particle). There was 100% agreement between results of the RT-LAMP and the nested PCR when testing 10-fold serial dilution of CCHFV RNA. The specificity studies indicated that there was no cross-reactivity with other related hemorrhagic fever viruses circulating in Sudan including, Rift Valley fever virus (RVFV), Dengue fever virus, and yellow fever virus. The RT-LAMP was performed under isothermal conditions at 63°C and no special apparatus was needed, which rendered the assay more economical and practical than real-time PCR in such developing countries, like Sudan. In addition, the RT-LAMP provides a valuable tool for rapid detection and differentiation of CCHFV during an outbreak of the disease in remote areas and in rural hospitals with resource-poor settings.

  20. One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV

    PubMed Central

    Lee, Se Hee; Baek, Yun Hee; Kim, Yang-Hoon; Choi, Young-Ki; Song, Min-Suk; Ahn, Ji-Young

    2017-01-01

    Due to the limitation of rapid development of specific antiviral drug or vaccine for novel emerging viruses, an accurate and rapid diagnosis is a key to manage the virus spread. We developed an efficient and rapid method with high specificity for the Middle East Respiratory Syndrome coronavirus (MERS-CoV), based on one-pot reverse transcription loop-mediated isothermal amplification (one-pot RT-LAMP). A set of six LAMP primers [F3, B3, FIP, BIP, LF (Loop-F), and LB (Loop-B)] were designed using the sequence of nucleocapsid (N) gene with optimized RT-LAMP enzyme conditions: 100 U M-MLV RTase and 4 U Bst polymerase, implying that the reaction was able to detect four infectious viral genome copies of MERS-CoV within a 60 min reaction time period. Significantly, EvaGreen dye has better signal read-out properties in one-pot RT-LAMP reaction and is more compatible with DNA polymerase than SYBR green I. Isothermally amplified specific N genes were further evaluated using field-deployable microchamber devices, leading to the specific identification of as few as 0.4 infectious viral genome copies, with no cross-reaction to the other acute respiratory disease viruses, including influenza type A (H1N1 and H3N2), type B, human coronavirus 229E, and human metapneumovirus. This sensitive, specific and feasible method provides a large-scale technical support in emergencies, and is also applied as a sample-to-detection module in Point of Care Testing devices. PMID:28119682

  1. One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV.

    PubMed

    Lee, Se Hee; Baek, Yun Hee; Kim, Yang-Hoon; Choi, Young-Ki; Song, Min-Suk; Ahn, Ji-Young

    2016-01-01

    Due to the limitation of rapid development of specific antiviral drug or vaccine for novel emerging viruses, an accurate and rapid diagnosis is a key to manage the virus spread. We developed an efficient and rapid method with high specificity for the Middle East Respiratory Syndrome coronavirus (MERS-CoV), based on one-pot reverse transcription loop-mediated isothermal amplification (one-pot RT-LAMP). A set of six LAMP primers [F3, B3, FIP, BIP, LF (Loop-F), and LB (Loop-B)] were designed using the sequence of nucleocapsid (N) gene with optimized RT-LAMP enzyme conditions: 100 U M-MLV RTase and 4 U Bst polymerase, implying that the reaction was able to detect four infectious viral genome copies of MERS-CoV within a 60 min reaction time period. Significantly, EvaGreen dye has better signal read-out properties in one-pot RT-LAMP reaction and is more compatible with DNA polymerase than SYBR green I. Isothermally amplified specific N genes were further evaluated using field-deployable microchamber devices, leading to the specific identification of as few as 0.4 infectious viral genome copies, with no cross-reaction to the other acute respiratory disease viruses, including influenza type A (H1N1 and H3N2), type B, human coronavirus 229E, and human metapneumovirus. This sensitive, specific and feasible method provides a large-scale technical support in emergencies, and is also applied as a sample-to-detection module in Point of Care Testing devices.

  2. Rapid and specific detection of porcine parvovirus by isothermal recombinase polymerase amplification assays.

    PubMed

    Yang, Yang; Qin, Xiaodong; Zhang, Wei; Li, Yanmin; Zhang, Zhidong

    2016-10-01

    Porcine parvovirus (PPV) is a major cause of swine reproductive failure and reported in many countries worldwide. Recombinase polymerase amplification (RPA) assays using a real-time fluorescent detection (PPV real-time RPA assay) and a lateral flow dipstick (PPV RPA LFD assay) were developed targeting PPV NS1 gene. The detection limit of PPV real-time RPA assay was 300 copies per reaction within 9 min at 38 °C, while the RPA LFD assay has a detection limit of 400 copies per reaction in less than 20 min at 38 °C. In both assays, there were no cross-reactions with porcine circovirus type 2, pseudorabies virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus, and foot-and-mouth disease virus. Based on a total of 128 clinical samples examined, the sensitivity and the specificity of the developed RPA assays for identification of PPV was 94.4% and 100%, respectively, when compared to real-time (qPCR) assay. Therefore, the RPA assay provides a rapid, sensitive and specific alternative for PPV detection.

  3. 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.

  4. 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.

  5. Rapid detection of Vibrio parahaemolyticus in raw oysters using immunomagnetic separation combined with loop-mediated isothermal amplification.

    PubMed

    Zeng, Jing; Wei, Haiyan; Zhang, Lei; Liu, Xuefeng; Zhang, Haiyu; Cheng, Jinxia; Ma, Dan; Zhang, Ximeng; Fu, Pubo; Liu, Li

    2014-03-17

    The objective of this study was to develop a method that combined nanoparticle-based immunomagnetic separation (IMS) with real-time loop-mediated isothermal amplification (LAMP) for the rapid detection of Vibrio parahaemolyticus. Magnetic nanoparticles were functionalized with monoclonal antibodies that were produced against flagella from V. parahaemolyticus to capture and separate the target cells from raw oysters. After optimization, the immunomagnetic nanoparticles (IMNPs) presented a capture efficiency of 87.3% for 10(5) colony-forming unit (CFU)/mL of V. parahaemolyticus using 2.5μg of IMNPs within 30min. Although a very low level of non-specific binding was seen among 8 non-V. parahaemolyticus Vibrio spp. and 5 non-Vibrio strains, the IMS-LAMP method identified 133 V. parahaemolyticus strains correctly without the amplification from 54 other strains. The detection limit was about 1.4×10(2)CFU/mL in pure culture and was unaffected by the presence of 10(8)CFU/mL of competing microflora. When applied in spiked oysters, the sensitivity was found to be 1.9×10(3)CFU/g without enrichment. After enrichment for 6-8h, the limit of detectability could be improved to 1.9 to 0.19CFU/g. Hence, the IMS-LAMP assay provided a rapid, simple, and cost-effective method for total V. parahaemolyticus detection. This method will have important implications in the rapid detection of contaminated food in the early stage before distribution.

  6. 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

  7. Most probable number - loop mediated isothermal amplification (MPN-LAMP) for quantifying waterborne pathogens in <25min.

    PubMed

    Ahmad, Farhan; Stedtfeld, Robert D; Waseem, Hassan; Williams, Maggie R; Cupples, Alison M; Tiedje, James M; Hashsham, Syed A

    2017-01-01

    We are reporting a most probable number approach integrated to loop mediated isothermal technique (MPN-LAMP) focusing on Gram-negative Escherichia coli and Gram-positive Enterococcus faecalis bacterial cells without nucleic acids extraction. LAMP assays for uidA from E. coli and gelE from E. faecalis were successfully performed directly on cells up to single digit concentration using a commercial real time PCR instrument. Threshold time values of LAMP assays of bacterial cells, heat treated bacterial cells (95°C for 5min), and their purified genomic DNA templates were similar, implying that amplification could be achieved directly from bacterial cells at 63°C. Viability of bacterial cells was confirmed by using propidium monoazide in a LAMP assay with E. faecalis. To check its functionality on a microfluidic platform, MPN-LAMP assays targeting <10CFU of bacteria were also translated onto polymeric microchips and monitored by a low-cost fluorescence imaging system. The overall system provided signal-to-noise (SNR) ratios up to 800, analytical sensitivity of <10CFU, and time to positivity of about 20min. MPN-LAMP assays were performed for cell concentrations in the range of 10(5)CFU to <10CFU. MPN values from LAMP assays confirmed that the amplifications were from <10CFU. The method described here, applicable directly on cells at 63°C, eliminates the requirement of complex nucleic acids extraction steps, facilitating the development of sensitive, rapid, low-cost, and field-deployable systems. This rapid MPN-LAMP approach has the potential to replace conventional MPN method for waterborne pathogens.

  8. Evaluation of loop-mediated isothermal amplification for the rapid identification of bacteria and resistance determinants in positive blood cultures.

    PubMed

    Rödel, J; Bohnert, J A; Stoll, S; Wassill, L; Edel, B; Karrasch, M; Löffler, B; Pfister, W

    2017-01-06

    The use of molecular assays to rapidly identify pathogens and resistance genes directly from positive blood cultures (BCs) contribute to shortening the time required for the diagnosis of bloodstream infections. In this work, loop-mediated isothermal amplification (LAMP) assays have been examined for their potential use in BC diagnosis. Three different assays were applied. The commercially available eazyplex® MRSA test detects Staphylococcus aureus, S. epidermidis, mecA, and mecC. Two in-house assays [Gram-positive (GP) and Gram-negative (GN)] have been developed for the detection of streptococci, enterococci, vanA, vanB, Pseudomonas spp., Enterobacteriaceae, and the bla CTX-M family. A total of 370 positive BCs were analyzed. LAMP test results were obtained within 30 min, including sample preparation. Amplification was measured by real-time fluorescence detection. The threshold time for fluorescence intensity values ranged from 6.25 to 13.75 min. The specificity and sensitivity of the assays varied depending on the target. Overall, from 87.7% of BCs, true-positive results were obtained, compared to routine standard diagnosis. Twenty-one tests were true-negative because of the lack of an appropriate target (5.7%). The concordance of positive test results for resistance genes with subsequent antibiotic susceptibility testing was 100%. From 15 BC bottles with mixed cultures, eazyplex® assays produced correct results in 73% of the cases. This study shows that LAMP assays are fast and cost-saving tools for rapid BC testing in order to expedite the diagnostic report and improve the antibiotic stewardship for sepsis patients.

  9. Comparison of the Efficacies of Loop-Mediated Isothermal Amplification, Fluorescence Smear Microscopy and Culture for the Diagnosis of Tuberculosis

    PubMed Central

    George, Geojith; Mony, Prem; Kenneth, John

    2011-01-01

    Background Despite the advent of novel diagnostic techniques, smear microscopy remains as the most practical test available in resource-limited settings for tuberculosis (TB) diagnosis. Due to the low sensitivity of microscopy and the long time required for culture, feasible and accessible rapid diagnostic methods are urgently needed. Loop-mediated Isothermal Amplification (LAMP) is a promising nucleic-acid amplification assay, which could be accessible, cost-effective and more suited for use with unpurified samples. Methodology/Principal Findings In the current study, the objective was to assess the efficacy of a LAMP assay for tuberculosis compared with fluorescence smear microscopy as well as Löwenstein-Jensen (LJ) and Mycobacteria Growth Indicator Tube (MGIT) cultures for the diagnosis of pulmonary tuberculosis using sputum samples. Smear microscopy and culture were performed for decontaminated and concentrated sputum from TB suspects and the LAMP was also performed on these specimens. The LAMP and smear microscopy were compared, in series and in parallel, to culture. LAMP and smear microscopy showed sensitivities of 79.5% and 82.1% respectively and specificities of 93.8% and 96.9% respectively, compared to culture. LAMP and smear in series had sensitivity and specificity of 79.5% and 100.0% respectively. LAMP and smear in parallel had sensitivity and specificity of 82.1% and 90.6% respectively. Conclusions/Significance The overall efficacies of LAMP and fluorescence smear microscopy in the current study were high and broadly similar. LAMP and smear in series had high specificity (100.0%) and can be used as a rule-in test combination. However, the performance of LAMP in smear negative samples was found to be insufficient. PMID:21695047

  10. 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.

  11. Development of a toxR-based loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus

    PubMed Central

    2010-01-01

    Background Vibrio parahaemolyticus is a leading cause of seafood-related bacterial gastroenteritis and outbreaks worldwide. Sensitive and specific detection methods are needed to better control V. parahaemolyticus infections. This study aimed at developing a highly specific and sensitive loop-mediated isothermal amplification (LAMP) assay for detecting V. parahaemolyticus in oysters. A set of five LAMP primers, two outer, two inner, and one loop were designed based on the published V. parahaemolyticus toxR sequence. Specificity of the assay was evaluated using a panel of 36 V. parahaemolyticus and 39 other strains. The assay sensitivity was determined using serial dilutions of V. parahaemolyticus ATCC 27969 culture ranging from 108 CFU/ml to extinction. The assay was also tested in experimentally inoculated oyster samples. Results The toxR-based LAMP assay was able to specifically detect all of the 36 V. parahaemolyticus strains without amplification from 39 other strains. The detection limit was 47-470 cells per reaction in pure culture, up to 100-fold more sensitive than that of toxR-PCR. When applied in spiked oysters, the assay was able to detect 1.1 × 105 V. parahaemolyticus cells per gram of oyster without enrichment, up to 100-fold more sensitive than that of toxR-PCR. Standard curves generated for detecting V. parahaemolyticus in both pure culture and spiked oyster samples showed good linear relationship between cell numbers and the fluorescence or turbidity signals. Conclusions The toxR-based LAMP assay developed in this study was sensitive, specific, and quantitative, holding great potential for future field detection of V. parahaemolyticus in raw oysters. PMID:20146814

  12. 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

  13. Precipitation of PEG/Carboxyl-Modified Gold Nanoparticles with Magnesium Pyrophosphate: A New Platform for Real-Time Monitoring of Loop-Mediated Isothermal Amplification.

    PubMed

    Qin, Ailin; Fu, Lok Tin; Wong, Jacky K F; Chau, Li Yin; Yip, Shea Ping; Lee, Thomas M H

    2017-03-29

    Gold nanoparticles have proven to be promising for decentralized nucleic acid testing by virtue of their simple visual readout and absorbance-based quantification. A major challenge toward their practical application is to achieve ultrasensitive detection without compromising simplicity. The conventional strategy of thermocycling amplification is unfavorable (because of both instrumentation and preparation of thermostable oligonucleotide-modified gold nanoparticle probes). Herein, on the basis of a previously unreported co-precipitation phenomenon between thiolated poly(ethylene glycol)/11-mercaptoundecanoic acid co-modified gold nanoparticles and magnesium pyrophosphate crystals (an isothermal DNA amplification reaction byproduct), a new ultrasensitive and simple DNA assay platform is developed. The binding mechanism underlying the co-precipitation phenomenon is found to be caused by the complexation of carboxyl and pyrophosphate with free magnesium ions. Remarkably, poly(ethylene glycol) does not hinder the binding and effectively stabilizes gold nanoparticles against magnesium ion-induced aggregation (without pyrophosphate). In fact, a similar phenomenon is observed in other poly(ethylene glycol)- and carboxyl-containing nanomaterials. When the gold nanoparticle probe is incorporated into a loop-mediated isothermal amplification reaction, it remains as a red dispersion for a negative sample (in the absence of a target DNA sequence) but appears as a red precipitate for a positive sample (in the presence of a target). This results in a first-of-its-kind gold nanoparticle-based DNA assay platform with isothermal amplification and real-time monitoring capabilities.

  14. Molecular epidemiology and a loop-mediated isothermal amplification method for diagnosis of infection with rabies virus in Zambia.

    PubMed

    Muleya, Walter; Namangala, Boniface; Mweene, Aaron; Zulu, Luke; Fandamu, Paul; Banda, Douglas; Kimura, Takashi; Sawa, Hirofumi; Ishii, Akihiro

    2012-01-01

    The National Livestock Epidemiology and Information Center (NALEIC) in Zambia reported over 132 cases of canine rabies diagnosed by the direct fluorescent antibody test (DFAT) from 2004 to 2009. In this study, the lineage of rabies virus (RABV) in Zambia was determined by phylogenetic analysis of the nucleoprotein (N) and glycoprotein (G) gene sequences. Total RNA was extracted from 87-DFAT brain specimens out of which only 35 (40%) were positive on nested reverse transcription polymerase chain reaction (RT-PCR) for each gene, and 26 being positive for both genes. Positive specimens for the N (n=33) and G (n=35) genes were used for phylogenetic analysis. Phylogenetic analysis of the N gene showed two phylogenetic clusters in Zambia belonging to the Africa 1b lineage present in eastern and southern Africa. While one cluster exclusively comprised Zambian strains, the other was more heterogeneous regarding the RABV origins and included strains from Tanzania, Mozambique and Zambia. Phylogenetic analysis of the G gene revealed similar RABV strains in different hosts and regions of Zambia. We designed primers for reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay from the consensus sequence of the N gene in an attempt to improve the molecular diagnosis of RABV in Zambia. The specificity and reproducibility of the RT-LAMP assay was confirmed with actual clinical specimens. Therefore, the RT-LAMP assay presented in this study may prove to be useful for routine diagnosis of rabies in Zambia.

  15. Rapid genotyping of carcinogenic human papillomavirus by loop-mediated isothermal amplification using a new automated DNA test (Clinichip HPV™).

    PubMed

    Satoh, Toyomi; Matsumoto, Koji; Fujii, Takuma; Sato, Osamu; Gemma, Nobuhiro; Onuki, Mamiko; Saito, Hiroshi; Aoki, Daisuke; Hirai, Yasuo; Yoshikawa, Hiroyuki

    2013-03-01

    This study was designed to evaluate the Clinichip HPV test, a new DNA test that detects carcinogenic human papillomavirus (HPV) rapidly by loop-mediated isothermal amplification and performs genotyping of all 13 carcinogenic types using automated DNA chip technology with an assay time 2.5h. Using this test, 247 Japanese women (109 with normal cytology, 43 with cervical intraepithelial neoplasia grade 1, 60 with cervical intraepithelial neoplasia grade 2/3 and 35 with invasive cervical cancer) were tested for carcinogenic HPV genotypes. The results were compared to those obtained by the polymerase chain reaction-amplified DNA sequencing using 13 type-specific primers. Overall, there was very good agreement for the detection of carcinogenic HPV between the Clinichip test and direct sequencing, with 95.5% total agreement and a kappa value of 0.91. Comparison of the detection of individual HPV types shows that the overall agreement was also high (range: 96.8-100%). In women with cervical intraepithelial neoplasia grade 2 or worse, the detection rate of carcinogenic HPV was 95.7% by both the Clinichip test and the direct-sequencing method, indicating complete agreement between the two methods. In conclusion, it was found that the Clinichip test is a promising new laboratory method for genotyping of carcinogenic HPV.

  16. Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Ralstonia solanacearum Phylotype I Mulberry Strains in China

    PubMed Central

    Huang, Wen; Zhang, Hao; Xu, Jingsheng; Wang, Shuai; Kong, Xiangjiu; Ding, Wei; Xu, Jin; Feng, Jie

    2017-01-01

    Ralstonia solanacearum phylotype I mulberry strains are causative agent of bacterial wilt of mulberry. Current diagnostic methods are not adopted to the mulberry wilt disease. In this study, we developed a rapid method, loop-mediated isothermal amplification (LAMP), to detect R. solanacearum phylotype I mulberry strains. A set of six primers was designed to target the clone MG67 sequence in this LAMP detection which can be completed in 20 min at 64°C. The results of the LAMP reaction could be observed with the naked eye due to magnesium pyrophosphate precipitate produced during the reaction or the color change after adding SYBR Green I. The specificity of the LAMP was confirmed using DNA from 46 representative strains of R. solanacearum and 7 other soil-borne bacteria strains. This method was also of high sensitivity and could be used to detect the presence of less than 160 fg genomic DNA or 2.2 × 102 CFU/ml of bacterial cells per 25 μl reaction volume, moreover, the presence of plant tissue fluid did not affect the sensitivity. Since it does not require expensive equipment or specialized techniques, this LAMP-based diagnostic method has the potential to be used under field conditions to make disease forecasting more accurate and efficient. PMID:28197157

  17. 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

  18. 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

  19. Rapid detection of the Marek's disease viral genome in chicken feathers by loop-mediated isothermal amplification.

    PubMed

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

    2012-03-01

    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.

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

    PubMed

    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.

  1. Rapid, Sensitive Detection of Bartonella quintana by Loop-Mediated Isothermal Amplification of the groEL Gene

    PubMed Central

    Hu, Shoukui; Niu, Lina; Luo, Lijuan; Song, Xiuping; Sun, Jimin; Liu, Qiyong

    2016-01-01

    Trench fever, caused by Bartonella quintana, is recognized as a re-emerging and neglected disease. Rapid and sensitive detection approaches are urgently required to monitor and help control B. quintana infections. Here, loop-mediated isothermal amplification (LAMP), which amplifies target DNA at a fixed temperature with high sensitivity, specificity and rapidity, was employed to detect B. quintana. Thirty-six strains, including 10 B. quintana, 13 other Bartonella spp., and 13 other common pathogens, were applied to verify and evaluate the LAMP assay. The specificity of the LAMP assay was 100%, and the limit of detection was 125 fg/reaction. The LAMP assay was compared with qPCR in the examination of 100 rhesus and 20 rhesus-feeder blood samples; the diagnostic accuracy was found to be 100% when LAMP was compared to qPCR, but the LAMP assay was significantly more sensitive (p < 0.05). Thus, LAMP methodology is a useful for diagnosis of trench fever in humans and primates, especially in low-resource settings, because of its rapid, sensitive detection that does not require sophisticated equipment. PMID:27916953

  2. Application of loop-mediated isothermal amplification (LAMP)-based technology for authentication of Catharanthus roseus (L.) G. Don.

    PubMed

    Chaudhary, Anis Ahmad; Hemant; Mohsin, Mohd; Ahmad, Altaf

    2012-04-01

    In this study, loop-mediated isothermal amplification (LAMP)-based molecular marker was developed for authentication of Catharanthus roseus, a medicinal plant. Samples of this plant were collected from different geographical locations in India. Random amplified polymorphic deoxyribonucleic acid (DNA) analysis of collected samples was carried out with 25 random primers. A 610-bp DNA fragment, common to all accessions, was eluted, cloned, and sequenced. Four LAMP primers were designed on the basis of sequence of 610 bp DNA fragment. LAMP reaction, containing 10× Bst DNA polymerase reaction buffer, Bst DNA polymerase, four in-house designed primers, dNTPs, MgSO(4), and betaine, was incubated at 65°C for 1 h. The resulting amplicon was visualized by adding SYBR Green I to the reaction tube. The data showed confirmatory results. Since the assay method is simple, sensitive, and cost-effective, it is a feasible method for identifying and authentication of C. roseus.

  3. 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

    2015-11-26

    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.

  4. A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cyprinid Herpesvirus 2 in Gibel Carp (Carassius auratus gibelio)

    PubMed Central

    Zhang, Hui; Zeng, Lingbing; Fan, Yuding; Zhou, Yong; Xu, Jin; Ma, Jie

    2014-01-01

    A rapid and sensitive loop-mediated isothermal amplification (LAMP) assay for Cyprinid herpesvirus 2 (CyHV-2) detection in gibel carp was developed. Following cloning and sequencing of the putative DNA helicase gene of CyHV-2 isolate from China, a set of four specific primers was designed based on the sequence. The MgCl2 concentration and the reaction temperature were optimized to 6 mM, 64°C, respectively. LAMP products were detected by visual inspection of a color change due to addition of SYBR Green I stain. The specificity and sensitivity of the LAMP assay were determined. No cross-reaction was observed with other fish DNA viruses including eel herpesvirus, koi herpesvirus, and Chinese giant salamander iridovirus. The LAMP assay was found to be equally sensitive as nested PCR. A comparative evaluation of 10 fish samples using LAMP and nested PCR assays showed an overall correlation in positive and negative results for CyHV-2. These results indicate that the LAMP assay is simple, sensitive, and specific and has a great potential use for CyHV-2 detection in the laboratory and field. PMID:24574914

  5. Development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of ostreid herpesvirus 1 DNA.

    PubMed

    Ren, Weicheng; Renault, Tristan; Cai, Yuyong; Wang, Chongming

    2010-12-01

    A loop-mediated isothermal amplification (LAMP) assay was developed for rapid, specific and sensitive detection of ostreid herpesvirus 1 (OsHV-1) DNA. A set of four primers was designed, based on the sequence of the ATPase subunit of the OsHV-1 DNA-packaging terminase gene. The reaction temperature and time were optimized to 64°C and 60min, 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 OsHV-1, and no cross-reaction was observed with other DNA viruses, such as White spot syndrome virus (WSSV), Penaeus stylirostris densovirus (PstDNV), Turbot reddish body iridovirus (TRBIV) and Lymphocystis disease virus (LCDV) found commonly in China. The lower detection limit of the LAMP assay was approximately 20 copies per reaction, and it was 100 times more sensitive than that of conventional PCR. A comparative evaluation of 10 oyster samples using LAMP and PCR assays showed overall correlation in positive and negative results for OsHV-1. These results indicate that the LAMP assay is a simple, rapid, sensitive, specific and reliable technique for the detection of OsHV-1. The LAMP technique has capacity for use for the detection of OsHV-1 both in the laboratory and on farms.

  6. Detection of Trypanosoma cruzi and T. rangeli Infections from Rhodnius pallescens Bugs by Loop-Mediated Isothermal Amplification (LAMP)

    PubMed Central

    Thekisoe, Oriel M. M.; Rodriguez, Carol V.; Rivas, Francisco; Coronel-Servian, Andrea M.; Fukumoto, Shinya; Sugimoto, Chihiro; Kawazu, Shin-Ichiro; Inoue, Noboru

    2010-01-01

    We have developed two loop-mediated isothermal amplification (LAMP) assays for specific detection of Trypanosoma cruzi and Trypanosoma rangeli based on the 18S ribosomal RNA (rRNA) and the small nucleolar RNA (snoRNA) genes, respectively. The detection limit of the assays is 100 fg and 1 pg for T. cruzi and T. rangeli, respectively, with reactions conducted in 60 minutes. The two LAMP assays were used in detection of T. cruzi and T. rangeli infections in comparison with polymerase chain reaction (PCR) for DNA samples extracted from Rhodnius pallescens bugs collected from palm trees in Panama. Out of a total of 52 DNA samples from R. pallescens bugs 17 (33%) and 14 (27%) were T. cruzi-positive by LAMP and PCR, respectively, while, 7 (13%) and 4 (8%) were T. rangeli-positive by LAMP and PCR, respectively. Further evaluation of these LAMP assays is needed, especially with specimens collected from human patients as well as blood kept for transfusion purposes. PMID:20439966

  7. Loop-mediated isothermal amplification (LAMP) assays for detection and identification of aquaculture pathogens: current state and perspectives.

    PubMed

    Biswas, Gouranga; Sakai, Masahiro

    2014-04-01

    Since its invention in 2000, loop-mediated isothermal amplification (LAMP) assay has been one of the most extensively used molecular diagnostic tools in bio-medical fields due to the rapidity, accuracy, and cost-effectiveness of the technique. This technique has also earned popularity in aquaculture disease diagnosis. Aquaculture, as a result of its rapid intensification and expansion, experiences increased infectious disease occurrences. For maintenance of economic viability, rapid, sensitive and efficient diagnosis of disease causing agents is an important step prior to undertaking effective prevention and control measures in aquaculture. Constraints on time and expertise required for conventional biochemical, serological and polymerase chain reaction (PCR)-based techniques offer avenues in adoption of the LAMP by the aquaculturists at field conditions. This assay has been successfully applied in detection of several bacterial, viral and parasitic pathogens causing serious diseases in aquaculture. In this review, we endeavored to accommodate the LAMP methodology with its different recent improvements and an overview of its application for the detection of aquaculture-associated pathogens.

  8. 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.

  9. 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

  10. 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.

  11. Real-Time Reverse-Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of Rift Valley Fever Virus▿

    PubMed Central

    Peyrefitte, Christophe N.; Boubis, Laetitia; Coudrier, Daniel; Bouloy, Michèle; Grandadam, Marc; Tolou, Hugues J.; Plumet, Sébastien

    2008-01-01

    The development and validation of a one-step, single-tube, real-time accelerated reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of the L RNA segment of Rift Valley fever virus (RVFV) are described. The assay was performed at a constant temperature (63°C), with a real-time follow-up using a LightCycler and a double-stranded-DNA-intercalating fluorochrome. The assay is highly sensitive and comparable to real-time RT-PCR, with a detection limit of ∼10 RNA copies per assay. However, the RT-LAMP assay is much faster than traditional RT-PCR and generates results in <30 min for most diluted samples. The specificity of the primers was established using other, related arboviruses as well as virus-containing and virus-free sera. The RT-LAMP assay reported here is thus a valuable tool for the rapid detection of RVFV in field diagnostic laboratories. PMID:18799705

  12. 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

  13. Diagnostic test accuracy of loop-mediated isothermal amplification assay for Mycobacterium tuberculosis: systematic review and meta-analysis

    PubMed Central

    Nagai, Kenjiro; Horita, Nobuyuki; Yamamoto, Masaki; Tsukahara, Toshinori; Nagakura, Hideyuki; Tashiro, Ken; Shibata, Yuji; Watanabe, Hiroki; Nakashima, Kentaro; Ushio, Ryota; Ikeda, Misako; Narita, Atsuya; Kanai, Akinori; Sato, Takashi; Kaneko, Takeshi

    2016-01-01

    Diagnostic test accuracy of the loop-mediated isothermal amplification (LAMP) assay for culture proven tuberculosis is unclear. We searched electronic databases for both cohort and case-control studies that provided data to calculate sensitivity and specificity. The index test was any LAMP assay including both commercialized kits and in-house assays. Culture-proven M. tuberculosis was considered a positive reference test. We included 26 studies on 9330 sputum samples and one study on 315 extra-pulmonary specimens. For sputum samples, 26 studies yielded the summary estimates of sensitivity of 89.6% (95% CI 85.6–92.6%), specificity of 94.0% (95% CI 91.0–96.1%), and a diagnostic odds ratio of 145 (95% CI 93–226). Nine studies focusing on Loopamp MTBC yielded the summary estimates of sensitivity of 80.9% (95% CI 76.0–85.1%) and specificity of 96.5% (95% CI 94.7–97.7%). Loopamp MTBC had higher sensitivity and lower specificity for smear-positive sputa compared to smear-negative sputa. In-house assays showed higher sensitivity and lower specificity compared to Loopamp MTBC. LAMP promises to be a useful test for the diagnosis of TB, however there is still need to improve the assay to make it simpler, cheaper and more efficient to make it competitive against other PCR methods already available. PMID:27958360

  14. 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

  15. Loop-mediated isothermal amplification combined with PCR and immunohistochemistry for detecting Porphyromonas gingivalis in periapical periodontitis.

    PubMed

    Kitano, Taiichi; Mikami, Yoshikazu; Iwase, Takashi; Asano, Masatake; Komiyama, Kazuo

    2016-01-01

    Porphyromonas gingivalis is important in the development of marginal periodontitis. However, the precise role and localization of P. gingivalis in chronic periapical periodontitis remain unclear. Thus, methods that can detect P. gingivalis in formalin-fixed and paraffin-embedded (FFPE) tissue samples are needed. We assessed a technique combining loop-mediated isothermal amplification (LAMP) with PCR (PCR-LAMP) for detection of P. gingivalis, using 110 FFPE tissue samples of chronic apical periodontitis. PCR-LAMP specifically detected P. gingivalis with high sensitivity in FFPE tissue samples, and the sensitivity of the technique was higher than that of PCR or LAMP alone. The results of immunohistochemistry (IHC) confirmed the specificity of PCR-LAMP. IHC showed that P. gingivalis was localized in a granular layer of chronic apical periodontitis, a region that correlated with the localization of macrophages. This is the first study to describe the localization of P. gingivalis in human periapical periodontitis. In conclusion, PCR-LAMP was an effective tool for detecting P. gingivalis in periapical periodontitis. In addition, IHC results improve our understanding of the role of P. gingivalis in the progression of periapical periodontitis. (J Oral Sci 58, 163-169, 2016).

  16. 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.

  17. 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.

  18. 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

  19. [Oligonucleotide derivatives in the nucleic acid hybridization analysis. II. Isothermal signal amplification in process of DNA analysis by minisequencing].

    PubMed

    Dmitrienko, E V; Khomiakova, E A; Pyshnaia; Bragin, A G; Vedernikov, V E; Pyshnyĭ, D V

    2010-01-01

    The isothermal amplification of reporter signal via limited probe extension (minisequencing) upon hybridization of nucleic acids has been studied. The intensity of reporter signal has been shown to increase due to enzymatic labeling of multiple probes upon consecutive hybridization with one DNA template both in homophase and heterophase assays using various kinds of detection signal: radioisotope label, fluorescent label, and enzyme-linked assay. The kinetic scheme of the process has been proposed and kinetic parameters for each step have been determined. The signal intensity has been shown to correlate with physicochemical characteristics of both complexes: probe/DNA and product/DNA. The maximum intensity has been observed at minimal difference between the thermodynamic stability of these complexes, provided the reaction temperature has been adjusted near their melting temperature values; rising or lowering the reaction temperature reduces the amount of reporting product. The signal intensity has been shown to decrease significantly upon hybridization with the DNA template containing single-nucleotide mismatches. Limited probe extension assay is useful not only for detection of DNA template but also for its quantitative characterization.

  20. 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

  1. 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.

  2. Microfluidic lab-on-a-foil for nucleic acid analysis based on isothermal recombinase polymerase amplification (RPA).

    PubMed

    Lutz, Sascha; Weber, Patrick; Focke, Max; Faltin, Bernd; Hoffmann, Jochen; Müller, Claas; Mark, Daniel; Roth, Günter; Munday, Peter; Armes, Niall; Piepenburg, Olaf; Zengerle, Roland; von Stetten, Felix

    2010-04-07

    For the first time we demonstrate a self-sufficient lab-on-a-foil system for the fully automated analysis of nucleic acids which is based on the recently available isothermal recombinase polymerase amplification (RPA). The system consists of a novel, foil-based centrifugal microfluidic cartridge including prestored liquid and dry reagents, and a commercially available centrifugal analyzer for incubation at 37 degrees C and real-time fluorescence detection. The system was characterized with an assay for the detection of the antibiotic resistance gene mecA of Staphylococcus aureus. The limit of detection was <10 copies and time-to-result was <20 min. Microfluidic unit operations comprise storage and release of liquid reagents, reconstitution of lyophilized reagents, aliquoting the sample into < or = 30 independent reaction cavities, and mixing of reagents with the DNA samples. The foil-based cartridge was produced by blow-molding and sealed with a self-adhesive tape. The demonstrated system excels existing PCR based lab-on-a-chip platforms in terms of energy efficiency and time-to-result. Applications are suggested in the field of mobile point-of-care analysis, B-detection, or in combination with continuous monitoring systems.

  3. Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Ralstonia solanacearum Phylotype I Mulberry Strains in China.

    PubMed

    Huang, Wen; Zhang, Hao; Xu, Jingsheng; Wang, Shuai; Kong, Xiangjiu; Ding, Wei; Xu, Jin; Feng, Jie

    2017-01-01

    Ralstonia solanacearum phylotype I mulberry strains are causative agent of bacterial wilt of mulberry. Current diagnostic methods are not adopted to the mulberry wilt disease. In this study, we developed a rapid method, loop-mediated isothermal amplification (LAMP), to detect R. solanacearum phylotype I mulberry strains. A set of six primers was designed to target the clone MG67 sequence in this LAMP detection which can be completed in 20 min at 64°C. The results of the LAMP reaction could be observed with the naked eye due to magnesium pyrophosphate precipitate produced during the reaction or the color change after adding SYBR Green I. The specificity of the LAMP was confirmed using DNA from 46 representative strains of R. solanacearum and 7 other soil-borne bacteria strains. This method was also of high sensitivity and could be used to detect the presence of less than 160 fg genomic DNA or 2.2 × 10(2) CFU/ml of bacterial cells per 25 μl reaction volume, moreover, the presence of plant tissue fluid did not affect the sensitivity. Since it does not require expensive equipment or specialized techniques, this LAMP-based diagnostic method has the potential to be used under field conditions to make disease forecasting more accurate and efficient.

  4. Rapid and Sensitive Detection of sFAT-1 Transgenic Pigs by Visual Loop-Mediated Isothermal Amplification.

    PubMed

    Tao, Chenyu; Yang, Yalan; Li, Xunbi; Zheng, Xinmin; Ren, Hongyan; Li, Kui; Zhou, Rong

    2016-07-01

    Genetically modified (GM) livestock have the potential to contribute to improving the environment and human health, with consumption of fewer resources and reduced waste production. However, the transgene process also poses risks. The safety assessment and control of transgenic animal products have drawn wide attention, and the relevant regulations and technology are being developed. Quick testing technology plays a significant role in on-site and customs sampling. Nowadays, loop-mediated isothermal amplification (LAMP) was widely applied in nucleic acid analysis because of its simplicity, rapidity, high efficiency and specificity. In this study, a specific, sensitive detection system for detecting sFAT-1 transgenic pigs was designed. A set of six primers including two loop primers was designed for the target sequence. The DNA samples were amplified in less than 1 h at the optimized temperature and detecting by both Nephelometer LA-320c and unaided eyes directly adding calcein. The detection limit of sFAT-1 LAMP was as low as 1.26 ng/μL. Furthermore, blind tests of transgenic and non-transgenic DNA samples were all correctly detected. Hence, the results in this study demonstrated that LAMP is a very useful tool for transgenic detection.

  5. Detection and identification of Trichophyton tonsurans from clinical isolates and hairbrush samples by loop-mediated isothermal amplification system.

    PubMed

    Yo, Ayaka; Yamamoto, Mikachi; Nakayama, Takako; Ishikawa, Jun; Makimura, Koichi

    2016-09-01

    Since the 1990s, there have been reports of the spread of dermatophytosis caused by Trichophyton tonsurans among contact sports athletes in several countries, including Japan. This study was performed to develop a loop-mediated isothermal amplification (LAMP) system for rapid and accurate detection and identification of T. tonsurans from clinical isolates or hairbrush samples for diagnosis and to prevent the spread of infection. A specific primer set was prepared by comparing the whole genome sequence of T. tonsurans with those of six other closely related dermatophytes. After confirming the sensitivity and specificity of this system, LAMP assay was performed using 37 clinical samples obtained from three healthy volunteers and 24 judo athletes. A total of 155 fungal isolates (56 strains of various standard fungi, 96 identified T. tonsurans isolates, three hairbrush-cultured isolates from judo athletes) and 37 hairbrush samples (34 samples from 24 judo athletes, and three samples from three healthy volunteers) were used for culture and LAMP assay, respectively. The assay showed no cross-reactivity to standard strains other than T. tonsurans. The detection limit was 100 copies of DNA template per tube. All of the 96 T. tonsurans isolates were amplified, and all samples from healthy volunteers showed negative results. Four of the 34 hairbrush samples obtained from judo athletes showed positive results in LAMP assay, and two of the four were positive in both culture and LAMP assay. We developed a rapid LAMP system with high specificity and sensitivity for diagnosis of T. tonsurans infection.

  6. 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.

  7. Rapid and sensitive detection of porcine torovirus by a reverse transcription loop-mediated isothermal amplification assay (RT-LAMP).

    PubMed

    Liu, Xiaowan; Zhou, Yuancheng; Yang, Fan; Liu, Pengjuan; Cai, Yuhan; Huang, Jianbo; Zhu, Ling; Xu, Zhiwen

    2016-02-01

    Porcine torovirus (PToV) is associated with swine gastroenteritis, but its pathogenesis is uncertain because there is limited information regarding PToV due to its difficulty to adapt in vitro. This study has developed a rapid one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of PToV. A set of four primers specific to six regions within the PToV's highly conserved fragment of the M gene was designed for use with the RT-LAMP assay. The RT-LAMP assay was sensitive with a detection limit of 1 × 10(1)copies/μL, which was 100-fold higher than reverse-transcription PCR. No cross-reaction was observed with other similar viruses. A total of 175 clinical specimens were collected from the Sichuan province, and PToV was detected by the established RT-LAMP assay with a positive rate of 39.2% (69/175). This study developed the first rapid, sensitive, simple, cost-effective and accurate method for the detection of PToV. The results show that the RT-LAMP assay is highly feasible in clinical settings.

  8. 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.

  9. One-step reverse transcription-loop-mediated isothermal amplification assay for sensitive and rapid detection of porcine kobuvirus.

    PubMed

    Li, Xinqiong; Zhou, Yuanchen; Ji, Hongwei; Xu, Zhiwen; Zhu, Ling

    2014-10-01

    Porcine kobuvirus (PKoV) is associated with swine gastroenteritis, but its pathogenesis is uncertain. In this study, a rapid one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method for the detection of PKoV is developed. A set of four primers specific to six regions within the PKoV 3D gene was designed for the RT-LAMP assay using total RNA extracted from PKoV-infected tissues. The reaction temperature and time for this assay were optimized. Compared with reverse-transcription PCR, RT-LAMP was able to detect PKoV at a 100-fold lower dilution. No cross-reaction was observed with other similar viruses, indicating that the assay is highly specific for PKoV. To investigate the prevalence of PKoV in symptomatic pigs in Sichuan province, the newly developed method was used to detect PKoV in a panel of clinical specimens, yielding a positive rate of 86.7% (144/166) in piglets. The results showed that the RT-LAMP assay is highly feasible in clinical settings. The data confirm that the RT-LAMP assay is rapid, simple and cost-effective and is particularly suitable for simple diagnosis of PKoV both in the field and in the laboratory.

  10. 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.

  11. Loop-mediated isothermal amplification (LAMP) assays for the species-specific detection of Eimeria that infect chickens.

    PubMed

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

    2015-02-20

    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.

  12. 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.

  13. 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.

  14. Real-time fluorescence Loop-Mediated Isothermal Amplification (LAMP) for rapid and reliable diagnosis of pulmonary tuberculosis.

    PubMed

    Cao, Donglin; Hu, Liangshan; Lin, Maorui; Li, Mingyou; Ye, Zebing; Sun, Hongtao; Huang, Jiwei; Yang, Huawen; Tian, Junzhang

    2015-02-01

    A reliable, simple and rapid diagnostic method that can be helpful in pulmonary tuberculosis diagnosis is urgently needed. Loop-mediated Isothermal Amplification (LAMP) allows DNA to be amplified rapidly at a constant temperature. In this study, real-time fluorescence LAMP was evaluated to rapidly detect Mycobacterium tuberculosis in sputum and was compared to the performance of real-time fluorescence quantitative PCR (Q-PCR). All the standard MTB strains were successfully detected and limit of detection (LOD) was 10(2)CFU/mL by real-time fluorescence LAMP within 20min. In light of MTB in sputum, the real-time fluorescence LAMP method yielded a sensitivity of 98.0% and a specificity of 78.3%, compared to Q-PCR assay, which yielded a sensitivity of 96.0% and a specificity of 82.6% for PTB diagnosis. There was an excellent overall agreement between LAMP and Q-PCR for PTB (κ=0.315) and non-PTB (κ=0.862). Therefore, the real-time fluorescence LAMP assay is a rapid, sensitive, and specific method to detect pulmonary tuberculosis.

  15. An isothermal, label-free, and rapid one-step RNA amplification/detection assay for diagnosis of respiratory viral infections.

    PubMed

    Koo, Bonhan; Jin, Choong Eun; Lee, Tae Yoon; Lee, Jeong Hoon; Park, Mi Kyoung; Sung, Heungsup; Park, Se Yoon; Lee, Hyun Jung; Kim, Sun Mi; Kim, Ji Yeun; Kim, Sung-Han; Shin, Yong

    2017-04-15

    Recently, RNA viral infections caused by respiratory viruses, such as influenza, parainfluenza, respiratory syncytial virus, coronavirus, and Middle East respiratory syndrome-coronavirus (MERS-CoV), and Zika virus, are a major public health threats in the world. Although myriads of diagnostic methods based on RNA amplification have been developed in the last decades, they continue to lack speed, sensitivity, and specificity for clinical use. A rapid and accurate diagnostic method is needed for appropriate control, including isolation and treatment of the patients. Here, we report an isothermal, label-free, one-step RNA amplification and detection system, termed as iROAD, for the diagnosis of respiratory diseases. It couples a one-step isothermal RNA amplification method and a bio-optical sensor for simultaneous viral RNA amplification/detection in a label-free and real-time manner. The iROAD assay offers a one-step viral RNA amplification/detection example to rapid analysis (<20min). The detection limit of iROAD assay was found to be 10-times more sensitive than that of real-time reverse transcription-PCR method. We confirmed the clinical utility of the iROAD assay by detecting viral RNAs obtained from 63 human respiratory samples. We envision that the iROAD assay will be useful and potentially adaptable for better diagnosis of emerging infectious diseases including respiratory diseases.

  16. 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.

  17. 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.

  18. Non-invasive diagnosis of cutaneous leishmaniasis by the direct boil loop-mediated isothermal amplification method and MinION™ nanopore sequencing.

    PubMed

    Imai, Kazuo; Tarumoto, Norihito; Amo, Kiyoko; Takahashi, Makoto; Sakamoto, Naoya; Kosaka, Atsushi; Kato, Yasuyuki; Mikita, Kei; Sakai, Jun; Murakami, Takashi; Suzuki, Yutaka; Maesaki, Shigefumi; Maeda, Takuya

    2017-03-11

    Cutaneous leishmaniasis (CL) is gaining attention as a public health problem. We present two cases of CL imported from Syria and Venezuela in Japan. We diagnosed them as CL non-invasively by the direct boil loop-mediated isothermal amplification method and an innovative sequencing method using the MinION™ sequencer. This report demonstrates that our procedure could be useful for the diagnosis of CL in both clinical and epidemiological settings.

  19. Evaluation of a real-time PCR and a loop-mediated isothermal amplification for detection of Xanthomonas arboricola pv. pruni in plant tissue samples.

    PubMed

    Palacio-Bielsa, Ana; López-Soriano, Pablo; Bühlmann, Andreas; van Doorn, Joop; Pham, Khanh; Cambra, Miguel A; Berruete, Isabel M; Pothier, Joël F; Duffy, Brion; Olmos, Antonio; López, María M

    2015-05-01

    Operational capacity of real-time PCR and loop-mediated isothermal amplification (LAMP) diagnostic assays for detection of Xanthomonas arboricola pv. pruni was established in a ring-test involving four laboratories. Symptomatic and healthy almond leaf samples with two methods of sample preparation were analyzed. Kappa coefficient, sensitivity, specificity, likelihood ratios and post-test probability of detection were estimated to manage the risk associated with the use of the two methods.

  20. 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.

  1. 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.

  2. Real-time colorimetric detection of target DNA using isothermal target and signaling probe amplification and gold nanoparticle cross-linking assay.

    PubMed

    Jung, Cheulhee; Chung, Ji Won; Kim, Un Ok; Kim, Min Hwan; Park, Hyun Gyu

    2011-01-15

    We describe a facile gold nanoparticle (AuNP)-mediated colorimetric method for real-time detection of target DNA in conjugation with our unique isothermal target and signaling probe amplification (iTPA) method, comprising novel ICA (isothermal chain amplification) and CPT (cycling probe technology). Under isothermal conditions, the iTPA simultaneously amplifies the target and signaling probe through two displacement events induced by a combination of four specially designed primers, the strand displacement activity of DNA polymerase, and the RNA degrading activity of RNase H. The resulting target amplicons are hybridized with gold nanoparticle cross-linking assay (GCA) probes having a DNA-RNA-DNA chimeric form followed by RNA cleavage by RNase H in the CPT step. The intact GCA probes were designed to cross-link two sets of DNA-AuNPs conjugates in the absence of target DNA, inducing aggregation (blue color) of AuNPs. On the contrary, the presence of target DNA leads to cleavage of the GCA probes in proportion to the amount of amplified target DNA and the solution remains red in color without aggregation of AuNPs. Relying on this strategy, 10(2) copies of target Chlamydia trachomatis plasmid were successfully detected in a colorimetric manner. Importantly, all the procedures employed up to the final detection of the target DNA were performed under isothermal conditions without requiring any detection instruments. Therefore, this strategy would greatly benefit convenient, real-time monitoring technology of target DNA under restricted environments.

  3. 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

  4. Clinical Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Neisseria meningitidis in Cerebrospinal Fluid

    PubMed Central

    Kilgore, Paul E.; Kim, Soon Ae; Takahashi, Hideyuki; Ohnishi, Makoto; Anh, Dang Duc; Dong, Bai Qing; Kim, Jung Soo; Tomono, Jun; Miyamoto, Shigehiko; Notomi, Tsugunori; Kim, Dong Wook; Seki, Mitsuko

    2015-01-01

    Background Neisseria meningitidis (Nm) is a leading causative agent of bacterial meningitis in humans. Traditionally, meningococcal meningitis has been diagnosed by bacterial culture. However, isolation of bacteria from patients’ cerebrospinal fluid (CSF) is time consuming and sometimes yields negative results. Recently, polymerase chain reaction (PCR)-based diagnostic methods of detecting Nm have been considered the gold standard because of their superior sensitivity and specificity compared with culture. In this study, we developed a loop-mediated isothermal amplification (LAMP) method and evaluated its ability to detect Nm in cerebrospinal fluid (CSF). Methodology/Principal Findings We developed a meningococcal LAMP assay (Nm LAMP) that targets the ctrA gene. The primer specificity was validated using 16 strains of N. meningitidis (serogroup A, B, C, D, 29-E, W-135, X, Y, and Z) and 19 non-N. meningitidis species. Within 60 min, the Nm LAMP detected down to ten copies per reaction with sensitivity 1000-fold more than that of conventional PCR. The LAMP assays were evaluated using a set of 1574 randomly selected CSF specimens from children with suspected meningitis collected between 1998 and 2002 in Vietnam, China, and Korea. The LAMP method was shown to be more sensitive than PCR methods for CSF samples (31 CSF samples were positive by LAMP vs. 25 by PCR). The detection rate of the LAMP method was substantially higher than that of the PCR method. In a comparative analysis of the PCR and LAMP assays, the clinical sensitivity, specificity, positive predictive value, and negative predictive value of the LAMP assay were 100%, 99.6%, 80.6%, and 100%, respectively. Conclusions/Significance Compared to PCR, LAMP detected Nm with higher analytical and clinical sensitivity. This sensitive and specific LAMP method offers significant advantages for screening patients on a population basis and for diagnosis in clinical settings. PMID:25853422

  5. 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

  6. Field Evaluation of a High Throughput Loop Mediated Isothermal Amplification Test for the Detection of Asymptomatic Plasmodium Infections in Zanzibar

    PubMed Central

    Morris, Ulrika; Ding, Xavier C.; Jovel, Irina; Msellem, Mwinyi I.; Bergman, Daniel; Islam, Atiqul; Ali, Abdullah S.; Polley, Spencer; Gonzalez, Iveth J.; Mårtensson, Andreas; Björkman, Anders

    2017-01-01

    Background New field applicable diagnostic tools are needed for highly sensitive detection of residual malaria infections in pre-elimination settings. Field performance of a high throughput DNA extraction system for loop mediated isothermal amplification (HTP-LAMP) was therefore evaluated for detecting malaria parasites among asymptomatic individuals in Zanzibar. Methods HTP-LAMP performance was evaluated against real-time PCR on 3008 paired blood samples collected on filter papers in a community-based survey in 2015. Results The PCR and HTP-LAMP determined malaria prevalences were 1.6% (95%CI 1.3–2.4) and 0.7% (95%CI 0.4–1.1), respectively. The sensitivity of HTP-LAMP compared to PCR was 40.8% (CI95% 27.0–55.8) and the specificity was 99.9% (CI95% 99.8–100). For the PCR positive samples, there was no statistically significant difference between the geometric mean parasite densities among the HTP-LAMP positive (2.5 p/μL, range 0.2–770) and HTP-LAMP negative (1.4 p/μL, range 0.1–7) samples (p = 0.088). Two lab technicians analysed up to 282 samples per day and the HTP-LAMP method was experienced as user friendly. Conclusions Although field applicable, this high throughput format of LAMP as used here was not sensitive enough to be recommended for detection of asymptomatic low-density infections in areas like Zanzibar, approaching malaria elimination. PMID:28095434

  7. 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.

  8. Rapid detection of viable salmonellae in produce by coupling propidium monoazide with loop-mediated isothermal amplification.

    PubMed

    Chen, Siyi; Wang, Fei; Beaulieu, John C; Stein, Rebecca E; Ge, Beilei

    2011-06-01

    Recent outbreaks linked to Salmonella-contaminated produce heightened the need to develop simple, rapid, and accurate detection methods, particularly those capable of determining cell viability. In this study, we examined a novel strategy for the rapid detection and quantification of viable salmonellae in produce by coupling a simple propidium monoazide sample treatment with loop-mediated isothermal amplification (PMA-LAMP). We first designed and optimized a LAMP assay targeting Salmonella. Second, the performance of PMA-LAMP for detecting and quantifying viable salmonellae was determined. Finally, the assay was evaluated in experimentally contaminated produce items (cantaloupe, spinach, and tomato). Under the optimized condition, PMA-LAMP consistently gave negative results for heat-killed Salmonella cells with concentrations up to 10(8) CFU/ml (or CFU/g in produce). The detection limits of PMA-LAMP were 3.4 to 34 viable Salmonella cells in pure culture and 6.1 × 10(3) to 6.1 × 10(4) CFU/g in spiked produce samples. In comparison, PMA-PCR was up to 100-fold less sensitive. The correlation between LAMP time threshold (T(T)) values and viable Salmonella cell numbers was high (R(2) = 0.949 to 0.993), with a quantification range (10(2) to 10(5) CFU/reaction in pure culture and 10(4) to 10(7) CFU/g in produce) comparable to that of PMA in combination with quantitative real-time PCR (PMA-qPCR). The complete PMA-LAMP assay took about 3 h to complete when testing produce samples. In conclusion, this rapid, accurate, and simple method to detect and quantify viable Salmonella cells in produce may present a useful tool for the produce industry to better control potential microbial hazards in produce.

  9. 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.

  10. 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

  11. Rapid identification of drug-type strains in Cannabis sativa using loop-mediated isothermal amplification assay.

    PubMed

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

    2017-01-01

    In Cannabis sativa L., tetrahydrocannabinol (THC) is the primary psychoactive compound and exists as the carboxylated form, tetrahydrocannabinolic acid (THCA). C. sativa is divided into two strains based on THCA content-THCA-rich (drug-type) strains and THCA-poor (fiber-type) strains. Both strains are prohibited by law in many countries including Japan, whereas the drug-type strains are regulated in Canada and some European countries. As the two strains cannot be discriminated by morphological analysis, a simple method for identifying the drug-type strains is required for quality control in legal cultivation and forensic investigation. We have developed a novel loop-mediated isothermal amplification (LAMP) assay for identifying the drug-type strains of C. sativa. We designed two selective LAMP primer sets for on-site or laboratory use, which target the drug-type THCA synthase gene. The LAMP assay was accomplished within approximately 40 min. The assay showed high specificity for the drug-type strains and its sensitivity was the same as or higher than that of conventional polymerase chain reaction. We also showed the effectiveness of melting curve analysis that was conducted after the LAMP assay. The melting temperature values of the drug-type strains corresponded to those of the cloned drug-type THCA synthase gene, and were clearly different from those of the cloned fiber-type THCA synthase gene. Moreover, the LAMP assay with simple sample preparation could be accomplished within 1 h from sample treatment to identification without the need for special devices or techniques. Our rapid, sensitive, specific, and simple assay is expected to be applicable to laboratory and on-site detection.

  12. Detection of HCV genotypes 1b and 2a by a reverse transcription loop-mediated isothermal amplification assay.

    PubMed

    Zhao, Na; Liu, Jinxia; Sun, Dianxing

    2016-12-09

    Hepatitis C virus (HCV) genotypes 1b and 2a are the major cause of liver disease in northern China; however, conventional detection tools are labor-consuming, technically demanding, and costly. Here, we assessed the specificity, sensitivity, and clinical utility of reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of HCV genotypes 1b and 2a. Firstly, clinical samples were collected from HCV genotype 1b and 2a infected patients and the RNA were extracted. Secondly, specificity of RT-LAMP assay for detection HCV genotypes 1b and 2a were tested against viral genomes of other hepatitis viruses. Sensitivity of RT-LAMP assay was determined using serial dilutions of standard HCV genotypes 1b and 2a. The amplified products were detected by both electrophoresis and calcein/Mn(2+) -dependent visual methods. Finally, we compared the clinical detection rate of RT-LAMP to that of real-time PCR. RT-LAMP assay showed high specificity to detect HCV genotypes 1b and 2b since there was no cross-reactivity with other hepatitis viruses. Sensitivity of RT-LAMP was 100 IU/mL for both genotypes detected by either electrophoresis or calcein/Mn(2+) -dependent visual methods. The detection rate of RT-LAMP assay in clinical samples was also comparable to that of real-time PCR without significant difference between the both assays. This study proposes a newly developed RT-LAMP assay for detection of HCV genotypes 1b and 2a. RT-LAMP is highly specific, sensitive, and simple diagnostic tool which would be useful for screening and early diagnosis of HCV especially in resource-limited environments.

  13. 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.

  14. Surveillance for Western equine encephalitis St. Louis encephalitis and West Nile viruses using reverse transcription loop-mediated isothermal amplification

    SciTech Connect

    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 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.

  15. Surveillance for Western equine encephalitis St. Louis encephalitis and West Nile viruses using reverse transcription loop-mediated isothermal amplification

    DOE PAGES

    Meagher, Robert J.; Ball, Cameron Scott; Langevin, Stanley A.; ...

    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

  16. Rapid Detection of Viable Salmonellae in Produce by Coupling Propidium Monoazide with Loop-Mediated Isothermal Amplification

    PubMed Central

    Chen, Siyi; Wang, Fei; Beaulieu, John C.; Stein, Rebecca E.; Ge, Beilei

    2011-01-01

    Recent outbreaks linked to Salmonella-contaminated produce heightened the need to develop simple, rapid, and accurate detection methods, particularly those capable of determining cell viability. In this study, we examined a novel strategy for the rapid detection and quantification of viable salmonellae in produce by coupling a simple propidium monoazide sample treatment with loop-mediated isothermal amplification (PMA-LAMP). We first designed and optimized a LAMP assay targeting Salmonella. Second, the performance of PMA-LAMP for detecting and quantifying viable salmonellae was determined. Finally, the assay was evaluated in experimentally contaminated produce items (cantaloupe, spinach, and tomato). Under the optimized condition, PMA-LAMP consistently gave negative results for heat-killed Salmonella cells with concentrations up to 108 CFU/ml (or CFU/g in produce). The detection limits of PMA-LAMP were 3.4 to 34 viable Salmonella cells in pure culture and 6.1 × 103 to 6.1 × 104 CFU/g in spiked produce samples. In comparison, PMA-PCR was up to 100-fold less sensitive. The correlation between LAMP time threshold (TT) values and viable Salmonella cell numbers was high (R2 = 0.949 to 0.993), with a quantification range (102 to 105 CFU/reaction in pure culture and 104 to 107 CFU/g in produce) comparable to that of PMA in combination with quantitative real-time PCR (PMA-qPCR). The complete PMA-LAMP assay took about 3 h to complete when testing produce samples. In conclusion, this rapid, accurate, and simple method to detect and quantify viable Salmonella cells in produce may present a useful tool for the produce industry to better control potential microbial hazards in produce. PMID:21498750

  17. Polymerase spiral reaction (PSR): a novel, visual isothermal amplification method for detection of canine parvovirus 2 genomic DNA.

    PubMed

    Gupta, Vikas; Chakravarti, Soumendu; Chander, Vishal; Majumder, Saurabh; Bhat, Shabir Ahmad; Gupta, Vivek Kumar; Nandi, Sukdeb

    2017-03-27

    Canine parvovirus-2 (CPV-2), which is ubiquitously distributed worldwide, causes severe and often fatal gastroenteritis in dogs. Accurate, differential and rapid diagnosis of canine parvoviral enteritis remains a challenge for clinicians. A recently developed isothermal amplification technique, polymerase spiral reaction (PSR), was optimized for the first time for a viral pathogen with reference recombinant plasmid standards from different CPV-2 antigenic variants (CPV-2, CPV-2a, CPV-2b and CPV-2c) and subsequently validated using clinical samples. Addition of chromogenic substrate SYBR Green I after the completion of the reaction resulted in bright green fluorescence in positive samples, while negative samples and a no-template control remained orange. These results were further substantiated through visualization of a laddering pattern of the PSR-amplified product in an agarose gel in positive cases and the absence of this pattern in no-template control and negative samples. The PSR assay was found to be highly specific, as it did not react with other putative canine pathogens (canine adenovirus 1 and canine distemper virus). The sensitivity of the newly developed PSR technique was compared with that of conventional PCR, real-time PCR and LAMP, using a serial tenfold dilution of canine parvovirus DNA. The detection limit of PSR was found to be at the femtogram level, which is comparable with that of real-time PCR and LAMP, which are ten times more sensitive than conventional PCR. The assay was validated using 90 clinical samples, of which 54 were found positive, while only 45 samples were positive in conventional PCR. This novel assay, which is fully compliant with the 'ASSURED' concept for disease diagnosis, provides a simple, rapid, specific, sensitive and cost-effective method for diagnosis of canine parvoviral enteritis in veterinary clinics.

  18. Rapid detection of all known ebolavirus species by reverse transcription-loop-mediated isothermal amplification (RT-LAMP).

    PubMed

    Oloniniyi, Olamide K; Kurosaki, Yohei; Miyamoto, Hiroko; Takada, Ayato; Yasuda, Jiro

    2017-03-26

    Ebola virus disease (EVD), a highly virulent infectious disease caused by ebolaviruses, has a fatality rate of 25-90%. Without a licensed chemotherapeutic agent or vaccine for the treatment and prevention of EVD, control of outbreaks requires accurate and rapid diagnosis of cases. In this study, five sets of six oligonucleotide primers targeting the nucleoprotein gene were designed for specific identification of each of the five ebolavirus species using reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay. The detection limits of the ebolavirus species-specific primer sets were evaluated using in vitro transcribed RNAs. The detection limit of species-specific RT-LAMP assays for Zaire ebolavirus, Sudan ebolavirus, Taï Forest ebolavirus, and Bundibugyo ebolavirus was 256 copies/reaction, while the detection limit for Reston ebolavirus was 64 copies/reaction, and the detection time for each of the RT-LAMP assays was 13.3±3.0, 19.8±4.6, 14.3±0.6, 16.1±4.7, and 19.8±2.4min (mean±SD), respectively. The sensitivity of the species-specific RT-LAMP assays were similar to that of the established RT-PCR and quantitative RT-PCR assays for diagnosis of EVD and are suitable for field or point-of-care diagnosis. The RT-LAMP assays were specific for the detection of the respective species of ebolavirus with no cross reaction with other species of ebolavirus and other viral hemorrhagic fever viruses such as Marburg virus, Lassa fever virus, and Dengue virus. The species-specific RT-LAMP assays developed in this study are rapid, sensitive, and specific and could be useful in case of an EVD outbreak.

  19. 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.

  20. 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

    2015-06-02

    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.

  1. 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.

  2. Evaluation of the Loop Mediated Isothermal DNA Amplification (LAMP) Kit for Malaria Diagnosis in P. vivax Endemic Settings of Colombia

    PubMed Central

    Vallejo, Andrés F.; Martínez, Nora L.; González, Iveth J.; Arévalo-Herrera, Myriam; Herrera, Sócrates

    2015-01-01

    Background Most commonly used malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections which are frequent in low transmission settings. Molecular methods such as polymerase chain reaction (PCR) are highly sensitive but remain too laborious for field deployment. In this study, the applicability of a malaria diagnosis kit based on loop-mediated isothermal amplification (mLAMP) was assessed in malaria endemic areas of Colombia with Plasmodium vivax predominance. Methodology/Principal Findings First, a passive case detection (PCD) study on 278 febrile patients recruited in Tierralta (department of Cordoba) was conducted to assess the diagnostic performance of the mLAMP method. Second, an active case detection (ACD) study on 980 volunteers was conducted in 10 sentinel sites with different epidemiological profiles. Whole blood samples were processed for microscopic and mLAMP diagnosis. Additionally RT-PCR and nested RT-PCR were used as reference tests. In the PCD study, P. falciparum accounted for 23.9% and P. vivax for 76.1% of the infections and no cases of mixed-infections were identified. Microscopy sensitivity for P. falciparum and P. vivax were 100% and 86.1%, respectively. mLAMP sensitivity for P. falciparum and P. vivax was 100% and 91.4%, respectively. In the ACD study, mLAMP detected 65 times more cases than microscopy. A high proportion (98.0%) of the infections detected by mLAMP was from volunteers without symptoms. Conclusions/Significance mLAMP sensitivity and specificity were comparable to RT-PCR. LAMP was significantly superior to microscopy and in P. vivax low-endemicity settings and under minimum infrastructure conditions, it displayed sensitivity and specificity similar to that of single-well RT-PCR for detection of both P. falciparum and P. vivax infections. Here, the dramatically increased detection of asymptomatic malaria infections by mLAMP demonstrates the usefulness of this

  3. Rapid and Sensitive Salmonella Typhi Detection in Blood and Fecal Samples Using Reverse Transcription Loop-Mediated Isothermal Amplification.

    PubMed

    Fan, Fenxia; Yan, Meiying; Du, Pengcheng; Chen, Chen; Kan, Biao

    2015-09-01

    Typhoid fever caused by Salmonella enterica serovar Typhi remains a significant public health problem in developing countries. Although the main method for diagnosing typhoid fever is blood culture, the test is time consuming and not always able to detect infections. Thus, it is very difficult to distinguish typhoid from other infections in patients with nonspecific symptoms. A simple and sensitive laboratory detection method remains necessary. The purpose of this study is to establish and evaluate a rapid and sensitive reverse transcription-based loop-mediated isothermal amplification (RT-LAMP) method to detect Salmonella Typhi infection. In this study, a new specific gene marker, STY1607, was selected to develop a STY1607-RT-LAMP assay; this is the first report of specific RT-LAMP detection assay for typhoid. Human-simulated and clinical blood/stool samples were used to evaluate the performance of STY1607-RT-LAMP for RNA detection; this method was compared with STY1607-LAMP, reverse transcription real-time polymerase chain reaction (rRT-PCR), and bacterial culture methods for Salmonella Typhi detection. Using mRNA as the template, STY1607-RT-LAMP exhibited 50-fold greater sensitivity than STY1607-LAMP for DNA detection. The STY1607-RT-LAMP detection limit is 3 colony-forming units (CFU)/mL for both the pure Salmonella Typhi samples and Salmonella Typhi-simulated blood samples and was 30 CFU/g for the simulated stool samples, all of which were 10-fold more sensitive than the rRT-PCR method. RT-LAMP exhibited improved Salmonella Typhi detection sensitivity compared to culture methods and to rRT-PCR of clinical blood and stool specimens from suspected typhoid fever patients. Because it can be performed without sophisticated equipment or skilled personnel, RT-LAMP is a valuable tool for clinical laboratories in developing countries. This method can be applied in the clinical diagnosis and care of typhoid fever patients as well as for a quick public health response.

  4. 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

  5. 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

  6. Rapid and ultrasensitive detection of microRNA by target-assisted isothermal exponential amplification coupled with poly (thymine)-templated fluorescent copper nanoparticles

    NASA Astrophysics Data System (ADS)

    Park, Kwan Woo; Batule, Bhagwan S.; Kang, Kyoung Suk; Park, Ki Soo; Park, Hyun Gyu

    2016-10-01

    We devised a novel method for rapid and ultrasensitive detection of target microRNA (miRNA) by employing target-assisted isothermal exponential amplification (TAIEA) combined with poly (thymine)-templated fluorescent copper nanoparticles (CuNPs) as signaling probes. The target miRNA hybridizes to the unimolecular template DNA and works as a primer for the extension reaction to form double-stranded product, which consequently generates two nicking endonuclease recognition sites. By simultaneous nicking and displacement reactions, exponential amplification generates many poly (thymine) strands as final products, which are employed for the synthesis of fluorescent CuNPs. Based on the fluorescent signal from CuNPs, target miRNA is detected as low as 0.27 fM around 1 h of total analysis time. The diagnostic capability of this system has been successfully demonstrated by reliably detecting target miRNA from different cell lysates, showing its great potential towards real clinical applications.

  7. Uracil-DNA glycosylase-treated reverse transcription loop-mediated isothermal amplification for rapid detection of avian influenza virus preventing carry-over contamination

    PubMed Central

    Kim, Eun-Mi; Jeon, Hyo-Sung; Kim, Ji-Jung; Shin, Yeun-Kyung; Lee, Youn-Jeong; Yeo, Sang-Geon

    2016-01-01

    Here, we describe a uracil-DNA glycosylase (UNG)-treated reverse transcription loop-mediated isothermal amplification (uRT-LAMP) for the visual detection of all subtypes of avian influenza A virus (AIV). The uRT-LAMP assay can prevent unwanted amplification by carryover contamination of the previously amplified DNA, although the detection limit of the uRT-LAMP assay is 10-fold lower than that of the RT-LAMP without a UNG treatment. To the best of our knowledge, this is the first successful application of deoxyuridine triphosphate/UNG strategy in RT-LAMP for AIV detection, and the assay can be applied for the rapid, and reliable diagnosis of AIVs, even in contaminated samples. PMID:26726027

  8. 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.

  9. Uracil-DNA glycosylase-treated reverse transcription loop-mediated isothermal amplification for rapid detection of avian influenza virus preventing carry-over contamination.

    PubMed

    Kim, Eun-Mi; Jeon, Hyo-Sung; Kim, Ji-Jung; Shin, Yeun-Kyung; Lee, Youn-Jeong; Yeo, Sang-Geon; Park, Choi-Kyu

    2016-09-30

    Here, we describe a uracil-DNA glycosylase (UNG)-treated reverse transcription loop-mediated isothermal amplification (uRT-LAMP) for the visual detection of all subtypes of avian influenza A virus (AIV). The uRT-LAMP assay can prevent unwanted amplification by carryover contamination of the previously amplified DNA, although the detection limit of the uRT-LAMP assay is 10-fold lower than that of the RT-LAMP without a UNG treatment. To the best of our knowledge, this is the first successful application of deoxyuridine triphosphate/UNG strategy in RT-LAMP for AIV detection, and the assay can be applied for the rapid, and reliable diagnosis of AIVs, even in contaminated samples.

  10. 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

  11. A loop-mediated isothermal amplification method for a differential identification of Taenia tapeworms from human: application to a field survey.

    PubMed

    Nkouawa, Agathe; Sako, Yasuhito; Li, Tiaoying; Chen, Xingwang; Nakao, Minoru; Yanagida, Tetsuya; Okamoto, Munehiro; Giraudoux, Patrick; Raoul, Francis; Nakaya, Kazuhiro; Xiao, Ning; Qiu, Jiamin; Qiu, Dongchuan; Craig, Philip S; Ito, Akira

    2012-12-01

    In this study, we applied a loop-mediated isothermal amplification method for identification of human Taenia tapeworms in Tibetan communities in Sichuan, China. Out of 51 proglottids recovered from 35 carriers, 9, 1, and 41 samples were identified as Taenia solium, Taenia asiatica and Taenia saginata, respectively. Same results were obtained afterwards in the laboratory, except one sample. These results demonstrated that the LAMP method enabled rapid identification of parasites in the field surveys, which suggested that this method would contribute to the control of Taenia infections in endemic areas.

  12. Semi-real time electrochemical monitoring for influenza virus RNA by reverse transcription loop-mediated isothermal amplification using a USB powered portable potentiostat.

    PubMed

    Nagatani, Naoki; Yamanaka, Keiichiro; Saito, Masato; Koketsu, Ritsuko; Sasaki, Tadahiro; Ikuta, Kazuyoshi; Miyahara, Toshiro; Tamiya, Eiichi

    2011-12-21

    In this paper, the semi-real time electrochemical monitoring method using a screen-printed electrode, which employs reverse transcription loop-mediated isothermal amplification (RT-LAMP) for influenza virus RNA, is presented. The amplified DNA combined with methylene blue (MB), which was used as an electroactive DNA intercalator, and the electrochemical signal was monitored using square wave voltammetry in the presence of RT-LAMP reagent components. MB molecules binding to amplified DNA caused the reduction of the peak current due to the slow diffusion of MB-amplified DNA complex to the electrode surface. We successfully monitored the amplification process of DNA on the basis of RT-LAMP by measuring and analyzing the electrochemical signal of MB with only one screen-printed electrode that connected with a USB powered portable potentiostat. The peak height of the current was related to the extent of amplification of DNA and the amount of input RNA. Since laborious probe immobilization is not required and both the amplification and the monitoring are possible in a single tube, our method does not suffer from potential cross-contamination. Furthermore, our method provides a new rote for the development of electrochemical hand held biosensors.

  13. 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.

  14. 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

  15. Development of a loop-mediated isothermal amplification assay combined with a lateral flow dipstick for rapid and simple detection of classical swine fever virus in the field.

    PubMed

    Chowdry, Vinay Kumar; Luo, Yuzi; Widén, Frederik; Qiu, Hua-Ji; Shan, Hu; Belák, Sándor; Liu, Lihong

    2014-03-01

    Classical swine fever (CSF) is a highly contagious viral disease and may cause heavy economic loss to farmers. The rapid, simple and accurate diagnosis of the disease at the frontline, for example on the farms of concern is crucial for disease control. This study describes the development and evaluation of a new loop-mediated isothermal amplification (LAMP) assay coupled with lateral flow dipstick (LFD) for the detection of classical swine fever virus (CSFV). This RT-LAMP-LFD assay combines the efficient one-step isothermal amplification of CSF viral RNA and the simplicity of the LFD to read the results within two to five minutes. Seven genotypes (1.1, 1.2, 1.3, 2.1, 2.2, 2.3 and 3.1), but not genotype 3.4, were successfully detected by the RT-LAMP-LFD assay, indicating that the method has a broad range of detection and can be applied in different geographical areas where CSFV strains belonging to these genotypes are present. The performance of this RT-LAMP-LFD assay was similar to that of the real-time RT-PCR. The analytical sensitivity was about 100copies per reaction when testing two genotypes (1.1 and 2.3). No cross-reactivity to non-CSFV pestiviruses was observed. This RT-LAMP-LFD assay can be a useful novel tool for the rapid, simple and economic diagnosis of classical swine fever in the field.

  16. 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

    2013-01-02

    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.

  17. Development and application of a reverse transcription loop-mediated isothermal amplification method for rapid detection of Duck hepatitis A virus type 1.

    PubMed

    Yang, Limin; Li, Jing; Bi, Yuhai; Xu, Lei; Liu, Wenjun

    2012-12-01

    We developed and evaluated a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detecting Duck hepatitis A virus type 1 (DHAV-1). The amplification could be finished in 1 h under isothermal conditions at 63 °C by employing a set of four primers targeting the 2C gene of DHAV-1. The RT-LAMP assay showed higher sensitivity than the RT-PCR with a detection limit of 0.1 ELD(50) 0.1 ml(-1) of DHAV-1. The RT-LAMP assay was highly specific; no cross-reactivity was observed from the samples of other related viruses, bacteria, allantoic fluid of normal chicken embryos, or the livers of uninfected ducks. Thirty clinical samples were subjected to detection by RT-LAMP, RT-PCR, and virus isolation, which obtained completely consistent, positive results. As a simple, rapid, and accurate detection method, this RT-LAMP assay has important potential applications in the clinical diagnosis of DHAV-1.

  18. Evaluation of rapid and sensitive reverse transcription loop-mediated isothermal amplification method for detecting infectious pancreatic necrosis virus in chum salmon (Oncorhynchus keta).

    PubMed

    Suebsing, Rungkarn; Oh, Myung-Joo; Kim, Jeong-Ho

    2011-07-01

    Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for detecting Infectious pancreatic necrosis virus (IPNV) in chum salmon (Oncorhynchus keta) in Korea. The RT-LAMP is a novel approach of nucleic acid gene amplification with high specificity, sensitivity, and rapidity under isothermal conditions. Based on the VP2/NS gene sequence of VR-299 and Jasper strains, a set of 6 IPNV-specific primers was designed to recognize 8 diverse sequences of the IPNV RNA. The assay was successfully optimized to detect IPNV at 65°C in 30 min. The detection limit was 0.075 tissue culture infectious dose infecting 50% of inoculated cultures per milliliter (TCID(50)/ml) from IPNV-infected rainbow trout gonad (RTG)-2 cells, whereas nested reverse transcription polymerase chain reaction (nRT-PCR) had a sensitivity of 7.5 TCID(50)/ml. Using RT-LAMP assay, field samples were analyzed and the results compared with those of nRT-PCR assay. Two hundred and sixty-six out of 659 (40.4%) samples were IPNV-positive by RT-LAMP, whereas 182 of 659 samples (27.6%) were IPNV-positive by nRT-PCR. The results indicate that RT-LAMP can be a useful tool for early field diagnosis of IPNV.

  19. Rapid detection of Bombyx mori nucleopolyhedrovirus (BmNPV) by loop-mediated isothermal amplification assay combined with a lateral flow dipstick method.

    PubMed

    Zhou, Yang; Wu, Jiege; Lin, Feng; Chen, Naifu; Yuan, Shaofei; Ding, Lina; Gao, Li; Hang, Bangxing

    2015-12-01

    The Bombyx mori nucleopolyhedrovirus (BmNPV) is a principal pathogen of the domestic silkworm. The disease often breaks out in sericultural countries and due to its high infectivity; it is difficult to control, resulting in heavy economic loss. In order to develop a rapid, sensitive visual detection and simple-to-use novel technology for detection of BmNPV, a loop-mediated isothermal amplification (LAMP) assay combined with a lateral flow dipstick (LFD) method was described. In this study, a set of four primers and a labeled probe were designed specifically to recognize six distinct regions of the BmNPV gp41 gene, and the LAMP for the detection of BmNPV was developed by isothermal amplification at 61 °C for 45 min, followed by hybridization with an FITC-labeled DNA probe for 5 min and detected by LFD within 5 min. The detection limit of LAMP-LFD was 0.2 pg DNA extracted from silkworm infected with BmNPV and was 100 times more sensitive than conventional PCR. No product was generated from silkworm infected with other viruses. Furthermore, we applied the technique to detect BmNPV in the hemolymph and feces at different intervals post infection (pi). In conclusion, the novel LAMP-LFD setup presented here is simple, rapid, reliable, and has the potential for future use in the detection of BmNPV.

  20. 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.

  1. 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

    2016-05-05

    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.

  2. Microscale loop-mediated isothermal amplification of viral DNA with real-time monitoring on solution-gated graphene FET microchip.

    PubMed

    Han, Dawoon; Chand, Rohit; Kim, Yong-Sang

    2017-07-15

    Rapid and reliable molecular analysis of DNA for disease diagnosis is highly sought-after. FET-based sensors fulfill the demands of future point-of-care devices due to its sensitive charge sensing and possibility of integration with electronic instruments. However, most of the FETs are unstable in aqueous conditions, less sensitive and requires conventional Ag/AgCl electrode for gating. In this work, we propose a solution-gated graphene FET (SG-FET) for real-time monitoring of microscale loop-mediated isothermal amplification of DNA. The SG-FET was fabricated effortlessly with graphene as an active layer, on-chip co-planar electrodes, and polydimethylsiloxane-based microfluidic reservoir. A linear response of about 0.23V/pH was seen when the buffers from pH 5-9 were analyzed on the SG-FET. To evaluate the performance of SG-FET, we monitored the amplification of Lambda phage gene as a proof-of-concept. During amplification, protons are released, which gradually alters the Dirac point voltage (VDirac) of SG-FET. The resulting device was highly sensitive with a femto-level limit of detection. The SG-FET could easily produce a positive signal within 16.5min of amplification. An amplification of 10ng/μl DNA for 1h produced a ∆VDirac of 0.27V. The sensor was tested within a range of 2×10(2) copies/μl (10 fg/μl) to 2×10(8) copies/μl (10ng/μl) of target DNA. Development of this sensing technology could significantly lower the time, cost, and complications of DNA detection.

  3. 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

  4. Rapid and sensitive detection of shrimp yellow head virus by loop-mediated isothermal amplification combined with a lateral flow dipstick.

    PubMed

    Khunthong, Sasiwarat; Jaroenram, Wansadaj; Arunrut, Narong; Suebsing, Rungkarn; Mungsantisuk, Idsada; Kiatpathomchai, Wansika

    2013-03-01

    Yellow head virus (YHV) is a highly virulent pathogen that has caused severe mortality in cultivated shrimp (Penaeus monodon and Penaeus vannamei) in Thailand. There are several technologies that are applied to detect YHV for further control of the disease. RT-PCR is currently widely used in the laboratory, but it has some disadvantages related to cost, time-consuming and complexity. An alternative assay combines RT with loop-mediated isothermal amplification (LAMP) that not only provides high specificity, sensitivity and rapidity, but is also cheaper and more suitable for field applications in shrimp aquaculture than the RT-PCR. RT-LAMP is performed under isothermal conditions with a set of four to six primers designed to recognize six to eight distinct target sequences, and it has been combined with a chromatographic lateral-flow dipstick (LFD) to detect LAMP amplified product, which avoids the use of gel electrophoresis. In this study, RT-LAMP for the detection of YHV was developed by isothermal amplification at 65 °C for 45 min, followed by hybridization with an FITC-labeled DNA probe for 5 min and detected by LFD within 5 min (time required approximately 55 min, excluding RNA extraction and preparation time). The detection limit of RT-LAMP-LFD was 0.1 pg RNA extracted from shrimp infected with YHV equivalent to the nested RT-PCR, and no cross reaction was observed with other common shrimp viral pathogens. The LAMP method described in this study showed a rapid, high sensitivity and specificity and it is recommended as user-friendly for diagnosis of YHV in the field.

  5. A fully disposable and integrated paper-based device for nucleic acid extraction, amplification and detection.

    PubMed

    Tang, Ruihua; Yang, Hui; Gong, Yan; You, MinLi; Liu, Zhi; Choi, Jane Ru; Wen, Ting; Qu, Zhiguo; Mei, Qibing; Xu, Feng

    2017-03-29

    Nucleic acid testing (NAT) has been widely used for disease diagnosis, food safety control and environmental monitoring. At present, NAT mainly involves nucleic acid extraction, amplification and detection steps that heavily rely on large equipment and skilled workers, making the test expensive, time-consuming, and thus less suitable for point-of-care (POC) applications. With advances in paper-based microfluidic technologies, various integrated paper-based devices have recently been developed for NAT, which however require off-chip reagent storage, complex operation steps and equipment-dependent nucleic acid amplification, restricting their use for POC testing. To overcome these challenges, we demonstrate a fully disposable and integrated paper-based sample-in-answer-out device for NAT by integrating nucleic acid extraction, helicase-dependent isothermal amplification and lateral flow assay detection into one paper device. This simple device allows on-chip dried reagent storage and equipment-free nucleic acid amplification with simple operation steps, which could be performed by untrained users in remote settings. The proposed device consists of a sponge-based reservoir and a paper-based valve for nucleic acid extraction, an integrated battery, a PTC ultrathin heater, temperature control switch and on-chip dried enzyme mix storage for isothermal amplification, and a lateral flow test strip for naked-eye detection. It can sensitively detect Salmonella typhimurium, as a model target, with a detection limit of as low as 10(2) CFU ml(-1) in wastewater and egg, and 10(3) CFU ml(-1) in milk and juice in about an hour. This fully disposable and integrated paper-based device has great potential for future POC applications in resource-limited settings.

  6. A label-free colorimetric isothermal cascade amplification for the detection of disease-related nucleic acids based on double-hairpin molecular beacon.

    PubMed

    Wu, Dong; Xu, Huo; Shi, Haimei; Li, Weihong; Sun, Mengze; Wu, Zai-Sheng

    2017-03-08

    K-Ras mutations at codon 12 play an important role in an early step of carcinogenesis. Here, a label-free colorimetric isothermal cascade amplification for ultrasensitive and specific detection of K-Ras point mutation is developed based on a double-hairpin molecular beacon (DHMB). The biosensor consists of DHMB probe and a primer-incorporated polymerization template (PPT) designed partly complementary to DHMB. In the presence of polymerase, target DNA is designed to trigger strand displacement amplification (SDA) via promote the hybridization of PPT with DHMB and subsequently initiates cascade amplification process with the help of the nicking endonuclease. During the hybridization and enzymatic reaction, G-quadruplex/hemin DNAzymes are generated, catalyzing the oxidation of ABTS(2-) by H2O2 in the presence of hemin. Utilizing the proposed facile colorimetric scheme, the target DNA can be quantified down to 4 pM with the dynamic response range of 5 orders of magnitude, indicating the substantially improved detection capability. Even more strikingly, point mutation in K-ras gene can be readily observed by the naked eye without the need for the labeling or expensive equipment. Given the high-performance for K-Ras analysis, the enhanced signal transduction capability associated with double-hairpin structure of DHMB provides a novel rout to screen biomarkers, and the descripted colorimetric biosensor seems to hold great promise for diagnostic applications of genetic diseases.

  7. Rapid and sensitive diagnoses of dry root rot pathogen of chickpea (Rhizoctonia bataticola (Taub.) Butler) using loop-mediated isothermal amplification assay

    PubMed Central

    Ghosh, Raju; Tarafdar, Avijit; Sharma, Mamta

    2017-01-01

    Dry root rot (DRR) caused by the fungus Rhizoctonia bataticola (Taub.) Butler, is an emerging disease in chickpea. The disease is often mistaken with other root rots like Fusarium wilt, collar rot and black root rot in chickpea. Therefore, its timely and specific detection is important. Current detection protocols are either based on mycological methods or on protocols involving DNA amplification by polymerase chain reaction (PCR). Here we report the rapid and specific detection of R. bataticola using loop-mediated isothermal amplification (LAMP) assay targeting fungal specific 5.8S rDNA sequence for visual detection of R. bataticola. The reaction was optimized at 63 °C for 75 min using minimum 10 fg of DNA. After adding SYBR Green I in LAMP products, the amplification was found to be highly specific in all the 94 isolates of R. bataticola collected from diverse geographical regions as well as DRR infected plants and sick soil. No reaction was found in other pathogenic fungi infecting chickpea (Fusarium oxysporum f. sp. ciceris, Rhizoctonia solani, Sclerotium rolfsii and Fusarium solani) and pigeonpea (Fusarium udum and Phytophthora cajani). The standardised LAMP assay with its simplicity, rapidity and specificity is very useful for the visual detection of this emerging disease in chickpea. PMID:28218268

  8. Rapid and sensitive diagnoses of dry root rot pathogen of chickpea (Rhizoctonia bataticola (Taub.) Butler) using loop-mediated isothermal amplification assay.

    PubMed

    Ghosh, Raju; Tarafdar, Avijit; Sharma, Mamta

    2017-02-20

    Dry root rot (DRR) caused by the fungus Rhizoctonia bataticola (Taub.) Butler, is an emerging disease in chickpea. The disease is often mistaken with other root rots like Fusarium wilt, collar rot and black root rot in chickpea. Therefore, its timely and specific detection is important. Current detection protocols are either based on mycological methods or on protocols involving DNA amplification by polymerase chain reaction (PCR). Here we report the rapid and specific detection of R. bataticola using loop-mediated isothermal amplification (LAMP) assay targeting fungal specific 5.8S rDNA sequence for visual detection of R. bataticola. The reaction was optimized at 63 °C for 75 min using minimum 10 fg of DNA. After adding SYBR Green I in LAMP products, the amplification was found to be highly specific in all the 94 isolates of R. bataticola collected from diverse geographical regions as well as DRR infected plants and sick soil. No reaction was found in other pathogenic fungi infecting chickpea (Fusarium oxysporum f. sp. ciceris, Rhizoctonia solani, Sclerotium rolfsii and Fusarium solani) and pigeonpea (Fusarium udum and Phytophthora cajani). The standardised LAMP assay with its simplicity, rapidity and specificity is very useful for the visual detection of this emerging disease in chickpea.

  9. Visual Detection of West Nile Virus Using Reverse Transcription Loop-Mediated Isothermal Amplification Combined with a Vertical Flow Visualization Strip.

    PubMed

    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 10(2) 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 10(1.5) TCID50/ml and 10(1.33) 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.

  10. Establishment of an on-site diagnostic procedure for detection of orf virus from oral lesions of Japanese serows (Capricornis crispus) by loop-mediated isothermal amplification

    PubMed Central

    INOSHIMA, Yasuo; TAKASU, Masaki; ISHIGURO, Naotaka

    2016-01-01

    Orf virus infection has been prevalent continuously in the population of wild Japanese serows (Capricornis crispus), goat-like grazing cloven-hoofed mammal species that live mainly in mountainous areas of Japan. Currently, definitive diagnosis of infection requires time-consuming laboratory work. To diagnose rapidly on-site, we developed a field-friendly procedure for the detection of orf virus from oral cavity lesions. DNA was extracted from goat saliva spiked with orf virus as a proxy for Japanese serows by a commercial kit without the use of electricity, and the quality of the extracted DNA was evaluated by conventional polymerase chain reaction (PCR). Extracted DNA was amenable to DNA amplification, the same as when extracted in a laboratory. Next, to find optimal conditions for DNA amplification by loop-mediated isothermal amplification (LAMP), Bst and Csa DNA polymerases and 3 colorimetric indicators for visual diagnosis, hydroxy naphthol blue (HNB), malachite green and D-QUICK, were compared using a portable cordless incubator. The combination of Bst or Csa DNA polymerase with HNB was found to be easiest for visual diagnosis by the naked eye, and viral DNA was successfully amplified from all orf virus strains used. These results suggest that the procedure established here can work completely on-site and can be useful for definitive diagnosis and differentiation of orf virus infection in Japanese serows in remote mountainous areas. PMID:27628591

  11. 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

    2016-04-05

    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.

  12. 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

  13. 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.

  14. Evaluation of Two Loop-mediated Isothermal Amplification Methods for the Detection of Salmonella Enteritidis and Listeria Monocytogenes in Artificially Contaminated Ready-to-Eat Fresh Produce

    PubMed Central

    Birmpa, Angeliki; Kalogeropoulos, Konstantinos; Kokkinos, Petros

    2015-01-01

    In the present study, the effectiveness of two loop-mediated isothermal amplification (LAMP) assays was evaluated. Samples of romaine lettuce, strawberries, cherry tomatoes, green onions and sour berries were inoculated with known dilutions (100-108 CFU/g of produce) of S. Enteritidis and L. monocytogenes. With LAMP, assay pathogens can be detected in less than 60 min. The limits of detection of S. Enteritidis and L. monocytogenes depended on the food sample tested and on the presence of enrichment step. After enrichment steps, all food samples were found positive even at low initial pathogen levels. The developed LAMP, assays, are expected to become a valuable, robust, innovative, powerful, cheap and fast monitoring tool, which can be extensively used for routine analysis, and screening of contaminated foods by the food industry and the Public Food Health Authorities. PMID:27800413

  15. 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

    2015-05-26

    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.

  16. 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.

  17. 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

  18. Visual and Real-Time Event-Specific Loop-Mediated Isothermal Amplification Based Detection Assays for Bt Cotton Events MON531 and MON15985.

    PubMed

    Randhawa, Gurinder Jit; Chhabra, Rashmi; Bhoge, Rajesh K; Singh, Monika

    2015-01-01

    Bt cotton events MON531 and MON15985 are authorized for commercial cultivation in more than 18 countries. In India, four Bt cotton events have been commercialized; more than 95% of total area under genetically modified (GM) cotton cultivation comprises events MON531 and MON15985. The present study reports on the development of efficient event-specific visual and real-time loop-mediated isothermal amplification (LAMP) assays for detection and identification of cotton events MON531 and MON15985. Efficiency of LAMP assays was compared with conventional and real-time PCR assays. Real-time LAMP assay was found time-efficient and most sensitive, detecting up to two target copies within 35 min. The developed real-time LAMP assays, when combined with efficient DNA extraction kit/protocol, may facilitate onsite GM detection to check authenticity of Bt cotton seeds.

  19. 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.

  20. Evaluation of a new rapid molecular diagnostic system for Plasmodium falciparum combined with DNA filter paper, loop-mediated isothermal amplification, and melting curve analysis.

    PubMed

    Yamamura, Mariko; Makimura, Koichi; Ota, Yasuo

    2009-01-01

    Falciparum malaria is a fatal infection without immediate diagnosability or treatment. There are shortages of clinicians and examiners skilled in the treatment of malaria in non-endemic countries, including Japan. This study was performed to evaluate a novel rapid molecular diagnostic system consisting of loop-mediated isothermal amplification (LAMP) combined with DNA filter paper (FTA card) and melting curve analysis. Combining LAMP with melting curve analysis enabled diagnosis of Plasmodium falciparum more accurately with relative ease. FTA cards could be used to clarify problems regarding storage, infectivity, and transportation. The LAMP assay was carried out at a constant temperature of 63 degrees C for 90 min. The diagnostic system (malaria-LAMP) accurately diagnosed malaria (47 samples from Thailand and 50 from Zimbabwe) with 97.8% sensitivity and 85.7% specificity as compared with microscopic methods, indicating the usefulness of this combined system.

  1. Development of a reverse transcription loop-mediated isothermal amplification assay for the rapid diagnosis of avian influenza A (H7N9) virus infection.

    PubMed

    Nakauchi, Mina; Takayama, Ikuyo; Takahashi, Hitoshi; Tashiro, Masato; Kageyama, Tsutomu

    2014-08-01

    A genetic diagnosis system for detecting avian influenza A (H7N9) virus infection using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) technology was developed. The RT-LAMP assay showed no cross-reactivity with seasonal influenza A (H3N2 and H1N1pdm09) or influenza B viruses circulating in humans or with avian influenza A (H5N1) viruses. The sensitivity of the RT-LAMP assay was 42.47 copies/reaction. Considering the high specificity and sensitivity of the assay for detecting the avian influenza A (H7N9) virus and that the reaction was completed within 30 min, the RT-LAMP assay developed in this study is a promising rapid diagnostic tool for avian influenza A (H7N9) virus infection.

  2. Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay.

    PubMed

    Parida, Manmohan; Horioke, Kouhei; Ishida, Hiroyuki; Dash, Paban Kumar; Saxena, Parag; Jana, Asha Mukul; Islam, Mohammed Alimul; Inoue, Shingo; Hosaka, Norimitsu; Morita, Kouichi

    2005-06-01

    The development and validation of a one-step, real-time, and quantitative dengue virus serotype-specific reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the 3' noncoding region for the rapid detection and differentiation of dengue virus serotypes are reported. The RT-LAMP assay is very simple and rapid, wherein the amplification can be obtained in 30 min under isothermal conditions at 63 degrees C by employing a set of four serotype-specific primer mixtures through real-time monitoring in an inexpensive turbidimeter. The evaluation of the RT-LAMP assay for use for clinical diagnosis with a limited number of patient serum samples, confirmed to be infected with each serotype, revealed a higher sensitivity by picking up 100% samples as positive, whereas 87% and 81% of the samples were positive by reverse transcription-PCR and virus isolation, respectively. The sensitivity and specificity of the RT-LAMP assay for the detection of viral RNA in patient serum samples with reference to virus isolation were 100% and 93%, respectively. The optimal assay conditions with zero background and no cross-reaction with other closely related members of the Flavivirus family (Japanese encephalitis, West Nile, and St. Louis encephalitis viruses) as well as within the four serotypes of dengue virus were established. None of the serum samples from healthy individuals screened in this study showed any cross-reaction with the four dengue virus serotype-specific RT-LAMP assay primers. These findings demonstrate that RT-LAMP assay has the potential clinical application for detection and differentiation of dengue virus serotypes, especially in developing countries.

  3. A CCD-based fluorescence imaging system for real-time loop-mediated isothermal amplification-based rapid and sensitive detection of waterborne pathogens on microchips.

    PubMed

    Ahmad, Farhan; Seyrig, Gregoire; Tourlousse, Dieter M; Stedtfeld, Robert D; Tiedje, James M; Hashsham, Syed A

    2011-10-01

    Rapid, sensitive, and low-cost pathogen diagnostic systems are needed for early disease diagnosis and treatment, especially in resource-limited settings. This study reports a low-cost charge-coupled device (CCD)-based fluorescence imaging system for rapid detection of waterborne pathogens by isothermal gene amplification in disposable microchips. Fluorescence imaging capability of this monochromatic CCD camera is evaluated by optimizing the gain, offset, and exposure time. This imaging system is validated for 12 virulence genes of major waterborne pathogens on cyclic olefin polymer (COP) microchips, using SYTO-82 dye and real time fluorescence loop-mediated isothermal amplification referred here as microRT(f)-LAMP. Signal-to-noise ratio (SNR) and threshold time (Tt) of microRT(f)-LAMP assays are compared with those from a commercial real-time polymerase chain reaction (PCR) instrument. Applying a CCD exposure of 5 s to 10(5) starting DNA copies of microRT(f)-LAMP assays increases the SNR by 8-fold and reduces the Tt by 9.8 min in comparison to a commercial real-time PCR instrument. Additionally, single copy level sensitivity for Campylobacter jejuni 0414 gene is obtained for microRT(f)-LAMP with a Tt of 19 min, which is half the time of the commercial real-time PCR instrument. Due to the control over the exposure time and the wide field imaging capability of CCD, this low-cost fluorescence imaging system has the potential for rapid and parallel detection of pathogenic microorganisms in high throughput microfluidic chips.

  4. An isothermal electrochemical biosensor for the sensitive detection of microRNA based on a catalytic hairpin assembly and supersandwich amplification.

    PubMed

    Zhang, Hua; Wang, Qing; Yang, Xiaohai; Wang, Kemin; Li, Qing; Li, Zhiping; Gao, Lei; Nie, Wenyan; Zheng, Yan

    2017-01-16

    A novel isothermal electrochemical biosensor was proposed for the sensitive detection of microRNA (miRNA) based on the ingenious combination of the target-catalyzed hairpin assembly (CHA) and supersandwich amplification strategies. Since miRNA-221 has been reported to be overexpressed in cancers and has been a potentially useful biomarker for the diagnosis of the related diseases, miRNA-221 was chosen as a model target miRNA. The target miRNA-221 triggered a toehold strand displacement assembly of the two hairpin substrates, which led to the cyclicality of the target miRNA and the CHA products. Subsequently, the CHA products hybridized with a capture probe on the electrode and the exposed stem of the CHA products was further used to propagate the supersandwich. After this, the signal probe was modified with horseradish peroxidase (HRP) to form a supersandwich multiplex HRP-DNA label, which could achieve an amplified electrochemical signal. Using the isothermal dual signal amplification strategies, miRNA-221 as low as 0.6 pM (3σ) could be detected. In addition, this biosensor showed high selectivity and could discriminate miRNA-221 from the homologous miRNAs. Note that human miRNA from cancer cells could also be detected and the results were in excellent agreement with those obtained using qRT-PCR. Given that the biosensor avoided the introduction of nanoparticles, the limitation of using the nanoparticles was overcome. The proposed biosensor has great potential for broad applications in the field of clinical analysis.

  5. 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.

  6. 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

  7. 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

    Kanitkar, Yogendra H; Stedtfeld, Robert D; Steffan, Robert J; Hashsham, Syed A; Cupples, Alison M

    2016-01-08

    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.

  8. Enzyme-free and isothermal detection of microRNA based on click-chemical ligation-assisted hybridization coupled with hybridization chain reaction signal amplification.

    PubMed

    Oishi, Motoi

    2015-05-01

    An enzyme-free and isothermal microRNA (miRNA) detection method has been developed based on click-chemical ligation-assisted hybridization coupled with hybridization chain reaction (HCR) on magnetic beads (MBs). The click-chemical ligation between an azide-modified probe DNA and a dibenzocyclooctyne-modified probe DNA occurred through the hybridization of target miRNA (miR-141). HCR on MBs was performed by the addition of DNA hairpin monomers (H1 and H2). After magnetic separation and denaturation/rehybridization of HCR products ([H1/H2] n ), the resulting HCR products were analyzed by the fluorescence emitted from an intercalative dye, allowing amplification of the fluorescent signal. The proposed assay had a limit of detection of 0.55 fmol, which was 230-fold more sensitive than that of the HCR on the MBs coupled with a conventional sandwich hybridization assay (without click-chemical ligation) (limit of detection 127 fmol). Additionally, the proposed assay could discriminate between miR-141 and other miR-200 family members. In contrast to quantitative reverse transcription polymerase chain reaction techniques using enzymes and thermal cycling, this is an enzyme-free assay that can be conducted under isothermal conditions and can specifically detect miR-141 in fetal bovine serum.

  9. Evaluation of a Loop-Mediated Isothermal Amplification Method as a Tool for Diagnosis of Infection by the Zoonotic Simian Malaria Parasite Plasmodium knowlesi▿

    PubMed Central

    Iseki, Hiroshi; Kawai, Satoru; Takahashi, Nobuyuki; Hirai, Makoto; Tanabe, Kazuyuki; Yokoyama, Naoaki; Igarashi, Ikuo

    2010-01-01

    Loop-mediated isothermal amplification (LAMP) is a novel method that rapidly amplifies target DNA with high specificity under isothermal conditions. It has been applied as a diagnostic tool for several infectious diseases, including viral, bacterial, and parasitic diseases. In the present study, we developed a LAMP method for the molecular diagnosis of Plasmodium knowlesi infection (PkLAMP) and evaluated its sensitivity, specificity, and clinical applicability. We designed three sets of PkLAMP primers for the species-specific β-tubulin gene. The primer sets for PkLAMP specifically amplified the autologous DNA extracts of P. knowlesi, and the sensitivity of the test was 100-fold that of single-PCR assay. These results indicate that our PkLAMP method can be used to efficiently distinguish between P. knowlesi and other malaria parasites. To evaluate the feasibility of using in vivo materials, comparisons of PkLAMP and the conventional nested PCR (nPCR) method and microscopic examination were made with blood samples from two experimentally infected monkeys. These studies showed that P. knowlesi infection can be identified much earlier with PkLAMP than with nPCR and microscopy. Moreover, the detection performance of PkLAMP using whole blood as the template was identical to that of PkLAMP when genomic DNA extracts were used. These results suggest that the PkLAMP method is a promising tool for molecular diagnosis of P. knowlesi infection in areas of endemicity. PMID:20444968

  10. Evaluation of a loop-mediated isothermal amplification method as a tool for diagnosis of infection by the zoonotic simian malaria parasite Plasmodium knowlesi.

    PubMed

    Iseki, Hiroshi; Kawai, Satoru; Takahashi, Nobuyuki; Hirai, Makoto; Tanabe, Kazuyuki; Yokoyama, Naoaki; Igarashi, Ikuo

    2010-07-01

    Loop-mediated isothermal amplification (LAMP) is a novel method that rapidly amplifies target DNA with high specificity under isothermal conditions. It has been applied as a diagnostic tool for several infectious diseases, including viral, bacterial, and parasitic diseases. In the present study, we developed a LAMP method for the molecular diagnosis of Plasmodium knowlesi infection (PkLAMP) and evaluated its sensitivity, specificity, and clinical applicability. We designed three sets of PkLAMP primers for the species-specific beta-tubulin gene. The primer sets for PkLAMP specifically amplified the autologous DNA extracts of P. knowlesi, and the sensitivity of the test was 100-fold that of single-PCR assay. These results indicate that our PkLAMP method can be used to efficiently distinguish between P. knowlesi and other malaria parasites. To evaluate the feasibility of using in vivo materials, comparisons of PkLAMP and the conventional nested PCR (nPCR) method and microscopic examination were made with blood samples from two experimentally infected monkeys. These studies showed that P. knowlesi infection can be identified much earlier with PkLAMP than with nPCR and microscopy. Moreover, the detection performance of PkLAMP using whole blood as the template was identical to that of PkLAMP when genomic DNA extracts were used. These results suggest that the PkLAMP method is a promising tool for molecular diagnosis of P. knowlesi infection in areas of endemicity.

  11. 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

  12. A rapid loop-mediated isothermal amplification (LAMP) method for detection of the macrolide-streptogramin type B resistance gene msrA in Staphylococcus aureus.

    PubMed

    Mu, Xiao-Qin; Liu, Bin-Bin; Hui, Ephraim; Huang, William; Yao, Li-Chen; Duo, Li-Bo; Sun, Wen-Ying; Li, Gui-Qiu; Wang, Fu-Xiang; Liu, Shu-Lin

    2016-12-01

    Macrolide-streptogramin type B resistance (the MSB phenotype) is a multidrug resistance phenotype in Staphylococcus aureus conferred by the resistance gene msrA. However, bacteria having the MSB phenotype are susceptible to lincosamides and 16-membered ring macrolides, which makes profiling resistance genes necessary and urgent for timely and appropriate use of antimicrobials. In this study, the loop-mediated isothermal amplification (LAMP) assay was optimized for prompt detection of the msrA gene. msrA gene sequences were obtained from the National Center for Biotechnology Information (NCBI) database and primers were designed using the LAMP primer designing software PrimerExplorer v.4, which together recognize seven distinct regions of the msrA gene. The specific LAMP primer set designed in this study could amplify the msrA gene within 25min at an isothermal temperature of 62°C. More importantly, the msrA gene could be detected at a sensitivity as low as 100pg. Furthermore, this optimized LAMP assay provided swift detection of the msrA gene even directly from human specimens. In conclusion, this assay may have great clinical application potential for detection of the msrA gene.

  13. Evaluation of a loop-mediated isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri.

    PubMed

    Yeh, Hung-Yueh; Shoemaker, Craig A; Klesius, Phillip H

    2005-10-01

    Channel catfish Ictalurus punctatus infected with Edwardsiella ictaluri results in 40--50 million dollars 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 amplification method (LAMP) that amplifies DNA with high specificity and rapidity at an isothermal condition was evaluated for rapid detection of E. ictaluri. A set of four primers, two outer and two inner, was designed specifically to recognize the eip 18 gene of this pathogen. The LAMP reaction mix was optimized. Reaction temperature and time of the LAMP assay for the eip 18 gene were also optimized at 65 degrees C for 60 min, respectively. Our results show that the ladder-like pattern of bands sizes from 234 bp specifically to the E. ictaluri gene was amplified. The detection limit of this LAMP assay was about 20 colony forming units. In addition, this optimized LAMP assay was used to detect the E. ictaluri eip 18 gene in brains of experimentally challenged channel catfish. Thus, we concluded that the LAMP assay can potentially be used for rapid diagnosis in hatcheries and ponds.

  14. 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.

  15. Loop-Mediated Isothermal Amplification Label-Based Gold Nanoparticles Lateral Flow Biosensor for Detection of Enterococcus faecalis and Staphylococcus aureus.

    PubMed

    Wang, Yi; Li, Hui; Wang, Yan; Zhang, Lu; Xu, Jianguo; Ye, Changyun

    2017-01-01

    The report describes a simple, rapid and sensitive assay for visual and multiplex detection of Enterococcus faecalis and Staphylococcus aureus based on multiple loop-mediated isothermal amplification (mLAMP) and lateral flow biosensor (LFB). Detection and differentiation of the Ef0027 gene (E. faecalis-specific gene) and nuc gene (S. aureus-specific gene) were determined using fluorescein (FITC)-and digoxin-modified primers in the mLAMP process. In the presence of biotin- and FITC-/digoxin-modified primers, the mLAMP yielded numerous biotin- and FITC-/digoxin-attached duplex products, which were detected by LFB through biotin/streptavidin interaction (biotin on the duplex and streptavidin on the gold nanoparticle) and immunoreactions (FITC/digoxin on the duplex and anti-FITC/digoxin on the LFB test line). The accumulation of gold nanoparticles generated a characteristic red line, enabling visual and multiplex detection of target pathogens without instrumentation. The limit of detection (LoD), analytical specificity and feasibility of LAMP-LFB technique were successfully examined in pure culture and blood samples. The entire procedure, including specimen (blood samples) processing (30 min), isothermal reaction (40 min) and result reporting (within 2 min), could be completed within 75 min. Thus, this assay offers a simple, rapid, sensitive and specific test for multiplex detection of E. faecalis and S. aureus strains. Furthermore, the LAMP-LFB strategy is a universal technique, which can be extended to detect various target sequences by re-designing the specific LAMP primers.

  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 multiplex loop-mediated isothermal amplification-RFLP (mLAMP-RFLP) to detect Salmonella spp. and Shigella spp. in milk.

    PubMed

    Shao, Yanchun; Zhu, Shengmei; Jin, Cuicui; Chen, Fusheng

    2011-08-02

    A multiplex loop-mediated isothermal amplification-RFLP (mLAMP-RFLP) was developed and validated for simultaneous detection of Salmonella strains and Shigella strains in milk. In this system, two sets of LAMP primers were designed to specifically target invA of Salmonella spp. and ipaH of Shigella spp. Under isothermal conditions at 63 °C, ladder pattern of DNA bands could be amplified within 60 min in the presence of genomic DNAs of Salmonella strains and Shigella strains, which could be distinguished between Salmonella spp. and Shigella spp. simultaneously based on the different ladder pattern of DNA bands and subsequent restriction enzyme analysis. The overall analysis time was approximately 20 h including the enrichment of the bacterial cells, which greatly saved detection time. The sensitivity of mLAMP was found to be 100 fg DNA/tube with genomic DNAs of Salmonella strains and Shigella strains, comparatively, multiplex PCR was 1 pg DNA/tube. The mLAMP allowed the detection of milk sample artificially contaminated by Salmonella strains and Shigella strains at initial inoculation levels of approximate 5CFU/10 mL. In conclusion, the mLAMP described here can potentially facilitate simultaneous monitoring of Salmonella and Shigella in a large number of food samples, which could be used as a primary screening method and as a supplement to classical detection method.

  18. 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.

  19. Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye.

    PubMed

    Cardoso, Tereza C; Ferrari, Heitor F; Bregano, Lívia C; Silva-Frade, Camila; Rosa, Ana Carolina G; Andrade, Alexandre L

    2010-12-01

    A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid visual detection of turkey coronavirus (TCoV) infection. The reaction is performed in one step in a single tube at 65 °C for 45 min, with hydroxynaphthol blue (HNB) dye added prior to amplification. The detection limit of the RT-LAMP assay was approximately 10(2) EID(50/50 μl) TCoV genome, and no cross-reaction with other avian viruses was observed. The assay was evaluated further in tissue suspensions prepared from the ileum and ileum-caecal junctions of infected turkey embryos; 100% of these samples were positive in the RT-LAMP assay. All individual feces samples collected in the field were considered positive by both conventional RT-PCR and RT-LAMP. In conclusion, RT-LAMP with HNB dye was shown to be a sensitive, simple assay for the rapid diagnosis of TCoV infection, either directly from feces or in association with virus isolation methods.

  20. 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.

  1. Rapid detection of Piper yellow mottle virus and Cucumber mosaic virus infecting black pepper (Piper nigrum) by loop-mediated isothermal amplification (LAMP).

    PubMed

    Bhat, A I; Siljo, A; Deeshma, K P

    2013-10-01

    The loop-mediated isothermal amplification (LAMP) assay for Piper yellow mottle virus and the reverse transcription (RT) LAMP assay for Cucumber mosaic virus each consisted of a set of five primers designed against the conserved sequences in the viral genome. Both RNA and DNA isolated from black pepper were used as a template for the assay. The results were assessed visually by checking turbidity, green fluorescence and pellet formation in the reaction tube and also by gel electrophoresis. The assay successfully detected both viruses in infected plants whereas no cross-reactions were recorded with healthy plants. Optimum conditions for successful amplification were determined in terms of the concentrations of magnesium sulphate and betaine, temperature, and duration. The detection limit for both LAMP and RT-LAMP was up to 100 times that for conventional PCR and up to one-hundredth of that for real-time PCR. The optimal conditions arrived at were validated by testing field samples of infected vines of three species from different regions.

  2. Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus

    PubMed Central

    Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

    2016-01-01

    Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products. PMID:27721691

  3. Evaluation of loop-mediated isothermal amplification for detection of Toxoplasma gondii in water samples and comparative findings by polymerase chain reaction and immunofluorescence test (IFT).

    PubMed

    Sotiriadou, Isaia; Karanis, Panagiotis

    2008-12-01

    The development and evaluation of a 1-step single-tube accelerated loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Toxoplasma in water samples is described. The method has been evaluated based on the amplification of B1 and TgOWP Toxoplasma genes, and it demonstrated a sensitivity detection limit of 0.1 tachyzoites' DNA for both genes. LAMP detection was evaluated and compared with nested polymerase chain reaction (PCR) in 26 water sample pellets spiked with known numbers of Toxoplasma oocysts. After DNA extraction, the detection sensitivity in spiked pellets was 100% by LAMP and 53.8% by PCR. Subsequently, 52 natural water samples of different origin were directly investigated by 3 assays: LAMP, PCR, and immunofluorescence test (IFT). Twenty-five (48%) of 52 have been found positive for Toxoplasma DNA by LAMP, whereas nested PCR products were generated in 7 of 52 (13.5%) water samples. All 52 water samples were negative for Toxoplasma by IFT. These data clearly indicate LAMP as a rapid, specific, and sensitive tool for the detection of Toxoplasma contamination in water samples.

  4. Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus.

    PubMed

    Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

    2016-10-01

    Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products.

  5. 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.

  6. 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.

  7. 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

  8. Reverse-transcriptase loop-mediated isothermal amplification as a rapid screening/monitoring tool for Salmonella enterica detection in liquid whole eggs.

    PubMed

    Techathuvanan, Chayapa; D'Souza, Doris Helen

    2012-04-01

    Reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) is a novel molecular detection method that is specific, fast, and simple. It is based on reverse transcription followed by DNA amplification using the Bst DNA polymerase large fragment requiring one temperature and a simple waterbath, without the need for any expensive equipment. Detection is by turbidity or agarose gel electrophoresis. Our objective was to apply this LAMP-based technology to rapidly and sensitively detect Salmonella enterica serovar Enteritidis in liquid whole eggs (LWEs) within 1 d. LWE were inoculated with S. Enteritidis and stomached in tetrathionate broth (TTB), and spread-plated on Xylose lysine tergitol 4 agar either immediately or after 6, 12, or 16-h enrichment. RNA was extracted from 5-mL TTB and the RT-LAMP assay was carried out using invA primers. After 16 and 12-h enrichment, improved Salmonella detection up to 10⁰ to 10¹ and 10⁴ CFU/25 mL LWE, respectively was obtained. Without enrichment, Salmonella could be detected at 10⁷ CFU/25 mL; however, after 6-h enrichment a 1-log improvement to 10⁶ CFU/25 mL was obtained. This RT-LAMP assay appears to be suitable as a potential screening/monitoring tool for Salmonella enterica from LWE products in routine settings with results obtainable within 24-h, which is significantly faster than traditional cultural assays.

  9. 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.

  10. 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.

  11. Establishment and application of a novel isothermal amplification assay for rapid detection of chloroquine resistance (K76T) in Plasmodium falciparum

    PubMed Central

    Chahar, Madhvi; Mishra, Neelima; Anvikar, Anup; Dixit, Rajnikant; Valecha, Neena

    2017-01-01

    Chloroquine (CQ) resistance in Plasmodium falciparum is determined by the mutations in the chloroquine resistance transporter (Pfcrt) gene. The point mutation at codon 76 (K76T), which has been observed in more than 91% of P. falciparum isolates in India, is the major determinant of CQ resistance. To overcome the limitations and challenges of traditional methods, in this investigation we developed an easy to use loop mediated isothermal amplification (LAMP) protocol for rapid detection of the K76T mutation associated with CQ resistance in P. falciparum with naked eye visualization. In- house designed primers were synthesized and optimized to specifically distinguish the CQ resistant mutants of P. falciparum. The LAMP reaction was optimal at 61 °C for 60 min and calcein dye was added prior to amplification to enable visual detection. We demonstrate the detection limit of <2 ng/μl respectively, supporting the high sensitivity of this calcein based LAMP method. To the best of our knowledge this is the first report on the establishment of an easy, reliable and cost effective LAMP assay for rapid and specific detection of highly CQ resistance in P. falciparum malaria. PMID:28134241

  12. Real-time target-specific detection of loop-mediated isothermal amplification for white spot syndrome virus using fluorescence energy transfer-based probes.

    PubMed

    Chou, Pin-Hsing; Lin, Yu-Chan; Teng, Ping-Hua; Chen, Chu-Liang; Lee, Pei-Yu

    2011-04-01

    Aiming to establish a target amplicon-specific detection system for loop-mediated isothermal amplification (LAMP), the fluorescent resonance energy transfer (FRET) probe technology was applied to develop the FRET LAMP platform. This report describes the development of the first FRET LAMP assay targeting white spot syndrome virus (WSSV) of penaeid shrimp. A successful accelerated WSSV LAMP was assembled first in a conventional oven and confirmed by gel electrophoresis and dot-blot hybridization. Subsequently, two additional FRET probes designed to target one loop region within WSSV LAMP amplicons were added to the same LAMP reaction. The reactions were carried out in a LightCycler (Roche) and significant FRET signals were detected in real time. Optimization of the reaction using plasmid DNA shortened the time for the detection of 10(2) copies of the target DNA to less than 70min. Cross reactivity was absent with WSSV-free or infectious hypodermal and hematopoietic necrosis virus-infected Penaeus vannamei samples. The performance of this system was comparable with that of a nested PCR assay from 21 WSSV-infected shrimp. Specifically detecting target amplicons and requiring no post-amplification manipulation, the novel FRET LAMP assay should allow indisputable detection of pathogens with minimized risks of amplicon contamination.

  13. Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Cascade Invasive Reaction and Nanoparticle Hybridization for Subtyping of Influenza A Virus

    PubMed Central

    Chi, Ying; Ge, Yiyue; Zhao, Kangchen; Zou, Bingjie; Liu, Bin; Qi, Xian; Bian, Qian; Shi, Zhiyang; Zhu, Fengcai; Zhou, Minghao; Cui, Lunbiao; Su, Chuan

    2017-01-01

    Considering the fatal human victims and economic loss caused by influenza virus infection every year, methodologies for rapid and on-site detection of influenza viruses are urgently needed. LAMP is the most commonly used nucleic acid isothermal amplification technology suitable for on-site use. However, for multiplex LAMP, differentiation of the amplicons derived from multiple targets is still challengeable currently. Here we developed a multiplex RT-LAMP assay for simultaneous amplification of three prominent subtypes of influenza viruses (A/H5, A/H7 and 2009A/H1). The amplicons were further identified by cascade invasive reaction and nanoparticle hybridization in separate target-specific detection tubes (referred to as mRT-LAMP-IRNH). The analytic sensitivities of the assay are 10 copies of RNA for all the three HA subtypes, and the specificity reached 100%. Clinical specimen analysis showed this assay had a combined sensitivity and specificity of 98.1% and 100%, respectively. Overall, the mRT-LAMP-IRNH assay can be used as a cost-saving method that utilizes a simple instrument to detect A/H5, A/H7, and 2009A/H1 influenza viruses, especially in resource-limited settings. PMID:28322309

  14. 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.

  15. Visualized detection of single-base difference in multiplexed loop-mediated isothermal amplification amplicons by invasive reaction coupled with oligonucleotide probe-modified gold nanoparticles.

    PubMed

    Lu, Yan; Ma, Xueping; Wang, Jianping; Sheng, Nan; Dong, Tianhui; Song, Qinxin; Rui, Jianzhong; Zou, Bingjie; Zhou, Guohua

    2017-04-15

    Loop-mediated isothermal amplification (LAMP) is a well-developed DNA amplification method with an ultra-high sensitivity, but it is difficult to recognize a single-base difference (like genotyping) in target-specific amplicons by conventional detection ways, such as the intercalation of dyes into dsDNA amplicons or the increase of solution turbidity along with the polymerization process. To allow genotyping based on LAMP suitable for POCT (point-of-care testing) or on-site testing, here we proposed a highly specific and cost-effective method for detecting a single-base difference in LAMP amplicons. The method includes three key steps, sequence amplifier to amplify multiple fragments containing the single nucleotide polymorphisms (SNPs) of interest, allele identifier to recognize a targeted base in the amplicons by invasive reaction, and signal generator to yield signals by hybridization-induced assembly of oligonucleotide probe-modified gold nanoparticles. Because the allele identifier is sensitive to one base difference, it is possible to use multiplexed LAMP (mLAMP) to generate amplicon mixtures for multiple SNP typing. Genotyping of 3 different SNPs (CYP2C19*2, CYP2C19*3 and MDR1-C3435T) for guiding the dosage of clopidogrel is successfully carried out in a 3-plex LAMP on real clinical samples. As our method relies on the naked-eye detection and constant-temperature reaction, no expensive instrument is required for both target amplification and sequence identification, thus much suitable for inexpensive gene-guided personalized medicine in source-limited regions.

  16. Feasibility and Operational Performance of Tuberculosis Detection by Loop-Mediated Isothermal Amplification Platform in Decentralized Settings: Results from a Multicenter Study

    PubMed Central

    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

    2016-01-01

    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

  17. Colorimetric tests for diagnosis of filarial infection and vector surveillance using non-instrumented nucleic acid loop-mediated isothermal amplification (NINA-LAMP)

    PubMed Central

    Poole, Catherine B.; Li, Zhiru; Alhassan, Andy; Guelig, Dylan; Diesburg, Steven; Tanner, Nathan A.; Zhang, Yinhua; Evans, Thomas C.; LaBarre, Paul; Wanji, Samuel; Burton, Robert A.; Carlow, Clotilde K. S.

    2017-01-01

    Accurate detection of filarial parasites in humans is essential for the implementation and evaluation of mass drug administration programs to control onchocerciasis and lymphatic filariasis. Determining the infection levels in vector populations is also important for assessing transmission, deciding when drug treatments may be terminated and for monitoring recrudescence. Immunological methods to detect infection in humans are available, however, cross-reactivity issues have been reported. Nucleic acid-based molecular assays offer high levels of specificity and sensitivity, and can be used to detect infection in both humans and vectors. In this study we developed loop-mediated isothermal amplification (LAMP) tests to detect three different filarial DNAs in human and insect samples using pH sensitive dyes for enhanced visual detection of amplification. Furthermore, reactions were performed in a portable, non-instrumented nucleic acid amplification (NINA) device that provides a stable heat source for LAMP. The efficacy of several strand displacing DNA polymerases were evaluated in combination with neutral red or phenol red dyes. Colorimetric NINA-LAMP assays targeting Brugia Hha I repeat, Onchocerca volvulus GST1a and Wuchereria bancrofti LDR each exhibit species-specificity and are also highly sensitive, detecting DNA equivalent to 1/10-1/5000th of one microfilaria. Reaction times varied depending on whether a single copy gene (70 minutes, O. volvulus) or repetitive DNA (40 min, B. malayi and W. bancrofti) was employed as a biomarker. The NINA heater can be used to detect multiple infections simultaneously. The accuracy, simplicity and versatility of the technology suggests that colorimetric NINA-LAMP assays are ideally suited for monitoring the success of filariasis control programs. PMID:28199317

  18. Higher specificity of nucleic acid sequence-based amplification isothermal technology than of real-time PCR for quantification of HIV-1 RNA on dried blood spots.

    PubMed

    Mercier-Delarue, Severine; Vray, Muriel; Plantier, Jean Christophe; Maillard, Theodora; Adjout, Zidan; de Olivera, Fabienne; Schnepf, Nathalie; Maylin, Sarah; Simon, Francois; Delaugerre, Constance

    2014-01-01

    Dried blood spots (DBS) are widely proposed as a plasma surrogate for monitoring antiretroviral treatment efficacy based on the HIV-1 RNA level (viral load [VL]) in resource-limited settings. Interfering coamplification of cell-associated HIV-1 DNA during reverse transcription (RT)-PCR can be avoided by using nucleic acid sequence-based amplification (NASBA) technology, which is based on an RNA template and isothermic conditions. We analyzed VL values obtained with DBS and plasma samples by comparing isothermic NASBA (NucliSENS EasyQ HIV-1 V2.0; bioMérieux) with real-time RT-PCR (Cobas TaqMan HIV-1 V2.0; Roche). Samples from 197 HIV-1-infected patients were tested (non-B subtypes in 51% of the cases). Nucleic acid extractions were performed by use of NucliSENS EasyMAG (bioMérieux) and Cobas AmpliPrep (Roche) before the NASBA and RT-PCR quantifications, respectively. Both quantification assays have lower limits of detection of 20 (1.3) and 800 (2.9) log10 copies/ml (log) in plasma and DBS, respectively. The mean (DBS minus plasma) differences were -0.39 and -0.46 log, respectively, for RT-PCR and NASBA. RT-PCR on DBS identified virological failure in 122 of 126 patients (sensitivity, 97%) and viral suppression in 58 of 70 patients (specificity, 83%), yielding 12 false-positive results (median, 3.2 log). NASBA on DBS identified virological failure in 85 of 96 patients (sensitivity, 89%) and viral suppression in 95 of 97 patients (specificity, 98%) and yielded 2 false-positive results (3.0 log for both). Both technologies detected HIV-1 RNA in DBS at a threshold of 800 copies/ml. This higher specificity of NASBA technology could avoid overestimation of poor compliance or the emergence of resistance when monitoring antiretroviral efficacy with the DBS method.

  19. Loop-Mediated Isothermal Amplification Label-Based Gold Nanoparticles Lateral Flow Biosensor for Detection of Enterococcus faecalis and Staphylococcus aureus

    PubMed Central

    Wang, Yi; Li, Hui; Wang, Yan; Zhang, Lu; Xu, Jianguo; Ye, Changyun

    2017-01-01

    The report describes a simple, rapid and sensitive assay for visual and multiplex detection of Enterococcus faecalis and Staphylococcus aureus based on multiple loop-mediated isothermal amplification (mLAMP) and lateral flow biosensor (LFB). Detection and differentiation of the Ef0027 gene (E. faecalis-specific gene) and nuc gene (S. aureus-specific gene) were determined using fluorescein (FITC)-and digoxin-modified primers in the mLAMP process. In the presence of biotin- and FITC-/digoxin-modified primers, the mLAMP yielded numerous biotin- and FITC-/digoxin-attached duplex products, which were detected by LFB through biotin/streptavidin interaction (biotin on the duplex and streptavidin on the gold nanoparticle) and immunoreactions (FITC/digoxin on the duplex and anti-FITC/digoxin on the LFB test line). The accumulation of gold nanoparticles generated a characteristic red line, enabling visual and multiplex detection of target pathogens without instrumentation. The limit of detection (LoD), analytical specificity and feasibility of LAMP-LFB technique were successfully examined in pure culture and blood samples. The entire procedure, including specimen (blood samples) processing (30 min), isothermal reaction (40 min) and result reporting (within 2 min), could be completed within 75 min. Thus, this assay offers a simple, rapid, sensitive and specific test for multiplex detection of E. faecalis and S. aureus strains. Furthermore, the LAMP-LFB strategy is a universal technique, which can be extended to detect various target sequences by re-designing the specific LAMP primers. PMID:28239371

  20. 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

  1. Loop-mediated isothermal amplification: Rapid and sensitive detection of the antibiotic resistance gene ISAba1-blaOXA-51-like in Acinetobacter baumannii.

    PubMed

    Mu, Xiaoqin; Nakano, Ryuichi; Nakano, Akiyo; Ubagai, Tsuneyuki; Kikuchi-Ueda, Takane; Tansho-Nagakawa, Shigeru; Kikuchi, Hirotoshi; Kamoshida, Go; Endo, Shiro; Yano, Hisakazu; Ono, Yasuo

    2016-02-01

    Carbapenem-resistant Acinetobacter baumannii, which are mainly induced by the production of OXA-type β-lactamases, are among the leading causes of nosocomial infections worldwide. Among the β-lactamase genes, the presence of the OXA-51-like gene carrying the upstream insertion sequence, ISAba1, was found to be one of the most prevalent carbapenem resistance mechanisms utilized by these bacteria. Consequently, it is necessary to develop a rapid detection method for ISAba1-blaOXA-51-like sequence for the timely and appropriate antibiotic treatment of A. baumannii infection. In this study, a loop-mediated isothermal amplification (LAMP) assay was optimized for ISAba1-blaOXA-51-like detection. The LAMP primer set was designed to recognize distinct sequences in the ISAba1-blaOXA-51-like gene and could amplify the gene within 25 min at an isothermal temperature of 60°C. This LAMP assay was able to detect the ISAba1-blaOXA-51-like gene with high specificity; in addition, no cross-reactivity was observed for other types of β-lactamase producers (OXA-23-like, OXA-40-like, OXA-58-like, and IMP-1), as indicated by the absence of false positive or false negative results. The detection limit for this assay was found to be 10(0)CFU per tube which was 100-fold more sensitive than a polymerase chain reaction assay for ISAba1-blaOXA-51-like detection. Furthermore, the LAMP assay provided swift detection of the ISAba1-blaOXA-51-like gene, even directly from clinical specimens. In summary, we have described a new, rapid assay for the detection of the ISAba1-blaOXA-51-like gene from A. baumannii that could be useful in a clinical setting. This method might facilitate epidemiological studies and allow monitoring of the emergence of drug resistant strains.

  2. 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

    2014-08-27

    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.

  3. Diagnostic accuracy of loop-mediated isothermal amplification (LAMP) for detection of Leishmania DNA in buffy coat from visceral leishmaniasis patients

    PubMed Central

    2012-01-01

    Background Visceral leishmaniasis (VL) remains as one of the most neglected tropical diseases with over 60% of the world’s total VL cases occurring in the Indian subcontinent. Due to the invasive risky procedure and technical expertise required in the classical parasitological diagnosis, the goal of the VL experts has been to develop noninvasive procedure(s) applicable in the field settings. Several serological and molecular biological approaches have been developed over the last decades, but only a few are applicable in field settings that can be performed with relative ease. Recently, loop-mediated isothermal amplification (LAMP) has emerged as a novel nucleic acid amplification method for diagnosis of VL. In this study, we have evaluated the LAMP assay using buffy coat DNA samples from VL patients in Bangladesh and compared its performance with leishmania nested PCR (Ln-PCR), an established molecular method with very high diagnostic indices. Methods Seventy five (75) parasitologically confirmed VL patients by spleen smear microcopy and 101 controls (endemic healthy controls −25, non-endemic healthy control-26, Tuberculosis-25 and other diseases-25) were enrolled in this study. LAMP assay was carried out using a set of four primers targeting L. donovani kinetoplast minicircle DNA under isothermal (62 °C) conditions in a heat block. For Ln-PCR, we used primers targeting the parasite’s small-subunit rRNA region. Results LAMP assay was found to be positive in 68 of 75 confirmed VL cases, and revealed its diagnostic sensitivity of 90.7% (95.84-81.14, 95% CI), whereas all controls were negative by LAMP assay, indicating a specificity of 100% (100–95.43, 95% CI). The Ln-PCR yielded a sensitivity of 96% (98.96-87.97, 95% CI) and a specificity of 100% (100–95.43, 95% CI). Conclusion High diagnostic sensitivity and excellent specificity were observed in this first report of LAMP diagnostic evaluation from Bangladesh. Considering its many fold advantages over

  4. A novel method of real-time reverse-transcription loop-mediated isothermal amplification developed for rapid and quantitative detection of human astrovirus.

    PubMed

    Wei, Haiyan; Zeng, Jing; Deng, Congliang; Zheng, Chengzhong; Zhang, Ximeng; Ma, Dan; Yi, Yong

    2013-03-01

    A one-step, real-time reverse-transcription loop-mediated isothermal amplification (rRT-LAMP) method targeting the 5' end of the capsid gene for rapid and quantitative detection of human astrovirus serotype 1 (HAstV 1) was developed. The assay is highly sensitive and comparable to real-time RT-PCR (rRT-PCR), with a detection limit of ∼100 RNA copies per assay. The specificity of the method was validated by the absence of any cross-reaction with RNA samples of HAstV 2-8 and other gastroenteritis viruses, followed by nucleotide sequencing of the amplified product. Fecal specimens (n=120) obtained from children under five years of age with gastroenteritis were tested by rRT-LAMP, rRT-PCR and transmission electron microscopy (TEM). Six (5%) of these samples were determined to be positive by both rRT-LAMP and rRT-PCR assay, and these two nucleic acid amplification methods resulted in a 200% increase in detection rates for HAstV infection compared with TEM alone. Furthermore, the rRT-LAMP assay is much more rapid than rRT-PCR and generates results in less than 20min for positive samples. The quantitation of viral load in stool specimens was determined from the standard curve plot of time-of-positivity versus initial RNA concentration. Most viral loads were determined to be within the range of 10(5)-10(8) copies. The results highlight the significance of the rapid rRT-LAMP method as a diagnostic and routine screening tool for the analysis of stool samples in hospital laboratories.

  5. 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

  6. 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.

  7. Sex determination in the wild: a field application of loop-mediated isothermal amplification successfully determines sex across three raptor species.

    PubMed

    Centeno-Cuadros, A; Abbasi, I; Nathan, R

    2017-03-01

    PCR-based methods are the most common technique for sex determination of birds. Although these methods are fast, easy and accurate, they still require special facilities that preclude their application outdoors. Consequently, there is a time lag between sampling and obtaining results that impedes researchers to take decisions in situ and in real time considering individuals' sex. We present an outdoor technique for sex determination of birds based on the amplification of the duplicated sex-chromosome-specific gene Chromo-Helicase-DNA binding protein using a loop-mediated isothermal amplification (LAMP). We tested our method on Griffon Vulture (Gyps fulvus), Egyptian Vulture (Neophron percnopterus) and Black Kite (Milvus migrans) (family Accipitridae). We introduce the first fieldwork procedure for sex determination of animals in the wild, successfully applied to raptor species of three different subfamilies using the same specific LAMP primers. This molecular technique can be deployed directly in sampling areas because it only needs a voltage inverter to adapt a thermo-block to a car lighter and results can be obtained by the unaided eye based on colour change within the reaction tubes. Primers and reagents are prepared in advance to facilitate their storage at room temperature. We provide detailed guidelines how to implement this procedure, which is simpler (no electrophoresis required), cheaper and faster (results in c. 90 min) than PCR-based laboratory methods. Our successful cross-species application across three different raptor subfamilies posits our set of markers as a promising tool for molecular sexing of other raptor families and our field protocol extensible to all bird species.

  8. 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

  9. Fast and simple detection methods for the 4-base pair deletion of canine MDR1/ ABCB1 gene by PCR and isothermal amplification.

    PubMed

    Stiedl, Cathrin P; Weber, Karin

    2017-03-01

    Dogs with a 4-bp deletion in the MDR1 (or ABCB1) gene show intolerance to certain drugs routinely used in veterinary medicine, such as ivermectin, vincristine, and doxorubicin. The mutation leads to a dysfunctional P-glycoprotein drug transporter, which results in drug accumulation in the brain and severe neurotoxicity. A rapid and accurate in-house test to determine the genotype of patients in cases of acute neurotoxic signs or in tumor patients is desirable. We describe a cost-effective detection method with simple technical equipment for veterinary practice. Two allele-specific methods are presented, which allow discrimination of all genotypes, require little hands-on time, and show the results within ~1 h after DNA sampling. DNA from buccal swabs of 115 dogs with known genotype (no mutation, n = 54; heterozygous for the mutation, n = 37; homozygous for the mutation, n = 24) was extracted either by using a column-based extraction kit or by heating swabs in a simple NaOH-Tris buffer. Amplification was performed either by allele-specific fast polymerase chain reaction or by allele-specific loop-mediated isothermal amplification (LAMP). Analysis was done either on agarose gels, by simple endpoint visualization using ultraviolet light, or by measuring the increase of fluorescence and time to threshold crossing. Commercial master mixes reduced the preparation time and minimized sources of error in both methods. Both methods allowed the discrimination of all 3 genotypes, and the results of the new methods matched the results of the previous genotyping. The presented methods could be used for fast individual MDR1/ ABCB1 genotyping with less equipment than existing methods.

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. Rapid, simple and direct detection of Meloidogyne hapla from infected root galls using loop-mediated isothermal amplification combined with FTA technology

    PubMed Central

    Peng, Huan; Long, Haibo; Huang, Wenkun; Liu, Jing; Cui, Jiangkuan; Kong, Lingan; Hu, Xianqi; Gu, Jianfeng; Peng, Deliang

    2017-01-01

    The northern root-knot nematode (Meloidogyne hapla) is a damaging nematode that has caused serious economic losses worldwide. In the present study, a sensitive, simple and rapid method was developed for detection of M. hapla in infested plant roots by combining a Flinders Technology Associates (FTA) card with loop-mediated isothermal amplification (LAMP). The specific primers of LAMP were designed based on the distinction of internal transcribed spacer (ITS) sequences between M. hapla and other Meloidogyne spp. The LAMP assay can detect nematode genomic DNA at concentrations low to 1/200 000, which is 100 times more sensitive than conventional PCR. The LAMP was able to highly specifically distinguish M. hapla from other closely related nematode species. Furthermore, the advantages of the FTA-LAMP assay to detect M. hapla were demonstrated by assaying infected root galls that were artificially inoculated. In addition, M. hapla was successfully detected from six of forty-two field samples using FTA-LAMP technology. This study was the first to provide a simple diagnostic assay for M. hapla using the LAMP assay combined with FTA technology. In conclusion, the new FTA-LAMP assay has the potential for diagnosing infestation in the field and managing the pathogen M. hapla. PMID:28368036

  15. Determining putative vectors of the Bogia Coconut Syndrome phytoplasma using loop-mediated isothermal amplification of single-insect feeding media

    PubMed Central

    Lu, Hengyu; Wilson, Bree A. L.; Ash, Gavin J.; Woruba, Sharon B.; Fletcher, Murray J.; You, Minsheng; Yang, Guang; Gurr, Geoff M.

    2016-01-01

    Phytoplasmas are insect vectored mollicutes responsible for disease in many economically important crops. Determining which insect species are vectors of a given phytoplasma is important for managing disease but is methodologically challenging because disease-free plants need to be exposed to large numbers of insects, often over many months. A relatively new method to detect likely transmission involves molecular testing for phytoplasma DNA in sucrose solution that insects have fed upon. In this study we combined this feeding medium method with a loop-mediated isothermal amplification (LAMP) assay to study 627 insect specimens of 11 Hemiptera taxa sampled from sites in Papua New Guinea affected by Bogia coconut syndrome (BCS). The LAMP assay detected phytoplasma DNA from the feeding solution and head tissue of insects from six taxa belonging to four families: Derbidae, Lophopidae, Flatidae and Ricaniidae. Two other taxa yielded positives only from the heads and the remainder tested negative. These results demonstrate the utility of combining single-insect feeding medium tests with LAMP assays to identify putative vectors that can be the subject of transmission tests and to better understand phytoplasma pathosystems. PMID:27786249

  16. Development and Evaluation of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Tylenchulus semipenetrans Using DNA Extracted from Soil

    PubMed Central

    Song, Zhi-Qiang; Cheng, Ju-E; Cheng, Fei-Xue; Zhang, De-Yong; Liu, Yong

    2017-01-01

    Tylenchulus semipenetrans is an important and widespread plant-parasitic nematode of citrus worldwide and can cause citrus slow decline disease leading to significant reduction in tree growth and yield. Rapid and accurate detection of T. semipenetrans in soil is important for the disease forecasting and management. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to detect T. semipenetrans using DNA extracted from soil. A set of five primers was designed from the internal transcribed spacer region (ITS1) of rDNA, and was highly specific to T. semipenetrans. The LAMP reaction was performed at 63°C for 60 min. The LAMP product was visualized directly in one reaction tube by adding SYBR Green I. The detection limit of the LAMP assay was 10−2 J2/0.5 g of soil, which was 10 times more sensitive than conventional PCR (10−1 J2/0.5 g of soil). Examination of 24 field soil samples revealed that the LAMP assay was applicable to a range of soils infested naturally with T. semipenetrans, and the total assay time was less than 2.5 h. These results indicated that the developed LAMP assay is a simple, rapid, sensitive, specific and accurate technique for detection of T. semipenetrans in field soil, and contributes to the effective management of citrus slow decline disease. PMID:28381965

  17. 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.

  18. 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

  19. Development and evaluation of a simple assay for Marburg virus detection using a reverse transcription-loop-mediated isothermal amplification method.

    PubMed

    Kurosaki, Yohei; Grolla, Allen; Fukuma, Aiko; Feldmann, Heinz; Yasuda, Jiro

    2010-07-01

    Marburg virus (MARV) causes a severe hemorrhagic fever in humans with a high mortality rate. The rapid and accurate identification of the virus is required to appropriately provide infection control and outbreak management. Here, we developed and evaluated a one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the rapid and simple detection of MARV. By combining two sets of primers specific for the Musoke and Ravn genetic lineages, a multiple RT-LAMP assay detected MARV strains of both lineages, and no cross-reactivity with other hemorrhagic fever viruses (Ebola virus and Lassa virus) was observed. The assay could detect 10(2) copies of the viral RNA per tube within 40 min by real-time monitoring of the turbidities of the reaction mixtures. The assay was further evaluated using viral RNA extracted from clinical specimens collected in the 2005 Marburg hemorrhagic fever outbreak in Angola and yielded positive results for samples containing MARV at greater than 10(4) 50% tissue culture infective doses/ml, exhibiting 78% (14 of 18 samples positive) consistency with the results of a reverse transcription-PCR assay carried out in the field laboratory. The results obtained by both agarose gel electrophoresis and naked-eye judgment indicated that the RT-LAMP assay developed in this study is an effective tool for the molecular detection of MARV. Furthermore, it seems suitable for use for field diagnostics or in laboratories in areas where MARV is endemic.

  20. Utility of Loop-mediated Isothermal Amplification Assay, Polymerase Chain Reaction, and ELISA for Diagnosis of Leptospirosis in South Indian Patients

    PubMed Central

    Sengupta, Mallika; Prabhakar, Abhilash Kundavaram Paul; Satyendra, Sowmya; Thambu, David; Abraham, Ooriapadickal Cherian; Balaji, Veeraraghavan; Chen, Hua-Wei; Chao, Chien-Chung; Ching, Wei-Mei; Prakash, John Antony Jude

    2017-01-01

    Background: Leptospirosis is a zoonotic disease which requires laboratory diagnosis for confirmation. Materials and Methods: In this study serum samples from adults with acute undifferentiated fever (duration ≤15 days) were tested for IgM antibodies to Leptospira by ELISA, PCR for rrs gene and loop-mediated isothermal amplification (LAMP) assay for LipL32 and LipL41. Results: Among the 150 sera tested, three were positive by PCR, LAMP and IgM ELISA/modified Faines’ criteria, two by only PCR; seven only by LAMP assay and forty fulfilled modified Faine's criteria (illness clinically compatible and IgM ELISA positive for leptospirosis). Clinical correlation revealed renal compromise, low platelet count and severe jaundice were significantly related to leptospirosis (P < 0.05). Conclusion: This study suggests that LAMP assay could be useful for diagnosis of leptospirosis during the 1st week of illness whereas IgM ELISA forms the mainstay of diagnosis from the 2nd week onward. Further studies especially community based, comparing ELISA, PCR, LAMP, culture and microscopic agglutination test are required to evaluate the veracity of these findings. PMID:28250618

  1. An integrated rotary microfluidic system with DNA extraction, loop-mediated isothermal amplification, and lateral flow strip based detection for point-of-care pathogen diagnostics.

    PubMed

    Park, Byung Hyun; Oh, Seung Jun; Jung, Jae Hwan; Choi, Goro; Seo, Ji Hyun; Kim, Do Hyun; Lee, Eun Yeol; Seo, Tae Seok

    2017-05-15

    Point-of-care (POC) molecular diagnostics plays a pivotal role for the prevention and treatment of infectious diseases. In spite of recent advancement in microfluidic based POC devices, there are still rooms for development to realize rapid, automatic and cost-effective sample-to-result genetic analysis. In this study, we propose an integrated rotary microfluidic system that is capable of performing glass microbead based DNA extraction, loop mediated isothermal amplification (LAMP), and colorimetric lateral flow strip based detection in a sequential manner with an optimized microfluidic design and a rotational speed control. Rotation direction-dependent coriolis force and siphon valving structures enable us to perform the fluidic control and metering, and the use of the lateral flow strip as a detection method renders all the analytical processes for nucleic acid test simplified and integrated without the need of expensive instruments or human intervention. As a proof of concept for point-of-care DNA diagnostics, we identified the food-borne bacterial pathogen which was contaminated in water or milk. Not only monoplex Salmonella Typhimurium but also multiplex Salmonella Typhimurium and Vibrio parahaemolyticus were analysed on the integrated rotary genetic analysis microsystem with a limit of detection of 50 CFU in 80min. In addition, three multiple samples were simultaneously analysed on a single device. The sample-to-result capability of the proposed microdevice provides great usefulness in the fields of clinical diagnostics, food safety and environment monitoring.

  2. Loop-mediated isothermal amplification assay for detection and discrimination of Toxocara canis and Toxocara cati eggs directly from sand samples.

    PubMed

    Macuhova, K; Kumagai, T; Akao, N; Ohta, N

    2010-12-01

    We developed a novel and simple method, using loop-mediated isothermal amplification (LAMP), for the detection and discrimination of Toxocara canis and Toxocara cati eggs. The new method employs 4 steps: (1) concentration of Toxocara eggs in a small amount of sand; (2) dissolution of the proteinaceous membrane of eggs and simultaneously separation of them from the sand using NaClO treatment; (3) extraction of DNA using NaOH treatment; and (4) detection of T. canis / T. cati DNA using a LAMP assay. All these steps are fast, easy to perform, and do not require expensive equipment or reagents. The novel method was tested both experimentally and in a field study. In the laboratory, we could reliably detect as few as 3 T. canis eggs in artificially contaminated sand, if the experiment was repeated twice. In the field trial, we were able to detect T. cati DNA from 4 natural sandpits having moderate to heavy contamination, although not in a single lightly contaminated sandpit. All of the examined sandpits were found to be contaminated with eggs of T. cati, but none appeared to contain T. canis. Our new method could extract DNA from T. canis and T. cati eggs directly from sand samples as well as detect and distinguish these 2 species in a few easy steps, with markedly reduced time and expense.

  3. The development of loop-mediated isothermal amplification (LAMP) assays for the rapid authentication of five forbidden vegetables in strict vegetarian diets

    PubMed Central

    Lee, Meng-Shiou; Su, Ting-Ying; Lien, Yi-Yang; Sheu, Shyang-Chwen

    2017-01-01

    Plant-based food ingredients such as garlic, Chinese leek, Chinese onion, green onion and onion are widely used in many cuisines around the world. However, these ingredients known as the “five forbidden vegetables” (FFVs) are not allowed in some vegetarian diets. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of FFVs using five respective LAMP primer sets. The specific primers targeted the ITS1-5.8S-ITS2 nuclear ribosomal DNA sequence regions among the five vegetables. The results demonstrated that the identification of FFVs using the newly developed LAMP assay is more sensitive than the traditional PCR method. Using pepper, basil, parsley, chili and ginger as references, established LAMP primer sets showed high specificity for the identification of the FFV species. Moreover, when FFVs were mixed with other plant ingredients at different ratios (100:0, 50:50, 20:80, 10:90, 5:95, 2:98, and 1:99), no cross-reactivity was evident using LAMP. Finally, genomic DNAs extracted from boiled and steamed FFVs in processed foods were used as templates; the performance of the LAMP reaction was not influenced using validated LAMP primers. Not only can FFV ingredients be identified but commercial foods containing FFVs can also be authenticated. This LAMP method will be useful for the authentication of FFVs in practical food markets in the future. PMID:28290475

  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.

  5. Loop-Mediated Isothermal Amplification of the sefA Gene for Rapid Detection of Salmonella Enteritidis and Salmonella Gallinarum in Chickens.

    PubMed

    Gong, Jiansen; Zhuang, Linlin; Zhu, Chunhong; Shi, Shourong; Zhang, Di; Zhang, Linji; Yu, Yan; Dou, Xinhong; Xu, Bu; Wang, Chengming

    2016-04-01

    Salmonella spp. pose a threat to both human and animal health, with more than 2600 serovars having been reported to date. Salmonella serovars are usually identified by slide agglutination tests, which are labor intensive and time consuming. In an attempt to develop a more rapid screening method for the major poultry Salmonella serovars, we developed a loop-mediated isothermal amplification (LAMP) assay, which directly detected the sefA gene, a fimbrial operon gene existing in several specific serovars of Salmonella enterica including the major poultry serovars, namely Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) and Salmonella enterica serovar Gallinarum (Salmonella Gallinarum). With the 177 bacterial strains we tested, positive reactions were only observed with 85 strains of serovar Salmonella Enteritidis and Salmonella Gallinarum. The detection limit of the LAMP assay was 4 CFU/reaction with genomic DNAs of Salmonella Enteritidis (ATCC 13076) from pure culture and 400 CFU/ reaction with DNA extracted from spiked chicken feces. The LAMP assay was more sensitive than conventional culture, especially without enrichment, in detecting Salmonella Enteritidis (CMCC 50041) in the spiked fecal samples. The results show the sefA LAMP method is a rapid, sensitive, specific, and practical method for directly detection of Salmonella Enteritidis and Salmonella Gallinarum in chickens. The sefA LAMP assay can potentially serve as new on-site diagnostics in the poultry industry.

  6. Evaluation of loop-mediated isothermal amplification method (LAMP) for pathogenic Leptospira spp. detection with leptospires isolation and real-time PCR

    PubMed Central

    SUWANCHAROEN, Duangjai; SITTIWICHEANWONG, Busara; WIRATSUDAKUL, Anuwat

    2016-01-01

    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

  7. Rapid detection of blaNDM, blaKPC, blaIMP, and blaVIM carbapenemase genes in bacteria by loop-mediated isothermal amplification.

    PubMed

    Cheng, Cancan; Zheng, Fen; Rui, Yongyu

    2014-12-01

    A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for rapid detection of blaKPC, blaNDM, blaIMP, and blaVIM carbapenemase genes. Six oligonucleotides, including outer, inner, and loop primers, were designed for eight distinct regions in each target gene. Two qualitative criteria were used to evaluate LAMP reactions: visual inspection of color change and real-time detection of fluorescence change. The lower detection limit was 10 colony forming units (CFU) per reaction for real-time detection and 100 CFU per reaction for visual inspection for each gene. Two hundred twenty-two carbapenem-resistant clinical isolates (including 100 Pseudomonas aeruginosa, 100 Acinetobacter sp., and 22 Enterobacteriaceae) were tested by LAMP assay. At the same time, these isolates were confirmed by conventional polymerase chain reaction (PCR) and sequencing analysis. In these clinical isolates, the results of 11 strains with blaNDM, 11 strains with blaKPC, 11 strains with blaVIM, and 2 strains with blaIMP obtained using LAMP assays were concordant with conventional PCR. The LAMP method reported here may be a useful and powerful tool for rapid detection of blaNDM, blaKPC, blaIMP, and blaVIM carbapenemase genes in bacteria.

  8. 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.

  9. A comparison of the reliability of two gene targets in loop-mediated isothermal amplification assays for detecting leptospiral DNA in canine urine.

    PubMed

    Gentilini, Fabio; Zanoni, Renato Giulio; Zambon, Elisa; Turba, Maria Elena

    2017-01-01

    We compared 2 novel loop-mediated isothermal amplification (LAMP) assays that target either the 16S ribosomal RNA ( rrs) gene or the gene encoding a 32-kDa leptospiral lipoprotein ( lipL32) in order to assess the effect of the target on the accuracy of the LAMP assays. The most sensitive assay was the rrs assay with a limit of detection (LOD) of 1.2 × 10(1) genome equivalents per reaction. The novel lipL32 assay showed an LOD of 1.2 × 10(2) genome equivalents per reaction. Both assays showed adequate specificity when tested against a collection of bacteria commonly found in voided canine urine. However, when field samples were assayed, the rrs assays gave many false-positive results and a poor positive predictive value of 8.33%. In conclusion, even if the LAMP assay is used in low prevalence areas, the lipL32 assay would be preferable. Conversely, the higher analytical sensitivity of the rrs assay could be effectively used as a screening test in endemic areas with high disease prevalence, followed by confirmation of the positive results using the lipL32 assay.

  10. Development and Evaluation of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Tylenchulus semipenetrans Using DNA Extracted from Soil.

    PubMed

    Song, Zhi-Qiang; Cheng, Ju-E; Cheng, Fei-Xue; Zhang, De-Yong; Liu, Yong

    2017-04-01

    Tylenchulus semipenetrans is an important and widespread plant-parasitic nematode of citrus worldwide and can cause citrus slow decline disease leading to significant reduction in tree growth and yield. Rapid and accurate detection of T. semipenetrans in soil is important for the disease forecasting and management. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to detect T. semipenetrans using DNA extracted from soil. A set of five primers was designed from the internal transcribed spacer region (ITS1) of rDNA, and was highly specific to T. semipenetrans. The LAMP reaction was performed at 63°C for 60 min. The LAMP product was visualized directly in one reaction tube by adding SYBR Green I. The detection limit of the LAMP assay was 10(-2) J2/0.5 g of soil, which was 10 times more sensitive than conventional PCR (10(-1) J2/0.5 g of soil). Examination of 24 field soil samples revealed that the LAMP assay was applicable to a range of soils infested naturally with T. semipenetrans, and the total assay time was less than 2.5 h. These results indicated that the developed LAMP assay is a simple, rapid, sensitive, specific and accurate technique for detection of T. semipenetrans in field soil, and contributes to the effective management of citrus slow decline disease.

  11. An improved method for detection of Edwardsiella tarda by loop-mediated isothermal amplification by targeting the EsrB gene

    NASA Astrophysics Data System (ADS)

    Xie, Guosi; Zhang, Qingli; Han, Nana; Shi, Chengyin; Wang, Xiuhua; Liu, Qinghui; Huang, Jie

    2012-07-01

    Edwardsiella tarda is a major pathogen in aquatic environments that can cause heavy economic losses. An improved method for quick and accurate detection of E. tarda by loop-mediated isothermal amplification (LAMP) with two additional loop primers was developed by targeting the EsrB gene ( EsrB — LAMP). In this method, the Mg2+ concentration, reaction temperature, and reaction time were optimized to 8 mmol/L, 61°C, and 40 min, respectively. The detection limit with the EsrB gene was as low as 10 copies, which is 100 times more sensitive than that of conventional polymerase chain reaction (PCR). The EsrB-LAMP assay was shown more sensitive and rapid than previously reported LAMP assays targeting the hemolysin gene ( hemolysin -LAMP) for detection of E. tarda. The EsrB -LAMP was also highly specific to E. tarda and had no cross-reaction with 13 other strains of bacteria. The assay can be carried out in a simple heating device and the EsrB-LAMP products can be visually detected by adding fluorescent dye to the reaction mixture. Taken together, the improved EsrB-LAMP diagnostic protocol has the potential for detection of E. tarda from indoor and outdoor samples.

  12. 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.

  13. Molecular Identification of an Invasive Wood-Boring Insect Lyctus brunneus (Coleoptera: Bostrichidae: Lyctinae) Using Frass by Loop-Mediated Isothermal Amplification and Nested PCR Assays.

    PubMed

    Ide, Tatsuya; Kanzaki, Natsumi; Ohmura, Wakako; Okabe, Kimiko

    2016-03-27

    Lyctus brunneus(Stephens) is one of the most destructive and worldwide invasive pests of seasoned woods for wooden products. This and other pestLyctusspecies have had their distribution expanded by international and domestic human transportation of infested wood and wood products. Rapid detection and accurate identification ofLyctusspecies are effective tools for helping to eradicate them in new introduction sites. The accurate species-level identification of adults requires expert knowledge about their morphology. However, it takes much time and effort to recover suitable adult specimens because they are borers inside wood. Frass ofLyctusspecies can easily be detected and recovered in and around infested wood. Thus, frass was tested to see if it was a suitable sample to allow development of a rapid and technically easy molecular detection and identification method forL.brunneus.Species-specific primers were designed from the cytochrome c oxidase subunit I region ofL.brunneusand used in development and testing of methods for successfully identifying them from their frass using the loop-mediated isothermal amplification (LAMP) or species-specific nested polymerase chain reaction (PCR) assays. The LAMP assay was faster and more sensitive for detecting the presence of DNA derived fromL.brunneusin their frass than the nested PCR assay. These methodologies will be applicable for the rapid detection and identification of other wood-boring invasive pests in regulatory applications.

  14. The evaluation of loop-mediated isothermal amplification-quartz crystal microbalance (LAMP-QCM) biosensor as a real-time measurement of HPV16 DNA.

    PubMed

    Jearanaikoon, Patcharee; Prakrankamanant, Preeda; Leelayuwat, Chanvit; Wanram, Surasak; Limpaiboon, Temduang; Promptmas, Chamras

    2016-03-01

    We have previously developed quartz crystal microbalance biosensor integrated with loop-mediated isothermal amplification (LAMP-QCM) for human papillomavirus (HPV) type58 DNA detection. Infection with HPV, particularly HPV16, remains a serious health problem due to its major risk factor contributing to cervical cancer. In the present study, LAMP-QCM biosensor was evaluated in terms of a quantitative assay for copy number of HPV16 DNA in cervical samples compared to quantitative PCR using TaqMan assay (TaqMan-qPCR). The detection limit of LAMP-QCM was found to be 10 fold more sensitive than TaqMan-qPCR with 100% specificity and 7.6% imprecision. Different plot of HPV16 DNA copy number using Bland-Altman analysis revealed 94% correlation between LAMP-QCM and qPCR. We therefore concluded that the developed LAMP-QCM biosensor provides a possible rapid and sensitive assay for HPV16 DNA quantification in a routine laboratory.

  15. 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.

  16. A loop-mediated isothermal amplification (LAMP) assay for Strongyloides stercoralis in stool that uses a visual detection method with SYTO-82 fluorescent dye.

    PubMed

    Watts, Matthew R; James, Gregory; Sultana, Yasmin; Ginn, Andrew N; Outhred, Alexander C; Kong, Fanrong; Verweij, Jaco J; Iredell, Jonathan R; Chen, Sharon C-A; Lee, Rogan

    2014-02-01

    An assay to detect Strongyloides stercoralis in stool specimens was developed using the loop-mediated isothermal amplification (LAMP) method. Primers were based on the 28S ribosomal subunit gene. The reaction conditions were optimized and SYTO-82 fluorescent dye was used to allow real-time and visual detection of the product. The product identity was confirmed with restriction enzyme digestion, cloning, and sequence analysis. The assay was specific when tested against DNA from bacteria, fungi and parasites, and 30 normal stool samples. Analytical sensitivity was to < 10 copies of target sequence in a plasmid and up to a 10(-2) dilution of DNA extracted from a Strongyloides ratti larva spiked into stool. Sensitivity was increased when further dilutions were made in water, indicative of reduced reaction inhibition. Twenty-seven of 28 stool samples microscopy and polymerase chain reaction positive for S. stercoralis were positive with the LAMP method. On the basis of these findings, the assay warrants further clinical validation.

  17. 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

  18. 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.

  19. Monte Carlo Modeling-Based Digital Loop-Mediated Isothermal Amplification on a Spiral Chip for Absolute Quantification of Nucleic Acids.

    PubMed

    Xia, Yun; Yan, Shuangqian; Zhang, Xian; Ma, Peng; Du, Wei; Feng, Xiaojun; Liu, Bi-Feng

    2017-03-21

    Digital loop-mediated isothermal amplification (dLAMP) is an attractive approach for absolute quantification of nucleic acids with high sensitivity and selectivity. Theoretical and numerical analysis of dLAMP provides necessary guidance for the design and analysis of dLAMP devices. In this work, a mathematical model was proposed on the basis of the Monte Carlo method and the theories of Poisson statistics and chemometrics. To examine the established model, we fabricated a spiral chip with 1200 uniform and discrete reaction chambers (9.6 nL) for absolute quantification of pathogenic DNA samples by dLAMP. Under the optimized conditions, dLAMP analysis on the spiral chip realized quantification of nucleic acids spanning over 4 orders of magnitude in concentration with sensitivity as low as 8.7 × 10(-2) copies/μL in 40 min. The experimental results were consistent with the proposed mathematical model, which could provide useful guideline for future development of dLAMP devices.

  20. 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.

  1. Development of Reverse Transcription Loop-Mediated Isothermal Amplification for Simple and Rapid Detection of Promyelocytic Leukemia–Retinoic Acid Receptor α mRNA

    PubMed Central

    Hashimoto, Yuki; Hatayama, Yuki; Kojima, Nao; Morishita, Shota; Matsumoto, Satoko; Hosoda, Yuzuru; Hara, Ayako; Motokura, Toru

    2016-01-01

    Background Acute promyelocytic leukemia (APL) is a disease characterized by expression of Promyelocytic Leukemia–Retinoic Acid Receptor α (PML-RARα) chimeric mRNA. Although APL is curable, early death due to hemorrhage is a major problem. Here, we report the development of a simple and rapid diagnostic method for APL based on reverse transcription loop-mediated isothermal amplification (RT-LAMP). Methods An RT-LAMP primer set was designed to detect three types of PML-RARα mRNA in a single reaction. Serial dilutions of plasmid DNA containing bcr1, bcr2, or bcr3 PML-RARα sequences and RNA extracted from bone marrow aspirates of 6 patients with APL were used to compare the results of RT-LAMP and nested PCR assays. Results Plasmid DNA was amplified by RT-LAMP, for which the reaction time was > 4 h shorter and the lower detection limit was higher than for nested RT-PCR. Six of 7 samples tested positive by both methods. Conclusion We developed an RT-LAMP assay for simple and rapid PML-RARα mRNA detection that may be clinically useful for point-of-care testing and APL diagnosis. PMID:28070163

  2. 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.

  3. Rapid detection and typing of influenza A and B by loop-mediated isothermal amplification: comparison with immunochromatography and virus isolation.

    PubMed

    Ito, Masahiro; Watanabe, Masahiro; Nakagawa, Naoko; Ihara, Toshiaki; Okuno, Yoshinobu

    2006-08-01

    A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was established for the detection of influenza A virus H1 and H3 subtype strains and influenza B virus strains specifically. The total procedure from RNA extraction to virus typing was completed within 3h. In terms of specificity, the representative AH1, AH3 and B strains were detected only by strain-specific primers respectively. No cross-detection was observed. In terms of sensitivity, virus was detected at a minimum concentration of 10 ffu/ml. Eighty-three nasopharyngeal aspirates obtained from children diagnosed clinically with influenza were tested by the RT-LAMP assay, along with commercially available immunochromatography rapid diagnostic tests and by virus isolation. Virus was isolated from 78 samples (94%) and the subtype was determined by the hemagglutination inhibition test. Although it took at least 3 days, the detection sensitivity was the best of the three methods. With two rapid assays, the detection sensitivity of the RT-LAMP assay (85.5%) was higher than that of immunochromatography tests (75.9%). In addition, the RT-LAMP assay can be used to differentiate emerging influenza virus subtypes by selecting appropriate primer sets.

  4. 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.

  5. 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

  6. Establishment of loop-mediated isothermal amplification (LAMP) for rapid detection of Brucella spp. and application to milk and blood samples.

    PubMed

    Song, Liuyan; Li, Juntao; Hou, Shuiping; Li, Xunde; Chen, Shouyi

    2012-09-01

    Brucella spp. are facultative intracellular bacteria that infect humans and animals. In this study, the loop-mediated isothermal amplification (LAMP) was used to detect the Brucella-specific gene omp25. Reaction conditions were optimized as temperature 65°C, reaction time 60 min, Mg(2+) concentration 8.0 mmol/L, polymerase content Bst DNA, 0.5 μL, deoxyribonucleotide concentration 1.6 mmol/L, and inner/outer primer ratio 1:8. The LAMP method was evaluated with 4 Brucella species and 29 non-Brucella bacteria species. Positive reactions were observed on all the 4 Brucella species but not on any non-Brucella species. The limit of detection of the LAMP method was 3.81 CFU Brucella spp. Using the LAMP method, 7 of 110 raw milk samples and 5 of 59 sheep blood samples were detected positive of Brucella spp. Results indicated that LAMP is a fast, specific, sensitive, inexpensive, and suitable method for diagnosis of Brucella spp. infection.

  7. Simple differentiation method of mumps Hoshino vaccine strain from wild strains by reverse transcription loop-mediated isothermal amplification (RT-LAMP).

    PubMed

    Yoshida, Naoko; Fujino, Motoko; Ota, Yoshinori; Notomi, Tsugunori; Nakayama, Tetsuo

    2007-01-26

    Mumps virus is still circulating and annual mumps outbreaks occur with fluctuating magnitudes in Japan. Aseptic meningitis has been reported after vaccination and it would be of importance to determine whether this was related to the vaccination. The objective of this study was to develop a sensitive, specific and rapid diagnostic method for the differentiation of the Hoshino vaccine strain from circulating wild types. We developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method of the hemagglutinin neuraminidase (HN) region for the detection of mumps virus genome from clinical samples. The typical ladder pattern disappeared after the LAMP products of the Hoshino vaccine strain were digested with ScaI, but those of wild types were not cut by ScaI. We obtained 19 cerebro spinal fluids (CSF) from the patients with aseptic meningitis and 17 salivary swab samples from the patients with acute parotitis after mumps vaccination, in which one case was complicated with orchitis. Mumps virus genome was detected in 18 CSF samples and in all NPS by RT-LAMP. The Hoshino vaccine strain was identified in 16 out of 18 CSF RT-LAMP positives and in 11 out of 17 NPS samples and the remaining samples were identified as wild types. RT-LAMP followed by ScaI digestion is a sensitive, simple and rapid differential method and useful for laboratory surveillance for vaccine-adverse events.

  8. Detection of microRNA in clinical tumor samples by isothermal enzyme-free amplification and label-free graphene oxide-based SYBR Green I fluorescence platform.

    PubMed

    Zhu, Debin; Zhang, Lan; Ma, Wenge; Lu, Suqin; Xing, Xiaobo

    2015-03-15

    MicroRNAs (miRNAs) are a kind of small molecules that involve in many important life activities. They have higher expression levels in many kinds of cancers. In this study, we developed an isothermal enzyme-free amplification (EFA) and label-free graphene oxide (GO)-based SYBR Green I fluorescence platform for detection of miRNA. MiRNA-21 was used as an example to demonstrate the feasibility of the method. Results show that the sensitivity of miRNA-21 is 1pM, and the linearity range is from 1pM to 1nM. The method can specifically discriminate miRNA-21 from miRNA-210 and miRNA-214. Three tumor cell lines of A549, HepG2 and MCF7 were detected by the method. The sensitivities of them were 10(2) cells, 10(3) cells and 10(3) cells respectively. Clinical tumor samples were also tested by this method, and 29 of 40 samples gave out positive signals. The method holds great promise in miRNA detection due to its convenience, rapidness, inexpensive and specificity.

  9. Development of loop-mediated isothermal amplification and SYBR green real-time PCR methods for the detection of Citrus yellow mosaic badnavirus in citrus species.

    PubMed

    Anthony Johnson, A M; Dasgupta, I; Sai Gopal, D V R

    2014-07-01

    Citrus yellow mosaic badnavirus (CMBV) is an important pathogen in southern India spread by infected citrus propagules. One of the measures to arrest the spread of CMBV is to develop methods to screen and certify citrus propagules as CMBV-free. The methods loop-mediated isothermal amplification (LAMP) and SYBR green real-time PCR (SGRTPCR) have been developed for the efficient detection of CMBV in citrus propagules. This paper compares the sensitivities of LAMP and SGRTPCR with polymerase chain reaction (PCR) for the detection of CMBV. Whereas PCR and LAMP were able to detect CMBV from a minimum of 10 ng of total DNA of infected leaf samples, SGRTPCR could detect the same from 1 ng of total DNA. Using SGRTPCR, the viral titres were estimated to be the highest in rough lemon and lowest in Nagpur Mandarin of the five naturally infected citrus species tested. The results will help in designing suitable strategies for the sensitive detection of CMBV from citrus propagules.

  10. The Novel Multiple Inner Primers-Loop-Mediated Isothermal Amplification (MIP-LAMP) for Rapid Detection and Differentiation of Listeria monocytogenes.

    PubMed

    Wang, Yi; Wang, Yan; Ma, Aijing; Li, Dongxun; Luo, Lijuan; Liu, Dongxin; Hu, Shoukui; Jin, Dong; Liu, Kai; Ye, Changyun

    2015-12-03

    Here, a novel model of loop-mediated isothermal amplification (LAMP), termed multiple inner primers-LAMP (MIP-LAMP), was devised and successfully applied to detect Listeria monocytogenes. A set of 10 specific MIP-LAMP primers, which recognized 14 different regions of target gene, was designed to target a sequence in the hlyA gene. The MIP-LAMP assay efficiently amplified the target element within 35 min at 63 °C and was evaluated for sensitivity and specificity. The templates were specially amplified in the presence of the genomic DNA from L. monocytogenes. The limit of detection (LoD) of MIP-LAMP assay was 62.5 fg/reaction using purified L. monocytogenes DNA. The LoD for DNA isolated from serial dilutions of L. monocytogenes cells in buffer and in milk corresponded to 2.4 CFU and 24 CFU, respectively. The amplified products were analyzed by real-time monitoring of changes in turbidity, and visualized by adding Loop Fluorescent Detection Reagent (FD), or as a ladder-like banding pattern on gel electrophoresis. A total of 48 pork samples were investigated for L. monocytogenes by the novel MIP-LAMP method, and the diagnostic accuracy was shown to be 100% when compared to the culture-biotechnical method. In conclusion, the MIP-LAMP methodology was demonstrated to be a reliable, sensitive and specific tool for rapid detection of L. monocytogenes strains.

  11. A loop-mediated isothermal amplification (LAMP) assay targeting 16S rRNA gene for rapid detection of Anaplasma phagocytophilum infection in sheep and goats.

    PubMed

    Ning, Changshen; Wang, Jinhong; Zhang, Yan; Wang, Xiaoxing; Cui, Yanyan; Yan, Yaqun; Wang, Rongjun; Jian, Fuchun; Zhang, Longxian

    2017-01-24

    Anaplasma phagocytophilum is a zoonotic pathogen and the causative agent of human granulocytic anaplasmosis (HGA) in humans and tick-borne fever in various kinds of animals. In the present study, a loop-mediated isothermal amplification (LAMP) assay for rapid detection of A. phagocytophilum was developed using primers specific to 16S rRNA gene of this organism. The LAMP assay was performed at 65 C for 60 min and terminated at 80 C for 10 min. The optimal reaction conditions, under which no cross-reaction was observed with other closely related tick borne parasites (Anaplasma bovis, Anaplasma ovis, Theileria luwenshuni, Babesia motasi and Schistosoma japonicum) was established. The assay exhibited much higher sensitivity when compared with conventional PCR (1 copy vs 1000 copies). To evaluate the applicability of the LAMP assay, 94 sheep field blood samples were analyzed for A. phagocytophilum infection using LAMP, nested PCR and conventional PCR assay at the same time. A positive LAMP result was obtained from 53 of the 94 samples (56.4%), while only 12 (12.8%) and 3 (3.2%) were tested positive by nested PCR and conventional PCR, respectively. In conclusion, this LAMP assay is a specific, sensitive, and rapid method for the detection of A. phagocytophilum in sheep.

  12. Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for the detection of Haemonchus contortus in goat fecal samples.

    PubMed

    Yang, Xin; Qi, Mingwei; Zhang, Zongze; Gao, Chong; Wang, Chunqun; Lei, Weiqiang; Tan, Li; Zhao, Junlong; Fang, Rui; Hu, Min

    2017-01-18

    Haemonchus contortus is one of the most significant strongylid nematodes infecting small ruminants and causes great economic losses to the livestock industry worldwide. Accurate diagnosis of H. contortus is crucial to control the infection. Traditional microscopic examinations are the most common methods for the diagnosis of H. contortus, but they are time-consuming and inaccurate. Molecular methods based on PCR are more accurate, but need expensive machines usually only used in the laboratory. Loop-mediated isothermal amplification (LAMP) is a rapid, simple, specific and sensitive method that has been widely used to detect viruses, bacteria and parasites. In the present study, a LAMP method targeting ribosomal ITS-2 gene for detection of the H. contortus in goat faecal samples has been established. The established LAMP method was H. contortus specific and the sensitivity of LAMP was the same as that of the H. contortus species-specific PCR with the lowest DNA level detected as being 1 pg. Examination of the clinical samples indicated that the positive rate of LAMP was higher than that of PCR, but no statistical difference was observed between LAMP and PCR (χ2=17.991, P=0.053). In conclusion, a LAMP assay with a high specificity and a good sensitivity has been developed to detect H. contortus infection in goats. The established LAMP assay is useful for clinical diagnosis of H. contortus.

  13. The development of loop-mediated isothermal amplification (LAMP) assays for the rapid authentication of five forbidden vegetables in strict vegetarian diets.

    PubMed

    Lee, Meng-Shiou; Su, Ting-Ying; Lien, Yi-Yang; Sheu, Shyang-Chwen

    2017-03-14

    Plant-based food ingredients such as garlic, Chinese leek, Chinese onion, green onion and onion are widely used in many cuisines around the world. However, these ingredients known as the "five forbidden vegetables" (FFVs) are not allowed in some vegetarian diets. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of FFVs using five respective LAMP primer sets. The specific primers targeted the ITS1-5.8S-ITS2 nuclear ribosomal DNA sequence regions among the five vegetables. The results demonstrated that the identification of FFVs using the newly developed LAMP assay is more sensitive than the traditional PCR method. Using pepper, basil, parsley, chili and ginger as references, established LAMP primer sets showed high specificity for the identification of the FFV species. Moreover, when FFVs were mixed with other plant ingredients at different ratios (100:0, 50:50, 20:80, 10:90, 5:95, 2:98, and 1:99), no cross-reactivity was evident using LAMP. Finally, genomic DNAs extracted from boiled and steamed FFVs in processed foods were used as templates; the performance of the LAMP reaction was not influenced using validated LAMP primers. Not only can FFV ingredients be identified but commercial foods containing FFVs can also be authenticated. This LAMP method will be useful for the authentication of FFVs in practical food markets in the future.

  14. 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.

  15. 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

    2015-12-22

    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.

  16. Rapid and Sensitive Detection of Vibrio parahaemolyticus and Vibrio vulnificus by Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification Technique.

    PubMed

    Wang, Yi; Li, Dongxun; Wang, Yan; Li, Kewei; Ye, Changyun

    2016-01-19

    Vibrio parahaemolyticus and Vibrio vulnificus are two marine seafood-borne pathogens causing severe illnesses in humans and aquatic animals. In this study, a recently developed novel multiple endonuclease restriction real-time loop-mediated isothermal amplification technology (MERT-LAMP) were successfully developed and evaluated for simultaneous detection of V. parahaemolyticus and V. vulnificus strains in only a single reaction. Two MERT-LAMP primer sets were designed to specifically target toxR gene of V. parahaemolyticus and rpoS gene of V. vulnificus. The MERT-LAMP reactions were conducted at 62 °C, and the positive results were produced in as short as 19 min with the genomic DNA templates extracted from the V. parahaemolyticus and V. vulnificus strains. The two target pathogens present in the same sample could be simultaneously detected and correctly differentiated based on distinct fluorescence curves in a real-time format. The sensitivity of MERT-LAMP assay was 250 fg and 125 fg DNA per reaction with genomic templates of V. parahaemolyticus and V. vulnificus strains, which was in conformity with conventional LAMP detection. Compared with PCR-based techniques, the MERT-LAMP technology was 100- and 10-fold more sensitive than that of PCR and qPCR methods. Moreover, the limit of detection of MERT-LAMP approach for V. parahaemolyticus isolates and V. vulnificus isolates detection in artificially-contaminated oyster samples was 92 CFU and 83 CFU per reaction. In conclusion, the MERT-LAMP assay presented here was a rapid, specific, and sensitive tool for the detection of V. parahaemolyticus and V. vulnificus, and could be adopted for simultaneous screening of V. parahaemolyticus and V. vulnificus in a wide variety of samples.

  17. 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.

  18. 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

  19. Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of the tdh and trh genes of Vibrio parahaemolyticus and related Vibrio species.

    PubMed

    Yamazaki, Wataru; Kumeda, Yuko; Misawa, Naoaki; Nakaguchi, Yoshitsugu; Nishibuchi, Mitsuaki

    2010-02-01

    Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the major virulence determinants of Vibrio parahaemolyticus. TRH is further differentiated into TRH1 and TRH2 on the basis of genetic and phenotypic differences. We developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for sensitive and rapid detection of the tdh, trh1, and trh2 genes of V. parahaemolyticus. The LAMP assay was designed for both combined and individual detection of the tdh, trh1, and trh2 genes and combined detection of the trh1 and trh2 genes. Our results showed that it gave the same results as DNA probes and conventional PCR assays for 125 strains of V. parahaemolyticus, 3 strains of Grimontia hollisae, and 2 strains of Vibrio mimicus carrying the tdh, trh1, and trh2 genes in various combinations. No LAMP products were detected for any of the 20 bacterial strains lacking the tdh, trh1, and trh2 genes. The sensitivities of the LAMP assay for detection of tdh-, trh1-, and trh2-carrying V. parahaemolyticus strains in spiked shrimp samples were 0.8, 21.3, and 5.0 CFU per LAMP reaction tube, respectively. Starting with DNA extraction from a single colony and from spiked shrimp samples, the LAMP assay required only 27 to 60 min and less than 80 min, respectively. This is the first report of a rapid and specific LAMP assay for detection and differentiation of the tdh, trh1, and trh2 genes of V. parahaemolyticus and related Vibrio species.

  20. Detection of Plasmodium vivax and Plasmodium falciparum DNA in human saliva and urine: loop-mediated isothermal amplification for malaria diagnosis.

    PubMed

    Ghayour Najafabadi, Zahra; Oormazdi, Hormozd; Akhlaghi, Lame; Meamar, Ahmad Reza; Nateghpour, Mehdi; Farivar, Leila; Razmjou, Elham

    2014-08-01

    This study investigated loop-mediated isothermal amplification (LAMP) detection of Plasmodium falciparum and Plasmodium vivax in urine and saliva of malaria patients. From May to November 2011, 108 febrile patients referred to health centers in Sistan and Baluchestan Province of south-eastern Iran participated in the study. Saliva, urine, and blood samples were analyzed with nested PCR and LAMP targeting the species-specific nucleotide sequence of small subunit ribosomal RNA gene (18S rRNA) of P. falciparum and P. vivax and evaluated for diagnostic accuracy by comparison to blood nested PCR assay. When nested PCR of blood is used as standard, microscopy and nested PCR of saliva and urine samples showed sensitivity of 97.2%, 89.4% and 71% and specificity of 100%, 97.3% and 100%, respectively. LAMP sensitivity of blood, saliva, and urine was 95.8%, 47% and 29%, respectively, whereas LAMP specificity of these samples was 100%. Microscopy and nested PCR of saliva and LAMP of blood were comparable to nested PCR of blood (к=0.95, 0.83, and 0.94, respectively), but agreement for nested PCR of urine was moderate (к=0.64) and poor to fair for saliva LAMP and urine LAMP (к=0.38 and 0.23, respectively). LAMP assay showed low sensitivity for detection of Plasmodium DNA in human saliva and urine compared to results with blood and to nested PCR of blood, saliva, and urine. However, considering the advantages of LAMP technology and of saliva and urine sampling, further research into the method is worthwhile. LAMP protocol and precise preparation protocols need to be defined and optimized for template DNA of saliva and urine.

  1. An immunoassay-based reverse-transcription loop-mediated isothermal amplification assay for the rapid detection of avian influenza H5N1 virus viremia.

    PubMed

    Tang, Yi; Yu, Xu; Chen, Hao; Diao, Youxiang

    2016-12-15

    Avian influenza virus (AIV) subtype H5N1 attracts particular consideration because it is a continuous threat to animals and public health systems. The viremia caused by AIV H5N1 infection may increase the risk of blood-borne transmission between humans. Therefore, there is a need to rapidly evaluate and implement screening measures for AIV H5N1 viremia that allows for rapid response to this potentially pandemic threat. The present report describes an immunoassay-based reverse-transcription loop-mediated isothermal amplification (immuno-RT-LAMP) assay for the rapid detection of AIV H5N1 in whole blood samples. Using PCR tubes coated with an H5 subtype monoclonal antibody, AIV H5N1 virions were specifically captured from blood samples. After a thermal lysis step, the released viral N1 gene was exponentially amplified using RT-LAMP on either a real-time PCR instrument for quantitative analysis, or in a water bath system for endpoint analysis. The detection limit of the newly developed immuno-RT-LAMP assay was as low as 1.62×10(1) 50% embryo infectious dose/mL of virus in both regular samples and simulated viremia samples. There were no cross-reactions with non-H5N1 influenza viruses or other avian viruses. The reproducibility of the assay was confirmed using intra- and inter-assay tests with variability ranging from 1.05% to 3.37%. Our results indicate that immuno-RT-LAMP is a novel, effective point-of-care virus identification solution for the rapid diagnosis and monitoring of AIV H5N1 in blood samples.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. Visual endpoint detection of Escherichia coli O157:H7 using isothermal Genome Exponential Amplification Reaction (GEAR) assay and malachite green.

    PubMed

    Jothikumar, Prithiviraj; Narayanan, Jothikumar; Hill, Vincent R

    2014-03-01

    Rapid and specific detection methods for bacterial agents in drinking water are important for disease prevention and responding to suspected contamination events. In this study, an isothermal Genome Exponential Amplification Reaction (GEAR) assay for Escherichia coli O157:H7 was designed specifically to recognize a 199-bp fragment of the lipopolysaccharide gene (rfbE) for rapid testing of water samples. The GEAR assay was found to be specific for E. coli O157:H7 using 10 isolates of E. coli O157:H7 and a panel of 86 bacterial controls. The GEAR assay was performed at a constant temperature of 65°C using SYTO 9 intercalating dye. Detection limits were determined to be 20 CFU for the GEAR assay. When SYTO 9 fluorescence was measured using a real-time PCR instrument, the assay had the same detection limit as when malachite green was added to the reaction mix and a characteristic blue color was visually observed in positive reactions. The study also found that 50 and 20 CFU of E. coli O157:H7 seeded into 100-liter of tap water could be detected by the GEAR assays after the sample was concentrated by hollow-fiber ultrafiltration (HFUF) and approximately 10% of HFUF concentrate was cultured using trypticase soy broth-novobiocin. When applied to 19 surface water samples collected from Tennessee and Kentucky, the GEAR assay and a published real-time PCR assay both detected E. coli O157:H7 in two of the samples. The results of this study indicate that the GEAR assay can be sensitive for rapid detection of E. coli O157:H7 in water samples using fluorometric instruments and visual endpoint determination.

  7. Development of a reverse transcription loop-mediated isothermal amplification assay for detecting nervous necrosis virus in olive flounder Paralichthys olivaceus.

    PubMed

    Suebsing, Rungkarn; Oh, Myung-Joo; Kim, Jeong-Ho

    2012-07-01

    In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid, sensitive, and inexpensive detection of nervous necrosis virus (NNV) in olive flounder, Paralichthys olivaceus, in Korea. A set of six specific primers was designed to target the RNA 2 gene encoding the coat protein of Korean NNV strains. The RT-LAMP reaction successfully detected NNV after 30 min at 65 degrees C. When the sensitivities among RT-LAMP, RT-PCR, and nested RTPCR were compared, the RT-LAMP was shown to be able to detect the RNA template at 2.58 × 10(-2) TCID50/ml, whereas the RT-PCR and nested RT-PCR were only able to detect the RNA template at 2.58 × 10(2) TCID50/ml and 2.58 TCID50/ml, respectively. Thus, the sensitivity of the RT-LAMP assay was higher than those of the RT-PCR assays. In the specificity test of the RT-LAMP, 2 genotypes of NNVs (SJNNV and RGNNV) were positive; however, no other fish viruses were positive with the primers, indicating that the RT-LAMP assay is only specific to NNV. A total of 102 olive flounder were collected from hatcheries between 2009 and 2011. The occurrence of NNV in olive flounder was determined to be 53.9% (55/ 102) by the RT-LAMP. On the other hand, the prevalence based on the nested RT-PCR and RT-PCR results was 33.8% (34/102) and 20.6% (21/102), respectively. This result indicates that the RT-LAMP assay developed in this study is suitable for early field diagnosis of NNV with high sensitivity.

  8. A nationwide survey of pathogenic leptospires in urine of cattle and buffaloes by Loop-mediated isothermal amplification (LAMP) method in Thailand, 2011–2013

    PubMed Central

    SUWANCHAROEN, Duangjai; LIMLERTVATEE, Supaluck; CHETIYAWAN, Philaiphon; TONGPAN, Phichet; SANGKAEW, Nongluck; SAWADDEE, Yaowarat; INTHAKAN, Kanya; WIRATSUDAKUL, Anuwat

    2016-01-01

    Leptospirosis is a worldwide distributed zoonosis which has long been endemic in Thailand. Cattle and buffaloes are important livestock species that live in close contact with humans, especially in rural areas. These animals may, therefore, act as long-term carriers of leptospirosis for humans and other livestock species. The present study employed loop-mediated isothermal amplification (LAMP) method to detect pathogenic leptospiral 16S rDNA in the urine of cattle and buffaloes for assessing associations between uroprevalence and species, sex, age and spatial distribution. A total of 3,657 urine samples were collected for laboratory diagnosis, and 312 of which turned positive to the test (true prevalence 5.90%; 95% CI 4.98–6.91). The highest true uroprevalence was found in lower northern region at 19.80% (95% CI 15.83–24.32) followed by upper and lower northeastern regions at 15.22% and 6.25%, respectively. However, the highest true uroprevalence in beef cattle, the majority of cattle in Thailand, was recorded in northeastern region which is the endemic area of human leptospirosis. The uroprevalence was not statistically different among species and types of examined animals. Male animals were over twice more likely to be infected compared to females. Excluding animals younger than one year of age due to small sample size, the uroprevalence upraised with increasing age. A collaborative investigation between veterinary and public health sectors is required to holistically explore the link between leptospirosis in humans and livestock, especially in high prevalent areas. PMID:27302016

  9. 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.

  10. Real-time loop-mediated isothermal amplification (LAMP) assay for group specific detection of important trichothecene producing Fusarium species in wheat.

    PubMed

    Denschlag, Carla; Rieder, Johann; Vogel, Rudi F; Niessen, Ludwig

    2014-05-02

    Trichothecene mycotoxins such as deoxynivaneol (DON), nivalenol (NIV) and T2-Toxin are produced by a variety of Fusarium spp. on cereals in the field and may be ingested by consumption of commodities and products made thereof. The toxins inhibit eukaryotic protein biosynthesis and may thus impair human and animal health. Aimed at rapid and sensitive detection of the most important trichothecene producing Fusarium spp. in a single analysis, a real-time duplex loop-mediated isothermal amplification (LAMP) assay was set up. Two sets of LAMP primers were designed independently to amplify a partial sequence of the tri6 gene in Fusarium (F.) graminearum and of the tri5 gene in Fusarium sporotrichioides, respectively. Each of the two sets detected a limited number of the established trichothecene producing Fusarium-species. However, combination of the two sets in one duplex assay enabled detection of F. graminearum, Fusarium culmorum, Fusarium cerealis, F. sporotrichioides, Fusarium langsethiae and Fusarium poae in a group specific manner. No cross reactions were detected with purified DNA from 127 other fungal species or with cereal DNA. To demonstrate the usefulness of the assay, 100 wheat samples collected from all over the German state of Bavaria were analyzed for the trichothecene mycotoxin DON by HPLC and for the presence of trichothecene producers by the new real-time duple