These are representative sample records from Science.gov related to your search topic.
For comprehensive and current results, perform a real-time search at Science.gov.
1

Helicase-dependent amplification of nucleic acids.  

PubMed

Helicase-dependent amplification (HDA) is a novel method for the isothermal in vitro amplification of nucleic acids. The HDA reaction selectively amplifies a target sequence by extension of two oligonucleotide primers. Unlike the polymerase chain reaction (PCR), HDA uses a helicase enzyme to separate the deoxyribonucleic acid (DNA) strands, rather than heat denaturation. This allows DNA amplification without the need for thermal cycling. The helicase used in HDA is a helicase super family II protein obtained from a thermophilic organism, Thermoanaerobacter tengcongensis (TteUvrD). This thermostable helicase is capable of unwinding blunt-end nucleic acid substrates at elevated temperatures (60° to 65°C). The HDA reaction can also be coupled with reverse transcription for ribonucleic acid (RNA) amplification. The products of this reaction can be detected during the reaction using fluorescent probes when incubations are conducted in a fluorimeter. Alternatively, products can be detected after amplification using a disposable amplicon containment device that contains an embedded lateral flow strip. PMID:24510297

Cao, Yun; Kim, Hyun-Jin; Li, Ying; Kong, Huimin; Lemieux, Bertrand

2013-01-01

2

Nucleic acid isothermal amplification technologies: a review.  

PubMed

Nucleic acid amplification technologies are used in the field of molecular biology and recombinant DNA technologies. These techniques are used as leading methods in detecting and analyzing a small quantity of nucleic acids. The polymerase chain reaction (PCR) is the most widely used method for DNA amplification for detection and identification of infectious diseases, genetic disorders and other research purposes. However, it requires a thermocycling machine to separate two DNA strands and then amplify the required fragment. Novel developments in molecular biology of DNA synthesis in vivo demonstrate the possibility of amplifying DNA in isothermal conditions without the need of a thermocycling apparatus. DNA polymerase replicates DNA with the aid of various accessory proteins. Recent identification of these proteins has enabled development of new in vitro isothermal DNA amplification methods, mimicking these in vivo mechanisms. There are several types of isothermal nucleic acid amplification methods such as transcription mediated amplification, nucleic acid sequence-based amplification, signal mediated amplification of RNA technology, strand displacement amplification, rolling circle amplification, loop-mediated isothermal amplification of DNA, isothermal multiple displacement amplification, helicase-dependent amplification, single primer isothermal amplification, and circular helicase-dependent amplification. In this article, we review these isothermal nucleic acid amplification technologies and their applications in molecular biological studies. PMID:18260008

Gill, Pooria; Ghaemi, Amir

2008-03-01

3

Miniaturized isothermal nucleic acid amplification, a review.  

PubMed

Micro-Total Analysis Systems (µTAS) for use in on-site rapid detection of DNA or RNA are increasingly being developed. Here, amplification of the target sequence is key to increasing sensitivity, enabling single-cell and few-copy nucleic acid detection. The several advantages to miniaturizing amplification reactions and coupling them with sample preparation and detection on the same chip are well known and include fewer manual steps, preventing contamination, and significantly reducing the volume of expensive reagents. To-date, the majority of miniaturized systems for nucleic acid analysis have used the polymerase chain reaction (PCR) for amplification and those systems are covered in previous reviews. This review provides a thorough overview of miniaturized analysis systems using alternatives to PCR, specifically isothermal amplification reactions. With no need for thermal cycling, isothermal microsystems can be designed to be simple and low-energy consuming and therefore may outperform PCR in portable, battery-operated detection systems in the future. The main isothermal methods as miniaturized systems reviewed here include nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), and strand displacement amplification (SDA). Also, important design criteria for the miniaturized devices are discussed. Finally, the potential of miniaturization of some new isothermal methods such as the exponential amplification reaction (EXPAR), isothermal and chimeric primer-initiated amplification of nucleic acids (ICANs), signal-mediated amplification of RNA technology (SMART) and others is presented. PMID:21387067

Asiello, Peter J; Baeumner, Antje J

2011-04-21

4

Isothermal DNA amplification in bioanalysis: strategies and applications.  

PubMed

Isothermal DNA amplification is an alternative to PCR-based amplification for point-of-care diagnosis. Since the early 1990s, the approach has been refined into a simple, rapid and cost-effective tool by means of several distinct strategies. Input signals have been diversified from DNA to RNA, protein or small organic molecules by translating these signals into input DNA before amplification, thus allowing assays on various classes of biomolecules. In situ detection of single biomolecules has been achieved using an isothermal method, leveraging localized signal amplification in an intact specimen. A few pioneering studies to develop a homogenous isothermal protein assay have successfully translated structure-switching of a probe upon target binding into input DNA for isothermal amplification. In addition to the detection of specific targets, isothermal methods have made whole-genome amplification of single cells possible owing to the unbiased, linear nature of the amplification process as well as the large size of amplified products given by ?29 DNA polymerase. These applications have been devised with the four isothermal amplification strategies covered in this review: strand-displacement amplification, rolling circle amplification, helicase-dependent amplification and recombinase polymerase amplification. PMID:21250850

Kim, Joonyul; Easley, Christopher J

2011-01-01

5

Thermophilic helicase-dependent DNA amplification using the IsoAmp™ SE experimental kit for rapid detection of Streptococcus equi subspecies equi in clinical samples.  

PubMed

A simple and portable assay for detection of Streptococcus equi subspecies equi has been developed based on amplification of S. equi-specific sequence using a thermophilic helicase-dependent reaction followed by visual detection of the amplicon in a disposable lateral flow cassette. An experimental kit (IsoAmp™ SE) was evaluated. Analytical sensitivity was 50 copies of S. equi genomic DNA per reaction. The IsoAmp SE assay had 100% specificity when applied to nasal swabs and washes. The assay was more sensitive than culture but less sensitive than nested polymerase chain reaction (PCR). The test requires neither expensive equipment nor extensive training of personnel, provides a practical alternative to culture or PCR assays for detection of S. equi in clinical samples, and expedites identification of atypical colonies of S. equi and Streptococcus zooepidemicus in the laboratory. PMID:21908346

Artiushin, Sergey; Tong, Yanhong; Timoney, John; Lemieux, Bertrand; Schlegel, Anne; Kong, Huimin

2011-09-01

6

Cross priming amplification: mechanism and optimization for isothermal DNA amplification.  

PubMed

CPA is a class of isothermal amplification reactions that is carried out by a strand displacement DNA polymerase and does not require an initial denaturation step or the addition of a nicking enzyme. At the assay temperature of 63°C, the formation of a primer-template hybrid at transient, spontaneous denaturation bubbles in the DNA template is favored over re-annealing of the template strands by the high concentration of primer relative to template DNA. Strand displacement is encouraged by the annealing of cross primers with 5' ends that are not complementary to the template strand and the binding of a displacement primer upstream of the crossing primer. The resulting exponential amplification of target DNA is highly specific and highly sensitive, producing amplicons from as few as four bacterial cells. Here we report on the basic CPA mechanism - single crossing CPA - and provide details on alternative mechanisms. PMID:22355758

Xu, Gaolian; Hu, Lin; Zhong, Huayan; Wang, Hongying; Yusa, Sei-Ichi; Weiss, Tristen C; Romaniuk, Paul J; Pickerill, Sam; You, Qimin

2012-01-01

7

Low Cost Extraction and Isothermal Amplification of DNA for Infectious Diarrhea Diagnosis  

PubMed Central

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

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

2013-01-01

8

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

PubMed

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

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

2014-11-01

9

In-field diagnostics using loop-mediated isothermal amplification.  

PubMed

Loop-mediated isothermal amplification (LAMP) is a method for amplification and detection of target organisms which, unlike polymerase chain reaction, does not require thermal cycling. LAMP assays can be developed in the laboratory for subsequent deployment in the field, where the simplicity of isothermal amplification makes LAMP a suitable method for rapid detection of phytoplasmas with levels of sensitivity and specificity approaching those of more complex and time-consuming laboratory methods. PMID:22987425

Tomlinson, Jenny

2013-01-01

10

Coupled isothermal polynucleotide amplification and translation system  

NASA Technical Reports Server (NTRS)

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.

Joyce, Gerald F. (Inventor)

1998-01-01

11

Isothermal Polymerase Amplification in a Centrifugal Microfluidic Foil Cartridge  

Microsoft Academic Search

Recombinase polymerase amplification (RPA) is a new isothermal DNA amplification method that runs at 37°C, amplifies single copies in less than 15 minutes, and allows real-time fluorescence detection. For the first time we automated this method by microfluidic integration into a centrifugal lab-on-a-chip system, comprising unit operations for reconstitution of reagents, mixing with the sample, and aliquoting to test cavities.

S. Lutz; P. Weber; M. Focke; B. Faltin; G. Roth; O. Piepenburg; N. Armes; D. Mark; R. Zengerle; F. von Stetten

2009-01-01

12

Genotyping SSR length variants by isothermal DNA amplification.  

PubMed

Loop-mediated isothermal DNA amplification (LAMP) is an alternative method for the amplification of DNA sequences. It has been applied primarily for the detection of specific targets. We demonstrate the novel use of LAMP to amplify SSR alleles in a set of rice varieties and show the results to be consistent with analysis performed by PCR. Furthermore, we test the sensitivity of the assay and show it to amplify from near single copy target. PMID:23004341

Lee, David; Ialicicco, Manuela; Akkinepalli, Harika; Morreale, Giacomo; Liu, Yan; Leung, Hei; Scippa, Gabriella S; Greenland, Andy; Mackay, Ian

2012-09-01

13

Isothermal amplified detection of DNA and RNA.  

PubMed

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

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

2014-05-01

14

Isothermal amplification method for next-generation sequencing.  

PubMed

We report an approach for generating immobilized monoclonal templates for next- generation sequencing applications. Our isothermal amplification method is based on a template walking mechanism using a pair of low-melting temperature (Tm) solid-surface homopolymer primers and a low-Tm solution phase primer. The method can generate more than one billion submicrometer-sized colonies in a single lane of a next-generation sequencing flowchip. An alternative paired-end sequencing method using interstrand DNA photo cross-linking to covalently link the complementary strands of the original templates to the solid surface is also demonstrated. PMID:23940326

Ma, Zhaochun; Lee, Raymond W; Li, Bin; Kenney, Paul; Wang, Yufang; Erikson, Jonathan; Goyal, Swati; Lao, Kaiqin

2013-08-27

15

Detection of SNP by the isothermal smart amplification method.  

PubMed

The complexity of molecular diagnostic assays is a significant barrier to employment in point-of-care diagnostics, despite the growing need for such technologies. We have developed a sensitive, accurate, rapid, and simple DNA amplification scheme that shows potential for translational medicine from pharmacogenomics-based drug discovery through to point-of-care diagnostics. Called the "Smart Amplification process 2" (SmartAmp 2), the method is isothermal, and employs a unique primer design and background suppression technology that can amplify target sequences from crude cell lysates. The specificity of the SmartAmp 2 assay enables detection of single-nucleotide differences such as somatic mutations in tumors and single nucleotide polymorphism (SNP) variants. Because mismatch amplification can be completely suppressed in SmartAmp 2, a reliable diagnostic result can be achieved based exclusively on amplification alone. Furthermore, mutation detection and SNP genotypes can be obtained in as little as 30-40 min from sample preparation of raw blood or tissue specimens. PMID:19768611

Lezhava, Alexander; Hayashizaki, Yoshihide

2009-01-01

16

Wolbachia detection in insects through LAMP: loop mediated isothermal amplification  

PubMed Central

Background The bacterium Wolbachia is a promising agent for the biological control of vector-borne diseases as some strains have the ability to block the transmission of key human disease-causing pathogens. Fast, accurate and inexpensive methods of differentiating between infected and uninfected insects will be of critical importance as field-based trials of Wolbachia-based bio-control become increasingly common. Findings We have developed a specific and sensitive method of detecting Wolbachia based on the isothermal DNA amplification. This technique can be performed in an ordinary heat block without the need for gel-based visualisation, and is effective for a wide variety of insect hosts. Conclusion Here we present the development of a rapid, highly sensitive and inexpensive method to detect Wolbachia in a variety of insect hosts, including key mosquito disease vectors. PMID:24885509

2014-01-01

17

Isothermal target and signaling probe amplification method, based on a combination of an isothermal chain amplification technique and a fluorescence resonance energy transfer cycling probe technology.  

PubMed

An iTPA (isothermal target and signaling probe amplification) method for the quantitative detection of nucleic acids, based on a combination of novel ICA (isothermal chain amplification) and fluorescence resonance energy transfer cycling probe technology (FRET CPT), is described. In the new ICA method, which relies on the strand displacement activity of DNA polymerase and the RNA degrading activity of RNase H, two displacement events occur in the presence of four specially designed primers. This phenomenon leads to powerful amplification of target DNA. Since the amplification is initiated only after hybridization of the four primers, the ICA method leads to high specificity for the target sequence. As part of the new ICA method, iTPA is achieved by incorporating FRET CPT to generate multiple fluorescence signals from a single target molecule. Using the resulting dual target and signaling probe amplification system, even a single copy level of a target gene can be successfully detected and quantified under isothermal conditions. PMID:20575518

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

2010-07-15

18

A phaseguided passive batch microfluidic mixing chamber for isothermal amplification.  

PubMed

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

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

2012-11-01

19

Quadratic isothermal amplification for the detection of microRNA.  

PubMed

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

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

2014-03-01

20

Accelerated reaction by loop-mediated isothermal amplification using loop primers  

Microsoft Academic Search

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand displacement activity. We have developed a method that accelerates the LAMP reaction by using additional primers, termed loop primers. Loop primers hybridize to

K. Nagamine; T. Hase; T. Notomi

2002-01-01

21

Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-  

E-print Network

Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch- suppression only occurred with a perfect primer match, amplification alone was sufficient to identify the target primer design. With these new tools, a high-precision and rapid DNA amplification technology becomes

Cai, Long

22

Detection of Loop-Mediated Isothermal Amplification Reaction by Turbidity Derived from Magnesium Pyrophosphate Formation  

Microsoft Academic Search

The loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that uses only one type of enzyme. One of the characteristics of the LAMP method is its ability to synthesize extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophosphate ion, is produced, yielding white precipitate of magnesium pyrophosphate in the reaction mixture. Judging the presence

Yasuyoshi Mori; Kentaro Nagamine; Norihiro Tomita; Tsugunori Notomi

2001-01-01

23

Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases.  

PubMed

Loop mediated isothermal amplification (LAMP) is a powerful innovative gene amplification technique emerging as a simple rapid diagnostic tool for early detection and identification of microbial diseases. The whole procedure is very simple and rapid wherein the amplification can be completed in less than 1 h under isothermal conditions employing a set of six specially designed primers spanning eight distinct sequences of a target gene, by incubating all the reagents in a single tube. Gene amplification products can be detected by agarose gel electrophoresis as well as by real-time monitoring in an inexpensive turbidimeter. Gene copy number can also be quantified with the help of a standard curve generated from different concentrations of gene copy number plotted against time of positivity with the help of a real-time turbidimeter. Alternatively, gene amplification can be visualised by the naked eye either as turbidity or in the form of a colour change when SYBR Green I, a fluorescent dsDNA intercalating dye, is employed. LAMP does not require a thermal cycler and can be performed simply with a heating block and/or water bath. Considering the advantages of rapid amplification, simple operation and easy detection, LAMP has potential applications for clinical diagnosis as well as surveillance of infectious diseases in developing countries without requiring sophisticated equipment or skilled personnel. PMID:18716992

Parida, Manmohan; Sannarangaiah, Santhosh; Dash, Paban Kumar; Rao, P V L; Morita, Kouichi

2008-01-01

24

Detection of genetically modified organisms (GMOs) using isothermal amplification of target DNA sequences  

Microsoft Academic Search

BACKGROUND: The most common method of GMO detection is based upon the amplification of GMO-specific DNA amplicons using the polymerase chain reaction (PCR). Here we have applied the loop-mediated isothermal amplification (LAMP) method to amplify GMO-related DNA sequences, 'internal' commonly-used motifs for controlling transgene expression and event-specific (plant-transgene) junctions. RESULTS: We have tested the specificity and sensitivity of the technique

David Lee; Maurizio La Mura; Theo R Allnutt; Wayne Powell

2009-01-01

25

Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances  

Microsoft Academic Search

To simplify the molecular detection of micro-organisms, we evaluated the tolerance of loop-mediated isothermal amplification (LAMP) to a culture medium and some biological substances. The sensitivity of LAMP was less affected by the various components of the clinical samples than was polymerase chain reaction (PCR); therefore, DNA purification from samples could be omitted.

Hisatoshi Kaneko; Takashi Kawana; Eiko Fukushima; Tatsuo Suzutani

2007-01-01

26

Three Isothermal Amplification Techniques for Rapid Identification of Cladophialophora carrionii, an Agent of Human Chromoblastomycosis.  

PubMed

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

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

2014-10-01

27

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

PubMed

Meeting the goal of providing point of care (POC) tests for molecular detection of pathogens in low resource settings places stringent demands on all aspects of the technology. OmniAmp DNA polymerase (Pol) is a thermostable viral enzyme that enables true POC use in clinics or in the field by overcoming important barriers to isothermal amplification. In this paper, we describe the multiple advantages of OmniAmp Pol as an isothermal amplification enzyme and provide examples of its use in loop-mediated isothermal amplification (LAMP) for pathogen detection. The inherent reverse transcriptase activity of OmniAmp Pol allows single enzyme detection of RNA targets in RT-LAMP. Common methods of nucleic acid amplification are highly susceptible to sample contaminants, necessitating elaborate nucleic acid purification protocols that are incompatible with POC or field use. OmniAmp Pol was found to be less inhibited by whole blood components typical in certain crude sample preparations. Moreover, the thermostability of the enzyme compared to alternative DNA polymerases (Bst) and reverse transcriptases allows pretreatment of complete reaction mixes immediately prior to amplification, which facilitates amplification of highly structured genome regions. Compared to Bst, OmniAmp Pol has a faster time to result, particularly with more dilute templates. Molecular diagnostics in field settings can be challenging due to the lack of refrigeration. The stability of OmniAmp Pol is compatible with a dry format that enables long term storage at ambient temperatures. A final requirement for field operability is compatibility with either commonly available instruments or, in other cases, a simple, inexpensive, portable detection mode requiring minimal training or power. Detection of amplification products is shown using lateral flow strips and analysis on a real-time PCR instrument. Results of this study show that OmniAmp Pol is ideally suited for low resource molecular detection of pathogens. PMID:25136338

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

2014-01-01

28

Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification.  

PubMed

A simple isothermal nucleic-acid amplification reaction, primer generation-rolling circle amplification (PG-RCA), was developed to detect specific nucleic-acid sequences of sample DNA. This amplification method is achievable at a constant temperature (e.g. 60 degrees C) simply by mixing circular single-stranded DNA probe, DNA polymerase and nicking enzyme. Unlike conventional nucleic-acid amplification reactions such as polymerase chain reaction (PCR), this reaction does not require exogenous primers, which often cause primer dimerization or non-specific amplification. Instead, 'primers' are generated and accumulated during the reaction. The circular probe carries only two sequences: (i) a hybridization sequence to the sample DNA and (ii) a recognition sequence of the nicking enzyme. In PG-RCA, the circular probe first hybridizes with the sample DNA, and then a cascade reaction of linear rolling circle amplification and nicking reactions takes place. In contrast with conventional linear rolling circle amplification, the signal amplification is in an exponential mode since many copies of 'primers' are successively produced by multiple nicking reactions. Under the optimized condition, we obtained a remarkable sensitivity of 84.5 ymol (50.7 molecules) of synthetic sample DNA and 0.163 pg (approximately 60 molecules) of genomic DNA from Listeria monocytogenes, indicating strong applicability of PG-RCA to various molecular diagnostic assays. PMID:19106144

Murakami, Taku; Sumaoka, Jun; Komiyama, Makoto

2009-02-01

29

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

PubMed

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. PMID:22898019

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

2012-10-01

30

Loop-mediated isothermal amplification for the detection of plant pathogens.  

PubMed

Loop-mediated isothermal amplification (LAMP) is a technique involving the use of four to six primers (two inner primers, two outer primers, and two loop primers) and the strand displacement activity of Bacillus subtilis-derived (Bst) DNA polymerase. The end result of strand displacement and loop formation and synthesis is the single-temperature amplification of a highly specific fragment from a DNA template at a much greater titre than that obtained with polymerase chain reaction. With LAMP, there are several methods to determine a positive reaction. Presented here are three alternative methods: gel electrophoresis, hydroxynaphthol blue colorimetric dye, and the fluorescent intercalating PicoGreen(®) reagent. PMID:22419496

Ward, Lisa I; Harper, Scott J

2012-01-01

31

IQPA: Isothermal nucleic acid amplification-based immunoassay using DNAzyme as the reporter system.  

PubMed

Immunoassays are often coupled to peroxidase activity for antigen detection. Sensitivity and speed of detection has been increased by the advent of hybrid methods such as immuno-PCR (polymerase chain reaction). However, a more simplified immunoassay that retains both colorimetric peroxidase detection and effective DNA amplification in a setting closer to field application conditions has been nonexistent. Here we describe a method that successfully combines a competitive immunoassay with the new isothermal quadruplex-primed amplification (QPA) to generate excess quadruplex reporter molecules with intrinsic peroxidase DNAzyme activity. PMID:24972268

Loh, Qiuting; Omar, Noorsharmimi; Glökler, Jörn; Lim, Theam Soon

2014-10-15

32

Development of Loop-Mediated Isothermal Amplification Assay for Detection of Entamoeba histolytica  

Microsoft Academic Search

A novel one-step, closed-tube, loop-mediated isothermal amplification (LAMP) assay for detecting Ent- amoeba histolytica, one of the leading causes of morbidity in developing countries, was developed. The sensitivity of the LAMP assay is 1 parasite per reaction. A total of 130 clinical samples were analyzed, and the results compared with those of conventional nested PCR to validate the practicability of

Shih-Yu Liang; Yun-Hsien Chan; Kan-Tai Hsia; Jing-Lun Lee; Ming-Chu Kuo; Kuo-Yuan Hwa; Chi-Wen Chan; Ting-Yi Chiang; Jung-Sheng Chen; Fang-Tzy Wu; Dar-Der Ji

2009-01-01

33

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

PubMed

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. PMID:23030060

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

2012-08-01

34

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

PubMed

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

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

2013-01-01

35

Sensitive and rapid detection of Campylobacter jejuni and Campylobacter coli using loop-mediated isothermal amplification.  

PubMed

Loop-mediated isothermal amplification (LAMP) is an established nucleic acid amplification method offering rapid, accurate, and cost-effective diagnosis of infectious diseases. From the beginning of DNA extraction to final detection of Campylobacter jejuni and Campylobacter coli, the assay requires less than 50 and 90 min from a colony on selective media, and human feces, respectively. For chicken meat samples, the assay requires approximately 24-48 h from the beginning of the enrichment culture to final detection. The sensitivity of the LAMP assay is tenfold higher than that of the equivalent PCR assay. LAMP amplification can be judged by both turbidimeter analysis and visual assessment with the unaided eye. The LAMP assay is a powerful tool for rapid, simple, and sensitive detection of C. jejuni and C. coli, which may facilitate the investigation of C. jejuni and C. coli contamination in chicken, as well as the early diagnosis of C. jejuni and C. coli infection in humans. PMID:23104296

Yamazaki, Wataru

2013-01-01

36

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

PubMed Central

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

Salant, Harold; Abbasi, Ibrahim; Hamburger, Joseph

2012-01-01

37

The development of a loop-mediated isothermal amplification method (LAMP) for Echinococcus granulosus [corrected] coprodetection.  

PubMed

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

Salant, Harold; Abbasi, Ibrahim; Hamburger, Joseph

2012-11-01

38

Loop-mediated isothermal amplification of a single DNA molecule in polyacrylamide gel-based microchamber.  

PubMed

Loop-mediated isothermal amplification (LAMP) is an original nucleic acid amplification method established by Notomi et al. LAMP is performed under isothermal condition, employing only a basic reaction protocol and minimal supporting electronics. These requirements prove to be viable for exploring the avenues to down-scale this biological reaction for Lab-on-a-chip application. Hence here, we developed a novel technique for fluorescent imaging of LAMP at a single molecule level. The experiment was conducted in a polyacrylamide (PAA) gel-based microchamber where a single DNA template, freely suspended in a solution containing primers and polymerase was initially encapsulated. In order to activate the amplification reaction, a microheater regulated by an automatic computerized feedback system was used for localized heating. This microchamber-based approach for LAMP demonstrated the effective exploitation of minute amount of templates and primers, and the overall reduction in LAMP detection time. An average efficiency of 80% was evaluated for conducting DNA amplification after 50 min of incubation at 65 degrees C. As the total time for reaction including detection can be completed in less than 1 h, this one-step, direct observation method displays the potential as a simple alternative to conventional techniques for genetic analysis and diagnosis in the clinical laboratory. PMID:18302022

Lam, Liza; Sakakihara, Shouichi; Ishizuka, Koji; Takeuchi, Shoji; Arata, Hideyuki F; Fujita, Hiroyuki; Noji, Hiroyuki

2008-08-01

39

[Colorimetric detection of HPV6 and HPV16 by loop mediated isothermal amplification].  

PubMed

A simple, rapid and sensitive colorimetric loop mediated isothermal amplification (LAMP) method was established to detect HPV6 and HPV 16 respectively. The method employed a set of four specially designed primers that recognized six distinct sequences of HPV6-E6 or HPV16-E7 for amplification of nucleic acid under isothermal conditions at 63 degrees C for one hour. The amplification process of 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 real-time turbidimeter and agarose electrophoresis. Thirteen cervical swab samples having single infection with 13 different HPV genotypes were examined to evaluate the specificity. A serial dilution of a cloned plasmid containing HPV-E6 or HPV-E7 gene was examined to evaluate the sensitivity. The results showed that no cross-reaction with other HPV genotypes was observed. The colorimetric LAMP assay could achieve a sensitivity of 1000 copies, 10-20 times lower than that of real-time PCR. The assay was further evaluated with 62 clinical specimens and consistent results were obtained compared with the detection using Kai Pu HPV Genotyping Kit. We concluded that this colorimetric LAMP assay had potential usefulness for the rapid screening of the HPV6 or HPV16 infection in the laboratories and hospitals of provincial and municipal region in China. PMID:21462508

Lu, Chun-bin; Luo, Le; Yang, Meng-jie; Nie, Kai; Wang, Miao; Ma, Xue-Jun

2011-01-01

40

Visual and Rapid Detection of Two Genetically Modified Soybean Events Using Loop-mediated Isothermal Amplification Method  

Microsoft Academic Search

To develop effective alternatives for detecting genetically modified organisms (GMOs), we reported one optimized visual loop-mediated\\u000a isothermal amplification (LAMP) method for the detection of exogenous DNA targets from two GM soybean events in this study.\\u000a This isothermal amplification can be performed within 40 min without polymerase chain reaction (PCR) equipment and the derived\\u000a LAMP products can be directly observed by naked

Xiaoyan Guan; Jinchao Guo; Ping Shen; Litao Yang; Dabing Zhang

2010-01-01

41

Rapid detection of Streptococcus pneumoniae by real-time fluorescence loop-mediated isothermal amplification  

PubMed Central

Background and aim of study A significant human pathogenic bacterium, Streptococcus pneumoniae was recognized as a major cause of pneumonia, and is the subject of many humoral immunity studies. Diagnosis is generally made based on clinical suspicion along with a positive culture from a sample from virtually any place in the body. But the testing time is too long. This study is to establish a rapid diagnostic method to identification of Streptococcus pneumoniae. Methods Our laboratory has recently developed a new platform called real-amp, which combines loop-mediated isothermal amplification (LAMP) with a portable tube scanner real-time isothermal instrument for the rapid detection of Streptococcus pneumonia. Two pairs of amplification primers required for this method were derived from a conserved DNA sequence unique to the Streptococcus pneumoniae. The amplification was carried out at 63 degree Celsius using SYBR Green for 60 minutes with the tube scanner set to collect fluorescence signals. Clinical samples of Streptococcus pneumoniae and other bacteria were used to determine the sensitivity and specificity of the primers by comparing with traditional culture method. Results The new set of primers consistently detected in laboratory-maintained isolates of Streptococcus pneumoniae from our hospital. The new primers also proved to be more sensitive than the published species-specific primers specifically developed for the LAMP method in detecting Streptococcus pneumoniae. Conclusions This study demonstrates that the Streptococcus pneumoniae LAMP primers developed here have the ability to accurately detect Streptococcus pneumoniae infections by real-time fluorescence LAMP. PMID:25276360

Guo, Xu-Guang; Zhou, Shan

2014-01-01

42

Comparison of nucleic acid sequence-based amplification and loop-mediated isothermal amplification for diagnosis of human African trypanosomiasis.  

PubMed

Diagnosis of human African trypanosomiasis (HAT) using molecular tests should ideally achieve high sensitivity without compromising specificity. This study compared 2 simplified tests, nucleic acid sequence-based amplification (NASBA) combined with oligochromatography (OC) and loop-mediated isothermal amplification (LAMP), executed on 181 blood samples from 65 Trypanosoma brucei gambiense HAT patients, 86 controls, and 30 serological suspects from Uganda. Basing on the composite reference standard, the diagnostic sensitivity and specificity of NASBA were 93.9% (95% confidence interval [CI] = 84.9-98.3%) and 100% (95% CI = 94.9-100%), respectively. The same parameters for LAMP were 76.9% (95% CI = 64.8-86.5%) and 100% (95% CI = 91.6-100%), respectively. The level of agreement between LAMP and microscopy was good with a kappa (?) value of 79.2% (95% CI = 69.4-88.9%), while that of NASBA-OC/microscopy was very good (? value 94.6%; 95% CI = 89.3-99.8%). The sensitivity of NASBA-OC was significantly higher than that of LAMP (Z = 2.723; P = 0.007). These tests have potential application to HAT surveillance. PMID:24439269

Mugasa, Claire M; Katiti, Diana; Boobo, Alex; Lubega, George W; Schallig, Henk D F H; Matovu, Enock

2014-02-01

43

Development of Loop-Mediated Isothermal Amplification Assay for Detection of Entamoeba histolytica?  

PubMed Central

A novel one-step, closed-tube, loop-mediated isothermal amplification (LAMP) assay for detecting Entamoeba histolytica, one of the leading causes of morbidity in developing countries, was developed. The sensitivity of the LAMP assay is 1 parasite per reaction. A total of 130 clinical samples were analyzed, and the results compared with those of conventional nested PCR to validate the practicability of this assay. No DNA was amplified from other diarrheal pathogens, such as other Entamoeba species, bacteria, and viruses. These results indicate that LAMP is a rapid, simple, and valuable diagnostic tool for epidemiological studies of amebiasis. PMID:19321720

Liang, Shih-Yu; Chan, Yun-Hsien; Hsia, Kan-Tai; Lee, Jing-Lun; Kuo, Ming-Chu; Hwa, Kuo-Yuan; Chan, Chi-Wen; Chiang, Ting-Yi; Chen, Jung-Sheng; Wu, Fang-Tzy; Ji, Dar-Der

2009-01-01

44

[Colorimetric detection of norovirus genotype GII by reverse transcription loop-mediated isothermal amplification].  

PubMed

A simple, rapid and sensitive colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was established to detect norovirus genotype GII. The method employed a set of six specially designed primers that recognized eight distinct sequences of RNA-dependant RNA polymerase and capsid protein gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for 60 minutes. 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 was validated by detecting several different diarrhea viruses including norovirus genotype GII. The sensitivity was determined by serial dilutions of RNA molecules from in vitro transcription of norovirus genotype GII in parallel with conventional RT-PCR detection. The assay was further evaluated with 93 clinical specimens of diarrhea patients. The results showed that the sensitivity of RT-LAMP was 1 000 copies/microL with a high specificity and the relative sensitivity was at the same level as that of conventional RT-PCR. Positive rate of RT-LAMP in analysis of clinical specimens was approximately the same as that of conventional RT-PCR as well. This colorimetric RT-LAMP assay was potential for rapid detection of norovirus genotype GII on spot due to the observation of visual result with high specificity and sensitivity, time-saving and cost benefit. PMID:22519179

Luo, Jian-Ming; Wu, Xi-Yang; Xu, Zi-Qian; Luo, Le; Nie, Kai; Yang, Meng-Jie; Zeng, Ya-Lan; Duan, Zhao-Jun; Ma, Xue-Jun

2012-03-01

45

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

PubMed Central

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 resource-limited settings. The system requires none or only little instrumentation for the nucleic acid amplification reaction and the read-out is possible with the naked eye. Showing the same sensitivity and specificity as comparable diagnostic methods but simultaneously increasing reaction speed and dramatically reducing assay requirements, the method has potential to become a true point-of-care test for the malaria parasite. PMID:24629133

2014-01-01

46

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

PubMed

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 ?l(-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 ?l(-1), which was 100 times lower than that of the polymerase chain reaction. Other pathogen strains were not amplified by the LAMP primers, indicating good specificity. SYBR Green I was added to visually detect the amplification products. Assay applicability was evaluated in 120 samples of Carassius auratus gibelio, and a positive rate of 92·5% was obtained. In conclusion, a conventional LAMP assay has high convenience, rapidity, sensitivity and specificity for detecting CyHV-2 in infected aquatic organisms. Significance and impact of the study: Herpesviral haematopoietic necrosis, caused by cyprinid herpesvirus 2 (CyHV-2), is a severe disease of goldfish and Prussian carp associated with high mortality. We developed a loop-mediated isothermal amplification (LAMP) assay to detect CyHV-2 at relatively low plasmid DNA copy levels. The results show that the LAMP assay has a number of advantages (simple, sensitive, rapid and specific) over the conventional polymerase chain reaction and can be applied in the laboratory and field. Particularly, the method is highly applicable to facilitate surveillance and early diagnosis of CyHV-2. PMID:24935791

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

2014-10-01

47

Detection of Mycobacterium ulcerans by the Loop Mediated Isothermal Amplification Method  

PubMed Central

Background Buruli ulcer (BU) caused by Mycobacterium ulcerans (M. ulcerans) has emerged as an important public health problem in several rural communities in sub-Saharan Africa. Early diagnosis and prompt treatment are important in preventing disfiguring complications associated with late stages of the disease progression. Presently there is no simple and rapid test that is appropriate for early diagnosis and use in the low-resource settings where M. ulcerans is most prevalent. Methodology We compared conventional and pocket warmer loop mediated isothermal amplification (LAMP) methods (using a heat block and a pocket warmer respectively as heat source for amplification reaction) for the detection of M. ulcerans in clinical specimens. The effect of purified and crude DNA preparations on the detection rate of the LAMP assays were also investigated and compared with that of IS2404 PCR, a reference assay for the detection of M. ulcerans. Thirty clinical specimens from suspected BU cases were examined by LAMP and IS2404 PCR. Principal Findings The lower detection limit of both LAMP methods at 60°C was 300 copies of IS2404 and 30 copies of IS2404 for the conventional LAMP at 65°C. When purified DNA extracts were used, both the conventional LAMP and IS2404 PCR concordantly detected 21 positive cases, while the pocket warmer LAMP detected 19 cases. Nine of 30 samples were positive by both the LAMP assays as well as IS2404 PCR when crude extracts of clinical specimens were used. Conclusion/Significance The LAMP method can be used as a simple and rapid test for the detection of M. ulcerans in clinical specimens. However, obtaining purified DNA, as well as generating isothermal conditions, remains a major challenge for the use of the LAMP method under field conditions. With further improvement in DNA extraction and amplification conditions, the pwLAMP could be used as a point of care diagnostic test for BU PMID:22509415

Ablordey, Anthony; Amissah, Diana Ackon; Aboagye, Isaac Frimpong; Hatano, Ben; Yamazaki, Toshio; Sata, Tetsutaro; Ishikawa, Koichi; Katano, Harutaka

2012-01-01

48

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

PubMed

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

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

2014-11-01

49

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

PubMed

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

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

2014-11-01

50

Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue.  

PubMed

Loop-mediated isothermal amplification (LAMP), a novel gene amplification method, enables the synthesis of larger amounts of both DNA and a visible byproduct--namely, magnesium pyrophosphate--without thermal cycling. A positive reaction is indicated by the turbidity of the reaction solution or the color change after adding an intercalating dye to the reaction solution, but the use of such dyes has certain limitations. Hydroxy naphthol blue (HNB), a metal indicator for calcium and a colorimetric reagent for alkaline earth metal ions, was used for a new colorimetric assay of the LAMP reaction. Preaddition of 120 microM HNB to the LAMP reaction solution did not inhibit amplification efficiency. A positive reaction is indicated by a color change from violet to sky blue. The LAMP reaction with HNB could also be carried out in a 96-well microplate, and the reaction could be measured at 650 nm with a microplate reader. The colorimetric LAMP method using HNB would be helpful for high-throughput DNA and RNA detection. PMID:19317660

Goto, Motoki; Honda, Eiichi; Ogura, Atsuo; Nomoto, Akio; Hanaki, Ken-Ichi

2009-03-01

51

Recombinase-Based Isothermal Amplification of Nucleic Acids with Self-Avoiding Molecular Recognition Systems (SAMRS).  

PubMed

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

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

2014-10-13

52

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

PubMed

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. PMID:24026689

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

2013-01-01

53

Isothermal nucleic acid amplification strategy by cyclic enzymatic repairing for highly sensitive microRNA detection.  

PubMed

Technologies enabling highly sensitive and selective detection of microRNAs (miRNAs) are critical for miRNA discovery and clinical theranostics. Here we develop a novel isothermal nucleic acid amplification technology based on cyclic enzymatic repairing and strand-displacement polymerase extension for highly sensitive miRNA detection. The enzymatic repairing amplification (ERA) reaction is performed via replicating DNA template using lesion bases by DNA polymerase and cleaving the DNA replicate at the lesions by repairing enzymes, uracil-DNA glycosylase, and endonuclease IV, to prime a next-round replication. By utilizing the miRNA target as the primer, the ERA reaction is capable of producing a large number of reporter sequences from the DNA template, which can then be coupled to a cyclic signal output reaction mediated by endonuclease IV. The ERA reaction can be configured as a single-step, close-tube, and real-time format, which enables highly sensitive and selective detection of miRNA with excellent resistance to contaminants. The developed technology is demonstrated to give a detection limit of 0.1 fM and show superb specificity in discriminating single-base mismatch. The results reveal that the ERA reaction may provide a new paradigm for efficient nucleic acid amplification and may hold the potential for miRNA expression profiling and related theranostic applications. PMID:24949808

Zhou, Dian-Ming; Du, Wen-Fang; Xi, Qiang; Ge, Jia; Jiang, Jian-Hui

2014-07-15

54

Strand Invasion Based Amplification (SIBA®): A Novel Isothermal DNA Amplification Technology Demonstrating High Specificity and Sensitivity for a Single Molecule of Target Analyte  

PubMed Central

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

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

2014-01-01

55

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

PubMed

Loop-mediated isothermal amplification (LAMP) of DNA is a simple, cost effective, and rapid method for the specific detection of genomic DNA using a set of six oligonucleotide primers with eight binding sites hybridizing specifically to different regions of a target gene, and a thermophilic DNA polymerase from Geobacillus stearothermophilus for DNA amplification. The method has been applied in various assays for the diagnosis of bacterial and viral infections of humans and animals, sexing of bovine and swine embryos, and in the detection of bacteria from environmental samples. Only recently, first applications for fungal organisms were published. During the current study a LAMP assay was developed for the specific detection of Fusarium graminearum, the major causative agent of Fusarium head blight of small cereals and producer of the mycotoxins deoxynivalenol, nivalenol, and zearalenone. The assay was based on the gaoA gene (galactose oxidase) of the fungus. Amplification of DNA during the reaction was indirectly detected in situ by using calcein fluorescence as a marker without the necessity of time-consuming electrophoretic analysis. The assay was optimized for rapidness, specificity, and sensitivity and was shown to detect the presence of less than 2pg of purified target DNA per reaction within 30 min. Within 132 fungal species tested, exclusively DNA isolated from cultures of F. graminearum (lineages 1-9) resulted in a fluorescent signal after amplification with the LAMP assay. The method was demonstrated to be useful in the analysis of fungal cultures by direct analysis of surface scrapings from agar plate cultures, direct testing of single infected barley grains, and detection of F. graminearum in total genomic DNA isolated from bulk samples of ground wheat grains. Results obtained indicate that LAMP offers an interesting new assay format for the rapid and specific DNA-based detection and identification of agriculturally important toxigenic fungi in pure cultures and in contaminated sample materials and therefore presents an alternative to PCR-based assays. PMID:20442002

Niessen, Ludwig; Vogel, Rudi F

2010-06-15

56

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

NASA Astrophysics Data System (ADS)

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).

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

2013-01-01

57

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

PubMed

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

Vaagt, Franziska; Haase, Ilka; Fischer, Markus

2013-02-27

58

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

NASA Astrophysics Data System (ADS)

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.

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

2013-11-01

59

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

PubMed

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

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

2014-01-01

60

Development of a reverse transcription loop-mediated isothermal amplification assay for detection of Porcine teschovirus.  

PubMed

Loop-mediated isothermal ampli?cation (LAMP) is a sensitive method for DNA ampli?cation. In the present report, the development of a single-tube, one-step, real-time accelerated reverse transcription (RT)-LAMP for the detection of Porcine teschovirus (PTV) is described. Six designed primers amplified target gene sequences successfully at constant temperature (65 °C) within 1 hr, and the amplification results could be visualized directly by the naked eye. The sensitivity of the LAMP was 10 times higher than that of conventional polymerase chain reaction, and no cross-reactivity was found when the genomes of other common swine pathogens were subjected to the RT-LAMP system. When 43 clinical samples were tested by the RT-LAMP method, results indicated that the test is simple, rapid, accurate, and sensitive for the detection of PTV. PMID:21908281

Wang, Bin; Wang, Yongqiang; Tian, Zhi-Jun; An, Tong-Qing; Peng, Jin-Mei; Tong, Guang-Zhi

2011-05-01

61

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

PubMed Central

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

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

2013-01-01

62

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

PubMed Central

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

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

2013-01-01

63

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

PubMed

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

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

2013-11-01

64

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

PubMed Central

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

2013-01-01

65

Detection of Acute Toxoplasmosis in Pigs Using Loop-Mediated Isothermal Amplification and Quantitative PCR  

PubMed Central

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

Wang, Guangxiang; Wang, Meng

2013-01-01

66

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

PubMed

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

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

67

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

PubMed Central

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

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

2012-01-01

68

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

PubMed

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

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

2010-04-01

69

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

PubMed

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

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

2012-01-01

70

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

PubMed Central

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

2014-01-01

71

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

PubMed

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

Fu, Zhongyu; Zhou, Xiaoming; Xing, Da

2013-12-15

72

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

PubMed

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

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

2014-01-01

73

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

PubMed

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/?L. The sensitivity of the LAMP was 100% compared to egg positive samples. While all microscopically positive samples were positive by LAMP, additionally 5 of 13 (38.5%) microscopically negative samples were also LAMP positive. The technique has great potential for differential diagnosis in endemic areas with mixed O. viverrini and intestinal fluke infections. As it is an easy and simple method, the LAMP is potentially applicable for point-of-care diagnosis. PMID:21871581

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

2012-03-01

74

Differential identification of three species of Curtovirus using loop-mediated isothermal amplification.  

PubMed

Rapid and sensitive detection methods for three species of Curtovirus were developed using a loop-mediated isothermal amplification (LAMP) technique. A universal primer set for detecting the three main species of Curtovirus at the same time, and three kinds of species-specific primer sets were designed and used for LAMP reactions. Results from the LAMP reactions were visualized both by color changes after adding SYBR Green I staining dye and by DNA laddering on agarose gel electrophoresis. The optimal conditions for the curtovirus LAMP reaction were confirmed at 60°C for the universal primers and at 62°C for the three species-specific primer sets. Amplification of curtoviruses by LAMP reaction was ten-fold more sensitive than that by polymerase chain reaction. Primers designed for curtovirus detection in this study did not anneal to or amplify DNA from other DNA or RNA viruses (tomato yellow leaf curl virus, tomato spotted wilt virus, and potato virus Y). Taken together, the primer sets and reaction conditions developed in this study show that the LAMP technique could be a useful tool to detect the three species of Curtovirus simultaneously and distinguish them in the laboratory and the field. PMID:24957721

Kil, E -J; Cho, S -H; Byun, H -S; Kim, J; Hwang, H -S; Auh, C -K; Heo, N -Y; Shin, Y -G; Lee, S

2014-06-01

75

Detection of new bunyavirus RNA by reverse transcription-loop-mediated isothermal amplification.  

PubMed

Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging and epidemic infectious disease in central and northeast China. It is caused by New Bunyavirus and carries an average 12% case fatality rate. Early and rapid detection is critical for prevention and control of New Bunyavirus infection, since no vaccine or antiviral drugs are currently available, and prevention requires careful attention to control of the suspected tick vector. In this study, a simple and sensitive reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid detection of New Bunyavirus. The detection limit of the RT-LAMP assay was approximately 10(3) 50% tissue culture infective doses/ml of New Bunyavirus in culture supernatants, and no cross-reactive amplification of other viruses known to cause similar clinical manifestations was observed. The assay was further evaluated using 138 specimens from clinically suspected SFTS and 40 laboratory-proven hantavirus infection with fever and renal syndrome patients, and the assay exhibited 97% agreement compared to real-time RT-PCR and conventional RT-PCR. Using real-time RT-PCR as the diagnostic gold standard, RT-LAMP was 99% sensitive and 100% specific. The RT-LAMP assay could become a useful alternative in clinical diagnosis of SFTS caused by New Bunyavirus, especially in resource-limited hospitals or rural clinics of China. PMID:24478484

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

2014-02-01

76

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

PubMed Central

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

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

2014-01-01

77

Development of a loop-mediated isothermal amplification assay for detection of Phytophthora sojae.  

PubMed

Phytophthora sojae is a devastating pathogen that causes soybean Phytophthora root rot. This study reports the development of a loop-mediated isothermal amplification (LAMP) assay targeting the A3aPro element for visual detection of P. sojae. The A3aPro-LAMP assay efficiently amplified the target element in < 80 min at 64 °C and was evaluated for specificity and sensitivity. The specificity was evaluated against P. sojae, Phytophthora spp., Pythium spp., and true fungi isolates. Magnesium pyrophosphate resulting from the LAMP of P. sojae could be detected by real-time measurement of turbidity. Phytophthora sojae DNA products were visualized as a ladder-like banding pattern on 2% gel electrophoresis. A positive colour (sky blue) was only observed in the presence of P. sojae with the addition of hydroxynaphthol blue prior to amplification, whereas none of other isolates showed a colour change. The detection limit of the A3aPro-specific LAMP assay for P. sojae was 10 pg ?L(-1) of genomic DNA per reaction. The assay also detected P. sojae from diseased soybean tissues and residues. These results suggest that the A3aPro-LAMP assay reported here can be used for the visual detection of P. sojae in plants and production fields. PMID:22697582

Dai, Ting-Ting; Lu, Chen-Chen; Lu, Jing; Dong, SuoMeng; Ye, WenWu; Wang, YuanChao; Zheng, XiaoBo

2012-09-01

78

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

PubMed

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

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

2014-08-01

79

Ultrasensitive detection of transcription factors using transcription-mediated isothermally exponential amplification-induced chemiluminescence.  

PubMed

Transcription factors (TFs) are important cellular components that modulate gene expression, and the malregulation of transcription will lead to a variety of diseases such as cancer and developmental syndromes. However, the conventional methods for transcription factor assay are generally cumbersome and costly with low sensitivity. Here, we develop a label-free strategy for ultrasensitive detection of transcription factors using a cascade signal amplification of RNA transcription, dual isothermally exponential amplification reaction (EXPAR), and G-quadruplex DNAzyme-driven chemiluminescence. Briefly, the specific binding of TF with the detecting probe prevents the cleavage of the detecting probe by exonuclease and subsequently facilitates the conversion of TF signal to abundant RNA triggers in the presence of T7 RNA polymerase. The obtained RNA triggers can initiate the strand displacement amplification to yield abundant DNAzymes and DNA triggers, and the released DNA triggers can further initiate the next rounds of EXPAR reaction. The synergistic operation of dual EXPAR reaction can produce large amounts of DNAzymes, which subsequently catalyze the oxidation of luminol by H2O2 to yield an enhanced chemiluminescence signal with the assistance of cofactor hemin. Conversely, in the absence of target TF, the naked detecting probes will be completely digested by exonucleases, leading to neither the transcription-mediated EXPAR nor the DNAzyme-driven chemiluminescence signal. This method has a low detection limit of as low as 6.03 × 10(-15) M and a broad dynamic range from 10 fM to 1 nM and can even measure the NF-?B p50 of crude cell nuclear extracts. Moreover, this method can be used to measure a variety of DNA-binding proteins by simply substituting the target-specific binding sequence in the detecting probes. PMID:24865817

Ma, Fei; Yang, Yong; Zhang, Chun-Yang

2014-06-17

80

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

Microsoft Academic Search

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

Siyi Chen; Beilei Ge

2010-01-01

81

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

PubMed Central

Background Contagious Bovine Pleuropneumonia (CBPP) is the most important chronic pulmonary disease of cattle on the African continent causing severe economic losses. The disease, caused by infection with Mycoplasma mycoides subsp. mycoides is transmitted by animal contact and develops slowly into a chronic form preventing an early clinical diagnosis. Because available vaccines confer a low protection rate and short-lived immunity, the rapid diagnosis of infected animals combined with traditional curbing measures is seen as the best way to control the disease. While traditional labour-intensive bacteriological methods for the detection of M. mycoides subsp. mycoides have been replaced by molecular genetic techniques in the last two decades, these latter approaches require well-equipped laboratories and specialized personnel for the diagnosis. This is a handicap in areas where CBPP is endemic and early diagnosis is essential. Results We present a rapid, sensitive and specific diagnostic tool for M. mycoides subsp. mycoides detection based on isothermal loop-mediated amplification (LAMP) that is applicable to field conditions. The primer set developed is highly specific and sensitive enough to diagnose clinical cases without prior cultivation of the organism. The LAMP assay detects M. mycoides subsp. mycoides DNA directly from crude samples of pulmonary/pleural fluids and serum/plasma within an hour using a simple dilution protocol. A photometric detection of LAMP products allows the real-time visualisation of the amplification curve and the application of a melting curve/re-association analysis presents a means of quality assurance based on the predetermined strand-inherent temperature profile supporting the diagnosis. Conclusion The CBPP LAMP developed in a robust kit format can be run on a battery-driven mobile device to rapidly detect M. mycoides subsp. mycoides infections from clinical or post mortem samples. The stringent innate quality control allows a conclusive on-site diagnosis of CBPP such as during farm or slaughter house inspections. PMID:23710975

2013-01-01

82

Elevated OPN, IP-10, and Neutrophilia in Loop-Mediated Isothermal Amplification Confirmed Tuberculosis Patients  

PubMed Central

Tuberculosis (TB) is the second most common cause of death from infectious diseases and results in high socioeconomic losses to many countries. Proper diagnosis is the first step in TB eradication. To develop a rapid, simple, and accurate diagnostic TB test and to characterize the prevalence of Mycobacterium tuberculosis (MTB) genotypes and immune profiles of TB patients, a total of 37?TB patients and 30 healthy control (HC) from Metro Manila were enrolled. Loop-mediated isothermal amplification (LAMP) reliably detected MTB infection. Manila genotype was identified by spoligotyping method in all TB patients. Osteopontin (OPN), interferon-?-induced protein 10?kDa (IP-10), and neutrophil counts were found to reflect the acute stage of MTB infection. The sensitivity and specificity were 94.6% and 93.3%, respectively, for both OPN and IP-10, and they were 83.8% and 78.6%, respectively, for neutrophils. The combination of OPN, IP-10, neutrophil count, IL-6, IL-8, TNF-?, MCP-1, platelets, galectin-9, and leukocyte count correctly identifies all the HC and 96.3% of TB patients. LAMP method may serve as a rapid, supportive method in addition to time-consuming culture methods. OPN, IP-10, and neutrophil counts are useful in detecting MTB infection and may have utility in monitoring the course of the disease. PMID:25378811

Leano, Susan; Nakajima, Chie; Niki, Toshiro; Ashino, Yugo; Suzuki, Yasuhiko; Telan, Elisabeth

2014-01-01

83

Detection of enterovirus 71 using reverse transcription loop-mediated isothermal amplification (RT-LAMP).  

PubMed

Reverse transcription loop-mediated isothermal amplification (RT-LAMP), which is a visual assay for nucleic acids, is performed in a single step using one tube at 65 °C for 1.5 h. In this study, RT-LAMP was established as a method for the detection of enterovirus 71 (EV71). The detection limit of the assay was approximately 10 copies, and no cross-reactivity was noted with Coxsackievirus A16, echovirus, human rotavirus (HRV) or norovirus. This assay, which offers greater sensitivity at a lower cost compared with the conventional reverse transcription polymerase chain reaction (RT-PCR), was validated using 252 clinical specimens that had been confirmed by laboratory diagnosis using RT-PCR. Both methods produced the same results with 52 positive samples. The RT-LAMP-based assay does not require specialised equipment, and therefore, it can be performed conveniently during an outbreak or under field conditions. In brief, the RT-LAMP-based assay provided a simple, rapid and efficient method for the detection of EV71 nucleic acid under field conditions. PMID:22155579

Wang, Xiang; Zhu, Jun-ping; Zhang, Qian; Xu, Zi-gang; Zhang, Fang; Zhao, Zhi-hui; Zheng, Wen-zhi; Zheng, Li-shu

2012-02-01

84

Development of a loop-mediated isothermal amplification assay for detection of Cronobacter spp. (Enterobacter sakazakii).  

PubMed

Contamination of Cronobacter spp. (Enterobacter sakazakii) in infant formulas and other food products is a severe problem. Here a loop-mediated isothermal amplification (LAMP) assay was developed for rapidly detecting Cronobacter spp. in powdered infant formula. Sequences of 16S/23S rDNA internal intergenic spacer of Cronobacter spp. were used as the target template to design LAMP primers. The detection outcome can be evaluated by the white precipitate or the fluorescence intensity under ultraviolet irradiation, both visible to naked eyes. The sensitivity and specificity of the LAMP assay was further analyzed in comparison with that of regular PCR and real time quantitative PCR. The results showed that all of Cronobacter spp. strains display positive reaction to the detections while all of the non-Cronobacter spp. strains were negative, and that the LAMP assay exhibits a high sensitivity of 9.1 fg/?L (The sensitivity of regular PCR and real time quantitative PCR is 91 and 9.1 pg/?L, respectively.). The amplified reaction could be accomplished in about 1 h, with the results visible to naked eyes. Hence, the LAMP assay developed by this study can provide a rapid and simple approach for the detection of Cronobacter spp. in infant formula. PMID:22805822

Liu, Xu; Fang, Jiehong; Zhang, Mingzhou; Wang, Xueyan; Wang, Weifen; Gong, Yunfei; Xi, Xi; Li, Mujie

2012-03-01

85

Four DNA extraction methods used in loop-mediated isothermal amplification for rapid adenovirus detection.  

PubMed

Loop-mediated isothermal amplification (LAMP) assays have become powerful tools for rapid diagnosis of infectious diseases. A more efficient, convenient and cheaper method for template preparation from the pellets or supernatants of nasopharyngeal aspirates was sought. Three DNA extraction methods (boiling, boiling in 1% Triton X-100, and treating with 0.02M NaOH) were compared with the commonly used DNAzol DNA extraction method. DNA preparations were then subjected to adenovirus (ADV) detection using LAMP assays and 119 clinical samples. The specificities for all three methods were 100% compared with the DNAzol method. The sensitivity of the boiling method was greater than that for the other two methods. The templates extracted from supernatants of nasopharyngeal aspirates using the boiling technique were further evaluated. Higher sensitivity (90.9%) and specificity (96.5%) were observed for LAMP assays compared with those from quantitative PCR assays. In conclusion, for template preparation, boiling supernatants of nasopharyngeal aspirates had comparable sensitivity and specificity with the DNAzol method. There were the added advantages that the boiling technique was simpler, cheaper, and had a shorter processing time. The boiling technique could become a suitable substitute for the DNAzol method when LAMP assays are used for ADV detection. PMID:24747588

Sun, Yu; Zhao, Linqing; Zhao, Meng; Zhu, Runan; Deng, Jie; Wang, Fang; Li, Fan; Ding, Yaxin; Tian, Run; Qian, Yuan

2014-08-01

86

Development of Double Loop-Mediated Isothermal Amplification to Detect Listeria monocytogenes in Food.  

PubMed

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

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

2014-12-01

87

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

PubMed Central

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

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

88

Rapid detection of Pseudomonas aeruginosa in mouse feces by colorimetric loop-mediated isothermal amplification.  

PubMed

A colorimetric loop-mediated isothermal amplification (LAMP) assay with hydroxy naphthol blue was designed to amplify a region in the outer membrane lipoprotein (oprL) gene of Pseudomonas aeruginosa. The LAMP assay showed 100% specificity for the serogroup and other bacteria, and the sensitivity was 10-fold higher than that of the PCR assays. The LAMP assay could detect P. aeruginosa inoculated in mouse feces at 130 colony-forming units (CFU)/0.1g feces (3.25 CFU/reaction). The assay was completed within 2h from DNA extraction. In a field trial, the LAMP assay revealed that none of the 27 samples was obtained from 2 specific pathogen-free (SPF) mouse facilities that were monitoring infection with P. aeruginosa; 1 out of 12 samples from an SPF mouse facility that was not monitoring infection with P. aeruginosa and 2 out of 7 samples from a conventional mouse facility were positive for P. aeruginosa. In contrast, P. aeruginosa was not detected in any of the samples by a conventional culture assay. Thus, this colorimetric LAMP assay is a simple and rapid method for P. aeruginosa detection. PMID:20298724

Goto, Motoki; Shimada, Kayo; Sato, Ayako; Takahashi, Eri; Fukasawa, Takafumi; Takahashi, Tomoki; Ohka, Seii; Taniguchi, Takahide; Honda, Eiichi; Nomoto, Akio; Ogura, Atsuo; Kirikae, Teruo; Hanaki, Ken-Ichi

2010-06-01

89

Development of a loop-mediated isothermal amplification for rapid detection of orf virus.  

PubMed

A loop-mediated isothermal amplification (LAMP) assay using six primers targeting a highly conserved region of the B2L gene has been developed to diagnose orf virus. The assay produces a ladder-like pattern of products on an agarose gel that can be specifically digested with BsrGI enzyme. The sensitivity of the LAMP assay, which was determined to be a single copy of the standard plasmid, was 100 fold and 10 fold higher than PCR and nested PCR, respectively; furthermore, no cross-reactivity was founded with the other tested viruses. By staining the products directly in the tube with PicoGreen or ethidium bromide, the products can be visualized with a similar sensitivity as by gel electrophoresis. Clinical samples were tested using PCR, nested PCR and LAMP assay and the positive rates were 60%, 70% and 70%, respectively. The LAMP assay allows easy, rapid, accurate and sensitive detection of infection with orf virus and is especially applicable in a resource-limited situation. PMID:19186192

Tsai, Su-Ming; Chan, Kun-Wei; Hsu, Wei-Li; Chang, Tien-Jye; Wong, Min-Liang; Wang, Chi-Young

2009-05-01

90

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

PubMed

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

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

2010-06-01

91

Rapid identification of virus-carrying mosquitoes using reverse transcription-loop-mediated isothermal amplification.  

PubMed

Mosquitoes are critical vectors in many arboviral transmission cycles. Considering the increasing incidence of arboviral infections throughout the world, monitoring of vector populations for the presence of an arbovirus could be considered an important initial step of risk assessment to humans and animals. In response to this need, increased efforts to develop rapid and reliable diagnostic techniques have been undertaken; a single-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect virus in vector mosquitoes (Aedes aegypti) using the Flock House Virus (FHV) as a model. The robustness of the RT-LAMP reaction was revealed by its ability to detect FHV from an "all-in-one" template using whole mosquito bodies within 30min. Furthermore, RT-LAMP identified successfully a mosquito carrying just a single FHV particle, a level easily overlooked in conventional analysis such as plaque forming assays. These observations suggest that RT-LAMP is more reliable and useful for routine diagnosis of vector mosquitoes in regions where the prevalence of vector-borne diseases such as West Nile fever or dengue fever are common. PMID:19027038

Perera, Namal; Aonuma, Hiroka; Yoshimura, Aya; Teramoto, Tokiyasu; Iseki, Hiroshi; Nelson, Bryce; Igarashi, Ikuo; Yagi, Takeshi; Fukumoto, Shinya; Kanuka, Hirotaka

2009-03-01

92

Loop-mediated isothermal amplification assays for screening of bacterial integrons  

PubMed Central

Background The occurrence and prevalence of integrons in clinical microorganisms and their role played in antimicrobial resistance have been well studied recently. As screening and detection of integrons are concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for integrons detection. Results In this study, three loop-mediated isothermal amplification (LAMP) assays targeting on class 1, 2 and 3 integrons were implemented and evaluated. Optimization of these detection assays were performed, including studing on the reaction temperature, volume, time, sensitivity and specificity (both primers and targets). Application of the established LAMP assays were further verified on a total of 1082 isolates (previously identified to be 397 integron-positive and 685 integron-negative strains). According to the results, the indispensability of each primer had been confirmed and the optimal reaction temperature, volume and time were found to be 65°C, 45 min and 25 ?L, respectively. As application was concerned, 361, 28 and 8 isolates carrying intI1, intI2 and intI3 yielded positive amplicons, respectively. Other 685 integron-negative bacteria were negative for the integron-screening LAMP assays, totaling the detection rate and specificity to be 100%. Conclusions The intI1-, intI2- and intI3-LAMP assays established in this study were demonstrated to be the valid and rapid detection methodologies for the screening of bacterial integrons.

2014-01-01

93

Loop-Mediated Isothermal Amplification (LAMP) for Rapid Identification of Anopheles gambiae and Anopheles arabiensis Mosquitoes  

PubMed Central

The main malaria vectors of sub-Saharan Africa, Anopheles gambiae sensu stricto and Anopheles arabiensis are morphologically indistinguishable, but often occur in sympatry and differ in feeding preference and vector competence. It is important to assess vector species identity for understanding the vectorial system and establishing appropriate vector control measures. The currently available species diagnosis methods for An. gambiae sensu latu require equipment to which public health practitioners in many African countries may not have access. This report describes a loop-mediated isothermal amplification technique (LAMP) for An. gambiae species diagnosis. The LAMP method was tested in single mosquito legs and whole body. The sensitivity and specificity of the LAMP method, in reference to the conventional rDNA-polymerse chain reaction (PCR) method, ranged from 0.93 to 1.00. The LAMP-based species identification method can be performed in a water bath and completed within 65 minutes, representing an alternative method for rapid and field applicable vector species diagnosis. PMID:19996433

Bonizzoni, Mariangela; Afrane, Yaw; Yan, Guiyun

2013-01-01

94

Detection of Pepino mosaic virus isolates from tomato by one-step reverse transcription loop-mediated isothermal amplification.  

PubMed

Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique for the amplification of DNA under isothermal conditions. For the first time, we applied this method to develop a simple and sensitive tool for the detection of Pepino mosaic virus (PepMV). PepMV is an emerging pathogen that causes yield and quality losses in tomato crops. Specific RT-LAMP primers for PepMV detection were designed based on triple gene block sequences. The reaction was performed in a single tube at 65 °C for 30 min. The RT-LAMP assay is easy to perform and inexpensive, and it may be applied in the rapid and specific diagnosis of PepMV. PMID:23605670

Hasiów-Jaroszewska, Beata; Borodynko, Natasza

2013-10-01

95

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

SciTech Connect

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.

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

2011-12-08

96

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

Microsoft Academic Search

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 ? C and real-time

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

2010-01-01

97

Rapid and Sensitive Detection of Mycobacterium ulcerans by Use of a Loop-Mediated Isothermal Amplification Test  

PubMed Central

This work reports the design and evaluation of a rapid loop-mediated isothermal amplification test for detecting Mycobacterium ulcerans DNA based on the multicopy insertion sequence IS2404. The test is robust and specific with a detection limit equivalent to 20 copies of the target sequence (0.01 to 0.1 genome). The test has potential for the diagnosis of Buruli ulcer under field conditions. PMID:22357495

Yeboah-Manu, Dorothy; Stinear, Timothy P.; Fyfe, Janet A. M.

2012-01-01

98

Loop-mediated isothermal amplification for rapid detection of the causal agents of cassava brown streak disease.  

PubMed

The causal agents of cassava brown streak disease have recently been identified as Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Primers have been developed for rapid detection of these viruses by reverse transcription loop-mediated isothermal amplification (RT-LAMP). Performance of the RT-LAMP assays compared favourably with published RT-PCR and real-time RT-PCR methods. Furthermore, amplification by RT-LAMP is completed in 40 min and does not require thermal cycling equipment. Modification of the RT-LAMP reactions to use labelled primers allowed rapid detection of amplification products using lateral flow devices containing antibodies specific to the incorporated labels, avoiding the need for fluorescence detection or gel electrophoresis. PMID:22820076

Tomlinson, J A; Ostoja-Starzewska, S; Adams, I P; Miano, D W; Abidrabo, P; Kinyua, Z; Alicai, T; Dickinson, M J; Peters, D; Boonham, N; Smith, J

2013-08-01

99

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

PubMed

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

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

2011-05-21

100

[Advance in loop-mediated isothermal amplification technique and its applications in point-of-care testing platforms].  

PubMed

Loop-mediated isothermal amplification (LAMP) is a novel in vitro nucleic acid amplification method conducted under isothermal conditions with the advantages of high specificity, sensitivity, rapidity and easy detection. Since it was established in 2000, it has been widely applied in various fields of analytical science including the diagnosis of a variety of pathogens, identification of embryo sex, detection of genetically modified organisms and cancer gene identification. Additionally, significant progress has been made in the optimization of the LAMP method, such as accelerated reactions, simplified sample processing, the realization of multiplex amplification, and the enhanced specificity of reaction and detection methods. LAMP technology also shows much potential to be adopted as part of point-of-care testing platforms by the micromation, automation and integration with other technologies such as Lab-on-a-Chip and digital nucleic acid amplification. This review summarizes the latest advances in the LAMP technique and its applications in developing point-of-care testing platforms. PMID:25272605

Guan, Li; Ma, Xue-Jun

2014-07-01

101

Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Capripoxviruses  

PubMed Central

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. PMID:22357504

Babiuk, Shawn; McIntosh, Michael T.

2012-01-01

102

Loop-mediated isothermal amplification (LAMP): Early detection of Toxoplasma gondii infection in mice  

PubMed Central

Background Toxoplasmosis is a widespread zoonotic parasitic disease that occurs in both animals and humans. Traditional molecular assays are often difficult to perform, especially for the early diagnosis of Toxoplasma gondii infections. Here, we established a novel loop-mediated isothermal amplification targeting the 529 bp repeat element (529 bp-LAMP) to detect T. gondii DNA in blood samples of experimental mice infected with tachyzoites of the RH strain. Findings The assay was performed with Bst DNA polymerase at 65°C for 1 h. The detection limit of the 529 bp-LAMP assay was as low as 0.6 fg of T. gondii DNA. The sensitivity of this assay was 100 and 1000 fold higher than that of the LAMP targeting B1 gene (B1-LAMP) and nested PCR targeting 529 bp repeat element (529 bp-nested PCR), respectively. The specificity of the 529 bp-LAMP assay was determined using the DNA samples of Trypanosoma evansi, Plasmodium falciparum, Paragonimus westermani, Schistosoma japonicum, Fasciola hepatica and Angiostrongylus cantonensis. No cross-reactivity with the DNA of any parasites was found. The assay was able to detect T. gondii DNA in all mouse blood samples at one day post infection (dpi). Conclusions We report the following findings: (i) The detection limit of the 529 bp-LAMP assay is 0.6 fg of T. gondii DNA; (ii) The assay does not involve any cross-reactivity with the DNA of other parasites; (iii) This is the first report on the application of the LAMP assay for early diagnosis of toxoplasmosis in blood samples from experimentally infected mice. Due to its simplicity, sensitivity and cost-effectiveness for common use, we suggest that this assay should be used as an early diagnostic tool for health control of toxoplasmosis. PMID:22214421

2012-01-01

103

Loop-mediated isothermal amplification for the detection of goose circovirus  

PubMed Central

Background Goose circovirus (GCV) presents an immunosuppressive problem in production of geese. The infection’s clinical symptoms include growth retardation or feathering disorders but the infection process may remain non-symptomatic what makes the infected birds more susceptible for secondary viral, bacterial and fungal infections. Diagnosis of GCV infection is made by histopathological examination, dot blot hybridization, polymerase chain reaction (PCR) and real-time PCR. However these techniques require application of thermocyclers and qualified staff which may be cost-consuming for some diagnostic units. The aim of this study was to develop loop-mediated isothermal amplification assay (LAMP) as a simple method of GCV detection. Results The presented study has shown LAMP as a rapid tool of detecting DNA of goose circovirus (GCV) as soon in 30 min time. The method used three sets of primers: two outer primers (F3 and B3), two inner primers (FIP and BIP) and two loop primers (FL and BL) to accelerate the reaction. The optimum reaction temperature and the time were 61°C for 30 min, respectively. The results were analysed using SYBR Green dye and GelRedTM solutions. Thirty-eight isolates of GCV collected from geese flocks in Poland were examined. For comparison, real-time polymerase chain reaction with F3 and B3 primers and SYBR Green dye was conducted. The obtained results have shown GCV-LAMP as a sensitive, rapid and specific assay and alternative for PCR-based methods. Conclusions The developed technique due to its simplicity may be applied by any veterinary laboratory or even mobile diagnostics units for the routine detection of GCV. PMID:22695123

2012-01-01

104

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

PubMed

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. PMID:22357504

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

2012-05-01

105

Quantitative analysis of zeptomole microRNAs based on isothermal ramification amplification.  

PubMed

To date, approximately 700 microRNA (miRNA) molecules have been identified in humans. Accurate and sensitive quantification of miRNA levels will help unveil their biological functions. Here, we extend the isothermal ramification amplification (RAM) approach to a sensitive and specific real-time assay for quantitative analysis of miRNA. This RAM miRNA assay is based on the threshold cycle (C(T)) principle similar to that of real-time PCR. It has a dynamic range of at least seven orders of magnitude, allowing for the quantification of miRNA input from 10(3) to 10(10) copies per reaction (10 nM to 1 fM). The capabilities of discriminating single-base mismatch and distinguishing mature miRNAs from their precursors are achieved by coupling the reverse-transcription of miRNA to the generation of a closed C-probe, rather than using expensive detection probes like in real-time PCR. Quantitative measurement of 5 miRNAs (mir-1, miR-122, mir-150, mir-143, and let-7a) across 12 mouse tissues is validated in total RNA samples without further purification. U6 snRNA, snoRNA 135, and miRNA-191 could be simultaneously quantified as endogenous controls. These results suggest that our RAM miRNA assay might provide a universal tool for miRNA detection and functional studies to meet the needs for bench examination, clinical diagnosis, and on-site detection. PMID:19620236

Yao, Bo; Li, Juan; Huang, Huang; Sun, Changhong; Wang, Zhao; Fan, Yu; Chang, Qing; Li, Shaolu; Xi, Jianzhong

2009-09-01

106

Application of loop-mediated isothermal amplification technique to rapid and direct detection of methicillin-resistant Staphylococcus aureus (MRSA) in blood cultures  

Microsoft Academic Search

Staphylococcus aureus is the most important pathogen in nosocomial infections, including bloodstream infections. Prompt identification of S. aureus from blood cultures and detection of methicillin resistance are essential in cases of suspected sepsis. A novel nucleic acid\\u000a amplification technique, loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63°C)\\u000a with high specificity, efficiency, and rapidity, was applied to detect

Yoshiki Misawa; Atsushi Yoshida; Ryoichi Saito; Honami Yoshida; Katsuko Okuzumi; Nobue Ito; Mitsumasa Okada; Kyoji Moriya; Kazuhiko Koike

2007-01-01

107

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

PubMed

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 range of applications. PMID:21476587

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

2011-05-01

108

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

PubMed

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

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

2014-11-20

109

Shrimp Taura syndrome virus detection by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick.  

PubMed

Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acid under isothermal conditions using four sets of specially designed primers that recognize six distinct target sequences with high specificity and sensitivity. In this report, a 60-min reverse transcription LAMP (RT-LAMP) method for amplification of Taura syndrome virus (TSV) cDNA using biotin-labeled primer was combined with a chromatographic lateral flow dipstick (LFD) for rapid and simple visual detection of TSV-specific amplicons. The LFD process involved a 5-min post RT-LAMP step for specific hybridization of cDNA with an FITC-labeled DNA probe that confirmed the presence of specific, biotin-labeled TSV amplicons. The resulting DNA duplexes could be visualized trapped at the LFD strip test line within 5min of sample exposure. Using the combined RT-LAMP and LFD system, the total assay interval was approximately 70min, excluding RNA extraction time. Detection sensitivity was comparable to other commonly used methods for nested RT-PCR detection of TSV. In addition to reduced assay time when compared to electrophoresis, combination of RT-LAMP with LFD confirms amplicon identity by hybridization and eliminates the need to handle carcinogenic ethidium bromide. PMID:18662723

Kiatpathomchai, Wansika; Jaroenram, Wansadaj; Arunrut, Narong; Jitrapakdee, Sarawut; Flegel, T W

2008-11-01

110

Embryo sexing and sex chromosomal chimerism analysis by loop-mediated isothermal amplification in cattle and water buffaloes.  

PubMed

In domestic animals of the family Bovidae, sex preselection of offspring has been demanded for convenience of milk/beef production and animal breeding. Development of the nonsurgical embryo transfer technique and sexing methods of preimplantation embryos made it possible. Sexing based on detection of Y chromosome-specific DNA sequences is considered the most reliable method to date. PCR enables amplification of a target sequence from a small number of blastomeres. However, it requires technical skill and is time consuming. Furthermore, PCR has the risk of false positives because of DNA contamination during handling of the PCR products in duplicate PCR procedures and/or electrophoresis. Therefore, for embryo sexing to become widely used in the cattle embryo transfer industry, a simple, rapid and precise sexing method needs to be developed. Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method, and the reaction is carried out under isothermal conditions (range, 60 to 65 C) using DNA polymerase with strand displacement activity. When the target DNA is amplified by LAMP, a white precipitate derived from magnesium pyrophosphate (a by-product of the LAMP reaction) is observed. It is noteworthy that LAMP does not need special reagents or electrophoresis to detect the amplified DNA. This review describes the development and application of an embryo sexing method using LAMP in cattle and water buffaloes. PMID:23965599

Hirayama, Hiroki; Kageyama, Soichi; Moriyasu, Satoru; Sawai, Ken; Minamihashi, Akira

2013-01-01

111

A loop-mediated isothermal amplification (LAMP) method for the identification of species within the Echinococcus granulosus complex.  

PubMed

To facilitate the specific identification of Echinococcus spp. isolates in endemic countries, a LAMP (loop-mediated isothermal amplification) assay was developed to detect the various agents known to cause cystic echinococcosis (E. granulosus s.s., E. equinus, E. ortleppi, E. canadensis and E. felidis). The infectivity of the different species and the severity of the disease in humans and livestock vary significantly among those species, and correct molecular identification of large numbers of field isolates is crucial to understand their epidemiology. However, funding constraints in many CE endemic countries often prevent PCR-based screening of field isolates. The LAMP method allows the amplification of DNA fragments under isothermal conditions which can be achieved using an ordinary waterbath, and the detection of amplification products only requires a UV light source. In the present study a LAMP assay was developed which allows the detection and differentiation of the 5 CE causing Echinococcus species. The diagnostic power was adjusted to species level, i.e. intraspecific strains (G1-3 within E. granulosus s.s., G6-10 within E. canadensis) are not discriminated. Wherever this would be necessary for epidemiological purposes, the method can be adjusted according to local requirements. The sensitivity of the assay was tested down to one fiftieth of a single protoscolex or egg, respectively. The present study describes a fast and simple method for the differentiation of CE causing Echinococcus species which can facilitate epidemiological studies in endemic countries. PMID:24418600

Wassermann, Marion; Mackenstedt, Ute; Romig, Thomas

2014-02-24

112

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

PubMed

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

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

2013-06-01

113

Increased Robustness of Single-Molecule Counting with Microfluidics, Digital Isothermal Amplification, and a Mobile Phone  

E-print Network

Amplification, and a Mobile Phone versus Real-Time Kinetic Measurements David A. Selck,¶ Mikhail A. Karymov with a consumer cell- phone camera, and to automated cloud-based processing of these images (R2 = 0.9997 vs true be imaged with the cell phone camera using flash as the excitation source. Many nonlinear amplification

Ismagilov, Rustem F.

114

Concurrent Reactivation of Herpes Simplex and Varicella Zoster Viruses Confirmed by the Loop-Mediated Isothermal Amplification Assay  

PubMed Central

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. PMID:24575004

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

2014-01-01

115

Evaluation of a loop-mediated isothermal amplification-based methodology to detect carbapenemase carriage in acinetobacter clinical isolates.  

PubMed

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-?-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

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

2014-12-01

116

A reverse transcription loop-mediated isothermal amplification assay to rapidly diagnose foot-and-mouth disease virus C  

PubMed Central

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to rapidly detect foot-and-mouth disease virus serotype C (FMDV C). By testing 10-fold serial dilutions of FMDV C samples, sensitivity of the FMDV C RT-LAMP was found to be 10 times higher than that of conventional reverse transcription-PCR (RT-PCR). No cross-reactivity with A, Asia 1, or O FMDV or swine vesicular disease virus (SVDV) indicated that FMDV C RT-LAMP may be an exciting novel method for detecting FMDV C. PMID:24690607

Ding, Yao-zhong; Zhou, Jian-hua; Ma, Li-na; Qi, Yan-ni; Wei, Gang

2014-01-01

117

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

PubMed Central

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

2011-01-01

118

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

PubMed

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. PMID:20480635

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

119

Development of a reverse transcription loop-mediated isothermal amplification assay for rapid detection of grass carp reovirus.  

PubMed

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a gene amplification method that can amplify the RNA template by isothermal incubation. This paper reports a rapid and sensitive RT-LAMP method which was developed for the detection of grass carp reovirus (GCRV). The present study concluded the optimal conditions for the LAMP reaction of which the Mg(2+) concentrations in the reaction mixtures, the incubation temperature, and the reaction time are at 8mM, 64°C, and 30 min, respectively. The analytical sensitivity of the RT-LAMP method was revealed as low as 7 copies of viral templates and 100-fold more sensitive than the published RT-PCR method. A visual inspection of in-tube LAMP products stained with a DNA fluorescent dye demonstrated that the positive and negative reactions exhibit distinct and different colors in daylight, which means that gel electrophoresis is not necessary to judge the positive or negative results. As the application of the method is rapid, easy, and no complicated instrument required, the GCRV-RT-LAMP method established in this study has great potential for the detection of GCRV in both the laboratory and the farm. PMID:23159672

Zhang, Qing-Li; Yan, Yi; Shen, Jin-Yu; Hao, Gui-Jie; Shi, Cheng-Yin; Wang, Qin-Tao; Liu, Hong; Huang, Jie

2013-02-01

120

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

PubMed Central

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 bacteria. The assay was capable of detecting CBC-causing strains from several geographical origins and pathotypes. Conclusions The CBC-LAMP technique is a simple, fast, sensitive and specific method for the diagnosis of Citrus Bacterial Canker. This method can be useful in the phytosanitary programs of the citrus industry worldwide. PMID:20565886

2010-01-01

121

A one-step reverse transcription loop-mediated isothermal amplification for detection and discrimination of infectious bursal disease virus  

PubMed Central

Background Infectious bursal disease (IBD) is a highly contagious immunosuppressive disease in young chickens caused by infectious bursal disease virus (IBDV). It causes huge economic losses to the poultry industry. The objective of this study is to develop a loop-mediated isothermal amplification (LAMP) method for the detection and discrimination of IBDV. Results In this study, we applied reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect IBDV in one simple step and further identified the very virulent strain from non-vvIBDVs with a simply post-amplification restriction enzyme analysis. Based on sequence analysis, a set of two inner, two outer and two loop primers were designed to target the VP5 gene and they showed great specificity with no cross reaction to the other common avian pathogens. The detection limit determined by both color change inspection and agarose gel electrophoresis was 28 copies viral RNA, which was almost as sensitive as a real-time RT-PCR previous developed in our laboratory. We also identified a unique Tfi I restriction site located exclusively in non-vvIBDVs, so very virulent strain could be distinguished from current vaccine strains. By screening a panel of clinical specimens, results showed that this method is high feasible in clinical settings, and it obtained results 100% correlated with real-time RT-PCR. Conclusion RT-LAMP is a rapid, simple and sensitive assay. In combination with the Tfi I restriction analysis, this method holds great promises not only in laboratory detection and discrimination of IBDV but also in large scale field and clinical studies. PMID:21385415

2011-01-01

122

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

PubMed Central

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

2011-01-01

123

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

PubMed Central

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 be controlled to ensure quantification at low target concentrations. PMID:22546148

2012-01-01

124

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

NASA Astrophysics Data System (ADS)

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/?L 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.

Liu, Hongxing; Xing, Da; Zhou, Xiaoming

2014-09-01

125

Development of loop-mediated isothermal amplification assay for specific and rapid detection of camelpox virus in clinical samples.  

PubMed

In this study, development of loop-mediated isothermal amplification (LAMP) assay based on ankyrin repeat protein gene (C18L) for specific and rapid detection of camelpox virus (CMLV) was carried out. The assay was optimized using viral genomic DNA (gDNA) extracted from density gradient purified CMLV and standard control recombinant DNA plasmid containing the target, which resulted in reliable amplification at 62°C for 60 min. The amplified LAMP product was identified by agarose gel electrophoresis and subsequent direct visualization under UV light or observation by naked-eye for the presence of turbidity and color change following the addition of SYBR Green I dye and hydroxy naphthol blue (HNB). The analytical specificity of LAMP and conventional PCR assays was evaluated using other related poxviruses namely buffalopox, goatpox, sheeppox, and orf viruses, which revealed only a specific amplification of CMLV. The LAMP assay was 10-fold more sensitive than the conventional PCR. Further, the assay was evaluated with DNA extracted from the cell culture isolates of CMLV (n=11) and clinical samples (n=23). These results proved that the developed LAMP is a simple, specific, sensitive, rapid and economical diagnostic tool for detection of CMLV from clinical materials. PMID:22575686

Venkatesan, G; Bhanuprakash, V; Balamurugan, V; Singh, R K; Pandey, A B

2012-07-01

126

Development of a loop-mediated isothermal amplification method for rapid detection of caprine arthritis-encephalitis virus proviral DNA.  

PubMed

A rapid detection assay based on loop-mediated isothermal amplification (LAMP) has been developed for detecting caprine arthritis-encephalitis (CAEV) proviral DNA. The LAMP assay utilized a set of five primers designed against highly conserved sequences located within the p25 gene region. The assay successfully detected CAEV proviral DNA in total DNA extracts originating from cell culture, whole blood samples and separated PBMCs. There was no cross-reaction with the negative control. Amplification was monitored using a Loopamp real-time turbidimeter; turbidity and the corresponding time were recorded. Amplification from CAEV-Shanxi DNA was detected as early as 17 min, with a maximum sensitivity of 0.0001 TCID(50), reached at 32 min. Sixty-eight animal blood samples were tested using AGID, PCR and LAMP assay, and the positive rates were 30.9 %, 33.8 % and 47.1 %, respectively. Whole blood can be used directly, eliminating the need for separation of PBMCs and nucleic acid extraction, reducing the overall procedure time to approximately 80 min. Therefore, the LAMP assay provides a specific and sensitive means for detecting CAEV proviral DNA in a simple, fast, and cost-effective manner and should be useful in eradication programs and epidemiological studies. Furthermore, the LAMP assay can be performed in less-well-equipped laboratories as well as in the field. PMID:22566005

Huang, Jinhai; Sun, Yuehui; Liu, Yebing; Xiao, Huazhi; Zhuang, Shiwen

2012-08-01

127

Rapid DNA amplification in a capillary tube by natural convection with a single isothermal heater.  

PubMed

Herein we describe a simple platform for rapid DNA amplification using convection. Capillary convective PCR (CCPCR) heats the bottom of a capillary tube using a dry bath maintained at a fixed temperature of 95°C. The tube is then cooled by the surrounding air, creating a temperature gradient in which a sample can undergo PCR amplification by natural convection through reagent circulation. We demonstrate that altering the melting temperature of the primers relative to the lowest temperature in the tube affects amplification efficiency; adjusting the denaturation temperature of the amplicon relative to the highest temperature in the tube affects maximum amplicon size, with amplicon lengths of ?500 bp possible. Based on these criteria, we successfully amplified DNA sequences from three different viral genomes in 30 min using CCPCR, with a sensitivity of ~30 copies per reaction. PMID:21231923

Chou, Wen Pin; Chen, Ping Hei; Miao, Ming; Kuo, Long Sheng; Yeh, Shiou Hwei; Chen, Pei Jer

2011-01-01

128

An Empirical Approach for Quantifying Loop-Mediated Isothermal Amplification (LAMP) Using Escherichia coli as a Model System  

PubMed Central

Loop mediated isothermal amplification (LAMP) is a highly efficient, selective and rapid DNA amplification technique for genetic screening of pathogens. However, despite its popularity, there is yet no mathematical model to quantify the outcome and no well-defined metric for comparing results that are available. LAMP is intrinsically complex and involves multiple pathways for gene replication, making fundamental modelling nearly intractable. To circumvent this difficulty, an alternate, empirical model is introduced that will allow one to extract a set of parameters from the concentration versus time curves. A simple recipe to deduce the time to positive, Tp - a parameter analogous to the threshold cycling time in polymerase chain reaction (PCR), is also provided. These parameters can be regarded as objective and unambiguous indicators of LAMP amplification. The model is exemplified on Escherichia coli strains by using the two gene fragments responsible for vero-toxin (VT) production and tested against VT-producing (O157 and O45) and non-VT producing (DH5 alpha) strains. Selective amplification of appropriate target sequences was made using well established LAMP primers and protocols, and the concentrations of the amplicons were measured using a Qubit 2.0 fluorometer at specific intervals of time. The data is fitted to a generalized logistic function. Apart from providing precise screening indicators, representing the data with a small set of numbers offers significant advantages. It facilitates comparisons of LAMP reactions independently of the sampling technique. It also eliminates subjectivity in interpretation, simplifies data analysis, and allows easy data archival, retrieval and statistical analysis for large sample populations. To our knowledge this work represents a first attempt to quantitatively model LAMP and offer a standard method that could pave the way towards high throughput automated screening. PMID:24979038

Subramanian, Sowmya; Gomez, Romel D.

2014-01-01

129

Development of a loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of Babesia canis infections.  

PubMed

Vector-borne diseases are rising in interest due to global warming, which is believed to impact on the distribution of vectors into new areas thus influencing the occurrence and epidemiology of vector-borne pathogens. Babesia canis belongs to the Piroplasmidae and there are three described subspecies, namely B. canis canis, B. canis rossi and B. canis vogeli. They are each transmitted by a different tick-species, Dermacentor reticulatus, Haemaphysalis leachi and Rhipicephalus sanguineus, respectively. There are also differences in the geographical distribution and pathogenicity to dogs of each subspecies. In this study, we aimed to establish a rapid and easy to perform DNA-based test using loop-mediated isothermal amplification to detect all three Babesia canis subspecies in one assay. PMID:20537107

Müller, H; Aysul, N; Liu, Z; Salih, D A; Karagenc, T; Beyer, D; Kullmann, B; Ahmed, J S; Seitzer, U

2010-04-01

130

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

PubMed

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 ?g 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. PMID:24952125

Metwally, L; Gomaa, N; Hassan, R

2014-04-01

131

Caprine arthritis encephalitis virus detection in blood by loop-mediated isothermal amplification (LAMP) assay targeting the proviral gag region.  

PubMed

Caprine arthritis encephalitis virus (CAEV), of the genus Lentivirus of the Retroviridae family, causes persistent disease, which is characterized by polyarthritis and mastitis in adult goats and progressive paresis (leukoencephalomyelitis) in kids. A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of CAEV in blood samples. Species-specific primers amplifying the gag gene region in the provirus were used for the detection of CAEV. The LAMP assay result was obtained 30 min after incubation on a constant temperature at 63 °C in a heat block. Resulting amplicons were visualized by addition of SYBR green dye after the reaction and checked by agarose gel electrophoresis. The sensitivity of LAMP assay was evaluated by comparing the result with the nested polymerase chain reaction. Based on the experiments, the result of the assay indicated a rapid and sensitive test for the detection of CAEV. PMID:24630755

Balbin, Michelle M; Belotindos, Lawrence P; Abes, Nancy S; Mingala, Claro N

2014-05-01

132

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

PubMed

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

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

2014-07-15

133

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

PubMed

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

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

2014-11-01

134

Design and application of a loop-mediated isothermal amplification assay for the rapid detection of Staphylococcus pseudintermedius.  

PubMed

Staphylococcus pseudintermedius is a commensal and opportunistic pathogen of dogs. It is mainly implicated in canine pyoderma, as well as other suppurative conditions of dogs. Although bacterial culture is routinely used for clinical diagnosis, molecular methods are required to accurately identify and differentiate S. pseudintermedius from other members of the Staphylococcus intermedius group. These methods, owing largely to their cost, are not easy to implement in nonspecialized laboratories or veterinary practices. In the current study, loop-mediated isothermal amplification (LAMP), a novel isothermal nucleic acid amplification procedure, was employed to develop a rapid, specific, and sensitive S. pseudintermedius assay. Different detection strategies, including the use of a lateral flow device, were evaluated. The assay was evaluated for cross-reactivity against 30 different bacterial species and validated on a panel of 108 S. pseudintermedius isolates, originating from different dog breeds and locations within the United Kingdom. The assay was specific, showing no cross-reactivity during in silico and in vitro testing. When tested using DNA extracts prepared directly from 35 clinical surgical site swabs, the assay could detect S. pseudintermedius in less than 15 min, with a diagnostic sensitivity of 94.6%, superior to that of a polymerase chain reaction method. The LAMP assay also had an analytical sensitivity in the order of 10(1) gene copies, and the amplified products were readily detected using a lateral flow device. The LAMP assay described in the present study is simple and rapid, opening up the possibility of its use as a diagnostic tool within veterinary practices. PMID:24398904

Diribe, Onyinye; North, Sarah; Sawyer, Jason; Roberts, Lisa; Fitzpatrick, Noel; La Ragione, Roberto

2014-01-01

135

Sensitive and Rapid Detection of the New Delhi Metallo-Beta-Lactamase Gene by Loop-Mediated Isothermal Amplification  

PubMed Central

New Delhi metallo-?-lactamase 1 (NDM-1), which is associated with resistance to carbapenem, was first reported in 2008. A sensitive and rapid molecular assay to detect the plasmid blaNDM-1 in clinical isolates is needed to control its spread. We describe a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of blaNDM-1 from pure culture and sputum, urine, and fecal samples. Eight sets of primers were designed to recognize six or eight distinct sequences on target blaNDM-1, and one set was selected as the most appropriate set of primers for its rapid detection. The specificity and sensitivity of the primers in the LAMP reactions for blaNDM-1 detection were determined. The sensitivity of the LAMP assay for blaNDM-1 detection in sputum, urine, and fecal samples was also tested. Two methods, namely, monitoring of turbidity and addition of calcein to the reaction tube, were used to determine negative and positive results. The results showed that target DNA was amplified and visualized by the two detection methods within 70 min at an isothermal temperature of 65°C. The sensitivity of LAMP, with a detection limit of 10.70 pg/?l DNA, was 100-fold greater than that of PCR. Thirteen infection bacterial strains without blaNDM-1 were selected for testing of specificity, and the results of the amplification were negative, which showed that the primers had good levels of specificity. The LAMP method reported here is demonstrated to be a potentially valuable means for the detection of blaNDM-1 and rapid clinical diagnosis, being fast, simple, and low in cost. PMID:22357496

Liu, Wei; Zou, Dayang; Li, Yan; Wang, Xuesong; He, Xiang; Wei, Xiao; Shao, Changlin; Li, Xuelian; Shang, Wei; Yu, Kaitao; Liu, Dawei; Li, Yunmei; Guo, Jing; Yin, Zhitao

2012-01-01

136

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

PubMed

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. PMID:24269205

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

2014-02-01

137

One-step reverse transcription loop-mediated isothermal amplification assay for rapid detection of Cymbidium mosaic virus.  

PubMed

Cymbidium mosaic virus (CymMV) is the most prevalent orchid virus. A single-tube one-step betaine-free reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assay was developed for the rapid and easy detection of orchid-infecting CymMV. Five sets of primers were designed based on the conserved regions among various virus isolates. The specificity and the sensitivity of the assay were then evaluated using the RT-LAMP reaction. Within 1h under isothermal conditions at 60°C the target viral gene was amplified successfully. This RT-LAMP assay was found to be quick, specific, sensitive and easy to perform assay that involved only one step and was simpler to carry out than alternative approaches. Thus this assay is an alternative for the rapid and easy detection of CymMV in orchids. This is first time that a RT-LAMP method for the detection of an orchid virus has been described. PMID:21237208

Lee, Meng-Shiou; Yang, Meng-Ja; Hseu, You-Cheng; Lai, Guan-Hua; Chang, Wen-Te; Hsu, Yau-Heiu; Lin, Ming-Kuem

2011-04-01

138

Development of isothermal TaqMan assays for detection of biothreat organisms.  

PubMed

TaqMan probe (dual-labeled DNA probe)-based real-time detection, one of the most sensitive and specific fluorescent detection methods, has been widely utilized in conjunction with polymerase chain reaction (PCR). Helicase-dependent amplification (HDA) is an isothermal amplification technology that has a similar reaction scheme to PCR, but replaces thermocycling with a helicase capable of unwinding a DNA duplex. Here we describe a novel isothermal real-time detection method (HDA-TaqMan) that combines the advantages of both HDA and a TaqMan assay. In this assay, the reactions of DNA unwinding, primer annealing, polymerization, probe hybridization, and subsequent hydrolysis by the polymerase are coordinated and synchronized to perform at a single temperature. It not only provides a useful tool for real-time detection of HDA, but also provides an isothermal format for the TaqMan system. With this platform, we have successfully developed rapid real-time isothermal assays for biodefense targets that include Vibrio cholerae and Bacillus anthracis. PMID:19007339

Tong, Yanhong; Tang, Wen; Kim, Hyun-Jin; Pan, Xiaojing; Ranalli, Tamara; Kong, Huimin

2008-11-01

139

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

PubMed Central

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. PMID:21731183

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

2011-01-01

140

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

PubMed

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

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

2014-08-01

141

Rapid Detection of Haptoglobin Gene Deletion in Alkaline-Denatured Blood by Loop-Mediated Isothermal Amplification Reaction  

PubMed Central

Anhaptoglobinemic patients run the risk of severe anaphylactic transfusion reaction because they produce serum haptoglobin antibodies. Being homozygous for the haptoglobin gene deletion allele (HPdel) is the only known cause of congenital anhaptoglobinemia, and detection of HPdel before transfusion is important to prevent anaphylactic shock. In this study, we developed a loop-mediated isothermal amplification (LAMP)-based screening for HPdel. Optimal primer sets and temperature for LAMP were selected for HPdel and the 5? region of the HP using genomic DNA as a template. Then, the effects of diluent and boiling on LAMP amplification were examined using whole blood as a template. Blood samples diluted 1:100 with 50 mmol/L NaOH without boiling gave optimal results as well as those diluted 1:2 with water followed by boiling. The results from 100 blood samples were fully concordant with those obtained by real-time PCR methods. Detection of the HPdel allele by LAMP using alkaline-denatured blood samples is rapid, simple, accurate, and cost effective, and is readily applicable in various clinical settings because this method requires only basic instruments. In addition, the simple preparation of blood samples using NaOH saves time and effort for various genetic tests. PMID:21497293

Soejima, Mikiko; Egashira, Kouichi; Kawano, Hiroyuki; Kawaguchi, Atsushi; Sagawa, Kimitaka; Koda, Yoshiro

2011-01-01

142

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

PubMed

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

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

2014-01-01

143

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

PubMed

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. PMID:21731183

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

2011-04-01

144

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

PubMed

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

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

2011-08-01

145

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

PubMed Central

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

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

2014-01-01

146

Loop-Mediated Isothermal Amplification Method Targeting the TTS1 Gene Cluster for Detection of Burkholderia pseudomallei and Diagnosis of Melioidosis  

Microsoft Academic Search

Melioidosis is a severe infection caused by Burkholderia pseudomallei. The timely implementation of effective antimicrobial treatment requires rapid diagnosis. Loop-mediated isothermal amplification (LAMP) targeting the TTS1 gene cluster was developed for the detection of B. pseudomallei. LAMP was sensitive and specific for the laboratory detection of this organism. The lower limit of detection was 38 genomic copies per reaction, and

Narisara Chantratita; Ella Meumann; Direk Limmathurotsakul; Vanaporn Wuthiekanun; Saran Wannapasni; Nicholas P. J. Day; Sharon J. Peacock

147

Comparison of Loop-Mediated Isothermal Amplification Assay and Conventional Culture Methods for Detection of Campylobacter jejuni and Campylobacter coli in Naturally Contaminated Chicken Meat Samples  

Microsoft Academic Search

We investigated the efficacy of a loop-mediated isothermal amplification (LAMP) assay for detection of chicken meat samples naturally contaminated with Campylobacter jejuni and Campylobacter coli. A total of 144 Preston enrichment broth cultures from chicken meat samples were assessed by using the LAMP assay and conventional culture methods, which consist of a combination of Preston enrichment culturing and plating onto

Wataru Yamazaki; Masumi Taguchi; Takao Kawai; Kentaro Kawatsu; Junko Sakata; Kiyoshi Inoue; Naoaki Misawa

2009-01-01

148

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

PubMed Central

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

2014-01-01

149

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

PubMed

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

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

2013-06-01

150

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

NASA Astrophysics Data System (ADS)

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.

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

2010-01-01

151

Detection of lead(II) ions with a DNAzyme and isothermal strand displacement signal amplification.  

PubMed

A DNAzyme based method for the sensitive and selective quantification of lead(II) ions has been developed. A DNAzyme that requires Pb(2+) for activation was selected. An RNA containing DNA substrate was cleaved by the DNAzyme in the presence of Pb(2+). The 2',3'-cyclic phosphate of the cleaved 5'-part of the substrate was efficiently removed by Exonuclease III. The remaining part of the single stranded DNA (9 or 13 base long) was subsequently used as the primer for the strand displacement amplification reaction (SDAR). The method is highly sensitive, 200 pM lead(II) could be easily detected. A number of interference ions were tested, and the sensor showed good selectivity. Underground water samples were also tested, which demonstrated the feasibility of the current approach for real sample applications. It is feasible that our method could be used for DNAzyme or aptazyme based new sensing method developments for the quantification of other target analytes with high sensitivity and selectivity. PMID:24144554

Li, Wenying; Yang, Yue; Chen, Jian; Zhang, Qingfeng; Wang, Yan; Wang, Fangyuan; Yu, Cong

2014-03-15

152

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

PubMed

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. PMID:23542058

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

2013-06-01

153

Rapid Visual Detection of Highly Pathogenic Streptococcus suis Serotype 2 Isolates by Use of Loop-Mediated Isothermal Amplification  

PubMed Central

Streptococcus suis serotype 2 (S. suis 2) is an important zoonotic pathogen that causes considerable economic losses to the pig industry and significantly threatens public health worldwide. The highly pathogenic S. suis 2, which contains the 89K pathogenicity island (PAI), has caused large-scale outbreaks of infections in humans, resulting in high mortality rates. In this study, we established two loop-mediated isothermal amplification (LAMP)-based assays that can rapidly detect S. suis 2 and the 89K PAI and can be performed simultaneously under the same conditions. Further, based on the findings of these two LAMP assays and using the same set of serially diluted DNA samples, we compared the sensitivities of different LAMP product detection methods, including SYBR green detection, gel electrophoresis, turbidimetry, calcein assays, and hydroxynaphthol blue detection. The results suggest that target genes can be amplified and detected within 48 min under 63°C isothermal conditions. The sensitivity of tests for S. suis 2 detection varies between detection methods and reaction systems, indicating that for each LAMP reaction system, multiple detection methods should be performed to select the optimal one. The sensitivities of the optimized methods (7.16 copies/reaction) in the present study were identical to those of the real-time PCR assay, and the test results for reference strains and clinical samples showed that these LAMP systems have high specificities. Thus, since the LAMP systems established in this study are simple, fast, and sensitive, they may have good clinical potential for detecting the highly pathogenic S. suis 2. PMID:23884995

Zhang, Jinhai; Zhu, Jing; Ren, Hao; Zhu, Shiying; Zhao, Ping; Zhang, Fengyu; Lv, Heng; Hu, Dan; Hao, Lina; Geng, Meiling; Gong, Xiufang; Pan, Xiuzhen

2013-01-01

154

Rapid detection of orf virus by loop-mediated isothermal amplification based on the DNA polymerase gene.  

PubMed

At present, there are no effective antiviral treatments available for contagious ecthyma, and rapid diagnosis is therefore critical for effective control of the disease. Recently, the invention of a novel LAMP technique that can rapidly amplify nucleic acids with high specificity and sensitivity under isothermal conditions has overcome some of the deficiencies of nucleic acid-based diagnostic tests and has made on-site diagnosis possible. To establish a flexible loop-mediated isothermal amplification (LAMP) assay for the rapid detection of orf virus, two pairs of primers, including outer primers F3/B3 and inner primers FIP/BIP, were designed to amplify the DNA polymerase gene. Optimal time and temperature conditions for LAMP were found to be 45 min and 62 °C, respectively. The LAMP assay was shown to be specific, with no cross-reactivity with sheeppox virus, goatpox virus, avian molluscum roup virus or vesicular stomatitis virus. Additionally, the sensitivity of the LAMP method was similar to that of real-time PCR and demonstrated greater sensitivity than a conventional polymerase chain reaction (PCR) assay. To assess the utility of LAMP in the detection of orf virus in clinical samples, a total of 35 samples collected from orf virus-infected sheep and goats were tested using the optimized LAMP assay, real-time PCR, and conventional PCR. Of the samples, 26 were found to be positive by LAMP, and 25 (74.3 %) were positive by real-time PCR, whereas only 18 (51.4 %) were positive by conventional PCR. Our results have shown that the LAMP assay developed in this study can be used for the rapid detection of orf virus. PMID:23183830

Li, Jida; Song, Deguang; He, Wenqi; Bao, Yingfu; Lu, Rongguang; Su, Gaoli; Wang, Gaili; Lu, Huijun; Zhao, Kui; Gao, Feng

2013-04-01

155

Development of Mitochondrial Loop-Mediated Isothermal Amplification for Detection of the Small Liver Fluke Opisthorchis viverrini (Opisthorchiidae; Trematoda; Platyhelminthes)  

PubMed Central

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

Nguyen, Nga Thi Bich; Truong, Nam Hai; De, Nguyen Van

2012-01-01

156

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

PubMed Central

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

2014-01-01

157

Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of Cronobacter sakazakii.  

PubMed

Cronobacter sakazakii is an emerging pathogen associated with the ingestion of contaminated reconstituted formula, which causes necrotizing enterocolitis, sepsis, and meningitis in low-birth-weight preterm neonatal infants. Sensitive and specific detection methods are needed to better control C. sakazakii infections. This study aims to develop a highly specific and sensitive loop-mediated isothermal amplification (LAMP) assay for detecting C. sakazakii in powdered infant formula (PIF). A set of four LAMP primers were designed based on the published C. sakazakii ompA gene sequence. Specificity of the assay was evaluated using a panel of 22 C. sakazakii, 27 Enterobacteriaceae family except C. sakazakii, and 25 other strains. Assay sensitivity was determined using serial dilutions of C. sakazakii American Type Culture Collection 51329 culture ranging from 10(6) colony-forming units (CFU)/mL to extinction. The assay was also tested in experimentally inoculated PIF samples. The ompA-based LAMP assay was able to detect specifically all of the 22 C. sakazakii strains without amplification from 52 non-C. sakazakii strains. The detection limit was 10(1) CFU/mL in pure culture, up to 10-fold more sensitive than that of the ompA-polymerase chain reaction (PCR). When applied to PIF, sensitivity was 10(2) CFU/mL, up to 10-fold that of the ompA-PCR. The ompA-based LAMP assay developed in this study was sensitive, specific, and low cost with great potential for future field detection of C. sakazakii in PIF. PMID:23199494

Fan, Hongying; Long, Beiguo; Wu, Xianbo; Bai, Yang

2012-12-01

158

Reverse transcription loop-mediated isothermal amplification for rapid detection of Japanese encephalitis virus in swine and mosquitoes.  

PubMed

Japanese encephalitis (JE) can infect many agriculturally important animals and humans, and has a high incidence in Asia. One of the natural hosts of the mosquito-borne JE virus (JEV) is domestic pigs, which act as amplifier hosts. Porcine infection results in fatal encephalitis, abortion, and stillbirth in pregnant sows, and hypospermia in boars. In this study, a rapid JEV detection method for swine and mosquitoes was developed based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) targeting the nucleocapsid (E) genes of JEV genotype I (lineage K94PO5), and genotype III (lineage SA14-14-2). Fifty-six swine blood samples and 20,000 mosquitoes were used to evaluate the method, compared to conventional RT-polymerase chain reaction (PCR) and real-time RT-PCR. RT-LAMP had detection limits of 2.57 and 2.34 copies/?L for JEV I and III, respectively. Assay sensitivity was similar to real-time RT-PCR, but was 10-fold higher than conventional RT-PCR. Assay specificity was high, showing no cross-reactivity to other flaviviruses. The results of virus isolation and identification of swine blood samples and mosquito samples were fully consistent with RT-LAMP. Finally, the JEV RT-LAMP assay was simpler and less time consuming than conventional RT-PCR or real-time RT-PCR, since the amplification step could be completed in a single tube within 50?min at 63°C. In conclusion, the newly-developed RT-LAMP assay is an accurate and convenient method for rapid and sensitive diagnosis of JEV in swine and mosquitoes, and may prove to be a practical molecular tool for surveillance and epidemiological investigations. PMID:23176446

Liu, Hao; Liu, Zhen-Jiang; Jing, Jie; Ren, Jing-Qiang; Liu, Yan-Yu; Guo, Huan-Huan; Fan, Min; Lu, Hui-Jun; Jin, Ning-Yi

2012-12-01

159

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

PubMed

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 2.5?g 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. PMID:24480190

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

2014-03-17

160

Rapid and sensitive detection of Plesiomonas shigelloides by loop-mediated isothermal amplification of the hugA gene.  

PubMed

Plesiomonas shigelloides is one of the causative agents of human gastroenteritis, with increasing number of reports describing such infections in recent years. In this study, the hugA gene was chosen as the target to design loop-mediated isothermal amplification (LAMP) assays for the rapid, specific, and sensitive detection of P. shigelloides. The performance of the assay with reference plasmids and spiked human stools as samples was evaluated and compared with those of quantitative PCR (qPCR). No false-positive results were observed for the 32 non-P. shigelloides strains used to evaluate assay specificity. The limit of detection for P. shigelloides was approximately 20 copies per reaction in reference plasmids and 5×10(3) CFU per gram in spiked human stool, which were more sensitive than the results of qPCR. When applied in human stool samples spiked with 2 low levels of P. shigelloides, the LAMP assays achieved accurate detection after 6-h enrichment. In conclusion, the LAMP assay developed in this study is a valuable method for rapid, cost-effective, and simple detection of P. shigelloides in basic clinical and field laboratories in the rural areas of China. PMID:23077478

Meng, Shuang; Xu, Jianguo; Xiong, Yanwen; Ye, Changyun

2012-01-01

161

Evaluation of Loop-Mediated Isothermal Amplification Suitable for Molecular Monitoring of Schistosome-Infected Snails in Field Laboratories  

PubMed Central

We previously described loop-mediated isothermal amplification (LAMP) for detection of Schistosoma haematobium and S. mansoni DNA in infected snails. In the present study, we adapted the LAMP assay for application in field laboratories in schistosomiasis-endemic areas. Isolation of DNA was simplified by blotting snail tissue (extracted in NaOH/sodium dodecyl sulfate) onto treated membranes, which enabled preservation at ambient temperatures. A ready-mix of LAMP reagents, suitable for shipment at ambient temperature and storage in minimal refrigeration, was used. Local survey teams without experience in molecular biology acquired operational expertise with this test within a few hours. Fifty-four field-caught snails were tested locally by LAMP and 59 were tested at similar conditions in Jerusalem. The LAMP results were consistent with those of a polymerase chain reaction; only four samples showed false-negative results. Results indicate that LAMP assays are suitable for detection of S. haematobium and S. mansoni in low-technology parasitology laboratories in which schistosomiasis elimination activities are undertaken. PMID:23208875

Hamburger, Joseph; Abbasi, Ibrahim; Kariuki, Curtis; Wanjala, Atsabina; Mzungu, Elton; Mungai, Peter; Muchiri, Eric; King, Charles H.

2013-01-01

162

Detection of Mycobacterium tuberculosis by using loop-mediated isothermal amplification combined with a lateral flow dipstick in clinical samples.  

PubMed

Tuberculosis (TB) is a communicable disease caused by the bacterium Mycobacterium tuberculosis (MTB) and is a persistent problem in the developing countries. Loop-mediated isothermal amplification (LAMP) allows DNA to be amplified rapidly at a constant temperature. Here, a LAMP method was combined with a chromatographic lateral-flow dipstick (LFD) to detect IS6110 gene of M. tuberculosis specifically and rapidly. The reaction was optimized at 63°C for 60?min, and the amplified DNA hybridized to an FITC-labeled oligonucleotide probe for 5?min was detected at the LFD test line 5?min after application. Excluding the step of DNA extraction, the test results could be generated approximately within 1?h. In addition to the advantage of short assay time, this technique could avoid the contact of carcinogenic ethidium bromide due to the exclusion of the electrophoresis analysis step. Furthermore, the data indicated that LAMP-LFD could detect M. tuberculosis genomic DNA as little as 5?pg. The technique showed a significant specificity since no cross-hybridization to M. intracellulare (MIC), M. fortuitum (MFT), M. avium (MAV), M. kansasii (MKS), and M. gordonae (MGD) genomic DNAs was observed. In the clinical unknown samples test, the sensitivity of LAMP-LFD was 98.92 ? % and the specificity was 100 ? % compared to those of the standard culture assay. Based on its sensitivity, specificity, rapidity, low cost, and convenience, LAMP-LFD could be applicable for use in both laboratories and epidemiological surveys of MTB. PMID:23555102

Kaewphinit, Thongchai; Arunrut, Narong; Kiatpathomchai, Wansika; Santiwatanakul, Somchai; Jaratsing, Pornpun; Chansiri, Kosum

2013-01-01

163

Loop mediated isothermal amplification combined with nucleic acid lateral flow strip for diagnosis of cyprinid herpes virus-3.  

PubMed

An improved loop mediated isothermal amplification (LAMP) assay for rapid, sensitive and specific detection of cyprinid herpes virus-3 (CyHV-3), also known as koi herpes virus (KHV), was developed. The lower detection limit of the CyHV-3-LAMP assay is 10 fg DNA which equivalent to 30 copies of CyHV-3 genome. Nucleic acid lateral flow assay was used for visual detection of the LAMP products. The LAMP- nucleic acid lateral flow assay relies on DNA hybridization technology and antigen-antibody reactions in combination with LAMP. For application of this assay, the biotinylated LAMP product was hybridized with a FITC-labelled specific probe for 5 min. The resulting DNA complex could be visualised as purple band at the strip test line within 5 min of sample exposure. The nucleic acid lateral flow analysis of the LAMP product was equivalent in sensitivity but more rapid than the conventional agarose gel electrophoresis. The combination of LAMP assay with the nucleic acid lateral flow analysis can simplify the diagnosis and screening of CyHV-3 as it is simple, requires very little training, does not require specialized equipment such as a thermal cycler, the results are read visually with no need to run a gel and has a high sensitivity and specificity. PMID:19781627

Soliman, Hatem; El-Matbouli, Mansour

2010-02-01

164

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

PubMed Central

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

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

2014-01-01

165

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

PubMed Central

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

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

2012-01-01

166

Development of loop-mediated isothermal amplification method for detection of Kudoa septempunctata (Myxozoa: Multivalvulida) in olive flounder (Paralichthys olivaceus).  

PubMed

A loop-mediated isothermal amplification (LAMP) assay was developed and validated for early, rapid, and sensitive detection of Kudoa septempunctata, a myxosporean parasite found in olive flounder (Paralichthys olivaceus). Recently, several outbreaks associated with ingestion of raw olive flounder muscles harboring mature K. septempunctata spores have been reported, and it is becoming obvious that fresh K. septempunctata spores can cause problems in humans when ingested. Thus, it is necessary to develop reliable detection method of K. septempunctata, to prevent outbreaks and ensure food safety. The LAMP assay has advantages over other molecular detection methods for detecting K. septempunctata in olive flounder muscle, in terms of simplicity, rapidity, and sensitivity. The reaction condition was optimized as 63 °C, 45 min, with three sets of specific primers. The results can be simply confirmed with the naked eye after adding SYBR Green I or by conventional electrophoresis followed by ethidium bromide staining. This LAMP assay did not show any cross-reaction with other kudoid myxosporeans (Kudoa lateolabracis, Kudoa thyrsites) can be found in olive flounder muscles and was validated by testing Kudoa septempunctata spore-spiked samples and field samples. The results showed that the LAMP assay is ten times more sensitive than the conventional polymerase chain reaction in this study and can be applied for early detection for monitoring and epidemiological studies of K. septempunctata in olive flounder aquaculture farms. PMID:24626774

Jeon, Chan-Hyeok; Wi, Seong; Song, Jun-Young; Choi, Hye-Sung; Kim, Jeong-Ho

2014-05-01

167

Visual Detection of Norovirus Genogroup II by Reverse Transcription Loop-Mediated Isothermal Amplification with Hydroxynaphthol Blue Dye.  

PubMed

A simple, rapid, specific, and sensitive colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay with hydroxynaphthol blue dye (HNB) was established, targeting RNA-dependent RNA polymerase and capsid protein gene for the detection of the dominant norovirus genogroup in China-NoV GII. The assay was carried out at 65 °C for 60 min with no cross-reactivity with other common gastroenteritis viruses. The sensitivity of this assay was 10(3) copies per reaction which is equivalent to the conventional RT-PCR test. The clinical test showed 94.83 % coincidence rate for NoV genogroup II detection compared with the results, confirmed by the Department of Viral Diarrhea of Chinese Center for Disease Control and Prevention via conventional RT-PCR. The HNB dye-based RT-LAMP could be a novel rapid screening method for prevalent norovirus genogroup II in China, especially in those resource-limited hospitals and rural local clinics. PMID:24752892

Luo, Jianming; Xu, Ziqian; Nie, Kai; Ding, Xiong; Guan, Li; Wang, Ji; Xian, Yuying; Wu, Xiyang; Ma, Xuejun

2014-09-01

168

Development of a loop-mediated isothermal amplification assay for rapid, sensitive and specific detection of a Campylobacter jejuni clone.  

PubMed

Loop-mediated isothermal amplification (LAMP) assay is a simple, rapid and specific detection method and has been used for detection and identification of different Campylobacter species. In this study, we develop a LAMP assay specific for detection of a particular clone (clone SA) of Campylobacter jejuni, associated with the vast majority of recent sheep abortions in the U.S. Using a set of specific primers for C. jejuni IA3902 (a clone SA isolate) and genomic DNA or boiled cell extract as template, the target DNA was amplified at 63 °C for 50 min in a water bath. A positive reaction was identified visually as white precipitate or fluorescence under UV, and confirmed by gel electrophoresis. Detection limit of the assay was comparable to that of conventional PCR. The LAMP was shown to be specific for detection of clone SA when tested on a number of C. jejuni strains of different genetic backgrounds. Applicability of the LAMP assay for specific detection of clone SA was demonstrated in animal tissues experimentally infected with IA3902 or genetically diverse C. jejuni strains. Since clone SA is the predominant cause of sheep abortions in the U.S. and is a zoonotic pathogen, the LAMP assay may be a valuable detection tool in future epidemiological studies. PMID:22188995

Luo, Yan; Sahin, Orhan; Dai, Lei; Sippy, Rachel; Wu, Zuowei; Zhang, Qijing

2012-05-01

169

Loop-Mediated Isothermal Amplification of Specific Endoglucanase Gene Sequence for Detection of the Bacterial Wilt Pathogen Ralstonia solanacearum  

PubMed Central

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

Pirc, Manca; Llop, Pablo; Ravnikar, Maja; Dreo, Tanja

2014-01-01

170

Molecular detection of nine rice viruses by a reverse-transcription loop-mediated isothermal amplification assay.  

PubMed

A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was established for the detection of nine viruses from infected rice plants, including rice black-streaked dwarf virus (RBSDV), rice dwarf virus (RDV), rice gall dwarf virus (RGDV), rice ragged stunt virus (RRSV), rice transitory yellowing virus (RTYV), rice stripe virus (RSV), rice grassy stunt virus (RGSV), rice tungro spherical virus (RTSV), and rice tungro bacilliform virus (RTBV). Virus-specific primer sets were designed from the genome sequences of these viruses. By the combination of RNA rapid extraction and RT-LAMP, these nine viruses could be detected within 2h from infected rice plants. The sensitivities of the assays were either higher than (for RSV, RTBV, and RTYV) or similar (for RDV) to those of one-step RT-PCR. Furthermore, RTBV and RTSV were detected not only in infected rice plants but also in viruliferous insect vectors. The RT-LAMP assays may facilitate studies on rice disease epidemiology, outbreak surveillance, and molecular pathology. PMID:20837064

Le, Dung Tien; Netsu, Osamu; Uehara-Ichiki, Tamaki; Shimizu, Takumi; Choi, Il-Ryong; Omura, Toshihiro; Sasaya, Takahide

2010-12-01

171

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

PubMed Central

Background Eimeria parasites can cause the disease coccidiosis in poultry and even subclinical infection can incur economic loss. Diagnosis of infection predominantly relies on traditional techniques including lesion scoring and faecal microscopy despite the availability of sensitive molecular assays, largely due to cost and the requirement for specialist equipment. Despite longstanding proven efficacy these traditional techniques demand time and expertise, can be highly subjective and may under-diagnose subclinical disease. Recognition of the tight economic margins prevailing in modern poultry production and the impact of avian coccidiosis on poverty in many parts of the world has highlighted a requirement for a panel of straightforward and sensitive, but cost-effective, Eimeria species-specific diagnostic assays. Results Loop-mediated isothermal amplification (LAMP) is an uncomplicated, quick and relatively inexpensive diagnostic tool. In this study we have developed a panel of species-specific LAMP assays targeting the seven Eimeria species that infect the chicken. Each assay has been shown to be genuinely species-specific with the capacity to detect between one and ten eimerian genomes, equivalent to less than a single mature schizont. Development of a simple protocol for template DNA preparation from tissue collected post mortem with no requirement for specialist laboratory equipment supports the use of these assays in routine diagnosis of eimerian infection. Preliminary field testing supports this hypothesis. Conclusions Development of a panel of sensitive species-specific LAMP assays introduces a valuable new cost-effective tool for use in poultry husbandry. PMID:22053893

2011-01-01

172

Development of a multiplex loop-mediated isothermal amplification assay to detect shiga toxin-producing Escherichia coli in cattle.  

PubMed

A multiplex loop-mediated isothermal amplification (mLAMP) assay was developed for simultaneous detection of the stx1 and stx2 genes and applied for detection of shiga toxin-producing Escherichia coli (STEC) in cattle farm samples. Two target genes were distinguished based on Tm values of 85.03 ± 0.54°C for stx1 and 87.47 ± 0.35°C for stx2. The mLAMP assay was specific (100% inclusivity and exclusivity), sensitive (with a detection limit as low as 10 fg/?L), and quantifiable (R² = 0.9313). The efficacy and sensitivity were measured to evaluate applicability of the mLAMP assay to cattle farm samples. A total of 12 (12/253; 4.7%) and 17 (17/253; 6.7%) STEC O157, and 11 (11/236; 4.7%) non-O157 STEC strains were isolated from cattle farm samples by conventional selective culture, immunomagnetic separation, and PCR-based culture methods, respectively. The coinciding multiplex PCR and mLAMP results for the types of shiga toxin revealed the value of the mLAMP assay in terms of accuracy and rapidity for characterizing shiga toxin genes. Furthermore, the high detection rate of specific genes from enrichment broth samples indicates the potential utility of this assay as a screening method for detecting STEC in cattle farm samples. PMID:24675834

Dong, Hee-Jin; Cho, Ae-Ri; Hahn, Tae-Wook; Cho, Seongbeom

2014-01-01

173

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

PubMed

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. PMID:20300675

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

2010-04-01

174

Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for the detection of the fathead minnow nidovirus.  

PubMed

Fathead minnow nidovirus (FHMNV) is a serious baitfish-pathogenic virus in North America. Studies to trace the spread of the virus and determine its host range are hampered by the absence of reliable diagnostic assays. In this study, a one-step, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed that targets a region in the FHMNV spike protein gene. The assay was optimized, and the best results were obtained at 8 mM of Mg(2+) with an incubation time of 40 min at 63 °C in the presence of calcein. The analytical sensitivity of the RT-LAMP method was estimated to be as low as 5 viral copies and was 1000-fold more sensitive than the conventional reverse transcription polymerase chain reaction (RT-PCR) method. The diagnostic sensitivity and specificity of the developed RT-LAMP assay versus the RT-PCR assay was 100% and 95.7%, respectively. A quantitative RT-LAMP of FHMNV with a high correlation coefficient (r(2)=0.9926) was also developed and the result of quantitation of viral copies in tissue samples of infected fish showed that the viral loads of the infected fish tissue samples reached up to 4.7×10(10) copies per mg. It is anticipated that the developed RT-LAMP and quantitative RT-LAMP methods will be instrumental for diagnosis and surveillance of FHMNV. PMID:24594288

Zhang, Qingli; Standish, Isaac; Winters, Andrew D; Puzach, Corey; Ulferts, Rachel; Ziebuhr, John; Faisal, Mohamed

2014-06-01

175

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

PubMed

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

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

2013-12-01

176

Isothermal and rapid detection of pathogenic microorganisms using a nano-rolling circle amplification-surface plasmon resonance biosensor.  

PubMed

Rolling circle amplification (RCA) of DNA is a sensitive and cost effective method for the rapid identification of pathogens without the need for sequencing. In this study, a surface plasmon resonance DNA biosensor based on RCA with a gold (Au) nanoparticle surface was established for isothermal identification of DNA. The probes included a specific padlock probe, a capture probe (CP), which is bound to biotin, and an Au nanoparticle-modified probe, which hybridizes with the RCA products. The CP was assembled on gold nanoparticles to increase its ability to bind and hybridize. The linear padlock probe, which was designed to circularize by ligation upon recognition of the bacterial pathogen-specific sequence in 16S rDNA, hybridizes to fully complementary sequences within the CP. Upon recognition, each target gene DNA is distinguished by localization onto the corresponding channel on the chip surface. Then, the immobilized CPs act as primers to begin the in situ solid-phase RCA reaction, which produces long single-stranded DNA. The RCA products fixed on the chip surface cause significant surface plasmon resonance angle changes. We demonstrated that six different bacterial pathogens can be identified simultaneously and that 0.5 pM of synthetic oligonucleotides and 0.5 pg ?l(-1) of genomic DNA from clinical samples can be detected by this method with low background signals. Therefore, the multiplex diagnostic method provides a highly sensitive and specific approach for the rapid identification of positive samples. PMID:25022511

Shi, Dachuan; Huang, Junfu; Chuai, Zhengran; Chen, Dong; Zhu, Xiaoyan; Wang, Huan; Peng, Jia; Wu, Haiyan; Huang, Qing; Fu, Weiling

2014-12-15

177

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

PubMed

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. PMID:24477385

Biswas, Gouranga; Sakai, Masahiro

2014-04-01

178

Rapid detection of duck hepatitis A virus genotype C using reverse transcription loop-mediated isothermal amplification.  

PubMed

A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was used and optimized to develop a rapid and sensitive detection system for duck hepatitis A virus genotype C (DHAV-C) RNA. A set of four specific primers was designed against highly conserved sequences located within the 3D gene from DHAV (strain GX1201). Under optimal reaction conditions, the sensitivity of DHAV-C-specific RT-LAMP was 100-fold higher than that of reverse transcriptase-polymerase chain reaction (RT-PCR), with a detection limit of 0.3pg (6.59×10(4) copies) per reaction. No cross-reactivity was observed from the samples of other duck viruses, which is in good accordance with RT-PCR. Furthermore, a positive reaction can be visually inspected by observing turbidity or color change after the addition of SYBR green I dye. The DHAV-C-specific RT-LAMP assay was applied to the samples and compared with RT-PCR. The positive-sample ratios were 26.7% (12 of 45) by RT-LAMP and 20% (9 of 45) by RT-PCR. Therefore, the newly developed RT-LAMP assay is a rapid, specific, sensitive, and cost-effective method of DHAV-C detection. This assay has potential applications in both clinical diagnosis and field surveillance of DHAV-C infection. PMID:24291148

Li, Chuanfeng; Chen, Zongyan; Meng, Chunchun; Liu, Guangqing

2014-02-01

179

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

PubMed

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

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

180

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

PubMed

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. PMID:24057244

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

2013-11-01

181

Real-time reverse-transcription loop-mediated isothermal amplification for rapid detection of rift valley Fever virus.  

PubMed

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 degrees 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 approximately 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

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

2008-11-01

182

Multiplex loop-mediated isothermal amplification detection by sequence-based barcodes coupled with nicking endonuclease-mediated pyrosequencing.  

PubMed

The loop-mediated isothermal amplification (LAMP) is a well-developed method for replicating a targeted DNA sequence with a high specificity, but multiplex LAMP detection is difficult because LAMP amplicons are very complicated in structure. To allow simultaneous detection of multiple LAMP products, a series of target-specific barcodes were designed and tagged in LAMP amplicons by FIP primers. The targeted barcodes were decoded by pyrosequencing on nicked LAMP amplicons. To enable the nicking reaction to occur just near the barcode regions, the recognition sequence of the nicking endonuclease (NEase) was also introduced into the FIP primer. After the nicking reaction, pyrosequencing started at the nicked 3' end when the added deoxyribonucleoside triphosphate (dNTP) was complementary to the non-nicked strand. To efficiently encode multiple targets, the barcodes were designed with a reporter base and two stuffer bases, so that the decoding of a target-specific barcode only required a single peak in a pyrogram. We have successfully detected the four kinds of pathogens including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and Treponema pallidum (TP), which are easily infected in blood, by a 4-plex LAMP in a single tube, indicating that barcoded LAMP coupled with NEase-mediated pyrosequencing is a simple, rapid, and reliable way in multiple target identification. PMID:22449174

Liang, Chao; Chu, Yanan; Cheng, Sijia; Wu, Haiping; Kajiyama, Tomoharu; Kambara, Hideki; Zhou, Guohua

2012-04-17

183

Rapid detection of peste des petits ruminants virus by a reverse transcription loop-mediated isothermal amplification assay.  

PubMed

Peste des petits ruminants virus (PPRV) is the causative agent of peste des petits ruminants (PPR), an economically important viral disease of small ruminants. In this report, a one-step, single-tube, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of PPRV. A set of six LAMP primers were designed based on the matrix gene sequence of PPRV to amplify the target RNA by incubation at 63°C for 60min with Bst DNA polymerase and reverse transcriptase. The amplified products could be observed by the naked eye. The specificity of the RT-LAMP assay was validated by amplifying eight strains of PPRV isolated in different geographical areas. No cross-reactivity with other related viruses, including rinderpest virus, canine distemper virus and measles virus, was detected. The sensitivity of the assay was similar to that of real-time reverse transcription polymerase chain reaction (RT-PCR) and 10-fold higher than that of conventional RT-PCR. Twenty clinical samples were evaluated by the RT-LAMP assay, and the results were consistent with those of real-time RT-PCR. As a simple, rapid and accurate detection method, this RT-LAMP assay has important potential applications in the clinical diagnosis of PPR and the surveillance of PPRV. PMID:20813134

Li, Lin; Bao, Jingyue; Wu, Xiaodong; Wang, Zhiliang; Wang, Junwei; Gong, Mingxia; Liu, Chunju; Li, Jinming

2010-12-01

184

Development of a single-tube loop-mediated isothermal amplification assay for detection of four pathogens of bacterial meningitis.  

PubMed

Several loop-mediated isothermal amplification (LAMP) assays have been developed to detect common causative pathogens of bacterial meningitis (BM). However, no LAMP assay is reported to detect Streptococcus agalactiae and Streptococcus suis, which are also among common pathogens of BM. Moreover, it is laborious and expensive by performing multiple reactions for each sample to detect bacterial pathogen. Thus, we aimed to design and develop a single-tube LAMP assay capable of detecting multiple bacterial species, based on the nucleotide sequences of the 16S rRNA genes of the bacteria. The nucleotide sequences of the 16S rRNA genes of main pathogens involved in BM were aligned to identify conserved regions, which were further used to design broad range specific LAMP assay primers. We successfully designed a set of broad range specific LAMP assay primers for simultaneous detection of four species including Staphylococcus aureus, Streptococcus pneumoniae, S. suis and S. agalactiae. The broad range LAMP assay was highly specific without cross-reactivity with other bacteria including Haemophilus influenzae, Neisseria meningitidis and Escherichia coli. The sensitivity of our LAMP assay was 100-1000 times higher compared with the conventional PCR assay. The bacterial species could be identified after digestion of the LAMP products with restriction endonuclease DdeI and HaeIII. PMID:22946506

Huy, Nguyen Tien; Hang, Le Thi Thuy; Boamah, Daniel; Lan, Nguyen Thi Phuong; Van Thanh, Phan; Watanabe, Kiwao; Huong, Vu Thi Thu; Kikuchi, Mihoko; Ariyoshi, Koya; Morita, Kouichi; Hirayama, Kenji

2012-12-01

185

Direct detection of Mycobacterium avium in environmental water and scale samples by loop-mediated isothermal amplification.  

PubMed

We previously demonstrated the colonization of Mycobacterium avium complex in bathrooms by the conventional culture method. In the present study, we aimed to directly detect M. avium organisms in the environment using loop-mediated isothermal amplification (LAMP), and to demonstrate the efficacy of LAMP by comparing the results with those obtained by culture. Our data showed that LAMP analysis has detection limits of 100 fg DNA/reaction for M. avium. Using an FTA(®) elute card, DNA templates were extracted from environmental samples from bathrooms in the residences of 29 patients with pulmonary M. avium disease. Of the 162 environmental samples examined, 143 (88%) showed identical results by both methods; 20 (12%) and 123 (76%) samples were positive and negative, respectively, for M. avium. Of the remaining 19 samples (12%), seven (5%) and 12 (7%) samples were positive by the LAMP and culture methods, respectively. All samples that contained over 20 colony forming units/primary isolation plate, as measured by the culture method, were also positive by the LAMP method. Our data demonstrate that the combination of the FTA elute card and LAMP can facilitate prompt detection of M. avium in the environment. PMID:24937215

Nishiuchi, Yukiko; Tamaru, Aki; Suzuki, Yasuhiko; Kitada, Seigo; Maekura, Ryoji; Tateishi, Yoshitaka; Niki, Mamiko; Ogura, Hisashi; Matsumoto, Sohkichi

2014-06-01

186

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

PubMed Central

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

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

2012-01-01

187

Rapid quantitative detection of Human immunodeficiency virus type 1 by a reverse transcription-loop-mediated isothermal amplification assay.  

PubMed

Accurate and rapid quantitation of Human immunodeficiency virus type 1 (HIV-1) RNA levels is a critical aspect in estimating the effect of antiviral therapy and establishing therapeutic schedule. Thus, for the first time, a rapid quantitative reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was designed to quantitate HIV-1 RNA. The results showed that the dynamic range was from 2.5×10(2) to 10(7) copies with a coefficient of determination (R(2)) of 0.991, and the limit of detection of RT-LAMP by Probit analysis at the 95% detection level was 196 copies. The intra-assay coefficient of variation (CV) ranged from 0.67% to 2.08% at 10(7) copies and 7.25% to 12.97% at 250 copies. The CVs of inter-assay were 2.39% and 13.93% for the high and low copy numbers, respectively. No cross-reaction with Human immunodeficiency virus type 2 (HIV-2), Human T lymphotrophic virus type 1 (HTLV-1) and Hepatitis C virus (HCV) was observed and a good agreement between the RT-LAMP method and the real-time reverse transcription-polymerase chain reaction (qRT-PCR) test was achieved. This proposed RT-LAMP method could be useful for rapid diagnosis of high risk group and pharmacodynamic assessment of anti-HIV drug, especially in less-equipped laboratories of impoverished areas. PMID:24630968

Zeng, Yalan; Zhang, Xiaoguang; Nie, Kai; Ding, Xiong; Ring, Brian Z; Xu, Lanying; Dai, Lei; Li, Xiying; Ren, Wei; Shi, Lei; Ma, Xuejun

2014-05-15

188

Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid and Sensitive Identification of Ostrich Meat  

PubMed Central

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

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

2014-01-01

189

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

PubMed

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

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

2014-12-01

190

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

Over the past few years, there has been an increasing demand for rapid and simple diagnostic tools that can be applied outside centralized laboratories by using transportable devices. In veterinary medicine, such mobile test systems would circumvent barriers associated with the transportation of samples and significantly reduce the time to diagnose important infectious animal diseases. Among a wide range of available technologies, high-speed real-time reverse transcriptase quantitative PCR (RT-qPCR) and the two isothermal amplification techniques loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) represent three promising candidates for integration into mobile pen-side tests. The aim of this study was to investigate the performance of these amplification strategies and to evaluate their suitability for field application. In order to enable a valid comparison, novel pathogen-specific assays have been developed for the detection of Schmallenberg virus and bovine viral diarrhea virus. The newly developed assays were evaluated in comparison with established standard RT-qPCR using samples from experimentally or field-infected animals. Even though all assays allowed detection of the target virus in less than 30 min, major differences were revealed concerning sensitivity, specificity, robustness, testing time, and complexity of assay design. These findings indicated that the success of an assay will depend on the integrated amplification technology. Therefore, the application-specific pros and cons of each method that were identified during this study provide very valuable insights for future development and optimization of pen-side tests. PMID:24648561

Aebischer, Andrea; Wernike, Kerstin; Hoffmann, Bernd; Beer, Martin

2014-06-01

191

A reverse-transcription, loop-mediated isothermal amplification assay for detection of bovine ephemeral fever virus in the blood of infected cattle  

Microsoft Academic Search

A novel reverse-transcription, loop-mediated isothermal amplification (RT-LAMP) assay for the detection of bovine ephemeral fever virus (BEFV) was developed and evaluated in this study. The RT-LAMP assay exhibited higher sensitivity when compared with conventional reverse-transcription polymerase chain reaction (RT-PCR) and virus isolation methods. The specificity of the assay was determined by digestion of the RT-LAMP products with restriction enzyme and

Fuying Zheng; Guozhen Lin; Jizhang Zhou; Guanghua Wang; Xiaoan Cao; Xiaowei Gong; Changqing Qiu

2011-01-01

192

Loop-mediated isothermal amplification assay for 16S rRNA methylase genes in Gram-negative bacteria.  

PubMed

Using the loop-mediated isothermal amplification (LAMP) method, we developed a rapid assay for detection of 16S rRNA methylase genes (rmtA, rmtB, and armA), and investigated 16S rRNA methylase-producing strains among clinical isolates. Primer Explorer V3 software was used to design the LAMP primers. LAMP primers were prepared for each gene, including two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB). Detection was performed with the Loopamp DNA amplification kit. For all three genes (rmtA, rmtB, and armA), 10(2) copies/tube could be detected with a reaction time of 60 min. When nine bacterial species (65 strains saved in National Institute of Infectious Diseases) were tested, which had been confirmed to possess rmtA, rmtB, or armA by PCR and DNA sequencing, the genes were detected correctly in these bacteria with no false negative or false positive results. Among 8447 clinical isolates isolated at 36 medical institutions, the LAMP method was conducted for 191 strains that were resistant to aminoglycosides based on the results of antimicrobial susceptibility tests. Eight strains were found to produce 16S rRNA methylase (0.09%), with rmtB being identified in three strains (0.06%) of 4929 isolates of Enterobacteriaceae, rmtA in three strains (0.10%) of 3284 isolates of Pseudomonas aeruginosa, and armA in two strains (0.85%) of 234 isolates of Acinetobacter spp. At present, the incidence of strains possessing 16S rRNA methylase genes is very low in Japan. However, when Gram-negative bacteria showing high resistance to aminoglycosides are isolated by clinical laboratories, it seems very important to investigate the status of 16S rRNA methylase gene-harboring bacilli and monitor their trends among Japanese clinical settings. PMID:25179393

Nagasawa, Mitsuaki; Kaku, Mitsuo; Kamachi, Kazunari; Shibayama, Keigo; Arakawa, Yoshichika; Yamaguchi, Keizo; Ishii, Yoshikazu

2014-10-01

193

Development of a Loop-Mediated Isothermal Amplification Assay for Rapid, Sensitive Detection of Campylobacter jejuni in Cattle Farm Samples.  

PubMed

Campylobacter jejuni is a leading cause of bacterial foodborne disease worldwide. The detection of this organism in cattle and their environment is important for the control of C. jejuni transmission and the prevention of campylobacteriosis. Here, we describe the development of a rapid and sensitive method for the detection of C. jejuni in naturally contaminated cattle farm samples, based on real-time loop-mediated isothermal amplification (LAMP) of the hipO gene. The LAMP assay was specific (100% inclusivity and exclusivity for 84 C. jejuni and 41 non-C. jejuni strains, respectively), sensitive (detection limit of 100 fg/?l), and quantifiable (R(2) = 0.9133). The sensitivity of the LAMP assay was then evaluated for its application to the naturally contaminated cattle farm samples. C. jejuni strains were isolated from 51 (20.7%) of 246 cattle farm samples, and the presence of the hipO gene was tested using the LAMP assay. Amplification of the hipO gene by LAMP within 30 min (mean ~10.8 min) in all C. jejuni isolates (n = 51) demonstrated its rapidity and accuracy. Next, template DNA was prepared from a total of 186 enrichment broth cultures of cattle farm samples either by boiling or using a commercial kit, and the sensitivity of detection of C. jejuni was compared between the LAMP and PCR assays. In DNA samples prepared by boiling, the higher sensitivity of the LAMP assay (84.4%) compared with the PCR assay (35.5%) indicates that it is less susceptible to the existence of inhibitors in sample material. In DNA samples prepared using a commercial kit, both the LAMP and PCR assays showed 100% sensitivity. We anticipate that the use of this rapid, sensitive, and simple LAMP assay, which is the first of its kind for the identification and screening of C. jejuni in cattle farm samples, may play an important role in the prevention of C. jejuni contamination in the food chain, thereby reducing the risk of human campylobacteriosis. PMID:25198853

Dong, Hee-Jin; Cho, Ae-Ri; Hahn, Tae-Wook; Cho, Seongbeom

2014-09-01

194

Visual detection of the human metapneumovirus using reverse transcription loop-mediated isothermal amplification with hydroxynaphthol blue dye  

PubMed Central

Background Human metapneumovirus (hMPV) is a major cause of acute respiratory infections ranging from wheezing to bronchiolitis and pneumonia in children worldwide. The objective of this study is to develop a visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of hMPV and applied to the clinical samples. Results In this study, visual RT-LAMP assay for hMPV was performed in one step with the addition of hydroxynaphthol blue (HNB), and were used to detect respiratory samples. Six primers, including two outer primers (F3 and B3), two inner primers (FIP, BIP) and two loop primers (LF and LB), were designed for hMPV N gene by the online software. Moreover, the RT-LAMP assay showed good specificity and no cross-reactivity was observed with human rhinovirus (HRV), human respiratory syncytial Virus (RSV), or influenza virus A/PR/8/34 (H1N1). The detection limit of the RT-LAMP assay was approximately ten viral RNA copies, lower than that of traditional reverse transcriptase polymerase chain reaction (RT-PCR) 100 RNA copies. In the 176 nasopharyngeal samples, 23 (13.1%) were conformed as hMPV positive by RT-LAMP, but 18 (10.2%) positive by RT-PCR. Conclusion Compared with conventional RT-PCR, the visual hMPV RT-LAMP assay performed well in the aspect of detect time, sensitivity, specificity and visibility. It is anticipated that the RT-LAMP will be used for clinical tests in hospital or field testing during outbreaks and in emergency. PMID:22838725

2012-01-01

195

Discriminating between varicella-zoster virus vaccine and wild-type strains by loop-mediated isothermal amplification.  

PubMed

The loop-mediated isothermal amplification (LAMP) method was developed to distinguish between the varicella-zoster virus (VZV) vaccine (vOka) strain and wild-type strains. Two single nucleotide polymorphisms (SNPs) (nucleotide [nt] 105705 for VR-1 VZV LAMP and nt 106262 for VR-2 VZV LAMP) located in the open reading frame 62 gene were selected as LAMP targets. Amplified vOka DNA demonstrated a typical ladder pattern; however, no LAMP product was detected in reactions performed with DNAs from other human herpesviruses by either VR-1 VZV LAMP or VR-2 VZV LAMP. This result was confirmed by a turbidity assay. The sensitivities of both VR-1 and VR-2 VZV LAMP determined by either the turbidity assay or agarose gel electrophoresis were 100 copies per reaction. To discriminate the vOka strain from wild-type strains, VR-1 and VR-2 VZV LAMP products were digested with the appropriate restriction enzymes (SacII for VR-1 LAMP and SmaI for VR-2 LAMP). The digested products were clearly different in the vOka strain and wild-type strains. To evaluate the utility of the LAMP methods for rapid differentiation, viral DNA (without DNA extraction) in swab samples was directly tested. Wild-type VZV DNA was detected in 20 swab samples by either VR-1 VZV LAMP or VR-2 VZV LAMP. Sequence analysis confirmed the expected SNPs in the LAMP products amplified from the vOka strain and the five wild-type strains. PMID:18550736

Higashimoto, Yuki; Ihira, Masaru; Ohta, Akane; Inoue, Shigeki; Usui, Chie; Asano, Yoshizo; Yoshikawa, Tetsushi

2008-08-01

196

Development of a rapid assay to detect the dinoflagellate Amyloodinium ocellatum using loop-mediated isothermal amplification (LAMP).  

PubMed

Amyloodinium ocellatum is a highly pathogenic dinoflagellate parasite with global distribution that causes high mortalities in the culture of tropical and sub-tropical marine and estuarine fishes. Diagnosis typically occurs through gross examination following the onset of morbidity, at which point treatment is of limited benefit. In the present study, a new molecular diagnostic tool for the rapid detection of A. ocellatum (AO) was developed using the loop-mediated isothermal amplification method (LAMP). The AO-LAMP assay designed is highly specific using a set of four primers - two outer and two inner primers targeting six different regions on the 5' end of the Small Subunit rDNA region (SSU rDNA) of A. ocellatum. The AO-LAMP assay, optimized for 25-30 min at 62°C, amplified the DNA from A. ocellatum extracted from both water and gill tissue samples and did not amplify DNA from four closely related dinoflagellate sp ecies. The detection limit of the AO-LAMP assay was 10 fg, exceptionally higher than the conventional PCR (1 pg). In addition, the standardized AO-LAMP assay was capable of detecting single tomonts and trophonts; the assay was not affected by the presence of possible inhibitory substances present in environmental water samples or gill samples. The AO-LAMP assay developed in the present study provides a novel useful tool for the simple, rapid and sensitive detection of A. ocellatum in water and gill tissue samples, which could assist in the early detection and improved control of A. ocellatum infections in aquaculture systems. PMID:23726415

Picón-Camacho, Sara M; Thompson, William P; Blaylock, Reginald B; Lotz, Jeffrey M

2013-09-23

197

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

PubMed Central

Background Malaria that is caused by Plasmodium vivax is the most widely distributed human malaria. Its recent resurgence in many parts of the world, including the Republic of Korea (ROK), emphasizes the importance of improved access to the early and accurate detection of P. vivax to reduce disease burden. In this study, a rapid and efficient loop-mediated isothermal amplification (LAMP)-based method was developed and validated using blood samples from malaria-suspected patients. Method A LAMP assay targeting the ?-tubulin gene for the detection of P. vivax was developed with six primers that recognize different regions of the target gene. The diagnostic performance of the ?-tubulin LAMP assay was compared to three other tests: microscopic examinations, rapid diagnostic tests (RDTs), and nested polymerase chain reactions (PCRs) using 177 whole blood specimens obtained from ROK military personnel from May to December 2011. Results The ?-tubulin LAMP assay was highly sensitive with a detection limit of 100 copies of P. vivax ?-tubulin gene per reaction within 50 min. It specifically amplified the target gene only from P. vivax. Validation of the ?-tubulin LAMP assay showed that the assay had the highest sensitivity (P?

2014-01-01

198

Rapid and sensitive detection of infectious spleen and kidney necrosis virus by loop-mediated isothermal amplification combined with a lateral flow dipstick.  

PubMed

Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acids under isothermal conditions. In this report, a 20-min LAMP amplification of the DPOL gene of infectious spleen and kidney necrosis virus (ISKNV) using a biotin-labeled primer was combined with lateral flow dipstick (LFD) chromatography for rapid and simple visual detection of ISKNV-specific amplicons. The LFD process involves a 5-min specific hybridization with an FITC-labeled DNA probe to confirm the presence of complement ISKNV amplicons that were biotinated in LAMP. The resulting DNA duplexes, consisting of labeled probes and amplicons, migrate along the LFD strip by chromatography for 5 min and are trapped at the test line and visualized by biotin labeling. The detection limit of ISKNV by LAMP-LFD was 10 copies. The results show that the LAMP-LFD method has the advantages of better sensitivity and speed and less dependence on equipment than the standard PCR for specifically detecting low levels of ISKNV DNA, and this can be useful in the field as a routine diagnostic tool. PMID:20107846

Ding, W C; Chen, Jiong; Shi, Y H; Lu, X J; Li, M Y

2010-03-01

199

Development of a reverse transcriptase loop-mediated isothermal amplification (LAMP) assay for the sensitive detection of Leishmania parasites in clinical samples.  

PubMed

Here we describe a generic, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) assay, for the identification of Leishmania species from clinical samples. LAMP is an isothermal reaction recently developed as a point-of-care diagnostic tool. Primers were designed in the conserved region of the 18S ribosomal RNA (rRNA) gene; amplification was visualized by the pre-amplification addition of fluorescent detection reagent (FDR) and a simple UV lamp. By using a reverse-transcriptase step, the system detected infections between 10 and 100 parasites per mL. The assay was tested on a range of nucleic acid extracts from Leishmania species, visceral leishmaniasis (VL) patients from Sudan, and cutaneous leishmaniasis (CL) patients from Suriname. The sensitivity of RT-LAMP from the blood of VL patients was 83% (N = 30) compared with microscopy of bone-marrow and lymph-node aspirates; for CL patients the observed sensitivity was 98% (N = 43). The potential to use LAMP as a diagnostic tool for leishmaniasis is discussed. PMID:20348505

Adams, Emily R; Schoone, Gerard J; Ageed, Al Farazdag; Safi, Sayda El; Schallig, Henk D F H

2010-04-01

200

A polymer microfluidic chip for quantitative detection of multiple water- and foodborne pathogens using real-time fluorogenic loop-mediated isothermal amplification.  

PubMed

Inexpensive, portable, and easy-to-use devices for rapid detection of microbial pathogens are needed to ensure safety of water and food. In this study, a disposable polymer microfluidic chip for quantitative detection of multiple pathogens using isothermal nucleic acid amplification was developed. The chip contains an array of 15 interconnected reaction wells with dehydrated primers for loop-mediated isothermal amplification (LAMP), and requires only a single pipetting step for dispensing of sample. To improve robustness of loading and amplification, hydrophobic air vents and microvalves were monolithically integrated in the multi-layered structure of the chip using an inexpensive knife plotter. For quantification, LAMP was performed with a highly fluorescent DNA binding dye (SYTO-82) and the reactions monitored in real-time using a low-cost fluorescence imaging system previously developed by our group (Ahmad et al., Biomed. Microdevices 13(5), 929-937). Starting from genomic DNA mixtures, the chip was successfully evaluated for rapid analysis of multiple virulence and marker genes of Salmonella, Campylobacter jejuni, Shigella, and Vibrio cholerae, enabling detection and quantification of 10-100 genomes per ?l in less than 20 min. It is anticipated that the microfluidic chip, along with the real-time imaging system, may be a key enabling technology for developing inexpensive and portable systems for on-site screening of multiple pathogens relevant to food and water safety. PMID:22566273

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

2012-08-01

201

Optimisation of reverse transcriptase loop-mediated isothermal amplification assay for rapid detection of Macrobrachium rosenbergii noda virus and extra small virus in Macrobrachium rosenbergii.  

PubMed

The standardisation and optimisation of a one step single tube reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) procedure is described for rapid diagnosis of white tail disease, a viral disease caused by Macrobrachium rosenbergii noda virus (MrNV) and extra small virus (XSV), in giant fresh water prawn, M. rosenbergii. Time, temperature and quantity of each reagent were optimised for the detection of the two viruses. This method was more sensitive than the conventional reverse transcriptase polymerase chain reaction (RT-PCR) for detecting the two viruses. The RT-LAMP reaction is highly suited for disease diagnosis in developing countries. Amplification of DNA can be detected without the use of agarose gel electrophoresis, by the production of a whitish precipitate of magnesium pyrophosphate as a by-product. The cost of RT-LAMP for one reaction is nearly 4 times less than that of RT-PCR. PMID:20307575

Haridas, Divya V; Pillai, Devika; Manojkumar, B; Nair, C Mohanakumaran; Sherief, P M

2010-07-01

202

Ultrasensitive Detection of Low-Abundance Surface-Marker Protein using Isothermal Rolling Circle Amplification in Microfluidic Nano-Liter Platform  

PubMed Central

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, we have applied a highly sensitive labeling method that translates antigen-antibody recognition processes into DNA detection event that can be greatly amplified via isothermal Rolling Circle Amplification (RCA). By merging the single-molecule detection power of RCA reaction with microfluidic technology we were able to demonstrate that identification of specific protein markers can be achieved on tumor cell surface in miniaturized nano-liter 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. PMID:21294269

Konry, Tania; Yarmush, Joel M.; Irimia, Daniel

2011-01-01

203

Rapid and Simple Method for Detecting the Toxin B Gene of Clostridium difficile in Stool Specimens by Loop-Mediated Isothermal Amplification  

Microsoft Academic Search

We applied the loop-mediated isothermal amplification (LAMP) assay to the detection of the toxin B gene (tcdB )o fClostridium difficile for identification of toxin B (TcdB)-positive C. difficile strains and detection of tcdB in stool specimens. tcdB was detected in all toxin A (TcdA)-positive, TcdB-positive (AB) and TcdA-negative, TcdB-positive (AB) C. difficile strains but not from TcdA-negative, TcdB-negative strains. Of

Haru Kato; Toshiyuki Yokoyama; Hideaki Kato; Yoshichika Arakawa

2005-01-01

204

Development of a sensitive loop-mediated isothermal amplification assay that provides specimen-to-result diagnosis of respiratory syncytial virus infection in 30 minutes.  

PubMed

Rapid isothermal amplification methods have recently been introduced, and some of these methods offer significant advantages over PCR. The objective of this study was to develop a rapid and sensitive multiplex loop-mediated isothermal amplification (M-LAMP) assay for the detection of respiratory syncytial virus subgroups A and B (RSV A and B). We designed six primers each for the matrix gene of RSV A and the polymerase gene of RSV B and developed an M-LAMP assay by using a commercially available master mix and a real-time fluorometer (Genie II; Optigene, United Kingdom) that displays real-time amplification, time to positivity, and amplicon annealing temperature (Tm). The M-LAMP was evaluated against PCR by testing 275 nasopharyngeal (NP) specimens. The final optimized M-LAMP assay had a mean amplification time of 14.2 min (compared with 90 to 120 min for PCR) and had an analytical sensitivity of 1 genome equivalent (ge) for both RSV A and B. Using PCR as a comparator, M-LAMP had a sensitivity of 100% (81/81) and specificity of 100% (194/194). We also evaluated a 3- to 10-min specimen processing method involving vortexing with glass beads and heating to 98°C in M-swab medium (Copan Italia, Brescia, Italy) and found that this rapid processing method allowed detection of 37/41 (90.2%) of positives when we used extracted nucleic acid. In summary, the M-LAMP assay had excellent sensitivity and specificity for detecting RSV A and B in NP specimens and, when coupled with a rapid specimen preparation method, could provide a specimen-to-result diagnosis time of 30 min. PMID:23761156

Mahony, James; Chong, Sylvia; Bulir, David; Ruyter, Alexandra; Mwawasi, Ken; Waltho, Daniel

2013-08-01

205

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

Quickness, simplicity, and effectiveness are the three major criteria for establishing a good molecular diagnosis method in many fields. Herein we report a novel detection system for genetically modified organisms (GMOs), which can be utilized to perform both on-field quick screening and routine laboratory diagnosis. In this system, a newly designed inexpensive DNA extraction device was used in combination with a modified visual loop-mediated isothermal amplification (vLAMP) assay. The main parts of the DNA extraction device included a silica gel membrane filtration column and a modified syringe. The DNA extraction device could be easily operated without using other laboratory instruments, making it applicable to an on-field GMO test. High-quality genomic DNA (gDNA) suitable for polymerase chain reaction (PCR) and isothermal amplification could be quickly isolated from plant tissues using this device within 15 min. In the modified vLAMP assay, a microcrystalline wax encapsulated detection bead containing SYBR green fluorescent dye was introduced to avoid dye inhibition and cross-contaminations from post-LAMP operation. The system was successfully applied and validated in screening and identification of GM rice, soybean, and maize samples collected from both field testing and the Grain Inspection, Packers, and Stockyards Administration (GIPSA) proficiency test program, which demonstrated that it was well-adapted to both on-field testing and/or routine laboratory analysis of GMOs. PMID:23181490

Zhang, Miao; Liu, Yinan; Chen, Lili; Quan, Sheng; Jiang, Shimeng; Zhang, Dabing; Yang, Litao

2013-01-01

206

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

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. PMID:24300833

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

2014-03-01

207

Development of an in-house loop-mediated isothermal amplification (LAMP) assay for detection of Mycobacterium tuberculosis and evaluation in sputum samples of Nepalese patients.  

PubMed

A number of nucleic acid amplification assays (NAAs) have been employed to detect tubercle bacilli in clinical specimens for tuberculosis (TB) diagnosis. Among these, loop-mediated isothermal amplification (LAMP) is an NAA possessing superior isothermal reaction characteristics. In the present study, a set of six specific primers targeting the Mycobacterium tuberculosis 16S rRNA gene with high sensitivity was selected and a LAMP system (MTB-LAMP) was developed. Using this system, a total of 200 sputum samples from Nepalese patients were investigated. The sensitivity of MTB-LAMP in culture-positive samples was 100 % (96/96), and the specificity in culture-negative samples was 94.2 % (98/104, 95 % confidence interval 90.5-97.9 %). The positive and negative predictive values of MTB-LAMP were 94.1 and 100 %, respectively. These results indicate that this MTB-LAMP method may prove to be a powerful tool for the early diagnosis of TB. PMID:18349362

Pandey, Basu Dev; Poudel, Ajay; Yoda, Tomoko; Tamaru, Aki; Oda, Naozumi; Fukushima, Yukari; Lekhak, Binod; Risal, Basista; Acharya, Bishnu; Sapkota, Bishwa; Nakajima, Chie; Taniguchi, Tooru; Phetsuksiri, Benjawan; Suzuki, Yasuhiko

2008-04-01

208

Development of a fluorescent-intercalating-dye-based reverse transcription loop-mediated isothermal amplification assay for rapid detection of seasonal Japanese B encephalitis outbreaks in pigs.  

PubMed

The standardization and validation of a one-step, single-tube, accelerated fluorescent-intercalating-dye-based reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the NS3 gene of Japanese B encephalitis virus (JEV) is described for rapid, simple, and high-throughput detection of JEV. The amplification can be completed in 35 min under isothermal conditions at 63°C by employing a set of six primers targeting the NS3 gene of JEV. The RT-LAMP assay described demonstrated high sensitivity for detecting JEV, with a detection limit in swine samples of 8.13 PFU/ml. The specificity of the selected primer sets was established by cross-reactivity studies with pathogens that exhibit similar clinical signs and testing of samples from healthy animals. The clinical applicability of the RT-LAMP assay was validated using either spiked samples or samples from seasonal outbreaks. The comparative evaluation of the RT-LAMP assay revealed 79.59 % concordance with conventional RT-PCR targeting the E gene of JEV. The RT-LAMP assay reported here is a valuable tool for rapid real-time and high-throughput seasonal infection surveillance and quarantine after outbreak through blood sampling by using ordinary real-time PCR thermocyclers without purchasing an expensive Loopamp real-time turbidimeter. PMID:22573187

Tian, C J; Lin, Z X; He, X M; Luo, Q; Luo, C B; Yu, H Q; Chen, R; Wu, X W; Zhu, D Z; Ren, Z J; Bi, Y Z; Ji, J

2012-08-01

209

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

PubMed

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. PMID:23219929

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

2013-03-01

210

Loop-mediated isothermal amplification of DNA (LAMP): a new diagnostic tool lights the world of diagnosis of animal and human pathogens: a review.  

PubMed

Diagnosis is an important part in case of animal husbandry as treatment of a disease depends on it. Advancement in molecular biology has generated various sophisticated tools like Polymerase Chain Reaction (PCR), its versions along with pen-side diagnostic techniques. Every diagnostic test however has both advantages and disadvantages; PCR is not an exception to this statement. To ease the odds faced by PCR several non-PCR techniques which can amplify DNA at a constant temperature has become the need of hour, thus generating a variety of isothermal amplification techniques including Nucleic Acid Sequence-Based Amplification (NASBA) along with Self-Sustained Sequence Replication (3SR) and Strand Displacement Amplification (SDA) and Loop mediated isothermal amplification (LAMP) test. LAMP stands out to be a good and effective diagnostic test for empowering in developing countries as it does not require sophisticated equipments and skilled personnel and proves to be cost-effective. Performance of LAMP mainly relies on crafting of six primers (including 2 loop primers) ultimately accelerating the reaction. LAMP amplifies DNA in the process pyrophosphates are formed causing turbidity that facilitates visualisation in a more effective way than PCR. The Bst and Bsm polymerase are the required enzymes for LAMP that does not possess 5'-3' exonuclease activity. Results can be visualized by adding DNA binding dye, SYBR green. LAMP is more stable than PCR and real-time PCR. Non-involvement of template DNA preparation and ability to generate 10(9) copies of DNA are added benefits that make it more effective than NASBA or 3SR and SDA. Thus, it fetches researcher's interest in developing various versions of LAMP viz., its combination with lateral flow assay or micro LAMP and more recently lyophilized and electric (e) LAMP. Availability of ready to use LAMP kits has helped diagnosis of almost all pathogens. LAMP associated technologies however needs to be developed as a part of LAMP platform rather than developing them as separate entities. This review deals with all these salient features of this newly developed tool that has enlightened the world of diagnosis. PMID:24783797

Dhama, Kuldeep; Karthik, K; Chakraborty, Sandip; Tiwari, Ruchi; Kapoor, Sanjay; Kumar, Amit; Thomas, Prasad

2014-01-15

211

Rapid and sensitive detection of Laem-Singh virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick.  

PubMed

Laem-Singh virus (LSNV) was discovered recently in Thailand in farmed Giant Tiger shrimp (Penaeus monodon) displaying signs of slow growth syndrome. Loop-mediated isothermal amplification (LAMP) allows DNA to be amplified rapidly at a constant temperature. Here a reverse transcription (RT)-LAMP method was combined with a chromatographic lateral-flow dipstick (LFD) to detect LSNV RNA rapidly and specifically. The reaction was optimized at 65°C for 30 min and amplified DNA hybridized to an FITC-labeled oligonucleotide probe for 5 min was detected at LFD test line 5 min after application. Including 10 min for rapid RNA extraction, test results could be generated within 1h and did not require electrophoresis. Compared to an existing RT-PCR method, the RT-LAMP-LFD was also ?1000-fold more sensitive in detecting LSNV RNA. PMID:21762729

Arunrut, Narong; Seetang-Nun, Yortyot; Phromjai, Jurairat; Panphut, Wattana; Kiatpathomchai, Wansika

2011-10-01

212

Bench-scale experiments for the development of a unified loop-mediated isothermal amplification (LAMP) assay for the in vitro diagnosis of Leishmania species' promastigotes.  

PubMed

We developed, in bench-scale experiments, a unified loop-mediated isothermal amplification (LAMP) assay for the detection of cutaneous, mucocutaneous and visceral leishmaniasis using DNA of cultivated promastigotes. Two primer sets for the LAMP assay were designed based on the 18S rRNA gene, and their sensitivity and specificity were tested and compared. Both of them were specific for Leishmania as the DNA of all ten Leishmania species tested was amplified, whereas the DNA of other parasites, including that of Trypanosoma, was not. The detection limit for primer set 1 ranged between 30 pg and 3·6 fg, depending on which Leishmania species tested. Primer set 2 showed high sensitivity, but was less sensitive than primer set 1. Our findings lead to the conclusion that the LAMP assay with primer set 1 is a promising and effective assay for the successful detection of a wide range of Leishmania infections using only a unified multiplex LAMP test. PMID:24168822

Karani, M; Sotiriadou, I; Plutzer, J; Karanis, P

2014-08-01

213

A reverse-transcription, loop-mediated isothermal amplification assay for detection of bovine ephemeral fever virus in the blood of infected cattle.  

PubMed

A novel reverse-transcription, loop-mediated isothermal amplification (RT-LAMP) assay for the detection of bovine ephemeral fever virus (BEFV) was developed and evaluated in this study. The RT-LAMP assay exhibited higher sensitivity when compared with conventional reverse-transcription polymerase chain reaction (RT-PCR) and virus isolation methods. The specificity of the assay was determined by digestion of the RT-LAMP products with restriction enzyme and detection of BEFV serogroup rabies virus (RV). Using RT-LAMP, RT-PCR and virus isolation methods, 36 blood samples were tested and the results indicated that RT-LAMP could detect early infection with BEFV. The RT-LAMP method is useful for the diagnosis of BEFV infection in blood samples. PMID:21093487

Zheng, Fuying; Lin, Guozhen; Zhou, Jizhang; Wang, Guanghua; Cao, Xiaoan; Gong, Xiaowei; Qiu, Changqing

2011-01-01

214

Comparison of a TaqMan real-time polymerase chain reaction assay with a loop-mediated isothermal amplification assay for detection of Gallid herpesvirus 1.  

PubMed

A TaqMan real-time polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) assay were developed to detect Gallid herpesvirus 1 (GaHV-1, formerly Infectious laryngotracheitis virus). The standard curve of real-time PCR was established, and the sensitivity reached 10 copies/?l. In the current study, the conversion between viral titer and GaHV-1 genomic copy number was constructed. Six primers for LAMP assay amplified target gene at 65°C within 45 min, and the detection limit was 60 copies/?l. The 6 primers were highly specific, sensitive, and reproducible for detection of GaHV-1. Although the sensitivity of LAMP was lower than that of real-time PCR, LAMP was faster, less expensive, and did not require a thermocycler. The LAMP assay would be a viable alternative assay in diagnostic laboratories that do not employ real-time PCR technology. PMID:22362944

Ou, Shan-Chia; Giambrone, Joseph J; Macklin, Kenneth S

2012-01-01

215

Specific detection of the toxic dinoflagellates Alexandrium tamarense and Alexandrium catenella from single vegetative cells by a loop-mediated isothermal amplification method.  

PubMed

In this study, we succeeded in developing a loop-mediated isothermal amplification (LAMP) method that enables sensitive and specific detection of the toxic marine dinoflagellates Alexandrium tamarense and Alexandrium catenella from single cells of both laboratory cultures and naturally blooming cells within 25 min, by monitoring the turbidimeter from the start of the LAMP reaction. The fluorescence intensity was strong enough to allow discrimination between positive and negative results by naked eye under a UV lamp, even in amplified samples from a single cell, by using the LAMP method. Unambiguous detection by naked eye was possible even in half the volume of LAMP cocktail recommended by the manufacturer, suggesting the potential to significantly reduce the cost of Alexandrium monitoring. Therefore, we can conclude that this method is one of the most convenient, sensitive, and cost-effective molecular tools for Alexandrium monitoring. PMID:22897962

Nagai, Satoshi; Itakura, Shigeru

2012-09-01

216

Detection of early and single infections of Schistosoma japonicum in the intermediate host snail, Oncomelania hupensis, by PCR and loop-mediated isothermal amplification (LAMP) assay.  

PubMed

Polymerase chain reaction (PCR) with the specific primer set amplifying 28S ribosomal DNA (rDNA) of Schistosoma japonicum was able to detect genomic DNA of S. japonicum, but not S. mansoni, at 100 fg. This procedure enabled us to detect the DNA from a single miracidium and a snail infected with one miracidium at just 1 day after infection. We compared these results with those from loop-mediated isothermal amplification (LAMP) targeting 28S rDNA and found similar results. The LAMP could amplify the specific DNA from a group of 100 normal snails mixed with one infected snail A PCR screening of infected snails from endemic regions in Anhui Province revealed schistosomal DNA even in snails found negative by microscopy. PCR and LAMP show promise for monitoring the early infection rate in snails, and they may be useful for predicting the risk of infection in the endemic places. PMID:20810818

Kumagai, Takashi; Furushima-Shimogawara, Rieko; Ohmae, Hiroshi; Wang, Tian-Ping; Lu, Shaohong; Chen, Rui; Wen, Liyong; Ohta, Nobuo

2010-09-01

217

Detection of Coconut cadang-cadang viroid (CCCVd) in oil palm by reverse transcription loop-mediated isothermal amplification (RT-LAMP).  

PubMed

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) detected Coconut cadang-cadang viroid (CCCVd) within 60 min at 60 °C in total nucleic acid extracted from oil palm leaves infected with CCCVd. Positive reactions showed colour change from orange to green in the reaction mix after the addition of fluorescent reagent, and a laddering pattern band on 2% agarose gel electrophoresis. Conventional RT-PCR with LAMP primers produced amplicons with a sequence identical to the 297-nt CCCVd oil palm variant with the primers being specific for CCCVd and not for other viroids such as PSTVd and CEVd. RT-LAMP was found to be rapid and specific for detecting oil palm CCCVd. PMID:24631346

Thanarajoo, Sathis Sri; Kong, Lih Ling; Kadir, Jugah; Lau, Wei Hongi; Vadamalai, Ganesan

2014-06-01

218

Detection of spring viraemia of carp virus (SVCV) by loop-mediated isothermal amplification (LAMP) in koi carp, Cyprinus carpio L  

USGS Publications Warehouse

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.

Shivappa, R. B.; Savan, R.; Kono, T.; Sakai, M.; Emmenegger, E.; Kurath, G.; Levine, J. F.

2008-01-01

219

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

PubMed

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. PMID:20444968

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

2010-07-01

220

Rapid and sensitive detection of infectious bursal disease virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick.  

PubMed

Infectious bursal disease (IBD), an immunosuppressive disease that affects all ages of chickens, results in significant losses in the poultry industry. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with a chromatographic lateral flow dipstick (LFD) for the detection of infectious bursal disease virus (IBDV) was developed. The whole process of testing can be completed in less than 70 min using biotin-labeled primers, an FITC-labeled DNA probe, and the LFD. The detection limits for IBDV using RT-LAMP and RT-LAMP-LFD were the same at 10(-1)plaque forming units (PFU). When other unrelated viruses and cells were tested, no false positive results were observed. In addition, the amplification efficiency of RT-LAMP was enhanced when a loop primer was used. The RT-LAMP-LFD product started to be detected after 40 min. Clinical samples were used to compare assays using RT-PCR, nested RT-PCR, RT-LAMP, and RT-LAMP-LFD and the positive rates were 16%, 40%, 40%, and 40%, respectively. In conclusion, this assay is an easy, rapid, accurate, and sensitive method for the detection of IBDV and will improve the screening of field samples, especially when veterinarians have limited resources. PMID:22353472

Tsai, Su-Ming; Liu, Hung-Jen; Shien, Jui-Hung; Lee, Long-Huw; Chang, Po-Chung; Wang, Chi-Young

2012-04-01

221

Development of a Reverse Transcription Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Subtype H7N9 Avian Influenza Virus  

PubMed Central

A novel influenza A (H7N9) virus has emerged in China. To rapidly detect this virus from clinical samples, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of the H7N9 virus. The minimum detection limit of the RT-LAMP assay was 0.01?PFU H7N9 virus, making this method 100-fold more sensitive to the detection of the H7N9 virus than conventional RT-PCR. The H7N9 virus RT-LAMP assays can efficiently detect different sources of H7N9 influenza virus RNA (from chickens, pigeons, the environment, and humans). No cross-reactive amplification with the RNA of other subtype influenza viruses or of other avian respiratory viruses was observed. The assays can effectively detect H7N9 influenza virus RNA in drinking water, soil, cloacal swab, and tracheal swab samples that were collected from live poultry markets, as well as human H7N9 virus, in less than 30?min. These results suggest that the H7N9 virus RT-LAMP assays were efficient, practical, and rapid diagnostic methods for the epidemiological surveillance and diagnosis of influenza A (H7N9) virus from different resource samples. PMID:24689044

Bao, Hongmei; Zhao, Yuhui; Wang, Yunhe; Xu, Xiaolong; Shi, Jianzhong; Zeng, Xianying; Wang, Xiurong; Chen, Hualan

2014-01-01

222

Use of Loop-Mediated Isothermal Amplification for Detection of Ophiostoma clavatum, the Primary Blue Stain Fungus Associated with Ips acuminatus  

PubMed Central

Loop-mediated isothermal amplification (LAMP) is an alternative amplification technology which is highly sensitive and less time-consuming than conventional PCR-based methods. Three LAMP assays were developed, two for detection of species of symbiotic blue stain fungi associated with Ips acuminatus, a bark beetle infesting Scots pine (Pinus sylvestris), and an additional assay specific to I. acuminatus itself for use as a control. In common with most bark beetles, I. acuminatus is associated with phytopathogenic blue stain fungi involved in the process of exhausting tree defenses, which is a necessary step for the colonization of the plant by the insect. However, the identity of the main blue stain fungus vectored by I. acuminatus was still uncertain, as well as its frequency of association with I. acuminatus under outbreak and non-outbreak conditions. In this study, we employed LAMP technology to survey six populations of I. acuminatus sampled from the Southern Alps. Ophiostoma clavatum was detected at all sampling sites, while Ophiostoma brunneo-ciliatum, reported in part of the literature as the main blue stain fungus associated with I. acuminatus, was not detected on any of the samples. These results are consistent with the hypothesis that O. clavatum is the main blue stain fungus associated with I. acuminatus in the Southern Alps. The method developed in the course of this work provides a molecular tool by which it will be easy to screen populations and derive important data regarding the ecology of the species involved. PMID:23396326

Tomlinson, Jennifer A.; Battisti, Andrea; Boonham, Neil; Capretti, Paolo

2013-01-01

223

Rapid diagnostic detection of plum pox virus in Prunus plants by isothermal AmplifyRP(®) using reverse transcription-recombinase polymerase amplification.  

PubMed

Plum pox virus (PPV) causes the most destructive viral disease known as plum pox or Sharka disease in stone fruit trees. As an important regulated pathogen, detection of PPV is thus of critical importance to quarantine and eradication of the spreading disease. In this study, the innovative development of two AmplifyRP(®) tests is reported for a rapid isothermal detection of PPV using reverse transcription-recombinase polymerase amplification. In an AmplifyRP(®) test, all specific recombination and amplification reactions occur at a constant temperature without thermal cycling and the test results are either recorded in real-time with a portable fluorescence reader or displayed using a lateral flow strip contained inside an amplicon detection chamber. The major improvement of this assay is that the entire test from sample preparation to result can be completed in as little as 20min and can be performed easily both in laboratories and in the field. The results from this study demonstrated the ability of the AmplifyRP(®) technique to detect all nine PPV strains (An, C, CR, D, EA, M, Rec, T, or W). Among the economic benefits to pathogen surveys is the higher sensitivity of the AmplifyRP(®) to detect PPV when compared to the conventional ELISA and ImmunoStrip(®) assays. This is the first report describing the use of such an innovative technique to detect rapidly plant viruses affecting perennial crops. PMID:25010790

Zhang, Shulu; Ravelonandro, Michel; Russell, Paul; McOwen, Nathan; Briard, Pascal; Bohannon, Seven; Vrient, Albert

2014-10-01

224

Loop-mediated isothermal amplification of vanA gene enables a rapid and naked-eye detection of vancomycin-resistant enterococci infection.  

PubMed

Vancomycin-resistant enterococci (VRE) are one of the leading causes of nosocomial infection at intensive care unit (ICU). A rapid and sensitive detection of VRE infection is in high demand for timely and suitable antibiotic treatment. Here, we optimized a distinct DNA-based diagnostic technique, loop-mediated isothermal amplification (LAMP) for a rapid detection of the presence of vanA gene, a critical component of the gene cluster required for vancomycin resistance. Amplification efficiency was optimal at 62°C and with 2mM MgSO4. The detection limit of the DNA template was 80pg and LAMP amplicons were detected within 40min; thereby suggesting a potential applicability of LAMP as a sensitive and urgent diagnostic method. Furthermore, positive LAMP reaction was directly detected with the naked-eye by monitoring the formation of a white precipitate or the color change induced by hydroxy naphthol blue (HNB) dye. Finally, 56 clinical isolates were successfully tested for the presence of vanA gene by LAMP, which was determined to be more sensitive than PCR. Together, our results clearly demonstrate the usefulness of LAMP for the diagnosis of VRE infection. PMID:24925601

Kim, Hye Jin; Kim, Yu Jin; Yong, Dong Eun; Lee, Kyungwon; Park, Jeon Han; Lee, Jae Myun; Yoon, Sang Sun

2014-09-01

225

Development and application of loop-mediated isothermal amplification for detection of the F167Y mutation of carbendazim-resistant isolates in Fusarium graminearum.  

PubMed

Resistance of Fusarium graminearum to carbendazim is caused by point mutations in the ?2-tubulin gene. The point mutation at codon 167 (TTT ? TAT, F167Y) occurs in more than 90% of field resistant isolates in China. To establish a suitable method for rapid detection of the F167Y mutation in F. graminearum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed and optimized to specially distinguish the F167Y mutation genotype. The LAMP reaction was optimal at 63°C for 60?min. When hydroxynaphthol blue dye (HNB) was added prior to amplification, samples with DNA of the F167Y mutation developed a characteristic sky blue color after the reaction but those without DNA or with different DNA did not. Results of HNB staining method were reconfirmed by gel electrophoresis. The developed LAMP had good specificity, stability and repeatability and was suitable for monitoring carbendazim-resistance populations of F. graminearum in agricultural production. PMID:25403277

Duan, Yabing; Zhang, Xiaoke; Ge, Changyan; Wang, Yong; Cao, Junhong; Jia, Xiaojing; Wang, Jianxin; Zhou, Mingguo

2014-01-01

226

Development and application of loop-mediated isothermal amplification for detection of the F167Y mutation of carbendazim-resistant isolates in Fusarium graminearum  

PubMed Central

Resistance of Fusarium graminearum to carbendazim is caused by point mutations in the ?2-tubulin gene. The point mutation at codon 167 (TTT ? TAT, F167Y) occurs in more than 90% of field resistant isolates in China. To establish a suitable method for rapid detection of the F167Y mutation in F. graminearum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed and optimized to specially distinguish the F167Y mutation genotype. The LAMP reaction was optimal at 63°C for 60?min. When hydroxynaphthol blue dye (HNB) was added prior to amplification, samples with DNA of the F167Y mutation developed a characteristic sky blue color after the reaction but those without DNA or with different DNA did not. Results of HNB staining method were reconfirmed by gel electrophoresis. The developed LAMP had good specificity, stability and repeatability and was suitable for monitoring carbendazim-resistance populations of F. graminearum in agricultural production. PMID:25403277

Duan, Yabing; Zhang, Xiaoke; Ge, Changyan; Wang, Yong; Cao, Junhong; Jia, Xiaojing; Wang, Jianxin; Zhou, Mingguo

2014-01-01

227

Use of loop-mediated isothermal amplification for detection of Ophiostoma clavatum, the primary blue stain fungus associated with Ips acuminatus.  

PubMed

Loop-mediated isothermal amplification (LAMP) is an alternative amplification technology which is highly sensitive and less time-consuming than conventional PCR-based methods. Three LAMP assays were developed, two for detection of species of symbiotic blue stain fungi associated with Ips acuminatus, a bark beetle infesting Scots pine (Pinus sylvestris), and an additional assay specific to I. acuminatus itself for use as a control. In common with most bark beetles, I. acuminatus is associated with phytopathogenic blue stain fungi involved in the process of exhausting tree defenses, which is a necessary step for the colonization of the plant by the insect. However, the identity of the main blue stain fungus vectored by I. acuminatus was still uncertain, as well as its frequency of association with I. acuminatus under outbreak and non-outbreak conditions. In this study, we employed LAMP technology to survey six populations of I. acuminatus sampled from the Southern Alps. Ophiostoma clavatum was detected at all sampling sites, while Ophiostoma brunneo-ciliatum, reported in part of the literature as the main blue stain fungus associated with I. acuminatus, was not detected on any of the samples. These results are consistent with the hypothesis that O. clavatum is the main blue stain fungus associated with I. acuminatus in the Southern Alps. The method developed in the course of this work provides a molecular tool by which it will be easy to screen populations and derive important data regarding the ecology of the species involved. PMID:23396326

Villari, Caterina; Tomlinson, Jennifer A; Battisti, Andrea; Boonham, Neil; Capretti, Paolo; Faccoli, Massimo

2013-04-01

228

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

At present, the sporadic occurrence of human rabies in Brazil can be attributed primarily to dog- and vampire bat-related rabies viruses. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was employed as a simultaneous detection method for both rabies field variants within 60 min. Vampire bat-related rabies viruses could be distinguished from dog variants by digesting amplicons of the RT-LAMP reaction using the restriction enzyme AlwI. Amplification and digestion could both be completed within 120 min after RNA extraction. In addition, the RT-LAMP assay also detected rabies virus in isolates from Brazilian frugivorous bats and Ugandan dog, bovine and goat samples. In contrast, there were false negative results from several Brazilian insectivorous bats and all of Chinese dog, pig, and bovine samples using the RT-LAMP assay. This study showed that the RT-LAMP assay is effective for the rapid detection of rabies virus isolates from the primary reservoir in Brazil. Further improvements are necessary so that the RT-LAMP assay can be employed for the universal detection of genetic variants of rabies virus in the field. PMID:20403387

Saitou, Yasumasa; Kobayashi, Yuki; Hirano, Shinji; Mochizuki, Nobuyuki; Itou, Takuya; Ito, Fumio H; Sakai, Takeo

2010-09-01

229

Comparative study of sensitivity, linearity, and resistance to inhibition of digital and nondigital polymerase chain reaction and loop mediated isothermal amplification assays for quantification of human cytomegalovirus.  

PubMed

Performing nucleic acid amplification techniques (NAATs) in digital format using limiting dilution provides potential advantages that have recently been demonstrated with digital polymerase chain reaction (dPCR). Key benefits that have been claimed are the ability to quantify nucleic acids without the need of an external calibrator and a greater resistance to inhibitors than real-time quantitative PCR (qPCR). In this study, we evaluated the performance of four NAATs, qPCR, dPCR, real-time quantitative loop mediated isothermal amplification (qLAMP), and digital LAMP (dLAMP), for the detection and quantification of human cytomegalovirus (hCMV). We used various DNA templates and inhibitors to compare the performance of these methods using a conventional real-time thermocycler platform (Bio-Rad CFX96) and a chip based digital platform (Fluidigm Biomark 12.765 Digital Array). dPCR performed well and demonstrated greater resistance to inhibitors than the other methods although this resistance did not apply equally to all inhibitors tested. dLAMP was found to be less sensitive than dPCR, but its quantitative performance was better than qLAMP, the latter being unable to quantify below 1000 copies. dLAMP was also more resistant to inhibitors than qLAMP. Unlike qPCR, both digital methods were able to quantify viral genomes without requiring a calibrator; however, neither can currently compete with the large reaction volumes, and thus the greater absolute sensitivity, of qPCR. With the introduction of digital instrumentation that will enable larger reaction volumes, digital amplification methods such as those evaluated in this study could potentially offer a robust alternative to qPCR for nucleic acid quantification. PMID:24684191

Nixon, Gavin; Garson, Jeremy A; Grant, Paul; Nastouli, Eleni; Foy, Carole A; Huggett, Jim F

2014-05-01

230

Improved detection limit in rapid detection of human enterovirus 71 and coxsackievirus A16 by a novel reverse transcription-isothermal multiple-self-matching-initiated amplification assay.  

PubMed

Rapid detection of human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) is important in the early phase of hand-foot-and-mouth disease (HFMD). In this study, we developed and evaluated a novel reverse transcription-isothermal multiple-self-matching-initiated amplification (RT-IMSA) assay for the rapid detection of EV71 and CVA16 by use of reverse transcriptase, together with a strand displacement DNA polymerase. Real-time RT-IMSA assays using a turbidimeter and visual RT-IMSA assays to detect EV71 and CVA16 were established and completed in 1 h, and the reported corresponding real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assays targeting the same regions of the VP1 gene were adopted as parallel tests. Through testing VP1 RNAs transcribed in vitro, the real-time RT-IMSA assays exhibited better linearity of quantification, with R(2) values of 0.952 (for EV71) and 0.967 (for CVA16), than the real-time RT-LAMP assays, which had R(2) values of 0.803 (for EV71) and 0.904 (for CVA16). Additionally, the detection limits of the real-time RT-IMSA assays (approximately 937 for EV71 and 67 for CVA16 copies/reaction) were higher than those of real-time RT-LAMP assays (approximately 3,266 for EV71 and 430 for CVA16 copies/reaction), and similar results were observed in the visual RT-IMSA assays. The new approaches also possess high specificities for the corresponding targets, with no cross-reactivity observed. In clinical assessment, compared to commercial reverse transcription-quantitative PCR (qRT-PCR) kits, the diagnostic sensitivities of the real-time RT-IMSA assays (96.4% for EV71 and 94.6% for CVA16) were higher than those of the real-time RT-LAMP assays (91.1% for EV71 and 90.8% for CVA16). The visual RT-IMSA assays also exhibited the same results. In conclusion, this proof-of-concept study suggests that the novel RT-IMSA assay is superior to the RT-LAMP assay in terms of detection limit and has the potential to rapidly detect EV71 and CVA16 viruses. PMID:24648558

Ding, Xiong; Nie, Kai; Shi, Lei; Zhang, Yong; Guan, Li; Zhang, Dan; Qi, Shunxiang; Ma, Xuejun

2014-06-01

231

Development and Application of Loop-Mediated Isothermal Amplification Assays for Rapid Visual Detection of cry2Ab and cry3A Genes in Genetically-Modified Crops  

PubMed Central

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

Li, Feiwu; Yan, Wei; Long, Likun; Qi, Xing; Li, Congcong; Zhang, Shihong

2014-01-01

232

Rapid and Simple Method for Detecting the Toxin B Gene of Clostridium difficile in Stool Specimens by Loop-Mediated Isothermal Amplification  

PubMed Central

We applied the loop-mediated isothermal amplification (LAMP) assay to the detection of the toxin B gene (tcdB) of Clostridium difficile for identification of toxin B (TcdB)-positive C. difficile strains and detection of tcdB in stool specimens. tcdB was detected in all toxin A (TcdA)-positive, TcdB-positive (A+B+) and TcdA-negative, TcdB-positive (A?B+) C. difficile strains but not from TcdA-negative, TcdB-negative strains. Of the 74 stool specimens examined, A+B+ or A?B+ C. difficile was recovered from 39 specimens, of which 38 specimens were LAMP positive and one was negative. Amplification was obtained in 10 specimens that were culture negative, indicating that LAMP is highly sensitive. The LAMP assay was applied to detection of tcdB in DNA extracted by a simple boiling method from 47 of those 74 specimens, which were cultured overnight in cooked-meat medium (CMM). Twenty-two of 24 culture-positive specimens were positive for LAMP on DNA from the culture in CMM. Four specimens were culture negative but positive by LAMP on DNA from CMM cultures. The LAMP assay is a reliable tool for identification of TcdB-positive C. difficile as well as for direct detection of tcdB in stool specimens with high sensitivity. Detection of tcdB by LAMP from overnight cultures in CMM could be an alternative method of diagnostic testing at clinical laboratories without special apparatus. PMID:16333105

Kato, Haru; Yokoyama, Toshiyuki; Kato, Hideaki; Arakawa, Yoshichika

2005-01-01

233

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

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 disease surveillance in schistosomiasis-endemic areas. PMID:25187956

Fernandez-Soto, Pedro; Gandasegui Arahuetes, Javier; Sanchez Hernandez, Alicia; Lopez Aban, Julio; Vicente Santiago, Belen; Muro, Antonio

2014-01-01

234

Identification of human DNA in forensic evidence by loop-mediated isothermal amplification combined with a colorimetric gold nanoparticle hybridization probe.  

PubMed

A DNA test based on loop-mediated isothermal amplification (LAMP) and colorimetric gold nanoparticle (AuNP) hybridization probe to detect the presence of human DNA in forensic evidence was developed. The LAMP primer set targeted eight regions of the human cytochrome b, and its specificity was verified against the DNA of 11 animal species, which included animals closely related to humans, such as chimpanzee and orangutan. By using the AuNP probe, sequence-specific LAMP product could be detected and the test result could be visualized through the change in color. The limit of detection was demonstrated with reproducibility to be as low as 718 fg of genomic DNA, which is equivalent to approximately 100 plasmid DNA copies containing the cytochrome b DNA target region. A simple DNA extraction method for the commonly found forensic biological samples was also devised to streamline the test process. This LAMP-AuNP human DNA test showed to be a robust, specific, and cost-effective tool for the forensic identification of human specimens without requiring sophisticated laboratory instruments. PMID:24827529

Watthanapanpituck, Khanistha; Kiatpathomchai, Wansika; Chu, Eric; Panvisavas, Nathinee

2014-11-01

235

The detection of T-Nos, a genetic element present in GMOs, by cross-priming isothermal amplification with real-time fluorescence.  

PubMed

An isothermal cross-priming amplification (CPA) assay for Agrobacterium tumefaciens nopaline synthase terminator (T-Nos) was established and investigated in this work. A set of six specific primers, recognizing eight distinct regions on the T-Nos sequence, was designed. The CPA assay was performed at a constant temperature, 63 °C, and detected by real-time fluorescence. The results indicated that real-time fluorescent CPA had high specificity, and the limit of detection was 1.06?×?10(3) copies of rice genomic DNA, which could be detected in 40 min. Comparison of real-time fluorescent CPA and conventional polymerase chain reaction (PCR) was also performed. Results revealed that real-time fluorescent CPA had a comparable sensitivity to conventional real-time PCR and had taken a shorter time. In addition, different contents of genetically modified (GM)-contaminated rice seed powder samples were detected for practical application. The result showed real-time fluorescent CPA could detect 0.5 % GM-contaminated samples at least, and the whole reaction could be finished in 35 min. Real-time fluorescent CPA is sensitive enough to monitor labeling systems and provides an attractive method for the detection of GMO. PMID:24748469

Zhang, Fang; Wang, Liu; Fan, Kai; Wu, Jian; Ying, Yibin

2014-05-01

236

Rapid and sensitive detection of Vibrio vulnificus by loop-mediated isothermal amplification combined with lateral flow dipstick targeted to rpoS gene.  

PubMed

A novel loop-mediated isothermal amplification (LAMP) combined with amplicon detection by chromatographic lateral flow dipstick (LFD) assay was developed and evaluated for the detection of Vibrio vulnificus. Biotinylated LAMP amplicons were produced by a set of six designed primers that recognized the V. vulnificus RNA polymerase subunit sigma factor S (rpoS) gene followed by hybridization with an FITC-labeled probe and LFD detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65 °C. The LAMP-LFD method accurately identified 14 isolates of V. vulnificus but did not detect 25 non-vulnificus Vibrio isolates and 37 non-Vibrio isolates. The sensitivity of LAMP-LFD for V. vulnificus detection in pure culture was 1.5 × 10(3) CFU ml(-1) or equivalent to 2.8 CFU per reaction. In the case of spiked oyster samples without enrichment, the detection limit for V. vulnificus was 1.2 × 10(4) CFU g(-1) or equivalent to 11 CFU per reaction. The results show that this method appears to be accurate, precise and valuable tool for identification of V. vulnificus and can be used efficiently for detection of V. vulnificus in contaminated food sample. PMID:21513793

Surasilp, Thanai; Longyant, Siwaporn; Rukpratanporn, Sombat; Sridulyakul, Pattarin; Sithigorngul, Paisarn; Chaivisuthangkura, Parin

2011-08-01

237

Rapid and sensitive detection of type II porcine reproductive and respiratory syndrome virus by reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip.  

PubMed

Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was combined with a vertical flow (VF) nucleic acid detection strip to develop a universal assay for the detection of type II porcine reproductive and respiratory syndrome virus (PRRSV). The loop primers were labeled separately with biotin and fluorescein isothiocyanate (FITC) in this assay. Using optimized parameters, the whole reaction could be completed in <50min in a completely enclosed environment. The detection limit of this assay was found to be 1pg RNA, 30 tissue culture infective dose 50 (TCID50) virus, or 230 copies of recombinant plasmid DNA, which is relatively higher than that of RT-LAMP analyzed by agarose gel, RT-LAMP visualized by calcein, and the conventional RT-polymerase chain reaction (PCR). No false-positive results were obtained in the specificity assay. The efficiency of the RT-LAMP method was tested by analyzing 43 clinical samples, and the results were compared with those obtained by RT-PCR analysis, with the respective positive rates of 32.56% and 27.91%. This result confirmed that the method described is a rapid, accurate, and sensitive method for universal type II PRRSV detection. Also, this method can be used for the rapid detection of type II PRRSV during the early phase of an outbreak, especially for rapid veterinary diagnosis on the spot and in rural areas. PMID:25241142

Gou, Hongchao; Deng, Jieru; Pei, Jingjing; Wang, Jiaying; Liu, Wenjun; Zhao, Mingqiu; Chen, Jinding

2014-12-01

238

Loop-Mediated Isothermal Amplification Assay for Detection of Generic and Verocytotoxin-Producing Escherichia coli among Indigenous Individuals in Malaysia  

PubMed Central

We have successfully developed a Loop-mediated isothermal amplification (LAMP) assay that could specifically detect generic Escherichia coli (E. coli). This assay was tested on 85 bacterial strains and successfully identified 54 E. coli strains (average threshold time, Tt = 21.26). The sensitivity of this assay was evaluated on serial dilutions of bacterial cultures and spiked faeces. The assay could detect 102?CFU/mL for bacterial culture with Tt = 33.30 while the detection limit for spiked faeces was 103?CFU/mL (Tt = 31.12). We have also detected 46 generic E. coli from 50 faecal samples obtained from indigenous individuals with 16% of the positive samples being verocytotoxin-producing E. coli (VTEC) positive. VT1/VT2 allele was present in one faecal sample while the ratio of VT1 to VT2 was 6?:?1. Overall, our study had demonstrated high risk of VTEC infection among the indigenous community and most of the asymptomatic infection occurred among those aged below 15 years. The role of asymptomatic human carriers as a source of dissemination should not be underestimated. Large scale screening of the VTEC infection among indigenous populations and the potential contamination sources will be possible and easy with the aid of this newly developed rapid and simple LAMP assay. PMID:24967435

Teh, Cindy Shuan Ju; Chua, Kek Heng; Lim, Yvonne Ai Lian; Lee, Soo Ching; Thong, Kwai Lin

2014-01-01

239

Visual Detection of High-Risk Human Papillomavirus Genotypes 16, 18, 45, 52, and 58 by Loop-Mediated Isothermal Amplification with Hydroxynaphthol Blue Dye?  

PubMed Central

A simple, rapid, sensitive, qualitative, colorimetric loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue dye (HNB) was established to detect high-risk human papillomavirus (HPV) genotypes 16, 18, 45, 52, and 58. All initial validation studies with the control DNA proved to be type specific. The colorimetric type-specific LAMP assay could achieve a sensitivity of 10 to 100 copies at 63°C for 65 min, comparable to that of real-time PCR. In order to evaluate the reliability of HPV type-specific LAMP, the assay was further evaluated with HPV DNAs from a panel of 294 clinical specimens whose HPV status was previously determined with a novel one-step typing method with multiplex PCR. The tested panel comprised 108 HPV DNA-negative samples and 186 HPV-DNA-positive samples of 14 genotypes. The results showed that the sensitivity of HPV type-specific LAMP for HPV types 16, 18, 45, 52, and 58 was 100%, 100%, 100%, 100%, and 100%, respectively, and the specificity was 100%, 98.5%, 100%, 98.8%, and 99.2%, respectively, compared with a novel one-step typing method with multiplex PCR. No cross-reactivity with other HPV genotypes was observed. In conclusion, this qualitative and colorimetric LAMP assay has potential usefulness for the rapid screening of HPV genotype 16, 18, 45, 52, and 58 infections, especially in resource-limited hospitals or rural clinics of provincial and municipal regions in China. PMID:21865423

Luo, Le; Nie, Kai; Yang, Meng-Jie; Wang, Miao; Li, Jin; Zhang, Chen; Liu, Hong-Tu; Ma, Xue-Jun

2011-01-01

240

Highly Sensitive Detection of Malaria Parasitemia in a Malaria-Endemic Setting: Performance of a New Loop-Mediated Isothermal Amplification Kit in a Remote Clinic in Uganda  

PubMed Central

Background.?Current malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections. Molecular methods such as polymerase chain reaction (PCR) are highly sensitive but remain too complex for field deployment. A new commercial molecular assay based on loop-mediated isothermal amplification (LAMP) was assessed for field use. Methods.?Malaria LAMP (Eiken Chemical, Japan) was evaluated for samples from 272 outpatients at a rural Ugandan clinic and compared with expert microscopy, nested PCR, and quantitative PCR (qPCR). Two technicians performed the assay after 3 days of training, using 2 alternative blood sample–preparation methods and visual interpretation of results by fluorescence assay. Results.?Compared with 3-well nested PCR, the sensitivity of both LAMP and single-well nested PCR was 90%; the microscopy sensitivity was 51%. For samples with a Plasmodium falciparum qPCR titer of ?2 parasites/µL, LAMP sensitivity was 97.8% (95% confidence interval, 93.7%–99.5%). Most false-negative LAMP results involved samples with parasitemia levels detectable by 3-well nested PCR but very low or undetectable by qPCR. Conclusions.?Malaria LAMP in a remote Ugandan clinic achieved sensitivity similar to that of single-well nested PCR in a United Kingdom reference laboratory. LAMP dramatically lowers the detection threshold achievable in malaria-endemic settings, providing a new tool for diagnosis, surveillance, and screening in elimination strategies. PMID:23633405

Hopkins, Heidi; Gonzalez, Iveth J.; Polley, Spencer D.; Angutoko, Patrick; Ategeka, John; Asiimwe, Caroline; Agaba, Bosco; Kyabayinze, Daniel J.; Sutherland, Colin J.; Perkins, Mark D.; Bell, David

2013-01-01

241

Development and Evaluation of a Simple Assay for Marburg Virus Detection Using a Reverse Transcription-Loop-Mediated Isothermal Amplification Method?  

PubMed Central

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 102 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 104 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. PMID:20421440

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

2010-01-01

242

Evaluation of reverse transcription loop-mediated isothermal amplification in conjunction with ELISA-hybridization assay for molecular detection of Mycobacterium tuberculosis.  

PubMed

Traditional culture, followed by a panel of biochemical tests for the diagnosis of tuberculosis (TB), is time-consuming, and rapid identification of Mycobacterium tuberculosis is crucial for the early administration of appropriate therapy. In this study, the reverse transcription loop-mediated isothermal amplification combined with enzyme-linked immunosorbent hybridization (RT-LAMP-ELISA-hybridization) assay has been designed for the rapid detection of 16S rRNA in clinical isolates of M. tuberculosis. This assay reproducibly detected a single copy, as opposed to 2000 copies of MTB 16S rRNA detected by conventional gel electrophoresis. Among the 150 specimens of sputum analysed, RT-LAMP-ELISA-hybridization assay had a sensitivity of 94.1% in the culture method, compared to the Amplified M. tuberculosis Direct Test (AMTD), 91.1% and the 88.2% sensitivity of acid-fast staining. Furthermore, RT-LAMP-ELISA-hybridization assay is more cost-effective when compared to the real-time TaqMan RT-PCR and AMTD assays. In conclusion, our results suggest that the RT-LAMP-ELISA-hybridization assay is a highly sensitive, low cost diagnostic tool useful for the rapid and accurate direct diagnosis of sputum specimens, and is suitable for routine clinical use. PMID:19022304

Lee, Mei-Feng; Chen, Yen-Hsu; Peng, Chien-Fang

2009-02-01

243

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

PubMed

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. PMID:21158640

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

2010-12-01

244

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

PubMed

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 duplex LAMP assay in parallel analyses. The LAMP assay showed positive results for all samples with a DON concentration exceeding 163ppb. The major advantage of the duplex LAMP assay is that the presence of six of the major trichothecene producing Fusarium spp. can be detected in a rapid and user-friendly manner with only one single assay. To our knowledge this is the first report of the use of a multiplex LAMP assay for fungal organisms. PMID:24631635

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

2014-05-01

245

Rapid and Sensitive Detection of H7N9 Avian Influenza Virus by Use of Reverse Transcription-Loop-Mediated Isothermal Amplification  

PubMed Central

An epidemic of human H7N9 influenza virus infection recently emerged in China whose clinical features include high mortality and which has also resulted in serious economic loss. The novel reassortant avian-origin influenza A (H7N9) virus which was the causative agent of this epidemic raised the possibility of triggering a large-scale influenza pandemic worldwide. It seemed likely that fast molecular detection assays specific for this virus would be in great demand. Here, we report a one-step reverse transcription–loop-mediated isothermal amplification (RT-LAMP) method for rapid detection of the hemagglutinin (HA) and neuraminidase (NA) genes of H7N9 virus, the minimum detection limit of which was evaluated using in vitro RNA transcription templates. In total, 135 samples from clinical specimens (from either patients or poultry) were tested using this method in comparison with the real-time PCR recommended by the World Health Organization (WHO). Our results showed that (i) RT-LAMP-based trials can be completed in approximately 12 to 23 min and (ii) the detection limit for the H7 gene is around 10 copies per reaction, similar to that of the real-time PCR, whereas the detection limit for its counterpart the N9 gene is 5 copies per reaction, a 100-fold-higher sensitivity than the WHO-recommended method. Indeed, this excellent performance of our method was also validated by the results for a series of clinical specimens. Therefore, we believe that the simple, fast, and sensitive method of RT-LAMP might be widely applied for detection of H7N9 infections and may play a role in prevention of an influenza pandemic. PMID:24006004

Zhang, Jinhai; Feng, Youjun; Hu, Dan; Lv, Heng; Zhu, Jing; Cao, Min; Zheng, Feng; Zhu, Jin; Gong, Xiufang; Hao, Lina; Srinivas, Swaminath; Ren, Hao; Qi, Zhongtian

2013-01-01

246

Loop-Mediated Isothermal Amplification for Rickettsia typhi (the Causal Agent of Murine Typhus): Problems with Diagnosis at the Limit of Detection  

PubMed Central

Murine typhus is a flea-borne disease of worldwide distribution caused by Rickettsia typhi. Although treatment with tetracycline antibiotics is effective, treatment is often misguided or delayed due to diagnostic difficulties. As the gold standard immunofluorescence assay is imperfect, we aimed to develop and evaluate a loop-mediated isothermal amplification (LAMP) assay. LAMP assays have the potential to fulfill the WHO ASSURED criteria (affordable, sensitive, specific, user friendly, robust and rapid, equipment free, deliverable to those who need them) for diagnostic methodologies, as they can detect pathogen-derived nucleic acid with low technical expenditure. The LAMP assay was developed using samples of bacterial isolates (n = 41), buffy coat specimens from R. typhi PCR-positive Lao patients (n = 42), and diverse negative controls (n = 47). The method was then evaluated prospectively using consecutive patients with suspected scrub typhus or murine typhus (n = 266). The limit of detection was ?40 DNA copies/LAMP reaction, with an analytical sensitivity of <10 DNA copies/reaction based on isolate dilutions. Despite these low cutoffs, the clinical sensitivity was disappointing, with 48% (95% confidence interval [95% CI], 32.5 to 62.7%) (specificity, 100% [95% CI, 100 to 100%]) in the developmental phase and 33% (95% CI, 9.2 to 56.8%) (specificity, 98.5% [95% CI, 97.0% to 100%]) in the prospective study. This low diagnostic accuracy was attributed to low patient R. typhi bacterial loads (median, 210 DNA copies/ml blood; interquartile range, 130 to 500). PCR-positive but LAMP-negative samples demonstrated significantly lower bacterial loads than LAMP-positive samples. Our findings highlight the diagnostic challenges for diseases with low pathogen burdens and emphasize the need to integrate pathogen biology with improved template production for assay development strategies. PMID:24371248

Dittrich, Sabine; Castonguay-Vanier, Josee; Moore, Catrin E.; Thongyoo, Narongchai; Newton, Paul N.

2014-01-01

247

Loop-Mediated Isothermal Amplification Method Targeting the TTS1 Gene Cluster for Detection of Burkholderia pseudomallei and Diagnosis of Melioidosis?  

PubMed Central

Melioidosis is a severe infection caused by Burkholderia pseudomallei. The timely implementation of effective antimicrobial treatment requires rapid diagnosis. Loop-mediated isothermal amplification (LAMP) targeting the TTS1 gene cluster was developed for the detection of B. pseudomallei. LAMP was sensitive and specific for the laboratory detection of this organism. The lower limit of detection was 38 genomic copies per reaction, and LAMP was positive for 10 clinical B. pseudomallei isolates but negative for 5 B. thailandensis and 5 B. mallei isolates. A clinical evaluation was conducted in northeast Thailand to compare LAMP to an established real-time PCR assay targeting the same TTS1 gene cluster. A total of 846 samples were obtained from 383 patients with suspected melioidosis, 77 of whom were subsequently diagnosed with culture-confirmed melioidosis. Of these 77 patients, a positive result was obtained from one or more specimens by PCR in 26 cases (sensitivity, 34%; 95% confidence interval [CI], 23.4 to 45.4%) and by LAMP in 34 cases (sensitivity, 44%; 95% CI, 32.8 to 55.9%) (P = 0.02). All samples from 306 patients that were culture negative for B. pseudomallei were negative by PCR (specificity, 100%; 95% CI, 98.8 to 100%), but 5 of 306 patients (1.6%) were positive by LAMP (specificity, 98.4%; 95% CI, 96.2 to 99.5%) (P = 0.03). The diagnostic accuracies of PCR and LAMP were 86.7% (95% CI, 82.9 to 89.9%) and 87.5% (95% CI, 83.7 to 90.6%), respectively (P = 0.47). Both assays were very insensitive when applied to blood samples; PCR and LAMP were positive for 0 and 1 of 44 positive blood cultures, respectively. The PCR and LAMP assays evaluated here are not sufficiently sensitive to replace culture in our clinical setting. PMID:18039797

Chantratita, Narisara; Meumann, Ella; Thanwisai, Aunchalee; Limmathurotsakul, Direk; Wuthiekanun, Vanaporn; Wannapasni, Saran; Tumapa, Sarinna; Day, Nicholas P. J.; Peacock, Sharon J.

2008-01-01

248

Visual detection of white spot syndrome virus using DNA-functionalized gold nanoparticles as probes combined with loop-mediated isothermal amplification.  

PubMed

The integration of loop-mediated isothermal amplification (LAMP) and DNA-functionalized AuNPs as visual detection probes (LAMP-AuNPs) was developed and applied for the detection of white spot syndrome virus (WSSV) from Penaeid shrimp in this study. The principle of this combination assay relies on the basis of stability characteristics of the DNA-functionalized AuNPs upon hybridization with the complementary target DNA toward salt-induced aggregation. If the detected target DNA is not complementary to the ssDNA probes, the DNA-functionalized AuNPs will be aggregated due to the screening effect of salt, resulting in the change of solution color from red to blue/gray and shift of the surface plasmon peak to longer wavelength. While the DNA-functionalized AuNPs are perfectly matched to the detected target DNA, the color of solution still remains red in color and no surface plasmon spectral shift. This assay provides simply technique, time-saving and its detection results could be achieved qualitatively and quantitatively by visualization using the naked eye due to the colorimetric change and by measurement using the UV-vis spectroscopy due to the surface plasmon spectral shift, respectively. In this study, LAMP-AuNPs assay was successfully developed with the detection of WSSV-LAMP generated product at 0.03 ?g/reaction, and showed the sensitivity of 2 × 10(2) copies WSSV plasmid DNA, that is comparable to the most sensitive method reported to date. The LAMP-AuNPs assay described in this study revealed a highly sensitive, rapid and reliable diagnostic protocol for detection of WSSV. This technique has a potential as a routine method for assessing the infectious diseases in Penaeid shrimp not only for WSSV, but also for other shrimp pathogens, and can be useful tool in field conditions for the diagnosis or surveillance programs. PMID:23211683

Seetang-Nun, Yortyot; Jaroenram, Wansadaj; Sriurairatana, Siriporn; Suebsing, Rungkarn; Kiatpathomchai, Wansika

2013-04-01

249

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

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 shellfish products. PMID:24988012

Tanaka, Natsuko; Iwade, Yoshito; Yamazaki, Wataru; Gondaira, Fumio; Vuddhakul, Varaporn; Nakaguchi, Yoshitsugu; Nishibuchi, Mitsuaki

2014-07-01

250

An aptamer-based bio-barcode assay with isothermal recombinase polymerase amplification for cytochrome-c detection and anti-cancer drug screening.  

PubMed

Based on a recently reported ultra-sensitive bio-barcode (BBC) assay, we have developed an aptamer-based bio-barcode (ABC) alternative to detect a cell death marker cytochrome-c (Cyto-c) and its subsequent application to screen anti-cancer drugs. Aptamer is a short single-stranded DNA selected from a synthetic DNA library by virtue of its high binding affinity and specificity to its target based on its unique 3D structure from the nucleotide sequence after folding. In the BBC assay, an antigen (Ag) in analytes is captured by a micro-magnetic particle (MMP) coated with capturing antibodies (Abs). Gold nanoparticles (NPs) with another recognition Ab against the same target and hundreds of identical DNA molecules of known sequence are subsequently added to allow the formation of sandwich structures ([MMP-Ab1]-Ag-[Ab2-NP-DNA]). After isolating the sandwiches by a magnetic field, the DNAs hybridized to their complementary DNAs covalently bound on the NPs are released from the sandwiches after heating. Acting as an Ag identification tag, these bio-barcode DNAs with known DNA sequence are then amplified by polymerase chain reaction (PCR) and detected by fluorescence. In our ABC assay, we employed a Cyto-c-specific aptamer to substitute both the recognition Ab and barcode DNAs on the NPs in the BBC assay; and a novel isothermal recombinase polymerase amplification for the time-consuming PCR. The detection limit of our ABC assay for the Cyto-c was found to be 10 ng/mL and this new assay can be completed within 3h. Several potential anti-cancer drugs have been tested in vitro for their efficacy to kill liver cancer with or without multi-drug resistance. PMID:24054573

Loo, Jacky F C; Lau, P M; Ho, H P; Kong, S K

2013-10-15

251

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

PubMed

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. PMID:24424127

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

2014-03-01

252

Comparison of loop-mediated isothermal amplification assay and conventional culture methods for detection of Campylobacter jejuni and Campylobacter coli in naturally contaminated chicken meat samples.  

PubMed

We investigated the efficacy of a loop-mediated isothermal amplification (LAMP) assay for detection of chicken meat samples naturally contaminated with Campylobacter jejuni and Campylobacter coli. A total of 144 Preston enrichment broth cultures from chicken meat samples were assessed by using the LAMP assay and conventional culture methods, which consist of a combination of Preston enrichment culturing and plating onto Butzler and modified charcoal cefoperazone deoxycholate agars. Compared with C. jejuni-C. coli isolation using the conventional culture test, the LAMP results showed 98.5% (67/68) and 97.4% (74/76) sensitivity and specificity, respectively, and the positive and negative predictive values were 97.1% (67/69) and 98.7% (74/75), respectively. The conventional culture test required more than 3 to 4 days to isolate and identify C. jejuni and C. coli in the Preston enrichment cultures. In contrast, the LAMP assay was markedly faster, requiring less than 90 min from the beginning of DNA extraction to final detection and differentiation of C. jejuni and C. coli. In total, the LAMP assay required 23.5 to 25.5 h from the beginning of the enrichment culture to final determination. These results suggest that our LAMP assay is a powerful tool for rapid, sensitive, and practical detection of C. jejuni and C. coli which may facilitate surveillance and control of C. jejuni-C. coli contamination in chicken, as well as investigations of food poisoning incidents caused by these organisms. This is the first report of a highly sensitive and specific LAMP assay to detect and differentiate C. jejuni and C. coli in chicken meat samples. PMID:19139242

Yamazaki, Wataru; Taguchi, Masumi; Kawai, Takao; Kawatsu, Kentaro; Sakata, Junko; Inoue, Kiyoshi; Misawa, Naoaki

2009-03-01

253

Highly sensitive detection of telomerase activity in tumor cells by cascade isothermal signal amplification based on three-way junction and base-stacking hybridization.  

PubMed

Herein, We report a simple and highly sensitive telomerase activity assay that integrates two consecutive isothermal signal amplification processes, namely, three-way junction triggered DNA-machine (3WJ-DNAM), and base-stacking hybridization assisted "biological circuit" DNA-machine (BSHBC-DNAM). In the presence of telomerase, the 3WJ are formed by the hybridization between the telomerase product and 3WJ-probes (3WJ-primer and 3WJ-template), which will initiate an autonomous 3WJ-DNAM by multiple processes of replication, nicking, and strand displacement, continuously generating short oligonucleotides as "triggers". These "triggers" will then provide additional stability for another two primers with a shared 5-bp complementary sequence at each 3'-end via base-stacking hybridization. And the BSHBC-DNAM are subsequently carried out by the strand-displacement induced circular utilization of "Trigger". Eventually, the single-stranded DNA (ssDNA) is generated in large quantities, and a significant fluorescence enhancement is observed due to the hybridization between the ssDNA and molecular beacons (MBs). In this way, per telomerase-mediated elongation event is efficiently and specifically converted into the greatly amplified fluorescence signals. This novel sensing strategy permits measurement of telomerase activity in cell extracts over the range of 3-5000 Hela cells, which is comparable or even superior to most previously reported methods. Using somatic and tumor cell lines, the selectivity and generality of the assay are investigated with satisfactory results. Furthermore, the inhibition effect of 3'-azido-3'-deoxythymidine (AZT) is also investigated, indicating its excellent performance in telomerase inhibitor screening. PMID:23122231

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

2013-03-15

254

Evaluation of a Loop-Mediated Isothermal Amplification Suite for the Rapid, Reliable, and Robust Detection of Shiga Toxin-Producing Escherichia coli in Produce  

PubMed Central

Shiga toxin-producing Escherichia coli (STEC) strains are a leading cause of produce-associated outbreaks in the United States. Rapid, reliable, and robust detection methods are needed to better ensure produce safety. We recently developed a loop-mediated isothermal amplification (LAMP) suite for STEC detection. In this study, the STEC LAMP suite was comprehensively evaluated against real-time quantitative PCR (qPCR) using a large panel of bacterial strains (n = 156) and various produce items (several varieties of lettuce, spinach, and sprouts). To simulate real-world contamination events, produce samples were surface inoculated with a low level (1.2 to 1.8 CFU/25 g) of individual STEC strains belonging to seven serogroups (O26, O45, O103, O111, O121, O145, and O157) and held at 4°C for 48 h before testing. Six DNA extraction methods were also compared using produce enrichment broths. All STEC targets and their subtypes were accurately detected by the LAMP suite. The detection limits were 1 to 20 cells per reaction in pure culture and 105 to 106 CFU per 25 g (i.e., 103 to 104 CFU per g) in produce, except for strains harboring the stx2c, eae-?, and eae-? subtypes. After 6 to 8 h of enrichment, the LAMP suite achieved accurate detection of low levels of STEC strains of various stx2 and eae subtypes in lettuce and spinach varieties but not in sprouts. A similar trend of detection was observed for qPCR. The PrepMan Ultra sample preparation reagent yielded the best results among the six DNA extraction methods. This research provided a rapid, reliable, and robust method for detecting STEC in produce during routine sampling and testing. The challenge with sprouts detection by both LAMP and qPCR calls for special attention to further analysis. PMID:24509927

Wang, Fei; Yang, Qianru; Qu, Yinzhi; Meng, Jianghong

2014-01-01

255

Homogenous, real-time duplex loop-mediated isothermal amplification using a single fluorophore-labeled primer and an intercalator dye: Its application to the simultaneous detection of Shiga toxin genes 1 and 2 in Shiga toxigenic Escherichia coli isolates.  

PubMed

We developed a completely homogeneous duplex loop-mediated isothermal amplification (LAMP) method. The present LAMP method employed a combination of a 6-carboxyfluorescein (FAM)-labeled primer (donor) for one target gene, a non-labeled primer for the other, and an intercalator ethidium bromide (EtBr) dye (acceptor) on the basis of fluorescence resonance energy transfer (FRET) between the FAM donor and EtBr acceptor. Measuring changes in fluorescence of FAM enabled the LAMP method to detect two different genes simultaneously. This method was used to detect Shiga toxin genes in Shiga toxigenic Escherichia coli isolates, demonstrating simultaneous detection of two different genes with rapidity and accuracy. PMID:20230890

Kouguchi, Yoshihiro; Fujiwara, Takako; Teramoto, Miki; Kuramoto, Mika

2010-08-01

256

Development of a multiplex polymerase chain reaction-sequence-specific primer method for NKG2D and NKG2F single-nucleotide polymorphism typing using isothermal multiple displacement amplification products.  

PubMed

Natural killer group 2 member D (NKG2D) on immune effector cells recognizes multiple stress-inducible ligands. NKG2D single-nucleotide polymorphism (SNP) haplotypes were related to the levels of cytotoxic activity of peripheral blood mononuclear cells. Indeed, these polymorphisms were also located in NKG2F. Isothermal multiple displacement amplification (IMDA) is used for whole genome amplification (WGA) that can amplify very small genomic DNA templates into microgram with whole genome coverage. This is particularly useful in the cases of limited amount of valuable DNA samples requiring multi-locus genotyping. In this study, we evaluated the quality and applicability of IMDA to genetic studies in terms of sensitivity, efficiency of IMDA re-amplification and stability of IMDA products. The smallest amount of DNA to be effectively amplified by IMDA was 200 pg yielding final DNA of approximately 16 µg within 1.5 h. IMDA could be re-amplified only once (second round of amplification), and could be kept for 5 months at 4°C and more than a year at -20°C without loosing genome coverage. The amplified products were used successfully to setup a multiplex polymerase chain reaction-sequence-specific primer for SNP typing of the NKG2D/F genes. The NKG2D/F multiplex polymerase chain reaction (PCR) contained six PCR mixtures for detecting 10 selected SNPs, including 8 NKG2D/F SNP haplotypes and 2 additional NKG2D coding SNPs. This typing procedure will be applicable in both clinical and research laboratories. Thus, our data provide useful information and limitations for utilization of genome-wide amplification using IMDA and its application for multiplex NKG2D/F typing. PMID:23530727

Kaewmanee, M; Phoksawat, W; Romphruk, A; Romphruk, A V; Jumnainsong, A; Leelayuwat, C

2013-06-01

257

Development of a Loop-Mediated Isothermal Amplification Method for Detecting Streptococcus equi subsp. zooepidemicus and Analysis of Its Use with Three Simple Methods of Extracting DNA from Equine Respiratory Tract Specimens.  

PubMed

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a dominant pathogenic bacterium in equine pneumonia. We developed a specific loop-mediated isothermal amplification (LAMP) method, which targets the gene encoding sorbitol-6-phosphate 2-dehydrogenase (sorD), for detecting S. zooepidemicus and examined the clinical efficacies of its use in combination with each of 3 DNA extraction methods easily used by veterinary practitioners, namely the Loopamp PURE DNA Extraction Kit, InstaGene Matrix and a conventional boiling method. The LAMP method plus the Loopamp PURE DNA Extraction Kit gave higher rates of positivity than the other combinations in both clinical and spiked samples containing clinically significant concentrations (>1 × 10(4) CFU/ml) of S. zooepidemicus. PMID:24871644

Kinoshita, Yuta; Niwa, Hidekazu; Katayama, Yoshinari

2014-10-01

258

Development of a Loop-Mediated Isothermal Amplification Method for Detecting Streptococcus equi subsp. zooepidemicus and Analysis of Its Use with Three Simple Methods of Extracting DNA from Equine Respiratory Tract Specimens  

PubMed Central

ABSTRACT Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a dominant pathogenic bacterium in equine pneumonia. We developed a specific loop-mediated isothermal amplification (LAMP) method, which targets the gene encoding sorbitol-6-phosphate 2-dehydrogenase (sorD), for detecting S. zooepidemicus and examined the clinical efficacies of its use in combination with each of 3 DNA extraction methods easily used by veterinary practitioners, namely the Loopamp PURE DNA Extraction Kit, InstaGene Matrix and a conventional boiling method. The LAMP method plus the Loopamp PURE DNA Extraction Kit gave higher rates of positivity than the other combinations in both clinical and spiked samples containing clinically significant concentrations (>1 × 104 CFU/ml) of S. zooepidemicus. PMID:24871644

KINOSHITA, Yuta; NIWA, Hidekazu; KATAYAMA, Yoshinari

2014-01-01

259

Increased robustness of single-molecule counting with microfluidics, digital isothermal amplification, and a mobile phone versus real-time kinetic measurements.  

PubMed

Quantitative bioanalytical measurements are commonly performed in a kinetic format and are known to not be robust to perturbation that affects the kinetics itself or the measurement of kinetics. We hypothesized that the same measurements performed in a "digital" (single-molecule) format would show increased robustness to such perturbations. Here, we investigated the robustness of an amplification reaction (reverse-transcription loop-mediated amplification, RT-LAMP) in the context of fluctuations in temperature and time when this reaction is used for quantitative measurements of HIV-1 RNA molecules under limited-resource settings (LRS). The digital format that counts molecules using dRT-LAMP chemistry detected a 2-fold change in concentration of HIV-1 RNA despite a 6 °C temperature variation (p-value = 6.7 × 10(-7)), whereas the traditional kinetic (real-time) format did not (p-value = 0.25). Digital analysis was also robust to a 20 min change in reaction time, to poor imaging conditions obtained with a consumer cell-phone camera, and to automated cloud-based processing of these images (R(2) = 0.9997 vs true counts over a 100-fold dynamic range). Fluorescent output of multiplexed PCR amplification could also be imaged with the cell phone camera using flash as the excitation source. Many nonlinear amplification schemes based on organic, inorganic, and biochemical reactions have been developed, but their robustness is not well understood. This work implies that these chemistries may be significantly more robust in the digital, rather than kinetic, format. It also calls for theoretical studies to predict robustness of these chemistries and, more generally, to design robust reaction architectures. The SlipChip that we used here and other digital microfluidic technologies already exist to enable testing of these predictions. Such work may lead to identification or creation of robust amplification chemistries that enable rapid and precise quantitative molecular measurements under LRS. Furthermore, it may provide more general principles describing robustness of chemical and biological networks in digital formats. PMID:24199852

Selck, David A; Karymov, Mikhail A; Sun, Bing; Ismagilov, Rustem F

2013-11-19

260

Development of a Real-Time Fluorescence Loop-Mediated Isothermal Amplification Assay for Rapid and Quantitative Detection of Fusarium oxysporum f. sp. cubense Tropical Race 4 In Soil  

PubMed Central

Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 103 spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China. PMID:24376590

Pu, Jinji; Qi, Yanxiang; Yu, Qunfang; Xie, Yixian; Peng, Jun

2013-01-01

261

The development of an accelerated reverse-transcription loop mediated isothermal amplification for the serotype specific detection of bluetongue virus 8 in clinical samples.  

PubMed

In 2006 bluetongue virus serotype 8 (BTV 8) was identified for the first time in the Netherlands causing a major epidemic in sheep and cattle that quickly spread to neighbouring Belgium, Germany and beyond to France and the UK. This resulted in severe animal health and welfare problems as well as substantial economic losses to the agrifood industries of these countries. Given that the early diagnosis of BTV infection 'in-the-field' is extremely useful to its subsequent management and control, this study was established to design a novel, sensitive and rapid nucleic acid diagnostic test for the serotype-specific detection of BTV 8, which could be used without the use of advanced laboratory support and equipment. Primers for the detection of BTV 8 were based on genome segment 2 of the virus, the VP2 gene. The assay was assessed using a full panel of BTV reference strains and clinical samples. Positive amplification was observed using a fluorescent detection reagent. The sensitivity of the RT-LAMP assay was 102 copies of RNA. The assay did not amplify the closely related orbivirus EHDV. This novel RT-LAMP offers a sensitive, specific and rapid method of detecting BTV 8. The approach is inexpensive and easy to use and could potentially be used in a 'pen-side' setting 'in the field' or by smaller less well-equipped laboratories in developing countries. PMID:24642243

Mulholland, Catherine; Hoffmann, Bernd; McMenamy, Michael J; Korthase, Christian; Earley, Bernadette; Markey, Bryan; Cassidy, Joseph P; McKillen, John; Allan, Gordon; Welsh, Michael D

2014-06-01

262

Simultaneous detection and differentiation of dengue virus serotypes 1-4, Japanese encephalitis virus, and West Nile virus by a combined reverse-transcription loop-mediated isothermal amplification assay  

PubMed Central

Background Rapid identification and differentiation of mosquito-transmitted flaviviruses in acute-phase sera of patients and field-caught vector mosquitoes are important for the prediction and prevention of large-scale epidemics. Results We developed a flexible reverse-transcription loop-mediated isothermal amplification (RT-LAMP) unit for the detection and differentiation of dengue virus serotypes 1-4 (DENV1-4), Japanese encephalitis virus (JEV), and West Nile virus (WNV). The unit efficiently amplified the viral genomes specifically at wide ranges of viral template concentrations, and exhibited similar amplification curves as monitored by a real-time PCR engine. The detection limits of the RT-LAMP unit were 100-fold higher than that of RT-PCR in 5 of the six flaviviruses. The results on specificity indicated that the six viruses in the assay had no cross-reactions with each other. By examining 66 viral strains of DENV1-4 and JEV, the unit identified the viruses with 100% accuracy and did not cross-react with influenza viruses and hantaviruses. By screening a panel of specimens containing sera of 168 patients and 279 pools of field-caught blood sucked mosquitoes, results showed that this unit is high feasible in clinical settings and epidemiologic field, and it obtained results 100% correlated with real-time RT-PCR. Conclusions The RT-LAMP unit developed in this study is able to quickly detect and accurately differentiate the six kinds of flaviviruses, which makes it extremely feasible for screening these viruses in acute-phase sera of the patients and in vector mosquitoes without the need of high-precision instruments. PMID:21777455

2011-01-01

263

Preliminary Validation of Direct Detection of Foot-And-Mouth Disease Virus within Clinical Samples Using Reverse Transcription Loop-Mediated Isothermal Amplification Coupled with a Simple Lateral Flow Device for Detection  

PubMed Central

Rapid, field-based diagnostic assays are desirable tools for the control of foot-and-mouth disease (FMD). Current approaches involve either; 1) Detection of FMD virus (FMDV) with immuochromatographic antigen lateral flow devices (LFD), which have relatively low analytical sensitivity, or 2) portable RT-qPCR that has high analytical sensitivity but is expensive. Loop-mediated isothermal amplification (LAMP) may provide a platform upon which to develop field based assays without these drawbacks. The objective of this study was to modify an FMDV-specific reverse transcription–LAMP (RT-LAMP) assay to enable detection of dual-labelled LAMP products with an LFD, and to evaluate simple sample processing protocols without nucleic acid extraction. The limit of detection of this assay was demonstrated to be equivalent to that of a laboratory based real-time RT-qPCR assay and to have a 10,000 fold higher analytical sensitivity than the FMDV-specific antigen LFD currently used in the field. Importantly, this study demonstrated that FMDV RNA could be detected from epithelial suspensions without the need for prior RNA extraction, utilising a rudimentary heat source for amplification. Once optimised, this RT-LAMP-LFD protocol was able to detect multiple serotypes from field epithelial samples, in addition to detecting FMDV in the air surrounding infected cattle, pigs and sheep, including pre-clinical detection. This study describes the development and evaluation of an assay format, which may be used as a future basis for rapid and low cost detection of FMDV. In addition it provides providing “proof of concept” for the future use of LAMP assays to tackle other challenging diagnostic scenarios encompassing veterinary and human health. PMID:25165973

Waters, Ryan A.; Fowler, Veronica L.; Armson, Bryony; Nelson, Noel; Gloster, John; Paton, David J.; King, Donald P.

2014-01-01

264

Development of an allele-specific, loop-mediated, isothermal amplification method (AS-LAMP) to detect the L1014F kdr-w mutation in Anopheles gambiae s. l.  

PubMed Central

Background Malaria control relies heavily on treated bed nets and indoor residual spraying with pyrethroid insecticides. Unfortunately, the resistance to pyrethroid insecticides, mainly due to the kdr mutation, is spreading in the main malaria vector Anopheles gambiae s.l., decreasing the insecticides’ efficacy. To manage the insecticide resistance rapidly and flexibly, simple and effective tools for the early detection of resistant mosquitoes are needed. This study aimed to develop an allele-specific, loop-mediated, isothermal amplification (AS-LAMP) method to detect the West African-type kdr mutation (kdr-w; L1014F) in field-collected mosquitoes. Methods DNA fragments of the wild-type and the mutated kdr gene were used to select the primers and develop the method. The primers were designed with the mutation at the 5’ end of the backward inner primer (BIP). The AS-LAMP method was compared to the AS-PCR method using the genomic DNA of 120 field-collected mosquitoes. Results The AS-LAMP method could discriminate between the wild-type homozygote, the heterozygote, and the kdr-w homozygote within 75?min. The AS-LAMP method has the advantage of being faster and at least as sensitive and specific as the AS-PCR method. Conclusions The AS-LAMP method can be used to detect the kdr mutation for quick decision-making, even in less well-equipped laboratories. PMID:22770418

2012-01-01

265

Evaluation of a direct reverse transcription loop-mediated isothermal amplification method without RNA extraction for the detection of human enterovirus 71 subgenotype C4 in nasopharyngeal swab specimens.  

PubMed

Human enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD) worldwide and has been associated with neurological complications which resulted in fatalities during recent outbreak in Asia pacific region. A direct reverse transcription loop-mediated isothermal amplification (direct RT-LAMP) assay using heat-treated samples without RNA extraction was developed and evaluated for the detection of EV71 subgenotype C4 in nasopharyngeal swab specimens. The analytical sensitivity and specificity of the direct RT-LAMP assay were examined. The detection limit of the direct RT-LAMP assays was 1.6 of a 50% tissue culture infective dose (TCID(50)) per reaction and no cross-reaction was observed with control viruses including Cosackievirus A (CVA) viruses (CVA2,4,5,7,9,10,14,16, and 24), Coxsackievirus B (CVB) viruses (CVB1,2,3,4, and 5) or ECHO viruses (ECHO3,6,11, and 19). The direct RT-LAMP assay was evaluated and compared to both RT-LAMP and quantitative real-time PCR (qRT-PCR) in detecting EV71 infection with 145 nasopharyngeal swab specimens. The clinical performance demonstrated the sensitivity and specificity of direct RT-LAMP was reported to be 90.3% and 100% respectively, compared to RT-LAMP, and 86.83% and 100% respectively, compared to qRT-PCR. These data demonstrated that the direct RT-LAMP assay can potentially be developed for the point of care screening of EV71 infection in China. PMID:23272248

Nie, Kai; Qi, Shun-Xiang; Zhang, Yong; Luo, Le; Xie, Yun; Yang, Meng-Jie; Zhang, Yi; Li, Jin; Shen, Hongwei; Li, Qi; Ma, Xue-Jun

2012-01-01

266

Evaluation of a Direct Reverse Transcription Loop-Mediated Isothermal Amplification Method without RNA Extraction for the Detection of Human Enterovirus 71 Subgenotype C4 in Nasopharyngeal Swab Specimens  

PubMed Central

Human enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD) worldwide and has been associated with neurological complications which resulted in fatalities during recent outbreak in Asia pacific region. A direct reverse transcription loop-mediated isothermal amplification (direct RT-LAMP) assay using heat-treated samples without RNA extraction was developed and evaluated for the detection of EV71 subgenotype C4 in nasopharyngeal swab specimens. The analytical sensitivity and specificity of the direct RT-LAMP assay were examined. The detection limit of the direct RT-LAMP assays was 1.6 of a 50% tissue culture infective dose (TCID50) per reaction and no cross-reaction was observed with control viruses including Cosackievirus A (CVA) viruses (CVA2,4,5,7,9,10,14,16, and 24), Coxsackievirus B (CVB) viruses (CVB1,2,3,4, and 5) or ECHO viruses (ECHO3,6,11, and 19). The direct RT-LAMP assay was evaluated and compared to both RT-LAMP and quantitative real-time PCR (qRT-PCR) in detecting EV71 infection with 145 nasopharyngeal swab specimens. The clinical performance demonstrated the sensitivity and specificity of direct RT-LAMP was reported to be 90.3% and 100% respectively, compared to RT-LAMP, and 86.83% and 100% respectively, compared to qRT-PCR. These data demonstrated that the direct RT-LAMP assay can potentially be developed for the point of care screening of EV71 infection in China. PMID:23272248

Luo, Le; Xie, Yun; Yang, Meng-jie; Zhang, Yi; Li, Jin; Shen, Hongwei; Li, Qi; Ma, Xue-jun

2012-01-01

267

Isothermal Confinement  

NASA Astrophysics Data System (ADS)

The concept of isothermal confinement is presented. The idea is a revival of the early magnetic fusion concepts with new insight. The plasma core is confined magnetically and is surrounded by a quasi-vacuum region. The temperature of the core is uniform and the turbulence associated with the temperature gradient is absent. The quasi-vacuum region is unstable against the pressure gradient and the turbulent transport rate is much larger than that of the core. Two modes of operation, pulsed and steady state, are considered. Recent experimental results in LHD and CDX-U appear to support the concept.

Ohkawa, Tihiro

268

Comparison of conventional RT-PCR, reverse-transcription loop-mediated isothermal amplification, and SYBR green I-based real-time RT-PCR in the rapid detection of bovine viral diarrhea virus nucleotide in contaminated commercial bovine sera batches.  

PubMed

Bovine viral diarrhea virus (BVDV) can contaminate biological products produced in bovine or porcine cells or manufactured using bovine sera. A rapid, specific, sensitive, and practical method of detecting BVDV in bio-products is needed. The purpose of this study was to compare three assays with respect to their ability to accurately detect BVDV in biological samples, namely reverse-transcription loop-mediated isothermal amplification (RT-LAMP), SYBR green I-based real-time RT-PCR, and conventional RT-PCR. All assays detected BVDV nucleotide and differentiated between BVDV-free and -contaminated bovine sera successfully. In addition, the results were specific to BVDV: the amplification of samples containing the closely related classical swine fever virus or other pathogenic bovine viruses yielded negative results. The lowest detection threshold, 10(1) copies, was displayed by the SYBR green I-based real-time RT-PCR and RT-LAMP assay. This assay was also the most effective in the detection of BVDV contamination in a set of commercially available bovine sera. The field conditions suggest that RT-LAMP is specific and sensitive to detecting BVDV in biological samples and may be used for quality control of biomaterials. PMID:25019170

Zhang, Shu-Qin; Tan, Bin; Li, Peng; Wang, Feng-Xue; Guo, Li; Yang, Yong; Sun, Na; Zhu, Hong-Wei; Wen, Yong-Jun; Cheng, Shi-Peng

2014-10-01

269

Isothermal and Adiabatic Measurements.  

ERIC Educational Resources Information Center

Describes the working of the Adiabatic Gas Law Apparatus, a useful tool for measuring the pressure, temperature, and volume of a variety of gases undergoing compressions and expansions. Describes the adaptation of this apparatus to perform isothermal measurements and discusses the theory behind the adiabatic and isothermal processes. (JRH)

McNairy, William W.

1996-01-01

270

nAture methods | VOL.10 NO.7 | JULY2013 | 641 We developed an integrated chip for real-time amplification  

E-print Network

amplification strategies: Pcr and isothermal amplification. using this platform, we genotyped and discriminated-time amplification and detection of nucleic acid using ph-sensing complementary metal-oxide semiconductor (cmos) technology. here we show an amplification-coupled detection method for directly measuring released hydrogen

Cai, Long

271

ORIGINAL ARTICLE Comparative genomics-guided loop-mediated isothermal  

E-print Network

ORIGINAL ARTICLE Comparative genomics-guided loop-mediated isothermal amplification sequencing and analytical techniques, genomic sequence data of prok- aryotes are accumulating at a very rapid pace. As of October 2008, there are 873 complete and pub- lished genome sequences, as well as 2025

Hsiang, Tom

272

ORIGINAL ARTICLE A loop-mediated isothermal amplification method  

E-print Network

of a double-stranded DNA genome [4]. Because of the severe losses of wild and farmed amphibian populations) and rainbow trout (Oncorhynchus mykiss) [8, 9]. STIV was isolated from soft-shelled turtle (Trionyx sinensis sequence of the STIV genome has been determined (Gen- Bank accession no. EU627010). ADIV is a newly discov

Gray, Matthew

273

Convenient detection of HPV virus in a clinical sample using concurrent rolling circle and junction probe amplifications.  

PubMed

Herein we show that two isothermal amplification strategies, rolling circle amplification and junction probe strategy, can be used in tandem in the same tube under isothermal conditions to detect HPV16 in clinical cervical swabs. It was discovered that the prior treatment of the clinical sample with a cocktail of restriction endonucleases (REAses) to digest the genomic DNA facilitated the isothermal detection assay. PMID:24852020

Yan, Lei; Liu, Kailong; Sintim, Herman O

2014-07-11

274

Generalized isothermic lattices  

NASA Astrophysics Data System (ADS)

We study multi-dimensional quadrilateral lattices satisfying simultaneously two integrable constraints: a quadratic constraint and the projective Moutard constraint. When the lattice is two dimensional and the quadric under consideration is the Möbius sphere one obtains, after the stereographic projection, the discrete isothermic surfaces defined by Bobenko and Pinkall by an algebraic constraint imposed on the (complex) cross-ratio of the circular lattice. We derive the analogous condition for our generalized isothermic lattices using Steiner's projective structure of conics, and we present basic geometric constructions which encode integrability of the lattice. In particular, we introduce the Darboux transformation of the generalized isothermic lattice and we derive the corresponding Bianchi permutability principle. Finally, we study two-dimensional generalized isothermic lattices, in particular geometry of their initial boundary value problem.

Doliwa, Adam

2007-10-01

275

Entropy-driven molecular switch and signal amplification for homogeneous SNPs detection.  

PubMed

An approach was developed to rapidly and sensitively detect SNP in homogeneous solution based on an entropy-driven molecular switch and isothermal polymerase amplification reaction without addition of exogenous primers. PMID:21240444

Shi, Chao; Zhao, Chunhui; Guo, Qingjie; Ma, Cuiping

2011-03-14

276

Nucleic acid amplification: Alternative methods of polymerase chain reaction  

PubMed Central

Nucleic acid amplification is a valuable molecular tool not only in basic research but also in application oriented fields, such as clinical medicine development, infectious diseases diagnosis, gene cloning and industrial quality control. A comperehensive review of the literature on the principles, applications, challenges and prospects of different alternative methods of polymerase chain reaction (PCR) was performed. PCR was the first nucleic acid amplification method. With the advancement of research, a no of alternative nucleic acid amplification methods has been developed such as loop mediated isothermal amplification, nucleic acid sequence based amplification, strand displacement amplification, multiple displacement amplification. Most of the alternative methods are isothermal obviating the need for thermal cyclers. Though principles of most of the alternate methods are relatively complex than that of PCR, they offer better applicability and sensitivity in cases where PCR has limitations. Most of the alternate methods still have to prove themselves through extensive validation studies and are not available in commercial form; they pose the potentiality to be used as replacements of PCR. Continuous research is going on in different parts of the world to make these methods viable technically and economically. PMID:24302831

Fakruddin, Md; Mannan, Khanjada Shahnewaj Bin; Chowdhury, Abhijit; Mazumdar, Reaz Mohammad; Hossain, Md. Nur; Islam, Sumaiya; Chowdhury, Md. Alimuddin

2013-01-01

277

RT-isoPCR: nested, high multiplex mRNA amplification.  

PubMed

RT-isoPCR provides multiplex amplification of mRNA targets using a first-stage multiplex RT-PCR reaction with subsequent isothermal amplification for individual target loci detection. We demonstrate detection of 24 mRNA targets with high specificity and sensitivity without compromising sample variation or introducing biases between targets. PMID:23964356

Søe, Martin Jensen; Warthoe, Peter

2013-10-21

278

A highly sensitive surface plasmon resonance sensor for the detection of DNA and cancer cells by a target-triggered multiple signal amplification strategy.  

PubMed

A sensitive and versatile surface plasmon resonance bioassay was proposed for the detection of DNA and Ramos cells by combining the target-triggered isothermal exponential amplification with the magnetic nanoparticle-based rolling circle amplification, producing remarkable amplification efficiency. PMID:25083516

He, Peng; Qiao, Wenping; Liu, Lijun; Zhang, Shusheng

2014-09-21

279

Non-instrumented nucleic acid amplification (NINA): Instrument-free molecular malaria diagnostics for low-resource settings  

Microsoft Academic Search

We have achieved the first complete, non-instrumented nucleic acid amplification test (NAAT) using a calcium oxide heat source thermally linked to an engineered phase change material. These two components alone maintain a thermal profile suitable for the loop-mediated isothermal amplification assay. Starting with computational fluid dynamics analysis, we identified nominal geometry for the exothermic reaction chamber, phase change material chamber,

Paul LaBarre; Jay Gerlach; Jared Wilmoth; Andrew Beddoe; Jered Singleton; Bernhard Weigl

2010-01-01

280

NASBA: A detection and amplification system uniquely suited for RNA  

SciTech Connect

The invention of PCR (polymerase chain reaction) has revolutionized our ability to amplify and manipulate a nucleic acid sequence in vitro. The commercial rewards of this revolution have driven the development of other nuclei acid amplification and detection methodologies. This has created an alphabet soup of technologies that use different amplification methods, including NASBA (nucleic acid sequence-based amplification), LCR (ligase chain reaction), SDA (strand displacement amplification), QBR (Q-beta replicase), CPR (cycling probe reaction), and bDNA (branched DNA). Despite the differences in their processes, these amplification systems can be separated into two broad categories based on how they achieve their goal: sequence-based amplification systems, such as PCR, NASBA, and SDA, amplify a target nucleic acid sequence. Signal-based amplification systems, such as LCR, QBR, CPR and bDNA, amplify or alter a signal from a detection reaction that is target-dependent. While the various methods have relative strengths and weaknesses, only NASBA offers the unique ability to homogeneously amplify an RNA analyte in the presence of homologous genomic DNA under isothermal conditions. Since the detection of RNA sequences almost invariably measures biological activity, it is an excellent prognostic indicator of activities as diverse as virus production, gene expression, and cell viability. The isothermal nature of the reaction makes NASBA especially suitable for large-scale manual screening. These features extend NASBA`s application range from research to commercial diagnostic applications. Field test kits are presently under development for human diagnostics as well as the burgeoning fields of food and environmental diagnostic testing. These developments suggest future integration of NASBA into robotic workstations for high-throughput screening as well. 17 refs., 1 tab.

Sooknanan, R.; Malek, L.T. [Cangen, Ontario (Canada)] [Cangen, Ontario (Canada)

1995-06-01

281

Non-instrumented nucleic acid amplification assay  

NASA Astrophysics Data System (ADS)

We have developed components of a diagnostic disposable platform that has the dual purpose of providing molecular diagnostics at the point of care (POC) as well as stabilizing specimens for further analysis via a centralized surveillance system. This diagnostic is targeted for use in low-resource settings by minimally trained health workers. The disposable device does not require any additional instrumentation and will be almost as rapid and simple to use as a lateral flow strip test - yet will offer the sensitivity and specificity of nucleic acid amplification tests (NAATs). The low-cost integrated device is composed of three functional components: (1) a sample-processing subunit that generates clean and stabilized DNA from raw samples containing nucleic acids, (2) a NA amplification subunit, and (3) visual amplicon detection sub-unit. The device integrates chemical exothermic heating, temperature stabilization using phase-change materials, and isothermal nucleic acid amplification. The aim of developing this system is to provide pathogen detection with NAAT-level sensitivity in low-resource settings where there is no access to instrumentation. If a disease occurs, patients would be tested with the disposable in the field. A nucleic acid sample would be preserved within the spent disposable which could be sent to a central laboratory facility for further analysis if needed.

Weigl, Bernhard H.; Domingo, Gonzalo; Gerlach, Jay; Tang, Dennis; Harvey, Darrel; Talwar, Nick; Fichtenholz, Alex; van Lew, Bill; LaBarre, Paul

2008-02-01

282

A mechanism for ramified rolling circle amplification  

PubMed Central

Background Amplification of single-stranded DNA circles has wide utility for a variety of applications. The two-primer ramified rolling circle amplification (RAM) reaction provides exponential DNA amplification under isothermal conditions, creating a regular laddered series of double-stranded DNA products. However, the molecular mechanism of the RAM reaction remains unexplained. Results A RAM reaction model predicts exponential accumulation of a double-stranded DNA product size series, and product-size ratios, that are consistent with observed RAM reaction products. The mechanism involves generation of a series of increasing size intermediate templates; those templates produce RAM products and recursively generate smaller intermediate templates. The model allows prediction of the number of rounds of circular template replication. Real-time RAM reaction data are consistent with the model. Analysis of RAM reaction products shows exponential growth limitation consistent with the model's predictions. Conclusions The model provides a rationale for the observed products of the RAM reaction, and the molecular yield among those products. Experimental results are consistent with the model. PMID:21138587

2010-01-01

283

Spectral Gap Amplification  

E-print Network

A large number of problems in science can be solved by preparing a specific eigenstate of some Hamiltonian H. The generic cost of quantum algorithms for these problems is determined by the inverse spectral gap of H for that eigenstate and the cost of evolving with H for some fixed time. The goal of spectral gap amplification is to construct a Hamiltonian H' with the same eigenstate as H but a bigger spectral gap, requiring that constant-time evolutions with H' and H are implemented with nearly the same cost. We show that a quadratic spectral gap amplification is possible when H satisfies a frustration-free property and give H' for these cases. This results in quantum speedups for optimization problems. It also yields improved constructions for adiabatic simulations of quantum circuits and for the preparation of projected entangled pair states (PEPS), which play an important role in quantum many-body physics. Defining a suitable black-box model, we establish that the quadratic amplification is optimal for frustration-free Hamiltonians and that no spectral gap amplification is possible, in general, if the frustration-free property is removed. A corollary is that finding a similarity transformation between a stoquastic Hamiltonian and the corresponding stochastic matrix is hard in the black-box model, setting limits to the power of some classical methods that simulate quantum adiabatic evolutions.

Rolando D. Somma; Sergio Boixo

2011-10-11

284

Stochastic amplification in epidemics  

E-print Network

REPORT Stochastic amplification in epidemics David Alonso1, *, Alan J. McKane2 and Mercedes Pascual, we present a stochastic theory for the major dynamical transitions in epidemics from regular in nonlinear ecological systems in general. Keywords: epidemics; oscillations; stochastic modelling; childhood

McKane, Alan

285

Amplification of Mitochondrial DNA  

NSDL National Science Digital Library

This laboratory activity, by the Biotechnology Education and Training Sequence Investment (BETSI) project at Southwestern College, walks students and educators through the procedure of amplifying mitochondrial DNA. The first section of the activity details the actual amplifying; the second section shows the procedure for DNA analysis by gel electrophoresis once amplification is complete.

2008-08-18

286

Improved DNA amplification technique  

SciTech Connect

A modification of the polymerase chain reaction (PCR) technique is described. The method allows the amplification of regions of DNA flanking a single region of known sequence, in contrast to standard PCR which requires two regions of known sequence at opposite ends of the fragment to be amplified. Various advantages of the new method are described.

Silveer, J.

1989-01-03

287

DNA amplification fingerprinting of bacteria  

Microsoft Academic Search

We have amplified short arbitrary stretches of total bacterial DNA to produce highly characteristic and complex DNA fingerprints. This DNA amplification fingerprinting (DAF) strategy involves enzymatic amplification of DNA directed by a single arbitrary oligonucleotide primer. Amplification produces a characteristic spectrum of products that is adequately resolved by polyacrylamide gel electrophoresis and visualized by silver staining. Although DAF is simple

Brant J. Bassam; Gustavo Caetano-Anollés; Peter M. Gresshoff

1992-01-01

288

Isothermal Dendritic Growth Experiment Video  

NASA Technical Reports Server (NTRS)

This video, captured during the Isothermal Dendritic Growth Experiment (IDGE) flown on STS-87 as a part of the fourth United States Microgravity payload, shows the growth of a dendrite, and the surface solidification that occurred on the front and back windows of the growth chamber. Dendrites are tiny, tree like structures that form as metals solidify.

1997-01-01

289

Predicting shaft torque amplification  

SciTech Connect

Shaft Torque Amplification (STA) is the form of SSR characterized by the higher system-model complexity and computational requirements its simulation, normally in a time-domain environment, demands. A multi-modal approach drawing the turbogenerator torsional response to electrical torque impulses applied to the machine air-gap is introduced in this article as an alternative STA analysis frame. Peak torque results from the proposed algorithm are compared with similar ones obtained from EMTP runs. Loss-of-life calculations and a capacitor-reinsertion application as STA control means, are included.

Achilles, R.A. [Achilles (R.A.), Neuquen (Argentina)] [Achilles (R.A.), Neuquen (Argentina)

1995-02-01

290

IDGE: Isothermal Dendritic Growth Experiment  

NASA Technical Reports Server (NTRS)

The Isothermal Dendritic Growth Experiment (IDGE) flew on STS-62 to study the microscopic, tree-like structures (dendrites) that form within metals as they solidify from molten materials. The size, shape, and orientation of these dendrites affect the strength and usefulness of metals. Data from this experiment will be used to test and improve the mathematical models that support the industrial production of metals.

1994-01-01

291

Isothermal combustion for improved efficiencies  

NASA Astrophysics Data System (ADS)

This theoretical effort proposes, and explains in detail, the concept of isothermal combustion for vastly improved efficiencies. The concept involves combustion at constant total temperatures in order to realize isothermal heat addition to the working fluid in typical power plants; it is rendered practical by extracting a precise amount of work from the flowing/expanding gases; the heat addition due to combustion will be balanced out to keep the total temperature constant. (In an oversimplified description, it might be said that this involves burning in the turbine stages, or burning during the expansion stroke.) Isothermal heat addition enables the thermodynamic cycle to approach the Carnot cycle more closely than the state-of-the-art Brayton, Otto, or Diesel cycles. A closed-form analytical expression is derived to explicitly show the cycle efficiency in terms of the pressure ratio and the overall temperature ratio. Thirty- to forty-percent efficiency increases are seen over the Brayton efficiency for the same overall temperature ratio. Some practical issues such as limits to pressure ratios, blade cooling, and service life are qualitatively discussed.

Ramohalli, Kumar N. R.

1987-06-01

292

Recombinase polymerase amplification assay for rapid detection of Francisella tularensis.  

PubMed

Several real-time PCR approaches to develop field detection for Francisella tularensis, the infectious agent causing tularemia, have been explored. We report the development of a novel qualitative real-time isothermal recombinase polymerase amplification (RPA) assay for use on a small ESEQuant Tube Scanner device. The analytical sensitivity and specificity were tested using a plasmid standard and DNA extracts from infected rabbit tissues. The assay showed a performance comparable to real-time PCR but reduced the assay time to 10 min. The rapid RPA method has great application potential for field use or point-of-care diagnostics. PMID:22518861

Euler, Milena; Wang, Yongjie; Otto, Peter; Tomaso, Herbert; Escudero, Raquel; Anda, Pedro; Hufert, Frank T; Weidmann, Manfred

2012-07-01

293

Recombinase Polymerase Amplification Assay for Rapid Detection of Francisella tularensis  

PubMed Central

Several real-time PCR approaches to develop field detection for Francisella tularensis, the infectious agent causing tularemia, have been explored. We report the development of a novel qualitative real-time isothermal recombinase polymerase amplification (RPA) assay for use on a small ESEQuant Tube Scanner device. The analytical sensitivity and specificity were tested using a plasmid standard and DNA extracts from infected rabbit tissues. The assay showed a performance comparable to real-time PCR but reduced the assay time to 10 min. The rapid RPA method has great application potential for field use or point-of-care diagnostics. PMID:22518861

Euler, Milena; Wang, Yongjie; Otto, Peter; Tomaso, Herbert; Escudero, Raquel; Anda, Pedro; Hufert, Frank T.

2012-01-01

294

Isothermal compressors for process gases  

SciTech Connect

This paper reports on isothermal compressors which are more efficient for all gases. The study of several representative gases considered stage efficiencies, pressure ratios and pressure losses of the intercoolers. Generally there are two ways to reduce power consumption of a gas compression process: minimize losses of the compressor or improve the thermodynamics of the process. But there are some new ways to reduce losses of turbocompressors. Losses of the impeller labyrinth seals and the balance piston labyrinth seal can be reduced by optimizing the labyrinth geometry and minimizing labyrinth clearances. Therefore, conventional labyrinth seals are still being studied and will be improved.

Wiederuh, E.; Meinhart, D. (FH Giessen-Friedberg, Giessen (Germany))

1992-09-01

295

Highly sensitive detection of gene expression of an intronless gene: amplification of mRNA, but not genomic DNA by nucleic acid sequence based amplification (NASBA)  

Microsoft Academic Search

NASBA is an isothermal nucleic acid amplification reaction that amplifies mRNA in a dsDNA background. Although similar to the sensitive reverse transcription\\/ polymerase chain reaction (RT-PCR) in mRNA detection, NASBA is not prone to give false positive results caused by genomic dsDNA. Therefore, NASBA is unique for sensitive detection of transcription of intronless genes, which preclude strategies such as intron

Albert Heim; Isabella Maria Grumbach; Stefanie Zeuke; Bert Top

1998-01-01

296

Nanoliter Reactors Improve Multiple Displacement Amplification  

E-print Network

Nanoliter Reactors Improve Multiple Displacement Amplification of Genomes from Single Cells Yann) Nanoliter reactors improve multiple displacement amplification of genomes from single cells. PLoS Genet 3

Quake, Stephen R.

297

Tips for Successful RNA Amplification  

NSDL National Science Digital Library

RNA amplification using the Van Gelder and Eberwine technique [1] is a multistep protocol comprised of several enzymatic and clean-up steps. Organization, technique, timing, and equipment are all critical to producing high quality amplified antisense RNA (aRNA). Since aRNA quality is key to obtaining reproducible array data, it is imperative that the techniques used for amplification are carried out with the utmost care. Here, we list our recommendations for performing consistent and reproducible RNA amplifications for microarray analysis.

Life Technologies Corporation (Life Technologies Corporation)

2011-01-01

298

Lunar ash flows - Isothermal approximation.  

NASA Technical Reports Server (NTRS)

Suggestion of the ash flow mechanism as one of the major processes required to account for some features of lunar soil. First the observational background and the gardening hypothesis are reviewed, and the shortcomings of the gardening hypothesis are shown. Then a general description of the lunar ash flow is given, and a simple mathematical model of the isothermal lunar ash flow is worked out with numerical examples to show the differences between the lunar and the terrestrial ash flow. The important parameters of the ash flow process are isolated and analyzed. It appears that the lunar surface layer in the maria is not a residual mantle rock (regolith) but a series of ash flows due, at least in part, to great meteorite impacts. The possibility of a volcanic contribution is not excluded. Some further analytic research on lunar ash flows is recommended.

Pai, S. I.; Hsieh, T.; O'Keefe, J. A.

1972-01-01

299

Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time  

PubMed Central

Background The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. Principal Findings Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs) enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART) continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi) produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi) produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. Conclusions The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a simple light detector, the iNAAT-BART combination is ideal for molecular diagnostic assays in both laboratory and low resource settings. PMID:21152399

Gandelman, Olga A.; Church, Vicki L.; Moore, Cathy A.; Kiddle, Guy; Carne, Christopher A.; Parmar, Surendra; Jalal, Hamid; Tisi, Laurence C.; Murray, James A. H.

2010-01-01

300

Cascade signal amplification for ultrasensitive electrochemical DNA detection.  

PubMed

In this work, by integrating multiple signal enhancement approaches, a new cascade signal amplification strategy is described to achieve highly sensitive electrochemical DNA detection. The presence of the target DNA leads to the unfolding of the biotin-modified hairpin probes on the sensor surface. With the addition of the primer sequences and polymerase, the target DNA is recycled and reused through isothermal strand-displacement polymerase reactions (ISDPR) to unfold a large number of the probes, which offer numerous binding sites to capture alkaline phosphatase (ALP)-loaded nanoparticle labels. These surface captured ALP enzymes catalyze the conversion of p-aminophenylphosphate to p-aminophenol, which generates amplified catalytic current responses due to the redox-recycling process during the potential sweep in the presence of the co-reactant NADH. With the synergistic signal amplifications by ISDPR-assisted target recycling, multi-ALP enzyme labels and redox-recycling, the proposed method offers highly sensitive detection of DNA down to 0.1 fM with single-base discrimination capability. Due to the significantly high sensitivity, the developed cascade signal amplification strategy can be potentially extended to detect various DNA targets at ultralow levels for early diagnoses of different diseases. PMID:24165824

Xu, Jin; Wang, Qiong; Xiang, Yun; Yuan, Ruo; Chai, Yaqin

2014-01-01

301

Ligation with nucleic acid sequence-based amplification.  

PubMed

This work presents a novel method for detecting nucleic acid targets using a ligation step along with an isothermal, exponential amplification step. We use an engineered ssDNA with two variable regions on the ends, allowing us to design the probe for optimal reaction kinetics and primer binding. This two-part probe is ligated by T4 DNA Ligase only when both parts bind adjacently to the target. The assay demonstrates that the expected 72-nt RNA product appears only when the synthetic target, T4 ligase, and both probe fragments are present during the ligation step. An extraneous 38-nt RNA product also appears due to linear amplification of unligated probe (P3), but its presence does not cause a false-positive result. In addition, 40 mmol/L KCl in the final amplification mix was found to be optimal. It was also found that increasing P5 in excess of P3 helped with ligation and reduced the extraneous 38-nt RNA product. The assay was also tested with a single nucleotide polymorphism target, changing one base at the ligation site. The assay was able to yield a negative signal despite only a single-base change. Finally, using P3 and P5 with longer binding sites results in increased overall sensitivity of the reaction, showing that increasing ligation efficiency can improve the assay overall. We believe that this method can be used effectively for a number of diagnostic assays. PMID:22449695

Ong, Carmichael; Tai, Warren; Sarma, Aartik; Opal, Steven M; Artenstein, Andrew W; Tripathi, Anubhav

2012-01-01

302

UNCORRECTED Microstructural characterization and isothermal oxidation behavior  

E-print Network

UNCORRECTED PROOF Microstructural characterization and isothermal oxidation behavior of hot of fine-scale microstructural characterization and isothermal oxidation of hot-pressed TiB2­ 10 10 wt.% Ti. The results of oxidation testing at 1200 °C for 12 h using a thermogravimetric analyzer illustrate near

Srivastava, Kumar Vaibhav

303

ISOTHERMAL AIR INGRESS VALIDATION EXPERIMENTS  

SciTech Connect

Idaho National Laboratory carried out air ingress experiments as part of validating computational fluid dynamics (CFD) calculations. An isothermal test loop was designed and set to understand the stratified-flow phenomenon, which is important as the initial air flow into the lower plenum of the very high temperature gas cooled reactor (VHTR) when a large break loss-of-coolant accident occurs. The unique flow characteristics were focused on the VHTR air-ingress accident, in particular, the flow visualization of the stratified flow in the inlet pipe to the vessel lower plenum of the General Atomic’s Gas Turbine-Modular Helium Reactor (GT-MHR). Brine and sucrose were used as heavy fluids, and water was used to represent a light fluid, which mimics a counter current flow due to the density difference between the stimulant fluids. The density ratios were changed between 0.87 and 0.98. This experiment clearly showed that a stratified flow between simulant fluids was established even for very small density differences. The CFD calculations were compared with experimental data. A grid sensitivity study on CFD models was also performed using the Richardson extrapolation and the grid convergence index method for the numerical accuracy of CFD calculations . As a result, the calculated current speed showed very good agreement with the experimental data, indicating that the current CFD methods are suitable for predicting density gradient stratified flow phenomena in the air-ingress accident.

Chang H Oh; Eung S Kim

2011-09-01

304

Biomedical Use of Isothermal Microcalorimeters  

PubMed Central

Isothermal microcalorimetry is becoming widely used for monitoring biological activities in vitro. Microcalorimeters are now able to measure heat production rates of less than a microwatt. As a result, metabolism and growth of relatively small numbers of cultured bacteria, protozoans, human cells and even small animals can be monitored continuously and extremely accurately at any chosen temperature. Dynamic effects on these organisms of changes in the culture environment—or of additions to it—are easily assessed over periods from hours to days. In addition microcalorimetry is a non-destructive method that does not require much sample preparation. It is also completely passive and thus allows subsequent evaluations of any kind on the undisturbed sample. In this review, we present a basic description of current microcalorimetry instruments and an overview of their use for various biomedical applications. These include detecting infections, evaluating effects of pharmaceutical or antimicrobial agents on cells, monitoring growth of cells harvested for tissue eingineering, and assessing medical and surgical device material physico-chemical stability and cellular biocompatibility. PMID:22163413

Braissant, Olivier; Wirz, Dieter; Göpfert, Beat; Daniels, A.U.

2010-01-01

305

The Isothermal Dendritic Growth Experiment  

NASA Technical Reports Server (NTRS)

The growth of dendrites is one of the commonly observed forms of solidification encountered when metals and alloys freeze under low thermal gradients, as occurs in most casting and welding processes. In engineering alloys, the details of the dendritic morphology directly relates to important material responses and properties. Of more generic interest, dendritic growth is also an archetypical problem in morphogenesis, where a complex pattern evolves from simple starting conditions. Thus, the physical understanding and mathematical description of how dendritic patterns emerge during the growth process are of interest to both scientists and engineers. The Isothermal Dendritic Growth Experiment (IDGE) is a basic science experiment designed to measure, for a fundamental test of theory, the kinetics and morphology of dendritic growth without complications induced by gravity-driven convection. The IDGE, a collaboration between Rensselaer Polytechnic Institute, in Troy NY, and NASA's Lewis Research Center (LeRC) was developed over a ten year period from a ground-based research program into a space flight experiment. Important to the success of this flight experiment was provision of in situ near-real-time teleoperations during the spaceflight experiment.

Glicksman, M. E.; Koss, M. B.; Malarik, D. C.

1998-01-01

306

Sequence independent amplification of DNA  

DOEpatents

The present invention is a rapid sequence-independent amplification procedure (SIA). Even minute amounts of DNA from various sources can be amplified independent of any sequence requirements of the DNA or any a priori knowledge of any sequence characteristics of the DNA to be amplified. This method allows, for example the sequence independent amplification of microdissected chromosomal material and the reliable construction of high quality fluorescent in situ hybridization (FISH) probes from YACs or from other sources. These probes can be used to localize YACs on metaphase chromosomes but also--with high efficiency--in interphase nuclei.

Bohlander, Stefan K. (Chicago, IL)

1998-01-01

307

Sequence independent amplification of DNA  

DOEpatents

The present invention is a rapid sequence-independent amplification procedure (SIA). Even minute amounts of DNA from various sources can be amplified independent of any sequence requirements of the DNA or any a priori knowledge of any sequence characteristics of the DNA to be amplified. This method allows, for example, the sequence independent amplification of microdissected chromosomal material and the reliable construction of high quality fluorescent in situ hybridization (FISH) probes from YACs or from other sources. These probes can be used to localize YACs on metaphase chromosomes but also--with high efficiency--in interphase nuclei. 25 figs.

Bohlander, S.K.

1998-03-24

308

Comprehensive human genome amplification using multiple displacement amplification  

Microsoft Academic Search

Fundamental to most genetic analysis is availability of genomic DNA of adequate quality and quantity. Because DNA yield from human samples is frequently limiting, much effort has been invested in developing methods for whole genome amplification (WGA) by random or degenerate oligonucleotide-primed PCR. However, existing WGA methods like degenerate oligonucleotide-primed PCR suffer from incomplete coverage and inadequate average DNA size.

Frank B. Dean; Seiyu Hosono; Linhua Fang; Xiaohong Wu; A. Fawad Faruqi; Patricia Bray-Ward; Zhenyu Sun; Qiuling Zong; Yuefen Du; Jing Du; Mark Driscoll; Wanmin Song; Stephen F. Kingsmore; Michael Egholm; Roger S. Lasken

2002-01-01

309

Isolation and amplification of mRNA within a simple microfluidic lab on a chip.  

PubMed

The major modules for realizing molecular biological assays in a micro-total analysis system (?TAS) were developed for the detection of pathogenic organisms. The specific focus was the isolation and amplification of eukaryotic mRNA within a simple, single-channel device for very low RNA concentrations that could then be integrated with detection modules. The hsp70 mRNA from Cryptosporidium parvum was used as a model analyte. Important points of study were surface chemistries within poly(methyl methacrylate) (PMMA) microfluidic channels that enabled specific and sensitive mRNA isolation and amplification reactions for very low mRNA concentrations. Optimal conditions were achieved when the channel surface was carboxylated via UV/ozone treatment followed by the immobilization of polyamidoamine (PAMAM) dendrimers on the surface, thus increasing the immobilization efficiency of the thymidine oligonucleotide, oligo(dT)25, and providing a reliable surface for the amplification reaction, importantly, without the need for blocking agents. Additional chemical modifications of the remaining active surface groups were studied to avoid nonspecific capturing of nucleic acids and hindering of the mRNA amplification at low RNA concentrations. Amplification of the mRNA was accomplished using nucleic acid sequence-based amplification (NASBA), an isothermal, primer-dependent technique. Positive controls consisting of previously generated NASBA amplicons could be diluted 10(15) fold and still result in successful on-chip reamplification. Finally, the successful isolation and amplification of mRNA from as few as 30 C. parvum oocysts was demonstrated directly on-chip and compared to benchtop devices. This is the first proof of successful mRNA isolation and NASBA-based amplification of mRNA within a simple microfluidic device in relevant analytical volumes. PMID:24328414

Reinholt, Sarah J; Behrent, Arne; Greene, Cassandra; Kalfe, Ayten; Baeumner, Antje J

2014-01-01

310

Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification.  

PubMed

Improved access to effective tests for diagnosing tuberculosis (TB) has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA) is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC) DNA in <20 minutes at 39 °C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110) and 20 fg (IS1081)were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9) and 86.1% (95%CI: 78.1, 94.1) respectively (n = 71). Specificities were 100% and 88.6% (95% CI: 80.8, 96.1) respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2) and 70.8% (95%CI: 62.9, 78.7) were obtained (n = 90). Specificities were 95.4 (95% CI: 92.3,98.1) and 88% (95% CI: 83.6, 92.4) respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB assays could be of use for integration into a point-of-care test for use in resource constrained settings. PMID:25118698

Boyle, David S; McNerney, Ruth; Teng Low, Hwee; Leader, Brandon Troy; Pérez-Osorio, Ailyn C; Meyer, Jessica C; O'Sullivan, Denise M; Brooks, David G; Piepenburg, Olaf; Forrest, Matthew S

2014-01-01

311

Rapid Detection of Mycobacterium tuberculosis by Recombinase Polymerase Amplification  

PubMed Central

Improved access to effective tests for diagnosing tuberculosis (TB) has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA) is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC) DNA in <20 minutes at 39°C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110) and 20 fg (IS1081)were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9) and 86.1% (95%CI: 78.1, 94.1) respectively (n?=?71). Specificities were 100% and 88.6% (95% CI: 80.8, 96.1) respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2) and 70.8% (95%CI: 62.9, 78.7) were obtained (n?=?90). Specificities were 95.4 (95% CI: 92.3,98.1) and 88% (95% CI: 83.6, 92.4) respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB assays could be of use for integration into a point-of-care test for use in resource constrained settings. PMID:25118698

Teng Low, Hwee; Leader, Brandon Troy; Perez-Osorio, Ailyn C.; Meyer, Jessica C.; O'Sullivan, Denise M.; Brooks, David G.; Piepenburg, Olaf; Forrest, Matthew S.

2014-01-01

312

Adsorption Isotherms and Surface Reaction Kinetics  

ERIC Educational Resources Information Center

Explains an error that occurs in calculating the conditions for a maximum value of a rate expression for a bimolecular reaction. The rate expression is derived using the Langmuir adsorption isotherm to relate gas pressures and corresponding surface coverages. (GS)

Lobo, L. S.; Bernardo, C. A.

1974-01-01

313

Double regenerative amplification of picosecond pulses  

NASA Astrophysics Data System (ADS)

An double Nd:YAG regenerative amplification picosecond pulse laser is demonstrated under the semiconductor saturable absorption mirror(SESAM) mode-locking technology and regenerative amplification technology, using BBO crystal as PC electro-optic crystal. The laser obtained is 20.71ps pulse width at 10 KHz repetition rate, and the energy power is up to 4W which is much larger than the system without pre-amplification. This result will lay a foundation for the following amplification.

Bai, Zhen-ao; Chen, Li-yuan; Bai, Zhen-xu; Chen, Meng; Li, Gang

2012-04-01

314

ON THE ISOTHERMALITY OF SOLAR PLASMAS  

SciTech Connect

Recent measurements have shown that the quiet unstructured solar corona observed at the solar limb is close to isothermal, at a temperature that does not appear to change over wide areas or with time. Some individual active region loop structures have also been found to be nearly isothermal both along their axis and across their cross section. Even a complex active region observed at the solar limb has been found to be composed of three distinct isothermal plasmas. If confirmed, these results would pose formidable challenges to the current theoretical understanding of the thermal structure and heating of the solar corona. For example, no current theoretical model can explain the excess densities and lifetimes of many observed loops if the loops are in fact isothermal. All of these measurements are based on the so-called emission measure (EM) diagnostic technique that is applied to a set of optically thin lines under the assumption of isothermal plasma. It provides simultaneous measurement of both the temperature and EM. In this work, we develop a new method to quantify the uncertainties in the technique and to rigorously assess its ability to discriminate between isothermal and multithermal plasmas. We define a formal measure of the uncertainty in the EM diagnostic technique that can easily be applied to real data. We here apply it to synthetic data based on a variety of assumed plasma thermal distributions and develop a method to quantitatively assess the degree of multithermality of a plasma.

Landi, E. [Department of Atmospheric, Oceanic, and Space Sciences, University of Michigan, Ann Arbor, MI 48109 (United States); Klimchuk, J. A. [NASA Goddard Space Flight Center, Greenbelt, MD 20771 (United States)

2010-11-01

315

Simultaneous DNA amplification and detection using a pH-sensing semiconductor system.  

PubMed

We developed an integrated chip for real-time amplification and detection of nucleic acid using pH-sensing complementary metal-oxide semiconductor (CMOS) technology. Here we show an amplification-coupled detection method for directly measuring released hydrogen ions during nucleotide incorporation rather than relying on indirect measurements such as fluorescent dyes. This is a label-free, non-optical, real-time method for detecting and quantifying target sequences by monitoring pH signatures of native amplification chemistries. The chip has ion-sensitive field effect transistor (ISFET) sensors, temperature sensors, resistive heating, signal processing and control circuitry all integrated to create a full system-on-chip platform. We evaluated the platform using two amplification strategies: PCR and isothermal amplification. Using this platform, we genotyped and discriminated unique single-nucleotide polymorphism (SNP) variants of the cytochrome P450 family from crude human saliva. We anticipate this semiconductor technology will enable the creation of devices for cost-effective, portable and scalable real-time nucleic acid analysis. PMID:23749303

Toumazou, Christofer; Shepherd, Leila M; Reed, Samuel C; Chen, Ginny I; Patel, Alpesh; Garner, David M; Wang, Chan-Ju A; Ou, Chung-Pei; Amin-Desai, Krishna; Athanasiou, Panteleimon; Bai, Hua; Brizido, Ines M Q; Caldwell, Benjamin; Coomber-Alford, Daniel; Georgiou, Pantelis; Jordan, Karen S; Joyce, John C; La Mura, Maurizio; Morley, Daniel; Sathyavruthan, Sreekala; Temelso, Sara; Thomas, Risha E; Zhang, Linglan

2013-07-01

316

Frequency domain optical parametric amplification  

PubMed Central

Today’s ultrafast lasers operate at the physical limits of optical materials to reach extreme performances. Amplification of single-cycle laser pulses with their corresponding octave-spanning spectra still remains a formidable challenge since the universal dilemma of gain narrowing sets limits for both real level pumped amplifiers as well as parametric amplifiers. We demonstrate that employing parametric amplification in the frequency domain rather than in time domain opens up new design opportunities for ultrafast laser science, with the potential to generate single-cycle multi-terawatt pulses. Fundamental restrictions arising from phase mismatch and damage threshold of nonlinear laser crystals are not only circumvented but also exploited to produce a synergy between increased seed spectrum and increased pump energy. This concept was successfully demonstrated by generating carrier envelope phase stable, 1.43?mJ two-cycle pulses at 1.8??m wavelength. PMID:24805968

Schmidt, Bruno E.; Thiré, Nicolas; Boivin, Maxime; Laramée, Antoine; Poitras, François; Lebrun, Guy; Ozaki, Tsuneyuki; Ibrahim, Heide; Légaré, François

2014-01-01

317

ExCyto PCR Amplification  

Microsoft Academic Search

BackgroundExCyto PCR cells provide a novel and cost effective means to amplify DNA transformed into competent bacterial cells. ExCyto PCR uses host E. coli with a chromosomally integrated gene encoding a thermostable DNA polymerase to accomplish robust, hot-start PCR amplification of cloned sequences without addition of exogenous enzyme.ResultsBecause the thermostable DNA polymerase is stably integrated into the bacterial chromosome, ExCyto

Vinay Dhodda; Ronald Godiska; Jeffrey D. Vanwye; David Mead; Rebecca Hochstein; Lynne Sheets; Sarah Vande Zande; Chris Niebauer; Douglas L. Crawford; Marjorie F. Oleksiak; Ching-Hong Yang

2010-01-01

318

Thermogravimetric studies of non-stoichiometric cerium oxides under isothermal and quasi-isothermal conditions  

Microsoft Academic Search

Quasi-isothermal thermogravimetry is a new technique in which the programmed heating of a furnace automatically ceases when the rate of a reaction taking place in a sample, which is indicated by the DTG-signal, exceeds a preset limit. In this way reactions can be studied under nearly isothermal conditions. In this paper the data obtained using this method during oxidation and

O. Toft Sørensen

1978-01-01

319

Rapid DNA detection by beacon-assisted detection amplification.  

PubMed

This protocol describes a new and rapid isothermal reaction process designed to amplify and detect a specific DNA sequence in purified DNA extracted from cultured cells. The protocol uses a DNA nanomachine that comprises two molecular switches that function in concert to isothermally amplify and detect a DNA target. First, a molecular beacon detection switch is 'activated' only if a DNA target sequence is present. A DNA primer and DNA polymerase are used to lock the beacon in an activated conformation. Second, an amplification and signal-transduction switch is initiated following successful activation. A nicking endonuclease and the DNA polymerase are used to replicate the DNA target. Both switches operate simultaneously at 40 °C in a single reaction to rapidly generate multiple copies of the DNA target in a cyclic polymerization reaction. This protocol enables femtomole amounts of a DNA target to be reproducibly amplified and detected in <40 min. We demonstrate the successful use of this protocol in assays containing synthetic DNA components and purified DNA extracted from biological samples. PMID:21637197

Connolly, Ashley R; Trau, Matt

2011-06-01

320

Loop-mediated amplification accelerated by stem primers.  

PubMed

Isothermal nucleic acid amplifications (iNAATs) have become an important alternative to PCR for in vitro molecular diagnostics in all fields. Amongst iNAATs Loop-mediated amplification (LAMP) has gained much attention over the last decade because of the simplicity of hardware requirements. LAMP demonstrates performance equivalent to that of PCR, but its application has been limited by the challenging primer design. The design of six primers in LAMP requires a selection of eight priming sites with significant restrictions imposed on their respective positioning and orientation. In order to relieve primer design constraints we propose an alternative approach which uses Stem primers instead of Loop primers and demonstrate the application of STEM-LAMP in assaying for Clostridium difficile, Listeria monocytogenes and HIV. Stem primers used in LAMP in combination with loop-generating and displacement primers gave significant benefits in speed and sensitivity, similar to those offered by Loop primers, while offering additional options of forward and reverse orientations, multiplexing, use in conjunction with Loop primers or even omission of one or two displacement primers, where necessary. Stem primers represent a valuable alternative to Loop primers and an additional tool for IVD assay development by offering more choices for primer design at the same time increasing assay speed, sensitivity, and reproducibility. PMID:22272122

Gandelman, Olga; Jackson, Rebecca; Kiddle, Guy; Tisi, Laurence

2011-01-01

321

Linear nicking endonuclease-mediated strand-displacement DNA amplification.  

PubMed

We describe a method for linear isothermal DNA amplification using nicking endonuclease-mediated strand displacement by a DNA polymerase. The nicking of one strand of a DNA target by the endonuclease produces a primer for the polymerase to initiate synthesis. As the polymerization proceeds, the downstream strand is displaced into a single-stranded form while the nicking site is also regenerated. The combined continuous repetitive action of nicking by the endonuclease and strand-displacement synthesis by the polymerase results in linear amplification of one strand of the DNA molecule. We demonstrate that DNA templates up to 5000 nucleotides can be linearly amplified using a nicking endonuclease with 7-bp recognition sequence and Sequenase version 2.0 in the presence of single-stranded DNA binding proteins. We also show that a mixture of three templates of 500, 1000, and 5000 nucleotides in length is linearly amplified with the original molar ratios of the templates preserved. Moreover, we demonstrate that a complex library of hydrodynamically sheared genomic DNA from bacteriophage lambda can be amplified linearly. PMID:21342654

Joneja, Aric; Huang, Xiaohua

2011-07-01

322

Equipment-Free Incubation of Recombinase Polymerase Amplification Reactions Using Body Heat  

PubMed Central

The development of isothermal amplification platforms for nucleic acid detection has the potential to increase access to molecular diagnostics in low resource settings; however, simple, low-cost methods for heating samples are required to perform reactions. In this study, we demonstrated that human body heat may be harnessed to incubate recombinase polymerase amplification (RPA) reactions for isothermal amplification of HIV-1 DNA. After measuring the temperature of mock reactions at 4 body locations, the axilla was chosen as the ideal site for comfortable, convenient incubation. Using commonly available materials, 3 methods for securing RPA reactions to the body were characterized. Finally, RPA reactions were incubated using body heat while control RPA reactions were incubated in a heat block. At room temperature, all reactions with 10 copies of HIV-1 DNA and 90% of reactions with 100 copies of HIV-1 DNA tested positive when incubated with body heat. In a cold room with an ambient temperature of 10 degrees Celsius, all reactions containing 10 copies or 100 copies of HIV-1 DNA tested positive when incubated with body heat. These results suggest that human body heat may provide an extremely low-cost solution for incubating RPA reactions in low resource settings. PMID:25372030

Richards-Kortum, Rebecca

2014-01-01

323

Equipment-free incubation of recombinase polymerase amplification reactions using body heat.  

PubMed

The development of isothermal amplification platforms for nucleic acid detection has the potential to increase access to molecular diagnostics in low resource settings; however, simple, low-cost methods for heating samples are required to perform reactions. In this study, we demonstrated that human body heat may be harnessed to incubate recombinase polymerase amplification (RPA) reactions for isothermal amplification of HIV-1 DNA. After measuring the temperature of mock reactions at 4 body locations, the axilla was chosen as the ideal site for comfortable, convenient incubation. Using commonly available materials, 3 methods for securing RPA reactions to the body were characterized. Finally, RPA reactions were incubated using body heat while control RPA reactions were incubated in a heat block. At room temperature, all reactions with 10 copies of HIV-1 DNA and 90% of reactions with 100 copies of HIV-1 DNA tested positive when incubated with body heat. In a cold room with an ambient temperature of 10 degrees Celsius, all reactions containing 10 copies or 100 copies of HIV-1 DNA tested positive when incubated with body heat. These results suggest that human body heat may provide an extremely low-cost solution for incubating RPA reactions in low resource settings. PMID:25372030

Crannell, Zachary Austin; Rohrman, Brittany; Richards-Kortum, Rebecca

2014-01-01

324

Detection of HIV cDNA point mutations with rolling-circle amplification arrays.  

PubMed

In this paper we describe an isothermal rolling-circle amplification (RCA) protocol to detect gene point mutations on chips. The method is based on an allele-specific oligonucleotide circularization mediated by a special DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and HIV cDNA gene. Mismatches around the ligation site can prevent probe circularization. The circularized probe (C-probe) can be amplified by rolling circle amplification to generate multimeric singlestranded DNA (ssDNA) under isothermal conditions. There are four sequence regions to bind respectively with fluorescent probe, RCA primer, solid probe and HIV cDNA template in the C-probe which we designed. These ssDNA products are hybridized with fluorescent probes and solid probes which are immobilized on a glass slide composing a regular microarray pattern. The fluorescence signals can be monitored by a scanner in the presence of HIV cDNA templates, whereas the probes cannot be circularized and signal of fluorescence cannot be found. The RCA array has capability of high-throughput detection of the point mutation and the single-nucleotide polymorphism (SNP).The development of C-probe-based technologies offers a promising prospect for situ detection, microarray, molecular diagnosis, single nucleotide polymorphism, and whole genome amplification. PMID:20335932

Wu, Lingwei; Liu, Quanjun; Wu, Zhongwei; Lu, Zuhong

2010-02-01

325

Measurement of Ferroelectric Polarization along Critical Isotherm  

NASA Astrophysics Data System (ADS)

A simple method is proposed for measuring the polarizations along the critical isotherm. A characteristic of the method is that the quasi-isothermal polarization can be measured while admitting the electrocaloric effect. The principle is that thermal equilibrium between a sample and its thermal bath can be achieved for a particular field E (i.e., P) by continuous application of a square-wave field of amplitude E. The results of the application of the method to triglycine sulfate are shown and are compared with the adiabatic results.

Imai, Kiyoyasu

1981-06-01

326

Evaporation Induced Isothermal Crystallization of Silicate Melt  

NASA Astrophysics Data System (ADS)

In order to investigate and role of evaporation and crystallization kinetics for silicate melt, isothermal vacuum experiments were carried out in the system MgO-SiO2. Due to successive evaporation, melt crystallized olivine at a fixed temperature. The evaporation rates and bulk chemical composition of residues varied with time, and reached a steady state. The pressure-composition phase diagram for the system at a fixed temperature well explains the experimental results. The results suggest a possibility of isothermal formation of chondrules (and some CAIs) at low pressures where evaporation takes place continuously.

Nagahara, H.

1996-03-01

327

Self-assembled DNA nanostructures prepared by rolling circle amplification for the delivery of siRNA conjugates.  

PubMed

Inspired by the isothermal enzymatic process of rolling circle amplification (RCA) of DNA strands, we have developed a system to achieve more than a 200-fold increase in the synthesis of DNA nanostructures using a single-stranded circular DNA template. The amplified DNA nanostructures have shown efficient delivery of folic acid (FA) conjugated siRNAs into KB cells with a dose dependent gene silencing. PMID:24967959

Hong, Cheol Am; Jang, Bora; Jeong, Eun Hye; Jeong, Hansaem; Lee, Hyukjin

2014-10-01

328

Dual channel sensitive detection of hsa-miR-21 based on rolling circle amplification and quantum dots tagging.  

PubMed

An isothermal, highly sensitive and specific assay for the detection of hsa-miR-21 with the integration of QDs tagging and rolling circle amplification was offered. In addition, a dual channel strategy for miRNA detection was proposed: anodic stripping voltammetry (ASV) and fluorescent method were both performed for the final Cd2+ signal readout. The designed strategy exhibited good specificity to hsa-miR-21 and presented comparable detection results by detection methods. PMID:24734513

Wangt, Dan-Chen; Hu, Li-Hui; Zhou, Yu-Hui; Huang, Yu-Ting; Li, Xinhua; Zhu, Jun-Jie

2014-04-01

329

A T7 exonuclease-assisted cyclic enzymatic amplification method coupled with rolling circle amplification: a dual-amplification strategy for sensitive and selective microRNA detection.  

PubMed

A T7 exonuclease-assisted cyclic enzymatic amplification method (CEAM) was combined with rolling circle amplification (RCA) to develop a RCA-CEAM dual amplification method for ultrasensitive detection of microRNA with excellent selectivity. PMID:24382471

Cui, Liang; Zhu, Zhi; Lin, Ninghang; Zhang, Huimin; Guan, Zhichao; Yang, Chaoyong James

2014-02-14

330

Chemical Amplification with Encapsulated Reagents  

NASA Technical Reports Server (NTRS)

Autocatalysis and chemical amplification are characteristic properties of living systems, and they give rise to behaviors such as increased sensitivity, responsiveness, and self-replication. Here we report a synthetic system in which a unique form of compartmentalization leads to nonlinear, autocatalytic behavior. The compartment is a reversibly formed capsule in which a reagent is sequestered. Reaction products displace the reagent from the capsule into solution and the reaction rate is accelerated. The resulting self-regulation is sensitive to the highly selective molecular recognition properties of the capsule.

Chen, Jian; Koemer, Steffi; Craig, Stephen; Lin, Shirley; Rudkevich, Dmitry M.; Rebek, Julius, Jr.

2002-01-01

331

Dynamics and Control of DNA Sequence Amplification  

E-print Network

DNA amplification is the process of replication of a specified DNA sequence \\emph{in vitro} through time-dependent manipulation of its external environment. A theoretical framework for determination of the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is presented based on first-principles biophysical modeling and control theory. Amplification of DNA is formulated as a problem in control theory with optimal solutions that can differ considerably from strategies typically used in practice. Using the Polymerase Chain Reaction (PCR) as an example, sequence-dependent biophysical models for DNA amplification are cast as control systems, wherein the dynamics of the reaction are controlled by a manipulated input variable. Using these control systems, we demonstrate that there exists an optimal temperature cycling strategy for geometric amplification of any DNA sequence and formulate optimal control problems that can be used to derive the optimal tempe...

Marimuthu, Karthikeyan

2014-01-01

332

The isothermal sulphation reactions of natural sorbents  

Microsoft Academic Search

The chemical reaction between sulphur dioxide and calcined limestone and dolomite particles was investigated using a thermogravimetric analyser. The samples used in this study originated from different parts of Turkey. Sulphation reactions were conducted under isothermal conditions in a gaseous mixture consisting of 15 vol% CO2, 0.35 vol% SO2 and a balance of dry air by volume. Before sulphation all

Ay?egül Er?oy-Meriçboyu; Sadriye Küçükbayrak

1995-01-01

333

Isothermal Titration Calorimetry in the Student Laboratory  

ERIC Educational Resources Information Center

Isothermal titration calorimetry (ITC) is the measurement of the heat produced by the stepwise addition of one substance to another. It is a common experimental technique, for example, in pharmaceutical science, to measure equilibrium constants and reaction enthalpies. We describe a stirring device and an injection pump that can be used with a…

Wadso, Lars; Li, Yujing; Li, Xi

2011-01-01

334

Apparatus to Measure Adiabatic and Isothermal Processes.  

ERIC Educational Resources Information Center

Describes a simple manual apparatus designed to serve as an effective demonstration of the differences between isothermal and adiabatic processes for the general or elementary physics student. Enables students to verify Boyle's law for slow processes and identify the departure from this law for rapid processes and can also be used to give a clear…

Lamb, D. W.; White, G. M.

1996-01-01

335

Performance Characteristics of an Isothermal Freeze Valve  

SciTech Connect

This document discusses performance characteristics of an isothermal freeze valve. A freeze valve has been specified for draining the DWPF melter at the end of its lifetime. Two freeze valve designs have been evaluated on the Small Cylindrical Melter-2 (SCM-2). In order to size the DWPF freeze valve, the basic principles governing freeze valve behavior need to be identified and understood.

Hailey, A.E.

2001-08-22

336

Thermocompression Engine Cycle with Isothermal Expansion  

Microsoft Academic Search

An engine cycle based on thermocompression, isothermal expansion, and isobaric reduction in volume is presented in this study. It is a closed cycle utilizing an external heat source; thus application to solar, geothermal, and waste heat utilization may be possible. One aspect of implementing the cycle allows for a heat exchanger located outside the working volume of the engine. This

RICHARD B. PETERSON

1998-01-01

337

Dynamics and control of DNA sequence amplification.  

PubMed

DNA amplification is the process of replication of a specified DNA sequence in vitro through time-dependent manipulation of its external environment. A theoretical framework for determination of the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is presented based on first-principles biophysical modeling and control theory. Amplification of DNA is formulated as a problem in control theory with optimal solutions that can differ considerably from strategies typically used in practice. Using the Polymerase Chain Reaction as an example, sequence-dependent biophysical models for DNA amplification are cast as control systems, wherein the dynamics of the reaction are controlled by a manipulated input variable. Using these control systems, we demonstrate that there exists an optimal temperature cycling strategy for geometric amplification of any DNA sequence and formulate optimal control problems that can be used to derive the optimal temperature profile. Strategies for the optimal synthesis of the DNA amplification control trajectory are proposed. Analogous methods can be used to formulate control problems for more advanced amplification objectives corresponding to the design of new types of DNA amplification reactions. PMID:25362284

Marimuthu, Karthikeyan; Chakrabarti, Raj

2014-10-28

338

Dynamics and control of DNA sequence amplification  

NASA Astrophysics Data System (ADS)

DNA amplification is the process of replication of a specified DNA sequence in vitro through time-dependent manipulation of its external environment. A theoretical framework for determination of the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is presented based on first-principles biophysical modeling and control theory. Amplification of DNA is formulated as a problem in control theory with optimal solutions that can differ considerably from strategies typically used in practice. Using the Polymerase Chain Reaction as an example, sequence-dependent biophysical models for DNA amplification are cast as control systems, wherein the dynamics of the reaction are controlled by a manipulated input variable. Using these control systems, we demonstrate that there exists an optimal temperature cycling strategy for geometric amplification of any DNA sequence and formulate optimal control problems that can be used to derive the optimal temperature profile. Strategies for the optimal synthesis of the DNA amplification control trajectory are proposed. Analogous methods can be used to formulate control problems for more advanced amplification objectives corresponding to the design of new types of DNA amplification reactions.

Marimuthu, Karthikeyan; Chakrabarti, Raj

2014-10-01

339

Trophic amplification of climate warming.  

PubMed

Ecosystems can alternate suddenly between contrasting persistent states due to internal processes or external drivers. It is important to understand the mechanisms by which these shifts occur, especially in exploited ecosystems. There have been several abrupt marine ecosystem shifts attributed either to fishing, recent climate change or a combination of these two drivers. We show that temperature has been an important driver of the trophodynamics of the North Sea, a heavily fished marine ecosystem, for nearly 50 years and that a recent pronounced change in temperature established a new ecosystem dynamic regime through a series of internal mechanisms. Using an end-to-end ecosystem approach that included primary producers, primary, secondary and tertiary consumers, and detritivores, we found that temperature modified the relationships among species through nonlinearities in the ecosystem involving ecological thresholds and trophic amplifications. Trophic amplification provides an alternative mechanism to positive feedback to drive an ecosystem towards a new dynamic regime, which in this case favours jellyfish in the plankton and decapods and detritivores in the benthos. Although overfishing is often held responsible for marine ecosystem degeneration, temperature can clearly bring about similar effects. Our results are relevant to ecosystem-based fisheries management (EBFM), seen as the way forward to manage exploited marine ecosystems. PMID:19740882

Kirby, Richard R; Beaugrand, Gregory

2009-12-01

340

Integrated Microcapillary for Sample-to-Answer Nucleic Acid Pretreatment, Amplification, and Detection.  

PubMed

This work develops an integrated microcapillary-based loop-mediated isothermal amplification (icLAMP) containing preloaded reagents and DNA extraction card, allowing for sample-to-answer screening of single nucleotide polymorphisms (SNPs) typing of the CYP2C19 gene from untreated blood samples with minimal user operation. With all reagents and the DNA extraction card preloaded inside the capillary, this icLAMP system can achieve on-site pretreatment, extraction, amplification, and detection of nucleic acids within 150 min, without the requirement for advanced instruments. As icLAMP technology carries many advantages such as disposability, easy operation, low cost, and reduced cross contamination and biohazard risks, we expect this system to have a great impact on point-of-care (POC) nucleic acid detection. PMID:25242282

Zhang, Lu; Zhang, Yi; Wang, Chunyan; Feng, Qiang; Fan, Fei; Zhang, Guojun; Kang, Xixiong; Qin, Xuzhen; Sun, Jiashu; Li, Yinghui; Jiang, Xingyu

2014-10-21

341

Rolling circle amplification: applications in nanotechnology and biodetection with functional nucleic acids.  

PubMed

Rolling circle amplification (RCA) is an isothermal, enzymatic process mediated by certain DNA polymerases in which long single-stranded (ss) DNA molecules are synthesized on a short circular ssDNA template by using a single DNA primer. A method traditionally used for ultrasensitive DNA detection in areas of genomics and diagnostics, RCA has been used more recently to generate large-scale DNA templates for the creation of periodic nanoassemblies. Various RCA strategies have also been developed for the production of repetitive sequences of DNA aptamers and DNAzymes as detection platforms for small molecules and proteins. In this way, RCA is rapidly becoming a highly versatile DNA amplification tool with wide-ranging applications in genomics, proteomics, diagnosis, biosensing, drug discovery, and nanotechnology. PMID:18680110

Zhao, Weian; Ali, M Monsur; Brook, Michael A; Li, Yingfu

2008-01-01

342

A target-triggered exponential amplification-based DNAzyme biosensor for ultrasensitive detection of folate receptors.  

PubMed

We develop a new method for ultrasensitive detection of folate receptors (FRs) using a target-triggered isothermally exponential amplification reaction (EXPAR)-based DNAzyme biosensor. This method exhibits excellent specificity and high sensitivity with a detection limit as low as 0.23 fM and a large dynamic range of 6 orders of magnitude from 1 fM to 1 nM. It might be further applied for the detection of various small molecule-binding proteins by simply changing the linked small molecule moiety of the hairpin probes. PMID:25350483

Wang, Li-Juan; Zhang, Yan; Zhang, Chun-Yang

2014-12-18

343

Instrument-free nucleic acid amplification assays for global health settings  

PubMed Central

Many infectious diseases that affect global health are most accurately diagnosed through nucleic acid amplification and detection. However, existing nucleic acid amplification tests are too expensive and complex for most low-resource settings. The small numbers of centralized laboratories that exist in developing countries tend to be in urban areas and primarily cater to the affluent. In contrast, rural area health care facilities commonly have only basic equipment and health workers have limited training and little ability to maintain equipment and handle reagents.1 Reliable electric power is a common infrastructure shortfall. In this paper, we discuss a practical approach to the design and development of non-instrumented molecular diagnostic tests that exploit the benefits of isothermal amplification strategies. We identify modular instrument-free technologies for sample collection, sample preparation, amplification, heating, and detection. By appropriately selecting and integrating these instrument-free modules, we envision development of an easy to use, infrastructure independent diagnostic test that will enable increased use of highly accurate molecular diagnostics at the point of care in low-resource settings. PMID:25089171

LaBarre, Paul; Boyle, David; Hawkins, Kenneth; Weigl, Bernhard

2014-01-01

344

Tsunami Amplification due to Focusing  

NASA Astrophysics Data System (ADS)

Tsunami runup measurements over the periphery of the Pacific Ocean after the devastating Great Japan tsunami of 11 March 2011 showed considerable variation in far-field and near-field impact. This variation of tsunami impact have been attributed to either directivity of the source or by local topographic effects. Directivity arguments alone, however, cannot explain the complexity of the radiated patterns in oceans with trenches and seamounts. Berry (2007, Proc. R. Soc. Lond. A 463, 3055-3071) discovered how such underwater features may concentrate tsunamis into cusped caustics and thus cause large local amplifications at specific focal points. Here, we examine focusing and local amplification, not by considering the effects of underwater diffractive lenses, but by considering the details of the dipole nature of the initial profile, and propose that certain regions of coastline are more at-risk, not simply because of directivity but because typical tsunami deformations create focal regions where abnormal tsunami wave height can be registered (Marchuk and Titov, 1989, Proc. IUGG/IOC International Tsunami Symposium, Novosibirsk, USSR). In this work, we present a new general analytical solution of the linear shallow-water wave equation for the propagation of a finite-crest-length source over a constant depth without any restriction on the initial profile. Unlike the analytical solution of Carrier and Yeh (2005, Comp. Mod. Eng. & Sci. 10(2), 113-121) which was restricted to initial conditions with Gaussian profiles and involved approximation, our solution is not only exact, but also general and allows the use of realistic initial waveform such as N-waves as defined by Tadepalli and Synolakis (1994, Proc. R. Soc. Lond. A 445, 99-112). We then verify our analytical solution for several typical wave profiles, both with the NOAA tsunami forecast model MOST (Titov and Synolakis, 1998, J. Waterw. Port Coast. Ocean Eng. 124(4), 157-171) which is validated and verified through (Synolakis et al., 2008, Pure Appl. Geophys. 165(11-12), 2197-2228), and with a Boussinesq model, to illustrate the role focusing can play for different initial conditions, and to show the robust nature of focusing with respect to dispersion. We also show how the focusing effect might have played a role in the 17 July 1998 Papua New Guinea and 17 July 2006 Java events, and also the 11 March 2011 Great Japan earthquake and tsunami. Our results strongly imply that focusing increases the shoreline amplification of the tsunami.; Schematic of focusing; initial displacement (upper left), wave evolution (upper right, lower left), maximum wave amplitude with focusing (lower right).

Moore, C. W.; Kanoglu, U.; Titov, V. V.; Aydin, B.; Spillane, M. C.; Synolakis, C. E.

2012-12-01

345

Inhaltsverzeichnis 1 Isothermal and Nonisothermal Multiphase Multicomponent Pro-  

E-print Network

Inhaltsverzeichnis 1 Isothermal and Nonisothermal Multiphase Multicomponent Pro- cesses Equations . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.2.1 General Multiphase Flow Di#11 1: Isothermal and Nonisothermal Multiphase Multicomponent Processes in the Subsurface Rainer Helmig

Cirpka, Olaf Arie

346

Gene synthesis by circular assembly amplification  

E-print Network

error-containing DNA10. We developed a one-cycle gene synthesis method that substan- tially improvesGene synthesis by circular assembly amplification Duhee Bang & George M Church Here we report the development of a gene-synthesis technology, circular assembly amplification. In this approach, we first

Church, George M.

347

Amplification, Technology, and Cochlear Implants for Infants.  

ERIC Educational Resources Information Center

Early amplification is crucial to efficient habilitation and development of oral communication skills in hearing-impaired infants. Initial evaluation and fitting of amplification is a joint effort by the audiologist, therapist, and parents, whether the child uses traditional hearing aids or cochlear implants, and should be supplemented by a…

Adam, Arlie J.

1993-01-01

348

Chemical amplification based on fluid partitioning  

Microsoft Academic Search

A system for nucleic acid amplification of a sample comprises partitioning the sample into partitioned sections and performing PCR on the partitioned sections of the sample. Another embodiment of the invention provides a system for nucleic acid amplification and detection of a sample comprising partitioning the sample into partitioned sections, performing PCR on the partitioned sections of the sample, and

Brian L. Anderson; Colston Jr. Billy W; Chris Elkin

2006-01-01

349

Third Sound Amplification and Detailed Balance  

SciTech Connect

Condensation of atoms from the vapor into a third sound resonance is expected to be capable of acoustic amplification. This results from normal to superfluid conversion that coherently accommodates atoms into the third sound velocity field. Consideration of third sound in light of the equilibrium detailed balance between vapor particles and the superfluid film provides further evidence that acoustic amplification is attainable.

Eddinger, J. D.; Ellis, F. M. [Department of Physics, Wesleyan University, Middletown, CT 06459 (United States)

2006-09-07

350

The prediction of moisture sorption isotherms for dairy powders  

Microsoft Academic Search

Moisture sorption isotherms were measured for whey protein isolate, high micellar casein and a milk protein concentrate powder. No temperature dependence was observed over the temperature range of 4–37°C. At 50°C the powders absorbed less moisture than observed at the lower temperatures. These isotherms were used to predict the isotherms for freeze-dried amorphous lactose\\/casein\\/whey protein powders. An isotherm for micellar

Kylie D. Foster; John E. Bronlund

2005-01-01

351

On the Isothermality of Solar Plasmas  

NASA Technical Reports Server (NTRS)

Recent measurements have shown that the quiet unstructured solar corona observed at the solar limb is close to isothermal, at a temperature that does not appear to change over wide areas or with time. Some in dividual active loop structures have also been found to be nearly iso thermal both along their axis and across their cross-section. Even a complex active region observed at the solar limb has been found to be composed of three distinct isothermal plasmas. If confirmed, these r esults would pose formidable challenges to the current theoretical understanding of the thermal structure and heating of the solar corona. For example, no current theoretical model can explain the excess dens ities and lifetimes of many observed loops if the loops are in fact i sothermal. All of these measurements are based on the so-called emiss ion measure (EM) diagnostic technique that is applied to a set of opt ically thin lines under the assumption of isothermal plasma. It provi des simultaneous measurement of both the temperature and EM. However, no study has ever been carried out to quantify the uncertainties in the technique and to rigorously assess its ability to discriminate bet ween isothermal and multithermal plasmas. Such a study is the topic o f the present work. We define a formal measure of the uncertainty in the EM diagnostic technique that can easily be applied to real data. We here apply it to synthetic data based on a variety of assumed plas ma thermal distributions, and develop a method to quantitatively asse ss the degree of multithermality of a plasma.

Landi, E.; Klimchuk, J. A.

2010-01-01

352

Isothermal isobaric molecular dynamics simulation of water  

Microsoft Academic Search

A series of isothermal isobaric molecular dynamics simulation are carried out on a system made up of 210 molecules at normal temperature and pressure (T=298K, P=1bar) and at high pressure (T=298K, P=2.1kbar). The physical model is based upon the tetrahedral cluster architecture composed of one central molecule (20% of the sample) and four outer molecules (80%). This theoretical approach of

M. Amrani; D. Bendeddouch; D. Bormann; A. Krallafa

2008-01-01

353

Post-Fragmentation Whole Genome Amplification-Based Method  

NASA Technical Reports Server (NTRS)

This innovation is derived from a proprietary amplification scheme that is based upon random fragmentation of the genome into a series of short, overlapping templates. The resulting shorter DNA strands (<400 bp) constitute a library of DNA fragments with defined 3 and 5 termini. Specific primers to these termini are then used to isothermally amplify this library into potentially unlimited quantities that can be used immediately for multiple downstream applications including gel eletrophoresis, quantitative polymerase chain reaction (QPCR), comparative genomic hybridization microarray, SNP analysis, and sequencing. The standard reaction can be performed with minimal hands-on time, and can produce amplified DNA in as little as three hours. Post-fragmentation whole genome amplification-based technology provides a robust and accurate method of amplifying femtogram levels of starting material into microgram yields with no detectable allele bias. The amplified DNA also facilitates the preservation of samples (spacecraft samples) by amplifying scarce amounts of template DNA into microgram concentrations in just a few hours. Based on further optimization of this technology, this could be a feasible technology to use in sample preservation for potential future sample return missions. The research and technology development described here can be pivotal in dealing with backward/forward biological contamination from planetary missions. Such efforts rely heavily on an increasing understanding of the burden and diversity of microorganisms present on spacecraft surfaces throughout assembly and testing. The development and implementation of these technologies could significantly improve the comprehensiveness and resolving power of spacecraft-associated microbial population censuses, and are important to the continued evolution and advancement of planetary protection capabilities. Current molecular procedures for assaying spacecraft-associated microbial burden and diversity have inherent sample loss issues at practically every step, particularly nucleic acid extraction. In engineering a molecular means of amplifying nucleic acids directly from single cells in their native state within the sample matrix, this innovation has circumvented entirely the need for DNA extraction regimes in the sample processing scheme.

Benardini, James; LaDuc, Myron T.; Langmore, John

2011-01-01

354

Spherical Isothermal Self-Similar Shock Flows  

E-print Network

We explore self-similar dynamical processes in a spherical isothermal self-gravitational fluid with an emphasis on shocks and outline astrophysical applications of such shock solutions. The previous similarity shock solutions of Tsai & Hsu and of Shu et al. may be classified into two types: Class I solutions with downstream being free-fall collapses and Class II solutions with downstream being Larson-Penston (LP) type solutions. By the analyses of Lou & Shen and Shen & Lou, we further construct similarity shock solutions in the `semi-complete space'. These general shock solutions can accommodate and model dynamical processes of radial outflows (wind), inflows (accretion or contraction), subsonic oscillations, and free-fall core collapses all with shocks in various settings such as star-forming molecular clouds, `champagne flows' in H{\\sevenrm II} regions around luminous massive OB stars or surrounding quasars, dynamical connection between the asymptotic giant branch phase to the proto-planetary nebula phase with a central hot white dwarf as well as accretion shocks around compact objects such as white dwarfs, neutron stars, and supermassive black holes. By a systematic exploration, we are able to construct families of infinitely many discrete Class I and Class II solutions matching asymptotically with a static outer envelope of singular isothermal sphere; the shock solutions of Tsai & Hsu form special subsets. These similarity shocks travel at either subsonic or supersonic constant speeds. We also construct twin shocks as well as an `isothermal shock' separating two fluid regions of two different yet constant temperatures.

Fu-Yan Bian; Yu-Qing Lou

2005-09-06

355

Isothermal Degradation of PVC\\/MBS Blends  

Microsoft Academic Search

The process of thermal degradation of poly(vinyl chloride)\\/poly(methyl methacrylate-butadiene-styrene) (PVC\\/MBS) blends was\\u000a investigated by means of isothermal thermogravimetry in nitrogen. The total mass loss was determined after 120 min. The kinetic\\u000a parameters of the degradation process were determined by applying two kinetic models: the model which assumes autocatalytic\\u000a degradation (Prout-Tompkins) and the model of two-dimensional diffusion. It was established that

B. Bari?; T. Kova?i?

1998-01-01

356

The Spatial Pattern of Cochlear Amplification  

PubMed Central

SUMMARY Sensorineural hearing loss, which stems primarily from the failure of mechanosensory hair cells, changes the traveling waves that transmit acoustic signals along the cochlea. However, the connection between cochlear mechanics and the amplificatory function of hair cells remains unclear. Using an optical technique that permits the targeted inactivation of prestin, a protein of outer hair cells that generates forces on the basilar membrane, we demonstrate that these forces interact locally with cochlear traveling waves to achieve enormous mechanical amplification. By perturbing amplification in narrow segments of the basilar membrane, we further show that a cochlear traveling wave accumulates gain as it approaches its peak. Analysis of these results indicates that cochlear amplification produces negative damping that counters the viscous drag impeding traveling waves; targeted photoinactivation locally interrupts this compensation. These results reveal the locus of amplification in cochlear traveling waves and connect the characteristics of normal hearing to molecular forces. PMID:23217746

Fisher, Jonathan A.N.; Nin, Fumiaki; Reichenbach, Tobias; Uthaiah, Revathy C.; Hudspeth, A.J.

2012-01-01

357

AMPLIFICATION OF RIBOSOMAL RNA SEQUENCES - Book Chapter  

EPA Science Inventory

This book chapter contains the following headings and subheadings: Introduction; Experimental Approach - Precautions, Template, Primers, Reaction Conditions, Enhancers, Post Amplification; Procedures - Template DNA, Basic PCR, Thermal Cycle Parameters, Enzyme Addition, Agarose Ge...

358

Amplification of Angular Rotations Using Weak Measurements  

NASA Astrophysics Data System (ADS)

We present a weak measurement protocol that permits a sensitive estimation of angular rotations based on the concept of weak-value amplification. The shift in the state of a pointer, in both angular position and the conjugate orbital angular momentum bases, is used to estimate angular rotations. This is done by an amplification of both the real and imaginary parts of the weak-value of a polarization operator that has been coupled to the pointer, which is a spatial mode, via a spin-orbit coupling. Our experiment demonstrates the first realization of weak-value amplification in the azimuthal degree of freedom. We have achieved effective amplification factors as large as 100, providing a sensitivity that is on par with more complicated methods that employ quantum states of light or extremely large values of orbital angular momentum.

Magaña-Loaiza, Omar S.; Mirhosseini, Mohammad; Rodenburg, Brandon; Boyd, Robert W.

2014-05-01

359

Amplification of Angular Rotations Using Weak Measurements  

E-print Network

We present a weak measurement protocol that permits a sensitive estimation of angular rotations based on the concept of weak-value amplification. The shift in the state of a pointer, in both angular position and the conjugate orbital angular momentum bases, is used to estimate angular rotations. This is done by an amplification of both the real and imaginary parts of the weak-value of a polarization operator that has been coupled to the pointer, which is a spatial mode, via a spin-orbit coupling. Our experiment demonstrates the first realization of weak-value amplification in the azimuthal degree of freedom. We have achieved effective amplification factors as large as 100, providing a sensitivity that is on par with more complicated methods that employ quantum states of light or extremely large values of orbital angular momentum.

Omar S. Magana-Loaiza; Mohammad Mirhosseini; Brandon Rodenburg; Robert W. Boyd

2013-12-10

360

Passive, Noiseless, Intensity Amplification of Repetitive Signals  

E-print Network

Amplification of signal intensity is essential for initiating physical processes, diagnostics, sensing, communications, and scientific measurement. During traditional amplification, the signal is amplified by multiplying the signal carriers through an active gain process using an external power source. However, for repetitive waveforms, sufficient energy for amplification often resides in the signal itself. In such cases, the unneeded external power is wasted, and the signal is additionally degraded by noise and distortions that accompany active gain processes. We show noiseless, intensity amplification of repetitive optical pulse waveforms with a gain from 2 to ~20 without using active gain, by recycling energy already stored in the input repetitive signal. This "green" method uses dispersion-induced self-imaging (Talbot) effects to precisely re-distribute the original signal energy into fewer replica waveforms. This approach simply requires a suitable manipulation of the input signal's phase profile along t...

Maram, R; Li, M; Azaña, J

2014-01-01

361

Amplification of chromosomal DNA in situ  

DOEpatents

Amplification of chromosomal DNA in situ to increase the amount of DNA associated with a chromosome or chromosome region is described. The amplification of chromosomal DNA in situ provides for the synthesis of Fluorescence in situ Hybridization (FISH) painting probes from single dissected chromosome fragments, the production of cDNA libraries from low copy mRNAs and improved in Comparative Genomic Hybridization (CGH) procedures.

Christian, Allen T. (Tracy, CA); Coleman, Matthew A. (Livermore, CA); Tucker, James D. (Livermore, CA)

2002-01-01

362

TOUCH-UP Gradient Amplification Method  

PubMed Central

We report a unique amplification technique that works efficiently and specifically over a temperature range, rather than at one specific temperature, throughout the amplification process. As bisulfite-modified DNA is one of the difficult to amplify templates, we used this technique to amplify regions of promoter-associated CpG island for 11 genes using this template. This technique amplified specific products for every gene without requiring any optimization. PMID:22468135

Rowther, F. B.; Kardooni, H.; Warr, T.

2012-01-01

363

Chemical amplification based on fluid partitioning  

DOEpatents

A system for nucleic acid amplification of a sample comprises partitioning the sample into partitioned sections and performing PCR on the partitioned sections of the sample. Another embodiment of the invention provides a system for nucleic acid amplification and detection of a sample comprising partitioning the sample into partitioned sections, performing PCR on the partitioned sections of the sample, and detecting and analyzing the partitioned sections of the sample.

Anderson, Brian L. (Lodi, CA); Colston, Jr., Billy W. (San Ramon, CA); Elkin, Chris (San Ramon, CA)

2006-05-09

364

Rolling circle amplification of metazoan mitochondrialgenomes  

SciTech Connect

Here we report the successful use of rolling circle amplification (RCA) for the amplification of complete metazoan mt genomes to make a product that is amenable to high-throughput genome sequencing techniques. The benefits of RCA over PCR are many and with further development and refinement of RCA, the sequencing of organellar genomics will require far less time and effort than current long PCR approaches.

Simison, W. Brian; Lindberg, D.R.; Boore, J.L.

2005-07-31

365

Reciprocating-flow ATP amplification system for increasing the number of amplification cycles.  

PubMed

We constructed a novel ATP amplification reactor using a reciprocating-flow system to increase the number of ATP amplification cycles without an increase in backpressure. We previously reported a continuous-flow ATP amplification system that effectively and quantitatively amplified ATP and increased the sensitivity of a quantitative bioluminescence assay. However, it was difficult to increase the number of amplification cycles due to backpressure in the system. Because addition of immobilized adenylate kinase (ADK) and pyruvate kinase (PK) columns increased backpressure, the maximum number of ATP amplification cycles within column durability was only 4. In this study, ATP amplification was performed using a reciprocating-flow system, and 10 cycles of ATP amplification could be achieved without an increase in backpressure. As a result, ATP was amplified more than 100-fold after 10 cycles of reciprocating flow. The gradient of ATP amplification was approximately 1.76(N). The backpressure on the columns was 0.03 MPa in 1-10 ATP amplification cycles, and no increases in backpressure were observed. PMID:19699705

Satoh, Tetsuya; Tsuruta, Kosuke; Shinoda, Yasuharu; Hirota, Ryuichi; Noda, Kenichi; Kuroda, Akio; Murakami, Yuji

2009-12-15

366

Gravitational instability of slowly rotating isothermal spheres  

E-print Network

We discuss the statistical mechanics of rotating self-gravitating systems by allowing properly for the conservation of angular momentum. We study analytically the case of slowly rotating isothermal spheres by expanding the solutions of the Boltzmann-Poisson equation in a series of Legendre polynomials, adapting the procedure introduced by Chandrasekhar (1933) for distorted polytropes. We show how the classical spiral of Lynden-Bell & Wood (1967) in the temperature-energy plane is deformed by rotation. We find that gravitational instability occurs sooner in the microcanonical ensemble and later in the canonical ensemble. According to standard turning point arguments, the onset of the collapse coincides with the minimum energy or minimum temperature state in the series of equilibria. Interestingly, it happens to be close to the point of maximum flattening. We determine analytically the generalization of the singular isothermal solution to the case of a slowly rotating configuration. We also consider slowly rotating configurations of the self-gravitating Fermi gas at non zero temperature.

P. -H. Chavanis

2002-04-14

367

Computer Modeling of Non-Isothermal Crystallization  

NASA Technical Reports Server (NTRS)

A realistic computer model for simulating isothermal and non-isothermal phase transformations proceeding by homogeneous and heterogeneous nucleation and interface-limited growth is presented. A new treatment for particle size effects on the crystallization kinetics is developed and is incorporated into the numerical model. Time-dependent nucleation rates, size-dependent growth rates, and surface crystallization are also included. Model predictions are compared with experimental measurements of DSC/DTA peak parameters for the crystallization of lithium disilicate glass as a function of particle size, Pt doping levels, and water content. The quantitative agreement that is demonstrated indicates that the numerical model can be used to extract key kinetic data from easily obtained calorimetric data. The model can also be used to probe nucleation and growth behavior in regimes that are otherwise inaccessible. Based on a fit to data, an earlier prediction that the time-dependent nucleation rate in a DSC/DTA scan can rise above the steady-state value at a temperature higher than the peak in the steady-state rate is demonstrated.

Kelton, K. F.; Narayan, K. Lakshmi; Levine, L. E.; Cull, T. C.; Ray, C. S.

1996-01-01

368

Isothermal and non-isothermal relaxation processes in dye-doped polystyrene  

NASA Astrophysics Data System (ADS)

The build-up of a polarized state and its isothermal and nonisothermal relaxation behavior in a NLO guest-host system obtained by doping of polystyrene (PS) with the disperse red 1 (DR1) dye molecules have been studied by the isothermal absorption currents measurements, the thermally stimulated polarization (TSP) and depolarization (TSD), the AC dielectric spectroscopy, as well as the Hamon method in order to cover wide ranges of temperatures and frequencies. The 20 micrometers -thick samples were pulsed by the 'sandwich' method. Analyzing isothermal polarization and depolarization and depolarization currents we proved the validity of the superposition principle. The Williams-Watts model supplemented with a concept of intrinsic time has been used to interpret the TSP curves, but it appeared that the isothermal behavior does not fully determine the TSP results. The a peak has been observed at dielectric loss curves with its position changing from 10 Hz at 100 degrees C to 3 X 104 Hz at 130 degrees C and narrowing with temperature. Temperature dependence of the relaxation time above Tg agreed with the WIlliams-Landel-Ferry model, while the Arrenius dependence was more appropriate at sub- Tg temperatures. It has been shown that the PS/DR1 system is not thermoreologically simple, i.e. the temperature-time superposition is not entirely valid.

Fedosov, S. N.; Giacometti, Jose A.; Costa, Mauro M.; Ferreira, G. F. Leal; Pissis, Polycarpos

1999-12-01

369

Numerical Model for Isothermal and Non-Isothermal Crystallization of Liquids and Glasses  

NASA Technical Reports Server (NTRS)

A new numerical model of isothermal and non-isothermal first order phase transformations, such as the crystallization of liquids and glasses, is presented. This model computes directly the volume fraction transformed, taking into account time-dependent nucleation rates and cluster-size-dependent growth velocities. The model is applied to the crystallization of lithium disilicate glass, using the appropriate kinetic and thermodynamic parameters. The model is used (1) to determine the validity of common methods for computing the volume fraction transformed as a function of time in isothermal experiments when a time-dependent nucleation rate is expected, (2) to simulate non-isothermal differential scanning calorimetry (DSC) studies of glass devitrification as a function of scan rate, and (3) to compute the effect of preannealing on the DSC peak parameters. A novel behavior of the nucleation rate with scan rate is predicted, arising because the relaxation of the cluster distribution cannot be described by a single relaxation time. Comparisons of the calculations with experimental data on this glass demonstrate the validity of the model.

Kelton, K. F.

1993-01-01

370

Isothermal and non-isothermal viscoelastic flow of PTT fluid in lid-driven polar cavity  

NASA Astrophysics Data System (ADS)

The isothermal and non-isothermal viscoelastic flow of Phan-Thien-Tanner (PTT) fluids is considered in liddriven polar cavity geometry, using a numerical solution method with parameter continuation technique. Thermoelastic effects, in terms of elastic/elongational effects and viscous dissipation, are demonstrated by the changes in vortical structure, temperature/stress distributions and heat transfer characteristics in the curved cavity. Central vortex/maximum temperature location shifts are observed under elastic and elongational (strain hardening and strain softening/shear thinning) effects for isothermal and non-isothermal conditions. The growth in size and strength of a secondary vortex is denoted in the downstream stationary corner of the cavity for the viscoelastic fluid under strain hardening, which also introduces an increase in stress gradients. Viscous heating is observed with elongational effects near the central vortex in the cavity. Stress components and their gradients decrease under viscous dissipation. The changes in temperature field and heat transfer properties in the cavity are revealed.

Mercan, Hatice; Atal?k, Kunt

2012-12-01

371

Heat induces gene amplification in cancer cells  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer This study discovered that heat exposure (hyperthermia) results in gene amplification in cancer cells. Black-Right-Pointing-Pointer Hyperthermia induces DNA double strand breaks. Black-Right-Pointing-Pointer DNA double strand breaks are considered to be required for the initiation of gene amplification. Black-Right-Pointing-Pointer The underlying mechanism of heat-induced gene amplification is generation of DNA double strand breaks. -- Abstract: Background: Hyperthermia plays an important role in cancer therapy. However, as with radiation, it can cause DNA damage and therefore genetic instability. We studied whether hyperthermia can induce gene amplification in cancer cells and explored potential underlying molecular mechanisms. Materials and methods: (1) Hyperthermia: HCT116 colon cancer cells received water-submerged heating treatment at 42 or 44 Degree-Sign C for 30 min; (2) gene amplification assay using N-(phosphoacetyl)-L-aspartate (PALA) selection of cabamyl-P-synthetase, aspartate transcarbarmylase, dihydro-orotase (cad) gene amplified cells; (3) southern blotting for confirmation of increased cad gene copies in PALA-resistant cells; (4) {gamma}H2AX immunostaining to detect {gamma}H2AX foci as an indication for DNA double strand breaks. Results: (1) Heat exposure at 42 or 44 Degree-Sign C for 30 min induces gene amplification. The frequency of cad gene amplification increased by 2.8 and 6.5 folds respectively; (2) heat exposure at both 42 and 44 Degree-Sign C for 30 min induces DNA double strand breaks in HCT116 cells as shown by {gamma}H2AX immunostaining. Conclusion: This study shows that heat exposure can induce gene amplification in cancer cells, likely through the generation of DNA double strand breaks, which are believed to be required for the initiation of gene amplification. This process may be promoted by heat when cellular proteins that are responsible for checkpoints, DNA replication, DNA repair and telomere functions are denatured. To our knowledge, this is the first study to provide direct evidence of hyperthermia induced gene amplification.

Yan, Bin, E-mail: yanbin@mercyhealth.com [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States) [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Mercy Cancer Center, Mercy Medical Center-North Iowa, Mason City, IA 50401 (United States); Ouyang, Ruoyun [Department of Respiratory Medicine, The Second Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410011 (China)] [Department of Respiratory Medicine, The Second Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410011 (China); Huang, Chenghui [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States) [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Department of Oncology, The Third Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410013 (China); Liu, Franklin [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States)] [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States); Neill, Daniel [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States)] [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Li, Chuanyuan [Dermatology, Duke University Medical Center, Durham, NC 27710 (United States)] [Dermatology, Duke University Medical Center, Durham, NC 27710 (United States); Dewhirst, Mark [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States)] [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States)

2012-10-26

372

Reverse Transcription Recombinase Polymerase Amplification Assay for the Detection of Middle East Respiratory Syndrome Coronavirus  

PubMed Central

The emergence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in the eastern Mediterranean and imported cases to Europe has alerted public health authorities. Currently, detection of MERS-CoV in patient samples is done by real-time RT-PCR. Samples collected from suspected cases are sent to highly-equipped centralized laboratories for screening. A rapid point-of-care test is needed to allow more widespread mobile detection of the virus directly from patient material. In this study, we describe the development of a reverse transcription isothermal Recombinase Polymerase Amplification (RT-RPA) assay for the identification of MERS-CoV. A partial nucleocapsid gene RNA molecular standard of MERS-coronavirus was used to determine the assay sensitivity. The isothermal (42°C) MERS-CoV RT-RPA was as sensitive as real-time RT-PCR (10 RNA molecules), rapid (3-7 minutes) and mobile (using tubescanner weighing 1kg). The MERS-CoV RT-RPA showed cross-detection neither of any of the RNAs of several coronaviruses and respiratory viruses affecting humans nor of the human genome. The developed isothermal real-time RT-RPA is ideal for rapid mobile molecular MERS-CoV monitoring in acute patients and may also facilitate the search for the animal reservoir of MERS-CoV. PMID:24459611

Abd El Wahed, Ahmed; Patel, Pranav; Heidenreich, Doris; Hufert, Frank T.; Weidmann, Manfred

2013-01-01

373

Reverse transcription recombinase polymerase amplification assay for the detection of middle East respiratory syndrome coronavirus.  

PubMed

The emergence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in the eastern Mediterranean and imported cases to Europe has alerted public health authorities. Currently, detection of MERS-CoV in patient samples is done by real-time RT-PCR. Samples collected from suspected cases are sent to highly-equipped centralized laboratories for screening. A rapid point-of-care test is needed to allow more widespread mobile detection of the virus directly from patient material. In this study, we describe the development of a reverse transcription isothermal Recombinase Polymerase Amplification (RT-RPA) assay for the identification of MERS-CoV. A partial nucleocapsid gene RNA molecular standard of MERS-coronavirus was used to determine the assay sensitivity. The isothermal (42°C) MERS-CoV RT-RPA was as sensitive as real-time RT-PCR (10 RNA molecules), rapid (3-7 minutes) and mobile (using tubescanner weighing 1kg). The MERS-CoV RT-RPA showed cross-detection neither of any of the RNAs of several coronaviruses and respiratory viruses affecting humans nor of the human genome. The developed isothermal real-time RT-RPA is ideal for rapid mobile molecular MERS-CoV monitoring in acute patients and may also facilitate the search for the animal reservoir of MERS-CoV. PMID:24459611

Abd El Wahed, Ahmed; Patel, Pranav; Heidenreich, Doris; Hufert, Frank T; Weidmann, Manfred

2013-01-01

374

Statistical Design in Isothermal Aging of Polyimide Resins  

NASA Technical Reports Server (NTRS)

Recent developments in research on polyimides for high temperature applications have led to the synthesis of many new polymers. Among the criteria that determines their thermal oxidative stability, isothermal aging is one of the most important. Isothermal aging studies require that many experimental factors are controlled to provide accurate results. In this article we describe a statistical plan that compares the isothermal stability of several polyimide resins, while minimizing the variations inherent in high-temperature aging studies.

Sutter, James K.; Jobe, Marcus; Crane, Elizabeth A.

1995-01-01

375

Isothermal and Nonisothermal Fatigue Behavior of a Metal Matrix Composite  

Microsoft Academic Search

The isothermal and nonisothermal fatigue resistance of a metal matrix com posite (MMC) consisting of Ti-15V-3Cr-3Al-3Sn (Ti-15-3) matrix reinforced by 33 v\\/o continuous SiC fibers was investigated. The fibers were nominally oriented parallel to the specimen axis. Isothermal fatigue tests were performed in air at 300 and 550°C. In non isothermal fatigue tests, each load excursion at 300°C or 550°C

T. P. Gabb; J. Gayda; R. A. Mackay

1990-01-01

376

Statistical design in isothermal aging of polyimide resins  

NASA Technical Reports Server (NTRS)

Recent developments in research on polyimides for high temperature applications have led to the synthesis of many new polymers. Among the criteria that determines their thermal oxidative stability, isothermal aging is one of the most important. Isothermal aging studies require that many experimental factors are controlled to provide accurate results. In this article we describe a statistical plan that compares the isothermal stability of several polyimide resins, while minimizing the variations inherent in high-temperature aging studies.

Sutter, James K.; Jobe, Marcus; Crane, Elizabeth A.

1995-01-01

377

Circle-to-circle amplification on a digital microfluidic chip for amplified single molecule detection.  

PubMed

We demonstrate a novel digital microfluidic nucleic acid amplification concept which is based on padlock probe mediated DNA detection and isothermal circle-to-circle amplification (C2CA). This assay platform combines two digital approaches. First, digital microfluidic manipulation of droplets which serve as micro-reaction chambers and shuttling magnetic particles between these droplets facilitates the integration of complex solid phase multistep assays. We demonstrate an optimized novel particle extraction and transfer protocol for superparamagnetic particles on a digital microfluidic chip that allows for nearly 100% extraction efficiencies securing high assay performance. Second, the compartmentalization required for digital single molecule detection is solved by simple molecular biological means, circumventing the need for complex microfabrication procedures necessary for most, if not all, other digital nucleic acid detection methods. For that purpose, padlock probes are circularized in a strictly target dependent ligation reaction and amplified through two rounds of rolling circle amplification, including an intermediate digestion step. The reaction results in hundreds of 500 nm sized individually countable DNA nanospheres per detected target molecule. We demonstrate that integrated miniaturized digital microfluidic C2CA results in equally high numbers of C2CA products ?L(-1) as off-chip tube control experiments indicating high assay performance without signal loss. As low as 1 aM synthetic Pseudomonas aeruginosa DNA was detected with a linear dynamic range over 4 orders of magnitude up to 10 fM proving excellent suitability for infectious disease diagnostics. PMID:24934991

Kühnemund, Malte; Witters, Daan; Nilsson, Mats; Lammertyn, Jeroen

2014-08-21

378

Electrochemical detection of Francisella tularensis genomic DNA using solid-phase recombinase polymerase amplification.  

PubMed

Solid-phase isothermal DNA amplification was performed exploiting the homology protein recombinase A (recA). The system was primarily tested on maleimide activated microtitre plates as a proof-of-concept and later translated to an electrochemical platform. In both cases, forward primer for Francisella tularensis holarctica genomic DNA was surface immobilised via a thiol or an amino moiety and then elongated during the recA mediated amplification, carried out in the presence of specific target sequence and reverse primers. The formation of the subsequent surface tethered amplicons was either colorimetrically or electrochemically monitored using a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the elongated strand. The amplification time was optimised to amplify even low amounts of DNA copies in less than an hour at a constant temperature of 37°C, achieving a limit of detection of 1.3×10(-13) M (4×10(6) copies in 50 ?L) for the colorimetric assay and 3.3×10(-14) M (2×10(5) copies in 10 ?L) for the chronoamperometric assay. The system was demonstrated to be highly specific with negligible cross-reactivity with non-complementary targets or primers. PMID:24334283

del Río, Jonathan Sabaté; Yehia Adly, Nouran; Acero-Sánchez, Josep Lluis; Henry, Olivier Y F; O'Sullivan, Ciara K

2014-04-15

379

The Amplification Model for Adaptive Mutation  

PubMed Central

It has been proposed that the lac revertants arising under selective conditions in the Cairns experiment do not arise by stress-induced mutagenesis of stationary phase cells as has been previously assumed. Instead, these revertants may arise within growing clones initiated by cells with a preexisting duplication of the weakly functional lac allele used in this experiment. It is proposed that spontaneous stepwise increases in lac copy number (amplification) allow a progressive improvement in growth. Reversion is made more likely primarily by the resultant increase in the number of mutational targets—more cells with more lac copies. The gene amplification model requires no stress-induced variation in the rate or target specificity of mutation and thus does not violate neo-Darwinian theory. However, it does require that a multistep process of amplification, reversion, and amplification segregation be completed within ?20 generations of growth. This work examines the proposed amplification model from a theoretical point of view, formalizing it into a mathematical framework and using this to determine what would be required for the process to occur within the specified period. The analysis assumes no stress-induced change in mutation rate and describes only the growth improvement occurring during the process of amplification and subsequent elimination of excess mutant lac copies. The dynamics of the system are described using Monte Carlo simulations and numerical integration of the deterministic equations governing the system. The results imply that the amplification model can account for the behavior of the system using biologically reasonable parameter values and thus can, in principle, explain Cairnsian adaptive mutation. PMID:15489536

Pettersson, Mats E.; Andersson, Dan I.; Roth, John R.; Berg, Otto G.

2005-01-01

380

Characterization of a novel close-to-root papillomavirus from a Florida manatee by using multiply primed rolling-circle amplification: Trichechus manatus latirostris papillomavirus type 1  

Microsoft Academic Search

By using an isothermal multiply primed rolling-circle amplification protocol, the complete genomic DNA of a novel papillomavirus was amplified from a skin lesion biopsy of a Florida manatee (Trichechus manatus latirostris), one of the most endangered marine mammals in United States coastal waters. The nucleotide sequence, genome organization, and phylogenetic position of the Trichechus manatus latirostris papillomavirus type 1 (TmPV-1)

Annabel Rector; Gregory D. Bossart; Shin-Je Ghim; John P. Sundberg; A. B. Jenson; R a Van

2004-01-01

381

Determination of Ibuprofen Isotherm Using Supercritical Fluid Chromatography.  

E-print Network

?? Chromatography is widely used to determine physiochemical properties data including adsorption isotherm. In the separation of enantiomers through sorptive processes, supercritical fluids allow efficient… (more)

Ho, Loi

2012-01-01

382

Determination of Ibuprofen Isotherm Using Supercritical Fluid Chromatography.  

E-print Network

??Chromatography is widely used to determine physiochemical properties data including adsorption isotherm. In the separation of enantiomers through sorptive processes, supercritical fluids allow efficient and… (more)

Ho, Loi

2012-01-01

383

Isothermal dendritic growth: A low gravity experiment  

NASA Technical Reports Server (NTRS)

The Isothermal Dendritic Growth Experiment is an active crystal growth experiment designed to test dendritic growth theory at low undercoolings where convection prohibits such studies at 1 g. The experiment will be essentially autonomous, though limited in-flight interaction through a computer interface is planned. One of the key components of the apparatus will be a crystal growth chamber capable of achieving oriented single crystal dendritic growth. Recent work indicates that seeding the chamber with a crystal of the proper orientation will not, in and of itself, be sufficient to meet this requirement. Additional flight hardware and software required for the STS flight experiment are currently being developed at NASA Lewis Research Center and at Rensselaer Polytechnic Institute.

Glicksman, M. E.; Hahn, R. C.; Lograsso, T. A.; Rubinstein, E. R.; Selleck, M. E.; Winsa, E.

1988-01-01

384

Isothermal Decomposition of Hydrogen Peroxide Dihydrate  

NASA Technical Reports Server (NTRS)

We present a new method of growing pure solid hydrogen peroxide in an ultra high vacuum environment and apply it to determine thermal stability of the dihydrate compound that forms when water and hydrogen peroxide are mixed at low temperatures. Using infrared spectroscopy and thermogravimetric analysis, we quantified the isothermal decomposition of the metastable dihydrate at 151.6 K. This decomposition occurs by fractional distillation through the preferential sublimation of water, which leads to the formation of pure hydrogen peroxide. The results imply that in an astronomical environment where condensed mixtures of H2O2 and H2O are shielded from radiolytic decomposition and warmed to temperatures where sublimation is significant, highly concentrated or even pure hydrogen peroxide may form.

Loeffler, M. J.; Baragiola, R. A.

2011-01-01

385

Gravitational instability of finite isothermal spheres  

NASA Astrophysics Data System (ADS)

We investigate the stability of bounded self-gravitating systems in the canonical ensemble by using a thermodynamical approach. Our study extends the earlier work of Padmanabhan (\\cite{pad89}) in the microcanonical ensemble. By studying the second variations of the free energy, we find that instability sets in precisely at the point of minimum temperature in agreement with the theorem of Katz (\\cite{kat78}). The perturbation that induces instability at this point is calculated explicitly; it has not a ``core-halo'' structure contrary to what happens in the microcanonical ensemble. We also study Jeans type gravitational instability of isothermal gaseous spheres described by Navier-Stokes equations. The introduction of a container and the consideration of an inhomogeneous distribution of matter avoids the Jeans swindle. We show analytically the equivalence between dynamical stability and thermodynamical stability and the fact that the stability of isothermal gas spheres does not depend on the viscosity. This confirms the findings of Semelin et al. (\\cite{sem01}) who used numerical methods or approximations. We also give a simpler derivation of the geometric hierarchy of scales inducing instability discovered by these authors. The density profiles that trigger these instabilities are calculated explicitly; high order modes of instability present numerous oscillations whose nodes also follow a geometric progression. This suggests that the system will fragment in a series of ``clumps'' and that these ``clumps'' will themselves fragment in substructures. The fact that both the domain sizes leading to instability and the ``clumps'' sizes within a domain follow a geometric progression with the same ratio suggests a fractal-like behavior. This gives further support to the interpretation of de Vega et al. (1996) concerning the fractal structure of the interstellar medium.

Chavanis, P. H.

2002-01-01

386

Experimental research of gas flows through isothermal and non-isothermal membranes  

NASA Astrophysics Data System (ADS)

In specialized test bench and in vacuum aerodynamic facilities VAT-2M TsAGI three types of a gas flows with observed kinetic effects were researched. Firstly, the flow through the membrane with uniform temperature was investigated. The dependence of flow rate through membranes on pressure drop across it was measured at various values of permeability. The experimental data at various flow regimes in the pores were compared with numerical data. The comparison gives the opportunity to associate the model perforated membrane with definite diameter of perforation channels and with definite permeability to each porous membrane with intricate pores. Flow rate through real and model membranes are the same ones for two limit regimes: the free-molecular regime and the Stokes ones. For experimental research of a gas flows induced by temperature difference across membrane the method of creation such temperature difference (uniform on membrane surface) was used. In this method thermoelectric effect is utilized. The dependence of thermo-transpiration flow rate and thermo-molecular pressure difference across non-isothermal membrane (for zero flow rate) on gas pressure were measured. The comparison of results of direct and indirect measurements of the velocity of thermo-transpiration was carried out. In the second case the flow rate of thermal transpiration was calculated by the experimental results on thermo-molecular pressure difference across non-isothermal membrane and the results of measurement of pressure driven flow through isothermal membrane.

Nikolskiy, Yu. V.; Friedlander, O. G.

2012-11-01

387

Loop-Mediated Isothermal Amplification Compared to Real-Time PCR and Enzyme Immunoassay for Toxigenic Clostridium difficile Detection  

PubMed Central

Clostridium difficile infection is the primary cause of health care-associated diarrhea. While most laboratories have been using rapid antigen tests for detecting C. difficile toxins, they have poor sensitivity; newer molecular methods offer rapid results with high test sensitivity and specificity. This study was designed to compare the performances of two molecular assays (Meridian illumigene and BD GeneOhm) and two antigen assays (Wampole Quik Chek Complete and TechLab Tox A/B II) to detect toxigenic C. difficile. Fecal specimens from hospitalized patients (n = 139) suspected of having C. difficile infection were tested by the four assays. Nine specimens were positive and 109 were negative by all four methods. After discrepant analysis by toxigenic culture (n = 21), the total numbers of stool specimens classified as positive and negative for toxigenic C. difficile were 21 (15%) and 118 (85%), respectively. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were as follows: GeneOhm (95.2%, 100%, 100%, and 99.2%), illumigene (95.2%, 96.6%, 83.3%, and 99.2%), Tox A/B II (52.4%, 97.5%, 78.6%, and 92.4%), and Quik Chek Complete (47.6%, 100%, 100%, and 91.9%). The illumigene assay performed comparably to the GeneOhm assay with a slight decrease in test specificity; the sensitivities of both far exceeded those of the antigen assays. The clinical characteristics of the concordant and discrepant study patients were similar, including stool consistency and frequency. In the era of rapid molecular-based tests for toxigenic C. difficile, toxin enzyme immunoassays (EIAs) should no longer be considered the standard of care. PMID:22189114

Sural, Preethi; Loomis, Caroline R.; Pesta, Christine; Gonzalez-Krellwitz, Laura; Robinson-Dunn, Barbara; Riska, Paul

2012-01-01

388

Amplification, Redundancy, and Quantum Chernoff Information  

NASA Astrophysics Data System (ADS)

Amplification was regarded, since the early days of quantum theory, as a mysterious ingredient that endows quantum microstates with macroscopic consequences, key to the "collapse of the wave packet," and a way to avoid embarrassing problems exemplified by Schrödinger's cat. Such a bridge between the quantum microworld and the classical world of our experience was postulated ad hoc in the Copenhagen interpretation. Quantum Darwinism views amplification as replication, in many copies, of the information about quantum states. We show that such amplification is a natural consequence of a broad class of models of decoherence, including the photon environment we use to obtain most of our information. This leads to objective reality via the presence of robust and widely accessible records of selected quantum states. The resulting redundancy (the number of copies deposited in the environment) follows from the quantum Chernoff information that quantifies the information transmitted by a typical elementary subsystem of the environment.

Zwolak, Michael; Riedel, C. Jess; Zurek, Wojciech H.

2014-04-01

389

Amplification, Redundancy, and the Quantum Chernoff Information  

E-print Network

Amplification was regarded, since the early days of quantum theory, as a mysterious ingredient that endows quantum microstates with macroscopic consequences, key to the "collapse of the wavepacket", and a way to avoid embarrassing problems exemplified by Schr\\"odinger's cat. Such a bridge between the quantum microworld and the classical world of our experience was postulated ad hoc in the Copenhagen Interpretation. Quantum Darwinism views amplification as replication, in many copies, of the information about quantum states. We show that such amplification is a natural consequence of a broad class of models of decoherence, including the photon environment we use to obtain most of our information. This leads to objective reality via the presence of robust and widely accessible records of selected quantum states. The resulting redundancy (the number of copies deposited in the environment) follows from the Quantum Chernoff Information that quantifies the information transmitted by a typical elementary subsystem of the environment.

Michael Zwolak; C. Jess Riedel; Wojciech H. Zurek

2013-12-18

390

Privacy Amplification in the Isolated Qubits Model  

E-print Network

Isolated qubits are a special class of quantum devices, which can be used to implement tamper-resistant cryptographic hardware such as one-time memories (OTM's). Unfortunately, these OTM constructions leak some information, and standard methods for privacy amplification cannot be applied here, because the adversary has advance knowledge of the hash function that the honest parties will use. In this paper we show a stronger form of privacy amplification that solves this problem, using a fixed hash function that is secure against all possible adversaries in the isolated qubits model. This allows us to construct single-bit OTM's which only leak an exponentially small amount of information. We then study a natural generalization of the isolated qubits model, where the adversary is allowed to perform a polynomially-bounded number of entangling gates, in addition to unbounded local operations and classical communication (LOCC). We show that our technique for privacy amplification is also secure in this setting.

Yi-Kai Liu

2014-10-15

391

Time varying arctic climate change amplification  

SciTech Connect

During the past 130 years the global mean surface air temperature has risen by about 0.75 K. Due to feedbacks -- including the snow/ice albedo feedback -- the warming in the Arctic is expected to proceed at a faster rate than the global average. Climate model simulations suggest that this Arctic amplification produces warming that is two to three times larger than the global mean. Understanding the Arctic amplification is essential for projections of future Arctic climate including sea ice extent and melting of the Greenland ice sheet. We use the temperature records from the Arctic stations to show that (a) the Arctic amplification is larger at latitudes above 700 N compared to those within 64-70oN belt, and that, surprisingly; (b) the ratio of the Arctic to global rate of temperature change is not constant but varies on the decadal timescale. This time dependence will affect future projections of climate changes in the Arctic.

Chylek, Petr [Los Alamos National Laboratory; Dubey, Manvendra K [Los Alamos National Laboratory; Lesins, Glen [DALLHOUSIE U; Wang, Muyin [NOAA/JISAO

2009-01-01

392

Adiabatic Versus Isothermal - Two Pictures of Galaxy Origin  

Microsoft Academic Search

The final word in favour of either the adiabatic or the isothermal case cannot be said. The author 'summarizes' the situation by simply saying that whereas later events (MWB fluctuation, n-point correlations etc.) seem to favour the isothermal picture the earlier events seem to favour the adiabatic picture from the point of view of the origin of the fluctuations.

S. A. Bonometto

1983-01-01

393

Isothermal reactivating Whiplash PCR for locally programmable molecular computation  

E-print Network

-009-9148-6 #12;Programmable molecular machines Á Polymerase chain reaction Á Isothermal computing Á Autocatalytic biomolecular computers Á State transition Abbreviations PCR Polymerase chain reaction WPCR Whiplash polymerase nucleic acid IR-WPCR Isothermal reactivating Whiplash polymerase chain reaction FRET Fluorescence

Reif, John H.

394

Financial Statement Audit Report of Isothermal Community College.  

ERIC Educational Resources Information Center

This report presents the results of the Isothermal Community College financial statement audit for the fiscal year ending on June 30, 1998. Isothermal Community College is a component of the State of North Carolina, thus the authority to audit is granted by Article 5A of G.S. 147. The accounts and operations of the institution were subject to…

Campbell, Ralph

395

Signal amplification by unidirectional coupling of oscillators  

E-print Network

We report our investigation on the input signal amplification<