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1

Multiplex isothermal helicase-dependent amplification assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae.  

PubMed

Thermophilic helicase dependent amplification (tHDA), which employs helicase to unwind double-stranded DNA at constant temperature, is a relatively new isothermal nucleic acid amplification technology. In this study, the development and optimization of a 4-plex tHDA assay for detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are described. tHDA is combined with sequence-specific sample preparation on magnetic beads and homogeneous endpoint fluorescence detection using dual-labeled probes. This 4-plex tHDA assay was applied to the detection of 2 genes on CT and a multicopy gene on NG in the presence of an internal control. The assay showed high analytical sensitivity and specificity of simultaneous CT/NG detection and is compatible with a wide variety of sample types and media. The isothermal reaction conditions and homogeneous endpoint detection utilized in this assay are well suited for laboratory automation and high-throughput screening applications as well as for point-of-care testing. PMID:22000085

Doseeva, Victoria; Forbes, Thomas; Wolff, John; Khripin, Yuri; O'Neil, Dominic; Rothmann, Thomas; Nazarenko, Irina

2011-10-14

2

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

Microsoft Academic Search

Since the development of polymerase chain reaction, amplification of nucleic acids has emerged as an elemental tool for molecular\\u000a biology, genomics, and biotechnology. Amplification methods often use temperature cycling to exponentially amplify nucleic\\u000a acids; however, isothermal amplification methods have also been developed, which do not require heating the double-stranded\\u000a nucleic acid to dissociate the synthesized products from templates. Among the

Yong-Joo Jeong; Dong-Eun Kim

2009-01-01

3

Application of Isothermal Helicase-Dependent Amplification with a Disposable Detection Device in a Simple Sensitive Stool Test for Toxigenic Clostridium difficile  

PubMed Central

Enzyme immunoassays (EIAs) are commonly used for the diagnosis of cases of Clostridium difficile-associated diarrhea (CDAD). However, these EIAs have high false-negative rates, even in patients with severe clinical disease. We have developed an IsoAmp CDAD test using a simple and user-friendly procedure to identify toxigenic C. difficile in feces. After DNA extraction from fecal samples, both the conserved sequence of the 5?-end fragment of the C. difficile tcdA toxin gene and competitive amplification internal control sequence were amplified using helicase-dependent amplification. Amplification products were detected using a novel amplicon-containment detection device. The analytical sensitivity of the assay was 20 copies of C. difficile genomic DNA per reaction. Evaluation of the clinical sensitivity and specificity of the IsoAmp CDAD test versus an EIA method using a PCR method as the reference standard revealed 100% sensitivity and 100% specificity for the IsoAmp CDAD test compared with 90.9% sensitivity and 100% specificity for the EIA method. Because the IsoAmp CDAD test requires no expensive equipments for nucleic acid amplification or detection and can be performed on a random access basis, the test provides a practical alternative to immunoassays for the diagnosis of CDAD with improved sensitivity.

Chow, Wing Huen A.; McCloskey, Cindy; Tong, Yanhong; Hu, Lin; You, Qimin; Kelly, Ciaran P.; Kong, Huimin; Tang, Yi-Wei; Tang, Wen

2008-01-01

4

Evaluation of Chlamydia trachomatis and Neisseria gonorrhoeae detection in urine, endocervical, and vaginal specimens by a multiplexed isothermal thermophilic helicase-dependent amplification (tHDA) assay.  

PubMed

We have developed a new research assay that combines sequence-specific sample preparation and isothermal amplification for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae infections. The assay targets both the omp gene and the cryptic plasmid of C. trachomatis and the multicopy opa gene of N. gonorrhoeae, which are amplified and detected in a single reaction. We evaluated the ability of the assay to detect C. trachomatis and N. gonorrhoeae infections in first-catch urine, swab, and liquid-based cytology samples. Total agreement between the new assay and APTIMA Combo 2 varied between 95.3% and 100%, depending on the sample type and target detected. Total agreement between the new assay and BD ProbeTec varied between 96.7% and 100%, depending on the sample type and target detected. The assay has a simple work flow, and endpoint results can be achieved in 3 h, including sample preparation. The assay described here was evaluated for research use and was compared to commercially available assays. PMID:21956990

O'Neil, Dominic; Doseeva, Victoria; Rothmann, Thomas; Wolff, John; Nazarenko, Irina

2011-09-28

5

A rapid and simple isothermal nucleic acid amplification test for detection of herpes simplex virus types 1 and 2  

Microsoft Academic Search

BackgroundA simple and rapid IsoAmp® HSV assay has been developed for qualitative detection of herpes simplex virus (HSV) types 1 and 2 from genital lesions. Sample preparation involved a simple dilution step and the diluted specimens were directly added to the device and amplified by isothermal helicase-dependent amplification (HDA). Amplification products were then detected by a DNA strip embedded in

Hyun-Jin Kim; Yanhong Tong; Wen Tang; Louisito Quimson; Vicki A. Cope; Xiaojing Pan; Aurelie Motre; Richard Kong; Jian Hong; Debbie Kohn; Nancy S. Miller; Melinda D. Poulter; Huimin Kong; Yi-Wei Tang; Belinda Yen-Lieberman

2011-01-01

6

Drawback of loop-mediated isothermal amplification  

Microsoft Academic Search

Loop-mediated isothermal amplification (LAMP) was a novel nucleic acid amplification method that amplified 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. In our research, LAMP was developed to detect foodborn Salmonella in raw milk, a set of six primers, two outer, two

Wang Deguo; Huo Guicheng; Wang Fugui; Li Yonggang; Ren Daxi

7

Compact optical diagnostic device for isothermal nucleic acids amplification  

Microsoft Academic Search

We recently reported the successful use of the loop-mediated isothermal amplification (LAMP) reaction for hepatitis B virus (HBV) DNA amplification and its optimal primer design method. In this study, we report the development of an integrated isothermal device for both amplification and detection of targeted HBV DNA. It has two major components, a disposable polymethyl methacrylate (PMMA) micro-reactor and a

Szu-Yuan Lee; Jhen-Gang Huang; Tsung-Liang Chuang; Jin-Chuan Sheu; Yi-Kuang Chuang; Mark Holl; Deirdre R. Meldrum; Chun-Nan Lee; Chii-Wann Lin

2008-01-01

8

Isothermal amplification system based on template-dependent extension.  

PubMed

A novel template-dependent extension based isothermal amplification (TEIA) system with high single-base discrimination capability is developed, where the interference caused by non-specific reaction in isothermal strand displacement amplification (SDA) technique is substantially avoided via using a functionalized template probe, showing potential value in the development and application of SDA based detection devices. PMID:23417075

Zhou, Hui; Xie, Su-Jin; Zhang, Song-Bai; Shen, Guo-Li; Yu, Ru-Qin; Wu, Zai-Sheng

2013-03-25

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

Quantitative Reverse Transcription Strand Displacement Amplification: Quantitation of Nucleic Acids Using an Isothermal Amplification Technique  

Microsoft Academic Search

Recent advances in nucleic acid amplification techniques have allowed for quantitation of viral nucleic acid levels in clinical specimens. The most prevalent testing is carried out for HIV viral load. Strand displacement amplification (SDA) is an isothermal DNA amplification system utilizing a restriction enzyme and a DNA polymerase with strand displacement properties. SDA was adapted for quantitative RNA amplification (QRT-SDA)

Colleen M. Nycz; Cheryl H. Dean; Perry D. Haaland; Catherine A. Spargo; G. Terrance Walker

1998-01-01

11

One new method of nucleic acid amplification—Loop-mediated isothermal amplification of DNA  

Microsoft Academic Search

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method, which amplifies DNA with high\\u000a specificity, sensitivity, rapidity and efficiency under isothermal conditions using a set of four specially designed primers\\u000a and a Bst DNA polymerase with strand displacement activity. The basic principle, characteristics, development of LAMP and its applications\\u000a are summarized in this article.

Xue-en Fang; Jian Li; Qin Chen

2008-01-01

12

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

13

Optimization of turn-back primers in isothermal amplification  

Microsoft Academic Search

The application of isothermal amplification technologies is rapidly expanding and currently covers different areas such as pathogen detection and SNP genotyping. Meanwhile, many of such technologies have complex reaction processes and often require a fine-tuned primer set where existing primer design tools are not sufficient. We have developed a primer selection system for one important primer, the turn-back primer (TP),

Yasumasa Kimura; Michiel J. L. de Hoon; Shintaro Aoki; Yuri Ishizu; Carsten O. Daub; Alexander Lezhava; Erik Arner; Yoshihide Hayashizaki; Yuki Kawai; Yasushi Kogo

2011-01-01

14

Rapid Diagnosis of Human Herpesvirus 6 Infection by a Novel DNA Amplification Method, Loop-Mediated Isothermal Amplification  

Microsoft Academic Search

A novel nucleic acid amplification method, termed loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions, may be a valuable tool for the rapid detection of infectious agents. LAMP was developed for human herpesvirus 6 (HHV-6), and its reliability was evaluated in this study. Although LAMP products were detected in HHV-6 B and

Masaru Ihira; Tetsushi Yoshikawa; Yoshihiko Enomoto; Shiho Akimoto; Masahiro Ohashi; Sadao Suga; Naoko Nishimura; Takao Ozaki; Yukihiro Nishiyama; Tsugunori Notomi; Yoshinori Ohta; Yoshizo Asano

2004-01-01

15

Isothermal amplification method for next-generation sequencing  

PubMed Central

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.

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

2013-01-01

16

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

17

Study on Loop-Mediated Isothermal Amplification Assay for Detection of Salmonella in Meat Products  

Microsoft Academic Search

A novel nucleic acid amplification method, termed Loop-mediated Isothermal Amplification (LAMP), which amplifies DNA with high specificity, efficiency, andrapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand displacement activity, may be a valuable tool for the rapid detection of infectious diseases. This study designed two pairs of gene-specific primers of conservative

Wang Yu; Zhang Wei; Yuan Yaowu; Ma Xiaoyan; Zhang Huiyan; Su Xudong; Wang Zhenqiang; Ma Bo

2009-01-01

18

Detection of periodontal pathogen Porphyromonas gingivalis by loop-mediated isothermal amplification method  

Microsoft Academic Search

A method for nucleic acid amplification, loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for periodontal pathogen, Porphyromonas gingivalis. A set of six primers was designed by targeting the 16S ribosomal RNA gene. By the detection system, target DNA was amplified and visualized on agarose gel within 30 min under isothermal condition at 64

Hiroshi Maeda; Susumu Kokeguchi; Chiyo Fujimoto; Ichiro Tanimoto; Wakako Yoshizumi; Fusanori Nishimura; Shogo Takashiba

2005-01-01

19

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

Microsoft Academic Search

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, sensitivity and rapidity, which can be applied for disease diagnosis in shrimp aquaculture. The method is performed under isothermal conditions with a set of four specially designed primers that recognize six distinct sequences of the target. In this study,

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

2007-01-01

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

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

Microsoft Academic Search

In this paper, we developed a metal micro-fluidic chip with advanced surface processing for ultra-small volume molecular isothermal amplification. This method takes advantages of the nucleic acid amplification with good stability and consistency, high sensitivity about 31 genomic DNA copies and bacteria specific gene identification. Based on the advanced surface processing, the bioreaction assays of nucleic acid amplification was dropped

Guoliang Huang; Li Ma; Xiaoyong Yang; Xu Yang

2011-01-01

22

Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products  

Microsoft Academic Search

As the human genome is decoded and its involvement in diseases is being revealed through postgenome research, increased adoption of genetic testing is expected. Critical to such testing methods is the ease of implementation and comprehensible presentation of amplification results. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, specific and cost-effective nucleic acid amplification method when compared to PCR, nucleic

Yasuyoshi Mori; Hidetoshi Kanda; Tsugunori Notomi; Norihiro Tomita

2008-01-01

23

Rapid and sensitive detection of Penaeus monodon nucleopolyhedrovirus by loop-mediated isothermal amplification  

Microsoft Academic Search

Loop-mediated isothermal amplification (LAMP) is a novel, sensitive and rapid method for amplification of nucleic acids under isothermal conditions. In this report, a LAMP method was developed for detection of Penaeus monodon nucleopolyhedrovirus (PemoNPV), known previously as monodon baculovirus (MBV), using a set of six primers designed to specifically recognize the PemoNPV polyhedrin gene. The optimized time and temperature conditions

Parin Chaivisuthangkura; Chutima Srisuk; Sombat Rukpratanporn; Siwaporn Longyant; Pattarin Sridulyakul; Paisarn Sithigorngul

2009-01-01

24

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

Microsoft Academic Search

Loop-mediated isothermal amplification (LAMP) is an original nucleic acid amplification method established by Notomi et al.\\u000a LAMP is performed under isothermal condition, employing only a basic reaction protocol and minimal supporting electronics.\\u000a These requirements prove to be viable for exploring the avenues to down-scale this biological reaction for Lab-on-a-chip application.\\u000a Hence here, we developed a novel technique for fluorescent imaging

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

2008-01-01

25

Development of loop-mediated isothermal amplification (LAMP) method for diagnosis of equine piroplasmosis  

Microsoft Academic Search

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid method whereby DNA is amplified with high specificity, efficiency, and rapidity under isothermal conditions using a set of four specifically designed primers and a DNA polymerase with strand displacement activity. In this study, we used LAMP primer sets designed from EMA-1 and Bc 48 genes for detection of Theileria equi and

Andy Alhassan; Oriel M. M. Thekisoe; Naoaki Yokoyama; Noboru Inoue; Makhosazana Y. Motloang; Peter A. Mbati; Hong Yin; Yoshinari Katayama; Toru Anzai; Chihiro Sugimoto; Ikuo Igarashi

2007-01-01

26

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

Microsoft Academic Search

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

Ludwig Niessen; Rudi F. Vogel

2010-01-01

27

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

NASA Astrophysics Data System (ADS)

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

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

2011-01-01

28

Monitoring the amplification of CATCH, a 3SR based cooperatively coupled isothermal amplification system, by fluorimetric methods  

Microsoft Academic Search

Three different types of fluorescence detection methods were employed to monitor amplification of a previously established isothermal cooperatively coupled amplification system as it can serve as a tool for the investigation of fundamental issues in evolutionary optimization. By using 54IRD-41 fluorescent labeled primers, the intercalating dye TOPRO-1 and a 54fluorescin\\/34DABCYL 4-(4-dimethy- lamino-phenylazo)benzoic acid labeled ss 24 nt DNA, evolving molecular

Ralf Ehricht; Thomas Kirner; Thomas Ellinger; Petra Foerster; John S. McCaskill

1997-01-01

29

Nucleic Acid Isothermal Amplification Technologies—A Review  

Microsoft Academic Search

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.

Pooria Gill; Amir Ghaemi

2008-01-01

30

Colorimetric detection of human papilloma virus by double isothermal amplification.  

PubMed

We developed a polymerase reaction free, low-cost and sensitive assay for the colorimetric detection of Human Papilloma Virus (HPV), based on the use of a smart design exploiting magnetic microbeads, chimeric RNA/DNAzyme oligonucleotides, and double signal amplification. This method allows obtaining a fast response with a detection limit of 10 pM, avoiding the amplification of the target via traditional PCR. PMID:24098886

Persano, Stefano; Valentini, Paola; Kim, Joong Hyun; Pompa, Pier Paolo

2013-10-17

31

Isothermal Amplification Using a Chemical Heating Device for Point-of-Care Detection of HIV-1  

PubMed Central

Background To date, the use of traditional nucleic acid amplification tests (NAAT) for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care (POC) settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. Methodology/Significant Findings In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid amplification (NINA) heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. Conclusion The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC.

Curtis, Kelly A.; Rudolph, Donna L.; Nejad, Irene; Singleton, Jered; Beddoe, Andy; Weigl, Bernhard; LaBarre, Paul; Owen, S. Michele

2012-01-01

32

Real-time detection of telomerase activity using the exponential isothermal amplification of telomere repeat assay.  

PubMed

As crucial pieces in the puzzle of cancer and human aging, telomeres and telomerase are indispensable in modern biology. Here we describe a novel exponential isothermal amplification of telomere repeat (EXPIATR) assay--a sensitive, simple, and reliable in vitro method for measuring telomerase activity in cell extracts. Through a strategically designed path of nucleic acid isothermal amplifications, EXPIATR abandons the expensive thermal cycling protocol and achieves ultrafast detection: telomerase activity equivalent to a single HeLa cancer cell can be detected in ?25 min. PMID:23186115

Tian, Leilei; Weizmann, Yossi

2012-12-03

33

Integrated glass microdevice for nucleic acid purification, loop-mediated isothermal amplification, and online detection.  

PubMed

A microdevice made of glass for genetic analysis has been fabricated, for the first time, for integration of extraction of nucleic acids and loop-mediated isothermal amplification (LAMP), followed by online fluorescence detection of amplification products on a single chip. The nucleic acid (NA) extraction region consists of a microfabricated serpentine channel in which micropillars were etched to increase the channel surface area and the capture efficiency of NAs. Nucleic acid molecules were bound to these pillars and channel surface in the presence of the chaotropic salt guanidine hydrochloride and eluted into a downstream amplification chamber with low ionic strength buffer where loop-mediated isothermal amplification was efficiently performed. Amplification can be detected online by the increase of fluorescence intensity at 540 nm when a low concentration of SYBR Green I, a fluorescent dsDNA intercalating dye, is employed. Flow control was accomplished by using laminar flow and differential channel flow resistances. Through passivation of the LAMP chamber and the channel between the extraction region and amplification domain, effective nucleic acid extraction and amplification were performed by just using a double-channel syringe pump and a heating block. By using this integrated microdevice, the purification of nucleic acids from complex biological matrixes and their subsequent amplification and detection online could be finished within 2 h. PMID:21456520

Wu, Qingqing; Jin, Wei; Zhou, Chao; Han, Sihai; Yang, Wenxiu; Zhu, Qiangyuan; Jin, Qinhan; Mu, Ying

2011-04-01

34

In Vitro Selection and Characterization of Cellulose-Binding RNA Aptamers Using isothermal Amplification  

Microsoft Academic Search

We sought to create new cellulose-binding RNA aptamers for use as modular components in the engineering of complex functional nucleic acids. We designed our in vitro selection strategy to incorporate self-sustained sequence replication (3SR), which is an isothermal nucleic acid amplification protocol that allows for the rapid amplification of RNAs with little manipulation. The best performing aptamer representative was chosen

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

2008-01-01

35

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

36

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

Microsoft Academic Search

A simple isothermal nucleic-acid amplification reaction, primer generation-rolling circle amplifica- tion (PG-RCA), was developed to detect specific nucleic-acid sequences of sample DNA. This ampli- fication method is achievable at a constant tem- perature (e.g. 608C) 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

Taku Murakami; Jun Sumaoka; Makoto Komiyama

2009-01-01

37

High sensitive, colorimetric, isothermal nucleic acids amplification: A versatile platform for protein biosensors  

Microsoft Academic Search

Colorimetric, in situ, isothermal rolling circle amplification (RCA)-based biosensor was developed as a novel, versatile nucleic acid-based amplification machine for platelet-derived growth factor (PDGF) detection. This antibody-free biosensor utilized nucleic acids immobilized on the plate and DNA aptamers to capture the target protein. As compared to the conventional fluorescence measurement [1], the reported colorimetric method can facilitate the development of

Ming-Yu Lin; Yu-Wei Chang; Yu-Ting Tai; Yuh-Shyong Yang; Yang-Tung Huang

2008-01-01

38

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

Microsoft Academic Search

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react under isothermal conditions with high specificity, efficiency, and rapidity. We used LAMP for detection of Mycobacterium tuberculosis complex, Mycobacterium avium, and Mycobacterium intracellulare directly from sputum specimens as well as for detection of culture isolates grown in a liquid medium (MGIT; Nippon Becton Dickinson Co.,

Tomotada Iwamoto; Toshiaki Sonobe; Kozaburo Hayashi

39

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

Microsoft Academic Search

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react under isothermal conditions with high specificity, efficiency, and rapidity. We used LAMP for detection of Mycobacterium tuberculosis complex, Mycobacterium avium, and Mycobacterium intracellulare directly from sputum specimens as well as for detection of culture isolates grown in a liquid medium (MGIT; Nippon Becton Dickinson Co.,

Tomotada Iwamoto; Toshiaki Sonobe; Kozaburo Hayashi

2003-01-01

40

Real-time detection of isothermal amplification reactions with thermostable catalytic hairpin assembly.  

PubMed

Catalytic hairpin assembly (CHA) is an enzyme-free amplification method that has previously proven useful in amplifying and transducing signals at the terminus of nucleic acid amplification reactions. Here, for the first time, we engineered CHA to be thermostable from 37 to 60 °C and in consequence have generalized its application to the real-time detection of isothermal amplification reactions. CHA circuits were designed and optimized for both high- and low-temperature rolling circle amplification (RCA) and strand displacement amplification (SDA). The resulting circuits not only increased the specificity of detection but also improved the sensitivity by as much as 25- to 10000-fold over comparable real-time detection methods. These methods have been condensed into a set of general rules for the design of thermostable CHA circuits with high signals and low noise. PMID:23647466

Jiang, Yu Sherry; Li, Bingling; Milligan, John N; Bhadra, Sanchita; Ellington, Andrew D

2013-05-09

41

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

42

Enzyme-Free Isothermal Exponential Amplification of Nucleic Acids and Nucleic Acid Analog Signals.  

National Technical Information Service (NTIS)

An enzyme-free, isothermal method of generating an amplification signal indicative of a target nucleic acid molecule is provided, as are compositions for performing such a method. An advantage of the detection system is that it is very sensitive, and can ...

B. Yurke D. Zhang E. Winfree

2004-01-01

43

Rapid and sensitive detection of Acinetobacter baumannii using loop-mediated isothermal amplification.  

PubMed

Here we report the design and evaluation of a loop-mediated isothermal amplification (LAMP) assay for detecting Acinetobacter baumannii DNA based on the 16S-23S rRNA intergenic spacer (ITS) sequence. The results showed that target DNA was amplified and visualized within 30min and with a detection limit 100-fold greater than PCR. PMID:23220188

Soo, Po-Chi; Tseng, Chun-Chieh; Ling, Siao-Ru; Liou, Ming-Li; Liu, Chih-Chin; Chao, Huei-Jen; Lin, Teng-Yi; Chang, Kai-Chih

2012-12-05

44

Nucleic acid sensing by regenerable surface-associated isothermal rolling circle amplification  

Microsoft Academic Search

A novel method for regenerating biosensors has been developed in which the highly specific detection of nucleic acid sequences is carried out using molecular padlock probe (MPP) technology and surface-associated rolling circle amplification (RCA). This technique has a low occurrence of false positive results when compared to polymerase chain reaction, and is an isothermal reaction, which is advantageous in systems

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

2007-01-01

45

Sensitive and Specific Detection of Yersinia pseudotuberculosis by Loop-Mediated Isothermal Amplification  

PubMed Central

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

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

2004-01-01

46

Mutation detection and single-molecule counting using isothermal rolling-circle amplification  

Microsoft Academic Search

Rolling-circle amplification (RCA) driven by DNA polymerase can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. In the presence of two primers, one hybridizing to the + strand, and the other, to the – strand of DNA, a complex pattern of DNA strand displacement ensues that generates 109 or more copies of each circle in

Xiaohua Huang; Zhengrong Zhu; Patricia Bray-Ward; David C. Thomas; Paul M. Lizardi; David C. Ward

1998-01-01

47

Sensitive and rapid detection of Trueperella pyogenes using loop-mediated isothermal amplification method.  

PubMed

Here we report the design and evaluation of a loop-mediated isothermal amplification (LAMP) assay for detecting Trueperella pyogenes DNA based on the plo gene sequence that encodes pyolysin. The results showed that target DNA was amplified specifically and with a detection limit 100-fold greater than PCR. PMID:23517678

Zhang, Wenlong; Meng, Xiangli; Wang, Junwei

2013-03-18

48

Detection and identification of Brettanomyces\\/ Dekkera sp. yeasts with a loop-mediated isothermal amplification method  

Microsoft Academic Search

Primer sets for a loop-mediated isothermal amplification (LAMP) method were developed to specifically identify each of the four Brettanomyces\\/Dekkera species, Dekkera anomala, Dekkera bruxellensis, Dekkera custersiana and Brettanomyces naardenensis. Each primer set was designed with target sequences in the ITS region of the four species and could specifically amplify the target DNA of isolates from beer, wine and soft drinks.

Nobuyuki Hayashi; Ritsuko Arai; Setsuzo Tada; Hiroshi Taguchi; Yutaka Ogawa

2007-01-01

49

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

PubMed Central

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 for use in GMO studies. Results show that detection of 0.01% GMO in equivalent background DNA was possible and dilutions of template suggest that detection from single copies of the template may be possible using LAMP. Conclusion This work shows that GMO detection can be carried out using LAMP for routine screening as well as for specific events detection. Moreover, the sensitivity and ability to amplify targets, even with a high background of DNA, here demonstrated, highlights the advantages of this isothermal amplification when applied for GMO detection.

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

2009-01-01

50

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

Microsoft Academic Search

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

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

2008-01-01

51

Robustness of a loop-mediated isothermal amplification reaction for diagnostic applications.  

PubMed

We evaluated the robustness of loop-mediated isothermal amplification (LAMP) of DNA for bacterial diagnostic applications. Salmonella enterica serovar Typhi was used as the target organism and compared with a real-time quantitative PCR (qPCR) for testing assay performance and reproducibly, as well as the impact of pH and temperature stability. This isothermal amplification method appeared to be particularly robust across 2 pH units (7.3-9.3) and temperature values (57-67 °C). The detection limit was comparable to that observed using optimized home-brew qPCR assays. The specificity of the amplification reaction remained high even at temperatures markedly different from the optimal one. Exposing reagents to the ambient temperature during the preparation of the reaction mixture as well as prolonging times for preparing the amplification reaction did not yield false-positive results. LAMP remained sensitive and specific despite the addition of untreated biological fluids such as stool or urine that commonly inhibit PCR amplification. Whereas the detection of microorganisms from whole blood or a blood-culture medium typically requires extensive sample purification and removal of inhibitors, LAMP amplification remained more sensitive than conventional qPCR when omitting such preparatory steps. Our results demonstrate that LAMP is not only easy to use, but is also a very robust, innovative and powerful molecular diagnostic method for both industrialized and developing countries. PMID:21276085

Francois, Patrice; Tangomo, Manuela; Hibbs, Jonathan; Bonetti, Eve-Julie; Boehme, Catharina C; Notomi, Tsugunori; Perkins, Mark D; Schrenzel, Jacques

2011-03-16

52

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

PubMed Central

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°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 (?60 molecules) of genomic DNA from Listeria monocytogenes, indicating strong applicability of PG–RCA to various molecular diagnostic assays.

Murakami, Taku; Sumaoka, Jun; Komiyama, Makoto

2009-01-01

53

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

NASA Astrophysics Data System (ADS)

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

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

54

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

55

Rapid Detection of Rabies Virus by Reverse Transcription Loop-Mediated Isothermal Amplification  

Microsoft Academic Search

SUMMARY: In this study, reverse transcription loop-mediated isothermal amplification (RT-LAMP) was established which can detect 103 copies of viral RNA corresponding to approximately 5 fg of RNA. RT-LAMP with the Phil primer set designed according to the nucleotide sequences obtained from a Kyoto patient who contracted rabies in the Philippines was able to amplify all 16 street viral sequences derived

Bazartseren Boldbaatar; Satoshi Inoue; Naoko Sugiura; Akira Noguchi; Jun Ryan; C. Orbin; Catalino Demetri; Mary Elizabeth Miranda; Akio Yamada

56

Detection of tomato yellow leaf curl virus by loop-mediated isothermal amplification reaction  

Microsoft Academic Search

The genomic DNA molecule of tomato yellow leaf curl virus (TYLCV), a whitefly-transmitted geminivirus, was amplified from total DNA extracts of TYLCV-infected tomato (Lycopersicon esculentum) by the use of loop-mediated isothermal amplification (LAMP). The procedure was also used to amplify TYLCV DNA from total DNA extracts of individual whiteflies (Bemisia tabaci) that had fed on TYLCV-infected plants. One of the

Shiro Fukuta; Shinro Kato; Keiko Yoshida; Yuko Mizukami; Akira Ishida; Junnichi Ueda; Michio Kanbe; Yoshiyuki Ishimoto

2003-01-01

57

A disposable, integrated loop-mediated isothermal amplification cassette with thermally actuated valves  

Microsoft Academic Search

An inexpensive, disposable, integrated, polymer-based cassette for loop-mediated isothermal amplification (LAMP) of target\\u000a nucleic acids was designed, fabricated, and tested. The LAMP chamber was equipped with single-use, thermally actuated valves\\u000a made with a composite consisting of a mixture of PDMS and expandable microspheres. The effect of the composite composition\\u000a on its expansion was investigated, and the valve’s performance was evaluated.

Changchun Liu; Michael G. Mauk; Haim H. Bau

2011-01-01

58

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

Microsoft Academic Search

A compact hand-held heated fluorometric instrument for performing real-time isothermal nucleic acid amplification and detection is described. The optoelectronic instrument combines a Printed Circuit Board\\/Micro Electro Mechanical Systems (PCB\\/MEMS) reaction detection\\/chamber containing an integrated resistive heater with attached miniature LED light source and photo-detector and a disposable glass waveguide capillary to enable a mini-fluorometer. The fluorometer is fabricated and assembled

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

2007-01-01

59

Use of reverse transcription loop-mediated isothermal amplification for the detection of Plum pox virus  

Microsoft Academic Search

A one step, accelerated reverse transcription loop-mediated isothermal amplification (RT-LAMP) procedure was developed for the detection of Plum pox virus (PPV). The six primers required for accelerated RT-LAMP were designed using a conserved region in the C-terminus of the coat protein coding region of PPV. RT-LAMP was used to detect isolates of five strains of PPV including the strains D,

Aniko Varga; Delano James

2006-01-01

60

Reverse Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of Transmissible Gastroenteritis Virus  

Microsoft Academic Search

Transmissible gastroenteritis virus (TGEV) is the causative agent of porcine transmissible gastroenteritis, and sensitive\\u000a detection methods are required for preventing the disease. In this article, reverse transcription-loop-mediated isothermal\\u000a amplification (RT-LAMP) was developed to detect TGEV. Three pairs of primers targeting the nucleocapsid (N) gene of TGEV were\\u000a synthesized and used in the RT-LAMP. The optimization, sensitivity, and specificity of the

Pengchong Li; Xiaofeng Ren

2011-01-01

61

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

PubMed

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

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

2009-02-26

62

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

PubMed

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

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

2012-07-04

63

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

64

Detection of Salmonella invA by isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) in Zambia  

Microsoft Academic Search

The isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) is a new isothermal DNA amplification method composed of exo Bca DNA polymerase, RNaseH and DNA–RNA chimeric primers. We detected invA of Salmonella from chicken carcasses, egg yolk and cattle fecal samples. Fifty-three of 59 isolates were invA-positive in ICAN-chromatostrip detection. The result was consistent with those obtained by standard

Emiko Isogai; Chitwambi Makungu; John Yabe; Patson Sinkala; Andrew Nambota; Hiroshi Isogai; Hideto Fukushi; Manda Silungwe; Charles Mubita; Michelo Syakalima; Bernard Mudenda Hang'ombe; Shunji Kozaki; Jun Yasuda

2005-01-01

65

Multiple strategies to improve sensitivity, speed and robustness of isothermal nucleic acid amplification for rapid pathogen detection  

Microsoft Academic Search

Background  In the past decades the rapid growth of molecular diagnostics (based on either traditional PCR or isothermal amplification\\u000a technologies) meet the demand for fast and accurate testing. Although isothermal amplification technologies have the advantages\\u000a of low cost requirements for instruments, the further improvement on sensitivity, speed and robustness is a prerequisite for\\u000a the applications in rapid pathogen detection, especially at

Yanhong Tong; Bertrand Lemieux; Huimin Kong

2011-01-01

66

Loop-Mediated Isothermal Amplification Method Targeting the lytA Gene for Detection of Streptococcus pneumoniae  

Microsoft Academic Search

It is difficult to separate Streptococcus pneumoniae from the genotypically similar species Streptococcus mitis and Streptococcus oralis, which are commensals of the human oral cavity. A novel nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63°C) with high specificity, efficiency, and rapidity, was examined regarding its applicability for detecting S. pneumoniae .A nS. pneumoniae-specific

Mitsuko Seki; Yoshihisa Yamashita; Hirotaka Torigoe; Hiromasa Tsuda; Setsuko Sato; Masao Maeno

2005-01-01

67

Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of West Nile Virus  

Microsoft Academic Search

A one-step, single tube, real-time accelerated reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting the envelope gene of West Nile (WN) virus. The RT-LAMP assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, efficiency, and rapidity under isothermal conditions with a set of six specially designed primers that recognize eight distinct

Manmohan Parida; Guillermo Posadas; Shingo Inoue; Futoshi Hasebe; Kouichi Morita

2004-01-01

68

Reverse transcription loop-mediated isothermal amplification for the detection of highly pathogenic porcine reproductive and respiratory syndrome virus  

Microsoft Academic Search

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the open reading frames 1a of highly pathogenic porcine reproductive and respiratory syndrome virus genome was developed. The 10 reference strains, 1 clinical isolation strain and 122 positive samples were tested. Positive reactions were confirmed for all strains and specimens by reverse transcription loop-mediated isothermal amplification and nested reverse transcription polymerase

Hao-tai Chen; Jie Zhang; De-hui Sun; Li-na Ma; Xiang-tao Liu; Kai Quan; Yong-sheng Liu

2008-01-01

69

Development and clinical validation of a loop-mediated isothermal amplification method for the rapid detection of Neisseria meningitidis  

Microsoft Academic Search

Loop-mediated isothermal amplification (LAMP) is an innovative technique that allows the rapid detection of target nucleic acid sequences under isothermal conditions without the need for complex instrumentation. The development, optimization, and clinical validation of a LAMP assay targeting the ctrA gene for the rapid detection of capsular Neisseria meningitidis were described. Highly specific detection of capsular N. meningitidis type strains

James P. McKenna; Derek J. Fairley; Michael D. Shields; Sara L. Cosby; Dorothy E. Wyatt; Conall McCaughey; Peter V. Coyle

2011-01-01

70

Direct detection of potato leafroll virus in potato tubers by immunocapture and the isothermal nucleic acid amplification method NASBA  

Microsoft Academic Search

NASBA, an isothermal amplification method for nucleic acids, was applied to the detection of RNA of potato leafroll virus (PLRV) in a single enzymatic reaction at 41 °C. A set of primers was selected from the coat protein open reading frame sequence of PLRV to allow amplification of viral RNA. The NASBA reaction products were visualized after electrophoresis by ethidium

G. Leone; H. B. van Schijndel; B. van Genien; C. D. Schoen

1997-01-01

71

Detection of Four Plasmodium Species by Genus and Species-Specific Loop-Mediated Isothermal Amplification for Clinical Diagnosis  

Microsoft Academic Search

Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the clinical detection of four species of human malaria parasites: Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. We evaluated the sensitivity and specificity of LAMP in comparison with the results of microscopic examination and nested PCR. LAMP showed a detection limit (analytical sensitivity) of 10

Eun-Taek Han; Risa Watanabe; Jetsumon Sattabongkot; Benjawan Khuntirat; Jeeraphat Sirichaisinthop; Hideyuki Iriko; Ling Jin; Satoru Takeo; Takafumi Tsuboi; Vector Borne

2007-01-01

72

Sensitive and rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimps by loop-mediated isothermal amplification  

Microsoft Academic Search

A method for nucleic acid amplification, loop-mediated isothermal amplification (LAMP) is a novel, sensitive and rapid technique, which can be applied for disease diagnosis in aquaculture. Using the LAMP method, a highly specific and sensitive diagnostic system for infectious hypodermal and hematopoietic necrosis virus (IHHNV) detection was designed. A set of four primers was designed by targeting the IHHNV genome

Zhao-Feng Sun; Chao-Qun Hu; Chun-Hua Ren; Qi Shen

2006-01-01

73

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

NASA Astrophysics Data System (ADS)

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

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

2005-11-01

74

A new accurate assay for Coxsackievirus A 16 by fluorescence detection of isothermal RNA amplification.  

PubMed

Coxsackievirus A 16 (CA16) is one of the most common causes of hand, foot, and mouth disease (HFMD) worldwide. Without a vaccine or antiviral drug early, rapid, and accurate detection is critical for preventing and controlling HFMD. A simultaneous amplification and testing (SAT) assay was developed for detecting CA16 based on isothermal RNA amplification with fluorescence using standard, real-time PCR equipment. Primers and probes were designed to target the VP1 region of CA16. Virus strains and clinical specimens were used to evaluate the diagnostic performance characteristics of the assay. The assay detected as few as 10 copies of CA16 RNA transcripts. Using real-time PCR plus sequencing as the reference standard, the sensitivity and specificity of the SAT-CA16 assay were 100% and 99.2%, respectively. These findings indicate that SAT-CA16 is a rapid and reliable method for detecting CA16. PMID:23872269

Xu, Jin; Cao, Lingfeng; Su, Liyun; Dong, Niuniu; Yu, Minghui; Ju, Jinliang

2013-07-18

75

Sensitive and Rapid Detection of Edwardsiellosis in Fish by a Loop-Mediated Isothermal Amplification Method  

PubMed Central

Here we report a rapid and sensitive method (using loop-mediated isothermal amplification [LAMP]) for the diagnosis of edwardsiellosis, a fish disease caused by Edwardsiella tarda, in Japanese flounder. A set of four primers was designed, and conditions for the detection were optimized for the detection of E. tarda in 45 min at 65°C. No amplification of the target hemolysin gene was detected in other related bacteria. When the LAMP primers were used, detection of edwardsiellosis in infected Japanese flounder kidney, and spleen and seawater cultures was possible. We have developed a rapid and sensitive diagnostic protocol for edwardsiellosis detection in fish. This is the first report of the application of LAMP for the diagnosis of a fish pathogen.

Savan, Ram; Igarashi, Arisa; Matsuoka, Satoru; Sakai, Masahiro

2004-01-01

76

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.

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

77

The rapid detection of Salmonella from food samples by loop-mediated isothermal amplification (LAMP).  

PubMed

Loop-mediated isothermal amplification (LAMP) assay was applied to the detection of Salmonella in food and human materials. It was possible for the assay to detect Salmonella within 60 min. All of 54 serovars of Salmonella tested were amplified, but all bacteria tested other than Salmonella were not. The LAMP assay could detect 10(2) cfu/ml levels of Salmonella. The specificity was similar to that of a PCR assay, but the sensitivity of LAMP was considered to be greater. Thus, the LAMP assay was confirmed to be a rapid, specific and sensitive detection method for Salmonella. PMID:19579659

Ueda, Shigeko; Kuwabara, Yoshihiro

2009-06-01

78

Loop-mediated isothermal amplification (LAMP): recent progress in research and development.  

PubMed

Loop-mediated isothermal amplification (LAMP) is an established technology that continues to attract the attention of researchers in many fields. Research and development efforts on LAMP technology in recent years have focused on two major areas; first, the study of its clinical application as an approved in vitro diagnostics tool in Japan and certain other countries; and second, research aimed at further simplifying the LAMP test process. This review provides an overview of the status of LAMP on these two topics by summarizing research work conducted, in the main, after our previous review article. PMID:23539453

Mori, Yasuyoshi; Kanda, Hidetoshi; Notomi, Tsugunori

2013-03-29

79

Simple, rapid, inexpensive platform for the diagnosis of malaria by loop mediated isothermal amplification (LAMP).  

PubMed

We attempted to improve the loop-mediated isothermal amplification (LAMP) method for malaria diagnosis by using a simple DNA extraction procedure, and a portable device performing both the amplification and detection of LAMP in one platform. Additionally, the device served as a heating block for the DNA preparation. We refer this method as LAMP-Tube scanner, and evaluated using 209 microscopically positive malaria samples and compared them to RDTs and LAMP-Thermocycler. Two most common human infecting Plasmodium species were detected. The LAMP-Tube scanner method is found to be simple and allowed real-time detection of DNA amplification. The time to amplification varied but was closely less than 60 min. Sensitivity and specificity of LAMP-Tube scanner in detecting Plasmodium falciparum were 95% and 93.3%, compared to microscopy and 98.3% and 100% respectively, compared to standard LAMP-Thermocycler. In addition, it showed a detection limit of 10 and 40 copies of the parasitemia for Plasmodium vivax and P. falciparum. Accordingly, in comparison to the results obtained by microscopy, the LAMP-Tube scanner had a less divergence in sensitivity and specificity, and yielded results similar to those of LAMP-Thermocycler. This method has the great potential as a field usable molecular tool for the diagnosis of malaria and is an alternative to conventional PCR-based diagnostic methods for field use. PMID:23562879

Surabattula, Rambabu; Vejandla, Manju Pradeep; Mallepaddi, Prudhvi Chand; Faulstich, Konrad; Polavarapu, Rathnagiri

2013-04-04

80

Rapid and sensitive detection of Penaeus monodon nucleopolyhedrovirus by loop-mediated isothermal amplification.  

PubMed

Loop-mediated isothermal amplification (LAMP) is a novel, sensitive and rapid method for amplification of nucleic acids under isothermal conditions. In this report, a LAMP method was developed for detection of Penaeus monodon nucleopolyhedrovirus (PemoNPV), known previously as monodon baculovirus (MBV), using a set of six primers designed to specifically recognize the PemoNPV polyhedrin gene. The optimized time and temperature conditions for the LAMP assay were 60 min at 63 degrees C. The sensitivity of LAMP for PemoNPV detection was approximately 50 viral copies ng(-1) genomic DNA (equivalent to 150 viral copies per reaction). Using a DNA template extracted from PemoNPV-infected shrimp by a viral nucleic acid kit, the detection limit of LAMP was 0.7 fg while that of nested PCR was 70 fg; therefore, the LAMP assay was 100 times more sensitive than nested PCR. The LAMP method did not amplify a product using nucleic acid extracted from shrimp infected with other viruses including yellow head virus (YHV), Taura syndrome virus (TSV), white spot syndrome virus (WSSV), Penaeus stylirostris densovirus (PstDNV) known previously as infectious hypodermal and hematopoietic necrosis virus (IHHNV), and Penaeus monodon densovirus (PmDNV) known previously as hepatopancreatic parvovirus (HPV). PMID:19703492

Chaivisuthangkura, Parin; Srisuk, Chutima; Rukpratanporn, Sombat; Longyant, Siwaporn; Sridulyakul, Pattarin; Sithigorngul, Paisarn

2009-08-22

81

Loop-mediated isothermal amplification for rapid and convenient detection of Mycoplasma hyopneumoniae.  

PubMed

Loop-mediated isothermal amplification (LAMP), a novel method of gene amplification, was employed in this study for detecting Mycoplasma hyopneumoniae in the respiratory tract or lungs of swine. The pathogen can be detected in LAMP reactions containing as few as 10 fg purified target DNA (10 copies of M. hyopneumoniae genome) within 30 min, which was comparable to real-time PCR. After 30-min reaction at 63 °C, the addition of a certain amount of dye (SYBR Green I and hydroxyl naphthol blue at a proper ratio) into the LAMP reaction system makes the results easily determined as positive or negative by visual inspection. In addition, the LAMP was able to distinguish between M. hyopneumoniae and other closely-related mycoplasma strains, indicating a high degree of specificity. The LAMP assay was more simple and cheap, since the reaction could be completed under isothermal conditions and less laboratorial infrastructure are required. And, it was proven reliable for M. hyopneumoniae diagnosis of nasal swab and lung samples from the field. PMID:23184577

Li, Jiahe; Minion, F Chris; Petersen, Andrew C; Jiang, Fei; Yang, Sheng; Guo, Panpan; Li, Jinxiang; Wu, Wenxue

2012-11-27

82

Combining isothermal rolling circle amplification and electrochemiluminescence for highly sensitive point mutation detection  

NASA Astrophysics Data System (ADS)

Many pathogenic and genetic diseases are associated with changes in the sequence of particular genes. We describe here a rapid and highly efficient assay for the detection of point mutation. This method is a combination of isothermal rolling circle amplification (RCA) and high sensitive electrochemluminescence (ECL) detection. In the design, a circular template generated by ligation upon the recognition of a point mutation on DNA targets was amplified isothermally by the Phi29 polymerase using a biotinylated primer. The elongation products were hybridized with tris (bipyridine) ruthenium (TBR)-tagged probes and detected in a magnetic bead based ECL platform, indicating the mutation occurrence. P53 was chosen as a model for the identification of this method. The method allowed sensitive determination of the P53 mutation from wild-type and mutant samples. The main advantage of RCA-ECL is that it can be performed under isothermal conditions and avoids the generation of false-positive results. Furthermore, ECL provides a faster, more sensitive, and economical option to currently available electrophoresis-based methods.

Su, Qiang; Zhou, Xiaoming

2008-12-01

83

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

84

Detection of Plasmodium vivax infection in the Republic of Korea by loop-mediated isothermal amplification (LAMP)  

Microsoft Academic Search

Loop-mediated isothermal amplification (LAMP) is a novel technique that rapidly amplifies target DNA in isothermal conditions. In a previous study, the sensitivities and specificities of LAMP, microscopy, and nested PCR were compared in the context of rapid malaria detection. In the present study, LAMP detected vivax malaria parasites in 115 of 117 microscopically positive samples (sensitivity, 98.3%; 95% CI, 97.4–100%),

Jun-Hu Chen; Feng Lu; Chae Seung Lim; Jung-Yeon Kim; Heui-June Ahn; In-Bum Suh; Satoru Takeo; Takafumi Tsuboi; Jetsumon Sattabongkot; Eun-Taek Han

2010-01-01

85

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

Microsoft Academic Search

Background  Infectious bursal disease (IBD) is a highly contagious immunosuppressive disease in young chickens caused by infectious bursal\\u000a disease virus (IBDV). It causes huge economic losses to the poultry industry. The objective of this study is to develop a\\u000a loop-mediated isothermal amplification (LAMP) method for the detection and discrimination of IBDV.\\u000a \\u000a \\u000a \\u000a \\u000a Results  In this study, we applied reverse transcription loop-mediated isothermal amplification

Yongqiang Wang; Zhonghui Kang; Honglei Gao; Yulong Gao; Liting Qin; Huan Lin; Fei Yu; Xiaole Qi; Xiaomei Wang

2011-01-01

86

Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases  

Microsoft Academic Search

Loop-mediated isothermal amplification (LAMP) is an established nucleic acid amplification method offering rapid, accurate,\\u000a and cost-effective diagnosis of infectious diseases. This technology has been developed into commercially available detection\\u000a kits for a variety of pathogens including bacteria and viruses. The current focus on LAMP methodology is as a diagnostic system\\u000a to be employed in resource-limited laboratories in developing countries, where

Yasuyoshi Mori; Tsugunori Notomi

2009-01-01

87

LAVA: An Open-Source Approach To Designing LAMP (Loop-Mediated Isothermal Amplification) DNA Signatures  

PubMed Central

Background We developed an extendable open-source Loop-mediated isothermal AMPlification (LAMP) signature design program called LAVA (LAMP Assay Versatile Analysis). LAVA was created in response to limitations of existing LAMP signature programs. Results LAVA identifies combinations of six primer regions for basic LAMP signatures, or combinations of eight primer regions for LAMP signatures with loop primers, which can be used as LAMP signatures. The identified primers are conserved among target organism sequences. Primer combinations are optimized based on lengths, melting temperatures, and spacing among primer sites. We compare LAMP signature candidates for Staphylococcus aureus created both by LAVA and by PrimerExplorer. We also include signatures from a sample run targeting all strains of Mycobacterium tuberculosis. Conclusions We have designed and demonstrated new software for identifying signature candidates appropriate for LAMP assays. The software is available for download at http://lava-dna.googlecode.com/.

2011-01-01

88

Loop-mediated isothermal amplification for the rapid detection of Salmonella.  

PubMed

Loop-mediated isothermal amplification (LAMP) assay detected Salmonella within 60 min. The 220 strains of 39 serotypes of Salmonella subsp. enterica and 7 strains of Salmonella enterica subsp. arizonae were amplified, but not 62 strains of 23 bacterial species other than Salmonella. The sensitivity of the LAMP assay was found to be >2.2 cfu/test tube using nine serotypes. The specificity was similar to that of a PCR assay, but the sensitivity of LAMP was greater. Both fluorescence and turbidity were able to detect the products in the LAMP assay. S. enteritidis in a liquid egg sample artificially inoculated with the organism was detected by the LAMP assay at 2.8 cfu/test tube, although negative by PCR assay. These results indicate that the LAMP assay is a rapid, specific and sensitive detection method for Salmonella. PMID:16242860

Hara-Kudo, Yukiko; Yoshino, Manabu; Kojima, Tadashi; Ikedo, Masanari

2005-10-07

89

Loop-mediated isothermal amplification for rapid detection of Newcastle disease virus.  

PubMed

We have evaluated a diagnostic system based on the loop-mediated isothermal amplification (LAMP) assay for the rapid, simple, and sensitive detection of Newcastle disease virus (NDV) directly from culture isolates as well as clinical samples. By using one set of specific primers targeting the fusion protein gene, the LAMP assay rapidly amplified the target gene within 2 h, requiring only a regular laboratory water bath or heat block for reaction. The results obtained from testing the genomes of 38 NDV strains, other different viruses, and clinical samples of experimentally infected chickens showed that LAMP was as sensitive and specific as nested PCR. All LAMP-positive samples were positive by nested PCR. The LAMP assay is faster than nested PCR, cost-effective, and easy to perform. Our results clearly demonstrate that the LAMP-based assay is a useful tool for the rapid and sensitive diagnosis of NDV infection. PMID:15814979

Pham, Hang Minh; Nakajima, Chie; Ohashi, Kazuhiko; Onuma, Misao

2005-04-01

90

Reverse transcription loop-mediated isothermal amplification for rapid detection of the newly emerged poultry Flavivirus.  

PubMed

Poultry Flavivirus (PF) was a recently emerged virus with high morbidity rates and mortality rates in China. It is the causative agent of egg drop syndrome at present. Development of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was the most efficient way to prevent and control the PF disease. The assay was performed at 64 °C for 45 min, using six specific primers that recognized eight targets of the PF E gene. The RT-LAMP assay, compared to conventional reverse transcription polymerase chain reaction, has 100-fold-greater sensitivity, with a detection limit of 1?×?10(-3) copies per ?L RNA and no cross-reaction with poultry other viruses. The RT-LAMP assay is a valuable tool for detected PF without requiring any sophisticated equipment, and the detection has potential usefulness for clinical diagnosis in the field. PMID:23152303

Li, Zhaolong; Chen, Shaoying; Wang, Shao; Chen, Shilong; Lin, Fengqian

2012-11-15

91

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

PubMed

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of Acanthamoeba. The sensitivity of the LAMP assay was tested using different copies of positive DNA. The specificity of the assay was tested using DNA extracted from Acanthamoeba, Pseudomonas aeruginosa, Candida albicans, herpes simplex virus-1 and human corneal epithelial cells. Its effectiveness was evaluated and compared with culture, corneal smear examination and real-time PCR in corneal samples from mice with Acanthamoeba keratitis. We also tested three corneal samples from patients with suspected Acanthamoeba or fungal infection using LAMP. Loop-mediated isothermal amplification was confirmed to be very sensitive, with the lowest detection limit being ten copies/tube of Acanthamoeba DNA. The LAMP primers only amplified Acanthamoeba DNA. During the development of Acanthamoeba keratitis in mice, almost all of the positive rates of LAMP at each time post-infection were higher than those of culture or corneal smear examination. The total positive rate of LAMP was significantly higher than those of culture and corneal smear examination (p <0.05), whereas the sensitivities of LAMP and real-time PCR were comparable. However, the trends of positive change in these different test methods were generally similar. Of the three clinical corneal specimens, two with suspected Acanthamoeba keratitis tested positive for Acanthamoeba using LAMP along with culture or corneal smear examination, whereas the other suspected fungal keratitis tested negative. The LAMP assay is a simple, rapid, highly specific and sensitive method for the diagnosis of keratitis caused by Acanthamoeba. PMID:23413965

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

2013-02-15

92

Reverse transcription loop-mediated isothermal amplification for rapid detection of transmissible gastroenteritis virus.  

PubMed

Transmissible gastroenteritis virus (TGEV) is the causative agent of porcine transmissible gastroenteritis, and sensitive detection methods are required for preventing the disease. In this article, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was developed to detect TGEV. Three pairs of primers targeting the nucleocapsid (N) gene of TGEV were synthesized and used in the RT-LAMP. The optimization, sensitivity, and specificity of the RT-LAMP were evaluated. Our results showed that the RT-LAMP amplified the N gene with high specificity, efficiency, and rapidity at isothermal condition. The optimal reaction condition was achieved at 60°C for 30 min. The RT-LAMP assay was more sensitive than gel-based RT-PCR and PCR. It had a higher sensitivity than enzyme-linked immunosorbent assay (ELISA) using the equal virus templates. In addition, the established RT-LAMP differentiated TGEV from porcine epidemic diarrhea virus, porcine rotavirus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus, and avian infectious bronchitis virus. The approach is suitable for detecting TGEV for field diagnostics or in less-equipped laboratories due to its convenience and simplicity. PMID:21127872

Li, Pengchong; Ren, Xiaofeng

2010-12-02

93

Rapid detection of herpes simplex virus DNA in cerebrospinal fluid: comparison between loop-mediated isothermal amplification and real-time PCR  

Microsoft Academic Search

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that amplifies DNA with high specificity, efficiency, and speed under isothermal conditions. To evaluate the usefulness of LAMP for diagnosing central nervous system infection with herpes simplex virus (HSV), we compared the LAMP method with real-time PCR, using samples that were previously tested by nested PCR. We examined 69

Hiroshi Kimura; Masaru Ihira; Yoshihiro Enomoto; Jun-ichi Kawada; Yoshinori Ito; Tsuneo Morishima; Tetsushi Yoshikawa; Yoshizo Asano

2005-01-01

94

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

Microsoft Academic Search

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is an important shrimp pathogen that causes mortality in Penaeus stylirostris and stunting (called runt deformity syndrome or RDS) in Penaeus vannamei. Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acids under isothermal conditions. It can be combined with a chromatographic lateral flow dipstick (LFD) for highly specific, rapid and simple visual

Narong Arunrut; Photchanathorn Prombun; Vanvimon Saksmerprome; Timothy W. Flegel; Wansika Kiatpathomchai

2011-01-01

95

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

PubMed Central

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

2012-01-01

96

Rapid detection of human herpesvirus 8 DNA using loop-mediated isothermal amplification.  

PubMed

The reliability of a loop-mediated isothermal amplification (LAMP) method for the detection of human herpesvirus 8 (HHV-8) DNA was evaluated. Although LAMP products were produced with the DNA sample extracted from BCP-1 cells, LAMP products were not produced with the DNAs from seven other human herpesviruses. The detection limit of the HHV-8 LAMP method was 100 copies of target sequence/tube. To determine whether the HHV-8 LAMP method could be used to quantify viral DNA, threshold times, which are defined as the time (in s) it takes to reach the threshold turbidity level (0.1), were measured for the amplification of serial dilutions of a DNA plasmid containing the target sequence. The standard curve possessed a correlation coefficient of 0.9428 with a slope of -84.079 and y-intercept value of 1936.2. Additionally, an attempt was made to detect viral DNA in 17 specimens collected from Kaposi's sarcomas and two cell lines obtained from primary effusion lymphomas. HHV-8 DNA was detected in 14 of the 17 Kaposi's sarcoma tissue samples and both of the primary effusion lymphoma cell lines. Viral DNA was not detected in HHV-8 LAMP-negative samples using the real-time PCR method. PMID:17512061

Kuhara, Tomoe; Yoshikawa, Tetsushi; Ihira, Masaru; Watanabe, Daisuke; Tamada, Yasuhiko; Katano, Harutaka; Asano, Yoshizo; Matsumoto, Yoshinari

2007-05-23

97

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

Microsoft Academic Search

Infectious myonecrosis virus (IMNV) has caused a slowly progressive disease with cumulative mortalities of up to 70% or more in cultured Penaeus (Litopenaeus) vannamei in Northeast Brazil and Indonesia. Rapid detection of viruses by loop-mediated isothermal amplification (LAMP) of genomic material with high specificity and sensitivity can be applied for diagnosis, monitoring and control of diseases in shrimp aquaculture. Using

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

2009-01-01

98

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

Microsoft Academic Search

The characteristics of loop-mediated isothermal amplification (LAMP) make it a promising platform for the molecular detection of tuberculosis (TB) in developing countries. Here, we report on the first clinical evaluation of LAMP for the detection of pulmonary TB in microscopy centers in Peru, Bangladesh, and Tanzania to determine its operational applicability in such settings. A prototype LAMP assay with simplified

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

99

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

PubMed

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

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

2013-08-21

100

Rapid and sensitive detection of white spot syndrome virus by loop-mediated isothermal amplification combined with a lateral flow dipstick  

Microsoft Academic Search

Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acids under isothermal conditions using a set of four specifically designed primers that recognize six distinct target sequences. It can be combined with a chromatographic lateral flow dipstick (LFD) for highly specific, rapid and simple visual detection of WSSV-specific amplicons. Using this protocol, a 30-min amplification followed by 5min hybridization with

Wansadaj Jaroenram; Wansika Kiatpathomchai; Timothy W. Flegel

2009-01-01

101

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

Microsoft Academic Search

Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acids under isothermal conditions. In\\u000a this report, a 20-min LAMP amplification of the DPOL gene of infectious spleen and kidney necrosis virus (ISKNV) using a biotin-labeled\\u000a primer was combined with lateral flow dipstick (LFD) chromatography for rapid and simple visual detection of ISKNV-specific\\u000a amplicons. The LFD process involves a 5-min specific

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

2010-01-01

102

Loop-mediated isothermal amplification for detection of HLA-B*58:01 allele.  

PubMed

Strong association of human leukocyte antigen (HLA)-B*58:01 allele with allopurinol-induced hypersensitivity was found worldwide, especially in the Han Chinese populations. This study aims to develop and evaluate a loop-mediated isothermal amplification (LAMP) assay for rapid detection of HLA-B*58:01. Two sets of LAMP primers targeting exons 2 and 3 of HLA-B*58:01 allele were designed and their annealing temperatures were optimized accordingly. The heating devices for LAMP assay were tested. The analytical sensitivities of the two sets of LAMP primers were determined by 1:10 serial dilution of a positive control with homozygous HLA-B*58:01 allele from 100?ng down to 1?fg. The analytical specificities of the LAMP primers were evaluated by 30 selected University of California, Los Angeles (UCLA) DNA Exchange Program samples with known HLA-B loci typings previously typed by sequencing. Both sets of LAMP primers targeting exons 2 and 3 amplified optimally at 67°C. Thermal cycler is essential in achieving a more precise and specific LAMP result. The sensitivity of the exon 2 LAMP primer set was found to be 1?pg, whereas it was 10?ng for the exon 3 primer set in a 60-min amplification. The LAMP primers were highly specific because LAMP results were perfectly concordant to the sequencing results. The HLA-B*58:01 LAMP assay has compatible sensitivity and specificity to routine genotyping assays, and it is potentially an alternative screening test for the detection of HLA-B*58:01 and ultimately allopurinol-induced hypersensitivity. PMID:23240628

Kwok, J; Kwong, K M

2012-12-13

103

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

PubMed

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

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

2013-05-27

104

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

2008-12-09

105

Detection of Group 1 Trypanosoma brucei gambiense by Loop-Mediated Isothermal Amplification?  

PubMed Central

Trypanosoma brucei gambiense group 1 is the major causative agent of the Gambian human African trypanosomiasis (HAT). Accurate diagnosis of Gambian HAT is still challenged by lack of precise diagnostic methods, low and fluctuating parasitemia, and generally poor services in the areas of endemicity. In this study, we designed a rapid loop-mediated isothermal amplification (LAMP) test for T. b. gambiense based on the 3? end of the T. b. gambiense-specific glycoprotein (TgsGP) gene. The test is specific and amplifies DNA from T. b. gambiense isolates and clinical samples at 62°C within 40 min using a normal water bath. The analytical sensitivity of the TgsGP LAMP was equivalent to 10 trypanosomes/ml using purified DNA and ?1 trypanosome/ml using supernatant prepared from boiled blood, while those of classical PCR tests ranged from 10 to 103 trypanosomes/ml. There was 100% agreement in the detection of the LAMP product by real-time gel electrophoresis and the DNA-intercalating dye SYBR green I. The LAMP amplicons were unequivocally confirmed through sequencing and analysis of melting curves. The assay was able to amplify parasite DNA from native cerebrospinal fluid (CSF) and double-centrifuged supernatant prepared from boiled buffy coat and bone marrow aspirate. The robustness, superior sensitivity, and ability to inspect results visually through color change indicate the potential of TgsGP LAMP as a future point-of-care test.

Njiru, Z. K.; Traub, R.; Ouma, J. O.; Enyaru, J. C.; Matovu, E.

2011-01-01

106

Detection of Mycoplasma pneumoniae by colorimetric loop-mediated isothermal amplification.  

PubMed

Mycoplasma pneumoniae (M. pneumoniae) is one of the most important pathogens that cause respiratory tract infection in children and adults. In this study, we describe a rapid and sensitive colorimetric loop mediated isothermal amplification (LAMP) method to detect M. pneumoniae. The specificity and sensitivity of this assay were detected with 21 common respiratory pathogens and 39 M. pneumoniae DNA. The sensitivity of LAMP was 100% among 39 M. pneumoniae isolates and the specificity was 100% among 9 members of other Mycoplasma and 12 common respiratory pathogens. The lowest detectable limit (LDL) of this assay was 102 copies, which detected by a series of standard M. pneumoniae DNA. To evaluate the clinical applicability of the LAMP assay, a total of 80 clinical samples were examined by conventional PCR, real-time PCR and the LAMP assays, respectively. The positive rates were 15.0%, 32.5% and 26.3%, respectively. This colorimetric LAMP assay demonstrated a high level of sensitivity comparable with that of conventional PCR for the detection of M. pneumoniae. It is a valuable method for simple, cost-effective and rapid detection of M. pneumoniae in the rural areas and basic clinical of China. PMID:23529294

Zhao, Fei; Liu, Zhong; Gu, Yixin; Yang, Yuelian; Xiao, Di; Tao, Xiaoxia; Meng, Fanliang; He, Lihua; Zhang, Jianzhong

2013-03-01

107

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

PubMed

Mouse hepatitis virus (MHV) is one of the most prevalent viruses detected in laboratory mouse colonies. Enterotropic strains predominate in natural infections, and molecular techniques for the detection of MHV shedding in feces are powerful enough to diagnose active infections. A reverse transcription-loop-mediated isothermal amplification (RT-LAMP) technique was developed for the detection of rodent coronaviruses within 90 min. The specificity of this technique was confirmed by its ability to detect all 17 different strains of MHV and 6 strains of rat coronaviruses as well as its failure to detect human, bovine, and porcine coronaviruses nonspecifically. The sensitivity of RT-LAMP was 3.2-fold higher than that of reverse transcription-polymerase chain reaction (RT-PCR) and 31.6-fold lower than that of nested RT-PCR. An evaluation of the diagnostic performance of RT-LAMP performed in duplicate using mouse fecal specimens showed that the sensitivity and specificity with respect to nested RT-PCR were 85.7% and 100%, respectively. RT-LAMP assays would be suitable for monitoring active MHV infection in mouse colonies. PMID:23123121

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

2012-10-30

108

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

PubMed

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; Hong, Yeonchul

2013-06-30

109

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

PubMed

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

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

2013-01-01

110

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

PubMed Central

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

2011-01-01

111

Rapid detection of wheat yellow mosaic virus by reverse transcription loop-mediated isothermal amplification  

PubMed Central

For the detection of wheat yellow mosaic virus (WYMV), we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. Using Primer Explorer software, four sets of primers were designed and RT-LAMP assay reaction conditions were optimized. The RT-LAMP was performed at different times by four primer sets. Agarose gel analysis showed that WYMV could be detected after 30 min with the primer set III and after 45 min with the other three primer sets, both under the 80-min reaction time. RT-LAMP had the same results with the four primer sets, thus primer set III and 65°C for 80 min reaction were selected for virus detection. There was no significant different when avian myeloblastosis virus (AMV) and moloney murine leukemia virus (M-MLV) RT-LAMP with the four primer sets and M-MLV was chosen due to its relatively cheap price. The result on specificity showed that the assay could amplify WYMV specifically, and the sensitivity comparison showed that the RT-LAMP was 100 times more sensitive than conventional reverse-transcriptase-polymerase chain reaction (RT-PCR). Overall, RT-LAMP was found to be a simple, specific, sensitive, convenient and time-saving method for WYMV detection.

2011-01-01

112

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

Microsoft Academic Search

We developed a rapid single nucleotide polymorphism (SNP) detection system named smart amplification process version 2 (SMAP 2). Because DNA amplification only occurred with a perfect primer match, amplification alone was sufficient to identify the target allele. To achieve the requisite fidelity to support this claim, we used two new and complementary approaches to suppress exponential background DNA amplification that

Yasumasa Mitani; Alexander Lezhava; Yuki Kawai; Takeshi Kikuchi; Atsuko Oguchi-Katayama; Yasushi Kogo; Masayoshi Itoh; Toru Miyagi; Hideki Takakura; Kanako Hoshi; Chiaki Kato; Takahiro Arakawa; Kazuhiro Shibata; Kenji Fukui; Ryoji Masui; Seiki Kuramitsu; Kazuma Kiyotani; Alistair Chalk; Katsuhiko Tsunekawa; Masami Murakami; Tetsuya Kamataki; Takanori Oka; Hiroshi Shimada; Paul E Cizdziel; Yoshihide Hayashizaki

2007-01-01

113

Detection of Salmonella invA by isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) in Zambia.  

PubMed

The isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) is a new isothermal DNA amplification method composed of exo Bca DNA polymerase, RNaseH and DNA-RNA chimeric primers. We detected invA of Salmonella from chicken carcasses, egg yolk and cattle fecal samples. Fifty-three of 59 isolates were invA-positive in ICAN-chromatostrip detection. The result was consistent with those obtained by standard PCR. Salmonella invA was detected in 12 of 14 carcass rinses by ICAN, while in 7 of 14 rinses by standard PCR. These results indicate that ICAN is an efficient, sensitive and simple system to detect invA of Salmonella species in developing countries such as Zambia. PMID:16293310

Isogai, Emiko; Makungu, Chitwambi; Yabe, John; Sinkala, Patson; Nambota, Andrew; Isogai, Hiroshi; Fukushi, Hideto; Silungwe, Manda; Mubita, Charles; Syakalima, Michelo; Hang'ombe, Bernard Mudenda; Kozaki, Shunji; Yasuda, Jun

2005-11-15

114

Detection of foot-and-mouth disease virus rna by reverse transcription loop-mediated isothermal amplification  

PubMed Central

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

2011-01-01

115

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

2013-04-21

116

Rapid and sensitive detection of “ Candidatus Liberibacter asiaticus” by cycleave isothermal and chimeric primer-initiated amplification of nucleic acids  

Microsoft Academic Search

We developed a detection method for “Candidatus Liberibacter asiaticus”, causal agent of citrus huanglongbing, using isothermal and chimeric primer-initiated amplification\\u000a of nucleic acids combined with cycling probe technology (Cycleave ICAN). With Cycleave ICAN, the reaction was done in one\\u000a tube in 1 h without the need for electrophoresis, and false positives were not generated. In addition, Cycleave ICAN method\\u000a was more

Naoya Urasaki; Shinji Kawano; Hiroyuki Mukai; Takashi Uemori; Osamu Takeda; Teruo Sano

2008-01-01

117

Sensitive and rapid detection of cholera toxin-producing Vibrio cholerae using a loop-mediated isothermal amplification  

Microsoft Academic Search

BACKGROUND: Vibrio cholerae is widely acknowledged as one of the most important waterborne pathogen causing gastrointestinal disorders. Cholera toxin (CT) is a major virulence determinant of V. cholerae. Detection of CT-producing V. cholerae using conventional culture-, biochemical- and immunological-based assays is time-consuming and laborious, requiring more than three days. Thus, we developed a novel and highly specific loop-mediated isothermal amplification

Wataru Yamazaki; Kazuko Seto; Masumi Taguchi; Masanori Ishibashi; Kiyoshi Inoue

2008-01-01

118

Sensitive and rapid detection of Vero toxin-producing Escherichia coli using loop-mediated isothermal amplification  

Microsoft Academic Search

A loop-mediated isothermal amplification (LAMP) assay was developed to detect Vero toxin (VT)-producing Escherichia coli rapidly (within 60 min). The 24 strains of VT-producing E. coli were successfully amplified, but 6 strains of non-VT-producing E. coli and 46 bacterial species other than E. coli were not. The sensitivity of the LAMP assay was found to be >0.7 c.f.u. per test

Yukiko Hara-Kudo; Jiro Nemoto; Kayoko Ohtsuka; Yuko Segawa; Kosuke Takatori; Tadashi Kojima; Masanari Ikedo

2007-01-01

119

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

Microsoft Academic Search

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

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

2010-01-01

120

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

Microsoft Academic Search

In this study, loop-mediated isothermal amplification (LAMP)-based molecular marker was developed for authentication of Catharanthus roseus, a medicinal plant. Samples of this plant were collected from different geographical locations in India. Random amplified\\u000a polymorphic deoxyribonucleic acid (DNA) analysis of collected samples was carried out with 25 random primers. A 610-bp DNA\\u000a fragment, common to all accessions, was eluted, cloned, and

Anis Ahmad Chaudhary; Hemant; Mohd Mohsin; Altaf Ahmad

121

Oligonucleotide derivatives in the hybridization analysis of nucleic acids: II. Isothermal signal amplification in DNA analysis by minisequencing  

Microsoft Academic Search

An isothermal amplification of a reporter signal during the analysis of the hybridization of nucleic acids was studied by\\u000a limited probe extension (minisequencing). The intensity of the reporter signal was shown to increase due to the multiple enzymatic\\u000a labeling of the probes during consecutive hybridization with one DNA template in both the homophase and heterophase assays\\u000a using various detection methods:

E. V. Dmitrienko; E. A. Khomyakova; I. A. Pyshnaya; A. G. Bragin; V. E. Vedernikov; D. V. Pyshnyi

2010-01-01

122

Use of loop-mediated isothermal amplification of DNA for the rapid detection of Mycobacterium tuberculosis in clinical specimens  

Microsoft Academic Search

Loop-mediated isothermal amplification (LAMP) is a recently developed molecular method that has been successfully implemented\\u000a in the detection of Mycobacterium tuberculosis in clinical specimens. LAMP has several advantages, such as rapidity, high sensitivity, ease of application and cost-effectiveness.\\u000a As a result, it is anticipated that its use for the detection of M. tuberculosis is likely to become widespread, especially in

I. K. Neonakis; D. A. Spandidos; E. Petinaki

123

Development of a New Method for Diagnosis of Rubella Virus Infection by Reverse Transcription-Loop-Mediated Isothermal Amplification  

Microsoft Academic Search

We developed a useful method for the detection of rubella virus genome RNA by reverse transcription loop-mediated isothermal amplification (RT-LAMP) and compared the sensitivity of RT-LAMP with that of other virological tests: reverse transcription-PCR (RT-PCR) and virus isolation. The rubella virus genome was amplified by RT-LAMP from clinical isolates obtained between 1987 and 2004 with similar sensitivities to the Takahashi

Nobuo Mori; Yoshie Motegi; Yasushi Shimamura; Takashi Ezaki; Tomo Natsumeda; Toshihiro Yonekawa; Yoshinori Ota; Tsugunori Notomi; Tetsuo Nakayama

2006-01-01

124

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

Microsoft Academic Search

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

Hatem Soliman; Mansour El-Matbouli

2010-01-01

125

Rapid detection of HIV1 by reverse-transcription, loop-mediated isothermal amplification (RT-LAMP)  

Microsoft Academic Search

A rapid, cost-effective diagnostic or confirmatory test for the detection of early HIV-1 infection is highly desired, especially for use in resource-poor or point-of-care settings. The reverse-transcription loop-mediated isothermal amplification (RT-LAMP) technology has been evaluated for the detection of HIV-1 DNA and RNA, using six RT-LAMP primers designed against highly conserved sequences located within the protease and p24 gene regions.

Kelly A. Curtis; Donna L. Rudolph; S. Michele Owen

2008-01-01

126

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

Microsoft Academic Search

A one step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of viral hemorrhagic septicaemia virus (VHS). A set of six primers were designed, based on the G-protein sequence of the VHS virus serotypes (He, F1, 23.75, Klapmolle and Rindsholm). The assay was optimised to amplify VHS RNA by incubation at 63°C for only 1h, and required

H. Soliman; M. El-Matbouli

2006-01-01

127

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

128

Development of reverse transcription loop-mediated isothermal amplification for rapid detection of porcine epidemic diarrhea virus  

Microsoft Academic Search

In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for detection of porcine epidemic diarrhea virus (PEDV). Six primers were designed to amplify the nucleocapsid (N) gene of PEDV. The optimization, sensitivity, and specificity\\u000a of the RT-LAMP were investigated. The results showed that the optimal reaction condition for RT-LAMP amplifying PEDV N gene\\u000a was achieved at 63°C

Xiaofeng Ren; Pengchong Li

2011-01-01

129

An isothermal method for whole genome amplification of fresh and degraded DNA for comparative genomic hybridization, genotyping and mutation detection.  

PubMed

Molecular genotyping has important biomedical and forensic applications. However, limiting amounts of human biological material often yield genomic DNA (gDNA) in insufficient quantity and of poor quality for a reliable analysis. This motivated the development of an efficient whole genome amplification method with quantitatively unbiased representation usable on fresh and degraded gDNA. Amplification of fresh frozen, formalin-fixed paraffin-embedded (FFPE) and DNase-degraded DNA using degenerate oligonucleotide-primed PCR or primer extension amplification using a short primer sequence bioinformatically optimized for coverage of the human genome was compared with amplification using current primers by chromosome-based and BAC-array comparative genomic hybridization (CGH), genotyping at short tandem repeats (STRs) and single base mutation detection. Compared with current primers, genome amplification using the bioinformatically optimized primer was significantly less biased on CGH in self-self hybridizations, and replicated tumour genome copy number aberrations, even from FFPE tissue. STR genotyping could be performed on degraded gDNA amplified using our technique but failed with multiple displacement amplification. Of the 18 different single base mutations 16 (89.5%) were correctly identified by sequencing gDNA amplified from clinical samples using our technique. This simple and efficient isothermal method should be helpful for genetic research and clinical and forensic applications. PMID:16766515

Lee, Cheryl I P; Leong, Siew Hong; Png, Adrian E H; Choo, Keng Wah; Syn, Christopher; Lim, Dennis T H; Law, Hai Yang; Kon, Oi Lian

2006-03-27

130

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

2012-07-20

131

A loop-mediated isothermal amplification method for the detection of members of the genus Ranavirus.  

PubMed

A loop-mediated isothermal amplification (LAMP) method was developed for detection of members of the genus Ranavirus. The optimum reaction mixture contained 2.5 ?L of each inner primer, RV-FIP (20 pmol/?L) and RV-BIP (20 pmol/?L), 0.5 ?L of each outer primer, RV-F3 (10 pmol/?L) and RV-B3 (10 pmol/?L), 1.25 ?L of each loop primer, RV-LF (20 pmol/?L) and RV-LB (20 pmol/?L), 3.5 ?L dNTP mix (10 mM each), 8 ?L MgSO4 (25 mM), 1 ?L of Bst DNA polymerase (8 U/mL, large fragment; New England Biolabs Inc., Beverly, MA, USA), 2.5 ?L 10 × supplied buffer, and 1 ?L of template DNA in a final volume of 25 ?L. The optimum reaction conditions were 63 °C for 60 min. This LAMP method could detect Andrias davidianus iridovirus (ADIV), soft-shelled turtle iridovirus (STIV), and epizootic hematopoietic necrosis virus (EHNV), all of which belong to the genus Ranavirus, but it could not detect other viruses such as koi herpes virus (KHV), channel catfish virus (CCV), infectious spleen and kidney necrosis virus (ISKNV) and white spot syndrome virus (WSSV). The detection limit of the LAMP method was 100 copies of STIV DNA segment, and the sensitivity was 10 times higher than that of the polymerase chain reaction (PCR) assay. The results could be estimated visually by eye when calcein was added. PMID:23665768

Min, Zhang; Hongli, Jing; Lifeng, Zhang; Na, Wang; Shaoqiang, Wu; Xiangmei, Lin

2013-05-12

132

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.

2012-01-01

133

Detecting Potentially Virulent Vibrio vulnificus Strains in Raw Oysters by Quantitative Loop-Mediated Isothermal Amplification ?  

PubMed Central

Vibrio vulnificus is a leading cause of seafood-related deaths in the United States. Sequence variations in the virulence-correlated gene (vcg) have been used to distinguish between clinical and environmental V. vulnificus strains, with a strong association between clinical ones and the C sequence variant (vcgC). In this study, vcgC was selected as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, sensitive, specific, and quantitative detection of potentially virulent V. vulnificus strains in raw oysters. No false-positive or false-negative results were generated among the 125 bacterial strains used to evaluate assay specificity. The detection limit was 5.4 CFU per reaction for a virulent V. vulnificus strain (ATCC 33815) in pure culture, 100-fold more sensitive than that of PCR. In spiked raw oysters, the assay was capable of detecting 2.5 × 103 CFU/g of V. vulnificus ATCC 33815, while showing negative results for a nonvirulent V. vulnificus strain (515-4c2) spiked at 107 CFU/g. After 6 h of enrichment, the LAMP assay could detect 1 CFU/g of the virulent V. vulnificus strain ATCC 33815. Standard curves generated in pure culture and spiked oysters suggested a good linear relationship between cell numbers of the virulent V. vulnificus strain and turbidity signals. In conclusion, the LAMP assay developed in this study could quantitatively detect potentially virulent V. vulnificus in raw oysters with high speed, specificity, and sensitivity, which may facilitate better control of V. vulnificus risks associated with raw oyster consumption.

Han, Feifei; Wang, Fei; Ge, Beilei

2011-01-01

134

Detecting salmonella serovars in shell eggs by loop-mediated isothermal amplification.  

PubMed

Shell eggs contaminated with Salmonella Enteritidis pose serious food safety and public health concerns. More vigilant product testing calls for rapid, accurate, and reliable detection methods for Salmonella. Two loop-mediated isothermal amplification (LAMP) assays targeting different regions of the Salmonella invasion protein (encoded by invA) have been reported. In this study, performance of the two LAMP assays was compared with that of PCR in detecting Salmonella Enteritidis and Salmonella Typhimurium strains in experimentally contaminated egg homogenates. Both LAMP assays were highly specific. The detection limits were approximately 1 CFU per reaction for both Salmonella serovars in pure culture, 100-fold more sensitive than that of PCR. Standard curves generated suggested a good linear relationship between Salmonella cell numbers and LAMP turbidity signals. In spiked egg homogenate, the LAMP assays could detect both Salmonella serovars down to 10(4) CFU/25 ml without enrichment and 10(0) CFU/25 ml with 8-h enrichment. In contrast, PCR was unable to detect either Salmonella serovar in egg homogenates spiked with less than 10(6) CFU/25 ml by direct testing and required at least 12 h of enrichment for samples spiked with 10(1) CFU/25 ml and 24 h for those with 10(0) CFU/25 ml. The complete LAMP assay took about 10 h (including 8 h of enrichment) to complete. In conclusion, the two LAMP assays were rapid, accurate, and reliable methods for detecting Salmonella serovars in shell eggs and may be adopted in routine egg testing for Salmonella to improve egg safety and protect public health. PMID:24112582

Yang, Qianru; Chen, Siyi; Ge, Beilei

2013-10-01

135

Combined Molecular Genetic and Cytogenetic Analysis from Single Cells after Isothermal Whole-Genome Amplification  

PubMed Central

BACKGROUND Analysis of chromosomal aberrations or single-gene disorders from rare fetal cells circulating in the blood of pregnant women requires verification of the cells’ genomic identity. We have developed a method enabling multiple analyses at the single-cell level that combines verification of the genomic identity of microchimeric cells with molecular genetic and cytogenetic diagnosis. METHODS We used a model system of peripheral blood mononuclear cells spiked with a colon adenocarcinoma cell line and immunofluorescence staining for cytokeratin in combination with DNA staining with the nuclear dye TO-PRO-3 in a preliminary study to define candidate microchimeric (tumor) cells in Cytospin preparations. After laser microdissection, we performed low-volume on-chip isothermal whole genome amplification (iWGA) of single and pooled cells. RESULTS DNA fingerprint analysis of iWGA aliquots permitted successful identification of all analyzed candidate microchimeric cell preparations (6 samples of pooled cells, 7 samples of single cells). Sequencing of 3 single-nucleotide polymorphisms was successful at the single-cell level for 20 of 32 allelic loci. Metaphase comparative genomic hybridization (mCGH) with iWGA products of single cells showed the gains and losses known to be present in the genomic DNA of the target cells. CONCLUSIONS This method may be instrumental in cell-based noninvasive prenatal diagnosis. Furthermore, the possibility to perform mCGH with amplified DNA from single cells offers a perspective for the analysis of nonmicrochimeric rare cells exhibiting genomic alterations, such as circulating tumor cells.

Kroneis, Thomas; Geigl, Jochen B.; El-Heliebi, Amin; Auer, Martina; Ulz, Peter; Schwarzbraun, Thomas; Dohr, Gottfried; Sedlmayr, Peter

2012-01-01

136

Detection of phytoplasma by loop-mediated isothermal amplification of DNA (LAMP).  

PubMed

Napier stunt phytoplasma (16SrXI and 16SrIII) in eastern Africa is a serious threat to the expansion of Napier grass (Pennisetum purpureum) farming in the region, where it is widely cultivated as fodder in zero grazing livestock systems. The grass has high potential for bio-fuel production, and has been adopted by farmers as a countermeasure to cereal stem borer Lepidoptera, since it attracts and traps the insect. Diagnosis of stunt phytoplasma have been largely by nested polymerase chain reaction (nPCR) targeting the 16S rRNA gene. However, the method is laborious, costly and technically demanding. This investigation has developed a simpler but effective phytoplasma diagnostic tool, called; loop-mediated isothermal amplification of DNA (LAMP). The assay was tested on 8 symptomatic and 8 asymptomatic plants, while its detection limit was compared to nested PCR using samples serially diluted from 3 ng/?l to 0.38 pg/?l. Molecular typing of LAMP products was determined by BsrI restriction digestion and Southern blot analysis. The assay sensitivity, positive and negative predictive values were estimated, while the specificity was tested on 11 phytoplasma groups. LAMP was specific to 5 phytoplasma groups: 16SrVI, X, XI and XVI. BsrI restriction digestion produced two predicted fragments, and there was specific binding of probe DNA to the LAMP amplicons in Southern blot analysis. The assay sensitivity was 100%, while the positive and negative predictive values were 63 and 100% respectively. LAMP was 20-fold more sensitive than nested PCR. This study validates LAMP for routine diagnosis of Napier stunt and other closely related phytoplasmas. PMID:21185882

Obura, E; Masiga, D; Wachira, F; Gurja, B; Khan, Z R

2010-12-24

137

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

138

An isothermal primer extension method for whole genome amplification of fresh and degraded DNA: applications in comparative genomic hybridization, genotyping and mutation screening  

Microsoft Academic Search

We describe a protocol that uses a bioinformatically optimized primer in an isothermal whole genome amplification (WGA) reaction. Overnight incubation at 37 °C efficiently generates several hundred- to several thousand-fold increases in input DNA. The amplified product retains reasonably faithful quantitative representation of unamplified whole genomic DNA (gDNA). We provide protocols for applying this isothermal primer extension WGA protocol in

Cheryl I P Lee; Siew Hong Leong; Adrian E H Png; Keng Wah Choo; Christopher Syn; Dennis T H Lim; Hai Yang Law; Oi Lian Kon

2006-01-01

139

Detection of virus mRNA within infected host cells using an isothermal nucleic acid amplification assay: marine cyanophage gene expression within Synechococcus sp  

Microsoft Academic Search

BACKGROUND: Signal-Mediated Amplification of RNA Technology (SMART) is an isothermal nucleic acid amplification technology, developed for the detection of specific target sequences, either RNA (for expression) or DNA. Cyanophages are viruses that infect cyanobacteria. Marine cyanophages are ubiquitous in the surface layers of the ocean where they infect members of the globally important genus Synechococcus. RESULTS: Here we report that

Susan D Wharam; Matthew J Hall; William H Wilson

2007-01-01

140

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

141

Sensitive and rapid detection of Paragonimus westermani infection in humans and animals by loop-mediated isothermal amplification (LAMP).  

PubMed

In the present study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated for the detection of Paragonimus westermani adults, metacercariae, and eggs in human and animal samples. The LAMP amplification can be finished in 45 min under isothermal condition at 60°C by employing a set of four species-specific primer mixtures and the results can be checked by naked-eye visualization. No amplification products were detected with deoxyribunucleic acid (DNA) of related trematode species including Fasciola hepatica, Fasciola gigantica, Clonorchis sinensis, Opisthorchis viverrini, Schistosoma mansoni, and Schistosoma japonicum. The method was further validated by examining P. westermani DNA in intermediate hosts including freshwater crabs and crayfish, as well as in sputum and pleural fluid samples from patients of paragonimiasis. These results indicated that the LAMP assay was highly specific, sensitive, and rapid, and it was approximately 100 times more sensitive than conventional specific PCR. The LAMP assay established in this study provides a rapid and sensitive tool for the detection of P. westermani DNA in freshwater crabs, crayfish, sputum, and pleural fluid samples, which has important implications for effective control of human paragonimiasis. PMID:21107864

Chen, M X; Ai, L; Zhang, R L; Xia, J J; Wang, K; Chen, S H; Zhang, Y N; Xu, M J; Li, X; Zhu, X Q; Chen, J X

2010-11-25

142

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

PubMed Central

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

Iwamoto, Tomotada; Sonobe, Toshiaki; Hayashi, Kozaburo

2003-01-01

143

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

PubMed

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

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

2012-07-11

144

Rapid detection of sacbrood virus (SBV) by one-step reverse transcription loop-mediated isothermal amplification assay  

PubMed Central

Background Sacbrood virus (SBV) primarily infects honeybee broods, and in order to deal with the problem cost effective detection methods are required. Findings A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid identification of SBV. The data demonstrated that, in a simple water bath, SBV RNA could be detected as early as 20 min at 65°C, and a positive amplification reaction was visible to the naked eye due to a color change brought on by the addition of nucleic acid stain SYBR Green. Conclusions The current study presents a method for the rapid and simple detection of SBV by RT-LAMP with high sensitivity and analytic specificity.

2012-01-01

145

Specific, simple and rapid detection of porcine circovirus type 2 using the loop-mediated isothermal amplification method  

Microsoft Academic Search

Background  Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS), and porcine\\u000a dermatitis and nephropathy syndrome (PDNS). It has caused heavy losses in global agriculture in recent decades. Rapid detection\\u000a of PCV2 is very important for the effective prophylaxis and treatment of PMWS.\\u000a \\u000a \\u000a \\u000a \\u000a Results  A loop-mediated isothermal amplification (LAMP) assay was used to detect PCV2 in

Kai Zhao; Wei Shi; Fangting Han; Yan Xu; Lianlong Zhu; Yong Zou; Xiao Wu; Hong Zhu; Furong Tan; Shiru Tao; Xueming Tang

2011-01-01

146

Loop-mediated isothermal amplification assay targeting the ompA gene for rapid detection of Riemerella anatipestifer.  

PubMed

A novel loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for the detection of Riemerella anatipestifer (RA) infection. The LAMP assay exhibited a higher sensitivity than conventional polymerase chain reaction (PCR) and microbial isolation. The specificity of the assay was determined by restriction enzyme digestion of the LAMP products and detection of Escherichia coli, Salmonella enterica and Pasteurella multocida. The LAMP assay was able to detect RA effectively in samples of the reference strains, isolated strains and infected duck brains. This assay is a useful tool for the diagnosis of RA infection in the clinical setting. PMID:21040782

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

2010-10-30

147

Rapid diagnosis of Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD) in salmonid fish by a novel DNA amplification method, loop-mediated isothermal amplification (LAMP).  

PubMed

A new molecular diagnostic assay was developed for detection of Tetracapsuloides bryosalmonae the causative agent of proliferative kidney disease (PKD) in salmonid fish using a loop-mediated isothermal amplification method (LAMP). The PKD-LAMP assay amplifies the T. bryosalmonae DNA extracted from infected kidney, under constant temperature of 65 degrees C within 1 h. The required equipment for DNA amplification is only a water bath. The amplification products were detected visually by using SYBR green I dye, which turns green in the presence of amplified products and remains orange in its absence, and by electrophoresis without any difference in the sensitivity of both methods. The developed PKD-LAMP assay demonstrated an exceptionally higher sensitivity than the conventional PCR. PKD-LAMP assay was found to be 100-fold more sensitive than the PCR assay. The developed assay is simple, rapid, cost-effective, specific and highly sensitive. The assay is also characterized by its field applicability, as it does not require the use of sophisticated equipment or skilled personnel. PMID:15895254

El-Matbouli, Mansour; Soliman, Hatem

2005-05-14

148

Proximity-dependent isothermal cycle amplification for small-molecule detection based on surface enhanced Raman scattering.  

PubMed

A novel proximity-dependent isothermal cycle amplification (PDICA) strategy has been proposed and successfully used for the determination of cocaine coupled with surface enhanced Raman scattering (SERS). For enhancing the SERS signal, Raman dye molecules modified bio-barcode DNA and gold nanoparticles (AuNPs) are used to prepare the Raman probes. Magnetic beads (MBs) are used as the carrier of amplification template and signal output products for circumventing the problem of high background induced by excess bio-barcode DNA. In the presence of target molecules, two label-free proximity probes can hybridize with each other and subsequently opens the hairpin connector-probe to perform the PDICA reaction including the target recycling amplification and strand-displacement amplification. As a result, abundant AuNPs Raman probes can be anchored on the surface of MBs and a low detection limit of 0.1nM for cocaine is obtained. This assay also exhibits an excellent selectivity and has been successfully performed in human serum, which confirms the reliability and practicality of this protocol. PMID:23994277

Li, Ying; Zeng, Yan; Mao, Yaning; Lei, Chengcun; Zhang, Shusheng

2013-08-06

149

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.

2011-01-01

150

Long-tail probe-mediated cycled strand displacement amplification: Label-free, isothermal and sensitive detection of nucleic acids.  

PubMed

We design a long-tail shaped DNA probe for the label-free, isothermal and sensitive detection of nucleic acids based on cycled strand displacement amplification (Ltail-CSDA). The long-tail probe, a stem-loop structure with a long poly(T) tail at 5'-termini, integrates target-binding and amplification and signaling within one multifunctional design. The specific binding between the long-tail probe and the target triggers a polymerization reaction, during which a long dsDNA product is synthesized and the hybridized target is displaced by the strand displacement activity of polymerase. The displaced target forms another specific probe-target binding and prompts cycled polymerization reactions. The proposed Ltail-CSDA has the distinct advantages of its isothermal nature, free-label, simplicity and attomolar sensitivity compared with other existing technologies. More significantly, the dynamic range of the method is extremely large, covering nine orders of magnitude. Using total RNA samples extracted from hepatitis C virus (HCV) as targets, we further demonstrate the detection capability of the method for complex nucleic acid samples, indicating its potential applicability for clinic molecular diagnostic assays. PMID:24148411

Su, Hongyan; Long, Jiabao; Guo, Qiuping; Meng, Xiaochun; Tan, Yongjun; Cai, Qingyun; Chen, Zhuo; Meng, Xiangxian

2013-05-30

151

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

2012-11-14

152

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.

2012-01-01

153

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.

2011-01-01

154

Real-Time Loop-Mediated Isothermal Amplification (RealAmp) for the Species-Specific Identification of Plasmodium vivax  

PubMed Central

Plasmodium vivax infections remain a major source of malaria-related morbidity and mortality. Early and accurate diagnosis is an integral component of effective malaria control programs. Conventional molecular diagnostic methods provide accurate results but are often resource-intensive, expensive, have a long turnaround time and are beyond the capacity of most malaria-endemic countries. Our laboratory has recently developed a new platform called RealAmp, which combines loop-mediated isothermal amplification (LAMP) with a portable tube scanner real-time isothermal instrument for the rapid detection of malaria parasites. Here we describe new primers for the detection of P. vivax using the RealAmp method. Three pairs of amplification primers required for this method were derived from a conserved DNA sequence unique to the P. vivax genome. The amplification was carried out at 64°C using SYBR Green or SYTO-9 intercalating dyes for 90 minutes with the tube scanner set to collect fluorescence signals at 1-minute intervals. Clinical samples of P. vivax and other human-infecting malaria parasite species were used to determine the sensitivity and specificity of the primers by comparing with an 18S ribosomal RNA-based nested PCR as the gold standard. The new set of primers consistently detected laboratory-maintained isolates of P. vivax from different parts of the world. The primers detected P. vivax in the clinical samples with 94.59% sensitivity (95% CI: 87.48–98.26%) and 100% specificity (95% CI: 90.40–100%) compared to the gold standard nested-PCR method. The new primers also proved to be more sensitive than the published species-specific primers specifically developed for the LAMP method in detecting P. vivax.

Patel, Jaymin C.; Oberstaller, Jenna; Xayavong, Maniphet; Narayanan, Jothikumar; DeBarry, Jeremy D.; Srinivasamoorthy, Ganesh; Villegas, Leopoldo; Escalante, Ananias A.; DaSilva, Alexandre; Peterson, David S.; Barnwell, John W.; Kissinger, Jessica C.; Udhayakumar, Venkatachalam; Lucchi, Naomi W.

2013-01-01

155

Adaptation of a visualized loop-mediated isothermal amplification technique for field detection of Plasmodium vivax infection  

PubMed Central

Background Loop-mediated isothermal amplification (LAMP) is a high performance method for detecting DNA and holds promise for use in the molecular detection of infectious pathogens, including Plasmodium spp. However, in most malaria-endemic areas, which are often resource-limited, current LAMP methods are not feasible for diagnosis due to difficulties in accurately interpreting results with problems of sensitive visualization of amplified products, and the risk of contamination resulting from the high quantity of amplified DNA produced. In this study, we establish a novel visualized LAMP method in a closed-tube system, and validate it for the diagnosis of malaria under simulated field conditions. Methods A visualized LAMP method was established by the addition of a microcrystalline wax-dye capsule containing the highly sensitive DNA fluorescence dye SYBR Green I to a normal LAMP reaction prior to the initiation of the reaction. A total of 89 blood samples were collected on filter paper and processed using a simple boiling method for DNA extraction, and then tested by the visualized LAMP method for Plasmodium vivax infection. Results The wax capsule remained intact during isothermal amplification, and released the DNA dye to the reaction mixture only when the temperature was raised to the melting point following amplification. Soon after cooling down, the solidified wax sealed the reaction mix at the bottom of the tube, thus minimizing the risk of aerosol contamination. Compared to microscopy, the sensitivity and specificity of LAMP were 98.3% (95% confidence interval (CI): 91.1-99.7%) and 100% (95% CI: 88.3-100%), and were in close agreement with a nested polymerase chain reaction method. Conclusions This novel, cheap and quick visualized LAMP method is feasible for malaria diagnosis in resource-limited field settings.

2011-01-01

156

Rapid isothermal detection assay: a probe amplification method for the detection of nucleic acids  

Microsoft Academic Search

Simple, accurate, and stable diagnostic tests are essential to control viral infectious diseases such as avian influenza virus. The current technologies are often inaccessible to people who need them, mainly because of the specialized equipment and the need for highly trained technologists. Here, we describe a rapid isothermal nucleic acid detection assay (RIDA) that can be used to detect both

Wenjuan Gao; Xiang Li; Lingwen Zeng; Tao Peng

2008-01-01

157

Rapid and sensitive detection of Penaeus monodon nucleopolyhedrovirus (PemoNPV) by loop-mediated isothermal amplification combined with a lateral-flow dipstick  

Microsoft Academic Search

Several methods such as traditional PCR or nested-PCR, immuno assay and histopathology have been developed for detection of Penaeus monodon nucleopolyhedrovirus (PemoNPV) formerly called monodon baculovirus (MBV). However, these methods have various disadvantages including low sensitivity, long assay time, use of toxic substances or unsuitability for field diagnosis. Loop-mediated isothermal amplification of target nucleotide sequences under isothermal conditions, combined with

Tongchai Nimitphak; Watcharachai Meemetta; Narong Arunrut; Saengchan Senapin; Wansika Kiatpathomchai

2010-01-01

158

Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of bovine Babesia parasites  

Microsoft Academic Search

A loop-mediated isothermal amplification (LAMP) technique has been used as a novel nucleic acid detection method, whereby the target DNA can be amplified with high specificity and sensitivity under an isothermal condition using a set of four specific primers. In this study, we designed two sets of the LAMP primers for rhoptry-associated protein-1 genes of Babesia bovis and B. bigemina,

Hiroshi Iseki; Andy Alhassan; Naomi Ohta; Oriel M. M. Thekisoe; Naoaki Yokoyama; Noboru Inoue; Andrew Nambota; Jun Yasuda; Ikuo Igarashi

2007-01-01

159

Rapid, sensitive, and quantitative detection of pathogenic DNA at the point of care through microfluidic electrochemical quantitative loop-mediated isothermal amplification.  

PubMed

Single-step DNA detection: a microfluidic electrochemical loop mediated isothermal amplification platform is reported for rapid, sensitive, and quantitative detection of pathogen genomic DNA at the point of care. DNA amplification was electrochemically monitored in real time within a monolithic microfluidic device, thus enabling the detection of as few as 16 copies of Salmonella genomic DNA through a single-step process in less than an hour. PMID:22488842

Hsieh, Kuangwen; Patterson, Adriana S; Ferguson, B Scott; Plaxco, Kevin W; Soh, H Tom

2012-04-04

160

Rapid, Sensitive, and Quantitative Detection of Pathogenic DNA at the Point of Care via Microfluidic Electrochemical Quantitative Loop-Mediated Isothermal Amplification (MEQ-LAMP)**  

PubMed Central

We present the Microfluidic Electrochemical Quantitative Loop-mediated isothermal AMPlification (MEQ-LAMP) platform for rapid, sensitive, and quantitative detection of pathogen genomic DNA at the point of care. DNA amplification is electrochemically monitored in real time within a monolithic microfluidic device, enabling the detection of as few as 16 copies of Salmonella genomic DNA via a single-step process in under an hour.

Hsieh, Kuangwen; Patterson, Adriana S.; Ferguson, B. Scott; Plaxco, Kevin W.

2012-01-01

161

Isothermal, In Vitro Amplification of Nucleic Acids by a Multienzyme Reaction Modeled After Retroviral Replication  

Microsoft Academic Search

A target nucleic acid sequence can be replicated (amplified) exponentially in vitro under isothermal conditions by using three enzymatic activities essential to retroviral replication: reverse transcriptase, RNase H, and a DNA-dependent RNA polymerase. By mimicking the retroviral strategy of RNA replication by means of cDNA intermediates, this reaction accumulates cDNA and RNA copies of the original target. Product accumulation is

John C. Guatelli; Kristina M. Whitfield; Deborah Y. Kwoh; Kevin J. Barringer; Douglas D. Richman; Thomas R. Gingeras

1990-01-01

162

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

163

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

PubMed

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

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

2009-12-01

164

Simple method for differentiating measles vaccine from wild-type strains using loop-mediated isothermal amplification.  

PubMed

Because of increasing measles vaccine coverage, the proportion of patients with modified measles has been increasing. Such patients have low-grade fever with very mild eruptions similar to vaccine-related adverse events. Differentiation between these two pathogenic conditions is required to improve the quality of laboratory-based measles surveillance. In this study, vaccine-specific and wild-type specific primer sets were designed for loop-mediated isothermal amplification in the N gene, and vaccine strains, C1, D3, D4, D5, D8, D9, G3 and H1 wild strains were examined. Three vaccine strains were efficiently amplified using a vaccine-specific primer set with an approximately 10-times higher sensitivity than wild-type primer. Modified measles was differentiated from vaccine-associated cases by this system, but limitations were encountered with the other genotypes. PMID:23489085

Nakayama, Tetsuo; Sawada, Akihito; Kubo, Hideyuki; Kaida, Atsushi; Tanaka, Toshimitsu; Shigemoto, Naoki; Komase, Katsuhiro; Takeda, Makoto

2013-03-01

165

Rapid typing of foot-and-mouth disease serotype Asia 1 by reverse transcription loop-mediated isothermal amplification  

PubMed Central

A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was rapidly used to detect serotype Asia 1 of foot-and-mouth disease virus (FMDV) within 45 min at 61°C. All FMDV serotype Asia 1 reference strains were positive by RT-LAMP, while other viruses such as FMDV serotypes O, C, A and classical swine fever virus, swine vesicular disease virus, porcine reproductive and respiratory syndrome virus and Japanese encephalitis virus remained negative. Furthermore, FMDV sreotype Asia 1 positive samples were able to detect by RT-LAMP assay. This RT-LAMP assay may be suitable particularly for diagnosis of FMDV serotype Asia 1 infection in field stations.

2011-01-01

166

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

PubMed

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

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

2013-06-12

167

Detection and identification of Brettanomyces/Dekkera sp. yeasts with a loop-mediated isothermal amplification method.  

PubMed

Primer sets for a loop-mediated isothermal amplification (LAMP) method were developed to specifically identify each of the four Brettanomyces/Dekkera species, Dekkera anomala, Dekkera bruxellensis, Dekkera custersiana and Brettanomyces naardenensis. Each primer set was designed with target sequences in the ITS region of the four species and could specifically amplify the target DNA of isolates from beer, wine and soft drinks. Furthermore, the primer sets differentiated strains of the target species from strains belonging to other species, even within the genus Brettanomyces/Dekkera. Moreover, the LAMP method with these primer sets could detect about 1 x 10(1) cfu/ml of Brettanomyces/Dekkera yeasts from suspensions in distilled water, wine and beer. This LAMP method with primer sets for the identification of Brettanomyces/Dekkera yeasts is advantageous in terms of specificity, sensitivity and ease of operation compared with standard PCR methods. PMID:17613376

Hayashi, Nobuyuki; Arai, Ritsuko; Tada, Setsuzo; Taguchi, Hiroshi; Ogawa, Yutaka

2007-02-04

168

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

PubMed

In this study, loop-mediated isothermal amplification (LAMP)-based molecular marker was developed for authentication of Catharanthus roseus, a medicinal plant. Samples of this plant were collected from different geographical locations in India. Random amplified polymorphic deoxyribonucleic acid (DNA) analysis of collected samples was carried out with 25 random primers. A 610-bp DNA fragment, common to all accessions, was eluted, cloned, and sequenced. Four LAMP primers were designed on the basis of sequence of 610 bp DNA fragment. LAMP reaction, containing 10× Bst DNA polymerase reaction buffer, Bst DNA polymerase, four in-house designed primers, dNTPs, MgSO(4), and betaine, was incubated at 65°C for 1 h. The resulting amplicon was visualized by adding SYBR Green I to the reaction tube. The data showed confirmatory results. Since the assay method is simple, sensitive, and cost-effective, it is a feasible method for identifying and authentication of C. roseus. PMID:21644004

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

2011-06-05

169

Development of reverse transcription loop-mediated isothermal amplification for rapid detection of porcine epidemic diarrhea virus.  

PubMed

In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for detection of porcine epidemic diarrhea virus (PEDV). Six primers were designed to amplify the nucleocapsid (N) gene of PEDV. The optimization, sensitivity, and specificity of the RT-LAMP were investigated. The results showed that the optimal reaction condition for RT-LAMP amplifying PEDV N gene was achieved at 63°C for 50 min. The RT-LAMP assay was more sensitive than gel-based RT-PCR and enzyme-linked immunosorbent assay. It was capable of detecting PEDV from clinical samples and differentiating PEDV from Porcine transmissible gastroenteritis virus, Porcine rotavirus, Porcine pseudorabies virus, Porcine reproductive and respiratory syndrome virus, and Avian infectious bronchitis virus. PMID:21286798

Ren, Xiaofeng; Li, Pengchong

2011-02-01

170

Novel Isothermal, Linear Nucleic Acid Amplification Systems for Highly Multiplexed Applications  

Microsoft Academic Search

Background: Global analysis of the genome, transcrip- tome, and proteome is facilitated by the recent develop- ment of tools for large-scale, highly parallel analysis. We describe a novel nucleic acid amplification system that generates products by several methods. 3-Ribo- SPIATM primes cDNA synthesis at the 3 polyA tail, and whole transcript (WT)-Ribo-SPIA primes cDNA synthe- sis across the full length

Nurith Kurn; Pengchin Chen; Joe Don Heath; Anne Kopf-Sill; Kathryn M. Stephens; Shenglong Wang

2005-01-01

171

An isothermal and sensitive nucleic acids assay by target sequence recycled rolling circle amplification.  

PubMed

Sequence-specific nucleic acid detection is playing a more and more important role in modern life sciences. Traditional rolling circle amplification (RCA) involves multiple distinct reaction steps and the experiment result is influenced by multiple factors. What's more, a main limitation of traditional RCA is that each target strand hybridizes with only one padlock probe, and this 1:1 hybridization ratio limits the sensitivity. Here we have proposed target sequence recycled rolling circle amplification (TR-RCA) to increase sensitivity by one step. We demonstrated that our method can not only make RCA occur, but also one target DNA can be reused and thus achieving self-recycle. In TR-RCA, the dumbbell probe recognizes the target DNA and hybridizes with it, and then the stem of the dumbbell probe is opened, after that the opened area anneals with the primer and triggers RCA. At the same time, after a target is displaced, it recognizes and hybridizes with another dumbbell probe, triggering the next cycle of RCA. This amplification method is achievable at a constant temperature simply by mixing dumbbell probes, target DNA, primers, and other chemical complexes together in one tube. Our method has significant advantages in ease of operation. And the results indicate that the target DNA can be detected at fM level with high specificity. PMID:23517825

Long, Yi; Zhou, Xiaoming; Xing, Da

2013-02-20

172

Loop-mediated isothermal amplification (LAMP) detection of Babesia orientalis in water buffalo (Bubalus babalis, Linnaeus, 1758) in China.  

PubMed

Loop-mediated isothermal amplification (LAMP) is a rapid method with high specificity and efficiency under isothermal condition using a set of four specifically designed primers that recognize six distinct sequences on the target gene. In this study, a LAMP method was developed for specific detection of Babesia orientalis in water buffalo (Bubalus babalis, Linnaeus, 1758). Four primers were designed from the V4 hypervariable region of the 18S rRNA gene of B. orientalis. Blood samples were collected from B. orientalis experimentally infected water buffalo as well as from 165 water buffalo from eight different regions of the Hubei province, south China. Genomic DNA was extracted, subjected to the LAMP assay and compared with results obtained using a previously described semi-nested PCR. The LAMP assay proofed to be B. orientalis specific and more sensitive than the semi-nested PCR. While previously B. orientalis had not been reported north of the Yangtse River, our results show that B. orientalis has spread to the north of the river. This could pose a serious threat to the water buffalo industry. PMID:19665847

He, Lan; Zhou, Yan-Qin; Oosthuizen, Marinda C; Zhao, Jun-Long

2009-07-04

173

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

PubMed

Rabbit hemorrhagic disease virus (RHDV) causes haemagglutination and severe liver damage, with a high mortality rate. To develop a rapid and sensitive method for the surveillance of RHDV, a one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was established using a set of four primers specific for the VP60 gene segment of RHDV. The established assay was performed at 64°C for 40 min under isothermal conditions, and the results were visualized directly by electrophoresis or as fluorescent signals under ultraviolet light. The detection limit of the RT-LAMP assay was 10 copies of viral RNA per reaction, which was comparable to quantitative real-time RT-PCR, and 100-fold more sensitive than standard RT-PCR. Furthermore, seven viral RNAs of field isolates in China could be detected successfully using this assay. Overall, the newly established RT-LAMP assay indicates the potential usefulness of the technique as a simple, rapid and sensitive procedure, and can visually detect RHDV infection without the need for any specialized equipment. PMID:23178586

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

2012-11-23

174

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.

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

2011-01-01

175

Development of a loop-mediated isothermal amplification method to rapidly detect porcine circovirus genotypes 2a and 2b  

PubMed Central

Background Porcine circovirus type 2 (PCV2), is nowadays associated with a number of diseases known as porcine circovirus-associated diseases (PCVAD), especially postweaning multisystemic wasting syndrome (PMWS). The epidemiological investigation of PCV2 infection was usually conducted by PCR, nested PCR, PCR-RFLP, TaqMan-based assay and nucleotide sequencing. However, there is still no rapid, sensitive and practical method for detecting PCV2 genotypes. As a novel nucleic acid amplification method, the loop-mediated isothermal amplification method (LAMP) has been used to detect a variety of pathogenic microorganisms. Results Herein, a LAMP method is developed to detect the genotypes of PCV2. The diagnostic sensitivity of LAMP is 1 copy/reaction for differentiating genotypes PCV2a and PCV2b. The reaction process was completed at 65°C for 1 hour in a water bath. Cross-reactivity assay shows that this method is specific for PCV2a and PCV2b and no reactive for PCV2c and other swine-origin viruses (i.e. CSFV, PRRSV, BVDV, TGEV and PEDV, etc). Identity between LAMP and nested PCR was 92.3% on 52 field clinical samples. Conclusions LAMP method provides a rapid, sensitive, reliable way to detect PCV2a and PCV2b, and a better means for the large scale investigation of PCV2a and PCV2b infection.

2012-01-01

176

Rapid diagnosis of turbot reddish body iridovirus in turbot using the loop-mediated isothermal amplification method.  

PubMed

Turbot reddish body iridovirus (TRBIV) is a new piscine iridovirus that infects the turbot, Scophthalmus maximus, cultured in northern China and can cause high mortality. In this study, a loop-mediated isothermal amplification (LAMP) method was developed for the specific detection of this virus using primers designed from an Msp I restriction DNA fragment of the TRBIV genome. Mg(2+) concentrations, the reaction temperature, and the reaction time of LAMP were optimized to 6mM, 65 degrees C, and 60 min, respectively. The detection limit of the LAMP method was as low as seven copies and was 100 times more sensitive than the conventional PCR technique. Visual inspection of LAMP amplifications 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 presence or absence of the virus. LAMP can be conducted in 1h and requires only a simple heating device for incubation. Thus, the LAMP-TRBIV detection protocol has great potential for use in the detection of TRBIV in both the laboratory and the farm. PMID:19187785

Zhang, Qingli; Shi, Chenyin; Huang, Jie; Jia, Kuntong; Chen, Xinhua; Liu, Hong

2009-01-31

177

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

PubMed

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of lymphocystis disease virus (LCDV). A set of five specific primers, two inner and two outer primers and a loop primer, were designed on the basis of the major capsid protein gene of LCDV. The reaction time and temperatures were optimized for 60 min at 63 degrees C, respectively. LAMP amplification products were detected by a ladder-like appearance on agarose gel electrophoresis or a naked-eye inspection of a color change in the reaction tube by addition of SYBR Green I. The assay was specific for LCDV, and there was no cross-reactivity with white spot syndrome virus (WSSV) or six other Iridoviridae viruses (epizootic hematopoietic necrosis virus, EHNV; tiger frog virus, TFV; Bohle iridovirus, BIV; soft-shelled turtle iridovirus, STIV; infectious spleen and kidney necrosis virus, ISKNV; red sea bream iridovirus, RSIV). The detection limit of the LAMP assay was 15 fg, which was similar to that of real-time quantitative polymerase chain reaction (PCR) and 10-fold higher than the conventional PCR. The LAMP assay was evaluated using 109 clinical samples, and the results indicated the suitability and simplicity of the test as a rapid, field diagnostic tool for detection of LCDV. The LCDV LAMP assay has potential for early diagnosis of LCDV infection. PMID:19891986

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

2009-11-02

178

Specific detection of DNA and RNA targets using a novel isothermal nucleic acid amplification assay based on the formation of a three-way junction structure  

Microsoft Academic Search

The formation of DNA three-way junction (3WJ) structures has been utilised to develop a novel isothermal nucleic acid amplification assay (SMART) for the detection of specific DNA or RNA targets. The assay consists of two oligonucleotide probes that hybridise to a specific target sequence and, only then, to each other forming a 3WJ structure. One probe (template for the RNA

Susan D. Wharam; Peter Marsh; John S. Lloyd; Trevor D. Ray; Graham A. Mock; René Assenberg; Julie E. McPhee; Philip Brown; Anthony Weston; Donald L. N. Cardy

2001-01-01

179

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

2012-11-27

180

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

PubMed

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; Wang, Changjun; Qi, Zhongtian

2013-07-24

181

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

182

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

PubMed

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

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

2012-02-08

183

Detection of Four Plasmodium Species by Genus- and Species-Specific Loop-Mediated Isothermal Amplification for Clinical Diagnosis?  

PubMed Central

Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the clinical detection of four species of human malaria parasites: Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. We evaluated the sensitivity and specificity of LAMP in comparison with the results of microscopic examination and nested PCR. LAMP showed a detection limit (analytical sensitivity) of 10 copies of the target 18S rRNA genes for P. malariae and P. ovale and 100 copies for the genus Plasmodium, P. falciparum, and P. vivax. LAMP detected malaria parasites in 67 of 68 microscopically positive blood samples (sensitivity, 98.5%) and 3 of 53 microscopically negative samples (specificity, 94.3%), in good agreement with the results of nested PCR. The LAMP reactions yielded results within about 26 min, on average, for detection of the genus Plasmodium, 32 min for P. falciparum, 31 min for P. vivax, 35 min for P. malariae, and 36 min for P. ovale. Accordingly, in comparison to the results obtained by microscopy, LAMP had a similar sensitivity and a greater specificity and LAMP yielded results similar to those of nested PCR in a shorter turnaround time. Because it can be performed with a simple technology, i.e., with heat-treated blood as the template, reaction in a water bath, and inspection of the results by the naked eye because of the use of a fluorescent dye, LAMP may provide a simple and reliable test for routine screening for malaria parasites in both clinical laboratories and malaria clinics in areas where malaria is endemic.

Han, Eun-Taek; Watanabe, Risa; Sattabongkot, Jetsumon; Khuntirat, Benjawan; Sirichaisinthop, Jeeraphat; Iriko, Hideyuki; Jin, Ling; Takeo, Satoru; Tsuboi, Takafumi

2007-01-01

184

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.

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

2012-01-01

185

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

PubMed Central

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

2010-01-01

186

Development of a loop-mediated Isothermal amplification assay for sensitive and rapid detection of Vibrio parahaemolyticus  

PubMed Central

Background Vibrio parahaemolyticus is a marine seafood-borne pathogen causing gastrointestinal disorders in humans. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are known as major virulence determinants of V. parahaemolyticus. Most V. parahaemolyticus isolates from the environment do not produce TDH or TRH. Total V. parahaemolyticus has been used as an indicator for control of seafood contamination toward prevention of infection. Detection of total V. parahaemolyticus using conventional culture- and biochemical-based assays is time-consuming and laborious, requiring more than three days. Thus, we developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the sensitive and rapid detection of Vibrio parahaemolyticus. Results The assay provided markedly more sensitive and rapid detection of V. parahaemolyticus strains than conventional biochemical and PCR assays. The assay correctly identified 143 V. parahaemolyticus strains, but did not detect 33 non-parahaemolyticus Vibrio and 56 non-Vibrio strains. Sensitivity of the LAMP assay for direct detection of V. parahaemolyticus in pure cultures and in spiked shrimp samples was 5.3 × 102 CFU per ml/g (2.0 CFU per reaction). The sensitivity of the LAMP assay was 10-fold more sensitive than that of the conventional PCR assay. The LAMP assay was markedly faster, requiring for amplification 13–22 min in a single colony on TCBS agar from each of 143 V. parahaemolyticus strains and less than 35 min in spiked shrimp samples. The LAMP assay for detection of V. parahaemolyticus required less than 40 min in a single colony on thiosulfate citrate bile salt sucrose (TCBS) agar and 60 min in spiked shrimp samples from the beginning of DNA extraction to final determination. Conclusion The LAMP assay is a sensitive, rapid and simple tool for the detection of V. parahaemolyticus and will facilitate the surveillance for control of contamination of V. parahaemolyticus in seafood.

Yamazaki, Wataru; Ishibashi, Masanori; Kawahara, Ryuji; Inoue, Kiyoshi

2008-01-01

187

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

PubMed

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

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

2013-05-09

188

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

2010-09-09

189

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

2012-10-15

190

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

PubMed

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

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

2011-12-14

191

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

PubMed

A loop-mediated isothermal amplification (LAMP) assay was developed for rapid, specific and sensitive detection of ostreid herpesvirus 1 (OsHV-1) DNA. A set of four primers was designed, based on the sequence of the ATPase subunit of the OsHV-1 DNA-packaging terminase gene. The reaction temperature and time were optimized to 64°C and 60min, respectively. LAMP products were detected by agarose gel electrophoresis or by visual inspection of a color change due to addition of fluorescent dye. The developed method was highly specific for detection of OsHV-1, and no cross-reaction was observed with other DNA viruses, such as White spot syndrome virus (WSSV), Penaeus stylirostris densovirus (PstDNV), Turbot reddish body iridovirus (TRBIV) and Lymphocystis disease virus (LCDV) found commonly in China. The lower detection limit of the LAMP assay was approximately 20 copies per reaction, and it was 100 times more sensitive than that of conventional PCR. A comparative evaluation of 10 oyster samples using LAMP and PCR assays showed overall correlation in positive and negative results for OsHV-1. These results indicate that the LAMP assay is a simple, rapid, sensitive, specific and reliable technique for the detection of OsHV-1. The LAMP technique has capacity for use for the detection of OsHV-1 both in the laboratory and on farms. PMID:20813133

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

2010-09-08

192

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

2013-09-22

193

Rapid, sensitive, and specific detection of the O4 group of Salmonella enterica by loop-mediated isothermal amplification.  

PubMed

The present study developed a loop-mediated isothermal amplification (LAMP) assay that amplifies the fragments of O4 Salmonella enterica-specific gene rfbJ and evaluates the potential use in detection of Salmonella enterica serovar Typhimurium (ST). The detection limit of the LAMP assay was 10(3) CFU/ml, which was lower than that of the PCR assay with the same target gene (10(5) CFU/ml), confirmed by electrophoresis. The increased turbidity of the final products of LAMP was also observed with more than 10(3) CFU/ml. Furthermore, the LAMP assay took only 60 min for a reaction, while the PCR assay needed 80-90 min for a reaction and approximately 30 min for the subsequent electrophoresis to confirm the specific band. The positive reaction was only observed for 55 strains of 11 serovars of O4 group Salmonella enterica. The LAMP assay developed in the present study is considered to be an effective method for specific detection of the O4 group Salmonella enterica serovars, including ST. PMID:19630227

Okamura, Masashi; Ohba, Yousuke; Kikuchi, Shuichi; Takehara, Kazuaki; Ikedo, Masanari; Kojima, Tadashi; Nakamura, Masayuki

2009-06-01

194

A simple and rapid method for detection of Goose Parvovirus in the field by loop-mediated isothermal amplification  

PubMed Central

Background Goose parvovirus (GPV) is a Dependovirus associated with latent infection and mortality in geese. Currently, in a worldwide scale, GPV severely affects geese production. The objective of this study is to develop a loop-mediated isothermal amplification (LAMP) method for the sensitive, rapid, and inexpensive detection of GPV in the field. Results A set of six specific primers was designed by targeting the GPV VP3 DNA. With Bst DNA polymerase large fragment, the target DNA could be amplified at 65°C as early as 20 min of incubation in a simple water bath. A positive reaction was identified through the detection of the LAMP product by color change visible to the naked eye. The detection limit of the assay was 28 copies/?l of plasmid pVP3, and with equal sensitivity and specificity to fluorescent quantitative real-time PCR (FQ-PCR). Conclusions The high sensitivity, specificity, and simplicity, as well as the high throughput, make this method suitable for specific detection of GPV infection in both field conditions and laboratory settings. The utilization of complicated equipment and conduct of technical training on the GPV LAMP were not necessary.

2010-01-01

195

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

PubMed Central

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

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

2010-01-01

196

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

2012-10-05

197

Bacteria screening, viability, and confirmation assays using bacteriophage-impedimetric/loop-mediated isothermal amplification dual-response biosensors.  

PubMed

Here, we integrate two complementary detection strategies for the identification and quantification of Escherichia coli based on bacteriophage T4 as a natural bioreceptor for living bacteria cells. The first approach involves screening and viability assays, employing bacteriophage as the recognition element in label-free electrochemical impedance spectroscopy. The complementary approach is a confirmation by loop-mediated isothermal amplification (LAMP) to amplify specifically the E. coli Tuf gene after lysis of the bound E. coli cells, followed by detection using linear sweep voltammetry. Bacteriphage T4 was cross-linked, in the presence of 1,4-phenylene diisothiocyanate, on a cysteamine-modified gold electrode. The impedimetric biosensor exhibits specific and reproducible detection with sensitivity over the concentration range of 10(3)-10(9) cfu/mL, while the linear response of the LAMP approach was determined to be 10(2)-10(7) cfu/mL. The limit of detection (LOD) of 8 × 10(2) cfu/mL in less than 15 min and 10(2) cfu/mL within a response time of 40 min were achieved for the impedimetric and LAMP method, respectively. This work provides evidence that integration of the T4-bacteriophage-modified biosensor and LAMP can achieve screening, viability, and confirmation in less than 1 h. PMID:23510137

Tlili, Chaker; Sokullu, Esen; Safavieh, Mohammadali; Tolba, Mona; Ahmed, Minhaz Uddin; Zourob, Mohammed

2013-04-26

198

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.

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

2012-01-01

199

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

PubMed

The isothermal amplification of reporter signal via limited probe extension (minisequencing) upon hybridization of nucleic acids has been studied. The intensity of reporter signal has been shown to increase due to enzymatic labeling of multiple probes upon consecutive hybridization with one DNA template both in homophase and heterophase assays using various kinds of detection signal: radioisotope label, fluorescent label, and enzyme-linked assay. The kinetic scheme of the process has been proposed and kinetic parameters for each step have been determined. The signal intensity has been shown to correlate with physicochemical characteristics of both complexes: probe/DNA and product/DNA. The maximum intensity has been observed at minimal difference between the thermodynamic stability of these complexes, provided the reaction temperature has been adjusted near their melting temperature values; rising or lowering the reaction temperature reduces the amount of reporting product. The signal intensity has been shown to decrease significantly upon hybridization with the DNA template containing single-nucleotide mismatches. Limited probe extension assay is useful not only for detection of DNA template but also for its quantitative characterization. PMID:21317947

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

200

A novel and more sensitive loop-mediated isothermal amplification assay targeting IS6110 for detection of Mycobacterium tuberculosis complex.  

PubMed

Developing improved tuberculosis (TB) diagnostics is one of the international research priorities, as TB remains globally a major health threat. Loop-mediated isothermal amplification (LAMP) is a new nucleic acid detection method that can be used in low-resource settings, because it does not require expensive or complex instruments. Using the repetitive insertion sequence IS6110 as a target gene, we developed an efficient LAMP assay, which specifically detects members of the Mycobacterium tuberculosis complex (MTBC). This assay proved 20 times more sensitive than IS6110-based conventional PCR. Moreover, its sensitivity was, respectively, 50 and 20 times higher than the one obtained with the two previously described LAMP assays for M. tuberculosis, based on gyrB and rrs, respectively. Identical sensitivities were obtained for LAMP and nested PCR, but the LAMP assay was more rapid and cost-effective than the latter. Although, our LAMP assay can successfully be performed using a non-denatured template, this results in a 200-fold reduction in the sensitivity of the assay. Moreover, by performing our LAMP assay on 15 clinical sputum samples from TB patients we were able to detect MTB. Taken together, our preliminary results indicate that IS6110-based MTBC-LAMP assay is a promising new TB-diagnostic test, with high sensitivity and that could easily be applied for the diagnosis of TB in a low-resource setting. PMID:19515543

Aryan, Ehsan; Makvandi, Manoochehr; Farajzadeh, Ahmad; Huygen, Kris; Bifani, Pablo; Mousavi, Seyed-Latif; Fateh, Abolfazl; Jelodar, Abbass; Gouya, Mohammad-Mehdi; Romano, Marta

2009-06-10

201

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

Microsoft Academic Search

Rapid, sensitive, and low-cost pathogen diagnostic systems are needed for early disease diagnosis and treatment, especially\\u000a in resource-limited settings. This study reports a low-cost charge-coupled device (CCD)-based fluorescence imaging system\\u000a for rapid detection of waterborne pathogens by isothermal gene amplification in disposable microchips. Fluorescence imaging\\u000a capability of this monochromatic CCD camera is evaluated by optimizing the gain, offset, and exposure

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

202

Single Rapid Real-Time Monitored Isothermal RNA Amplification Assay for Quantification of Human Immunodeficiency Virus Type 1 Isolates from Groups M, N, and O  

Microsoft Academic Search

Because human immunodeficiency virus type 1 (HIV-1) subtypes and circulating recombinant forms (CRFs) are spreading rapidly worldwide and are becoming less confined to a geographical area, RNA assays that can detect and quantify all HIV-1 isolates reliably are in demand. We have developed a fast, real-time monitored RNA assay based on an isothermal nucleic acid sequence-based amplification technology that amplifies

MICHEL P. DE BAAR; MAAIKE W. VAN DOOREN; ESTHER DE ROOIJ; MARGREET BAKKER; BOB VAN GEMEN; JAAP GOUDSMIT; ANTHONY DE RONDE

203

Real-time loop-mediated isothermal amplification for the CaMV-35S promoter as a screening method for genetically modified organisms  

Microsoft Academic Search

A method has been developed to detect genetically modified organisms (GMOs). The detection method is based on the loop-mediated isothermal amplification (LAMP) reaction. Cauliflower mosaic virus 35S (CaMV-35S) promoter gene, a widespread genetic element, was amplified by a set of LAMP primers. One of the characteristics of the LAMP method is its ability to synthesize an extremely large amount of

Shiro Fukuta; Yuko Mizukami; Akira Ishida; Junichi Ueda; Mayumi Hasegawa; Izumi Hayashi; Minako Hashimoto; Michio Kanbe

2004-01-01

204

Specific detection of reverse transcription-loop-mediated isothermal amplification amplicons for Taura syndrome virus by colorimetric dot–blot hybridization  

Microsoft Academic Search

The goal of this study was to develop a field diagnosis system based on isothermal reverse transcription-loop-mediated amplification (RT-LAMP) for shrimp Taura syndrome virus (TSV), placing emphasis on specific and simple detection of the LAMP amplicons. After a single-tube RT-LAMP reaction for TSV was established, colorimetric dot–blot hybridization (DBH) was adopted to detect signals only from the target-derived amplicons. The

Ping-Hua Teng; Chu-Liang Chen; Ping-Feng Sung; Fu-Chun Lee; Bor-Rung Ou; Pei-Yu Lee

2007-01-01

205

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

206

Loop-mediated isothermal amplification for rapid detection and differentiation of wild-type pseudorabies and gene-deleted virus vaccines  

Microsoft Academic Search

A loop-mediated isothermal amplification (LAMP) assay was developed specifically for detection and differentiation of pseudorabies virus (PRV). One group of primers was designed to detect wild-type strains (i.e., strains with the gE gene) and the other group of primers was designed to detect both PRV gE-vaccine and wild-type strains (i.e., strains with the gG gene and with or without the

Chao-fan Zhang; Shang-jin Cui; Chao Zhu

2010-01-01

207

Differential detection of Wheat yellow mosaic virus, Japanese soil-borne wheat mosaic virus and Chinese wheat mosaic virus by reverse transcription loop-mediated isothermal amplification reaction.  

PubMed

A differential detection method for three wheat viruses: Wheat yellow mosaic virus (WYMV), Japanese soil-borne mosaic virus (JSBWMV) and Chinese wheat mosaic virus (CWMV) using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction was developed. All three primer sets, which were designed from the genome sequences of WYMV, JSBWMV and CWMV respectively, worked most efficiently at 65 °C and could detect each virus RNA within 10 min by fluorescence monitoring using an isothermal DNA amplification and fluorescence detection device. Furthermore, these primer sets showed unique annealing curves. The peak denaturing temperatures of WYMV, JSBWMV and CWMV primer sets were 87.6 °C, 84.8 °C and 86.4 °C, respectively and were clearly distinguished by the isothermal DNA amplification and fluorescence detection device. The RT-LAMP assay including all three primer sets was found to be 100 times more sensitive than RT-PCR for WYMV and JSBWMV and as sensitive as RT-PCR for CWMV. The RT-LAMP method was validated for the simultaneous detection of these viruses in wheat and barley leaves. PMID:23523736

Fukuta, Shiro; Tamura, Masaru; Maejima, Hidekazu; Takahashi, Reiko; Kuwayama, Sachiko; Tsuji, Takako; Yoshida, Tomofumi; Itoh, Koji; Hashizume, Hajime; Nakajima, Yasunori; Uehara, Yasushi; Shirako, Yukio

2013-03-21

208

Development and application of loop-mediated isothermal amplification assays on rapid detection of various types of staphylococci strains  

PubMed Central

A loop-mediated isothermal amplification (LAMP) method for rapid detection of various Staphylococcus strains and associated antibiotic resistance determinant had been developed and evaluated in this study. Six primers, including outer primers, inner primers and loop primers, were specially designed for recognizing eight distinct sequences on three targets: 16SrRNA, femA and mecA.. Forty-one reference strains, including various species of gram-negative and -positive isolates, were included in this study to evaluate and optimize LAMP assays. The optimal reaction condition was found to be 65 °C for 45 min, with detection limits at 100 fg DNA/tube and 10 CFU/reaction for 16S rRNA, 100 fg DNA/tube and 10 CFU/reaction for femA, 1 pg DNA/tube and 100 CFU/reaction for mecA, respectively. Application of LAMP assays were performed on 118 various types of Staphylococcus isolates, the detection rate of LAMP assays for the 16SrRNA, femA and mecA was 100% (118/118), 98.5% (64/65) and 94.3% (66/70), and the negative predictive value (NPV) was 100%, 98.1% and 92.3% respectively; with a 100% positive predictive value (PPV) for all three targets. In conclusion, LAMP assays were demonstrated to be useful and powerful tools for rapid detection of various Staphylococcus strains, and undoubtedly, the rapidness, technical simplicity, and cost-effectiveness of LAMP assays will demonstrate broad application for bacteriological detection of food-borne Methicillin-resistant Staphylococcus (MRS) isolates.

Xu, Zhenbo; Li, Lin; Chu, Jin; Peters, Brian M.; Harris, Megan L.; Li, Bing; Shi, Lei; Shirtliff, Mark E.

2011-01-01

209

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.

2012-01-01

210

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

2013-04-20

211

Detection of Salmonella enterica in naturally contaminated liquid eggs by loop-mediated isothermal amplification, and characterization of Salmonella isolates.  

PubMed

Loop-mediated isothermal amplification (LAMP) assay was effective in detecting Salmonella enterica in naturally contaminated liquid egg samples. Salmonella was detected in 110 samples taken from four egg-breaking plants. The egg samples were pre-enriched in buffered peptone water (BPW) at 37 degrees C for 20 h. The selective enrichment was done in Rappaport-Vassiliadis or tetrathionate broth and plated onto xylose lysine deoxycholate agar and brilliant green agar, modified. In addition, the PCR assay was used to detect Salmonella after pre-enrichment in BPW at 37 degrees C for 20 h. The culture method and PCR assay were compared to the LAMP assay, which was also performed after pre-enrichment in BPW. PCR failed to detect Salmonella in 10% of 110 samples, whereas the culture method and LAMP assay successfully identified Salmonella in all samples. However, the LAMP assay was found to be much more rapid than the culture method and as sensitive in detecting Salmonella from liquid eggs. In all of the egg-breaking plants studied, Salmonella was isolated on most tested days. The positive samples showed that more than 75% of the Salmonella strains had identical genetic patterns when analyzed by pulsed-field gel electrophoresis. This suggests that the same Salmonella strains having survived long periods of time in the plants were contaminating the production line. The LAMP assay is rapid, specific, and sensitive for Salmonella detection in liquid eggs and is able to monitor Salmonella contamination in egg-handling plants more reliably. PMID:16269703

Ohtsuka, Kayoko; Yanagawa, Keiko; Takatori, Kosuke; Hara-Kudo, Yukiko

2005-11-01

212

Sensitive and rapid detection of cholera toxin-producing Vibrio cholerae using a loop-mediated isothermal amplification  

PubMed Central

Background Vibrio cholerae is widely acknowledged as one of the most important waterborne pathogen causing gastrointestinal disorders. Cholera toxin (CT) is a major virulence determinant of V. cholerae. Detection of CT-producing V. cholerae using conventional culture-, biochemical- and immunological-based assays is time-consuming and laborious, requiring more than three days. Thus, we developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the sensitive and rapid detection of cholera toxin (CT)-producing Vibrio cholerae. Results The assay provided markedly more sensitive and rapid detection of CT-producing V. cholerae strains than conventional biochemical and PCR assays. The assay correctly identified 34 CT-producing V. cholerae strains, but did not detect 13 CT non-producing V. cholerae and 53 non-V. cholerae strains. Sensitivity of the LAMP assay for direct detection of CT-producing V. cholerae in spiked human feces was 7.8 × 102 CFU per g (1.4 CFU per reaction). The sensitivity of the LAMP assay was 10-fold more sensitive than that of the conventional PCR assay. The LAMP assay for detection of CT-producing V. cholerae required less than 35 min with a single colony on thiosulfate citrate bile salt sucrose (TCBS) agar and 70 min with human feces from the beginning of DNA extraction to final determination. Conclusion The LAMP assay is a sensitive, rapid and simple tool for the detection of CT-producing V. cholerae and will be useful in facilitating the early diagnosis of human V. cholerae infection.

Yamazaki, Wataru; Seto, Kazuko; Taguchi, Masumi; Ishibashi, Masanori; Inoue, Kiyoshi

2008-01-01

213

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

PubMed

Recent outbreaks linked to Salmonella-contaminated produce heightened the need to develop simple, rapid, and accurate detection methods, particularly those capable of determining cell viability. In this study, we examined a novel strategy for the rapid detection and quantification of viable salmonellae in produce by coupling a simple propidium monoazide sample treatment with loop-mediated isothermal amplification (PMA-LAMP). We first designed and optimized a LAMP assay targeting Salmonella. Second, the performance of PMA-LAMP for detecting and quantifying viable salmonellae was determined. Finally, the assay was evaluated in experimentally contaminated produce items (cantaloupe, spinach, and tomato). Under the optimized condition, PMA-LAMP consistently gave negative results for heat-killed Salmonella cells with concentrations up to 10(8) CFU/ml (or CFU/g in produce). The detection limits of PMA-LAMP were 3.4 to 34 viable Salmonella cells in pure culture and 6.1 × 10(3) to 6.1 × 10(4) CFU/g in spiked produce samples. In comparison, PMA-PCR was up to 100-fold less sensitive. The correlation between LAMP time threshold (T(T)) values and viable Salmonella cell numbers was high (R(2) = 0.949 to 0.993), with a quantification range (10(2) to 10(5) CFU/reaction in pure culture and 10(4) to 10(7) CFU/g in produce) comparable to that of PMA in combination with quantitative real-time PCR (PMA-qPCR). The complete PMA-LAMP assay took about 3 h to complete when testing produce samples. In conclusion, this rapid, accurate, and simple method to detect and quantify viable Salmonella cells in produce may present a useful tool for the produce industry to better control potential microbial hazards in produce. PMID:21498750

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

2011-04-15

214

Study of DNA extraction methods for use in loop-mediated isothermal amplification detection of single resting cysts in the toxic dinoflagellates Alexandrium tamarense and A. catenella.  

PubMed

In a previous study, we experienced instable amplification and a low amplification success in loop-mediated isothermal amplification (LAMP) reactions from naturally occurring vegetative cells or resting cysts of the toxic dinoflagellates Alexandrium tamarense and Alexandrium catenella. In this study, we examined 4 methods for extracting DNA from single resting cysts of A. tamarense and A. catenella to obtain more stable and better amplification success and to facilitate unambiguous detection using the LAMP method. Apart from comparing the 4 different DNA extraction methods, namely, (1) boiling in Tris-EDTA (TE) buffer, (2) heating at 65 °C in hexadecyltrimethylammonium bromide buffer, (3) boiling in 0.5% Chelex buffer, and (4) boiling in 5% Chelex buffer, we also examined the need for homogenization to crush the resting cysts before DNA extraction in each method. Homogenization of resting cysts was found to be essential for DNA extraction in all 4 methods. The detection time was significantly shorter in 5% Chelex buffer than in the other buffers and the amplification success was 100% (65/65), indicating the importance of DNA extraction and the effectiveness of 5% Chelex buffer in the Alexandrium LAMP. PMID:22897963

Nagai, Satoshi; Yamamoto, Keigo; Hata, Naotugu; Itakura, Shigeru

2012-04-20

215

Loop-mediated isothermal amplification (LAMP) for malarial parasites of humans: would it come to clinical reality as a point-of-care test?  

PubMed

Loop-mediated isothermal amplification (LAMP) is a novel molecular method that accelerates and facilitates DNA amplification and detection under isothermal conditions. It represents a revolution in molecular biology by reducing the high cost, turnaround time and technicality of polymerase chain reaction and other amplification methods. It has been applied for the diagnosis of a variety of viral, bacterial, parasitic and other diseases in the biomedical field. LAMP has been involved in studies concerning the diagnosis of malaria which is still a major cause of morbidity and mortality in different parts of the world. For the success attained with this technology to diagnose human malaria, is it time to think that LAMP-based point-of-care diagnostics come to application to support the diagnosis of clinical malaria cases? The present review deals with the use of LAMP in the diagnosis of malaria and related investigations to make a view on what has been investigated and highlights the future perspectives regarding the possible applications of LAMP in diagnosis of the disease. PMID:22366670

Abdul-Ghani, Rashad; Al-Mekhlafi, Abdulsalam M; Karanis, Panagiotis

2012-02-16

216

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

217

Detection of virus mRNA within infected host cells using an isothermal nucleic acid amplification assay: marine cyanophage gene expression within Synechococcus sp  

PubMed Central

Background Signal-Mediated Amplification of RNA Technology (SMART) is an isothermal nucleic acid amplification technology, developed for the detection of specific target sequences, either RNA (for expression) or DNA. Cyanophages are viruses that infect cyanobacteria. Marine cyanophages are ubiquitous in the surface layers of the ocean where they infect members of the globally important genus Synechococcus. Results Here we report that the SMART assay allowed us to differentiate between infected and non-infected host cultures. Expression of the cyanophage strain S-PM2 portal vertex gene (g20) was detected from infected host Synechococcus sp. WH7803 cells. Using the SMART assay, we demonstrated that g20 mRNA peaked 240 – 360 minutes post-infection, allowing us to characterise this as a mid to late transcript. g20 DNA was also detected, peaking 10 hours post-infection, coinciding with the onset of host lysis. Conclusion The SMART assay is based on isothermal nucleic acid amplification, allowing the detection of specific sequences of DNA or RNA. It was shown to be suitable for differentiating between virus-infected and non-infected host cultures and for the detection of virus gene expression: the first reported use of this technology for such applications.

Wharam, Susan D; Hall, Matthew J; Wilson, William H

2007-01-01

218

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.

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

2011-01-01

219

Lab in a tube: ultrasensitive detection of microRNAs at the single-cell level and in breast cancer patients using quadratic isothermal amplification.  

PubMed

Through rational design of a functional molecular probe with high sequence specificity that takes advantage of sensitive isothermal amplification with simple operation, we developed a one-pot hairpin-mediated quadratic enzymatic amplification strategy for microRNA (miRNA) detection. Our method exhibits ultrahigh sensitivity toward miR-21 with detection limits of 10 fM at 37 °C and 1 aM at 4 °C, which corresponds to nine strands of miR-21 in a 15 ?L sample, and it is capable of distinguishing among miRNA family members. More importantly, the proposed approach is also sensitive and selective when applied to crude extractions from MCF-7 and PC3 cell lines and even patient tissues from intraductal carcinoma and invasive ductal carcinoma of the breast. PMID:23445447

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

2013-03-13

220

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

PubMed

In this study, we applied a loop-mediated isothermal amplification method for identification of human Taenia tapeworms in Tibetan communities in Sichuan, China. Out of 51 proglottids recovered from 35 carriers, 9, 1, and 41 samples were identified as Taenia solium, Taenia asiatica and Taenia saginata, respectively. Same results were obtained afterwards in the laboratory, except one sample. These results demonstrated that the LAMP method enabled rapid identification of parasites in the field surveys, which suggested that this method would contribute to the control of Taenia infections in endemic areas. PMID:22698671

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

2012-06-11

221

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

2013-06-12

222

Application of loop-mediated isothermal amplification assay for the sensitive and rapid diagnosis of visceral leishmaniasis and post-kala-azar dermal leishmaniasis.  

PubMed

Loop-mediated isothermal amplification (LAMP) is at the forefront in the search for innovative diagnostics for rapid and specific amplification of target DNA under isothermal conditions. We have applied LAMP assay using SYBR Green for clear-cut naked eye detection of Leishmania (Leishmania) donovani in 200 clinical samples of visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL). The assay was positive in 53/55 VL blood samples (sensitivity, 96.4%; 95% confidence interval [CI], 87.7-99%), 15/15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI, 79.6-100%), 60/62 PKDL tissue biopsy samples (sensitivity, 96.8%; 95% CI, 88.9-99.1%), and 1/68 control samples (specificity, 98.5%; 95% CI, 92.1-99.7%). The assay was specific for L. (L.) donovani, the causative species for VL and negative for L. (L.) infantum, L. (L.) tropica, and L. (L.) major. This is the first comprehensive clinical study demonstrating the applicability of the LAMP assay for a rapid and reliable molecular diagnosis of VL and PKDL. PMID:23433714

Verma, Sandeep; Avishek, Kumar; Sharma, Vanila; Negi, Narendra Singh; Ramesh, Venkatesh; Salotra, Poonam

2013-02-19

223

An Isothermal Method for Whole Genome Amplification of Fresh and Degraded DNA for Comparative Genomic Hybridization, Genotyping and Mutation Detection  

Microsoft Academic Search

Molecular genotyping has important biomedical and forensic applications. However, limiting amounts of human biological material often yield genomic DNA (gDNA) in insufficient quantity and of poor quality for a reliable analysis. This motivated the development of an efficient whole genome amplification method with quantitatively unbiased representation usable on fresh and degraded gDNA. Amplification of fresh frozen, formalin-fixed paraffin-embedded (FFPE) and

Cheryl I. P. Lee; Siew Hong Leong; Adrian E. H. Png; Keng Wah Choo; Christopher Syn; Dennis T. H. Lim; Hai Yang Law; Oi Lian Kon

2006-01-01

224

Dumbbell probe-mediated cascade isothermal amplification: a novel strategy for label-free detection of microRNAs and its application to real sample assay.  

PubMed

Considering the great significance of microRNAs (miRNAs) in cancer detection and typing, the development of sensitive, specific, quantitative, and low-cost methods for the assay of expression levels of miRNAs is desirable. We describe a highly efficient amplification platform for ultrasensitive analysis of miRNA (taking let-7a miRNA as a model analyte) based on a dumbbell probe-mediated cascade isothermal amplification (DP-CIA) strategy. The method relies on the circularization of dumbbell probe by binding target miRNA, followed by rolling circle amplification (RCA) reaction and an autonomous DNA machine performed by nicking/polymerization/displacement cycles that continuously produces single-stranded G-quadruplex to assemble with hemin to generate a color signal. In terms of the high sensitivity (as low as 1 zmol), wide dynamic range (covering 9 orders of magnitude), good specificity (even single-base difference) and easy operation (one probe and three enzymes), the proposed label-free assay is successfully applied to direct detection of let-7a miRNA in real sample (total RNA extracted from human lung tissue), demonstrating an attractive alternative for miRNA analysis for gene expression profiling and molecular diagnostics, particularly for early cancer diagnosis. PMID:23265735

Bi, Sai; Cui, Yangyang; Li, Li

2012-11-16

225

Loop-mediated isothermal amplification assay targeting the femA gene for rapid detection of Staphylococcus aureus from clinical and food samples.  

PubMed

In this study, a loop-mediated isothermal amplification (LAMP) method to rapidly detect Staphylococcus aureus strains was developed and evaluated by extensively applying a large number of S. aureus isolates from clinical and food samples. Six primers were specially designed for recognizing eight distinct sequences on the species-specific femA gene of S. aureus. The detection limits were 100 fg DNA/tube and 10(4) CFU/ml. The LAMP assay was applied to 432 S. aureus strains isolated from 118 clinical and 314 food samples. Total detection rates for the LAMP and polymerase chain reaction assays were 98.4% (306/311) and 89.4% (278/311), respectively. PMID:23412068

Zhao, Xihong; Li, Yanmei; Park, Myoungsu; Wang, Jun; Zhang, Youhong; He, Xiaowei; Forghani, Fereidoun; Wang, Li; Yu, Guangchao; Oh, Deog-Hwan

2013-02-01

226

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

PubMed

Falciparum malaria is a fatal infection without immediate diagnosability or treatment. There are shortages of clinicians and examiners skilled in the treatment of malaria in non-endemic countries, including Japan. This study was performed to evaluate a novel rapid molecular diagnostic system consisting of loop-mediated isothermal amplification (LAMP) combined with DNA filter paper (FTA card) and melting curve analysis. Combining LAMP with melting curve analysis enabled diagnosis of Plasmodium falciparum more accurately with relative ease. FTA cards could be used to clarify problems regarding storage, infectivity, and transportation. The LAMP assay was carried out at a constant temperature of 63 degrees C for 90 min. The diagnostic system (malaria-LAMP) accurately diagnosed malaria (47 samples from Thailand and 50 from Zimbabwe) with 97.8% sensitivity and 85.7% specificity as compared with microscopic methods, indicating the usefulness of this combined system. PMID:19168954

Yamamura, Mariko; Makimura, Koichi; Ota, Yasuo

2009-01-01

227

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

PubMed

Rapid, sensitive, and low-cost pathogen diagnostic systems are needed for early disease diagnosis and treatment, especially in resource-limited settings. This study reports a low-cost charge-coupled device (CCD)-based fluorescence imaging system for rapid detection of waterborne pathogens by isothermal gene amplification in disposable microchips. Fluorescence imaging capability of this monochromatic CCD camera is evaluated by optimizing the gain, offset, and exposure time. This imaging system is validated for 12 virulence genes of major waterborne pathogens on cyclic olefin polymer (COP) microchips, using SYTO-82 dye and real time fluorescence loop-mediated isothermal amplification referred here as microRT(f)-LAMP. Signal-to-noise ratio (SNR) and threshold time (Tt) of microRT(f)-LAMP assays are compared with those from a commercial real-time polymerase chain reaction (PCR) instrument. Applying a CCD exposure of 5 s to 10(5) starting DNA copies of microRT(f)-LAMP assays increases the SNR by 8-fold and reduces the Tt by 9.8 min in comparison to a commercial real-time PCR instrument. Additionally, single copy level sensitivity for Campylobacter jejuni 0414 gene is obtained for microRT(f)-LAMP with a Tt of 19 min, which is half the time of the commercial real-time PCR instrument. Due to the control over the exposure time and the wide field imaging capability of CCD, this low-cost fluorescence imaging system has the potential for rapid and parallel detection of pathogenic microorganisms in high throughput microfluidic chips. PMID:21720851

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

2011-10-01

228

Rapid Detection and Differentiation of Dengue Virus Serotypes by a Real-Time Reverse Transcription-Loop-Mediated Isothermal Amplification Assay  

PubMed Central

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

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

2005-01-01

229

Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus  

Microsoft Academic Search

Summary.  Speed is paramount in the diagnosis of foot-and-mouth disease (FMD) and simplicity is required if a test is to be deployed\\u000a in the field. The development of a one-step, reverse transcription loop-mediated amplification (RT-LAMP) assay enables FMD\\u000a virus (FMDV) to be detected in under an hour in a single tube without thermal cycling. A fragment of the 3D RNA polymerase

J. P. Dukes; D. P. King; S. Alexandersen

2006-01-01

230

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

231

Rapid and sensitive detection of Penaeus monodon nucleopolyhedrovirus (PemoNPV) by loop-mediated isothermal amplification combined with a lateral-flow dipstick.  

PubMed

Several methods such as traditional PCR or nested-PCR, immuno assay and histopathology have been developed for detection of Penaeus monodon nucleopolyhedrovirus (PemoNPV) formerly called monodon baculovirus (MBV). However, these methods have various disadvantages including low sensitivity, long assay time, use of toxic substances or unsuitability for field diagnosis. Loop-mediated isothermal amplification of target nucleotide sequences under isothermal conditions, combined with amplicon detection by chromatographic lateral-flow dipsticks allows for more efficient, field friendly detection within 75 min (not including DNA preparation time). In this study, the LAMP amplicon was biotinylated via an inner LAMP primer designed from a BamHI fragment B, a hypothetical protein gene of PemoNPV under isothermal condition at 63 degrees C for 1 h. Next, the LAMP product was hybridized at 63 degrees C for 5 min with an optimal FITC-labeled probe that was designed specifically for the LAMP amplicons. The FITC-labeled biotinylated LAMP product picked up gold-labeled, anti-FITC near the LFD origin and the whole, triple-labeled complex was captured by an immobilized biotin-binding protein to yield a red nano-gold stripe at the LFD test line. With a DNA template extracted from PemoNPV-infected shrimp, the LAMP-LFD detection limit was 0.1 pg, whereas one-step PCR and nested-PCR followed with gel electrophoresis was 1 pg. The LAMP-LFD method gave negative test results with buffer and DNA from shrimp infected with other common shrimp DNA viruses including, Penaeus monodon densovirus (PmDNV) formerly called hepatopancreatic parvovirus (HPV), white spot syndrome virus (WSSV) and Penaeus stylirostris densovirus (PstDNV) formerly called infectious hypodermal and hematopoietic necrosis virus (IHHNV). The test platform can be adapted easily for rapid detection of other shrimp viruses, since the LAMP-LFD combination system was a highly sensitive, specific, convenient, and does not require sophisticated instruments. PMID:19818396

Nimitphak, Tongchai; Meemetta, Watcharachai; Arunrut, Narong; Senapin, Saengchan; Kiatpathomchai, Wansika

2009-10-08

232

Development of a loop-mediated isothermal amplification procedure as a sensitive and rapid method for detection of 'candidatus Liberibacter solanacearum' in potatoes and Psyllids.  

PubMed

This study reports the development of a loop-mediated isothermal amplification procedure (LAMP) for polymerase chain reaction (PCR)-based detection of 'Candidatus Liberibacter solanacearum', the bacterial causal agent of potato zebra chip (ZC) disease. The 16S rDNA gene of 'Ca. Liberibacter solanacearum' was used to design a set of six primers for LAMP PCR detection of the bacterial pathogen in potato plants and the psyllid vector. The advantage of the LAMP method is that it does not require a thermocycler for amplification or agarose gel electrophoresis for resolution. Positive LAMP results can be visualized directly as a precipitate. The LAMP strategy reported here reliably detected 'Ca. Liberibacter solanacearum' and the closely related species 'Ca. Liberibacter asiaticus', the causative agent of huanglongbing disease of citrus, in plant DNA extracts. Although not as sensitive as quantitative real-time PCR, LAMP detection was equivalent to conventional PCR in tests of ZC-infected potato plants from the field. Thus, the LAMP method shows strong promise as a reliable, rapid, and cost-effective method of detecting 'Ca. Liberibacter' pathogens in psyllids and field-grown potato plants and tubers. PMID:22881872

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

2012-09-01

233

Detection of Schistosoma mansoni and Schistosoma haematobium DNA by Loop-Mediated Isothermal Amplification: Identification of Infected Snails from Early Prepatency  

PubMed Central

Monitoring post-control transmission of schistosomes by examining humans becomes less effective as infection rates among humans decrease. Molecular monitoring of prepatent schistosome infection in snails by the polymerase chain reaction (PCR) has been used for studying human-to-snail transmission, and snail prepatent infection rates were found to correspond to infection prevalence and average intensity in human populations contacting the sites studied. We have now developed loop-mediated isothermal amplification (LAMP) assays for identifying Schistosoma mansoni and S. haematobium to facilitate large-scale evaluation of post-intervention transmission potential. LAMP primers were designed based on the Sm1-7 and DraI repeated sequences of the corresponding schistosomes, and amplification by LAMP of these 121-basepair highly abundant sequences provided a detection sensitivity of 0.1 fg of genomic DNA. When these LAMP assays were applied for examining infected laboratory snails, it was possible to identify infection from the first day after exposure to miracidia. The potential advantages of these assays are discussed.

Abbasi, Ibrahim; King, Charles H.; Muchiri, Eric M.; Hamburger, Joseph

2010-01-01

234

[Visual detection of H1 subtype and identification of N1, N2 subtype of avian influenza virus by reverse transcription loop-mediated isothermal amplification assay].  

PubMed

In order to visually detect H1, N1 and N2 subtype of avian influenza virus (AIV), three reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed. According to the sequences of AIV gene available in GenBank, three degenerate primer sets specific to HA gene of H1 subtype AIV, NA gene of N1 and N2 subtype AIV were designed, and the reaction conditions were optimized. The results showed that all the assays had no cross-reaction with other subtype AIV and other avian respiratory pathogens, and the detection limit was higher than that of conventional RT-PCR. These assays were performed in water bath within 50 minutes. Without opening tube, the amplification result could be directly determined by inspecting the color change of reaction system as long as these assays were fin-ished. Fourteen specimens of H1N1 subtype and eight specimens of H1N2 subtype of AIV were identified from the 120 clinical samples by RT-LAMP assays developed, which was consistent with that of virus isolation. These results suggested that the three newly developed RT-LAMEP assays were simple, specific and sensitive and had potential for visual detection of H1, N1 and N2 subtype of AIV in field. PMID:23757846

Peng, Yi; Xie, Zhi-Xun; Guo, Jie; Zhou, Chen-Yu; Liu, Jia-Bo; Pang, Yao-Shan; Deng, Xian-Wen; Xie, Zhi-Qin; Xie, Li-Ji; Fan, Qing; Luo, Si-Si

2013-03-01

235

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

PubMed

Reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) is a novel molecular detection method that is specific, fast, and simple. It is based on reverse transcription followed by DNA amplification using the Bst DNA polymerase large fragment requiring one temperature and a simple waterbath, without the need for any expensive equipment. Detection is by turbidity or agarose gel electrophoresis. Our objective was to apply this LAMP-based technology to rapidly and sensitively detect Salmonella enterica serovar Enteritidis in liquid whole eggs (LWEs) within 1 d. LWE were inoculated with S. Enteritidis and stomached in tetrathionate broth (TTB), and spread-plated on Xylose lysine tergitol 4 agar either immediately or after 6, 12, or 16-h enrichment. RNA was extracted from 5-mL TTB and the RT-LAMP assay was carried out using invA primers. After 16 and 12-h enrichment, improved Salmonella detection up to 10? to 10¹ and 10? CFU/25 mL LWE, respectively was obtained. Without enrichment, Salmonella could be detected at 10? CFU/25 mL; however, after 6-h enrichment a 1-log improvement to 10? CFU/25 mL was obtained. This RT-LAMP assay appears to be suitable as a potential screening/monitoring tool for Salmonella enterica from LWE products in routine settings with results obtainable within 24-h, which is significantly faster than traditional cultural assays. PMID:22352954

Techathuvanan, Chayapa; D'Souza, Doris Helen

2012-02-21

236

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.

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

2013-01-01

237

Loop-mediated isothermal amplification for rapid detection and differentiation of wild-type pseudorabies and gene-deleted virus vaccines.  

PubMed

A loop-mediated isothermal amplification (LAMP) assay was developed specifically for detection and differentiation of pseudorabies virus (PRV). One group of primers was designed to detect wild-type strains (i.e., strains with the gE gene) and the other group of primers was designed to detect both PRV gE-vaccine and wild-type strains (i.e., strains with the gG gene and with or without the gE gene). After amplification by Bst enzyme at a constant temperature of 65 degrees C, a laddering of bright products was visible following electrophoresis on a 2% agarose gel. LAMP was 100-1000-fold more sensitive than the standard PCR. The assay was specific in that it did not amplify other porcine viruses including porcine parvovirus, porcine circovirus type 1, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, classical swine fever virus, swine transmissible gastroenteritis coronavirus, and porcine epidemic diarrhea virus. Because of its sensitivity, specificity, and simplicity, the LAMP assay could be a useful method for early and rapid differentiation of swine vaccinated with PRV gE-deleted vaccine from swine infected with wild virus. PMID:20691214

Zhang, Chao-Fan; Cui, Shang-Jin; Zhu, Chao

2010-08-04

238

Erwinia amylovora loop-mediated isothermal amplification (LAMP) assay for rapid pathogen detection and on-site diagnosis of fire blight.  

PubMed

Several molecular methods have been developed for the detection of Erwinia amylovora, the causal agent of fire blight in pear and apple, but none are truly applicable for on-site use in the field. We developed a fast, reliable and field applicable detection method using a novel target on the E. amylovora chromosome that we identified by applying a comparative genomic pipeline. The target coding sequences (CDSs) are both uniquely specific for and all-inclusive of E. amylovora genotypes. This avoids potential false negatives that can occur with most commonly used methods based on amplification of plasmid gene targets, which can vary among strains. Loop-mediated isothermal AMPlification (LAMP) with OptiGene Genie II chemistry and instrumentation proved to be an exceptionally rapid (under 15 min) and robust method for detecting E. amylovora in orchards, as well as simple to use in the plant diagnostic laboratory. Comparative validation results using plant samples from inoculated greenhouse trials and from natural field infections (of regional and temporal diverse origin) showed that our LAMP had an equivalent or greater performance regarding sensitivity, specificity, speed and simplicity than real-time PCR (TaqMan), other LAMP assays, immunoassays and plating, demonstrating its utility for routine testing. PMID:23275135

Bühlmann, Andreas; Pothier, Joël F; Rezzonico, Fabio; Smits, Theo H M; Andreou, Michael; Boonham, Neil; Duffy, Brion; Frey, Jürg E

2012-12-28

239

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

PubMed Central

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

2012-01-01

240

Simple and rapid detection of swine hepatitis E virus by reverse transcription loop-mediated isothermal amplification.  

PubMed

Hepatitis E virus (HEV) is an enteric pathogen of humans and animals, and pigs have been considered an important reservoir of this virus. Recent evidence has indicated the cross-species transmission of hepatitis E virus (HEV) from pigs to humans, causing zoonosis, mostly via consumption of uncooked or undercooked animal meat/viscera. In this study, we have developed a one-step RT-LAMP assay for rapid detection of swine HEV. Specific primer sets targeting the ORF3 gene were designed. The sensitivity of the RT-LAMP assay was 10(1) copies/?l of RNA template, which was tenfold higher than that of RT-nPCR. The specificity of this assay was demonstrated by the lack of amplification of DNA/RNA from other swine viruses. Furthermore, a total of 41 bile samples were subjected to RT-LAMP and RT-nPCR. Eighteen positive samples were detected by RT-nPCR, while 36 positive samples were detected by RT-LAMP, indicating that the sensitivity of the RT-LAMP assay was higher than that of the conventional RT-nPCR assay. The RT-LAMP assay reported here may be used for diagnosis of swine HEV, not only in laboratories but also under field conditions. PMID:22855125

Zhang, Liang-Quan; Zhao, Fu-Rong; Liu, Zhi-Gang; Kong, Wei-Li; Wang, Heng; Ouyang, Yun; Liang, Huan-Bin; Zhang, Chao-Yi; Qi, Hai-Tao; Huang, Chang-Li; Guo, Si-Hu; Zhang, Gui-Hong

2012-08-02

241

One-step detection of Bean pod mottle virus in soybean seeds by the reverse-transcription loop-mediated isothermal amplification  

PubMed Central

Background Bean pod mottle virus (BPMV) is a wide-spread and destructive virus that causes huge economic losses in many countries every year. A sensitive, reliable and specific method for rapid surveillance is urgently needed to prevent further spread of BPMV. Methods A degenerate reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer set was designed on the conserved region of BPMV CP gene. The reaction conditions of RT-LAMP were optimized and the feasibility, specificity and sensitivity of this method to detect BPMV were evaluated using the crude RNA rapidly extracted from soybean seeds. Results The optimized RT-LAMP parameters including 6?mM MgCl2, 0.8?M betaine and temperature at 62.5-65°C could successfully amplify the ladder-like bands from BPMV infected soybean seeds. The amplification was very specific to BPMV that no cross-reaction was observed with other soybean viruses. Inclusion of a fluorescent dye makes it easily be detected in-tube by naked eye. The sensitivity of RT-LAMP assay is higher than the conventional RT-PCR under the conditions tested, and the conventional RT-PCR couldn’t be used for detection of BPMV using crude RNA extract from soybean seeds. Conclusion A highly efficient and practical method was developed for the detection of BPMV in soybean seeds by the combination of rapid RNA extraction and RT-LAMP. This RT-LAMP method has great potential for rapid BPMV surveillance and will assist in preventing further spread of this devastating virus.

2012-01-01

242

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

PubMed

A one-step, real-time reverse-transcription loop-mediated isothermal amplification (rRT-LAMP) method targeting the 5' end of the capsid gene for rapid and quantitative detection of human astrovirus serotype 1 (HAstV 1) was developed. The assay is highly sensitive and comparable to real-time RT-PCR (rRT-PCR), with a detection limit of ?100 RNA copies per assay. The specificity of the method was validated by the absence of any cross-reaction with RNA samples of HAstV 2-8 and other gastroenteritis viruses, followed by nucleotide sequencing of the amplified product. Fecal specimens (n=120) obtained from children under five years of age with gastroenteritis were tested by rRT-LAMP, rRT-PCR and transmission electron microscopy (TEM). Six (5%) of these samples were determined to be positive by both rRT-LAMP and rRT-PCR assay, and these two nucleic acid amplification methods resulted in a 200% increase in detection rates for HAstV infection compared with TEM alone. Furthermore, the rRT-LAMP assay is much more rapid than rRT-PCR and generates results in less than 20min for positive samples. The quantitation of viral load in stool specimens was determined from the standard curve plot of time-of-positivity versus initial RNA concentration. Most viral loads were determined to be within the range of 10(5)-10(8) copies. The results highlight the significance of the rapid rRT-LAMP method as a diagnostic and routine screening tool for the analysis of stool samples in hospital laboratories. PMID:23274752

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

2012-12-26

243

Highly sensitive detection of microRNAs based on isothermal exponential amplification-assisted generation of catalytic G-quadruplex DNAzyme.  

PubMed

It is well-known that microRNAs (miRNAs) have become an ideal class of biomarker candidates for clinical diagnosis of cancers, thus sensitive and selective detection of microRNAs is of great significance in understanding biological functions of miRNAs, early-phase diagnosis of cancers, as well as discovery of new targets for drugs. In this work, we have developed a sensitive method for microRNAs detection based on isothermal exponential amplification-assisted generation of catalytic G-quadruplex DNAzyme, and demonstrated its practical application in biological sample of cell lysate. The assay involves a combination of polymerase strand extension, single-strand nicking and catalytic reaction of G-quadruplex/hemin complex. It is designed such that, the target miRNA initiates the efficient synthesis of two kinds of short oligonucleotide fragments in the continuous cycle of the polymerization, nicking and displacement reactions, by means of thermostable polymerase and nicking endonuclease. One fragment has the same sequence as the target miRNA, except that the deoxyribonucleotides and thymine replace the ribonucleotides and uridine in the miRNA, to activate new cyclic chain reactions of polymerization, nicking and displacement reactions as the target miRNA. The other is the signal molecule of horseradish peroxidase (HRP)-mimicking G-quadruplex DNAzyme. With such designed signal amplification processes, the proposed assay showed a quantitative analysis of sequence-specific miRNAs in a wide range from 1 fM to 100 nM with a low detection limit of 1 fM. Moreover, this assay demonstrated excellent differentiation ability for the mismatch miRNAs targets and good performance in biological samples. PMID:23202342

Wang, Xin-Ping; Yin, Bin-Cheng; Wang, Ping; Ye, Bang-Ce

2012-11-07

244

Detection of Haemophilus parasuis isolates from South China by loop-mediated isothermal amplification and isolate characterisation.  

PubMed

Haemophilus parasuis is the etiological agent of Glässer's disease, which is characterised by ?brinous polyserositis, meningitis and polyarthritis, causing severe economic losses to the swine industry. In this study, a loop-mediated isothermal ampli?cation (LAMP) test was developed to improve the speci?city, facility and speed of diagnosis of H. parasuis isolates. The LAMP assay rapidly ampli?ed the target gene within 50 min incubation at 63 °C in a laboratory water bath. The LAMP amplicon could be visualised directly in the reaction tubes following the addition of SYBR Green I dye. The detection limit of this LAMP method was 10 CFU/mL, which was 10 times more sensitive than the earlier 16S rRNA polymerase chain reaction (PCR) test conducted by Oliveira, Galina and Pijoan (2001), and no cross-reactivity was observed from other non-H. parasuis strains. This LAMP test was evaluated further on 187 clinical specimens from pigs suspected of being infected with H. parasuis. Forty-three were found positive by bacterial isolation of H. parasuis, as well as by the 16S rRNA PCR and LAMP tests. The 43 H. parasuis isolates were classi?ed into 9 serovars and had 37 genetic patterns when analysed by pulsed-?eld gel electrophoresis (PFGE). This displayed that various H. parasuis serovars and genotypes were widely distributed in South China. Therefore, the speed, speci?city and sensitivity of the LAMP test, the lack of a need for expensive equipment, and the visual readout showed great potential for a correct clinical diagnosis of H. parasuis in favour of controlling Glässer's disease. PMID:23327310

Zhang, Jian-Min; Shen, Hai-Yan; Liao, Ming; Ren, Tao; Guo, Li-Li; Xu, Cheng-Gang; Feng, Sai-Xiang; Fan, Hui-Ying; Li, Jing-Yi; Chen, Ji-Dang; Zhang, Bin

2012-04-24

245

Development and evaluation of loop-mediated isothermal amplification (LAMP) for rapid detection of Clonorchis sinensis from its first intermediate hosts, freshwater snails.  

PubMed

Clonorchiasis, caused by Clonorchis sinensis, is a key foodborne zoonosis, which is mainly found in China, Korea and Vietnam. Detection of this parasite from the second intermediate host, the freshwater fish is the common method for epidemiological surveys of this parasite, but is time consuming, labour intensive and easily leads to misdiagnosis. In this study, we have developed a rapid, sensitive and reliable molecular method for the diagnosis of C. sinensis from its first intermediate hosts, freshwater snails, based on a loop-mediated isothermal amplification (LAMP) method. The specific amplified fragment from genomic DNA of C. sinensis did not cross-react with those from other relevant trematodes and a range of hosts (freshwater fish, shrimps and snails) of C. sinensis living in similar environments. The detection limit of the LAMP method was as low as 10 fg which was 1000 times more sensitive than conventional PCR, which was also demonstrated by successful application to field samples. These results show that the LAMP method is a more sensitive tool than conventional PCR for the detection of C. sinensis infection in the first intermediate hosts and, due to a simpler protocol, is an ideal molecular method for field-based epidemiological surveys of this parasite. PMID:23870065

Chen, Y; Wen, T; Lai, D-H; Wen, Y-Z; Wu, Z-D; Yang, T-B; Yu, X-B; Hide, G; Lun, Z-R

2013-07-22

246

Loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria annulata infection in China targeting the 18S rRNA and ITS sequences.  

PubMed

We have developed two loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria annulata, an economically important cattle disease in China that occurs in subtropical and tropical areas. These assays target the ribosomal RNA (18S rRNA) and ITS LAMP sequences. The primer set for each gene target consists of four primers, and each set recognizes six distinct regions on the target gene to allow for the highly specific detection of T. annulata. The specific ladder bands were amplified from the autologous genomic DNA of four Chinese-laboratory-preserved standard T. annulata stocks, and there were no cross-reactions with the genomic DNA of normal bovine blood and other protozoan species. The LAMP assays were sufficiently sensitive to detect 0.1 pg/?l of genomic DNA. Furthermore, DNA extracted from blood collected from cattle experimentally infected with T. annulata (18-105 days post-infection) was amplified, demonstrating the high sensitivity of these primers. Of the 351 field samples collected from China, 24.5% were positively detected by two LAMP primers, and 18.2% were found to be positive for T. annulata infection by PCR. These results indicate that the LAMP assay could be a potential diagnostic tool for epidemiological studies of T. annulata infection in China. PMID:22370125

Liu, Aihong; Guan, Guiquan; Du, Pengfei; Liu, Zhijie; Gou, Huitian; Liu, Junlong; Yang, Jifei; Li, Youquan; Ma, Milin; Niu, Qinli; Ren, Qiaoyun; Bai, Qi; Yin, Hong; Luo, Jianxun

2012-02-19

247

An Optimized Protocol for DNA Extraction from Wheat Seeds and Loop-Mediated Isothermal Amplification (LAMP) to Detect Fusarium graminearum Contamination of Wheat Grain.  

PubMed

A simple, rapid, and efficient method for isolating genomic DNA from germinated seeds of wheat that is free from polysaccharides and polyphenols is reported. DNA was extracted, treated with RNase, measured and tested for completeness using agarose gel electrophoresis. DNA purification from wheat grains yielded abundant, amplifiable DNA with yields typically between 100 and 200 ng DNA/mg. The effectiveness and reliability of the method was tested by assessing quantity and quality of the isolated DNA using three PCR-based markers. Inter-simple sequence repeats (ISSRs) were used to assess the genetic diversity between different wheat varieties. Specific PCR primer pair Tox5-1/Tox5-2 and a loop-mediated isothermal amplification (LAMP) procedure were used to detect genomic DNA of Fusarium graminearum in contaminated wheat seeds. In this method there is no need to use liquid nitrogen for crushing germinated seedlings. The protocol takes approximately one hour to prepare high quality DNA. In combination with the LAMP assay it is a fast and cost-effective alternative to traditional diagnostic methods for the early detection of toxigenic fusaria in cereals. PMID:21747688

Abd-Elsalam, Kamel; Bahkali, Ali; Moslem, Mohamed; Amin, Osama E; Niessen, Ludwig

2011-06-01

248

Development and evaluation of a loop-mediated isothermal amplification method in conjunction with an enzyme-linked immunosorbent assay for specific detection of Salmonella serogroup D.  

PubMed

Loop-mediated isothermal amplification in conjunction with enzyme-linked immunosorbent assay (LAMP-ELISA) provides a sensitive, specific and cost-effective method for detection of etiological causes of infections. The present study developed a reliable LAMP-ELISA diagnostic kit for identification of Salmonella serogroup D strains and evaluated its potential use in the detection of Salmonella serovars Enteritidis and Typhi. The LAMP-ELISA assay used a serogroup D/A-specific primer set to amplify a region of the prt gene, followed by hybridization of the digoxigenin-labeled products to a highly specific oligonucleotide probe for exact identification of serogroup D serovars. Among the bacteria tested, a positive reaction was only observed for strains belong to Salmonella serogroup D. The detection limit of the LAMP-ELISA assay was 4 CFU per tube, which was lower than PCR-ELISA assay with the same target gene (50 CFU per tube). Finally, the technique was successfully applied to an artificially contaminated meat sample with a detection limit 10(3) CFU mL(-1), which was 10 times more sensitive than PCR-ELISA. Overall, the LAMP-ELISA assay could be used as a sensitive alternative method to PCR-ELISA for the specific detection of Salmonella serogroup D serovars in routine food microbiology or clinical laboratories worldwide. PMID:22704377

Ravan, Hadi; Yazdanparast, Razieh

2012-05-02

249

Loop-mediated isothermal amplification for the rapid, sensitive, and specific detection of the O9 group of Salmonella in chickens.  

PubMed

In the present study, the loop-mediated isothermal amplification (LAMP) assay was developed to amplify the fragments of the O9 Salmonella-specific insertion element and evaluated in the laboratory for its potential use in a field situation, such as poultry farms. Among the bacteria tested, a positive reaction was observed only for 128 strains of 6 serovars of the O9 group Salmonella, such as Enteritidis (SE) and Pullorum. The detection limit of the LAMP assay was 10(3)CFU/ml, which was more sensitive than that of the polymerase chain reaction (PCR) assay with the same target gene (10(6)CFU/ml). The final results were obtained within 30 min for the LAMP assay, while the PCR assay needed a total of 120 min. When the LAMP assay was applied to the enrichment broth mixed with cecal dropping samples either spiked with SE in vitro or excreted by SE-inoculated hens, the results were comparable to those of the conventional plating method including 2 separate enrichments. In conclusion, the LAMP assay developed in the present study is an effective method for the specific detection of the O9 group Salmonella serovars, including SE. PMID:18538511

Okamura, Masashi; Ohba, Yousuke; Kikuchi, Shuichi; Suzuki, Akiko; Tachizaki, Hajime; Takehara, Kazuaki; Ikedo, Masanari; Kojima, Tadashi; Nakamura, Masayuki

2008-04-30

250

Development and clinical verification of a loop-mediated isothermal amplification method for detection of Salmonella species in suspect infected ducks.  

PubMed

To detect Salmonella spp. in suspect infected ducks, a loop-mediated isothermal amplification (LAMP) assay was developed. With the help of this assay, we can detect Salmonella enterica serovar Enteriditis and Salmonella enterica serovar Anatis above 6.0 cfu/test and 4.8 cfu/test, respectively, in pure-culture conditions, even in the existence of 0.01 g of duck liver or spleen homogenates; the detection thresholds were still achieved at 6.0 cfu per test tube. Further experiments of the test strains indicated the high specificity of this LAMP assay. In the detection of clinical specimens, a total of 115 Salmonella suspect infected clinical samples was analyzed by traditional culture method, LAMP, and PCR. The results suggested that 11 samples were positive by LAMP and culture methods; however, only 8 specimens were positive by PCR. In consideration of the high sensitivity and fast scanning speed of our LAMP method in clinical specimens, it was more suitable for field application and epidemiological investigation. PMID:22399738

Tang, T; Cheng, A; Wang, M; Li, X; He, Q; Jia, R; Zhu, D; Chen, X

2012-04-01

251

Development of a new loop-mediated isothermal amplification assay for prt (rfbS) gene to improve the identification of Salmonella serogroup D.  

PubMed

Loop-mediated isothermal amplification (LAMP) is a promising nucleic acid assay for rapid and cost-effective detection of pathogen-specific sequences within a sample. Development of an appropriate taxonomic group-specific LAMP assay highly relies on the design of proper primers to cover all major members of the taxon. Regarding this fact, we designed and evaluated a new LAMP primer set specific to prt (rfbS) gene for rapid identification of Salmonella serogroup D serotypes. Unlike the previously reported LAMP assay for serogroup D which detects solely the non-typhoidal serotypes; the new LAMP primers set detects both typhoidal and non-typhoidal serotypes of this serogroup with a detection limit of 10 CFU/rection. Furthermore, the technique was successfully applied to artificially contaminated meat samples with an inoculation level of 1-5 CFU/250 ml of Salmonella Enteritidis, following a 5-h pre-enrichment step in tryptic soy broth. Overall, the new LAMP assay and its optimized setup would be useful for fast diagnosis of food poisoning incidents caused by these bacteria. PMID:22806032

Ravan, Hadi; Yazdanparast, Razieh

2012-02-09

252

A loop-mediated isothermal amplification method targets the phoP gene for the detection of Salmonella in food samples.  

PubMed

A loop-mediated isothermal amplification (LAMP) assay was developed and validated for the specific detection of Salmonella in food. The assay used specifically designed primers to target within the phoP gene and correctly identified all 66 strains of Salmonella and 73 non-Salmonella strains tested. The phoP gene regulates the expression of genes involved in virulence and the survival of Salmonella from destruction by macrophage. The probability of detection was 100% when a Salmonella cell suspension containing 10(1) CFU/ml was used as a template in the LAMP assay. Prior to the LAMP assay, a sample preparation protocol was applied that included a pre-enrichment step in buffered peptone water, followed by extraction and purification of DNA. In this way, 85 various food samples were investigated for Salmonella including minced meat of pig and raw milk. The diagnostic accuracy was shown to be 100% when compared to the traditional culture method. This combination of sample enrichment, and LAMP assay can detect 35 CFU per 250 ml of prepared food samples. The overall analysis time for the LAMP assay method was approximately 24 h. This is in contrast to 5 to 7 days of analysis time required for the traditional culture method. Consequently, the LAMP described here has the potential to become a standardized method for the rapid detection of Salmonella in diagnostic laboratories once further validated by inter-laboratory studies. PMID:19540609

Li, Xuefei; Zhang, Shu; Zhang, Hongwei; Zhang, Lihuai; Tao, Haitao; Yu, Jia; Zheng, Wenjie; Liu, Chenghu; Lü, Dan; Xiang, Rong; Liu, Yin

2009-06-01

253

Loop-mediated isothermal amplification (LAMP) method based on two species-specific primer sets for the rapid identification of Chinese Babesia bovis and B. bigemina.  

PubMed

Bovine babesiosis is a tick-transmitted hemoprotozoan disease that is mainly caused by Babesia bovis and/or Babesia bigemina and is characterized by significant morbidity and mortality worldwide. This disease is widespread in most parts of China. However, it is difficult to rapidly discriminate between the B. bovis and B. bigemina species. To detect and distinguish these species, a loop-mediated isothermal amplification (LAMP) platform that targets specific sequences of the internal transcribed spacer (ITS) genes was developed. Specificity testing revealed that there was no cross-reaction with the other tick-borne parasites B. ovate, B. major, unnamed bovine Babesia, Theileria annulata, Theileria sinensis, Theileria sergenti, and Anaplasma marginale, or with bovine white blood cells. The sensitivity of the LAMP method was 0.1 pg DNA for both B. bovis and B. bigemina, which was superior to that of the classical PCR methods. This assay was evaluated for its diagnostic utility using blood samples collected from experimentally and naturally infected cattle in China. These findings indicate that the Babesia species-specific LAMP assay may have potential clinical application in the detection and differentiation of Babesia species, particularly in countries in which babesiosis is endemic. PMID:22849831

Liu, Aihong; Guan, Guiquan; Du, Pengfei; Gou, Huitian; Liu, Zhijie; Liu, Junlong; Ma, Milin; Yang, Jifei; Li, Youquan; Niu, Qinli; Ren, Qiaoyun; Bai, Qi; Yin, Hong; Luo, Jianxun

2012-07-28

254

Detection of Mycobacterium tuberculosis using a capillary-array microsystem with integrated DNA extraction, loop-mediated isothermal amplification, and fluorescence detection.  

PubMed

This article reports for the first time a high-throughput microfluidic system with fully integrated loop-mediated isothermal amplification (LAMP) analysis. With the developed system, parallel Mycobacterium tuberculosis detections were implemented in polytetrafluoroethylene capillaries through the utilization of droplet technology coupled with magnetic beads. During the analysis, liquid plugs containing different types of sample or reagents are sequentially introduced into the capillaries and made to form droplets therein. The whole analytical process, including DNA extraction, LAMP, and detection of the amplified products were conducted in such droplets. The developed microsystem is able to process 10 samples in parallel. The entire diagnostic procedure, from sample-in to answer-out, can be automatically completed within 50 min with a limit of detection (LOD) of 10 bacteria. This microsystem was evaluated by analyzing clinical samples, and a clinical sensitivity (positive detection rate) of 96.8% and specificity (negative detection rate) of 100% were achieved. The presented capillary LAMP assay features high-throughput and low-cost and thus is a promising tool for rapid tuberculosis diagnosis. PMID:23521496

Liu, Dayu; Liang, Guangtie; Zhang, Qiong; Chen, Bin

2013-04-10

255

Development and evaluation of an IS711-based loop mediated isothermal amplification method (LAMP) for detection of Brucella spp. on clinical samples.  

PubMed

DNA-based methods have emerged as an additional tool for Brucella infection-confirmation at a herd level. However, their implementation may require the use of specialized equipment. In this context the recently developed loop-mediated isothermal amplification (LAMP) technique may constitute an additional and cost-effective tool for rapid and specific DNA detection, especially in low income areas. In the present study the usefulness of a newly developed LAMP assay aiming at the multicopy-IS711 sequence was assessed on a variety of clinical samples (n=81 from abortions and ewes; cattle, n=3; swine, n=4) that were analyzed in parallel using real-time PCR and bacteriology. Although overall sensitivities obtained with the three methods were comparable (p>0.05), our results highlighted the complementarity between bacteriology and molecular-based methods for increased sensitivity. Significant differences (p<0.05) were observed with all techniques depending on the nature of the sample. Our results demonstrate the potential of the IS711-LAMP technique for direct Brucella detection. PMID:23714043

Pérez-Sancho, M; García-Seco, T; Arrogante, L; García, N; Martínez, I; Diez-Guerrier, A; Perales, A; Goyache, J; Domínguez, L; Alvarez, J

2013-05-25

256

Evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Vibrio parahaemolyticus in naturally contaminated seafood samples.  

PubMed

We investigated the efficacy of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of seafood samples naturally contaminated with Vibrio parahaemolyticus. A total of 171 seafood samples enriched in alkaline peptone water (APW) were assessed by LAMP assay and conventional culture methods, which consist of a combination of APW enrichment culture and plating onto CHROMagar Vibrio and TCBS agars. Compared with V. parahaemolyticus isolation using the conventional culture test, LAMP results showed 100% (30/30) and 90.8% (128/141) sensitivity and specificity, respectively. The conventional culture test required more than 3 days to isolate and identify V. parahaemolyticus in the APW enrichment culture. In contrast, the LAMP assay was markedly faster, requiring less than 60 min from the beginning of DNA extraction to final detection of V. parahaemolyticus. In total, the LAMP assay required 17-19 h from the beginning of enrichment culture to final determination. This is the first report of the LAMP assay for rapid screening of seafood samples naturally contaminated by V. parahaemolyticus. PMID:21645826

Yamazaki, Wataru; Kumeda, Yuko; Uemura, Ryoko; Misawa, Naoaki

2011-05-05

257

Development and evaluation of a loop-mediated isothermal amplification assay for the rapid detection of porcine cytomegalovirus under field conditions  

PubMed Central

Background Porcine cytomegalovirus (PCMV) induces silent infection in adult pigs but more frequently causes fatal, generalized infection in newborn piglets. This study aimed to develop a new loop-mediated isothermal amplification (LAMP) method for the sensitive, rapid, and inexpensive detection of PCMV under field conditions. Methods Tissue obtained from nine-week-old PCMV-free Landrace pigs or pig samples from postmortem examinations were analyzed. The samples were found to have clinical signs and lesions consistent with inclusion body rhinitis. Six specific primers were designed by targeting the PCMV DNA polymerase (DPOL) DNA. The LAMP reaction was optimized in a water bath. The sensitivity and specificity of LAMP and polymerase chain reaction (PCR) were compared. Results PCMV DNA was amplified at 65°C, and the result could be detected as early as 30 min into the reaction. Positive reactions could be visualized by the naked eye as a color change brought on by the addition of SYBR Green. The sensitivity and specificity of LAMP were found to be similar to those of the PCR. Conclusions LAMP is a high-throughput technique for the detection of PCMV and has a high specificity, sensitivity and simplicity; these factors make it suitable for detection of PCMV under field conditions.

2012-01-01

258

Pepino mosaic virus genotype shift in North America and development of a loop-mediated isothermal amplification for rapid genotype identification  

PubMed Central

Background Pepino mosaic, once an emerging disease a decade ago, has become endemic on greenhouse tomatoes worldwide in recent years. Three distinct genotypes of Pepino mosaic virus (PepMV), including EU, US1 and CH2 have been recognized. Our earlier study conducted in 2006–2007 demonstrated a predominant EU genotype in Canada and United States. The objective of the present study was to monitor the dynamic of PepMV genetic composition and its current status in North America. Results Through yearly monitoring efforts in 2009–2012, we detected a dramatic shift in the prevalent genotype of PepMV from the genotype EU to CH2 in North America since early 2010, with another shift from CH2 to US1 occurring in Mexico only two years later. Through genetic diversity analysis using the coat protein gene, such genotype shifting of PepMV in North America was linked to the positive identification of similar sequence variants in two different commercial tomato seed sources used for scion and rootstock, respectively. To allow for a quick identification, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) system was developed and demonstrated to achieve a rapid identification for each of the three genotypes of PepMV, EU, US1 and CH2. Conclusion Through systemic yearly monitoring and genetic diversity analysis, we identified a linkage between the field epidemic isolates and those from commercial tomato seed lots as the likely sources of initial PepMV inoculum that resulted in genetic shifting as observed on greenhouse tomatoes in North America. Application of the genotype-specific RT-LAMP system would allow growers to efficiently determine the genetic diversity on their crops.

2013-01-01

259

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 Central

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.

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

2009-01-01

260

Detection of Panton-Valentine Leukocidin DNA from methicillin-resistant Staphylococcus aureus by resistive pulse sensing and loop-mediated isothermal amplification with gold nanoparticles.  

PubMed

This report describes a novel diagnostic assay for rapid detection of the Panton-Valentine Leukocidin (PVL) toxin of methicillin-resistant Staphylococcus aureus (MRSA) utilizing resistive pulse sensing (RPS), loop-mediated isothermal DNA amplification (LAMP) in combination with gold nanoparticles (AuNPs). The PVL DNA from MRSA was specifically amplified by LAMP using four primers at one temperature (65 °C). The DNA products with biotin were then conjugated to a first AuNP1 (55±2 nm) through biotin-avidin binding. A second AuNP2 (30±1.5 nm) coated with a specific DNA probe hybridized with the LAMP DNA products at the loop region to enhance assay sensitivity and specificity, to generate supra-AuNP1-DNA-AuNP2 assemblies. Scanning electron microscopy confirmed the presence of these supra-assemblies. Using RPS, detection and quantitation of the agglomerated AuNPs were performed by a tunable fluidic nanopore sensor. The results demonstrate that the LAMP-based RPS sensor is sensitive and rapid for detecting the PVL DNA. This technique could achieve a limit of detection (LOD) up to about 500 copies of genomic DNA from the bacteria MRSA MW2 and the detection can be completed within two hours with a straightforward signal-to-readout setup. It is anticipated that this LAMP-based AuNP RPS may become an effective tool for MRSA detection and a potential platform in clinical laboratory to report the presence or absence of other types of infectious agents. PMID:23708283

Yang, Alice Kar Lai; Lu, Haifei; Wu, Shu Yuen; Kwok, Ho Chin; Ho, Ho Pui; Yu, Samuel; Cheung, Anthony Ka Lun; Kong, Siu Kai

2013-04-19

261

Comparison of loop-mediated isothermal amplification and PCR for the detection and differentiation of Marek's disease virus serotypes 1, 2, and 3.  

PubMed

The previously conducted study on loop-mediated isothermal amplification (LAMP) has shown its usefulness for the detection of Marek's disease virus (MDV) virulent field strains. The current study improves the previously designed LAMP method with an additional pair of loop primers, which accelerates the reaction, and describes two other LAMP procedures for the specific detection of FC126 strain of turkey herpesvirus and nonpathogenic SB-1 strain. The developed LAMP procedures were also confirmed and compared with PCR. Each LAMP reaction used three pairs of specific primers designed to target the nucleotide sequence of the very virulent MDV strain, the SB-1 strain of MDV-2, and turkey herpesvirus, respectively. All LAMP reactions were flexible and provided reliable results at a wide range of incubation temperatures from 54.0 to 62.3 C in 15 to 90 min. LAMP does not need any thermocyclers, because all assays were conducted in a water bath. The green fluorescence signal was recorded under ultraviolet illumination in LAMP samples containing virulent MDV and turkey herpesvirus where SYBR Green was added to the reaction mixture, whereas the SB-1-positive samples presented orange illumination after GelRed staining solution. The sensitivity of the three LAMP reactions ranged from 2 log10 plaque-forming units (PFU)/ml of the virulent MDV HPRS-16 strain and turkey herpesvirus (HVT) to 3 log10 PFU/ml of the SB-1 nonpathogenic strain. The sensitivity of the compared PCR was lower by 1-2 log10 PFU/ml. The conducted studies have shown that developed LAMP methods may be used instead of PCR for the detection and differentiation of virulent and nonpathogenic MDV strains used in prophylaxis against MD. LAMP may be conducted without access to thermocyclers. PMID:23901773

Wo?niakowski, Grzegorz; Samorek-Salamonowicz, Elzbieta; Kozdru?, Wojciech

2013-06-01

262

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

PubMed

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; Li, Bingjun; Wang, Changjun

2013-09-04

263

Application of loop-mediated isothermal amplification for malaria diagnosis during a follow-up study in S?o Tom?  

PubMed Central

Background A reliable and simple test for the detection of malaria parasite is crucial in providing effective treatment and therapeutic follow-up, especially in malaria elimination programmes. A comparison of four methods, including nested polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) were used for the malaria diagnosis and treatment follow-up in São Tomé and Príncipe, during a successful pre-elimination campaign. Method During the period September to November 2009, blood samples from 128 children (five to 14 years old) with temperature ?38°C (tympanic) in the District of Agua Grande were examined using four different methods, i.e., histidine-rich protein 2 (HRP-2) based rapid diagnostic tests (HRP-2-RDTs), optical microscopy, nested PCR, and LAMP. First-line treatment with artesunate-amodiaquine was given for uncomplicated malaria and intravenous quinine was given for complicated malaria. Children with persistent positivity for malaria by microscopy, or either by nested PCR, or by LAMP on day 7 were given second-line treatment with artemether-lumefantrine. Treatment follow-up was made weekly, for up to four weeks. Results On day 0, positive results for HRP-2-RDTs, microscopy, nested PCR, and LAMP, were 68(53%), 47(37%), 64(50%), and 65(51%), respectively. When nested PCR was used as a reference standard, only LAMP was comparable; both HRP-2-RDTs and microscopy had moderate sensitivity; HRP-2-RDTs had poor positive predictive value (PPV) and a moderate negative predictive value (NPV) for the treatment follow-up. Seventy-one children with uncomplicated malaria and eight children with complicated falciparum malaria were diagnosed based on at least one positive result from the four tests as well as clinical criteria. Twelve of the 79 children receiving first-line treatment had positive results by nested PCR on day 7 (nested PCR-corrected day 7 cure rate was 85%). After the second-line treatment, nested PCR/LAMP-corrected day 28 cure rate was 83% for these 12 children. Conclusions HRP-2-RDTs have similar sensitivity as microscopy but less specificity. However, as compared to nested PCR, the poor sensitivity of HRP-2-RDTs indicates that low parasitaemia may not be detected after treatment, as well as the low specificity of HRP-2-RDTs indicates it cannot be applied for treatment follow-up. LAMP has similar sensitivity and specificity to nested PCR. With high PPV and NPV, LAMP is simpler and faster as compared to nested PCR with the advantage of detecting low parasitaemia becoming a potential point-of-care test for treatment follow-up.

2012-01-01

264

Efficient Identification of Clinically Relevant Candida Yeast Species by Use of an Assay Combining Panfungal Loop-Mediated Isothermal DNA Amplification with Hybridization to Species-Specific Oligonucleotide Probes?  

PubMed Central

The occurrence of invasive mycoses has progressively increased in recent years. Yeasts of the genus Candida remain the leading etiologic agents of those infections. Early identification of opportunistic yeasts may contribute significantly to improved disease management and the selection of appropriate antifungal therapy. We developed a rapid and reliable molecular identification system for clinically relevant yeasts that makes use of nonspecific primers to amplify a region of the 26S rRNA gene, followed by reverse hybridization of the digoxigenin-labeled products to a panel of species-specific oligonucleotide probes arranged on a nylon membrane macroarray format. DNA amplification was achieved by the recently developed loop-mediated isothermal DNA amplification technology, a promising option for the development of improved laboratory diagnostic kits. The newly developed method was successful in distinguishing among the major clinically relevant yeasts associated with bloodstream infections by using simple, rapid, and cost-effective procedures and equipment.

Inacio, Joao; Flores, Orfeu; Spencer-Martins, Isabel

2008-01-01

265

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

2013-03-27

266

Development of a Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Detection of Pandemic (H1N1) 2009 Virus as a Novel Molecular Method for Diagnosis of Pandemic Influenza in Resource-Limited Settings?  

PubMed Central

This paper reports on the development of a one-step, real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the hemagglutinin (HA) gene for the rapid molecular-based detection of pandemic (H1N1) 2009 virus. The detection limit of the pandemic (H1N1) 2009 virus HA-specific RT-LAMP assay was same as that of the currently used real-time reverse transcription-PCR method. The assay detected the pandemic (H1N1) 2009 virus HA gene in 136 RNA samples extracted from nasopharyngeal swab specimens from Japanese and Vietnamese patients. No cross-reactive amplification with the RNA of other seasonal influenza viruses was observed, and the detection of specific viral genome targets in clinical specimens was achieved in less than 40 min. The sensitivity and specificity of the pandemic (H1N1) 2009 virus HA-specific RT-LAMP assay obtained in this study were 97.8% and 100%, respectively. Use of the (H1N1) 2009 virus HA-specific RT-LAMP assay will enable the faster and easier diagnosis of pandemic (H1N1) 2009 virus infection, especially in resource-limited situations in developing countries.

Kubo, Toru; Agoh, Masanobu; Mai, Le Q.; Fukushima, Kiyoyasu; Nishimura, Hidekazu; Yamaguchi, Akinori; Hirano, Manabu; Yoshikawa, Akira; Hasebe, Futoshi; Kohno, Shigeru; Morita, Kouichi

2010-01-01

267

Comparative evaluation of conventional polymerase chain reaction (PCR), with loop-mediated isothermal amplification and SYBR green I-based real-time PCR for the quantitation of porcine circovirus-1 DNA in contaminated samples destined for vaccine production.  

PubMed

Porcine circovirus type1 (PCV1), described initially as a contaminant of a porcine kidney cell line, is ubiquitous within the swine population The presence of PCV1 in porcine cell lines can lead to contamination during both human and porcine vaccine production. Therefore, a rapid, specific, sensitive and practical method is needed for the detection of PCV1 in bio-products. The aim of this study was to compare three assays in their ability to accurately quantify PCV1 virus in biological samples, namely loop-mediated isothermal amplification (LAMP), SYBR green I-based real-time polymerase chain reaction (PCR) and conventional PCR. All assays yielded successful quantitation of PCV1 DNA and differentiated between PCV1-free and-contaminated cells. In addition, the results were specific for PCV1, since amplification of samples containing closely-related PCV2 or other pathogenic swine viruses yielded negative results. The lowest detection threshold of 10(2) copies was displayed by the SYBR green I-based real-time PCR assay. In addition, this assay was the most effective in detecting PCV1 contamination in a set of commercially available porcine vaccines. Therefore we conclude that SYBR green I-based real-time PCR is specific and sensitive for detecting PCV1 in biological samples and maybe used for quality control of vaccine and biomaterial production. PMID:23538038

Yang, Bo-Chao; Wang, Feng-Xue; Zhang, Shu-Qin; Song, Ni; Li, Jian-Xi; Yang, Zhi-Qiang; Wen, Yong-Jun; Wu, Hua

2013-03-26

268

Comparison of real-time PCR, reverse transcriptase real-time PCR, loop-mediated isothermal amplification, and the FDA conventional microbiological method for the detection of Salmonella spp. in produce.  

PubMed

Contamination of foods, especially produce, with Salmonella spp. is a major concern for public health. Several methods are available for the detection of Salmonella in produce, but their relative efficiency for detecting Salmonella in commonly consumed vegetables, often associated with outbreaks of food poisoning, needs to be confirmed. In this study, the effectiveness of three molecular methods for detection of Salmonella in six produce matrices was evaluated and compared to the FDA microbiological detection method. Samples of cilantro (coriander leaves), lettuce, parsley, spinach, tomato, and jalapeno pepper were inoculated with Salmonella serovars at two different levels (10(5) and <10(1) CFU/25 g of produce). The inoculated produce was assayed by the FDA Salmonella culture method (Bacteriological Analytical Manual) and by three molecular methods: quantitative real-time PCR (qPCR), quantitative reverse transcriptase real-time PCR (RT-qPCR), and loop-mediated isothermal amplification (LAMP). Comparable results were obtained by these four methods, which all detected as little as 2 CFU of Salmonella cells/25 g of produce. All control samples (not inoculated) were negative by the four methods. RT-qPCR detects only live Salmonella cells, obviating the danger of false-positive results from nonviable cells. False negatives (inhibition of either qPCR or RT-qPCR) were avoided by the use of either a DNA or an RNA amplification internal control (IAC). Compared to the conventional culture method, the qPCR, RT-qPCR, and LAMP assays allowed faster and equally accurate detection of Salmonella spp. in six high-risk produce commodities. PMID:21803916

Zhang, Guodong; Brown, Eric W; González-Escalona, Narjol

2011-07-29

269

Comparison of Real-Time PCR, Reverse Transcriptase Real-Time PCR, Loop-Mediated Isothermal Amplification, and the FDA Conventional Microbiological Method for the Detection of Salmonella spp. in Produce ? †  

PubMed Central

Contamination of foods, especially produce, with Salmonella spp. is a major concern for public health. Several methods are available for the detection of Salmonella in produce, but their relative efficiency for detecting Salmonella in commonly consumed vegetables, often associated with outbreaks of food poisoning, needs to be confirmed. In this study, the effectiveness of three molecular methods for detection of Salmonella in six produce matrices was evaluated and compared to the FDA microbiological detection method. Samples of cilantro (coriander leaves), lettuce, parsley, spinach, tomato, and jalapeno pepper were inoculated with Salmonella serovars at two different levels (105 and <101 CFU/25 g of produce). The inoculated produce was assayed by the FDA Salmonella culture method (Bacteriological Analytical Manual) and by three molecular methods: quantitative real-time PCR (qPCR), quantitative reverse transcriptase real-time PCR (RT-qPCR), and loop-mediated isothermal amplification (LAMP). Comparable results were obtained by these four methods, which all detected as little as 2 CFU of Salmonella cells/25 g of produce. All control samples (not inoculated) were negative by the four methods. RT-qPCR detects only live Salmonella cells, obviating the danger of false-positive results from nonviable cells. False negatives (inhibition of either qPCR or RT-qPCR) were avoided by the use of either a DNA or an RNA amplification internal control (IAC). Compared to the conventional culture method, the qPCR, RT-qPCR, and LAMP assays allowed faster and equally accurate detection of Salmonella spp. in six high-risk produce commodities.

Zhang, Guodong; Brown, Eric W.; Gonzalez-Escalona, Narjol

2011-01-01

270

Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Leishmania infantum in canine leishmaniasis based on cysteine protease B genes.  

PubMed

We developed a Leishmania infantum specific LAMP assay that was carried out using a set of, six primers targeting the cysteine protease B multi copy gene of L. infantum. Our result shows that we, successfully detect the L. infantum DNA and that amplification is specific as no cross reaction was seen, with L. major, L. tropica, L. turanica, L. aethiopica, L. tarentolae, L. gerbilii, Trypanosoma cruzi or, human genomic DNA. When compared to conventional cpb based PCR, the sensitivity of LAMP assay, was higher with a detection limit of 50fg/?l of genomic L. infantum parasite DNA. Accurate and rapid, diagnosis of canine leishmaniasis (CanL) is an important issue that allows early treatment and, prevents transmission. Our developed LAMP assay was used to evaluate occurrences of Leishmania infantum in seventy five (75) dogs from the field. Blood samples were used to perform LAMP assay, classical PCR, IFAT and microscopy that was used as gold standard. The IFAT in addition to, microscopy, are the basic techniques used for CanL diagnosis at the School of Veterinary Medicine, where we obtained our samples. Compared to molecular methods, the serology (IFAT) test shows the, best sensitivity (88.57%) with, however, a much lower specificity (52.5%) due to a relatively high, number of false-positive results (22 animals). The PCR assay shows a low sensitivity (37.14%) and, specificity around (82.5%). Our LAMP assay shows a suitable sensitivity (54%) and a good specificity, (80%), with however, positive (70%) and negative (66%) predictive values. Furthermore, the best, positive likelihood ratio (LR+) was obtained by LAMP assay (2.7). This technique presents the highest, kappa value (with a fair agreement of 0.34). Moreover, the relative stability of the reagents indicates, that LAMP may be a good alternative to a conventional PCR, especially under field conditions. Finally in, a brief cost evaluation, the LAMP assay compares favorably with other molecular diagnostic tests. This, is the first study that evaluates the L. infantum specific LAMP alongside other diagnostics tools for, CanL. Our results indicate a suitable sensitivity and specificity for the developed LAMP assay that could, has usefulness application on dogs and human L. infantum diagnosis. PMID:23972768

Chaouch, Melek; Mhadhbi, Moez; Adams, Emily R; Schoone, Gerard J; Limam, Sassi; Gharbi, Zyneb; Darghouth, Mohamed Aziz; Guizani, Ikram; Benabderrazak, Souha

2013-08-03

271

Loop-mediated isothermal amplification assays for detection of Equid herpesvirus 1 and 4 and differentiating a gene-deleted candidate vaccine strain from wild-type Equid herpesvirus 1 strains.  

PubMed

Loop-mediated isothermal amplification (LAMP) is a novel method for the rapid and sensitive detection of DNA without the need for expensive equipment. In the present study, LAMP assays were developed for the specific detection of Equid herpesvirus 1 and 4 (EHV-1 and EHV-4, respectively) and for the differentiation of glycoprotein E (gE)-deleted EHV-1 (DeltagE) strain, a candidate strain for a live vaccine, from field EHV-1 strains. Specific primer sets were designed for the gC and gE genes of EHV-1 and for the gC gene of EHV-4. The analytical sensitivities of the LAMP assays were compared with those of polymerase chain reaction (PCR). The detection limits of LAMP for EHV-1 gC and gE and PCR for EHV-1 gC were 1 plaque-forming unit (PFU)/tube, and those of LAMP and PCR for EHV-4 gC were 0.1 PFU/tube. The DeltagE strain could be differentiated from wild-type EHV-1 strains based on the reactivity in the LAMP for EHV-1 gC in combination with the LAMP for EHV-1 gE. The analytical specificities of the LAMP for EHV-1 and EHV-4 were examined by using several equine pathogens, and no cross-reactions were observed. The LAMP detection abilities for EHV-1 and EHV-4 on nasal swab samples collected from experimentally infected horses were in good agreement with that of PCR for EHV-1 and EHV-4, respectively. The LAMP assays developed in the current study were sensitive and specific for EHV-1 and EHV-4, and should provide a valuable alternative to PCR for use in clinical laboratories in the field. PMID:20093679

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

2010-01-01

272

Rapid and sensitive detection of novel avian-origin influenza A (H7N9) virus by reverse transcription loop-mediated isothermal amplification combined with a lateral-flow device.  

PubMed

A severe disease in humans caused by a novel avian-origin influenza A (H7N9) virus emerged in China recently, which has caused at least 128 cases and 26 deaths. Rapid detection of the novel H7N9 virus is urgently needed to differentiate the disease from other infections, and to facilitate infection control as well as epidemiologic investigations. In this study, a reverse transcription loop-mediated isothermal amplification combined with a lateral flow device (RT-LAMP-LFD) assay to rapidly detect H7N9 virus was developed and evaluated. The RT-LAMP primers were designed to target the haemagglutinin (HA) and neuraminidase (NA) genes of H7N9 virus. Results of 10-fold dilution series assays showed that analysis of RT-LAMP products by the LFD method was as sensitive as real-time turbidity detection, and that the analytic sensitivities of the HA and NA RT-LAMP assays were both 10 copies of synthetic RNA. Furthermore, both the assays showed 100% clinical specificity for identification of H7N9 virus. The performance characteristics of the RT-LAMP-LFD assay were evaluated with 80 clinical specimens collected from suspected H7N9 patients. The NA RT-LAMP-LFD assay was more sensitive than real time RT-PCR assay. Compared with a combination of virus culture and real-time RT-PCR, the sensitivity, specificity, positive predictive value, and negative predictive value of the RT-LAMP-LFD assay were all 100%. Overall, The RT-LAMP-LFD assay established in this study can be used as a reliable method for early diagnosis of the avian-origin influenza A (H7N9) virus infection. PMID:23936359

Ge, Yiyue; Wu, Bin; Qi, Xian; Zhao, Kangchen; Guo, Xiling; Zhu, Yefei; Qi, Yuhua; Shi, Zhiyang; Zhou, Minghao; Wang, Hua; Cui, Lunbiao

2013-08-01

273

Rapid and Sensitive Detection of Novel Avian-Origin Influenza A (H7N9) Virus by Reverse Transcription Loop-Mediated Isothermal Amplification Combined with a Lateral-Flow Device  

PubMed Central

A severe disease in humans caused by a novel avian-origin influenza A (H7N9) virus emerged in China recently, which has caused at least 128 cases and 26 deaths. Rapid detection of the novel H7N9 virus is urgently needed to differentiate the disease from other infections, and to facilitate infection control as well as epidemiologic investigations. In this study, a reverse transcription loop-mediated isothermal amplification combined with a lateral flow device (RT-LAMP-LFD) assay to rapidly detect H7N9 virus was developed and evaluated. The RT-LAMP primers were designed to target the haemagglutinin (HA) and neuraminidase (NA) genes of H7N9 virus. Results of 10-fold dilution series assays showed that analysis of RT-LAMP products by the LFD method was as sensitive as real-time turbidity detection, and that the analytic sensitivities of the HA and NA RT-LAMP assays were both 10 copies of synthetic RNA. Furthermore, both the assays showed 100% clinical specificity for identification of H7N9 virus. The performance characteristics of the RT-LAMP-LFD assay were evaluated with 80 clinical specimens collected from suspected H7N9 patients. The NA RT-LAMP-LFD assay was more sensitive than real time RT-PCR assay. Compared with a combination of virus culture and real-time RT-PCR, the sensitivity, specificity, positive predictive value, and negative predictive value of the RT-LAMP-LFD assay were all 100%. Overall, The RT-LAMP-LFD assay established in this study can be used as a reliable method for early diagnosis of the avian-origin influenza A (H7N9) virus infection.

Qi, Xian; Zhao, Kangchen; Guo, Xiling; Zhu, Yefei; Qi, Yuhua; Shi, Zhiyang; Zhou, Minghao; Wang, Hua; Cui, Lunbiao

2013-01-01

274

Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) system for a highly sensitive detection of enterovirus in the stool samples of acute flaccid paralysis cases  

PubMed Central

Background In the global eradication program for poliomyelitis, the laboratory diagnosis plays a critical role by isolating poliovirus (PV) from the stool samples of acute flaccid paralysis (AFP) cases. In this study, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) system for a rapid and highly sensitive detection of enterovirus including PV to identify stool samples positive for enterovirus including PV. Methods A primer set was designed for RT-LAMP to detect enterovirus preferably those with PV-like 5'NTRs of the viral genome. The sensitivity of RT-LAMP system was evaluated with prototype strains of enterovirus. Detection of enterovirus from stool extracts was examined by using RT-LAMP system. Results We detected at least 400 copies of the viral genomes of PV(Sabin) strains within 90 min by RT-LAMP with the primer set. This RT-LAMP system showed a preference for Human enterovirus species C (HEV-C) strains including PV, but exhibited less sensitivity to the prototype strains of HEV-A and HEV-B (detection limits of 7,400 to 28,000 copies). Stool extracts, from which PV, HEV-C, or HEV-A was isolated in the cell culture system, were mostly positive by RT-LAMP method (positive rates of 15/16 (= 94%), 13/14 (= 93%), and 4/4 (= 100%), respectively). The positive rate of this RT-LAMP system for stool extracts from which HEV-B was isolated was lower than that of HEV-C (positive rate of 11/21 (= 52%)). In the stool samples, which were negative for enterovirus isolation by the cell culture system, we found that two samples were positive for RT-LAMP (positive rates of 2/38 (= 5.3%)). In these samples, enterovirus 96 was identified by sequence analysis utilizing a seminested PCR system. Conclusions RT-LAMP system developed in this study showed a high sensitivity comparable to that of the cell culture system for the detection of PV, HEV-A, and HEV-C, but less sensitivity to HEV-B. This RT-LAMP system would be useful for the direct detection of enterovirus from the stool extracts.

2009-01-01

275

Nucleic Acid Sequence-Based Amplification  

Microsoft Academic Search

\\u000a Nucleic acid sequence-based amplification (NASBA; bioMérieux, Boxtel, The Netherlands) is a commercially available amplification\\u000a procedure that uses RNA as the target. It makes use of the simultaneous enzymatic activities of avian myeloblastosis virus\\u000a reverse transcriptase (AMV-RT), RNase H, and T7 RNA polymerase, under isothermal conditions. The constant temperature maintained\\u000a throughout the amplification reaction allows each step of the reaction to

Katherine Loens; D. Ursi; H. Goossens; M. Ieven

276

General purpose adsorption isotherms  

Microsoft Academic Search

The fitting of adsorption isotherm equations to experimental data is often an important aspect of data analysis. If the Langmuir and Freundlich isotherms are used, then consideration must be given to the proper weighting of the observations. Preferably nonlinear regression (nonlinear least squares) should be used since this enables these isotherms to be fitted directly and also enables other isotherms

David G. Kinniburgh

1986-01-01

277

Noninstrumented 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

2008-03-01

278

Nucleic acid sequence-based amplification (NASBA) detection of medically important Candida species  

Microsoft Academic Search

Nucleic Acid Sequence Based Amplification (NASBA), an isothermal amplification technique for nucleic acids, was evaluated for the identification of medically important Candida species using primers selected from 18S rRNA sequences conserved in fungi. An RNA fragment of 257 nucleotides was amplified for Candida albicans. Nineteen different fungi were tested for rRNA amplification with the NASBA. All were positive when analyzed

Myra N. Widjojoatmodjo; Annemarie Borst; Rianne A. F. Schukkink; Adrienne T. A. Box; Nicole M. M. Tacken; Bob Van Gemen; Jan Verhoef; Bert Top; Ad. C. Fluit

1999-01-01

279

Evaluation of Phi29-based whole-genome amplification for microarray-based comparative genomic hybridisation  

Microsoft Academic Search

For the optimal performance of high throughput genomic technologies sufficient yields of high-quality DNA are crucial. Following microdissection, most samples fail to produce sufficient quantities of DNA for genome-wide experiments. Various PCR-based amplification methods have been used, but these usually produce nonuniform representations of the genome. Bacteriophage Phi29 DNA polymerase random-primed DNA amplification is based on isothermal multiple displacement amplification.

Edurne Arriola; Maryou B K Lambros; Chris Jones; Tim Dexter; Alan Mackay; David S P Tan; Narinder Tamber; Kerry Fenwick; Alan Ashworth; Mitch Dowsett; Jorge S Reis-Filho

2007-01-01

280

Isothermal spherical Couette flow  

Microsoft Academic Search

We summarise different types of instabilities and flow patterns in isothermal spherical Couette flows as a function of the\\u000a aspect ratio. The flow of a viscous incompressible fluid in the gap between two concentric spheres was investigated for the\\u000a case, that only the inner sphere rotates and the outer one is stationary. Flow visualisation studies were carried out for\\u000a a

Markus Junk; Christoph Egbers

2000-01-01

281

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

282

Isothermal process solar collector panel  

Microsoft Academic Search

An isothermal process solar collector panel is disclosed. The panel includes a collector plate for absorbing radiant heat; and a plurality of isothermal process heat pipes in an array over a surface of the collector plate. Each heat pipe is closed at both ends and contains thermodynamic working fluid for transferring heat energy from the collector plate to a second

Watt

1978-01-01

283

Rapid, isothermal DNA self-replication induced by a destabilizing lesion.  

PubMed

You spin me round: Using a destabilizing abasic site and high concentration of ligase, rapid DNA self-replication in an isothermal ligase chain reaction (LCR) was produced. Both destabilization and rapid ligation are essential for proper LCR replication. This method also provides insight into prebiotic nucleotide replication and is a potential amplification method for biodiagnostics. PMID:23922255

Kausar, Abu; Mitran, Catherine J; Li, Yimeng; Gibbs-Davis, Julianne M

2013-08-06

284

Miniaturized nucleic acid amplification systems for rapid and point-of-care diagnostics: a review.  

PubMed

Point-of-care (POC) genetic diagnostics critically depends on miniaturization and integration of sample processing, nucleic acid amplification, and detection systems. Polymerase chain reaction (PCR) assays have extensively applied for the diagnosis of genetic markers of disease. Microfluidic chips for microPCR with different materials and designs have been reported. Temperature cycling systems with varying thermal masses and conductivities, thermal cycling times, flow-rates, and cross-sectional areas, have also been developed to reduce the nucleic acid amplification time. Similarly, isothermal amplification techniques (e.g., loop-mediated isothermal amplification or LAMP), which are still are emerging, have a better potential as an alternative to PCR for POC diagnostics. Isothermal amplification techniques have: (i) moderate incubation temperature leading to simplified heating and low power consumption, (ii) yield high amount of amplification products, which can be detected either visually or by simple detectors, (iii) allow direct genetic amplification from bacterial cells due to the superior tolerance to substances that typically inhibit PCR, (iv) have high specificity, and sensitivity, and (v) result in rapid detection often within 10-20 min. The aim of this review is to provide a better understanding of the advantages and limitations of microPCR and microLAMP systems for rapid and POC diagnostics. PMID:22704369

Ahmad, Farhan; Hashsham, Syed A

2012-05-03

285

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)

1995-06-01

286

Centrosome amplification in tumorigenesis.  

PubMed

With regard to cancer development the centrosome has been the center of attraction of scientists for already more than a 100 years. After the initial assumption that amplified centrosomes and abnormal mitotic arrangements might be a cause of cancer at the beginning of the last century, enormous efforts have been undertaken to clarify the relevance of centrosome amplification in tumorigenesis. In the meantime, centrosome amplification has been observed in most, both solid and hematological, cancer entities and by now is viewed as a "hallmark" of cancer cells. In this review we summarize basics in centrosome biology and what is known about the emergence of amplified centrosomes. In addition, we discuss how centrosome amplification might cause aneuploidy thereby leading to malignant transformation of cells. Furthermore, we present recent insights into the role of centrosome amplification in tumor formation based on work in model systems. PMID:22342684

Anderhub, Simon J; Krämer, Alwin; Maier, Bettina

2012-02-14

287

Alternative molecular tests for virological diagnosis.  

PubMed

Several nucleic acid amplification techniques (NAATs), particularly PCR and real-time PCR, are currently used in the routine clinical laboratories. Such approaches have allowed rapid diagnosis with a high degree of sensitivity and specificity. However, conventional PCR methods have several intrinsic disadvantages such as the requirement for temperature cycling apparatus, and sophisticated and costly analytical equipments. Therefore, amplification at a constant temperature is an attractive alternative method to avoid these requirements. A new generation of isothermal amplification techniques are gaining a wide popularity as diagnostic tools due to their simple operation, rapid reaction and easy detection. The main isothermal methods reviewed here include loop-mediated isothermal amplification, nucleic acid sequence-based amplification, and helicase-dependent amplification. In this review, design criteria, potential of amplification, and application of these alternative molecular tests will be discussed and compared to conventional NAATs. PMID:22484997

Sidoti, Francesca; Bergallo, Massimiliano; Costa, Cristina; Cavallo, Rossana

2013-03-01

288

Detection DNA Point Mutation with Rolling-Circle Amplification Chip  

Microsoft Academic Search

We present a protocol with isothermal rolling-circle amplification (RCA) to detect DNA point mutation on chip. The basic principle of the method is an allele-specific oligonucleotide circularization mediated by special DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and target DNA. Mismatches around the ligation site can prevent probe circularization. The circularized probe (C-probe)

Lingwei Wu; Yun Ling; Anshu Yang; Shuixing Wang

2010-01-01

289

Parametric Amplification of Atoms  

Microsoft Academic Search

We have observed parametric generation and amplification of ultracold atom pairs. A 87Rb Bose-Einstein condensate was loaded into a one-dimensional optical lattice with quasimomentum k0 and spontaneously scattered into two final states with quasimomenta k1 and k2 . Furthermore, when a seed of atoms was first created with quasimomentum k1 we observed parametric amplification of scattered atoms pairs in states

Gretchen K. Campbell; Jongchul Mun; Micah Boyd; Erik W. Streed; Wolfgang Ketterle; David E. Pritchard

2005-01-01

290

Simultaneous Strand Displacement Amplification and Fluorescence Polarization Detection of Chlamydia trachomatisDNA  

Microsoft Academic Search

Strand displacement amplification (SDA) is an isothermal DNA amplification technology that uses a restriction enzyme and polymerase. We have developed a target-specific method which allows simultaneous SDA and detection in a homogeneous format. This is accomplished by including a detector oligodeoxynucleotide labeled with 5-(4,6-dichlorotriazin-2-yl)amino fluorescein in the SDA reaction. Fluorescence polarization is used to monitor hybridization of the detector probe

Patricia A. Spears; C. Preston Linn; Dan L. Woodard; G. Terrance Walker

1997-01-01

291

Detection of Mycoplasma pneumoniae by Real-Time Nucleic Acid Sequence-Based Amplification  

Microsoft Academic Search

Real-time isothermal nucleic acid sequence-based amplification (RT-NASBA) was applied to the detection of Mycoplasma pneumoniae. In vitro-generated M. pneumoniae RNA was used to assess the sensitivity of the assay. The 95% hit rate was 148 molecules of M. pneumoniae RNA in the amplification and 104 molecules of in vitro-generated RNA after nucleic acid extraction. The sensitivity of the RT-NASBA and

K. Loens; M. Ieven; D. Ursi; T. Beck; M. Overdijk; P. Sillekens; H. Goossens

2003-01-01

292

Typing of Mycoplasma pneumoniae by nucleic acid sequence-based amplification, NASBA®  

Microsoft Academic Search

Nucleic acid sequence-based amplification, NASBA®, is an isothermal amplification technique for nucleic acids and was used for typing a collection of 24Mycoplasma pneumoniaestrains. A set of primers was chosen from the 16S rRNA sequence alignment ofMycoplasmaspecies. The nucleotide sequences of the (?)RNA amplicons were determined forM. pneumoniaestrains M15\\/83 (type 1) and FH (type 2), and revealed a one-point difference at

C. Ovyn; D. van Strijp; M. leven; D. Ursi; B. van Gemen; H. Goossens

1996-01-01

293

Nucleic Acid Sequence-Based Amplification of Aspergillus RNA in Blood Samples  

Microsoft Academic Search

Nucleic acid sequence-based amplification (NASBA), an isothermal amplification technique, was established and evaluated for the detection of Aspergillus RNA and compared with a previously published, well-defined real-time PCR assay amplifying a region of the Aspergillus 18S rRNA gene. NASBA showed a lower detection limit of 1 CFU and detected RNA from five different clinically relevant Aspergillus species, including Aspergillus fumigatus.

JUERGEN LOEFFLER; HOLGER HEBART; PHILIPP COX; NICOLE FLUES; ULRIKE SCHUMACHER; HERMANN EINSELE; Medizinische Klinik

2001-01-01

294

Characteristics and applications of nucleic acid sequence-based amplification (NASBA)  

Microsoft Academic Search

Nucleic acid sequence-based amplification (NASBA) is a sensitive, isothermal, transcription-based amplification system specifically\\u000a designed for the detection of RNA targets. In some NASBA systems, DNA is also amplified though very inefficiently and only\\u000a in the absence of the corresponding RNA target or in case of an excess (>1000-fold) of target DNA over RNA. As NASBA is primer-dependent\\u000a and amplicon detection

Birgit Deiman; Pierre van Aarle; Peter Sillekens

2002-01-01

295

Real-Time, Sequence-Specific Detection of Nucleic Acids during Strand Displacement Amplification  

Microsoft Academic Search

Strand displacement amplification (SDA) is an isothermal nucleic acid amplification method based on the primer-directed nicking activity of a restriction enzyme and the strand displacement activity of an exonuclease-deficient polymerase. Here we describe fluorogenic reporter probes that permit real-time, sequence-specific detection of targets amplified during SDA. The new probes possess the single-strand half of a BsoBI recognition sequence flanked on

James G. Nadeau; J. Bruce Pitner; C. Preston Linn; James L. Schram; Cheryl H. Dean; Colleen M. Nycz

1999-01-01

296

Hyperbranching rolling circle amplification, an improved protocol for discriminating between closely related fungal species.  

PubMed

Hyperbranching Rolling Circle Amplification (HRCA) is a technique derived from Rolling Circle Amplification (RCA) in which DNA polymerase replicates circularized oligonucleotide probes under isothermal conditions with either linear or geometric kinetics. Since its first introduction HRCA has been proven to be a robust DNA amplification technique for detecting pathogenic fungi and other applications, allowing rapid detection of nucleic-acid sequences with high specificity. Here we describe an improved protocol of HRCA, which is both specific and sensitive for detecting low copy numbers of template DNA. The test can be performed within one working day in routine molecular labs. PMID:23296894

Sun, Jiufeng; de Hoog, Sybren

2013-01-01

297

Isothermal cavity, blackbody radiation source.  

PubMed

A prototype blackbody radiation source has been developed which incorporates a double cone cavity of extremely uniform surface temperature. This uniformity of surface temperature is the result of a unique method of supplying heat to the cavity. The principle of operation is based on heat pipe techniques which transfer heat by change of phase and mass transfer. The cavity is heated by serving as the condenser section of the heat pipe which is inherently a nearly isothermal device. Optical tests show that the isothermal cavity has a lambertian distribution over an inclusive angle of better than 60 degrees while operating between 419 degrees C and 760 degrees C. PMID:20094191

Bliss, F E; Davis, S; Stein, B

1970-09-01

298

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

299

Detection of Dengue Viral RNA Using a Nucleic Acid Sequence-Based Amplification Assay  

Microsoft Academic Search

Faster techniques are needed for the early diagnosis of dengue fever and dengue hemorrhagic fever during the acute viremic phase of infection. An isothermal nucleic acid sequence-based amplification (NASBA) assay was optimized to amplify viral RNA of all four dengue virus serotypes by a set of universal primers and to type the amplified products by serotype-specific capture probes. The NASBA

SHUENN-JUE L. WU; EUN MI LEE; RAVITHAT PUTVATANA; ROXANNE N. SHURTLIFF; KEVIN R. PORTER; WURYADI SUHARYONO; DOUGLAS M. WATTS; CHWAN-CHUEN KING; GERALD S. MURPHY; CURTIS G. HAYES; JOSEPH W. ROMANO; APO AP; APO AA

2001-01-01

300

Isothermal compressors for process gases  

Microsoft Academic Search

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

E. Wiederuh; D. Meinhart

1992-01-01

301

Coronal loops: isothermal or multithermal?  

NASA Astrophysics Data System (ADS)

Are coronal loops isothermal? A controversy over this question has arisen recently because different investigators using different techniques have obtained very different answers. Analysis of SOHO-EIT and TRACE data using narrowband filter ratios to obtain temperature maps has produced several key publications that suggest that coronal loops may be isothermal. We have constructed a multi-thermal distribution for several pixels along a relatively isolated coronal loop on the southwest limb of the solar disk using spectral line data from SOHO-CDS taken on 1998 Apr 20. These distributions are clearly inconsistent with isothermal plasma along either the line of sight or the length of the loop, and suggested rather that the temperature increases from the footpoints to the loop top. We speculated originally that these differences could be attributed to pixel size -- CDS pixels are larger, and more `contaminating' material would be expected along the line of sight. To test this idea, we used CDS iron line ratios from our data set to mimic the isothermal results from the narrowband filter instruments. These ratios indicated that the temperature gradient along the loop was flat, despite the fact that a more complete analysis of the same data showed this result to be false! The CDS pixel size was not the cause of the discrepancy; rather, the problem lies with the isothermal approximation used in EIT and TRACE analysis. These results should serve as a strong warning to anyone using this simplistic method to obtain temperature. This warning is echoed on the EIT web page: ``Danger! Enter at your own risk!'' In other words, values for temperature may be found, but they may have nothing to do with physical reality.

Schmelz, J.; Cirtain, J.

302

Generalized privacy amplification  

Microsoft Academic Search

This paper provides a general treatment of privacy amplification by public discussion, a concept introduced by Bennett, Brassard and Robert (1988) for a special scenario. The results have applications to unconditionally-secure secret-key agreement protocols, quantum cryptography and to a non-asymptotic and constructive treatment of the secrecy capacity of wire-tap and broadcast channels, even for a considerably strengthened definition of secrecy

C. H. Bennett; G. Brassard; C. Crepeau; U. M. Maurer

1994-01-01

303

Some consideration on the Langmuir isotherm equation  

Microsoft Academic Search

The Langmuir isotherm equation has been widely used in adsorption process for decades. This note analyzed application constrain of the Langmuir isotherm equation. In a strict theoretical sense, the concentration of adsorbate used in the Langmuir isotherm equation must be expressed as its molar concentration. However, in the literature of adsorption research, the volumetric concentration of adsorbate has been commonly

Yu Liu

2006-01-01

304

Isothermal and non-isothermal crystallization in amorphous sucrose and lactose at low moisture contents  

Microsoft Academic Search

Differential scanning calorimetry has been used in isothermal and non-isothermal modes to provide information on the crystallization of sucrose and lactose at low water contents. Using approaches previously applied to polymer crystallization an attempt has been made to combine the isothermal and non-isothermal data into a single curve. This is achieved by the use of appropriate shift factors in the

Claire J Kedward; William MacNaughtan; John R Mitchell

2000-01-01

305

A New Method of Determining Moisture Flow Coefficients for both Isothermal and Non-isothermal Conditions  

Microsoft Academic Search

SUMMARY: The calculation of isothermal moisture transport requires a moisture flow coefficient, D ?(?,T), for isothermal flow withas the moisture state variable. In the non-isothermal case, a second flow coefficient, D T?(?,T), is required to account for the temperature gradient. This means that a number of isothermal measurements are required for a few different temperature levels. It also means that

Stephen Burke; Johan Claesson; Jesper Arfvidsson

306

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

307

RNA pre-amplification enables large-scale RT-qPCR gene-expression studies on limiting sample amounts  

Microsoft Academic Search

BACKGROUND: The quantitative polymerase chain reaction (qPCR) is a widely utilized method for gene-expression analysis. However, insufficient material often compromises large-scale gene-expression studies. The aim of this study is to evaluate an RNA pre-amplification method to produce micrograms of cDNA as input for qPCR. FINDINGS: The linear isothermal Ribo-SPIA pre-amplification method (WT-Ovation; NuGEN) was first evaluated by measuring the expression

Joëlle Vermeulen; Stefaan Derveaux; Steve Lefever; Els De Smet; Katleen De Preter; Nurten Yigit; Anne De Paepe; Filip Pattyn; Frank Speleman; Jo Vandesompele

2009-01-01

308

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.

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

2012-01-01

309

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

310

Non-Dispersion-Managed Soliton Amplification Using Distributed Raman Amplification  

Microsoft Academic Search

Optical soliton, amplified by the distributed Raman amplification (DRA), is numerically studied using more accurate method than previous works. In our work, the DRA gain is actually designed by numerically solving the DRA equations, which include both the pump depletion and the soliton depletion. Our results predict that only the non-adiabatic region exists for the soliton amplification by DRA. The

Pasu Kaewplung; Ekachai Kongboonsod; Preeda Jarupoom

2006-01-01

311

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

312

Amplification of Sound by Gas Phase Reactions.  

National Technical Information Service (NTIS)

A four year study of sound propagation in chemically reacting mixtures has led to experimental observation of sound amplification. Photo-initiated C12-H2-inert gas reactions provided the energy for the amplification observed. Amplification experiments wer...

H. E. Bass R. M. Detsch

1983-01-01

313

Isothermal Titration Calorimetry of RNA  

PubMed Central

Isothermal titration calorimetry (ITC) is a fast and robust method to study the physical basis of molecular interactions. A single well-designed experiment can provide complete thermodynamic characterization of a binding reaction, including Ka, ?G, ?H, ?S and reaction stoichiometry (n). Repeating the experiment at different temperatures allows determination of the heat capacity change (?CP) of the interaction. Modern calorimeters are sensitive enough to probe even weak biological interactions making ITC a very popular method among biochemists. Although ITC has been applied to protein studies for many years, it is becoming widely applicable in RNA biochemistry as well, especially in studies which involve RNA folding and RNA-interactions with small molecules, proteins and with other RNAs. This review focuses on best practices for planning, designing, and executing effective ITC experiments when one or more of the reactants is an RNA.

Salim, Nilshad N.; Feig, Andrew L.

2009-01-01

314

Isothermal and non-isothermal crystallisation kinetics of pCBT and PBT  

Microsoft Academic Search

The crystallisation behaviour of in situ polymerised cyclic butylene terephthalates (pCBT) and poly(butylene terephthalate)s\\u000a (PBT) were studied by differential scanning calorimetry (DSC) both under isothermal and non-isothermal conditions. The crystallisation\\u000a was analysed by adopting the Avrami, Ozawa and Kissinger methods for the isothermal and non-isothermal crystallisations, respectively.\\u000a An Avrami exponent n between 2 and 3 was found for the pCBTs

B. Lehmann; J. Karger-Kocsis

2009-01-01

315

Prediction of Bacillus subtilis spore survival after a combined non-isothermal-isothermal heat treatment  

Microsoft Academic Search

Different inactivation kinetics data have been used to predict the number of survivors exposed to a heat treatment and, in consequence, to design thermal processes for the food industry. In this work, spores of an acidophilic strain of Bacillus subtilis were heated under isothermal and non-isothermal conditions. Experimental results obtained after isothermal treatments were analysed using the classical two-step linear

Raquel Conesa; Paula M. Periago; Arturo Esnoz; Antonio López; Alfredo Palop

2003-01-01

316

Radiometric and equivalent isothermal surface temperatures  

Microsoft Academic Search

The analytical solution for the heat flux from an anisothermal canopy developed from K theory by Brutsaert and Sugita [1996] (hereinafter referred to as B&S) has been extended to provide a parameterization of the difference between the radiometric and the equivalent isothermal surface temperature. The latter is the isothermal temperature at which a canopy would give the correct sensible heat

Richard D. Crago

1998-01-01

317

Genome amplification of single sperm using multiple displacement amplification  

PubMed Central

Sperm typing is an effective way to study recombination rate on a fine scale in regions of interest. There are two strategies for the amplification of single meiotic recombinants: repulsion-phase allele-specific PCR and whole genome amplification (WGA). The former can selectively amplify single recombinant molecules from a batch of sperm but is not scalable for high-throughput operation. Currently, primer extension pre-amplification is the only method used in WGA of single sperm, whereas it has limited capacity to produce high-coverage products enough for the analysis of local recombination rate in multiple large regions. Here, we applied for the first time a recently developed WGA method, multiple displacement amplification (MDA), to amplify single sperm DNA, and demonstrated its great potential for producing high-yield and high-coverage products. In a 50 ?l reaction, 76 or 93% of loci can be amplified at least 2500- or 250-fold, respectively, from single sperm DNA, and second-round MDA can further offer >200-fold amplification. The MDA products are usable for a variety of genetic applications, including sequencing and microsatellite marker and single nucleotide polymorphism (SNP) analysis. The use of MDA in single sperm amplification may open a new era for studies on local recombination rates.

Jiang, Zhengwen; Zhang, Xingqi; Deka, Ranjan; Jin, Li

2005-01-01

318

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.

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

319

Nucleic Acid Amplification in Yeast.  

National Technical Information Service (NTIS)

Plasmid DNA from single yeast colonies was efficiently amplified using rolling circle amplification (RCA). The amplified DNA was directly used for restriction digestion, DNA sequencing, and yeast transformation. The RCA of plasmid DNA from single yeast co...

W. Farmerie W. Y. Song X. Ding

2004-01-01

320

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

321

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.

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

2010-01-01

322

ISOFIT - A PROGRAM FOR FITTING SORPTION ISOTHERMS TO EXPERIMENTAL DATA  

EPA Science Inventory

Isotherm expressions are important for describing the partitioning of contaminants in environmental systems. ISOFIT (ISOtherm FItting Tool) is a software program that fits isotherm parameters to experimental data via the minimization of a weighted sum of squared error (WSSE) obje...

323

Assessment, by Transcription-Mediated Amplification, of Virologic Response in Patients with Chronic Hepatitis C Virus Treated with Peginterferon a-2a  

Microsoft Academic Search

Transcription-mediated amplification (TMA) is an isothermal, autocatalytic target amplification method which has the potential to detect less than 50 hepatitis C virus (HCV) RNA copies\\/ml (10 IU\\/ml). The TMA assay was used to assess the presence of residual HCV RNA in plasma from patients treated with polyethylene glycol-modified interferon a-2a (peginterferon a-2a) who showed a virologic relapse after the end

CHRISTOPH SARRAZIN; DAVID A. HENDRICKS; FARHAD SEDARATI; STEFAN ZEUZEM

324

Gas adsorption isotherm for dealuminated zeolites  

SciTech Connect

Adsorption is a current technique to remove volatile organic compounds from process gas streams. In industrial applications hydrophobic dealuminated zeolites are often used as adsorbents, because of their specific advantages in comparison to activated carbon or polar zeolites. A new equation for complex adsorption isotherms was developed by using a mathematical method well-known in control engineering. On the basis of this analogy, it is possible to describe the transitional region between Langmuir isotherms and isotherms following Henry`s law. Development and verification of the equation are based on experimental data of the system zeolite DAY-ethanol-air.

Bathen, D.; Schmidt-Traub, H.; Simon, M. [Univ. of Dortmund (Germany). Dept. of Chemical Engineering

1997-09-01

325

Detection of Toxoplasma gondii oocysts in different water resources by Loop Mediated Isothermal Amplification (LAMP).  

PubMed

Human toxoplasmosis is potentially contracted due to consumption of contaminated drinking water and represents an increasing public health risk worldwide. Toxoplasma gondii oocysts can be resistant to standard disinfection processes, including UV radiation. Increased awareness of the risk of waterborne toxoplasmosis outbreaks has led to an increase in research interest in the detection of oocysts in environmental water systems. Ninety-five environmental water samples from the Lower Rhine area in Germany have been included in the study and examined for the presence of Toxoplasma. Water samples were filtered or flocculated by aluminum sulfate and purified by sucrose density gradient. DNA was then extracted, and the DNA samples were then examined by LAMP analysis. T. gondii DNA was detected in eight out of 83 (9.6%) influent and effluent samples obtained from wastewater treatment plants. All samples (n=12) from the surface, ground, raw and tap waters tested negative. The purpose of this work was to investigate the occurrence and distribution of Toxoplasma oocysts on the Lower Rhine in Germany. Our study provides evidence that the assay is a sensitive, specific, rapid and cost effective method for the detection of T. gondii and is useful for both the investigations of cases of waterborne outbreaks and for identifying the source of contamination. PMID:23088835

Gallas-Lindemann, Carmen; Sotiriadou, Isaia; Mahmoodi, Mohammad Reza; Karanis, Panagiotis

2012-10-23

326

Ultra-Sensitive Detection of Prion Protein in Blood Using Isothermal Amplification Technology.  

National Technical Information Service (NTIS)

The detection of the pathologic prion protein that is Implicated in transmissible spongiform encephalopathies (TSEs) is necessary to diagnose the disease. Presently, the Western Blot or ELISA are used to test the brain stem in cattle for the presence of p...

N. T. Constantine

2004-01-01

327

Multichange isothermal mutagenesis: a new strategy for multiple site-directed mutations in plasmid DNA.  

PubMed

Multichange ISOthermal (MISO) mutagenesis is a new technique allowing simultaneous introduction of multiple site-directed mutations into plasmid DNA by leveraging two existing ideas: QuikChange-style primers and one-step isothermal (ISO) assembly. Inversely partnering pairs of QuikChange primers results in robust, exponential amplification of linear fragments of DNA encoding mutagenic yet homologous ends. These products are amenable to ISO assembly, which efficiently assembles them into a circular, mutagenized plasmid. Because the technique relies on ISO assembly, MISO mutagenesis is additionally amenable to other relevant DNA modifications such as insertions and deletions. Here we provide a detailed description of the MISO mutagenesis concept and highlight its versatility by applying it to three experiments currently intractable with standard site-directed mutagenesis approaches. MISO mutagenesis has the potential to become widely used for site-directed mutagenesis. PMID:23654272

Mitchell, Leslie A; Cai, Yizhi; Taylor, Martin; Noronha, Anne Marie; Chuang, James; Dai, Lixin; Boeke, Jef D

2013-03-11

328

Non-crosslinking gold nanoprobes for detection of nucleic acid sequence-based amplification products.  

PubMed

Lack of an appropriate detection method for isothermal RNA amplification technique, known as nucleic acid sequence-based amplification (NASBA), is considered as a major defect for its vast applications. In that regard, novel detection methods as fast, specific, and sensitive as the gold nanoprobe detection technique are highly demanded and non-crosslinking gold nanoprobes are regarded as the ideal choice. In this study, we attempted to integrate these two techniques (RNA amplification and nanoprobe detection) into a single detection-associated amplification method. In that line, essential adjustments such as amplicon dilution, disturbing reagent extraction, ion adjustment, and modification of the hybridization protocol were needed due to the ribonucleic acid nature of NASBA products and the presence of some interfering reagents in the amplification reaction environment. The adjustments successfully resulted in the gold nanoparticle-based detection of NASBA products with naked eyes in a whole operational time of less than 3.5h (including nucleic acid extraction, amplification, and detection). Furthermore, the developed assay was successfully applied to detect dnaK messenger RNA of Salmonella typhimurium. The developed colorimetric method facilitated the detection step of NASBA leading to an ideal methodology for rapid assays and serves as an ideal alternative to the highly expensive reverse transcription polymerase chain reaction (RT-PCR) approach. PMID:22449495

Mollasalehi, Hamidreza; Yazdanparast, Razieh

2012-03-23

329

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

330

Isothermal Forging of HIP'ed Superalloy.  

National Technical Information Service (NTIS)

Ni-base superalloy powders were consolidated by HIP for an isothermal, superplastic forging. As a result, the forging billet having enough superplasticity was obtained by HIPing the powder at a temperature below gamma-prime solvus. The superplastic behavi...

H. Takigawa

1985-01-01

331

Isothermal and non-isothermal shrinkage behaviors of highly oriented PET yarns  

Microsoft Academic Search

The isothermal and non-isothermal shrinkage behaviors of highly oriented Poly(ethylene terephthalate) yarns were investigated.\\u000a In isothermal measurements, shrinkage and shrinkage stress firstly monotonously increased due to more and more activated frozen\\u000a molecular segments with increasing time and temperature, and then relaxed at high temperature resulting from intermolecular\\u000a slipping of micro-fibrils. According to the different contributions of amorphous and crystalline regions

Xian Zhang; Xingyou Tian; Xiayin Yao; Jin Zheng; Wentao Liu; Yong Li; Ping Cui

2008-01-01

332

Pyrolysis characteristics of coal and RDF blends in non-isothermal and isothermal conditions  

Microsoft Academic Search

Pyrolysis characteristics of the sub-bituminous coal, Refuse Derived Fuel (RDF) and their blends were determined at non-isothermal (15°C\\/min) and isothermal (500–800°C) conditions in thermo-gravimetric analyzer (TGA) and a thermobalance reactor. The decomposition rates of coal and RDF blends are higher than the calculated values of each fuel at the lower temperature ranges (300–385°C). In the isothermal condition, activation energies of

Myung Won Seo; Sang Done Kim; See Hoon Lee; Jae Goo Lee

2010-01-01

333

Comprehensive kinetic studies on isothermal and non-isothermal reactions of some aluminium compounds  

Microsoft Academic Search

Residual differences after model fitting were investigated in both isothermal and non-isothermal kinetics in order to make\\u000a numerical comparisons between several models and various parameter-estimating methods. Data from two independent experimental\\u000a series were evaluated.\\u000a \\u000a A large data set, collected earlier under isothermal conditions from decompositions and hydrothermal reactions of aluminium\\u000a hydroxides and oxides, was processed first. It showed that mechanical

J. Madarász; G. Pokol; C. Novák; H. Moselhy; S. Gál

1993-01-01

334

On the Isothermality of Solar Plasmas  

NASA Astrophysics Data System (ADS)

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.; Klimchuk, J. A.

2010-11-01

335

Isothermal and non-isothermal crystallization in amorphous sucrose and lactose at low moisture contents.  

PubMed

Differential scanning calorimetry has been used in isothermal and non-isothermal modes to provide information on the crystallization of sucrose and lactose at low water contents. Using approaches previously applied to polymer crystallization an attempt has been made to combine the isothermal and non-isothermal data into a single curve. This is achieved by the use of appropriate shift factors in the time and temperature domains. This was successful for sucrose but not for lactose. It was suggested that this was because lactose crystallizes into multiple forms whereas sucrose crystallizes in a single form. PMID:11117325

Kedward, C J; MacNaughtan, W; Mitchell, J R

2000-11-01

336

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

337

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

338

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

339

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

2013-06-09

340

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

2011-05-12

341

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

2011-12-08

342

Rapid detection of Marek's disease virus in feather follicles by loop-mediated amplification.  

PubMed

Marek's disease (MD) remains a serious problem in the production of poultry. The disease is caused by Marek's disease virus (MDV), and despite the ubiquitous use of vaccination to control losses, MD still affects poultry farming worldwide. The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) method for the simple and inexpensive detection of MDV in feather tips of chickens. Two pairs of specific primers complementary to the meq oncogene of MDV were designed, targeting the sequence of the very virulent MDV strain, RB1B. Bst polymerase was used for the isothermal amplification of viral DNA at 65 C for 90 min in a water bath. The fluorescence signal was identified in MDV-positive samples after the addition of SYBR Green and ultraviolet (UV) illumination. The sensitivity of LAMP was 2 log 10 plaque-forming units (PFU)/ml of HPRS16 and 10(3) copies/il of plasmid containing the target gene (meq) and was equal in sensitivity to PCR amplification. Due to the use of three sets of primers, LAMP was highly specific for MDV-1 DNA. The developed LAMP technique is a rapid and simple tool for the specific detection of MDV in samples of feathers taken from live chickens. Since the use of thermocyclers is not necessary for LAMP assay, it can be conducted by small laboratories and even field veterinarians. PMID:22017048

Wo?niakowski, Grzegorz; Samorek-Salamonowicz, Elzbieta; Kozdru?, Wojciech

2011-09-01

343

Magnetogravity waves in an isothermal conductive atmosphere  

SciTech Connect

An analysis is given of the propagation of all types of nonadiabatic MHD waves in a compressible, isothermal, conductive atmosphere with a uniform vertical magnetic field. Analytic solutions of the problem are obtained in terms of higher transcendental functions. Several cases of linear transformation of the waves are considered, and the possiblity that such transformations may occur in the solar atmosphere is discussed.

Zhugzhda, Y.D.

1979-01-01

344

STUDY ON SORPTION ISOTHERMS OF SHRIMP BEADS  

Microsoft Academic Search

Sorption isotherms of shrimp heads ( adsorption and desorption ) were analyzed by the microclimate method as temperatures of 30, 45 and 60°C and conditions for shrimp heads conservation by drying were determined. Equilibrium time was reduced from normally 2–3 weeks to only 5–8 days by employing sulfuric acid at different concentrations and vacuum. The results were adjusted by Henderson

M. S. Salgado Cervantes; I. Andrade Gonzalez; K. N. Waliszewski Kubiak; M. A. Garcia Alvarado

1993-01-01

345

Thermodynamic analysis on one isothermal expansion process  

NASA Astrophysics Data System (ADS)

One isothermal expansion process of the ideal gas contained in one tank is analyzed in detailed from the viewpoint of thermodynamics. Simultaneously, we give an insight into why the irreversible process achieves impossibly more work than the reversible process between the same initial and final equilibrium states.

Liu, Juanfang; Chen, Qinghua

2013-10-01

346

Rapid isothermal processing of silicon wafers  

Microsoft Academic Search

The reduction in the size of semiconductor devices has not only increased their speed and the number that can be fitted on a chip but has also led to the need for very accurately controlled thermal processing of the semiconductor wafers. Conventional furnaces are used as standard in IC processing but now, however, alternative rapid isothermal processing technologies are gaining

S. S. Gill

1986-01-01

347

Rapid isothermal process technology for optoelectronic applications  

Microsoft Academic Search

Rapid isothermal processing (RIP) based on incoherent sources of light is emerging as a reduced thermal budget (product of processing time and temperature) processing technique. As compared to a stand-alone annealing unit, the integration of RIP with other processing units leading to integrated RIP systems is very attractive for the next generation of devices and circuits. From cost and performance

Rajendra Singh

1991-01-01

348

Simulated water adsorption isotherms in carbon nanopores  

NASA Astrophysics Data System (ADS)

Water adsorption isotherms are calculated by grand canonical Monte Carlo simulations for the SPC/E water model in carbon nanopores at 298 K. The pores are of slit or cylindrical morphology. Carbon-slit pores are of widths 0.8, 1.0 and 1.6 nm. The simulated single-walled carbon nanotubes are of 1.4 and 2.7 nm diameter ((10:10) and (20:20) respectively). In all cases considered, the adsorption isotherms are characterized by negligible adsorption at low pressures, pore filling by a capillary-condensation-like mechanism and adsorption-desorption hysteresis loops. For both pore morphologies considered, the relative pressures at which pore filling occurs, and the width of the adsorption-desorption hysteresis loop decrease with decreasing pore size. Adsorption isotherms simulated for water in carbon nanotubes show pore filling at lower relative pressures and narrower adsorption-desorption hysteresis loops when compared to adsorption isotherms simulated in carbon-slit pores of similar sizes. By using representative simulation snapshots, the mechanisms of pore filling and pore emptying are discussed. Pore filling happens by growth of hydrogen-bonded clusters of adsorbed water molecules, without the formation of monolayers as observed in the adsorption of simple fluids. Pore emptying occurs by the formation of bubbles, often in contact with the hydrophobic surface, followed by the coalescence and growth of these bubbles.

Striolo, Alberto; Gubbins, Keith E.; Chialvo, Ariel A.; Cummings, Peter T.

349

Titanium alloy blade isothermal adjustment die design  

Microsoft Academic Search

TC11 titanium alloy blade is one of aircraft engine important parts, its shape is complex and the quality requirement is high. The warps and the forgings may be discard due to the formed forging's remaining stress and the temperature stress. But it is possible to use the isothermal adjustment process to reduce the warp distortion and make the blade shape

Zhang Hui; Feng Xiaoming; Zhang Changming; Guo Congsheng

2010-01-01

350

Water Sorption Isotherms of Pistachio Nuts  

Microsoft Academic Search

The adsorption and desorption isotherms of pistachio nuts (kernel and shell) were measured at 15, 25 and 40°C using a gravimetric method. Hysteresis was more pronounced in the shell than in the kernel and reduced as the temperature increased. The isosteric heat of desorption, calculated from the slope of a lnawvs. 1\\/Tplot, was higher than the isosteric heat of adsorption.

S. Yanniotis; I. Zarmboutis

1996-01-01

351

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

352

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

353

Coronal Loops: Evolving Beyond the Isothermal Approximation  

NASA Astrophysics Data System (ADS)

Are coronal loops isothermal? A controversy over this question has arisen recently because different investigators using different techniques have obtained very different answers. Analysis of SOHO-EIT and TRACE data using narrowband filter ratios to obtain temperature maps has produced several key publications that suggest that coronal loops may be isothermal. We have constructed a multi-thermal distribution for several pixels along a relatively isolated coronal loop on the southwest limb of the solar disk using spectral line data from SOHO-CDS taken on 1998 Apr 20. These distributions are clearly inconsistent with isothermal plasma along either the line of sight or the length of the loop, and suggested rather that the temperature increases from the footpoints to the loop top. We speculated originally that these differences could be attributed to pixel size -- CDS pixels are larger, and more `contaminating' material would be expected along the line of sight. To test this idea, we used CDS iron line ratios from our data set to mimic the isothermal results from the narrowband filter instruments. These ratios indicated that the temperature gradient along the loop was flat, despite the fact that a more complete analysis of the same data showed this result to be false! The CDS pixel size was not the cause of the discrepancy; rather, the problem lies with the isothermal approximation used in EIT and TRACE analysis. These results should serve as a strong warning to anyone using this simplistic method to obtain temperature. This warning is echoed on the EIT web page: ``Danger! Enter at your own risk!'' In other words, values for temperature may be found, but they may have nothing to do with physical reality. Solar physics research at the University of Memphis is supported by NASA grant NAG5-9783. This research was funded in part by the NASA/TRACE MODA grant for Montana State University.

Schmelz, J. T.; Cirtain, J. W.; Allen, J. D.

2002-05-01

354

Isothermal and Non-Isothermal Techniques in Kinetic Studies of Reactions in a Photoactive Molecular Material  

Microsoft Academic Search

The kinetics of thermally driven reactions in a crystalline photochromic solid, 1-methyl-2,4,4,6-tetraphenyl-1,4-dihydropyridine (DHP), has been studied. The processes have been monitored by measuring the isothermal bleaching of coloured species produced upon UV irradiation of DHP, and by measuring the heat flow in non-isothermal differential scanning calorimetry experiments. Signatures of two processes were detected; a combination of quantum-chemical calculations and spectroscopic

Ewa ?liwi?ska; ANDRZEJ OLSZOWSKI; JULIUSZ SWORAKOWSKI

1999-01-01

355

Viscosity of bulk free radical polymerizing systems under near-isothermal and non-isothermal conditions  

Microsoft Academic Search

A viscometer-reactor assembly is used to generate data on the viscosity, ?(t), of an example polymerizing system exhibiting the Trommsdorff effect, namely, the bulk free radical polymerization of methyl methacrylate (MMA), at different temperature conditions [near-isothermal and non-isothermal (near-step increase and near-step decrease in temperature)] and at two different initiator, 2,2?-azoisobutyronitrile (AIBN), concentrations. Two types of cup and bob assemblies,

Jitendra S. Sangwai; Deoki N. Saraf; Santosh K. Gupta

2006-01-01

356

Evaluation of surface excess isotherms in liquid chromatography.  

PubMed

Methods are proposed to calculate surface excess isotherms and to use them to derive adsorption isotherms in liquid chromatography. The consequences of these methods are discussed. The excess isotherm of isopropyl alcohol from its aqueous solutions on a C18 adsorbent was obtained using the minor disturbance method. The slope of the inflection tangent of the excess isotherm provides the position of the plane separating the adsorbed layer and the bulk phase, from which the adsorption isotherm was derived. At low concentrations of isopropyl alcohol, frontal analysis was used to derive the adsorption isotherm on the same adsorbent using an independent method. The isotherm was thus derived from both frontal analysis data and the minor disturbance method. The results obtained are compared. Our results show that the use of the same concentration unit for the calculation and the representation of the data is the only correct way to calculate the excess isotherms in practical applications of liquid chromatography. PMID:23601291

Vajda, Péter; Felinger, Attila; Guiochon, Georges

2013-03-29

357

High-performance isotherm extraction for infrared satellite cloud image  

Microsoft Academic Search

The isotherm is an important feature of infrared satellite cloud images (ISCI), which can directly reveal substantial information of cloud systems. The isotherm extraction of ISCI can remove the redundant information and therefore helps to compress the information of ISCI. In this paper, an isotherm extraction method is presented. The main aggregate of clouds can be segmented based on mathematical

Zhengguang Liu; Bing Wu; Yong Liu; Yuan Liu

2001-01-01

358

Strategies for signal amplification in nucleic acid detection  

Microsoft Academic Search

Many aspects of molecular genetics necessitate the detection of nucleic acid sequences. Current approaches involving target\\u000a amplification (in situ PCR, Primed in situ Labeling, Self-Sustained Sequence Replication, Strand Displacement Amplification), probe amplification (Ligase Chain Reaction,\\u000a Padlock Probes, Rolling Circle Amplification) and signal amplification (Tyramide Signal Amplification, Branched DNA Amplification)\\u000a are summarized in the present review, together with their advantages and

S. Calin Andras; J. Brian Power; Edward C. Cocking; Michael R. Davey

2001-01-01

359

Moisture Sorption Isotherms of Red Chillies  

Microsoft Academic Search

The equilibrium moisture contents were determined for red chillies using the static method at 25, 35 and 45°C over a range of relative humidities from 0·115 to 0·865. The sorption capacity of chillies decreased with an increase in temperature at constant relative humidity. The sorption isotherms exhibited the phenomenon of hysteresis, in which the equilibrium moisture content was higher at

S Kaleemullah; R Kailappan

2004-01-01

360

Sorption isotherm characteristics of aonla flakes  

Microsoft Academic Search

The equilibrium moisture content was determined for un-osmosed and osmosed (salt osmosed and sugar osmosed) aonla flakes using\\u000a the static method at temperatures of 25, 40,50, 60 and 70 °C over a range of relative humidities from 20 to 90%. The sorption\\u000a capacity of aonla decreased with an increase in temperature at constant water activity. The sorption isotherms exhibited hysteresis,\\u000a in

Amarjit Singh

2011-01-01

361

Unified desorption isotherms for porcelain bodies  

Microsoft Academic Search

The isotherms Up =U(~p) were constructed for five experimental points for each of nine values of ~p, altering in the range (p = 5-90~e. The experimental data were processed for each value of ~p for the reliability coefficient a=0.95. The resulting mean square error for the series of measurements ASu=0.0144 with a Student coefficient value t~ = 2.78 enabled us

A. A. Shershnev; I. A. Elagina; G. I. Panichev; M. A. Lukashevich

1979-01-01

362

Isothermal coarsening of primary particles during rheocasting  

Microsoft Academic Search

The isothermal coarsening behavior of primary solid particles in A356 aluminum alloy semi-solid slurry produced by angular oscillation (AO) technique was investigated. The comparison between the calculation and experimental results shows good quantitative agreement with Lifshitz-Slyozov-Wagner theory. The results show that the variation in shape factor and solid fraction is not significant, the average particle size increases with increasing holding

Hong-min GUO; Xue-quan LUO; Ai-sheng ZHANG; Xiang-jie YANG

2010-01-01

363

Intensity isotherms and distributions on oligonucleotide microarrays  

Microsoft Academic Search

We describe a physico-chemical model relating measured fluorescence intensities on oligonucleotide microarrays to the underlying specific target concentration in the hybridised solution via a hyperbolic isotherm response function. The model includes various chemical reactions occurring at the microarray surface and in bulk solution during hybridisation, including specific and non-specific hybridisation, and also the effects of probe-target dissociation during the post

Conrad J. Burden

2009-01-01

364

Synthesis and langmuir isotherms of difluorostearic acids  

Microsoft Academic Search

The CF2 containing 6,6- and 10,10-difluorooctadecanoic acids 7 and 8, respectively have been synthesised and their Langmuir isotherms evaluated. In marked contrast to the behaviour of octadecanoic acid (stearic acid) 1 it is demonstrated that spreading on an aqueous subphase generates an unstable monolayer which reorganises to a more stable bilayer structure. Grazing incidence X-ray scattering and ellipsometric measurements after

David O'Hagan; Itsumaro Kumadaki; Michael Petty; Hiroaki Takaya; Christopher Pearson

1998-01-01

365

Nucleic Acid Sequence-Based Amplification of Aspergillus RNA in Blood Samples  

PubMed Central

Nucleic acid sequence-based amplification (NASBA), an isothermal amplification technique, was established and evaluated for the detection of Aspergillus RNA and compared with a previously published, well-defined real-time PCR assay amplifying a region of the Aspergillus 18S rRNA gene. NASBA showed a lower detection limit of 1 CFU and detected RNA from five different clinically relevant Aspergillus species, including Aspergillus fumigatus. All 77 blood samples tested by PCR and NASBA showed identical results in both assays. Results with the NASBA technique were obtained within 6 h. Thus, the NASBA technique provided a valuable tool for sensitive, specific, fast, and reliable detection of Aspergillus RNA with potential for routine diagnosis, including the possibility to test the viability of cells.

Loeffler, Juergen; Hebart, Holger; Cox, Philipp; Flues, Nicole; Schumacher, Ulrike; Einsele, Hermann

2001-01-01

366

Kinetics of isothermal and non-isothermal crystallization of poly(ethylene oxide) (PEO) in PEO\\/fatty acid blends  

Microsoft Academic Search

In this work isothermal and non-isothermal crystallization kinetics of poly(ethylene oxide) (PEO) and PEO in PEO\\/fatty acid (lauric and stearic acid) blends, that are used as thermal energy storage materials, was studied using differential scanning calorimetry (DSC) data. The Avrami equation was adopted to describe isothermal crystallization of PEO and non-isothermal crystallization was analyzed using both the modified Avrami approach

Kinga Pielichowska; Krzysztof Pielichowski

2011-01-01

367

Detection of Salmonella in chicken meat by insulated isothermal PCR.  

PubMed

Consumption of Salmonella-contaminated foods, such as poultry and fresh eggs, is known to be one of the main causes of salmonellosis. Conventional PCR methods, including real-time PCR for rapid detection of Salmonella, in general require skilled technicians and costly instruments. A recently developed novel convective PCR, insulated isothermal PCR (iiPCR), is carried out in polycarbonate capillary tubes. In this study, we designed TaqMan probes and PCR primers based on the yrfH gene encoding a heat shock protein for the iiPCR detection of Salmonella in chicken meat samples. The TaqMan probe was labeled with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine at the 5' and 3' ends, respectively. The PCR amplicon was 133 bp. A typical run of this iiPCR assay was completed within 1 h. Specific PCR products were obtained for 148 strains representing 49 serotypes of Salmonella tested. Under the same conditions, false-positive results were not obtained for 98 non-Salmonella strains tested, including strains of Enterobacteriaceae closely related to Salmonella. For chicken meat samples, with a 5-h enrichment step Salmonella at as low as 10? CFU/g of poultry meat could be detected. Because the amplification signals from the probes are detectable at 520 nm, identification of the PCR products by gel electrophoresis is not required. Compared with conventional PCR, the iiPCR system requires less expertise and provides an economical, reliable, and rapid tool for result interpretation. Detection results can be obtained within 8 h, including the enrichment and DNA extraction steps. PMID:23905786

Tsen, Hau-Yang; Shih, Chia-Ming; Teng, Ping-Hua; Chen, Hsin-Yen; Lin, Chia-Wei; Chiou, Chien-Shun; Wang, Hwa-Tang Thomas; Chang, Hsiao-Fen Grace; Chung, Te-Yu; Lee, Pei-Yu; Chiang, Yu-Cheng

2013-08-01

368

Instrument-free nucleic acid amplification assays for global health settings  

NASA Astrophysics Data System (ADS)

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.

Labarre, Paul; Boyle, David; Hawkins, Kenneth; Weigl, Bernhard

2011-05-01

369

Linked Linear Amplification: A New Method for the Amplification of DNA  

Microsoft Academic Search

Background: Linked Linear Amplification (LLA) is a new nucleic acid amplification method that uses multi- ple cycles of primer extension reactions. The presence of nonreplicable elements in LLA primers renders primer extension products unusable as templates for further amplification, leading to linear accumulation of prod- ucts. Through the use of nested primers, linear reactions can be \\

Antonio A. Reyes; Luis A. Ugozzoli; Jimmie D. Lowery; John W. Breneman; Craig S. Hixson; R. Bruce Wallace

2001-01-01

370

The MDM2 gene amplification database.  

PubMed Central

The p53 tumor suppressor gene is inactivated in human tumors by several distinct mechanisms. The best characterized inactivation mechanisms are: (i) gene mutation; (ii) p53 protein association with viral proteins; (iii) p53 protein association with the MDM2 cellular oncoprotein. The MDM2 gene has been shown to be abnormally up-regulated in human tumors and tumor cell lines by gene amplification, increased transcript levels and enhanced translation. This communication presents a brief review of the spectrum of MDM2 abnormalities in human tumors and compares the tissue distribution of MDM2 amplification and p53 mutation frequencies. In this study, 3889 samples from tumors or xenografts from 28 tumor types were examined for MDM2 amplification from previously published sources. The overall frequency of MDM2 amplification in these human tumors was 7%. Gene amplification was observed in 19 tumor types, with the highest frequency observed in soft tissue tumors (20%), osteosarcomas (16%) and esophageal carcinomas (13%). Tumors which showed a higher incidence of MDM2 amplification than p53 mutation were soft tissue tumors, testicular germ cell cancers and neuro-blastomas. Data from studies where both MDM2 amplification and p53 mutations were analyzed within the same samples showed that mutations in these two genes do not generally occur within the same tumor. In these studies, 29 out of a total of 33 MDM2 amplification-positive tumors had wild-type p53. We hypothesize that heretofore uncharacterized carcinogens favor MDM2 amplification over p53 mutations in certain tumor types. A database listing the MDM2 gene amplifications is available on the World Wide Web at http://www. infosci.coh.org/mdm2 . Charts of MDM2 amplification frequencies and comparisons with p53 genetic alterations are also available at this Web site.

Momand, J; Jung, D; Wilczynski, S; Niland, J

1998-01-01

371

Classroom Amplification Technology: Theory and Practice.  

ERIC Educational Resources Information Center

|This article reviews some relevant events in the development of acoustical standards for classrooms, describes classroom challenges to providing clear acoustical signals to children in classrooms, and outlines amplification solutions to some of those classroom challenges. Solutions include personal amplification devices and use of signal-to-noise…

Crandell, Carl C.; Smaldino, Joseph J.

2000-01-01

372

Amplification of chromosomal DNA in situ  

Microsoft Academic Search

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

Allen T. Christian; Matthew A. Coleman; James D. Tucker

2002-01-01

373

Amplification in the Cochlear Apex  

NASA Astrophysics Data System (ADS)

It is well established that an active process, the 'cochlear amplifier' increases auditory sensitivity and frequency resolution in the basal, high frequency regions of the inner ear. The amplifier depends critically on the endocochlear potential, the value of which will greatly influence hair cell transduction and amplification. In the apical, low frequency regions of the cochlea, the situation is much less clear. We therefore preformed a series of experiments on cochlear explants. A glass microelectrode inserted through Reissner's membrane allowed rapid changes in the endocochlear potential during simultaneous sound stimulation. Changes in cochlear vibrations were assessed with laser interferometry and resampled confocal imaging. At low sound pressures, displacements were in the range 1 - 10 nm. Positive current injected through the pipette resulted in substantial enhancement of vibration amplitudes, an effect that vanished at higher stimulus levels. However, the increase in amplitude was not accompanied by substantial changes in frequency tuning or response phase.

Fridberger, Anders; Jacob, Stefan

2009-02-01

374

Non-isothermal creep of metallic glasses  

SciTech Connect

Metallic glass creep investigations under linear heating were started quite long ago and have been continued hitherto. It was determined that at low stresses metallic glasses (MGs) display the Newtonian flow and the creep strain rate increases with the heating rate. A quantitative model of MGs non-isothermal creep is developed. The experiment specially carried out has shown the validity of the obtained flow law. It is revealed that temperature dependence of MGs Newtonian viscosity is determined by the heating rate and by the energy spectrum of irreversible structural relaxation.

Khonik, V.A.; Mikhailov, V.A. [State Pedagogical Univ., Voronezh (Russian Federation). Dept. of General Physics; Safonov, I.A. [State Technical Univ., Voronezh (Russian Federation). Dept. of General Physics

1997-10-01

375

Isothermal adsorption kinetics on heterogeneous surfaces  

NASA Astrophysics Data System (ADS)

Adsorption kinetics on energetically heterogeneous surfaces under isothermal conditions is analyzed using the uniform energy distribution model. Considering the quasi-equilibrium of surface diffusion between the adsorption sites with different energy, the kinetic equations d?/dt=(kp-AK)(1-?) for first-order adsorption and d?/dt=kp1-AK?(1-?) for dissociative adsorption are obtained, where K is a coefficient describing the surface diffusion equilibrium, which depends on the coverage and the energy distribution. Under isochoric conditions with p decreasing due to adsorption, surface diffusion accelerates the rate towards equilibrium significantly, as observed in static calorimetric adsorption experiments. An approximate solution in Lagergren form is derived for this condition.

Xia, Xinyu; Naumann D'Alnoncourt, Raoul; Strunk, Jennifer; Litvinov, Sergey; Muhler, Martin

2007-04-01

376

Radiation from a homogeneous isothermal sphere.  

PubMed

The theory of electromagnetic fluctuations as developed by Rytov was used to calculate the radiant power as a function of frequency radiated from a homogeneous isothermal sphere. The formula obtained is valid for all radii, frequencies, and complex dielectric constants. It is also shown that the emission coefficient computed from this theory is precisely equal to the absorption coefficient computed from the Mie theory. The formula obtained is readily adaptable to numerical calculations, and results are presented for the case of a good conducting sphere with a wide range of size parameters. PMID:20094340

Kattawar, G W; Eisner, M

1970-12-01

377

Nonlinear periodic solutions for isothermal magnetostatic atmospheres  

SciTech Connect

Magnetohydrodynamic equilibria for a plasma in a gravitational field are investigated analytically. For equilibria with one ignorable spatial coordinate, the equations reduce to a single nonlinear elliptic equation for the magnetic potential A, known as the Grad-Shafranov equation. Specifying the arbitrary functions in the latter equation, one obtains three types of nonlinear elliptic equations (a Liouville equation, a sinh Poisson equation, and a generalization of those with a sum of exponentials). Analytical solutions are obtained using the tanh method; this is elaborated in the Appendix. The solutions are adequate to describe an isothermal atmosphere in a uniform gravitational field showing parallel filaments of diffuse, magnetized plasma suspended horizontally in equilibrium.

Khater, A. H.; Kamel, E. S. [Department of Mathematics, Faculty of Science, Beni-Suef University, Beni-Suef 62511 (Egypt); Callebaut, D. K. [Department of Physics, CGB, University of Antwerp, B-2020 Antwerp (Belgium)

2008-12-15

378

Maximum work from isothermal chemical engines  

NASA Astrophysics Data System (ADS)

Upper bounds are derived for the work that can be extracted from simple isothermal chemical converters (chemical engines) that operate from a finite chemical potential reservoir. The incremental gains that stem from adding an arbitrary number of engines is derived. Results are analogous to those for heat engines fed by a finite heat capacity reservoir. Systems of practical interest include chemical reactions, electrochemical cells, and solid-state converters. The effectiveness of introducing more engines to utilize unexploited chemical potential is expressed in terms of the specific relation between mass transfer and chemical potential difference, often referred to as the device's current-voltage curve.

Gordon, J. M.

1993-01-01

379

Isothermal deformation of gamma titanium aluminide  

SciTech Connect

Gamma titanium aluminide has received considerable attention in recent years from the automotive industry as a potential material for making rotating and reciprocating components to produce a quieter and more efficient engine. The objectives of this study were to identify processing routes for the manufacture of automobile valves from gamma titanium aluminide. The issues considered were microstructure and composition of the material, and processing parameters such as deformation rates, temperatures, and total deformation. This paper examines isothermal deformation of gamma titanium aluminide in order to develop a processing window for this type of material.

Srinivasan, R.; Singh, J.P.; Tuval, E.; Weiss, I. [Wright State Univ., Dayton, OH (United States). Mechanical and Materials Engineering Dept.

1996-04-15

380

Determining enzyme kinetics via isothermal titration calorimetry.  

PubMed

Isothermal titration calorimetry (ITC) has emerged as a powerful tool for determining the thermodynamic properties of chemical or physical equilibria such as protein-protein, ligand-receptor, and protein-DNA binding interactions. The utility of ITC for determining kinetic information, however, has not been fully recognized. Methods for collecting and analyzing data on enzyme kinetics are discussed here. The step-by-step process of converting the raw heat output rate into the kinetic parameters of the Michaelis-Menten equation is explicitly stated. The hydrolysis of sucrose by invertase is used to demonstrate the capability of the instrument and method. PMID:23423886

Demarse, Neil A; Killian, Marie C; Hansen, Lee D; Quinn, Colette F

2013-01-01

381

Proposed novel dynamic equations for direct determination of activation energy in non-isothermal isothermal systems  

Microsoft Academic Search

A new mathematical approach for the estimation of apparent activation energy is proposed for non-isothermal dynamic process in both weight loss and weight gain conditions and is tested in different organic and inorganic systems. Satisfactory agreement between the new approach and the classical Arrhenius equation is observed. The proposed equations are applicable to a particular change occurring at two specified

Dipu Borah; Mrinal K. Baruah

2005-01-01

382

Some Remarks on the Classification of Water Vapor Sorption Isotherms and Blahovec and Yanniotis Isotherm Equation  

Microsoft Academic Search

Mathematical analysis was performed on equations describing water vapor sorption isotherms. It was shown that an identity exists between the Blahovec and Yanniotis (BY), D'Arcy and Watt (DW), and generalized D'Arcy and Watt (GDW) models. The original BY model has been modified to account for temperature dependence of the model parameters. This modification enables the application of this model to

Sylwester Furmaniak; Artur P. Terzyk; Piotr A. Gauden

2011-01-01

383

Comparison of isothermal and non-isothermal pipeline gas flow models  

Microsoft Academic Search

The transient flow of gas in pipes can be adequately described by a one-dimensional approach. Basic equations describing the transient flow of gas in pipes are derived from an equation of motion (or momentum), an equation of continuity, equation of energy and state equation.In much of the literature, either an isothermal or an adiabatic approach is adopted. For the case

Andrzej J. Osiadacz; Maciej Chaczykowski

2001-01-01

384

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

385

Isothermal Shocks in Fully Relativistic Accretion Disks  

NASA Astrophysics Data System (ADS)

The coupled hydrodynamic equations governing equatorial flows in thin disk accretion in the Schwarzschild metric are developed and studied under isothermal conditions. The transonic solutions representing physically allowed accretion onto black holes and the location of the sonic point are investigated. For a certain range of angular momentum, there exist two physical sonic points. A shock can occur between the two sonic points. The entire solution with a shock still satisfies the condition for black hole accretion, which requires supersonic flow near the black hole horizon. The jump condition across the shock will be modified by the relativistic effects only when the sound speed is comparable to the speed of light. The necessary condition for the presence of a shock is also changed from the classical condition M>1 where M is the Mach number to the relativistic condition M>Mc where Mc>1 is a value depending on the ratio of the sound speed to the speed of light. Stability analysis shows that only one of the two possible shocks between the two sonic points is stable. Isothermal shocks are used to explain the QPO behavior in black hole candidates such as Cygnus X-1, Nova Muscae and GX339-4.

Yang, R.; Kafatos, M.; Becker, P. A.

1993-12-01

386

Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection.  

PubMed

A rapid assay operable under isothermal or nonisothermal conditions is described, where the sensitivity of a typical molecular beacon (MB) system is improved by using thermostable RNase H to enzymatically cleave an MB composed of a DNA stem and an RNA loop (R/D-MB). On hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (~5.7× above background) due to an opening of the probe and a concomitant reduction in the Förster resonance energy transfer efficiency. The addition of thermostable RNase H resulted in the cleavage of the RNA loop, which eliminated energy transfer. The cleavage step also released bound target DNA, enabling it to bind to another R/D-MB probe and rendering the approach a cyclic amplification scheme. Full processing of R/D-MBs maximized the fluorescence signal to the fullest extent possible (12.9× above background), resulting in an approximately 2- to 2.8-fold increase in the signal-to-noise ratio observed isothermally at 50 °C following the addition of RNase H. The probe was also used to monitor real-time polymerase chain reactions by measuring enhancement of donor fluorescence on R/D-MB binding to amplified pUC19 template dilutions. Hence, the R/D-MB-RNase H scheme can be applied to a broad range of nucleic acid amplification methods. PMID:23000602

Jacroux, Thomas; Rieck, Daniel C; Cui, Rong; Ouyang, Yexin; Dong, Wen-Ji

2012-09-19

387

Sequence-specific detection of nucleic acids utilizing isothermal enrichment of G-quadruplex DNAzymes.  

PubMed

G-quadruplex DNAzymes are peroxidase-like complexes formed by nucleic acid G-quadruplexes and hemin. Various chemical sensors and biosensors have been developed, based on such DNAzymes. Here we report a novel, specific nucleic acid detection method utilizing the isothermal amplification strategy of G-quadruplex DNAzymes. In this method, an unlabeled oligonucleotide probe was used. The probing sequence of the oligonucleotide was in the form of a stem-loop structure. A G-rich sequence, containing three GGG repeats, was linked to the 5'-end of the stem-loop structure. In the presence of target, the probing sequence hybridized to the target, and a G(n) (n?2) repeat was extended from its 3'-end. This G(n) repeat, together with the three GGG repeats at the 5'-end, folded into a G-quadruplex, and displayed enhanced peroxidase acitivity upon hemin binding. Utilizing the dynamic binding interaction between the probe and its target, the enrichment of G-quadruplex DNAzymes was achieved. Using this method, simple, rapid and cost-effective nucleic acid detection could be achieved. This method displayed high target-length tolerance and good detection specificity; one-base mismatch could be judged easily, even by visual inspection. This method may be used as an auxiliary tool for amplified detection of specific DNA targets in some situations, in which isothermal detection is desirable. PMID:22595435

Xiao, Hao-Jie; Hak, Ho Chol; Kong, De-Ming; Shen, Han-Xi

2012-04-21

388

A methylation-stimulated DNA machine: an autonomous isothermal route to methyltransferase activity and inhibition analysis.  

PubMed

The operation of DNA nanomachines is generally triggered by either conformational changes of DNA nanostructure or external environmental stimuli. In the present study, we demonstrate an alternative driving force, DNA methylation, to stimulate DNA machine operation. DNA methylation changes neither DNA sequence and conformation nor external environment, however, blocks its cleavage by corresponding methylation-sensitive restriction endonuclease. We thus designed a strand displacement amplification DNA machine, which could be stimulated upon DNA methylation and then autonomously generates accumulated amounts of peroxidase-mimicking DNAzyme signaling machine products in an isothermal manner. The machine product DNAzyme could catalyze the H(2)O(2)-mediated oxidation of 2,2'-azino-bis(3-ethylbenzo thiazoline-6-sulfonic acid) (ABTS(2-)) to a colored product ABTS(·-). This methylation-stimulated DNA machine was further used as a colorimetric assay for analysis of methyltransferases activities and screening of methylation inhibitors. As compared with classical methylation assay, this facile isothermal DNA machine avoids the introduction of methylation-specific polymerase chain reaction and radioactive labels, which might be employed as an effective tool for DNA methylation analysis. PMID:20803193

Zhu, Changfeng; Wen, Yanqin; Peng, Hongzhen; Long, Yitao; He, Yao; Huang, Qing; Li, Di; Fan, Chunhai

2010-08-29

389

Moisture sorption isotherms of cereals at different temperatures.  

PubMed

In this research, moisture sorption isotherms of wheat (Kink and Lancer) barley, rye, oat and corn were determined at 20, 25, 35, 50 and 70 degrees C. The sorption isotherm curves of all cereal samples showed the characteristics of type II isotherm. This indicated that the adsorption occurred in cereal samples was a multilayer adsorption and cereal samples were of a microcapillary structure. In addition, the adsorption in cereal samples decreased as temperature increased. PMID:10795577

Ertugay, M F; Certel, M

2000-04-01

390

Quasi-isothermal measurement by TMDSC in isotactic polypropylene  

Microsoft Academic Search

We measure the frequency dependences of complex heat flows for isothermally crystallized isotactic polypropylene (iPP) by\\u000a the quasi-isothermal TMDSC. Regarding the quasi-isothermal melting processes as a kind of the single relaxation process, we\\u000a analyze them by the Debye model. The resultant heat capacity of iPP is larger (about 11%) than usual thermodynamic heat capacity.\\u000a We also found that the excess

H. Kaneko; T. Osada; M. Iijima

2008-01-01

391

Advanced backward Raman amplification seeding  

NASA Astrophysics Data System (ADS)

Next generations of ultrapowerful laser pulses, reaching exawatt and zetawatt powers within reasonably compact facilities, might be based on the backward Raman amplification (BRA) in plasmas. Amplified pulse intensities hundreds times higher than the pump intensity are already observed experimentally. More advanced BRA stages should produce even higher intensities. The largest nonfocused intensity, limited primarily by instabilities associated with the relativistic electron nonlinearity of the amplified laser pulse, is, roughly speaking, 0.1 of the fully relativistic value. It corresponds to the amplified pulse final (and shortest) duration be about the electron plasma wave period. The needed seed pulse should be at least that short then to stay ahead of the amplified pulse, rather than be shadowed by it (which would much reduce the seeding efficiency). However, at earlier BRA stages, when the amplified pulse is longer, the optimal duration of the seed pulse is also longer. This work proposes the use of self-contracting seed pulses for further optimizing the advanced BRA.

Malkin, Vladimir; Fisch, Nathaniel

2010-11-01

392

Isothermal and non-isothermal crystallization behavior of poly( l-lactic acid): Effects of stereocomplex as nucleating agent  

Microsoft Academic Search

The effects of incorporated poly(d-lactic acid) (PDLA) as poly(lactic acid) (PLA) stereocomplex crystallites on the isothermal and non-isothermal crystallization behavior of poly(l-lactic acid) (PLLA) from the melt were investigated for a wide PDLA contents from 0.1 to 10wt%. In isothermal crystallization from the melt, the radius growth rate of PLLA spherulites (crystallization temperature (Tc)?125°C), the induction period for PLLA spherulite

Hideto Tsuji; Hiroki Takai; Swapan Kumar Saha

2006-01-01

393

Isothermal and non-isothermal characterization of catalytic nylon membranes chemically grafted: dependence on the grafting percentage  

Microsoft Academic Search

The behaviour of five different hydrophobic ?-galactosidase derivatives, obtained by grafting different amount of butylmethacrylate (BMA) on planar nylon membranes, has been studied under isothermal and non-isothermal conditions.Under isothermal conditions the effect of the grafting percentage on the enzyme activity has been studied as a function of pH, temperature and substrate concentration. Independently from the parameters under observation, the yield

M. M El-Masry; A De Maio; M Portaccio; S Di Martino; U Bencivenga; S Rossi; F. S Gaeta; D. G Mita

2001-01-01

394

Isothermal and non-isothermal crystallization kinetics of modified rape straw flour\\/high-density polyethylene composites  

Microsoft Academic Search

The isothermal and non-isothermal crystallization kinetics of modified rape straw flour\\/high-density polyethylene (MRSF\\/HDPE) composites were investigated by means of differential scanning calorimetry (DSC). The Avrami model was applied to describe the process of isothermal crystallization. According to Hoffman–Weeks theory, the values of the equilibrium melting point (Tm0) increased with an increase in the content of MSRF in the composites. The

Peng Zou; Shangwen Tang; Zizheng Fu; Hanguo Xiong

2009-01-01

395

Artificial neural network modeling of phase volume fraction of Ti alloy under isothermal and non-isothermal hot forging conditions  

Microsoft Academic Search

An artificial neural network (ANN) model was applied to simulate the phase volume fraction of titanium alloy under isothermal\\u000a and non-isothermal hot forging condition. For isothermal hot forging process, equilibrium phase volume fraction at specific\\u000a temperature was predicted. For this purpose, chemical composition of six alloy elements (i.e. Al, V, Fe, O, N, and C) and\\u000a specimen temperature were chosen

J. H. Kim; N. S. Reddy; J. T. Yeom; C. S. Lee; N. K. Park

2007-01-01

396

Non-isothermal decomposition kinetics of diosgenin  

NASA Astrophysics Data System (ADS)

The thermal stability and kinetics of isothermal decomposition of diosgenin were studied by thermogravimetry (TG) and Differential Scanning Calorimeter (DSC). The activation energy of the thermal decomposition process was determined from the analysis of TG curves by the methods of Flynn-Wall-Ozawa, Doyle, Šatava-Šesták and Kissinger, respectively. The mechanism of thermal decomposition was determined to be Avrami-Erofeev equation ( n = 1/3, n is the reaction order) with integral form G(?) = [-ln(1 - ?)]1/3 (? = 0.10-0.80). E a and log A [s-1] were determined to be 44.10 kJ mol-1 and 3.12, respectively. Moreover, the thermodynamics properties of ? H ?, ? S ?, and ? G ? of this reaction were 38.18 kJ mol-1, -199.76 J mol-1 K-1, and 164.36 kJ mol-1 in the stage of thermal decomposition.

Chen, Fei-xiong; Fu, Li; Feng, Lu; Liu, Chuo-chuo; Ren, Bao-zeng

2013-10-01

397

Isothermal decomposition of hydrogen peroxide dihydrate.  

PubMed

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 H(2)O(2) and H(2)O are shielded from radiolytic decomposition and warmed to temperatures where sublimation is significant, highly concentrated or even pure hydrogen peroxide may form. PMID:21545167

Loeffler, M J; Baragiola, R A

2011-05-05

398

Balanced amplification: a new mechanism of selective amplification of neural activity patterns  

PubMed Central

In cerebral cortex, ongoing activity absent a stimulus can resemble stimulus-driven activity in size and structure. In particular, spontaneous activity in cat primary visual cortex (V1) has structure significantly correlated with evoked responses to oriented stimuli. This suggests that, from unstructured input, cortical circuits selectively amplify specific activity patterns. Current understanding of selective amplification involves elongation of a neural assembly’s lifetime by mutual excitation among its neurons. We introduce a new mechanism for selective amplification without elongation of lifetime: “balanced amplification”. Strong balanced amplification arises when feedback inhibition stabilizes strong recurrent excitation, a pattern likely to be typical of cortex. Thus, balanced amplification should ubiquitously contribute to cortical activity. Balanced amplification depends on the fact that individual neurons project only excitatory or only inhibitory synapses. This leads to a hidden feedforward connectivity between activity patterns. We show in a detailed biophysical model that this can explain the cat V1 observations.

Murphy, Brendan K.; Miller, Kenneth D.

2009-01-01

399

Analytical Solutions of Singular Isothermal Quadrupole Lens  

NASA Astrophysics Data System (ADS)

Using an analytical method, we study the singular isothermal quadrupole (SIQ) lens system, which is the simplest lens model that can produce four images. In this case, the radial mass distribution is in accord with the profile of the singular isothermal sphere lens, and the tangential distribution is given by adding a quadrupole on the monopole component. The basic properties of the SIQ lens have been studied in this Letter, including the deflection potential, deflection angle, magnification, critical curve, caustic, pseudo-caustic, and transition locus. Analytical solutions of the image positions and magnifications for the source on axes are derived. We find that naked cusps will appear when the relative intensity k of quadrupole to monopole is larger than 0.6. According to the magnification invariant theory of the SIQ lens, the sum of the signed magnifications of the four images should be equal to unity, as found by Dalal. However, if a source lies in the naked cusp, the summed magnification of the left three images is smaller than the invariant 1. With this simple lens system, we study the situations where a point source infinitely approaches a cusp or a fold. The sum of the magnifications of the cusp image triplet is usually not equal to 0, and it is usually positive for major cusps while negative for minor cusps. Similarly, the sum of magnifications of the fold image pair is usually not equal to 0 either. Nevertheless, the cusp and fold relations are still equal to 0 in that the sum values are divided by infinite absolute magnifications by definition.

Chu, Zhe; Lin, W. P.; Yang, Xiaofeng

2013-06-01

400

Isothermal vapour flow in extremely dry soils  

NASA Astrophysics Data System (ADS)

In dry soils hydraulic connectivity within the liquid water phase decreases and vapour flow becomes a significant transport mechanism for water. The temperature or solute concentration of the liquid phase affects the vapour pressure of the surrounding air, thus temperature or solute gradients can drive vapour flows. However, in extremely dry soils where water is retained by adsorptive forces rather than capillarity, vapour flows can also occur. In such soils tiny changes in water content significantly affect the equilibrium vapour pressure in the soil, and hence small differences in water content can initiate vapour pressure gradients. In many field conditions this effect may be negligible compared to vapour flows driven by other factors. However, flows of this type are particularly significant in a new type of subsurface irrigation system which uses pervaporation, via a polymer tubing, as the mechanism for water supply. In this system, water enters the soil in vapour phase. Experiments were performed in laboratory conditions using marine sand that had previously been oven dried and cooled. This dry sand was used to represent the desert conditions in which this irrigation system is intended for use. Experimental results show that isothermal vapour flows can significantly affect the performance of such irrigation systems due to the rapid transport of water through the soil via the vapour phase. When the irrigation pipe was buried at a depth of 10cm a vapour flow from the soil surface was observed in less than 2 hours. These flows therefore affect the loss of mass into the atmosphere and thus must be considered when evaluating the availability of water for the irrigated crop. The experiments also provide a rare opportunity to observe isothermal vapour flows initiating from a subsurface source. Such experiments allow the significance of these flows to be quantified and potentially applied to other areas of arid zone hydrology.

Todman, L. C.; Ireson, A. M.; Butler, A. P.; Templeton, M. R.

2012-04-01

401

The spatial pattern of cochlear amplification.  

PubMed

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

2012-12-01

402

Coupled amplification and sequencing of genomic DNA  

SciTech Connect

Addition of dideoxyribonucleotides during the exponential phase of the PCR should result in the synthesis of two complementary sequence ladders. The authors have explored this hypothesis to develop coupled amplification and sequencing of genomic DNA. Coupled amplification and sequencing is a biphasic method for sequencing both strands of template as they are amplified. Stage I selects and amplifies a single target form the genomic DNA sample. Stage II accomplishes the sequencing as well as additional amplification of the target using aliquots from the stage I reaction mixed with end-labeled primer and dideoxynucleotiodes. They have successfully applied coupled amplification and sequencing to a 300-base-pair fragment 4 kilobases upstream from HOX2B directly from human whole genomic DNA.

Ruano, G.; Kidd, K.K. (Yale Univ. School of Medicine, New Haven, CT (United States))

1991-04-01

403

Reflection Amplification with Active Two-Ports.  

National Technical Information Service (NTIS)

This report contains the results obtained from a study of reflection amplification with active two-ports. A design theory of the transistor reflection amplifiers based on the fundamental properties of active two-ports and broadband equlization of prescrib...

W. Ku P. Ver Planck C. Chao J. Anderson

1967-01-01

404

Hormonal Involvement in Breast Cancer Gene Amplification.  

National Technical Information Service (NTIS)

We propose to map origins of replication in the breast cancer genome and compare these sites with genomic positions that bind the estrogen receptor (ER) and genomic regions of DNA amplification. Correlations will support our hypothesis that ER adjacent to...

A. Brodsky B. Raphael S. A. Gerbi

2008-01-01

405

Parametric Oscillation and Amplification at Optical Frequencies.  

National Technical Information Service (NTIS)

The report is based on Soviet open sources published 1962-1966 and one Western source published in 1966. The fourth in a series, the present report summarizes Soviet research on parametric amplification of light. The Introduction presents background infor...

C. Shishkevish

1966-01-01

406

Development of Real-Time Multiplex Nucleic Acid Sequence-Based Amplification for Detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in Respiratory Specimens  

Microsoft Academic Search

Received 27 February 2007\\/Returned for modification 21 June 2007\\/Accepted 5 November 2007 Real-time multiplex isothermal nucleic acid sequence-based amplification (NASBA) was developed to detect Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in respiratory specimens using the NucliSens Basic Kit (bioMerieux, Boxtel, The Netherlands). Oligonucleotide primers were derived from the M. pneumoniae, C. pneumoniae, and Legionella pneumophila 16S rRNA. For real-time

K. Loens; T. Beck; D. Ursi; M. Overdijk; P. Sillekens; H. Goossens; M. Ieven

2008-01-01

407

Whole genome amplification from plant cell colonies of somatic hybrids using strand displacement amplification  

Microsoft Academic Search

The application of strand displacement amplification (SDA) is demonstrated for whole genome amplification from nanograms to\\u000a micrograms for DNA isolated from small plant cell colonies. Secondary digest amplified fragment length polymorphism (SD-AFLP)\\u000a analysis confirmed that the amplified genome is a representative of the entire genome. This approach allows the amplification\\u000a of DNA isolated from small cell colonies of putative somatic

S. V. Raikar; C. Bryant; R. Braun; A. J. Conner; M. C. Christey

2007-01-01

408

Backward Raman Amplification in a Plasma Waveguide  

NASA Astrophysics Data System (ADS)

Backward Raman amplification of a short laser pulse in a plasma waveguide is demonstrated. With a guided seed pulse of 0.8-?J energy and a pump pulse of 345-mJ energy in a 9-mm-long optically preformed plasma waveguide, 910-fold energy amplification is achieved. Heating of the plasma by the long pump pulse is identified to be a key issue for plasma-waveguide-based backward Raman amplifiers.

Pai, C.-H.; Lin, M.-W.; Ha, L.-C.; Huang, S.-T.; Tsou, Y.-C.; Chu, H.-H.; Lin, J.-Y.; Wang, J.; Chen, S.-Y.

2008-08-01

409

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

410

The PCP theorem by gap amplification  

Microsoft Academic Search

We present a new proof of the PCP theorem that is based on a combinatorial amplification lemma. The unsat value of a set of constraints C = (c1,...,cn), denoted UNSAT(C), is the smallest fraction of unsatisfied constraints, ranging over all possible assignments for the underlying variables.We describe a new combinatorial amplification transformation that doubles the unsat-value of a constraint-system, with

Irit Dinur

2006-01-01

411

Fluorescence Signal Amplification for Ultrasensitive DNA Detection  

Microsoft Academic Search

\\u000a Ultrasensitive and reliable DNA detection tools are currently needed for the diagnostic of infectious and genetic diseases.\\u000a To achieve the required sensitivity, one can amplify either the target DNA or the signal generated by each target molecule\\u000a detected. Since enzymatic amplification of DNA is prone to contamination or inhibition, sensors that can benefit from signal\\u000a amplification are usually more robust.

Kim Doré; Mario Leclerc; Denis Boudreau

412

Privacy Amplification Secure Against Active Adversaries  

Microsoft Academic Search

. Privacy amplification allows two parties Alice and Bob knowinga partially secret string S to extract, by communication over a publicchannel, a shorter, highly secret string S0. Bennett, Brassard, Cr'epeau,and Maurer showed that the length of S0can be almost equal to the conditionalR'enyi entropy of S given an opponent Eve's knowledge. All previousresults on privacy amplification assumed that Eve has

Ueli M. Maurer; Stefan Wolf

1997-01-01

413

Parametric Amplification of Scattered Atom Pairs  

Microsoft Academic Search

We have observed parametric generation and amplification of ultracold atom pairs. A Rb87 Bose-Einstein condensate was loaded into a one-dimensional optical lattice with quasimomentum k0 and spontaneously scattered into two final states with quasimomenta k1 and k2. Furthermore, when a seed of atoms was first created with quasimomentum k1 we observed parametric amplification of scattered atoms pairs in states k1

Gretchen K. Campbell; Jongchul Mun; Micah Boyd; Erik W. Streed; Wolfgang Ketterle; David E. Pritchard

2006-01-01

414

Continuous phase amplification with a Sagnac interferometer  

SciTech Connect

We describe a phase-amplification technique using a Sagnac interferometer. We monitor the relative phase between two paths of a precisely misaligned interferometer by measuring the average position of a split-Gaussian mode in the dark port. Although we monitor only the dark port, we show that the signal varies linearly with phase and that we can obtain similar sensitivity to balanced homodyne detection. We derive the source of the amplification using classical wave optics.

Starling, David J.; Dixon, P. Ben; Williams, Nathan S.; Jordan, Andrew N.; Howell, John C. [Department of Physics and Astronomy, University of Rochester, Rochester, New York 14627 (United States)

2010-07-15

415

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

416

Powdered Crystalline Silicotitanate (CST) Isotherms for SRS Wastes  

SciTech Connect

One of the primary inputs for modeling an ion exchange column is the equilibrium driving force for mass transfer between the solution and the solid phase. The equilibrium relationship is typically known as an isotherm. This document contains the predicted isotherms for the various Savannah River Site (SRS) waste types in equilibrium with powdered (ungranulated) crystalline silicotitanate (CST).

Jacobs, R.A.

1999-02-26

417

Effects of Schwarzschild Geometry on Isothermal Plasma Wave Dispersion  

Microsoft Academic Search

The behavior of isothermal plasma waves has been analyzed near the Schwarzschild horizon. We consider a non-rotating background with non-magnetized and magnetized plasmas. The general relativistic magnetohydrodynamical equations for the Schwarzschild planar analogue spacetime with an isothermal state of the plasma are formulated. The perturbed form of these equations is linearized and Fourier analyzed by introducing simple harmonic waves. The

M. Sharif; Umber Sheikh

2008-01-01

418

Isothermal calorimetry for biological applications in food science and technology  

Microsoft Academic Search

All physical, chemical and biological processes produce heat and isothermal calorimetry is a general measurement technique to study all kinds of processes by the heat they produce. This paper gives several examples of studies of biological processes in the food area using isothermal calorimetry. It is for example shown how different unit operations influence respiration of vegetable tissue, how the

Lars Wadsö; Federico Gómez Galindo

2009-01-01

419

Adsorption Isotherms on Aminoantipyrine Imprinted Polymer Stationary Phase  

Microsoft Academic Search

Summary The molecular imprinted technique was applied for the preparation of a polymer selector by using methacrylic acid as the functional monomer, EDMA as the crosslinker, AIBN as the initiator and aminoantipyrine as the template. The adsorption isotherms of aminoantipyrine, antipyrine, pyrazolone and the competitive adsorption isotherms of aminoantipyrine and antipyrine on the imprinted stationary phase were determined using rectangular

G. Yang; D. Wang; Z. Li; S. Zhou; Y. Chen

2003-01-01

420

Isothermal storage of solar energy in building construction  

Microsoft Academic Search

The role of advanced isothermal heat storage systems in buildings is discussed. A storage system encapsulated with phase change materials in which energy is absorbed in the hot period and released in the cold period is analyzed. The thermal behaviour of isothermal heat storage composites is examined using numerical techniques.Two methods of heat transfer with latent heat storage are described

Dariusz Heim

2010-01-01

421

Calculation of single adsorption isotherms from gravimetrically measured binary gas mixture adsorption isotherms on activated carbon at high pressures  

Microsoft Academic Search

A method is developed for the calculation of single-component adsorption isotherms from gravimetrically measured binary gas mixture adsorption isotherms at high pressures, at two temperatures and for different mole fractions of the gas phase. The adsorption of nitrogen\\/methane on active carbon Norit R1 is taken as an experimental example.

P. Braeuer; M. Salem; P. Harting; K. Quitzsch

1997-01-01

422

Adsorption isotherms in ion exchange reactions. Further treatments and remarks on the application of the Langmuir isotherm  

Microsoft Academic Search

In a previous work, the author gave an adsorption isotherm for the general case of single-site heterovalent exchange and simpler approximate equations having linear forms involving the same plot used for the Langmuir isotherm. The present work gives a substantiation of these equations by calculations involving some potential practical examples. It also extends the equations to the case of two-site

Nasr Z. Misak

1995-01-01

423

Comparison Between Water Vapor Sorption Isotherms Obtained Using The New Dynamic Dewpoint Isotherm Method and those Obtained Using The Standard Saturated Salt Slurry Method  

Microsoft Academic Search

The Dynamic Dewpoint isotherm method directly determines sample aw using chilled-mirror technology, while changes in sample weight are tracked gravimetrically. The objective of this research was to compare the Dynamic Dewpoint isotherms to saturated salt slurry isotherms for five materials: dent corn starch, isolated soy protein, microcrystalline cellulose, crystalline sucrose, and corn flakes. The Dynamic Dewpoint isotherms were obtained using

Shelly J. Schmidt; Joo Won Lee

2012-01-01

424

COMPARISON BETWEEN WATER VAPOR SORPTION ISOTHERMS OBTAINED USING THE NEW DYNAMIC DEWPOINT ISOTHERM METHOD AND THOSE OBTAINED USING THE STANDARD SATURATED SALT SLURRY METHOD  

Microsoft Academic Search

The Dynamic Dewpoint isotherm method directly determines sample aw using chilled-mirror technology, while changes in sample weight are tracked gravimetrically. The objective of this research was to compare the Dynamic Dewpoint isotherms to saturated salt slurry isotherms for five materials: dent corn starch, isolated soy protein, microcrystalline cellulose, crystalline sucrose, and corn flakes. The Dynamic Dewpoint isotherms were obtained using

Shelly J. Schmidt; Joo Won Lee

2011-01-01

425

Flux amplification in helicity injected spherical tori  

NASA Astrophysics Data System (ADS)

An important measure of the effective current drive by helicity injection into spheromaks and spherical tori is provided by the flux amplification factor, defined as the ratio between the closed poloidal flux in the relaxed mean field and the initial injector vacuum poloidal flux. Flux amplification in magnetic helicity injection is governed by a resonant behavior for Taylor-relaxed plasmas satisfying j=kB. Under the finite net toroidal flux constraint in a spherical torus (ST), the constrained linear resonance k1c is upshifted substantially from the primary Jensen-Chu resonance k1 that was known to be responsible for flux amplification in spheromak formation. Standard coaxial helicity injection into a ST operates at large M, with M the characteristic dimensionless parameter defined as the ratio between the toroidal flux in the discharge chamber and the injector poloidal flux. Meaningful flux amplification for ST plasmas is limited by a critical kr at which edge toroidal field reverses its direction. The kr is downshifted from k1 by a small amount inversely proportional to M. The maximum flux amplification factor r?(k=kr) scales linearly with M. At the other end of k, substantial flux amplification (k=ko)~1 becomes available for ko that scales inversely proportional to M, a significant departure from that in spheromak formation. These important parameters follow the inequality koamplification factor in a ST to be smaller than M. The scaling laws are given analytically in the asymptotic limit of M>>1, but numerical solutions indicate that they are useful even for M~1.

Tang, X. Z.; Boozer, A. H.

2005-04-01

426

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

Microsoft Academic Search

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

Luciano A Rigano; María R Marano; Atilio P Castagnaro; Alexandre Morais Do Amaral; Adrian A Vojnov

2010-01-01

427

USE OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION FOR RAPID DETECTION OF CHANNEL CATFISH ICTALURUS PUNCTATUS IMPORTANT BACTERIAL PATHOGEN  

Technology Transfer Automated Retrieval System (TEKTRAN)

Channel catfish Ictalurus punctatus infected with Edwardsiella ictaluri results in $40 - 50 million annual losses in profits to catfish producers. Early detection of this pathogen will be necessary for disease control and reduction of economic loss. In this communication, the loop-mediated isother...

428

Efficient, specific, compact hepatitis B diagnostic device: Optical detection of the hepatitis B virus by isothermal amplification  

Microsoft Academic Search

Hepatitis B virus (HBV) is a common viral pathogen that infects over 370 million people worldwide. It has been proven that HBV infection is associated with other clinical problems ranging from chronic hepatitis to cirrhosis or hepatocellular carcinoma. A miniature biosensor that can diagnose infectious diseases rapidly and precisely will be invaluable and in high demand. In this study, we

Szu-Yuan Lee; Chun-Nan Lee; Holl Mark; Deirdre R. Meldrum; Chii-Wann Lin

2007-01-01

429

An inexpensive and rapid diagnostic method of Koi Herpesvirus (KHV) infection by loop-mediated isothermal amplification  

Microsoft Academic Search

BACKGROUND: Koi Herpesvirus (KHV) affects both juvenile and adult common carp and koi, and is especially lethal to fry. The high mortalities caused by the disease have had a negative impact on the international koi trade. Different diagnostic techniques have been used to detect KHV, including: isolation of the virus in cell culture, electron microscopy, several PCR tests, ELISA and

Hatem Soliman; Mansour El-Matbouli

2005-01-01

430

Isothermal Dilatometric Study of Sintering in Kaolin  

NASA Astrophysics Data System (ADS)

Solid-state sintering for kaolin samples was studied by dilatometric measurements in the isothermal regime in the temperature range from 600 °C to 1100 °C. The relative expansion was measured for a period of 10h. For the temperatures up to 850 °C, we observed only a small shrinkage (less than 0.5 %), most of which took place within the first 3h of the measurements. For the temperatures above 850 °C, a significant shrinkage occurred for the whole measured time interval and reached up to 2.7 %. Anomalous behavior—a decrease in the shrinkage with the temperature—was observed in the range from 700 °C to 850 °C. The dilatometric measurements are supplemented by porosity distribution measurements. The standard spherical-grains microscopic model was applied to determine that for the initial stages of the sintering process, grain boundary diffusion was the dominant mechanism at lower temperatures (600 °C to 850 °C), whereas lattice diffusion was dominant at higher temperatures (900 °C, 1050 °C, and 1100 °C).

Ondruška, Ján; Trník, Anton; Keppert, Martin; Medved', Igor; Vozár, Libor

2012-10-01

431

Thermocompression engine cycle with isothermal expansion  

SciTech Connect

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 would permit a much larger heat transfer area than current Stirling cycle engines can accommodate, thus leading to higher practical efficiency without the penalty of increasing the dead volume of the device. Expressions for the ideal thermal efficiency and maximum work output point are derived. Results show a lower ideal efficiency than the Carnot cycle for all relevant expansion ratios, although for moderate expansion ratios, the deviation is not large if the temperature ratio exceeds 3. Furthermore, the efficiency at which most engines operate, the so-called maximum work output point, is similar to that derived from finite-rate analysis of Otto and Brayton cycles.

Peterson, R.B. [Oregon State Univ., Corvallis, OR (United States). Dept. of Mechanical Engineering

1998-04-01

432

Primase-based whole genome amplification  

PubMed Central

In vitro DNA amplification methods, such as polymerase chain reaction (PCR), rely on synthetic oligonucleotide primers for initiation of the reaction. In vivo, primers are synthesized on-template by DNA primase. The bacteriophage T7 gene 4 protein (gp4) has both primase and helicase activities. In this study, we report the development of a primase-based Whole Genome Amplification (pWGA) method, which utilizes gp4 primase to synthesize primers, eliminating the requirement of adding synthetic primers. Typical yield of pWGA from 1 ng to 10 ng of human genomic DNA input is in the microgram range, reaching over a thousand-fold amplification after 1 h of incubation at 37°C. The amplification bias on human genomic DNA is 6.3-fold among 20 loci on different chromosomes. In addition to amplifying total genomic DNA, pWGA can also be used for detection and quantification of contaminant DNA in a sample when combined with a fluorescent reporter dye. When circular DNA is used as template in pWGA, 108-fold of amplification is observed from as low as 100 copies of input. The high efficiency of pWGA in amplifying circular DNA makes it a potential tool in diagnosis and genotyping of circular human DNA viruses such as human papillomavirus (HPV).

Li, Ying; Kim, Hyun-Jin; Zheng, Chunyang; Chow, Wing Huen A.; Lim, Jeonghwa; Keenan, Brendan; Pan, Xiaojing; Lemieux, Bertrand; Kong, Huimin

2008-01-01

433

Strand displacement amplification as an in vitro model for rolling-circle replication: deletion formation and evolution during serial transfer.  

PubMed Central

Strand displacement amplification is an isothermal DNA amplification reaction based on a restriction endonuclease nicking its recognition site and a polymerase extending the nick at its 3' end, displacing the downstream strand. The reaction resembles rolling-circle replication of single-stranded phages and small plasmids. The displaced sense strand serves as target for an antisense reaction and vice versa, resulting in exponential growth and the autocatalytic nature of this in vitro reaction as long as the template is the limiting agent. We describe the optimization of strand displacement amplification for in vitro evolution experiments under serial transfer conditions. The reaction was followed and controlled by use of the fluorescent dye thiazole orange binding to the amplified DNA. We were able to maintain exponential growth conditions with a doubling time of 3.0 min throughout 100 transfers or approximately 350 molecular generations by using an automatic handling device. Homology of in vitro amplification with rolling-circle replication was mirrored by the occurring evolutionary processes. Deletion events most likely caused by a slipped mispairing mechanism as postulated for in vivo replication took place. Under our conditions, the mutation rate was high and a molecular quasi-species formed with a mutant lacking internal hairpin formation ability and thus outgrowing all other species under dGTP/dCTP deficiency. Images

Walter, N G; Strunk, G

1994-01-01

434

Adsorption isotherm studies of methyl chloride on MgO  

SciTech Connect

The wetting properties of methyl chloride (CH{sub 3}Cl) on MgO (100) surfaces have been investigated between 132 and 180 K using high-resolution adsorption isotherms. At low temperatures only one adsorption step is observed. At higher temperatures, i.e., T>158.9 K, a second isotherm step appears, signaling the presence of a layering transition. Unlike adsorption behavior on graphite, however, there is no evidence of a low-to-high-density transition in the monolayer phase. In addition to the layering properties, the isothermal compressibility and isosteric heat of adsorption of the adsorbed films are calculated. (c) 2000 The American Physical Society.

Sprung, Michael [Chemistry Department, Brookhaven National Laboratory, Upton, New York, 11973-5000 (United States); Larese, J. Z. [Chemistry Department, Brookhaven National Laboratory, Upton, New York, 11973-5000 (United States)

2000-05-15

435

Monte Carlo simulations in generalized isobaric-isothermal ensembles.  

PubMed

We present three generalized isobaric-isothermal ensemble Monte Carlo algorithms, which we refer to as the multibaric-multithermal, multibaric-isothermal, and isobaric-multithermal algorithms. These Monte Carlo simulations perform random walks widely in volume space and/or in potential energy space. From only one simulation run, one can calculate isobaric-isothermal-ensemble averages in wide ranges of pressure and temperature. We demonstrate the effectiveness of these algorithms by applying them to the Lennard-Jones 12-6 potential system with 500 particles. PMID:15447615

Okumura, Hisashi; Okamoto, Yuko

2004-08-13

436

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

437

Parametric Amplification of Scattered Atom Pairs  

NASA Astrophysics Data System (ADS)

We have observed parametric generation and amplification of ultracold atom pairs. A ^87Rb Bose-Einstein condensate was loaded into a one-dimensional optical lattice with quasimomentum k0 and spontaneously scattered into two final states with quasimomenta k1 and k2 . Furthermore, when a seed of atoms was first created with quasimomentum k1 we observed parametric amplification of scattered atoms pairs in states k1 and k2 when the phase-matching condition was fulfilled. This process is analogous to optical parametric generation (OPG) and amplification (OPA) of photons and could be used to efficiently create entangled pairs of atoms. Furthermore, these results could explain the dynamic instability of condensates in moving lattices observed in recent experiments.

Campbell, Gretchen K.; Mun, Jongchul; Boyd, Micah; Streed, Erik W.; Ketterle, Wolfgang; Pritchard, David E.

2006-05-01

438

Phase-coherent amplification of matter waves  

PubMed

Phase-coherent matter-wave amplification was demonstrated using Bose- Einstein-condensed rubidium-87 atoms. A small seed matter wave was created with coherent optical Bragg diffraction. Amplification of this seed matter wave was achieved by using the initial condensate as a gain medium through the superradiance effect. The coherence properties of the amplified matter wave, studied with a matter-wave interferometer, were shown to be locked to those of the initial seed wave. The active matter-wave device demonstrated here has great potential in the fields of atom optics, atom lithography, and precision measurements. PMID:10600733

Kozuma; Suzuki; Torii; Sugiura; Kuga; Hagley; Deng

1999-12-17

439

On sensitivity amplification in intracellular signaling cascades  

NASA Astrophysics Data System (ADS)

Sensitivity amplification has long been regarded as a virtually universal property of signal transduction cascades, yet a comprehensive parameter analysis remains a challenge even for relatively simple networks. We use a fast and accurate method to compute properties of multilevel cascades of activation-inactivation cycles and show that the monocyclic cascades amplify sensitivity only under specific conditions. In particular, it is found that efficient sensitivity amplification in a cascade, relative to the sensitivities of individual cycles, requires asymmetry in saturation of converter enzymes, with inhibitors much more saturated than activators.

Rácz, Éva; Slepchenko, Boris M.

2008-09-01

440

Amplification of tension in branched macromolecules.  

PubMed

We propose a method for the design of branched macromolecules that are capable of building up high tension ( approximately nN) in their covalent bonds without applying an external force. The tension is self-generated due to repulsion between branches and depends on the molecular architecture leading to amplification and focusing of tension in specific bonds. The simplest architecture is a pom-pom composed of a linear spacer and two z-arm stars at its ends resulting in z-fold tension amplification. Adsorption of those macromolecules on a substrate results in further increase in tension as compared to molecules in solution. PMID:19392489

Panyukov, Sergey V; Sheiko, Sergei S; Rubinstein, Michael

2009-04-07