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1

Nanoparticle based DNA biosensor for tuberculosis detection using thermophilic helicase-dependent isothermal amplification.  

PubMed

The present study describes the development of a DNA based biosensor to detect Mycobacterium tuberculosis using thermophilic helicase-dependent isothermal amplification (tHDA) and dextrin coated gold nanoparticles (AuNPs) as electrochemical reporter. The biosensor is composed of gold nanoparticles (AuNPs) and amine-terminated magnetic particles (MPs) each functionalized with a different DNA probe that specifically hybridize with opposite ends of a fragment within the IS6110 gene, which is M. tuberculosis complex (MTC) specific. After hybridization, the formed complex (MP-target-AuNP) is magnetically separated from the solution and the AuNPs are electrochemically detected on a screen printed carbon electrode (SPCE) chip. The obtained detection limit is 0.01 ng/?l of isothermally amplified target (105 bp). This biosensor system can be potentially implemented in peripheral laboratories with the use of a portable, handheld potentiostat. PMID:21616654

Torres-Chavolla, Edith; Alocilja, Evangelyn C

2011-07-15

2

Development of a novel one-tube isothermal reverse transcription thermophilic helicase-dependent amplification platform for rapid RNA detection.  

PubMed

The high complexity and cost of polymerase chain reaction-based molecular diagnostics sometimes limits their use in the clinical diagnostics setting. A new helicase-based isothermal amplification method offers an alternative to standard polymerase chain reaction, allowing amplification and detection of specific DNA sequences at a constant reaction temperature without thermocycling equipment. Herein, we describe the development of a novel one-tube isothermal reverse transcription-thermophilic helicase-dependent amplification (RT-tHDA) platform for RNA target detection based on the already established tHDA system. The RT-tHDA platform is highly sensitive and specific for a variety of RNA targets tested, including purified RNA molecules, armored RNA particles, and RNA virus. Moreover, rapid one-step RT-tHDA can be achieved by inclusion of an extreme thermostable single-stranded DNA binding protein in the reaction, resulting in one millionfold amplification of Ebola virus-armored RNA in less than 10 minutes. This RT-tHDA method expands on the known methods to amplify specific RNA targets and results in an easily prepared and contained platform. PMID:17975029

Goldmeyer, James; Kong, Huimin; Tang, Wen

2007-11-01

3

Microfluidic devices harboring unsealed reactors for real-time isothermal helicase-dependent amplification  

Microsoft Academic Search

High-throughput microchip devices used for nucleic-acid amplification require sealed reactors. This is to prevent evaporative\\u000a loss of the amplification mixture and cross-contamination, which may occur among fluidically connected reactors. In most high-throughput\\u000a nucleic-acid amplification devices, reactor sealing is achieved by microvalves. Additionally, these devices require micropumps\\u000a to distribute amplification mixture into an array of reactors, thereby increasing the device cost,

Naveen Ramalingam; Tong Chee San; Teo Jin Kai; Matthew Yew Mun Mak; Hai-Qing Gong

2009-01-01

4

Helicase-dependent amplification of nucleic acids.  

PubMed

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

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

2013-01-01

5

An integrated disposable device for DNA extraction and helicase dependent amplification.  

PubMed

Here we report the demonstration of an integrated microfluidic chip that performs helicase dependent amplification (HDA) on samples containing live bacteria. Combined chip-based sample preparation and isothermal amplification are attractive for world health applications, since the need for instrumentation to control flow rate and temperature changes are reduced or eliminated. Bacteria lysis, nucleic acid extraction, and DNA amplification with a fluorescent reporter are incorporated into a disposable polymer cartridge format. Smart passive fluidic control using a flap valve and a hydrophobic vent (with a nanoporous PTFE membrane) with a simple on-chip mixer eliminates multiple user operations. The device is able to detect as few as ten colony forming units (CFU) of E. coli in growth medium. PMID:20066496

Mahalanabis, Madhumita; Do, Jaephil; ALMuayad, Hussam; Zhang, Jane Y; Klapperich, Catherine M

2010-04-01

6

Diagnostic Devices for Isothermal Nucleic Acid Amplification  

PubMed Central

Since the development of the polymerase chain reaction (PCR) technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling. The application of diagnostic devices for isothermal DNA amplification has recently been studied extensively. In this paper, we describe the basic concepts of several isothermal amplification approaches and review recent progress in diagnostic device development.

Chang, Chia-Chen; Chen, Chien-Cheng; Wei, Shih-Chung; Lu, Hui-Hsin; Liang, Yang-Hung; Lin, Chii-Wann

2012-01-01

7

Molecular Test Based on Isothermal Helicase-Dependent Amplification for Detection of the Clostridium difficile Toxin A Gene.  

PubMed

The AmpliVue Clostridium difficile assay and a glutamate dehydrogenase (GDH)-illumigene algorithm were evaluated using 308 diarrheal stool specimens of patients suspected of having C. difficile infection. Compared to the enriched toxigenic culture method, the sensitivities, specificities, and positive and negative predictive values of the AmpliVue C. difficile assay and the GDH-illumigene-based algorithm were 91.7% (95% confidence interval [CI], 76.4 to 97.8), 100% (95% CI, 98.3 to 100), 100% (95% CI, 87 to 100), and 98.9% (95% CI, 96.6 to 99.7), respectively. PMID:24759714

Eckert, Catherine; Holscher, Eleonore; Petit, Amandine; Lalande, Valérie; Barbut, Frédéric

2014-07-01

8

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

PubMed Central

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

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

2013-01-01

9

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

PubMed

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

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

2013-01-01

10

Loop-Mediated Isothermal Amplification for Detection of African Trypanosomes  

Microsoft Academic Search

While PCR is a method of choice for the detection of African trypanosomes in both humans and animals, the expense of this method negates its use as a diagnostic method for the detection of endemic trypanosomiasis in African countries. The loop-mediated isothermal amplification (LAMP) reaction is a method that amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions

Noritaka Kuboki; Noboru Inoue; Tatsuya Sakurai; Francescopaolo Di Cello; Dennis J. Grab; Hiroshi Suzuki; Chihiro Sugimoto; Ikuo Igarashi

2003-01-01

11

Coupled isothermal polynucleotide amplification and translation system  

NASA Technical Reports Server (NTRS)

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

Joyce, Gerald F. (Inventor)

1998-01-01

12

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

13

Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification  

PubMed Central

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

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

2012-01-01

14

Wolbachia detection in insects through LAMP: loop mediated isothermal amplification  

PubMed Central

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

2014-01-01

15

Strand displacement amplification--an isothermal, in vitro DNA amplification technique.  

PubMed Central

Strand Displacement Amplification (SDA) is an isothermal, in vitro nucleic acid amplification technique based upon the ability of HincII to nick the unmodified strand of a hemiphosphorothioate form of its recognition site, and the ability of exonuclease deficient klenow (exo- klenow) to extend the 3'-end at the nick and displace the downstream DNA strand. Exponential amplification results from coupling sense and antisense reactions in which strands displaced from a sense reaction serve as target for an antisense reaction and vice versa. In the original design (G. T. Walker, M. C. Little, J. G. Nadeau and D. D. Shank (1992) Proc. Natl. Acad. Sci 89, 392-396), the target DNA sample is first cleaved with a restriction enzyme(s) in order to generate a double-stranded target fragment with defined 5'- and 3'-ends that can then undergo SDA. Although effective, target generation by restriction enzyme cleavage presents a number of practical limitations. We report a new target generation scheme that eliminates the requirement for restriction enzyme cleavage of the target sample prior to amplification. The method exploits the strand displacement activity of exo- klenow to generate target DNA copies with defined 5'- and 3'-ends. The new target generation process occurs at a single temperature (after initial heat denaturation of the double-stranded DNA). The target copies generated by this process are then amplified directly by SDA. The new protocol improves overall amplification efficiency. Amplification efficiency is also enhanced by improved reaction conditions that reduce nonspecific binding of SDA primers. Greater than 10(7)-fold amplification of a genomic sequence from Mycobacterium tuberculosis is achieved in 2 hours at 37 degrees C even in the presence of as much as 10 micrograms of human DNA per 50 microL reaction. The new target generation scheme can also be applied to techniques separate from SDA as a means of conveniently producing double-stranded fragments with 5'- and 3'-sequences modified as desired. Images

Walker, G T; Fraiser, M S; Schram, J L; Little, M C; Nadeau, J G; Malinowski, D P

1992-01-01

16

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

17

Rapid sexing of bovine preimplantation embryos using loop-mediated isothermal amplification  

Microsoft Academic Search

Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The product of LAMP is detected by the turbidity of the reaction mixture without electrophoresis. The objective of this study was to develop a rapid sexing method for bovine preimplantation embryos using LAMP. The first experiment was conducted to optimize the

Hiroki Hirayama; Soichi Kageyama; Satoru Moriyasu; Ken Sawai; Sadao Onoe; Yoshiyuki Takahashi; Seiji Katagiri; Keiko Toen; Keiko Watanabe; Tsugunori Notomi; Hidenari Yamashina; Sigenori Matsuzaki; Akira Minamihashi

2004-01-01

18

Real-Time Detection of HIV-2 by Reverse Transcription-Loop-Mediated Isothermal Amplification.  

PubMed

Currently, there are no FDA-approved nucleic acid amplification tests (NAATs) for the detection or confirmation of HIV-2 infection. Here, we describe the development of a real-time assay for the detection of HIV-2 DNA and RNA using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and the ESEQuant tube scanner, a portable isothermal amplification/detection device. PMID:24789187

Curtis, Kelly A; Niedzwiedz, Philip L; Youngpairoj, Ae S; Rudolph, Donna L; Owen, S Michele

2014-07-01

19

Shortening distance of forward and reverse primers for nucleic acid isothermal amplification.  

PubMed

Abstract Existent nucleic acid isothermal detection techniques for clinical diseases are difficult to promote greatly due to limitations in such aspects as methodology, costs of detection, amplification efficiency and conditions for operation. There is therefore an urgent need for a new isothermal amplification method with the characteristics of high accuracy, easy operation, short time of detection and low costs. We have devised a new method of nucleic acid isothermal amplification using Bst DNA polymerase under isothermal conditions (60-65°C). We call this method of amplification by shortening the distance between forward and reverse primers for nucleic acid isothermal amplification SDAMP. The results demonstrated that this technique is highly sensitive, specific and has short reaction times (40-60 min). Results of sequencing show that the products of SDAMP amplification are mainly polymers formed by series connection of monomers formed through linkage of forward primer and complementary sequences in reverse primer via a few bases. The method is different from current methods of nucleic acid amplification. Our study shows, however, that it is a specific method of nucleic acid isothermal amplification depending on interactions between primers and DNA template. PMID:24713573

Haitao, Qu; Wenchao, Zhang; Xiaohui, Zhang; Xiujun, Wang; Sulong, Li

2014-06-01

20

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

PubMed

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

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

2014-11-15

21

Detection of Bacteria Carrying the stx2 Gene by In Situ Loop-Mediated Isothermal Amplification  

Microsoft Academic Search

A new in situ DNA amplification technique for microscopic detection of bacteria carrying a specific gene is described. Loop-mediated isothermal amplification (LAMP) was used to detect stxA2 in Escherichia coli O157:H7 cells. The mild permeabilization conditions and low isothermal temperature used in the in situ LAMP method caused less cell damage than in situ PCR. It allowed use of fluorescent

Fumito Maruyama; Takehiko Kenzaka; Nobuyasu Yamaguchi; Katsuji Tani; Masao Nasu

2003-01-01

22

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

23

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

Microsoft Academic Search

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

Yasuyoshi Mori; Kentaro Nagamine; Norihiro Tomita; Tsugunori Notomi

2001-01-01

24

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

25

Ultrasensitive detection of microRNAs based on hairpin fluorescence probe assisted isothermal amplification.  

PubMed

A hairpin fluorescence probe assisted isothermal amplification strategy was used for microRNAs (miRNAs) detection. The fluorescence hairpin probe was rationally designed by software NUPACK to reduce background signal. This isothermal amplification method consisted of two circuits. The amplification strategy not only could detect miRNA, but also amplified and reversely transcribed miRNA into DNA to enhance the stability of the target. The approach was ultrasensitive and as low as 8.5×10(-15)mol/L miR-Let-7a, corresponding to 8.5×10(-20)mol miR-Let-7a in 10µL, was able to be detected within 20min at 37°C. Moreover, successful detection of miR-Let-7a in a total RNA sample was also achieved. Thus, the rapid, simple, isothermal, and highly sensitive approach should be a promising tool for on-the-spot detection. PMID:24613970

Ma, Cuiping; Liu, Sen; Shi, Chao

2014-08-15

26

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

2013-02-01

27

Isolation of Single-Stranded DNA from Loop-Mediated Isothermal Amplification Products  

Microsoft Academic Search

Loop-mediated isothermal amplification (LAMP), in which a specific DNA sequence can be directly amplified under isothermal conditions, yields DNA in large quantities of more than 500 ?g\\/ml. We have developed a method to isolate single-stranded DNA fragments from LAMP products that are stem–loop DNAs with several inverted repeats of the target DNA. This method requires the TspRI restriction enzyme, a

Kentaro Nagamine; Yoko Kuzuhara; Tsugunori Notomi

2002-01-01

28

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

Microsoft Academic Search

Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique that rapidly amplifies target DNA under isothermal conditions. In the present study, a LAMP test was designed from the serum resistance-associated (SRA) gene of Trypanosoma brucei rhodesiense, the cause of the acute form of African sleeping sickness, and used to detect parasite DNA from processed and heat-treated infected blood samples.

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

2008-01-01

29

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

30

Specific versus Nonspecific Isothermal DNA Amplification through Thermophilic Polymerase and Nicking Enzyme Activities†  

PubMed Central

Rapid isothermal nucleic acid amplification technologies can enable diagnosis of human pathogens and genetic variations in a simple, inexpensive, user-friendly format. The isothermal exponential amplification reaction (EXPAR) efficiently amplifies short oligonucleotides called triggers in less than 10 min by means of thermostable polymerase and nicking endonuclease activities. We recently demonstrated that this reaction can be coupled with upstream generation of trigger oligonucleotides from a genomic target sequence, and with downstream visual detection using DNA-functionalized gold nanospheres. The utility of EXPAR in clinical diagnostics is, however, limited by a nonspecific background amplification phenomenon, which is further investigated in this report. We found that nonspecific background amplification includes an early phase and a late phase. Observations related to late phase background amplification are in general agreement with literature reports of ab initio DNA synthesis. Early phase background amplification, which limits the sensitivity of EXPAR, differs however from previous reports of nonspecific DNA synthesis. It is observable in the presence of single-stranded oligonucleotides following the EXPAR template design rules and generates the trigger sequence expected for the EXPAR template present in the reaction. It appears to require interaction between the DNA polymerase and the single-stranded EXPAR template. Early phase background amplification can be suppressed or eliminated by physically separating the template and polymerase until the final reaction temperature has been reached, thereby enabling detection of attomolar starting trigger concentrations.

Tan, Eric; Erwin, Barbara; Dames, Shale; Ferguson, Tanya; Buechel, Megan; Irvine, Bruce; Voelkerding, Karl; Niemz, Angelika

2014-01-01

31

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

32

Loop-Mediated Isothermal Amplification Method for Rapid Detection of the Periodontopathic Bacteria Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola  

Microsoft Academic Search

Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the rapid detection of the major periodontal pathogens Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. The LAMP method 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. In this

Akihiro Yoshida; Shiori Nagashima; Toshihiro Ansai; Masayo Tachibana; Hiroaki Kato; Hajime Watari; Tsugunori Notomi; Tadamichi Takehara

2005-01-01

33

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

34

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

35

Rapid detection of tobacco viruses by reverse transcription loop-mediated isothermal amplification.  

PubMed

Tobacco viruses may cause a wide range of diseases that heavily reduce tobacco quality and yield worldwide. In order to detect viral diseases in tobacco fields, a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was established. Nucleotide amplification could be observed clearly after adding SYBR Green I, within 60 min under isothermal conditions, at 63-65 °C with a set of primers targeting the viral coat protein (CP) genes of tobacco viruses including cucumber mosaic virus (CMV), potato virus Y (PVY), tobacco etch virus (TEV), tobacco mosaic virus (TMV) and tobacco vein banding mosaic virus (TVBMV). This method has high specificity and sensitivity. The sensitivity of the RT-LAMP was 10 to 100 times higher than that of the conventional RT-PCR method. The RT-LAMP assay was proven reliable for virus diagnosis of tobacco samples from the field. PMID:22886186

Zhao, Lei; Cheng, Julong; Hao, Xingan; Tian, Xiaoman; Wu, Yunfeng

2012-12-01

36

Detection of gp43 of Paracoccidioides brasiliensis by the loop-mediated isothermal amplification (LAMP) method  

Microsoft Academic Search

Paracoccidioidomycosis is a deep mycosis caused by the thermo-dependent dimorphic fungus Paracoccidioides brasiliensis and is prevalent in Latin American countries. We detected the species specific gp43 gene of P. brasiliensis by loop-mediated isothermal amplification (LAMP) in 22 clinical and seven armadillo-derived isolates. The amplified DNA appeared as a ladder with a specific banding pattern. The advantage of the LAMP method

Shigeo Endo; Takashi Komori; Giannina Ricci; Ayako Sano; Koji Yokoyama; Akira Ohori; Katsuhiko Kamei; Marcello Franco; Makoto Miyaji; Kazuko Nishimura

2004-01-01

37

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

PubMed

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

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

2013-01-01

38

Simple System for Isothermal DNA Amplification Coupled to Lateral Flow Detection  

PubMed Central

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

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

2013-01-01

39

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

2009-05-01

40

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

PubMed

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

Yamazaki, Wataru

2013-01-01

41

Mitochondrial DNA Targets Increase Sensitivity of Malaria Detection Using Loop-Mediated Isothermal Amplification ?  

PubMed Central

Loop-mediated isothermal amplification (LAMP) of DNA offers the ability to detect very small quantities of pathogen DNA following minimal tissue sample processing and is thus an attractive methodology for point-of-care diagnostics. Previous attempts to diagnose malaria by the use of blood samples and LAMP have targeted the parasite small-subunit rRNA gene, with a resultant sensitivity for Plasmodium falciparum of around 100 parasites per ?l. Here we describe the use of mitochondrial targets for LAMP-based detection of any Plasmodium genus parasite and of P. falciparum specifically. These new targets allow routine amplification from samples containing as few as five parasites per ?l of blood. Amplification is complete within 30 to 40 min and is assessed by real-time turbidimetry, thereby offering rapid diagnosis with greater sensitivity than is achieved by the most skilled microscopist or antigen detection using lateral flow immunoassays.

Polley, Spencer D.; Mori, Yasuyoshi; Watson, Julie; Perkins, Mark D.; Gonzalez, Iveth J.; Notomi, Tsugunori; Chiodini, Peter L.; Sutherland, Colin J.

2010-01-01

42

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

PubMed Central

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

Salant, Harold; Abbasi, Ibrahim; Hamburger, Joseph

2012-01-01

43

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

PubMed

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

Salant, Harold; Abbasi, Ibrahim; Hamburger, Joseph

2012-11-01

44

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

45

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

46

Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification  

PubMed Central

Rolling-circle amplification (RCA) and ramification amplification (RAM, also known as hyperbranched RCA) are isothermal nucleic acid amplification technologies that have gained a great application in in situ signal amplification, DNA and protein microarray assays, single nucleotide polymorphism detection, as well as clinical diagnosis. Real-time detection of RCA or RAM products has been a challenge because of most real-time detection systems, including Taqman and Molecular Beacon, are designed for thermal cycling-based DNA amplification technology. In the present study, we describe a novel fluorescent probe construct, termed molecular zipper, which is specially designed for quantifying target DNA by real-time monitoring RAM reactions. Our results showed that the molecular zipper has very low background fluorescence due to the strong interaction between two strands. Once it is incorporated into the RAM products its double strand region is opened by displacement, therefore, its fluorophore releases a fluorescent signal. Applying the molecular zipper in RAM assay, we were able to detect as few as 10 molecules within 90 min reaction. A linear relationship was observed between initial input of targets and threshold time (R2 = 0.985). These results indicate that molecular zipper can be applied to real-time monitoring and qualification of RAM reaction, implying an amenable method for automatic RAM-based diagnostic assays.

Yi, Jizu; Zhang, Wandi; Zhang, David Y.

2006-01-01

47

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

PubMed

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

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

2014-01-01

48

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

PubMed

In this work, isothermal circular strand-displacement polymerization amplification assay is developed for highly specific and sensitive detection of adenosine triphosphate (ATP). The amplification process consists of circular common target molecule-displacement polymerization (CCDP) and circular nucleic acid strand-displacement polymerization (CNDP). In the presence of ATP, the complementary strand was released from the aptamer by the target recognition of ATP, and catalyzed the subsequent cycle reaction. With the polymerase and primer, the displaced target triggers the process of CCDP. With the involvement of nicking endonuclease, the released complementary strand triggers the CNDP. Combined CCDP with CNDP, the exponentially produced fluorescence probes are obtained, achieving a detection limit of ATP as low as 2.6×10(-10)M. Moreover, the proposed strategy exhibits an excellent specificity and is successfully applied in real sample assay which demonstrates potential application in practical samples. PMID:24851721

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

2014-11-15

49

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

2013-11-01

50

Towards rapid genotyping of resistant malaria parasites: could loop-mediated isothermal amplification be the solution?  

PubMed Central

Loop-mediated isothermal amplification (LAMP) is an innovative molecular technique that has been validated for point-of-care testing to diagnose malaria. Molecular detection and tracking of anti-malarial drug resistance is mainly based on highly sophisticated, costly and time-consuming techniques. With the validation of resistance-associated gene mutations in malaria parasites, there is a need to develop rapid, easy-to-use molecular tests for anti-malarial drug resistance genotyping. LAMP could be further developed as a point-of-care test to rapidly detect anti-malarial drug resistance-associated molecular markers, thereby help detecting and monitoring drug resistance in surveillance studies.

2014-01-01

51

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

2013-06-01

52

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

PubMed Central

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

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

2013-01-01

53

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

PubMed Central

Background Nucleic acid amplification is the most sensitive and specific method to detect Plasmodium falciparum. However the polymerase chain reaction remains laboratory-based and has to be conducted by trained personnel. Furthermore, the power dependency for the thermocycling process and the costly equipment necessary for the read-out are difficult to cover in resource-limited settings. This study aims to develop and evaluate a combination of isothermal nucleic acid amplification and simple lateral flow dipstick detection of the malaria parasite for point-of-care testing. Methods A specific fragment of the 18S rRNA gene of P. falciparum was amplified in 10 min at a constant 38°C using the isothermal recombinase polymerase amplification (RPA) method. With a unique probe system added to the reaction solution, the amplification product can be visualized on a simple lateral flow strip without further labelling. The combination of these methods was tested for sensitivity and specificity with various Plasmodium and other protozoa/bacterial strains, as well as with human DNA. Additional investigations were conducted to analyse the temperature optimum, reaction speed and robustness of this assay. Results The lateral flow RPA (LF-RPA) assay exhibited a high sensitivity and specificity. Experiments confirmed a detection limit as low as 100 fg of genomic P. falciparum DNA, corresponding to a sensitivity of approximately four parasites per reaction. All investigated P. falciparum strains (n?=?77) were positively tested while all of the total 11 non-Plasmodium samples, showed a negative test result. The enzymatic reaction can be conducted under a broad range of conditions from 30-45°C with high inhibitory concentration of known PCR inhibitors. A time to result of 15 min from start of the reaction to read-out was determined. Conclusions Combining the isothermal RPA and the lateral flow detection is an approach to improve molecular diagnostic for P. falciparum in resource-limited settings. The system requires none or only little instrumentation for the nucleic acid amplification reaction and the read-out is possible with the naked eye. Showing the same sensitivity and specificity as comparable diagnostic methods but simultaneously increasing reaction speed and dramatically reducing assay requirements, the method has potential to become a true point-of-care test for the malaria parasite.

2014-01-01

54

Identification of fungal pathogens by visible microarray system in combination with isothermal gene amplification.  

PubMed

The increasing incidence of infectious diseases caused by fungi in immunocompromised patients has encouraged researchers to develop rapid and accurate diagnosis methods. Identification of the causative fungal species is critical in deciding the appropriate treatment, but it is not easy to get satisfactory results due to the difficulty of fungal cultivation and morphological identification from clinical samples. In this study, we established a microarray system that can identify 42 species from 24 genera of clinically important fungal pathogens by using a chemical color reaction in the detection process. The array uses the internal transcribed spacer region of the rRNA gene for identification of fungal DNA at the species level. The specificity of this array was tested against a total of 355 target and nontarget fungal species. The fungal detection was succeeded directly from 10(3) CFU/ml for whole blood samples, and 50 fg DNA per 1 ml of serum samples indicating that the array system we established is sensitive to identify infecting fungi from clinical sample. Furthermore, we conducted isothermal amplification in place of PCR amplification and labeling. The successful identification with PCR-amplified as well as isothermally amplified target genes demonstrated that our microarray system is an efficient and robust method for identifying a variety of fungal species in a sample. PMID:24952715

Sakai, Kanae; Trabasso, Plinio; Moretti, Maria Luiza; Mikami, Yuzuru; Kamei, Katsuhiko; Gonoi, Tohru

2014-08-01

55

Detection of mink enteritis virus by loop-mediated isothermal amplification (LAMP).  

PubMed

Loop-mediated isothermal amplification (LAMP) method was discovered in the last decade but only used for the first time in the diagnosis of mink enteritis virus (MEV) infection in this study. The amplification could be completed within 60 min, under isothermal condition at 65°C, by employing a set of four primers targeting the VP2 gene of MEV. The LAMP was more sensitive than the conventional PCR, with a detection limit of 10(-1) median tissue culture infective doses (TCID(50))/ml per reaction, compared with 10 TCID(50)/ml for PCR analysis. No cross reactivity was observed for other related viruses, including canine distemper virus (CDV) and Aleutian mink disease parvovirus (AMDV). Eighty four of 230 clinical samples were found to be positive for MEV, which is higher than that determined by using the conventional PCR method (68). The results indicate the LAMP can be potentially used to determine MEV as a simple, rapid procedure. This assay would be an available alternative to PCR analysis for the diagnosis of MEV infection in mink, particularly in less well-equipped laboratories and in rural settings where resources are limited. PMID:23183142

Wang, Jianke; Cheng, Shipeng; Yi, Li; Cheng, Yuening; Yang, Shen; Xu, Hongli; Li, Zhenguang; Shi, Xinchuan; Wu, Hua; Yan, Xijun

2013-02-01

56

Loop-mediated isothermal amplification (LAMP): methods for plant species identification in food.  

PubMed

Loop-mediated isothermal amplification (LAMP) is a DNA-based analytical method that can be used as an isothermal alternative to polymerase chain reaction (PCR). In comparison to PCR, the advantage of LAMP is the possibility to perform the isothermal reaction without any sophisticated technical equipment; only a water bath is needed, and naked eye detection is sufficient. Up to now, an application of LAMP methods for the detection of even closely related plant species in food or feed matrices has not been described, whereas a large number of PCR methods for that topic are cited in the literature. The aim of the study was the evaluation of LAMP-based methods for plant species identification with respect to method parameters such as R(2), LOD, and LOQ. An existing (real-time) PCR method (for the detection of spices) was used for comparison. It could be shown that the developed LAMP methods have potential as alternative strategies to PCR in DNA-based analysis. PMID:23432417

Focke, Felix; Haase, Ilka; Fischer, Markus

2013-03-27

57

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

58

Isothermal Nucleic Acid Amplification Strategy by Cyclic Enzymatic Repairing for Highly Sensitive MicroRNA Detection.  

PubMed

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

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

2014-07-15

59

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

NASA Astrophysics Data System (ADS)

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

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

2013-01-01

60

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

PubMed Central

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

2013-01-01

61

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

PubMed Central

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

Wang, Guangxiang; Wang, Meng

2013-01-01

62

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

PubMed

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

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

2013-11-01

63

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

PubMed Central

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

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

2013-01-01

64

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

PubMed

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

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

2013-01-01

65

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

PubMed

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

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

2014-07-01

66

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

PubMed

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

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

2014-07-15

67

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

PubMed

Suspected cases of hemorrhagic enteritis associated with hemorrhagic enteritis virus (HEV) are becoming more frequent among yellow chickens in the Guangdong Province of China. In this study, we have developed a one-step, ecumenical, real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay for the rapid diagnosis of HEV. The RealAmp assay was performed at 63°C and reduced the assay time to 15min, using a simple and portable device, the ESE-Quant Tube Scanner. The detection limit of DNA was 1fg/?l, and the detection was specific only to HEV. We also used nested PCR to evaluate the application of the RealAmp assay. The coincidence rate of the two methods was 100%. Our data indicated that the RealAmp assay provides a sensitive, specific, and user-friendly diagnostic tool for the identification and quantification of HEV for field diagnosis and in laboratory research. PMID:24487182

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

2014-04-01

68

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

NASA Astrophysics Data System (ADS)

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

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

2013-11-01

69

Real-Time Isothermal Detection of Shiga Toxin-Producing Escherichia coli Using Recombinase Polymerase Amplification.  

PubMed

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

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

2014-07-01

70

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

2013-11-01

71

Detection of murine norovirus by reverse transcription loop-mediated isothermal amplification.  

PubMed

Murine norovirus (MNV) has considerable genetical and biological diversity and is recognized worldwide as the most common contaminant in laboratory mouse colonies. This study developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method with the potential to detect a broad range of MNV. RT-LAMP, using a set of five primers containing mixed bases, obtained results under isothermal conditions at 62°C for 90min. Sensitivity of RT-LAMP was 50-fold less than that of two-step TaqMan real-time reverse transcription-polymerase chain reaction (TaqMan RT-PCR). Diagnostic performance of RT-LAMP on RNA extracted from mouse fecal specimens was compared with TaqMan RT-PCR and nested RT-PCR. MNV was detected in 54 of 120 mouse fecal specimens by RT-LAMP, and RT-LAMP had an estimated sensitivity and specificity of 96.4% and 100% compared with TaqMan RT-PCR, and 94.7% and 100% compared with nested RT-PCR. RT-LAMP, which does not require expensive instruments, might be useful for the screening of mice actively or persistently infected with MNV. PMID:24717164

Hanaki, Ken-Ichi; Ike, Fumio; Kajita, Ayako; Yasuno, Wataru; Yanagiba, Misato; Goto, Motoki; Sakai, Kouji; Ami, Yasushi; Kyuwa, Shigeru

2014-08-01

72

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

2011-03-01

73

Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of BK Virus?  

PubMed Central

Loop-mediated isothermal amplification (LAMP) is a novel method for rapid amplification of DNA. Its advantages include rapidity and minimal equipment requirement. The LAMP assay was developed for BK virus (BKV), which is a leading cause of morbidity in renal transplant recipients. The characteristics of the assay, including its specificity and sensitivity, were evaluated. BKV LAMP was performed using various incubation times with a variety of specimens, including unprocessed urine and plasma samples. A ladder pattern on gel electrophoresis, typical of successful LAMP reactions, was observed specifically only for BKV and not for other viruses. The sensitivity of the assay with 1 h of incubation was 100 copies/tube of a cloned BKV fragment. Additionally, a positive reaction was visually ascertained by a simple color reaction using SYBR green dye. BKV LAMP was also successful for urine and plasma specimens without the need for DNA extraction. Due to its simplicity and specificity, the LAMP assay can potentially be developed for “point of care” screening of BKV.

Bista, Bipin Raj; Ishwad, Chandra; Wadowsky, Robert M.; Manna, Pradip; Randhawa, Parmjeet Singh; Gupta, Gaurav; Adhikari, Meena; Tyagi, Rakhi; Gasper, Gina; Vats, Abhay

2007-01-01

74

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

PubMed Central

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

2014-01-01

75

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

PubMed

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

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

2012-03-01

76

A real-time microfluidic multiplex electrochemical loop-mediated isothermal amplification chip for differentiating bacteria.  

PubMed

This report shows that loop-mediated isothermal amplification (LAMP) of nucleic acid can be integrated on a laser etched indium tin oxide (ITO) electrode-based multiplex microfluidic chip for real-time quantitative differentiation of bacteria; we call this technique microfluidic multiplex electrochemical LAMP (?ME-LAMP) system. Three important acute upper respiratory tract infections (URTI) related bacteria, namely Mycobacterium tuberculosis (MTB), Haemophilus influenza (HIN), and Klebsiella pneumonia (KPN) were chosen for this study. We monitored the amplification process by measuring and analyzing the electrochemical signal of methylene blue (MB) through eight etched ITO electrochemical reactors. The results indicated that this assay with the ability of analyzing multiple genes qualitatively and quantitatively is highly specific, operationally simple, and cost/time effective. It exhibits high sensitivity with detection limits of 28, 17, and 16copies?L(-1) for MTB, HIN, and KPN, respectively. The whole differentiation can be finished in a short time of 45min, which has the potential to apply in clinical diagnosis. PMID:24787122

Luo, Juan; Fang, Xueen; Ye, Daixing; Li, Huixiang; Chen, Hui; Zhang, Song; Kong, Jilie

2014-10-15

77

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

PubMed

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

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

2014-02-01

78

Development and comparison of a rapid isothermal nucleic acid amplification test for typing of herpes simplex virus types 1 and 2 on a portable fluorescence detector.  

PubMed

We have developed a rapid and simple molecular test, the IsoGlow HSV Typing assay, for the detection and typing of herpes simplex virus (type 1 and 2) from genital or oral lesions. Clinical samples suspended in viral transport mediums are simply diluted and then added to a helicase-dependent amplification master mix. The amplification and detection were performed on a portable fluorescence detector called the FireFly instrument. Detection of amplification products is based on end-point analysis using cycling probe technology. An internal control nucleic acid was included in the amplification master mix to monitor the presence of amplification inhibitors in the samples. Because the device has only two fluorescence detection channels, two strategies were developed and compared to detect the internal control template: internal control detected by melting curve analysis using a dual-labeled probe, versus internal control detection using end-point fluorescence release by a CPT probe at a lower temperature. Both have a total turnaround time of about 1 hour. Clinical performance relative to herpes viral culture was evaluated using 176 clinical specimens. Both formats of the IsoGlow HSV typing assay had sensitivities comparable to that of the Food and Drug Administration-cleared IsoAmp HSV (BioHelix Corp., Beverly MA) test and specificity for the two types of HSV comparable to that of ELVIS HSV (Diagnostic Hybrids, Athens, OH). PMID:22951487

Tong, Yanhong; McCarthy, Kaitlin; Kong, Huimin; Lemieux, Bertrand

2012-11-01

79

Loop-mediated isothermal amplification-based diagnostic assay for monkeypox virus infections.  

PubMed

Monkeypox virus (MPXV) causes a smallpox-like disease in non-human primates and humans. This infection is endemic to central and western Africa. MPXV is divided into two genetically different groups, Congo Basin and West African MPXV, with the former being the more virulent. A real-time quantitative MPXV genome amplification system was developed for the diagnosis of MPXV infections using loop-mediated isothermal amplification (LAMP) technology. Primers used for genome amplification of Congo Basin (C-LAMP), West African (W-LAMP), and both Congo Basin and West African (COM-LAMP) MPXV by LAMP were designed according to the nucleotide sequences of the Congo Basin-specific D14L gene, the West African-specific partial ATI gene, and the partial ATI gene that is shared by both groups, respectively. The sensitivity and specificity of the LAMP were evaluated with nested PCR using peripheral blood and throat swab specimens collected from Congo Basin MPXV or West African MPXV-infected monkeys. The sensitivity and specificity of COM-LAMP, C-LAMP, and W-LAMP were 80% (45/56) and 100% (64/64); 79% (19/24) and 100% (24/24); and 72% (23/32) and 100% (40/40), respectively. The viremia level determined by LAMP assays increased with increases in the severity of the monkeypox-associated symptoms. The newly developed LAMP assay was confirmed to be a rapid, quantifiable, and highly sensitive and specific system effective in the diagnosis of MPXV infections. The LAMP assays made it possible to discriminate between Congo Basin and West African MPXV. The LAMP developed in this study is useful not only for diagnosis of but also for the assessment of MPXV infections. PMID:19382264

Iizuka, Itoe; Saijo, Masayuki; Shiota, Tomoyuki; Ami, Yasushi; Suzaki, Yuriko; Nagata, Noriyo; Hasegawa, Hideki; Sakai, Kouji; Fukushi, Shuetsu; Mizutani, Tetsuya; Ogata, Momoko; Nakauchi, Mina; Kurane, Ichiro; Mizuguchi, Masashi; Morikawa, Shigeru

2009-06-01

80

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

81

Reporter-triggered isothermal exponential amplification strategy in ultrasensitive homogeneous label-free electrochemical nucleic acid biosensing.  

PubMed

A simple and novel reporter-triggered isothermal exponential amplification reaction (R-EXPAR) integrated with a miniaturized electrochemical device was developed, which achieved excellent improvement (five orders of magnitude) of sensitivity toward reporter, G-quadruplex. This R-EXPAR strategy has been successfully implemented to construct a homogeneous label-free electrochemical sensor for ultrasensitive DNA detection. PMID:24781659

Nie, Ji; Zhang, De-Wen; Zhang, Fang-Ting; Yuan, Fang; Zhou, Ying-Lin; Zhang, Xin-Xiang

2014-06-14

82

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

Microsoft Academic Search

The reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is a sensitive nucleic acid diagnostic method that can amplify rapidly a target template; it can be applied for the diagnosis of viral disease in grouper aquaculture. In this study, two outer and two inner primers were designed from nervous necrosis virus (NNV) coat protein gene sequence. The reaction temperature and time

Chia-Hsuan Sung; Jenn-Kan Lu

2009-01-01

83

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

Microsoft Academic Search

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.

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

2011-01-01

84

A New Loop-Mediated Isothermal Amplification Method for Rapid, Simple, and Sensitive Detection of Leptospira spp. in Urine  

PubMed Central

We developed a new loop-mediated isothermal amplification (LAMP) method to detect rrs, a 16S rRNA gene of pathogenic Leptospira spp. in urine. The method enables detection of two leptospiral cells per reaction mixture following boiling of urine specimens. The sensitivity of this method is higher than that of culture or of flaB nested PCR.

Nakajima, Chie; Harunari, Tsunehito; Tanikawa, Tsutomu; Tokiwa, Toshihiro; Uchimura, Eriko; Furuya, Tokujiro; Mingala, Claro Niegos; Villanueva, Marvin Ardeza; Ohnishi, Makoto; Suzuki, Yasuhiko

2012-01-01

85

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

PubMed Central

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

2013-01-01

86

Sensitive and Rapid Detection of Giardia lamblia Infection in Pet Dogs using Loop-Mediated Isothermal Amplification  

PubMed Central

Giardia lamblia is recognized as one of the most prevalent parasites in dogs. The present study aimed to establish a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of G. lamblia from dogs. The fecal samples were collected and prepared for microscopic analysis, and then the genomic DNA was extracted directly from purified cysts. The concentration of DNA samples of G. lamblia were diluted by 10-fold serially ranging from 10-1 to 10-5 ng/µl for LAMP and PCR assays. The LAMP assay allows the amplification to be finished within 60 min under isothermal conditions of 63? by employing 6 oligonucleotide primers designed based on G. lamblia elongation factor 1 alpha (EF1?) gene sequence. Our tests showed that the specific amplification products were obtained only with G. lamblia, while no amplification products were detected with DNA of other related protozoans. Sensitivity evaluation indicated that the LAMP assay was sensitive 10 times more than PCR. It is concluded that LAMP is a rapid, highly sensitive and specific DNA amplification technique for detection of G. lamblia, which has implications for effective control and prevention of giardiasis.

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

2013-01-01

87

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

2013-02-01

88

Loop-Mediated Isothermal Amplification for Rapid and Reliable Diagnosis of Tuberculous Meningitis?  

PubMed Central

Diagnosis of tuberculous meningitis (TBM) is often difficult. A reliable, simple, and rapid diagnostic test that can be performed in any standard laboratory could be helpful in TBM diagnosis. In this study, a loop-mediated isothermal amplification assay (LAMP) was evaluated to rapidly detect and diagnose TBM infection and was compared to the performance of nested PCR. Six specific primers were used to recognize the IS6110 genomic sequence from Mycobacterium tuberculosis, which included one forward outer primer, one reverse outer primer, two respective inner primers, and two loop primers. The optimum reaction temperature and time were 63°C and 60 min, respectively. Nested PCR was performed targeting the IS6110 region from M. tuberculosis using a commercial kit. The LAMP method yielded a sensitivity of 88.23% and a specificity of 80%, compared to the nested-PCR assay, which yielded a sensitivity of 52.9% and a specificity of 90% for TBM diagnosis. Comparative experiments showed that the LAMP assay is a rapid, sensitive, and specific method to detect TBM infection and that it is superior to the nested-PCR assay. LAMP is very simple, and it can be performed in any laboratory and in rural settings.

Nagdev, Khushboo J.; Kashyap, Rajpal S.; Parida, Manmohan M.; Kapgate, Rajkumar C.; Purohit, Hemant J.; Taori, Girdhar M.; Daginawala, Hatim F.

2011-01-01

89

Detection of two exogenous genes in transgenic cattle by loop-mediated isothermal amplification.  

PubMed

Nucleotide-based analytical approaches are indispensable and effective, targeting for the transgenic ingredients in biotechnical products in terms of safety assessment. In this study, a loop-mediated isothermal amplification method was developed for the specific detection of exogenous nucleic acids of hLTF/hLALBA-induced transgenic cattle. The detection limit of the LAMP method was proved to be as low as 10 copies of target molecules in optimized systems, and to be 10-100 times more sensitive than the conventional PCR. Furthermore, fluorescent dye SYBR Green I was used to visualize the color changes of LAMP products by naked eyes in daylight, which resulted in distinct colors between positive and negative reactions. For the detection of transgenes, all the transgenic samples collected from hLTF and hLALBA-induced cattle were amplified by LAMP in 1 h, followed by direct visual SYBR Green I dying or gel electrophoresis. Results showed that transgenic and non-transgenic samples exhibited distinct properties in colors or electrophoresis profiles. Thus, all the results indicated that the LAMP assay was a simple and convenient method for the test of transgenic animals. PMID:22684613

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

2012-12-01

90

Development of a loop-mediated isothermal amplification assay for the detection of porcine hokovirus.  

PubMed

Hokoviruses have recently been detected as pathogens belonging to the family Parvoviridae, which comprises porcine hokovirus (PHoV) and bovine hokovirus (BHoV). In this study, we developed a loop-mediated isothermal amplification (LAMP) assay for the rapid, specific and sensitive detection of PHoV. A set of four primers specific for six regions within the PHoV VP1/2 genes was designed using online software. The reaction temperature and time were optimized at 65°C and 60 min, respectively. LAMP products were detected by agarose gel electrophoresis or by visual inspection of a color change caused by a fluorescent dye. The method was highly specific for PHoV, and no cross-reaction was observed with porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine bocavirus (PBoV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), classic swine fever virus (CSFV), or Japanese encephalitis virus (JEV). The detection limit was approximately 10 copies per reaction, which was 10 times more sensitive than conventional PCR. Furthermore, the efficiency of detection of PHoV in clinical samples was comparable to that of PCR and sequencing. These results show that the LAMP assay is a simple, rapid, sensitive and specific method for detecting PHoV. It does not require specialized equipment and can be used to detect PHoV both in the laboratory and in the field. PMID:23850717

Li, Bin; Sun, Bing; Du, Lu-ping; Mao, Ai-hua; Wen, Li-bin; Ni, Yan-xiu; Zhang, Xue-han; He, Kong-wang

2013-11-01

91

Rapid and simple detection of Ebola virus by reverse transcription-loop-mediated isothermal amplification.  

PubMed

Ebola virus (EBOV) causes severe hemorrhagic fever in humans and nonhuman primates with high mortality rates. Rapid identification of the virus is required to prevent spread of the infection. In this study, we developed and evaluated a one-step simple reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for the rapid detection of Zaire ebolavirus (ZEBOV), the most virulent species of EBOV, targeting the trailer region of the viral genome. The assay could detect 20 copies of the artificial ZEBOV RNA in 26 min with a real time-monitoring detection, and also detect 10(-3) FFU of the cell-culture propagated viruses. The reaction time needed to detect 10(4) FFU of ZEBOV was only 20 min. In addition, the assay was highly specific for ZEBOV. The RT-LAMP assay developed in this study is rapid, simple, highly specific, and sensitive for the detection of ZEBOV, and so may be an effective diagnostic tool. Furthermore, as this technique does not require sophisticated instrumentation, it seems very suitable for diagnosis in the field or laboratories in Ebola outbreak areas such as Central Africa. PMID:17194485

Kurosaki, Yohei; Takada, Ayato; Ebihara, Hideki; Grolla, Allen; Kamo, Naoki; Feldmann, Heinz; Kawaoka, Yoshihiro; Yasuda, Jiro

2007-04-01

92

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

93

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

PubMed

Bovine herpes virus-1 (BoHV-1) is a serious viral pathogen of domestic and wild cattle. Herein, we report development of a new molecular diagnostic assay for rapid and sensitive detection of BoHV-1 utilizing the loop-mediated isothermal amplification (LAMP) technique. BoHV-1-LAMP assay was optimized to amplify the target DNA by incubation the Bst-DNA polymerase enzyme with a set of specially constructed six primers, based on the gE-gene of BoHV-1 virus, at 65°C for 60min. BoHV-1-LAMP products were detected by visual inspection using SYBR Green-I stain and had a ladder-like appearance by gel electrophoresis analysis. Negative results obtained with DNA from other tested fish viruses confirmed the specificity of the assay. The analytical sensitivity of the BoHV-1-LAMP assay was 1fg of BoHV-1 DNA (dilution of 10(6)). The developed assay could successfully detect BoVH-1 DNA from clinical samples. Results of this study indicate that the developed BoHV-1-LAMP is rapid and highly sensitive assay not only for detection of BoHV-1 in clinical samples, but also for differentiation between wild-type (gE-positive) and gE-negative BoHV-1 viruses, which will improve the control programs of BoHV-1 in Egypt. PMID:24769199

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

2014-08-01

94

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

PubMed

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

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

2014-08-01

95

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

PubMed

Papaya leaf distortion mosaic virus (PLDMV) can infect transgenic papaya resistant to a related pathogen, Papaya ringspot virus (PRSV), posing a substantial threat to papaya production in China. Current detection methods, however, are unable to be used for rapid detection in the field. Here, a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of PLDMV, using a set of four RT-LAMP primers designed based on the conserved sequence of PLDMV CP. The RT-LAMP method detected specifically PLDMV and was highly sensitive, with a detection limit of 1.32×10(-6) ?g of total RNA per reaction. Indeed, the reaction was 10 times more sensitive than one-step RT-PCR, while also requiring significantly less time and equipment. The effectiveness of RT-LAMP and one-step RT-PCR in detecting the virus were compared using 90 field samples of non-transgenic papaya and 90 field samples of commercialized PRSV-resistant transgenic papaya from Hainan Island. None of the non-transgenic papaya tested positive for PLDMV using either method. In contrast, 19 of the commercialized PRSV-resistant transgenic papaya samples tested positive by RT-LAMP assay, and 6 of those tested negative by RT-PCR. Therefore, the PLDMV-specific RT-LAMP is a simple, rapid, sensitive, and cost-effective tool in the field diagnosis and control of PLDMV. PMID:24100065

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

2014-01-01

96

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

PubMed

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

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

2012-02-01

97

Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Staphylococcus aureus  

PubMed Central

Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is an important human pathogen that produces a variety of toxins and causes a wide range of infections, including soft-tissue infections, bacteremia, and staphylococcal food poisoning. A loop-mediated isothermal amplification (LAMP) assay targeting the arcC gene of S. aureus was developed and evaluated with 119 S. aureus and 25 non-S. aureus strains. The usefulness of the assay was compared with the PCR method that targets spa and arcC genes. The optimal temperature for the LAMP assay was 58.5°C with a detection limit of 2.5?ng/?L and 102?CFU/mL when compared to 12.5?ng/?L and 103?CFU/mL for PCR (spa and arcC). Both LAMP and PCR assays were 100% specific, 100% sensitive, 100% positive predictive value (PPV), and 100% negative predictive value (NPV). When tested on 30 spiked blood specimens (21 MRSA, eight non-S. aureus and one negative control), the performance of LAMP and PCR was comparable: 100% specific, 100% sensitive, 100% PPV, and 100% NPV. In conclusion, the LAMP assay was equally specific with a shorter detection time when compared to PCR in the identification of S. aureus. The LAMP assay is a promising alternative method for the rapid identification of S. aureus and could be used in resource-limited laboratories and fields.

Lim, King Ting; Teh, Cindy Shuan Ju; Thong, Kwai Lin

2013-01-01

98

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

PubMed

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

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

2014-08-01

99

[Development of the detection system for Mycobacterium tuberculosis DNA by using the isothermal DNA amplification method ICAN].  

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 developed the detection system, combined the ICAN with luminescence detection by a probe hybridization, for Mycobacterium tuberculosis DNA targeting the IS6110 insertion element. We examined performance tests of the system. This system was able to detect one copy of Mycobacterium tuberculosis DNA for only 3.5 hours, and performance of the system was equivalent or better to the Roche PCR system. We also examined a detection system by using magnetic beads, which system could shorten detection time for 2.5 hours. It was shown that the ICAN system was an efficient and sensitive detection system for Mycobacterium tuberculosis DNA from mass samples. PMID:12078054

Shimada, Masamitsu; Hino, Fumitsugu; Sagawa, Hiroaki; Mukai, Hiroyuki; Asada, Kiyozo; Kato, Ikunoshin

2002-05-01

100

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

2005-01-01

101

Evaluation of a Loop-Mediated Isothermal Amplification Method Using Fecal Specimens for Differential Detection of Taenia Species from Humans? ‡  

PubMed Central

We compared the performance of loop-mediated isothermal amplification (LAMP) with that of a multiplex PCR method for differential detection of human Taenia parasites in fecal specimens from taeniasis patients. The LAMP method, with no false positives, showed a higher sensitivity (88.4%) than the multiplex PCR (37.2%). Thus, it is expected that the LAMP method has a high value for molecular diagnosis of taeniasis.

Nkouawa, Agathe; Sako, Yasuhito; Li, Tiaoying; Chen, Xingwang; Wandra, Toni; Swastika, I. Kadek; Nakao, Minoru; Yanagida, Tetsuya; Nakaya, Kazuhiro; Qiu, Dongchuan; Ito, Akira

2010-01-01

102

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

103

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

104

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

PubMed

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

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

2011-05-21

105

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

106

Quantitative analysis of zeptomole microRNAs based on isothermal ramification amplification.  

PubMed

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

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

2009-09-01

107

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

PubMed

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

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

2014-06-01

108

Real-time quantitative LAMP (loop-mediated isothermal amplification of DNA) as a simple method for monitoring ammonia-oxidizing bacteria  

Microsoft Academic Search

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 quantitative monitoring method for environmental microorganisms targeting amoA gene in ammonia-oxidizing bacteria. Quantitative analysis was performed first by measuring fluorescence derived from an intercalation dye using a real-time thermal

Yoshiteru Aoi; Mariko Hosogai; Satoshi Tsuneda

2006-01-01

109

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

2011-05-01

110

Embryo Sexing and Sex Chromosomal Chimerism Analysis by Loop-Mediated Isothermal Amplification in Cattle and Water Buffaloes  

PubMed Central

Abstract 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.

HIRAYAMA, Hiroki; KAGEYAMA, Soichi; MORIYASU, Satoru; SAWAI, Ken; MINAMIHASHI, Akira

2013-01-01

111

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

PubMed

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

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

2008-11-01

112

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

PubMed

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

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

2013-03-31

113

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

PubMed

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

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

2013-01-21

114

A successful diagnostic case of Pneumocystis pneumonia by the loop-mediated isothermal amplification method in a patient with dermatomyositis.  

PubMed

Pneumocystis pneumonia (PCP) can occur in patients with many causes of the immunocompromised state other than human immunodeficiency virus (HIV). It is quite difficult to diagnose PCP without HIV because there is no method for detecting Pneumocystis jirovecii. Thus, non-HIV PCP continues to have high mortality. Recently, loop-mediated isothermal amplification (LAMP) is becoming an established nucleic acid amplification method offering rapid, accurate, and cost-effective diagnosis of infectious diseases. We report a non-HIV PCP case successfully diagnosed by the LAMP method. It was previously reported that PCR in BALF specimens had been the most sensitive method in the diagnosis of PCP without HIV. The LAMP method would be more sensitive than conventional PCR and an effective tool in the early diagnosis of PCP. PMID:22526388

Asai, Nobuhiro; Aoshima, Masahiro; Ohkuni, Yoshihiro; Kobayashi, Haruki; Matsunuma, Ryo; Nakashima, Kei; Katsurada, Naoko; Nakano, Hiroto; Otsuka, Yoshihito; Kawamura, Yasutaka; Matsui, Kazuo; Kaneko, Norihiro

2012-12-01

115

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

PubMed

Concurrent reactivation of herpes simplex and varicella zoster viruses is rare. Here, we describe the case of an elderly patient with herpes labialis and herpes zoster manifesting as a right-side facial eruption with vesicles and crusting. The loop-mediated isothermal amplification (LAMP) assay demonstrated the presence of both herpes simplex virus type 1 and varicella zoster virus in swab samples taken from the face, which was confirmed by real-time PCR, suggesting concurrent reactivation of both viruses. The use of the LAMP assay in the present case indicates its usefulness in the diagnosis of atypical herpes infections. PMID:24575004

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

2014-01-01

116

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

2011-02-01

117

The loop-mediated isothermal amplification assay for rapid diagnosis of Babesia canis canis infections in dogs.  

PubMed

The aim of this study was to use a rapid and easy DNA-based test, the loop-mediated isothermal amplification (LAMP), for diagnosis of Babesia canis canis infections in dogs. 10 DNA samples of 18S RNA-A and 10 DNA samples of 18S RNA-B of B. canis canis were used in the study. LAMP method could successfully detect DNA in all examined samples down to 0.1 pg dilution. Obtained results suggest that this method has high specificity and sensitivity and can be applied in analytical laboratories in diagnosis of canine babesiosis. PMID:23691588

Adaszek, ?; Jankowska, M; Kalinowski, M; Banach, T; Wu?upek, D; Winiarczyk, S

2013-01-01

118

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

119

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

PubMed

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

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

2013-11-01

120

Rapid and real-time detection of hepatitis A virus by reverse transcription loop-mediated isothermal amplification assay.  

PubMed

A one-step, single tube, real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting sequences of the untranslated region in the genome of hepatitis A virus (HAV). The RT-LAMP assay reported in this study was very simple and rapid; the HAV-specific amplification was obtained in 50 min under isothermal conditions at 62.5 degrees C by employing a set of seven primers. The RNAs of three cell-adapted HAV strains belonging to different subgenotypes (IA, IB and IIIB) were equally well amplified. The detection limits of the RT-LAMP assay for these HAV strains were 0.4-0.8 focus forming units (FFU)/reaction. The results of the calibration using the WHO international standard indicated that the RT-LAMP assay had similar sensitivity to the conventional RT-PCR method. A comparison of the results from the RT-LAMP and the LightCycler PCR assay using clinical samples in feces revealed that the findings were similar between the two methods. Although several genotypes remain to be tested, it is concluded that the new real-time RT-LAMP assay is very suitable for detection and quantitation of most prevalent genotypes of HAV in diagnostic laboratories. PMID:17604128

Yoneyama, Tetsuo; Kiyohara, Tomoko; Shimasaki, Noriko; Kobayashi, Gen; Ota, Yoshinori; Notomi, Tsugunori; Totsuka, Atsuko; Wakita, Takaji

2007-11-01

121

Real-time loop-mediated isothermal amplification (RealAmp) for the species-specific identification of Plasmodium vivax.  

PubMed

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. PMID:23349994

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

122

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

123

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

PubMed Central

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

2012-01-01

124

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

PubMed

The Chinese giant salamander (Andrias davidianus) iridovirus (GSIV) is an emerging infectious pathogen responsible for severe hemorrhagic disease and high mortality in cultured Chinese giant salamanders. A loop-mediated isothermal amplification (LAMP) assay based on the major caspid protein (MCP) gene has been developed to detect this virus. Primer pairs for the LAMP assay were designed based on the GSIV MCP gene sequence. Amplification results indicate that under optimized conditions the LAMP assay has the ability to specifically detect the virus in both diseased animals and infected epithelioma papilloma cyprinid (EPC) cells. The assay was shown to be 10-fold more sensitive than nested PCR and was able to detect concentrations of 10(-9) (approximately 0.01 pg/?L). The LAMP assay is relatively easy to perform in situ and the amplification products can be observed directly under UV light or via staining with SYBR Green I. The LAMP assay is also rapid and cost-effective. This study establishes the use of a LAMP assay for rapid detection of GSIV, which is a novel and important tool for the diagnosis of GSIV infection in laboratory or farmed Chinese giant salamanders. PMID:24025343

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

2013-12-01

125

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

Microsoft Academic Search

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

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

2005-01-01

126

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

PubMed Central

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

Subramanian, Sowmya; Gomez, Romel D.

2014-01-01

127

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

128

An Isothermal System that Couples Ligand-dependent Catalysis to Ligand-independent Exponential Amplification  

PubMed Central

A system was devised that enables quantitative, ligand-dependent exponential amplification for various ligands that can be recognized by an RNA aptamer. The aptamer is linked to an RNA enzyme that catalyzes the joining of two oligonucleotide substrates. The product of this reaction is another RNA enzyme that undergoes self-sustained replication at constant temperature, increasing in copy number exponentially. The concentration of the ligand determines the amount of time required for the replication products to reach a threshold concentration. A standardized plot of time to threshold versus ligand concentration can be used to determine the concentration of ligand in an unknown sample. This system is analogous to quantitative PCR, linking rare recognition events to subsequent exponential amplification, but unlike PCR can be applied to the quantitative detection of non-nucleic-acid ligands.

Lam, Bianca J.; Joyce, Gerald F.

2011-01-01

129

Detection and identification of IHN and ISA viruses by isothermal DNA amplification in microcapillary tubes  

Microsoft Academic Search

Unique base sequences derived from RNA of both infectious hematopoietic necrosis virus (IHNV) and infectious salmon anemia\\u000a virus (ISAV) were detected and identified using a combination of surface-associated molecular padlock DNA probes (MPPs) and\\u000a rolling circle amplification (RCA) in microcapillary tubes. DNA oligonucleotides with base sequences identical to RNA obtained\\u000a from IHNV or ISAV were recognized by MPPs. Circularized MPPs

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

2006-01-01

130

Rapid detection of Betacoronavirus 1 from clinical fecal specimens by a novel reverse transcription loop-mediated isothermal amplification assay.  

PubMed

Betacoronavirus 1 (BCoV-1) is an important pathogen causing diarrhea in calves. In the current study, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of BCoV-1 was successfully developed. The primers were designed to target the highly conserved fragment of BCoV-1 nucleocapsid gene. The assay displayed high specificity detecting only BCoV-1 with no cross reaction with other viruses. When 418 clinical samples from 6 different geographical areas of Xinjiang province were tested by the RT-LAMP method, the results indicated that this test is a simple, rapid, accurate, and sensitive method for the detection of BCoV-1. PMID:22362949

Qiao, Jun; Meng, Qingling; Cai, Xuepeng; Chen, Chuangfu; Zhang, Zaichao; Tian, Zhenzhong

2012-01-01

131

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

PubMed

We evaluated the utility of 2 methods for detection of methicillin-resistant Staphylococcus aureus (MRSA) directly from signal-positive blood culture bottles: loop-mediated isothermal amplification (LAMP) assay, and direct cefoxitin disk diffusion (DCDD) test using a 30 ?g cefoxitin disk. In parallel, standard microbiological identification and oxacillin susceptibility testing with MecA PCR was performed. Of 60 blood cultures positive for Gram-positive cocci in clusters, LAMP (via detection of the FemA and MecA genes) showed 100% sensitivity and specificity for identification of MRSA/MSSA. When coagulase-negative staphylococci were tested, sensitivity for detection of methicillin resistance was 91.7% and specificity was 100%. DCDD along with direct tube coagulase assay detected only 80.6% of MRSA/MSSA. LAMP showed higher diagnostic accuracy although DCDD was more cost-effective and did not require additional reagents or supplies. PMID:24952125

Metwally, L; Gomaa, N; Hassan, R

2014-04-01

132

One-step reverse transcription loop mediated isothermal amplification assay for sensitive and rapid detection of Cucurbit chlorotic yellows virus.  

PubMed

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of Cucurbit chlorotic yellows virus (CCYV). In this procedure, a set of four primers matching a total of six sequences in the coat protein gene region of CCYV was synthesized for the RT-LAMP assay using total RNA extracted from CCYV-infected melon leaf tissues, and the optimum reaction temperature and assay time were determined. The sensitivity assay showed that the virus was detectable in RT-LAMP reactions at dilutions of 1×10(-11), which was 10(5) times more sensitive than the RT-PCR assay. The RT-LAMP assay for CCYV and Sweet potato chlorotic stunt virus (SPCSV) exhibited high specificity for CCYV. This simple and sensitive method has potential for detection of CCYV in samples collected in the field. PMID:24056260

Wang, Zhenyue; Gu, Qinsheng; Sun, Hu; Li, Honglian; Sun, Bingjian; Liang, Xiangzhi; Yuan, Yuan; Liu, Ruili; Shi, Yan

2014-01-01

133

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

2012-04-01

134

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

PubMed

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

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

2014-05-01

135

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

PubMed

Papaya (Carica papaya L.) is established as a cash crop throughout the tropical and subtropical regions due to its easy adaptation to diverse agricultural conditions, high yields, and prompt returns. The sex types of papaya plants are hermaphrodite, male, and female. Among them, hermaphroditic plants are the major type in papaya production, because the fruit has commercial advantages over that of the other sexes. Sex inheritance in papaya is determined by the M and M(h) dominant alleles in males and hermaphrodites, respectively, and a recessive m allele in females. Currently, all hermaphrodite seeds are not available due to the lethality of dominant homozygosity. Therefore, in this study, six male-hermaphrodite-specific markers were developed for a rapid sex identification using multiplex loop-mediated isothermal amplification (mLAMP) to efficiently and precisely select hermaphroditic individuals in the seedling or early growth stage. The LM1-LAMP assay consisted of two sex-LAMP reactions for amplifying two male-specific markers (T12 and Cpsm90) in one reaction, and showed several advantages in terms of a rapid reaction time (<1 h), isothermal conditions (less equipment required), a high efficiency (0.5 ng of DNA required in the reaction mixture), and an economical reaction system (5 ?l in volume). The established method can be easily performed in the field by visual inspection and facilitates the selection of all hermaphroditic individuals in papaya production. PMID:22706662

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

2012-10-01

136

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

PubMed

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 and clinical isolates was demonstrated, with no cross-reactivity with other Neisseria spp. or with a comprehensive panel of other common human pathogens. The lower limit of detection was 6 ctrA gene copies detectable in 48 min, with positive reactions readily identifiable visually via a simple color change. Higher copy numbers could be detected in as little as 16 min. When applied to a total of 394 clinical specimens, the LAMP assay in comparison to a conventional TaqMan® based real-time polymerase chain reaction system demonstrated a sensitivity of 100% and a specificity of 98.9% with a ? coefficient of 0.942. The LAMP method represents a rapid, sensitive, and highly specific technique for the detection of N. meningitidis and has the potential to be used as a point-of-care molecular test and in resource-poor settings. PMID:21251556

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

2011-02-01

137

Rapid and real-time detection of Porcine Sapelovirus by reverse transcription loop-mediated isothermal amplification assay.  

PubMed

The present study describes the development and validation of a one-step, single-tube, and real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) detecting Porcine Sapelovirus. RT-LAMP characterized by one strand displacement reaction with the specific stem-loop structure and Bst DNA polymerase could be finished in 60 min under isothermal condition at 63 °C. RT-LAMP assay showed higher sensitivity with 10(1) copies/?L than RT-PCR for the detection of Sapelovirus. The specificity of RT-LAMP assay was validated by the absence of any cross-reaction with other closely related virus in Picornaviridae group and other common virus causing porcine diarrhea. 7 positive Sapelovirus infection out of 63 fecal samples were identified using RT-LAMP, while 5 positive samples were determined by a conventional RT-PCR. A cost-effective method for Saplovirus detection with high sensitivity and specificity was developed and evaluated. PMID:24667302

Wang, Chunyan; Yu, Dayi; Cui, Li; Hua, Xiuguo; Yuan, Congli; Sun, Huan; Liu, Yuxiao

2014-07-01

138

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

PubMed

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

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

2011-04-01

139

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

2010-02-01

140

Development of loop-mediated isothermal amplification (LAMP) targeting the GroEL gene for rapid detection of Riemerella anatipestifer.  

PubMed

Riemerella anatipestifer (RA) infections cause major economic losses in the duck industry. Detection of RA using conventional assays is time-consuming and laborious. In this study, a simple and rapid assay for the detection of RA was established based on the GroEL gene sequence of RA using loop-mediated isothermal amplification (LAMP) with a set of six primers (two outer primers, two inner primers, and two loop primers). This assay was able to detect all the tested RA strains with different serotypes. A minimum of 10 colony-forming units (CFU) of RA was detected, which represents 50-fold higher sensitivity than that of the standard polymerase chain reaction (PCR) method. This assay showed good specificity to RA strains and did not react with any other species of bacteria. The assay is rapidly completed and the amplification is achieved at a minimum of 20 min at 65 C. Furthermore, the assay successfully detected RA in the liver samples of ducklings infected with RA, suggesting that the assay could be used for the clinical diagnosis of RA infection. PMID:22017034

Han, Xiangan; Ding, Chan; He, Liang; Hu, Qinghai; Yu, Shengqing

2011-09-01

141

Rapid detection of human rotavirus using NSP4 gene specific reverse transcription loop-mediated isothermal amplification assay.  

PubMed

The seasonal outbreaks of human rotavirus (RV) infection occur every winter. Most patients are diagnosed clinically by a rapid latex agglutination detection kit or polymerase chain reaction assays for RV from stool samples, but some problems have been reported on the specificity and sensitivity of such rapid detection assays. To ratify these issues, a sensitive, specific, simple, and rapid nucleic acid based diagnostic method is expected to be introduced and the reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed to detect the RV in human stool samples by incubation at 60 °C for 1 h and amplification was confirmed by electrophoretic laddering, restriction enzyme digestion, and hydroxynapthol blue discoloration. The assay established in this study was found to detect only the RVs and no cross-reaction with other viruses, demonstrating its high specificity. By using serial samples dilution as template, the detection limit of LAMP was 10 times more than that of PCR. The results showed the potential clinical feasibility of RT-LAMP as a useful diagnostic tool for the detection of RV with high sensitivity in comparison to conventional RT-PCR. PMID:24426285

Malik, Yashpal Singh; Sharma, Kuldeep; Kumar, Naveen; Shivachandra, Sathish B; Rawat, Vinita; Rakholia, Ritu; Ranjan, Rajeev; Ganesh, Balasubramanian; Parida, Manmohan

2013-09-01

142

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

PubMed Central

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

Noren, Torbjorn; Alriksson, Ingegard; Andersson, Josefin; Akerlund, Thomas; Unemo, Magnus

2011-01-01

143

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

Microsoft Academic Search

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

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

2009-01-01

144

Highly efficient isothermal DNA amplification system using three elements of 5'-DNA-RNA-3' chimeric primers, RNaseH and strand-displacing DNA polymerase.  

PubMed

We developed an efficient method of isothermally amplifying DNA termed ICAN, Isothermal and Chimeric primer-initiated Amplification of Nucleic acids. This method allows the amplification of target DNA under isothermal conditions at around 55 degrees C using only a pair of 5'-DNA-RNA-3' chimeric primers, a thermostable RNaseH and a DNA polymerase with strong strand-displacing activity. ICAN is capable of amplifying DNA at least several times greater than the amount produced with PCR by increasing primer concentration. This method would be applicable for on-site DNA detection including gene diagnosis, and would also be suitable for 'real time' detection when combined with a cycling probe. PMID:17720718

Mukai, Hiroyuki; Uemori, Takashi; Takeda, Osamu; Kobayashi, Eiji; Yamamoto, Junko; Nishiwaki, Kazue; Enoki, Tatsuji; Sagawa, Hiroaki; Asada, Kiyozo; Kato, Ikunoshin

2007-08-01

145

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

PubMed Central

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

2014-01-01

146

Ultrasensitive detection of microRNA with isothermal amplification and a time-resolved fluorescence sensor.  

PubMed

MicroRNAs (miRNAs) play important roles in a plethora of biological and cellular processes. The levels of miRNAs can be useful biomarkers for cellular events or disease diagnosis, thus the method for sensitive and selective detection of miRNAs is imperative to miRNA discovery, study, and clinical diagnosis. Here we develop a novel method to quantify miRNA expression levels as low as attomolar sensitivity by two-stage exponential amplification reaction (EXPAR) and a time-resolved fluorescence sensor in real samples. The method reveals superior sensitivity with a detection limit of miRNA of 0.1 aM under pure conditions. The method also shows the high selectivity for discriminating differences between miRNA family members, thus providing a promising alternative to standard approaches for quantitative detection of miRNA. PMID:24561522

Wang, Ke; Zhang, Kai; Lv, Zhuwu; Zhu, Xue; Zhu, Ling; Zhou, Fanfan

2014-07-15

147

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

PubMed

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

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

2009-11-01

148

Visual detection of Potato Leafroll virus by loop-mediated isothermal amplification of DNA with the GeneFinder™ dye.  

PubMed

The most common virus affecting potatoes in the field worldwide is Potato Leafroll virus (PLRV), belonging to the family Luteoviridae, genius Plerovirus. There are several molecular methods to detect PLRV including polymerase chain reaction (PCR), Multiplex AmpliDet RNA and double antibody sandwich ELISA (DAS-ELISA). But these techniques take a long time for 3h to two days, requiring sophisticated tools. The aim of this study was to reduce the time required to detect PLRV, using a newly designed loop-mediated isothermal amplification (LAMP) technique requiring only an ordinary water bath or thermoblock. PLRV RNA was extracted from overall 80 infected naturally potato leaves. A set of six novel primers for the LAMP reaction was designed according to the highly conserved sequence of the viral coat protein (CP) gene. LAMP was carried out under isothermal conditions, applying the Bst DNA polymerase enzyme; the LAMP products were detected visually using the GeneFinder™ florescence dye. A positive result using the GeneFinder™ dye was a color change from the original orange to green. Results confirmed LAMP with GeneFinder™ provides a rapid and safe assay for detection of PLRV. Since with other molecular methods, equipping laboratories with a thermocycler or expensive detector systems is unavoidable, this assay was found to be a simple, cost-effective molecular method that has the potential to replace other diagnostic methods in primary laboratories without the need for expensive equipment or specialized techniques. It can also be considered as a reliable alternative viral detection system in further investigations. PMID:23680094

Almasi, Mohammad Amin; Erfan Manesh, Maryam; Jafary, Hossein; Dehabadi, Seyed Mohammad Hosseini

2013-09-01

149

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

PubMed

Crimean-Congo hemorrhagic fever (CCHF) virus (CCHFV) activity has been detected in Kordufan region of the Sudan in 2008 with high case-fatality rates in villages and rural hospitals in the region. Therefore, in the present study, a reverse transcription (RT) loop-mediated isothermal amplification (RT-LAMP) assay was developed and compared to nested RT-PCR for rapid detection of CCHFV targeting the small (S) RNA segment. A set of RT-LAMP primers, designed from a highly conserved region of the S segment of the viral genome, was employed to identify all the Sudanese CCHFV strains. The sensitivity studies indicated that the RT-LAMP detected 10fg of CCHFV RNA as determined by naked eye turbidity read out, which is more likely the way it would be read in a resource-poor setting. This level of sensitivity is good enough to detect most acute cases. Using agarose gel electrophoresis, the RT-LAMP assay detected as little as 0.1fg of viral RNA (equivalent to 50 viral particle). There was 100% agreement between results of the RT-LAMP and the nested PCR when testing 10-fold serial dilution of CCHFV RNA. The specificity studies indicated that there was no cross-reactivity with other related hemorrhagic fever viruses circulating in Sudan including, Rift Valley fever virus (RVFV), Dengue fever virus, and yellow fever virus. The RT-LAMP was performed under isothermal conditions at 63°C and no special apparatus was needed, which rendered the assay more economical and practical than real-time PCR in such developing countries, like Sudan. In addition, the RT-LAMP provides a valuable tool for rapid detection and differentiation of CCHFV during an outbreak of the disease in remote areas and in rural hospitals with resource-poor settings. PMID:23542058

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

2013-06-01

150

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

151

Development of a Highly Sensitive Loop-Mediated Isothermal Amplification (LAMP) Method for the Detection of Loa loa  

PubMed Central

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

Fernandez-Soto, Pedro; Mvoulouga, Prosper Obolo; Akue, Jean Paul; Aban, Julio Lopez; Santiago, Belen Vicente; Sanchez, Miguel Cordero; Muro, Antonio

2014-01-01

152

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

PubMed

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

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

2014-01-01

153

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

PubMed

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

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

2012-12-01

154

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

155

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

156

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

157

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

PubMed Central

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

2014-01-01

158

Rapid genome detection of schmallenberg virus and bovine viral diarrhea virus by use of isothermal amplification methods and high-speed real-time reverse transcriptase PCR.  

PubMed

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

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

2014-06-01

159

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

160

Rapid Identification of Chikungunya and Dengue Virus by a Real-Time Reverse Transcription-Loop-Mediated Isothermal Amplification Method  

PubMed Central

Both Chikungunya and Dengue virus belong to the acute arthropod-borne viruses. Because of the lack of specific symptoms, it is difficult to distinguish the two infections based on clinical manifestations. To identify and quantitatively detect Chikungunya and Dengue viruses, a real-time accelerated reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) platform was developed, and 26-confirmed RNA samples, 42 suspects, and 18 healthy serum samples were evaluated by the method. The RT-polymerase chain reaction (PCR) and cDNA sequencing were used as references. The results showed that it could identify the Chikungunya and Dengue virus RNA correctly in all antibody-positive samples within 1 hour, without any cross-reactions. The virus load of the positive samples was quantitatively detected with a turbidimeter. The sensitivity was 100% and specificity was 95.25%. The findings indicate that the RT-LAMP is an effective method for rapid quantity detection of Chikungunya virus and Dengue virus in serum samples with convenient operation, high specificity, and high sensitivity.

Lu, Xi; Li, Xiaobo; Mo, Ziyao; Jin, Faguang; Wang, Boliang; Zhao, Hongbo; Shan, Xiaoxiao; Shi, Lei

2012-01-01

161

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

2013-05-21

162

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

PubMed

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

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

2012-01-01

163

Rapid and Sensitive Detection of Plesiomonas shigelloides by Loop-Mediated Isothermal Amplification of the hugA Gene  

PubMed Central

Plesiomonas shigelloides is one of the causative agents of human gastroenteritis, with increasing number of reports describing such infections in recent years. In this study, the hugA gene was chosen as the target to design loop-mediated isothermal amplification (LAMP) assays for the rapid, specific, and sensitive detection of P. shigelloides. The performance of the assay with reference plasmids and spiked human stools as samples was evaluated and compared with those of quantitative PCR (qPCR). No false-positive results were observed for the 32 non-P. shigelloides strains used to evaluate assay specificity. The limit of detection for P. shigelloides was approximately 20 copies per reaction in reference plasmids and 5×103 CFU per gram in spiked human stool, which were more sensitive than the results of qPCR. When applied in human stool samples spiked with 2 low levels of P. shigelloides, the LAMP assays achieved accurate detection after 6-h enrichment. In conclusion, the LAMP assay developed in this study is a valuable method for rapid, cost-effective, and simple detection of P. shigelloides in basic clinical and field laboratories in the rural areas of China.

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

2012-01-01

164

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

PubMed

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

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

2014-05-01

165

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

PubMed Central

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 vaccine strain. The detection limit of RT-LAMP was compared with that of RT-PCR using the Takahashi vaccine strain. We detected rubella virus genome material corresponding to 30 PFU/ml in a culture fluid sample by RT-LAMP within 60 min after the extraction of RNA with equal sensitivity to RT-nested PCR. The positive result rates of RT-LAMP, RT-PCR, and virus isolation were also compared using throat swabs obtained from patients who were clinically diagnosed with acute rubella virus infection in 2004 in Tochigi, Japan. Among nine patients with clinical rubella, the positive result rates were three/nine (33.3%) for virus isolation, six/nine (66.7%) for RT-PCR, and seven/nine (77.8%) for RT-LAMP. Consequently, RT-LAMP for rubella virus would be expected to be a reliable rapid diagnostic tool in the clinical setting.

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

2006-01-01

166

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

167

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

PubMed Central

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

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

2014-01-01

168

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

PubMed

Since its invention in 2000, loop-mediated isothermal amplification (LAMP) assay has been one of the most extensively used molecular diagnostic tools in bio-medical fields due to the rapidity, accuracy, and cost-effectiveness of the technique. This technique has also earned popularity in aquaculture disease diagnosis. Aquaculture, as a result of its rapid intensification and expansion, experiences increased infectious disease occurrences. For maintenance of economic viability, rapid, sensitive and efficient diagnosis of disease causing agents is an important step prior to undertaking effective prevention and control measures in aquaculture. Constraints on time and expertise required for conventional biochemical, serological and polymerase chain reaction (PCR)-based techniques offer avenues in adoption of the LAMP by the aquaculturists at field conditions. This assay has been successfully applied in detection of several bacterial, viral and parasitic pathogens causing serious diseases in aquaculture. In this review, we endeavored to accommodate the LAMP methodology with its different recent improvements and an overview of its application for the detection of aquaculture-associated pathogens. PMID:24477385

Biswas, Gouranga; Sakai, Masahiro

2014-04-01

169

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

PubMed

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

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

2014-12-15

170

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

PubMed

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

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

2014-01-01

171

Establishment and application of a loop-mediated isothermal amplification (LAMP) system for detection of cry1Ac transgenic sugarcane  

PubMed Central

To meet the demand for detection of foreign genes in genetically modified (GM) sugarcane necessary for regulation of gene technology, an efficient method with high specificity and rapidity was developed for the cry1Ac gene, based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed using the sequence of cry1Ac along with optimized reaction conditions: 5.25?mM of Mg2+, 4:1 ratio of inner primer to outer primer, 2.0?U of Bst DNA polymerase in a reaction volume of 25.0??L. Three post-LAMP detection methods (precipitation, calcein (0.60?mM) with Mn2+ (0.05?mM) complex and SYBR Green I visualization), were shown to be effective. The sensitivity of the LAMP method was tenfold higher than that of conventional PCR when using templates of the recombinant cry1Ac plasmid or genomic DNA from cry1Ac transgenic sugarcane plants. More importantly, this system allowed detection of the foreign gene on-site when screening GM sugarcane without complex and expensive instruments, using the naked eye. This method can not only provide technological support for detection of cry1Ac, but can also further facilitate the use of this detection technique for other transgenes in GM sugarcane.

Zhou, Dinggang; Guo, Jinlong; Xu, Liping; Gao, Shiwu; Lin, Qingliang; Wu, Qibin; Wu, Luguang; Que, Youxiong

2014-01-01

172

Establishment and application of a loop-mediated isothermal amplification (LAMP) system for detection of cry1Ac transgenic sugarcane.  

PubMed

To meet the demand for detection of foreign genes in genetically modified (GM) sugarcane necessary for regulation of gene technology, an efficient method with high specificity and rapidity was developed for the cry1Ac gene, based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed using the sequence of cry1Ac along with optimized reaction conditions: 5.25?mM of Mg(2+), 4:1 ratio of inner primer to outer primer, 2.0?U of Bst DNA polymerase in a reaction volume of 25.0??L. Three post-LAMP detection methods (precipitation, calcein (0.60?mM) with Mn(2+) (0.05?mM) complex and SYBR Green I visualization), were shown to be effective. The sensitivity of the LAMP method was tenfold higher than that of conventional PCR when using templates of the recombinant cry1Ac plasmid or genomic DNA from cry1Ac transgenic sugarcane plants. More importantly, this system allowed detection of the foreign gene on-site when screening GM sugarcane without complex and expensive instruments, using the naked eye. This method can not only provide technological support for detection of cry1Ac, but can also further facilitate the use of this detection technique for other transgenes in GM sugarcane. PMID:24810230

Zhou, Dinggang; Guo, Jinlong; Xu, Liping; Gao, Shiwu; Lin, Qingliang; Wu, Qibin; Wu, Luguang; Que, Youxiong

2014-01-01

173

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

PubMed

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

Lenar?i?, Rok; Morisset, Dany; Pirc, Manca; Llop, Pablo; Ravnikar, Maja; Dreo, Tanja

2014-01-01

174

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

PubMed Central

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

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

2014-01-01

175

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

PubMed

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

Soliman, Hatem; El-Matbouli, Mansour

2010-02-01

176

Development of Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Azumiobodo hoyamushi (Kinetoplastea)  

PubMed Central

Ascidian soft tunic syndrome (AsSTS) caused by Azumiobodo hoyamushi (A. hoyamushi) is a serious aquaculture problem that results in mass mortality of ascidians. Accordingly, the early and accurate detection of A. hoyamushi would contribute substantially to disease management and prevention of transmission. Recently, the loop-mediated isothermal amplification (LAMP) method was adopted for clinical diagnosis of a range of infectious diseases. Here, the authors describe a rapid and efficient LAMP-based method targeting the 18S rDNA gene for detection of A. hoyamushi using ascidian DNA for the diagnosis of AsSTS. A. hoyamushi LAMP assay amplified the DNA of 0.01 parasites per reaction and detected A. hoyamushi in 10 ng of ascidian DNA. To validate A. hoyamushi 18S rDNA LAMP assays, AsSTS-suspected and non-diseased ascidians were examined by microscopy, PCR, and by using the LAMP assay. When PCR was used as a gold standard, the LAMP assay showed good agreement in terms of sensitivity, positive predictive value (PPV), and negative predictive value (NPV). In the present study, a LAMP assay based on directly heat-treated samples was found to be as efficient as DNA extraction using a commercial kit for detecting A. hoyamushi. Taken together, this study shows the devised A. hoyamushi LAMP assay could be used to diagnose AsSTS in a straightforward, sensitive, and specific manner, that it could be used for forecasting, surveillance, and quarantine of AsSTS.

Song, Su-Min; Sylvatrie-Danne, Dinzouna-Boutamba; Joo, So-Young; Shin, Yun Kyung; Yu, Hak Sun; Lee, Yong-Seok; Jung, Ji-Eon; Inoue, Noboru; Lee, Won Kee; Goo, Youn-Kyoung; Chung, Dong-Il

2014-01-01

177

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

PubMed

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

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

2013-12-01

178

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

PubMed

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

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

2014-06-01

179

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

PubMed

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

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

2014-06-15

180

Isothermal amplification of genetically modified DNA sequences directly from plant tissues lowers the barriers to high-throughput and field-based genotyping.  

PubMed

DNA extractions are a major cost for high-throughput genotyping. The loop-mediated isothermal amplification (LAMP) assay has been used for the detection of two genetically modified (GM) related sequences. The amplification of target DNA sequences from leaf and maize seed tissues prepared with minimum preparative treatment (disruption in water) demonstrates the ability of LAMP to work in conditions normally inhibitive to PCRs. The wide dynamic range of detection in these samples suggests that LAMP is highly sensitive even when the target is presented in such a crude form. LAMP offers a means of reducing genotyping costs as well as simplifying testing procedures. PMID:19772345

Lee, David; La Mura, Maurizio; Allnutt, Theo; Powell, Wayne; Greenland, Andy

2009-10-28

181

Development and Clinical Evaluation of sdaA Loop-Mediated Isothermal Amplification Assay for Detection of Mycobacterium tuberculosis with an Approach To Prevent Carryover Contamination.  

PubMed

A rapid and sensitive loop-mediated isothermal amplification assay for the sdaA gene of Mycobacterium tuberculosis was developed using a dUTP-uracil-N-glycosylase (dUTP-UNG) strategy to prevent carryover contamination. Evaluation of the assay using clinical specimens (n = 648) showed high specificity (97.2%) and sensitivity (100%), demonstrating its potential as a diagnostic test for tuberculosis, especially in resource-limited settings. PMID:24789191

Nimesh, Manoj; Joon, Deepali; Varma-Basil, Mandira; Saluja, Daman

2014-07-01

182

Development and Evaluation of a Novel Loop-Mediated Isothermal Amplification Method for Rapid Detection of Severe Acute Respiratory Syndrome Coronavirus  

Microsoft Academic Search

The development and evaluation of a one-step single-tube accelerated real-time quantitative reverse tran- scription (RT) loop-mediated isothermal amplification (LAMP) assay is reported for rapid detection of the severe acute respiratory syndrome coronavirus (SARS-CoV) replicase gene. A total of 49 samples (15 throat washes, 13 throat swabs, and 21 combined throat and nasal swabs) collected from patients admitted to the Hanoi-French

H. T. C. Thai; M. Q. Le; Cuong Duc Vuong; Manmohan Parida; Harumi Minekawa; Tsugunori Notomi; Futoshi Hasebe; Kouichi Morita

2004-01-01

183

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

184

Performance of reversed transcription loop-mediated isothermal amplification technique detecting EV71: a systematic review with meta-analysis.  

PubMed

Human enterovirus 71 (EV71) is the major etiological agent of hand, foot and mouth disease (HFMD), which is a common infectious disease in young children. Studies in the past have shown that reversed transcription loop-mediated isothermal amplification (RT-LAMP) was a rapid approach for the detection of EV71 in HFMD. This meta-analysis study is to evaluate the diagnostic role of RT-LAMP in detecting EV71 infection. A comprehensive literature research of PubMed, Embase, Wan Fang Data, and Chinese National Knowledge Infrastructure databases was conducted on articles aiming at the diagnostic performance of RT-LAMP in EV71 detection published before February 10, 2014. Data from selected studies were pooled to yield the summary sensitivity, specificity, positive and negative likelihood ratio (PLR, NLR), diagnostic odds ratio (DOR), and receiver operating characteristic (SROC) curve by using STATA VERSION 12.0 software. Ten studies including a total of 907 clinical samples were of high quality in this meta-analysis. Overall, the pooled sensitivity, specificity, PLR, NLR, DOR, and the area under the SROC curve was 0.99 (0.97, 1.00), 0.97 (0.94, 1.00), 5.90 (95% CI: 3.90-8.94), 0.20 (95% CI: 0.14-0.29), and 1.00 (95% CI: 0.99-1.00), respectively. The univariate analysis of potential variables showed some changes in the diagnostic performance, but none of the differences reached statistical significance. Despite inter-study variability, the test performance of RT-LAMP was consistent with real-time RT-PCR in detecting EV71. This meta-analysis suggests that RT-LAMP is a useful diagnostic tool with high sensitivity and specificity for detecting EV71. PMID:24815384

Lei, Xiaoying; Wen, Hongling; Zhao, Li; Yu, Xuejie

2014-04-01

185

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

186

Loop-Mediated Isothermal Amplification (LAMP) assay for the identification of Echinococcus multilocularis infections in canine definitive hosts  

PubMed Central

Background Alveolar echinococcosis, caused by the metacestode larval stage of Echinococcus multilocularis, is a zoonosis of public health significance and is highly prevalent in northwest China. To effectively monitor its transmission, we developed a new rapid and cheap diagnostic assay, based on loop-mediated isothermal amplification (LAMP), to identify canine definitive hosts infected with E. multilocularis. Methods The primers used in the LAMP assay were based on the mitochondrial nad5 gene of E. multilocularis and were designed using Primer Explorer V4 software. The developed LAMP assay was compared with a conventional PCR assay, using DNA extracted from the feces of dogs experimentally infected with E. multilocularis, on 189 dog fecal samples collected from three E. multilocularis-endemic regions in Qinghai province, the People’s Republic of China, and 30 negative control copro-samples from dogs from an area in Gansu province that had been subjected to an intensive de-worming program. Light microscopy was also used to examine the experimentally obtained and field collected dog copro-samples for the presence of E. multilocularis eggs. Results The E. multilocularis-positivity rates obtained for the field-collected fecal samples were 16.4% and 5.3% by the LAMP and PCR assays, respectively, and all samples obtained from the control dogs were negative. The LAMP assay was able to detect E. multilocularis DNA in the feces of experimentally infected dogs at 12 days post-infection, whereas the PCR assay was positive on the 17th day and eggs were first detectable by light microscopy at day 44 post-challenge. Conclusion The earlier specific detection of an E. multilocularis infection in dog copro-samples indicates that the LAMP assay we developed is a realistic alternative method for the field surveillance of canines in echinococcosis-endemic areas.

2014-01-01

187

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

PubMed Central

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

2014-01-01

188

Etiologic Diagnosis of Lower Respiratory Tract Bacterial Infections Using Sputum Samples and Quantitative Loop-Mediated Isothermal Amplification  

PubMed Central

Etiologic diagnoses of lower respiratory tract infections (LRTI) have been relying primarily on bacterial cultures that often fail to return useful results in time. Although DNA-based assays are more sensitive than bacterial cultures in detecting pathogens, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. Here we report a nationwide cohort study on 2986 suspected LRTI patients across P. R. China. We compared the performance of a DNA-based assay qLAMP (quantitative Loop-mediated isothermal AMPlification) with that of standard bacterial cultures in detecting a panel of eight common respiratory bacterial pathogens from sputum samples. Our qLAMP assay detects the panel of pathogens in 1047(69.28%) patients from 1533 qualified patients at the end. We found that the bacterial titer quantified based on qLAMP is a predictor of probability that the bacterium in the sample can be detected in culture assay. The relatedness of the two assays fits a logistic regression curve. We used a piecewise linear function to define breakpoints where latent pathogen abruptly change its competitive relationship with others in the panel. These breakpoints, where pathogens start to propagate abnormally, are used as cutoffs to eliminate the influence of contaminations from normal flora. With help of the cutoffs derived from statistical analysis, we are able to identify causative pathogens in 750 (48.92%) patients from qualified patients. In conclusion, qLAMP is a reliable method in quantifying bacterial titer. Despite the fact that there are always latent bacteria contaminated in sputum samples, we can identify causative pathogens based on cutoffs derived from statistical analysis of competitive relationship. Trial Registration ClinicalTrials.gov NCT00567827

Peng, Peichao; Cheng, Xiaoxing; Wang, Guoqing; Qian, Minping; Gao, Huafang; Han, Bei; Chen, Yusheng; Hu, Yinghui; Geng, Rong; Hu, Chengping; Zhang, Wei; Yang, Jingping; Wan, Huanying; Yu, Qin; Wei, Liping; Li, Jiashu; Tian, Guizhen; Wang, Qiuyue; Hu, Ke; Wang, Siqin; Wang, Ruiqin; Du, Juan; He, Bei; Ma, Jianjun; Zhong, Xiaoning; Mu, Lan; Cai, Shaoxi; Zhu, Xiangdong; Xing, Wanli; Yu, Jun; Deng, Minghua; Gao, Zhancheng

2012-01-01

189

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

190

Toehold-mediated strand displacement reaction triggered isothermal DNA amplification for highly sensitive and selective fluorescent detection of single-base mutation.  

PubMed

Highly sensitive and selective detection strategy for single-base mutations is essential for risk assessment of malignancy and disease prognosis. In this work, a fluorescent detection method for single-base mutation was proposed based on high selectivity of toehold-mediated strand displacement reaction (TSDR) and powerful signal amplification capability of isothermal DNA amplification. A discrimination probe was specially designed with a stem-loop structure and an overhanging toehold domain. Hybridization between the toehold domain and the perfect matched target initiated the TSDR along with the unfolding of the discrimination probe. Subsequently, the target sequence acted as a primer to initiate the polymerization and nicking reactions, which released a great abundant of short sequences. Finally, the released strands were annealed with the reporter probe, launching another polymerization and nicking reaction to produce lots of G-quadruplex DNA, which could bind the N-methyl mesoporphyrin IX to yield an enhanced fluorescence response. However, when there was even a single base mismatch in the target DNA, the TSDR was suppressed and so subsequent isothermal DNA amplification and fluorescence response process could not occur. The proposed approach has been successfully implemented for the identification of the single-base mutant sequences in the human KRAS gene with a detection limit of 1.8pM. Furthermore, a recovery of 90% was obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of this detection strategy for single-base mutations in biological samples. PMID:24742973

Zhu, Jing; Ding, Yongshun; Liu, Xingti; Wang, Lei; Jiang, Wei

2014-09-15

191

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

PubMed

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

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

2010-03-01

192

Rapid detection of fluoroquinolone resistance by isothermal chimeric primer-initiated amplification of nucleic acids from clinical isolates of Neisseria gonorrhoeae.  

PubMed

To ensure a complete response to fluoroquinolone therapy against Neisseria gonorrhoeae infections, rapid susceptibility determinations are required. We assessed a new approach, an isothermal chimeric primer-initiated amplification of nucleic acids (ICAN)/hybrid-chromatography method to detect rapidly fluoroquinolone resistance in N. gonorrhoeae. Comparison of the amplification results with fluoroquinolone minimum inhibitory concentrations (MICs), which were determined by an agar dilution method, showed that the new method accurately determined fluoroquinolone resistance in all ciprofloxacin- and/or gatifloxacin-resistant isolates, but agreed with results based on MICs in only 6 of 8 (75.0%) ciprofloxacin-susceptible and 7 of 12 (58.3%) gatifloxacin-susceptible isolates. Our results suggest that this method can rapidly and reliably detect point mutations in the gyrA gene as well as fluoroquinolone resistance in resistant isolates of N. gonorrhoeae. PMID:16278026

Horii, Toshinobu; Monji, Akio; Uemura, Keiichi; Nagura, Osanori

2006-06-01

193

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

PubMed

In this paper, the semi-real time electrochemical monitoring method using a screen-printed electrode, which employs reverse transcription loop-mediated isothermal amplification (RT-LAMP) for influenza virus RNA, is presented. The amplified DNA combined with methylene blue (MB), which was used as an electroactive DNA intercalator, and the electrochemical signal was monitored using square wave voltammetry in the presence of RT-LAMP reagent components. MB molecules binding to amplified DNA caused the reduction of the peak current due to the slow diffusion of MB-amplified DNA complex to the electrode surface. We successfully monitored the amplification process of DNA on the basis of RT-LAMP by measuring and analyzing the electrochemical signal of MB with only one screen-printed electrode that connected with a USB powered portable potentiostat. The peak height of the current was related to the extent of amplification of DNA and the amount of input RNA. Since laborious probe immobilization is not required and both the amplification and the monitoring are possible in a single tube, our method does not suffer from potential cross-contamination. Furthermore, our method provides a new rote for the development of electrochemical hand held biosensors. PMID:22010112

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

2011-12-21

194

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

PubMed

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

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

2013-08-01

195

[Evaluation of the simultaneous detection system for Chlamydia trachomatis/Neisseria gonorrhoeae DNA by the isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN)].  

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 developed the simultaneous detection system for Chlamydia trachomatis/Neisseria gonorrhoeae DNA, combined with luminescence detection by a probe hybridization. In the performance tests, this system was able to detect 10 to 100 copies of C. trachomatis/N. gonorrhoeae DNA for only 3.5 hours, and was highly specific to C. trachomatis/N. gonorrhoeae without any cross-reaction to C. pneumoniae, N. lactamica, N. sicca or N. meningitidis. When we tested 60 clinical samples of urine and cervical swabs, the interpretive results were completely consistent with those obtained by Roche PCR system. Of 13 positive samples by the ICAN and PCR systems for C. trachomatis, four were negative by EIA method(IDEIA Chlamydia). These results indicate that the ICAN system is an efficient and sensitive system to simultaneously detect C. trachomatis/N. gonorrhoeae DNA. PMID:14679783

Shimada, Masamitsu; Hino, Fumitsugu; Yamamoto, Junko; Mukai, Hiroyuki; Hosobe, Takahide; Onodera, Shoichi; Hoshina, Sadayori; Machida, Katsuhiko

2003-11-01

196

Real-time quantitative LAMP (loop-mediated isothermal amplification of DNA) as a simple method for monitoring ammonia-oxidizing bacteria.  

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 quantitative monitoring method for environmental microorganisms targeting amoA gene in ammonia-oxidizing bacteria. Quantitative analysis was performed first by measuring fluorescence derived from an intercalation dye using a real-time thermal cycler, and then by measuring the turbidity of the reaction solution using a real-time turbidimeter. As a result, it was possible to quantify the initial amoA DNA concentration from an environment with a sensitivity down to 10(2) DNA copies of target DNA and a dynamic range of 7-9 orders in magnitude. Background DNA from nontargeted bacteria (Pseudomonas denitrificans) that does not encode amoA gene did not affect the quantitative capability of LAMP. Over results suggested that the real-time LAMP is effective for monitoring microorganisms and their gene expression in environments. PMID:16790287

Aoi, Yoshiteru; Hosogai, Mariko; Tsuneda, Satoshi

2006-10-01

197

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

PubMed

Yellow head virus (YHV) is a highly virulent pathogen that has caused severe mortality in cultivated shrimp (Penaeus monodon and Penaeus vannamei) in Thailand. There are several technologies that are applied to detect YHV for further control of the disease. RT-PCR is currently widely used in the laboratory, but it has some disadvantages related to cost, time-consuming and complexity. An alternative assay combines RT with loop-mediated isothermal amplification (LAMP) that not only provides high specificity, sensitivity and rapidity, but is also cheaper and more suitable for field applications in shrimp aquaculture than the RT-PCR. RT-LAMP is performed under isothermal conditions with a set of four to six primers designed to recognize six to eight distinct target sequences, and it has been combined with a chromatographic lateral-flow dipstick (LFD) to detect LAMP amplified product, which avoids the use of gel electrophoresis. In this study, RT-LAMP for the detection of YHV was developed by isothermal amplification at 65 °C for 45 min, followed by hybridization with an FITC-labeled DNA probe for 5 min and detected by LFD within 5 min (time required approximately 55 min, excluding RNA extraction and preparation time). The detection limit of RT-LAMP-LFD was 0.1 pg RNA extracted from shrimp infected with YHV equivalent to the nested RT-PCR, and no cross reaction was observed with other common shrimp viral pathogens. The LAMP method described in this study showed a rapid, high sensitivity and specificity and it is recommended as user-friendly for diagnosis of YHV in the field. PMID:23219929

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

2013-03-01

198

Loop-mediated isothermal amplification as an emerging technology for detection of Yersinia ruckeri the causative agent of enteric red mouth disease in fish  

PubMed Central

Background Enteric Redmouth (ERM) disease also known as Yersiniosis is a contagious disease affecting salmonids, mainly rainbow trout. The causative agent is the gram-negative bacterium Yersinia ruckeri. The disease can be diagnosed by isolation and identification of the causative agent, or detection of the Pathogen using fluorescent antibody tests, ELISA and PCR assays. These diagnostic methods are laborious, time consuming and need well trained personnel. Results A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for detection of Y. ruckeri the etiological agent of enteric red mouth (ERM) disease in salmonids. The assay was optimised to amplify the yruI/yruR gene, which encodes Y. ruckeri quorum sensing system, in the presence of a specific primer set and Bst DNA polymerase at an isothermal temperature of 63°C for one hour. Amplification products were detected by visual inspection, agarose gel electrophoresis and by real-time monitoring of turbidity resulted by formation of LAMP amplicons. Digestion with HphI restriction enzyme demonstrated that the amplified product was unique. The specificity of the assay was verified by the absence of amplification products when tested against related bacteria. The assay had 10-fold higher sensitivity compared with conventional PCR and successfully detected Y. ruckeri not only in pure bacterial culture but also in tissue homogenates of infected fish. Conclusion The ERM-LAMP assay represents a practical alternative to the microbiological approach for rapid, sensitive and specific detection of Y. ruckeri in fish farms. The assay is carried out in one hour and needs only a heating block or water bath as laboratory furniture. The advantages of the ERM-LAMP assay make it a promising tool for molecular detection of enteric red mouth disease in fish farms.

Saleh, Mona; Soliman, Hatem; El-Matbouli, Mansour

2008-01-01

199

Development and application of loop-mediated isothermal amplification methods targeting the seM gene for detection of Streptococcus equi subsp. equi.  

PubMed

Loop-mediated isothermal amplification (LAMP) constitutes a potentially valuable diagnostic tool for rapid diagnosis of contagious diseases. In this study, we developed a novel LAMP method (seM-LAMP) to detect the seM gene of Streptococcus equi subsp. equi (S. equi), the causative agent of strangles in equids. The seM-LAMP successfully amplified the target sequence of the seM gene at 63°C within 60 min. The sensitivity of the seM-LAMP was slightly lower than the 2nd reaction of the seM semi-nested PCR. To evaluate the species specificity of the seM-LAMP, we tested 100 S. equi and 189 non-S. equi strains. Significant amplification of the DNA originating from S. equi was observed within 60 min incubation, but no amplification of non-S. equi DNA occurred. The results were identical to those of seM semi-nested PCR. To investigate the clinical usefulness of the methods, the seM-LAMP and the seM semi-nested PCR were used to screen 590 nasal swabs obtained during an outbreak of strangles. Both methods showed that 79 and 511 swabs were S. equi positive and negative, respectively, and the results were identical to those of the culture examination. These results indicate that the seM-LAMP is potentially useful for the reliable routine diagnosis of Streptococcus equi subsp. equi infections. PMID:22032896

Hobo, Seiji; Niwa, Hidekazu; Oku, Kazuomi

2012-03-01

200

Rapid Detection of Norovirus from Fecal Specimens by Real-Time Reverse Transcription-Loop-Mediated Isothermal Amplification Assay  

Microsoft Academic Search

In this study, we developed a one-step, single-tube genogroup-specific reverse transcription-loop-mediated iso- thermal amplification (RT-LAMP) assay for the detection of norovirus (NoV) genomes targeting from the C terminus of the RNA-dependent RNA polymerase gene to the capsid N-terminal\\/shell domain region. This is the first report on the development of an RT-LAMP assay for the detection of NoV genomes. Because of

Shinji Fukuda; Shinichi Takao; Masaru Kuwayama; Yukie Shimazu; Kazuo Miyazaki

2006-01-01

201

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

PubMed

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

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

2014-06-15

202

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

PubMed

A genetic diagnosis system for detecting avian influenza A (H7N9) virus infection using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) technology was developed. The RT-LAMP assay showed no cross-reactivity with seasonal influenza A (H3N2 and H1N1pdm09) or influenza B viruses circulating in humans or with avian influenza A (H5N1) viruses. The sensitivity of the RT-LAMP assay was 42.47 copies/reaction. Considering the high specificity and sensitivity of the assay for detecting the avian influenza A (H7N9) virus and that the reaction was completed within 30 min, the RT-LAMP assay developed in this study is a promising rapid diagnostic tool for avian influenza A (H7N9) virus infection. PMID:24747008

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

2014-08-01

203

Development of a method for the detection of infectious myonecrosis virus by reverse-transcription loop-mediated isothermal amplification and nucleic acid lateral flow hybrid assay.  

PubMed

We report the development of a reverse-transcription loop-mediated isothermal amplification and nucleic acid lateral flow method (RT-LAMP-NALF) for detection of infectious myonecrosis virus (IMNV). The RT-LAMP-NALF method combines simplified nucleic acid extraction, a reverse-transcription loop-mediated isothermal amplification platform, and one-step visual colorimetric confirmation of the IMNV amplified sequences using a generic NALF qualitative detection test strip. The sensitivity of RT-LAMP (using two and three primer pairs) and nested RT-LAMP (using three primer pairs) was compared by real-time reverse-transcription-polymerase chain reaction (RT-PCR) using TaqMan probe. The detection of RT-LAMP (three primer pairs) products was accomplished by using a NALF-test strip. The RT-LAMP-NALF showed equivalent sensitivity to RT-LAMP (using three primer pairs), and it was found to be 100 and 10 times more sensitive than one-step RT-PCR and RT-LAMP (two primer pairs), respectively. On the other hand, the RT-LAMP-NALF was 10 and 100 times less sensitive than nested RT-PCR and real-time RT-PCR, respectively. The simplified RNA extraction method ranged from 4.4 x 10(6) to 2.2 x 10(8) IMNV copy numbers microL(-1) RNA, and it was similar with the standard RNA extraction (from 1.2 x 10(6) to 6.3 x 10(7) IMNV copy numbers microL(-1) RNA). These results clearly demonstrate that the RT-LAMP-NALF method is specific, sensitive, can shorten the time for analysis, and has potential application for IMNV diagnosis in resource-poor diagnostic settings. PMID:19531063

Andrade, T P D; Lightner, D V

2009-11-01

204

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

205

Loop-mediated isothermal amplification method for rapid detection of the toxic dinoflagellate Alexandrium, which causes algal blooms and poisoning of shellfish.  

PubMed

The marine dinoflagellate genus Alexandrium includes a number of species that produce potent neurotoxins responsible for paralytic shellfish poisoning, which in humans may cause muscular paralysis, neurological symptoms and, in extreme cases, death. Because of the genetic diversity of different genera and species, molecular tools may help to detect the presence of target microorganisms in marine field samples. Here we employed a loop-mediated isothermal amplification (LAMP) method for the rapid and simple detection of toxic Alexandrium species. A set of four primers were designed based upon the conserved region of the 5.8S rRNA gene of members of the genus Alexandrium. Using this detection system, toxic Alexandrium genes were amplified and visualized as a ladder-like pattern of bands on agarose gels under isothermal condition within 60 min. The LAMP amplicons were also directly visualized by eye in the reaction tube by the addition of SYBR Green I. This LAMP assay was 10-fold more sensitive than a conventional PCR method with a detection limit of 5 cells per tube when targeting DNA from Alexandrium minutum. The LAMP assay reported here indicates the potential usefulness of the technique as a valuable simple, rapid alternative procedure for the detection of target toxic Alexandrium species during coastal water monitoring. PMID:18355290

Wang, Li; Li, Lin; Alam, M J; Geng, Yuhuan; Li, Zhiyong; Yamasaki, Shinji; Shi, Lei

2008-05-01

206

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

2013-03-01

207

[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

208

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

209

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

210

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

PubMed

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

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

2014-05-01

211

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

PubMed

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

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

2014-06-01

212

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

213

Higher Specificity of Nucleic Acid Sequence-Based Amplification Isothermal Technology than of Real-Time PCR for Quantification of HIV-1 RNA on Dried Blood Spots  

PubMed Central

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

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

2014-01-01

214

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

215

Establishment and application of a loop-mediated isothermal amplification method for simple, specific, sensitive and rapid detection of Toxoplasma gondii.  

PubMed

The Loop-mediated isothermal amplification (LAMP) method amplifies DNA with high simply, specificity, sensitivity and rapidity. In this study, A LAMP assay with 6 primers targeting a highly conserved region of the GRA1 gene was developed to diagnose Toxoplasma gondii. The reaction time of the LAMP assay was shortened to 30 min after optimizing the reaction system. The LAMP assay was found to be highly specific and stable. The detection limit of the LAMP assay was 10 copies, the same as that of the conventional PCR. We used the LAMP assay to develop a real-time fluorogenic protocol to quantitate T. gondii DNA and generated a log-linear regression plot by plotting the time-to-threshold values against genomic equivalent copies. Furthermore, the LAMP assay was applied to detect T. gondii DNA in 423 blood samples and 380 lymph node samples from 10 pig farms, and positive results were obtained for 7.8% and 8.2% of samples, respectively. The results showed that the LAMP method is slightly more sensitive than conventional PCR (6.1% and 7.6%). Positive samples obtained from 6 pig farms. The LAMP assay established in this study resulted in simple, specific, sensitive and rapid detection of T. gondii DNA and is expected to play an important role in clinical detection of T. gondii. PMID:23965849

Cao, Lili; Cheng, Ronghua; Yao, Lin; Yuan, Shuxian; Yao, Xinhua

2014-01-01

216

Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. niveum in soil.  

PubMed

Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (Fon) is one of the major limiting factors for watermelon production worldwide. Rapid and accurate detection of the causal pathogen is the cornerstone of integrated disease management. In this paper, a real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay was developed for the rapid and quantitative detection of Fon in soil. Positive products were amplified only from Fon isolates and not from any other species or formae speciales of F. oxysporum tested, showing a high specificity of the primer sets. The detection limit of the RealAmp assay was 1.2 pg ?L(-1) genomic DNA or 10(3) spores g(-1) of artificially inoculated soil, whereas real-time PCR could detect as low as 12 fg ?L(-1) or 10(2) spores g(-1). The RealAmp assay was further applied to detect eight artificially inoculated and 85 field soil samples. No significant differences were found between the results tested by the RealAmp and real-time PCR assays. The RealAmp assay is a simple, rapid and effective technique for the quantitative detection and monitoring of Fon in soil under natural conditions. PMID:24256412

Peng, Jun; Zhan, Yuanfeng; Zeng, Fanyun; Long, Haibo; Pei, Yuelin; Guo, Jianrong

2013-12-01

217

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

NASA Astrophysics Data System (ADS)

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

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

2012-07-01

218

Loop-mediated isothermal amplification assay for detection of Histomonas meleagridis infection in chickens targeting the 18S rRNA sequences.  

PubMed

Histomonas meleagridis is the causative agent of histomonosis, a disease of gallinaceous fowl characterized by necrotic typhlitis, hepatitis, and high mortality. To develop a rapid and sensitive method for specific detection of H. meleagridis, an assay based on loop-mediated isothermal amplification (LAMP) targeting the 18S rRNA gene was established. The detection limit of the LAMP assay was 10 copies for standard plasmids containing an 18S rRNA gene fragment, which was superior to that of a classical PCR method. Specificity tests revealed that there was no cross-reaction with other protozoa such as Trichomonas gallinae, Blastocytis sp, Tetratrichomonas gallinarum, Plasmodium gallinaceum, Toxoplasma gondii, Eimeria tenella, Leucocytozoon caulleryi and Leucocytozoon sabrazesi. The assay was evaluated for its diagnostic utility using liver and caeca samples collected from suspected field cases, the detection rate was 100 and 97.92%, respectively. These results indicate that the LAMP assay may be a useful tool for rapid detection and identification of H. meleagridis in poultry. PMID:24320623

Xu, Jinjun; Qu, Chanbao; Tao, Jianping

2014-01-01

219

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

PubMed

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

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

2014-05-01

220

Visual Detection of Canine Parvovirus Based on Loop-Mediated Isothermal Amplification Combined with Enzyme-Linked Immunosorbent Assay and with Lateral Flow Dipstick  

PubMed Central

ABSTRACT Loop-mediated isothermal amplification (LAMP) combined with enzyme-linked immunosorbent assay (LAMP–ELISA) and with lateral flow dipstick (LAMP–LFD) are rapid, sensitive and specific methods for the visual detection of clinical pathogens. In this study, LAMP–ELISA and LAMP–LFD were developed for the visual detection of canine parvovirus (CPV). For LAMP, a set of four primers (biotin-labeled forward inner primers) was designed to specifically amplify a region of the VP2 gene of CPV. The optimum time and temperature for LAMP were 60 min and 65°C, respectively. The specific capture oligonucleotide probes, biotin-labeled CPV probe for LAMP–ELISA and fluorescein isothiocyanate-labeled CPV probe for LAMP–LFD were also designed for hybridization with LAMP amplicons on streptavidin-coated wells and LFD strips, respectively. For the comparison of detection sensitivity, conventional PCR and LAMP for CPV detection were also performed. The CPV detection limits by PCR, PCR–ELISA, LAMP, LAMP–ELISA and LAMP–LFD were 102, 102, 10?1, 10?1 and 10?1 TCID50/ml, respectively. In tests using artificially contaminated dog fecal samples, the samples with CPV inoculation levels of ?1 TCID50/ml gave positive results by both LAMP–ELISA and LAMP–LFD. Our data indicated that both LAMP–ELISA and LAMP–LFD are promising as rapid, sensitive and specific methods for an efficient diagnosis of CPV infection.

SUN, Yu-Ling; YEN, Chon-Ho; TU, Ching-Fu

2013-01-01

221

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

PubMed Central

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

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

2013-01-01

222

Development of the visual loop-mediated isothermal amplification assays for seven genetically modified maize events and their application in practical samples analysis.  

PubMed

As more and more genetically modified (GM) crops are approved for commercialization and planting, the development of quick and on-spot methods for GM crops and their derivates is required. Herein, we established the polymerase chain reaction and agarose gel electrophoresis-free system for the identification of seven GM maize events (DAS-59122-7, T25, BT176, TC1507, MON810, BT11, and MON863) employing a loop-mediated isothermal amplification (LAMP) technique. The LAMP assay was performed using a set of four specific primers at 60-65 °C in less than 40 min, and the results were observed by direct visual observation. In these developed assays, the specificity targeted at each GM maize event based on the event-specific sequence was well confirmed, and the limits of detection were as low as four copies of maize haploid genomic DNA with an exception of 40 copies for MON810 assay. Furthermore, these developed assays were successfully used to test six practical samples with different GM maize events and contents (ranged from 0.0 to 2.0%). All of the results indicated that the established event-specific visual LAMP assays are more convenient, rapid, and low-cost for GM maize routine analysis. PMID:21520936

Chen, Lili; Guo, Jinchao; Wang, Qidi; Kai, Guoyin; Yang, Litao

2011-06-01

223

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

PubMed

Marburg virus (MARV) causes a severe hemorrhagic fever in humans with a high mortality rate. The rapid and accurate identification of the virus is required to appropriately provide infection control and outbreak management. Here, we developed and evaluated a one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the rapid and simple detection of MARV. By combining two sets of primers specific for the Musoke and Ravn genetic lineages, a multiple RT-LAMP assay detected MARV strains of both lineages, and no cross-reactivity with other hemorrhagic fever viruses (Ebola virus and Lassa virus) was observed. The assay could detect 10(2) copies of the viral RNA per tube within 40 min by real-time monitoring of the turbidities of the reaction mixtures. The assay was further evaluated using viral RNA extracted from clinical specimens collected in the 2005 Marburg hemorrhagic fever outbreak in Angola and yielded positive results for samples containing MARV at greater than 10(4) 50% tissue culture infective doses/ml, exhibiting 78% (14 of 18 samples positive) consistency with the results of a reverse transcription-PCR assay carried out in the field laboratory. The results obtained by both agarose gel electrophoresis and naked-eye judgment indicated that the RT-LAMP assay developed in this study is an effective tool for the molecular detection of MARV. Furthermore, it seems suitable for use for field diagnostics or in laboratories in areas where MARV is endemic. PMID:20421440

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

2010-07-01

224

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

PubMed

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

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

2014-08-01

225

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

PubMed

Citrus yellow mosaic badnavirus (CMBV) is an important pathogen in southern India spread by infected citrus propagules. One of the measures to arrest the spread of CMBV is to develop methods to screen and certify citrus propagules as CMBV-free. The methods loop-mediated isothermal amplification (LAMP) and SYBR green real-time PCR (SGRTPCR) have been developed for the efficient detection of CMBV in citrus propagules. This paper compares the sensitivities of LAMP and SGRTPCR with polymerase chain reaction (PCR) for the detection of CMBV. Whereas PCR and LAMP were able to detect CMBV from a minimum of 10 ng of total DNA of infected leaf samples, SGRTPCR could detect the same from 1 ng of total DNA. Using SGRTPCR, the viral titres were estimated to be the highest in rough lemon and lowest in Nagpur Mandarin of the five naturally infected citrus species tested. The results will help in designing suitable strategies for the sensitive detection of CMBV from citrus propagules. PMID:24675064

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

2014-07-01

226

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

PubMed Central

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

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

2014-01-01

227

Visual Detection and Evaluation of Latent and Lytic Gene Expression during Epstein-Barr Virus Infection Using One-Step Reverse Transcription Loop-Mediated Isothermal Amplification  

PubMed Central

Epstein-Barr virus (EBV)-associated disease exhibits distinct gene expression patterns characterized by the transcription of EBV nuclear antigen (EBNA) 1, EBNA2, latent membrane protein (LMP) 1, LMP2A, and BZLF1 (Zebra). A series of visual reverse transcript loop-mediated isothermal amplification (RT-LAMP) assays were performed to examine the expression of EBNA1, EBNA2, LMP1, LMP2A and BZLF1. The sensitivity of RT-LAMP for these transcripts was approximately equivalent to real-time RT-PCR (RT-qPCR), which was developed to quantify relative levels of EBV transcripts, and 10 to 100-fold more sensitive than conventional RT-PCR. Cross-reactions to other viruses were not observed upon examination of cell lines infected with herpes simplex viruses-1 and -2 (HSV-1 and -2), varicella zoster virus (VZV), human cytomegalovirus (HCMV) or Kaposi’s sarcoma-associated herpesvirus. When applied to 146 specimens, RT-LAMP exhibited high clinical sensitivity and specificity, with an excellent agreement (? > 0.92) compared to RT-qPCR. These assays are convenient for rapid early diagnosis and for surveillance of EBV-infected individuals by evaluating the EBV transcriptional profile, because the results can be visualized with the naked eye. These assays may be employed in further investigations because they can aid the design of improved therapeutic regimens and can be used specifically in resource-poor settings.

Liu, Xiaoying; Tang, Jingfeng; Wang, Man; Ma, Qiang; Wang, Yefu

2013-01-01

228

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

PubMed

An assay to detect Strongyloides stercoralis in stool specimens was developed using the loop-mediated isothermal amplification (LAMP) method. Primers were based on the 28S ribosomal subunit gene. The reaction conditions were optimized and SYTO-82 fluorescent dye was used to allow real-time and visual detection of the product. The product identity was confirmed with restriction enzyme digestion, cloning, and sequence analysis. The assay was specific when tested against DNA from bacteria, fungi and parasites, and 30 normal stool samples. Analytical sensitivity was to < 10 copies of target sequence in a plasmid and up to a 10(-2) dilution of DNA extracted from a Strongyloides ratti larva spiked into stool. Sensitivity was increased when further dilutions were made in water, indicative of reduced reaction inhibition. Twenty-seven of 28 stool samples microscopy and polymerase chain reaction positive for S. stercoralis were positive with the LAMP method. On the basis of these findings, the assay warrants further clinical validation. PMID:24323513

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

2014-02-01

229

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

PubMed Central

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.

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

2011-01-01

230

Isolation and detection of extended spectrum ?-lactamase (ESBL)-producing enterobacteriaceae from meat using chromogenic agars and isothermal loop-mediated amplification (LAMP) assays.  

PubMed

The aim of this work was to develop a molecular method using loop-mediated isothermal amplification (LAMP) for detection of extended spectrum ?-lactamase (ESBL)-producing Enterobacteriaceae from meat, and to compare it with different isolation agars and microarrays. LAMP assays were developed for CTX-M groups 1, 2, and 9 and OXA-10-like genes. Chicken, lamb, beef, pork, and turkey samples were spiked with 10, 100, and 1,000 cfu/gram using 8 strains of ESBL-producing Enterobacteriaceae (CTX-M sequence types 1, 2, 3, 14, 15, OXA-11, SHV-2, TEM-52) +/- a mix of competitor organisms. Samples were enriched overnight in buffered peptone water (BPW) +/- antibacterials before plating to CHROMagar CTX, OXOID ESBL Brilliance agar, and MacConkey agar with 1 mg/L cefotaxime. Selected BPW broths were also tested using LAMP assays, microarrays and using cefpodoxime discs on agar. For isolation/detection of ESBL producers from beef, pork, lamb, and turkey spiked with 10 or 100 cfu/gram ESBL (natural flora only), all agars and the LAMP assays showed 100% sensitivity and specificity for ESBL spike strains. For chicken samples, both LAMP and chromogenic agars showed improved sensitivity and specificity for isolation of ESBLs compared with MacConkey agar, particularly with competitor bacteria added. In comparison, the cefpodoxime disc method and microarray showed reduced sensitivity. PMID:24329955

Anjum, M F; Lemma, F; Cork, D J; Meunier, D; Murphy, N; North, S E; Woodford, N; Haines, J; Randall, L P

2013-12-01

231

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

2009-08-15

232

Assessment of the loop-mediated isothermal amplification assay for rapid diagnosis of Mycoplasma pneumoniae in pediatric community-acquired pneumonia.  

PubMed

Rapid diagnosis of Mycoplasma pneumoniae pneumonia is required for timely treatment with effective antibiotics; however, PCR-based methods are often too expensive and technologically intensive for general use in clinical practice. In this study, the efficacy of the loop-mediated isothermal amplification (LAMP) assay for diagnosis of M. pneumoniae pneumonia in clinical practice was prospectively evaluated. From July 2011 to March 2012, 531 children hospitalized for community-acquired pneumonia were enrolled. In all patients, throat swabs were obtained on admission for the detection of M. pneumoniae DNA, and paired serum samples were obtained to assay M. pneumoniae particle agglutination (PA) antibody titers. M. pneumoniae pneumonia was diagnosed by either a positive LAMP assay or an increase of 4-fold or greater in the PA titer. Overall, 271 children (51.0% of the patients with pneumonia) were diagnosed with M. pneumoniae pneumonia. Among these, 258 (95.2%) and 248 (91.5%) were identified by the LAMP assay and serological tests, respectively. When the results of serological tests were considered as standard, the sensitivity, specificity, and positive and negative predictive values of the LAMP assay were 94.8%, 91.9%, and 91.1% and 95.2%, respectively. The median duration of pharyngeal carriage, as measured by the LAMP assay, was 9.5 days. Thus, the LAMP assay is useful in the rapid diagnosis of M. pneumoniae pneumonia. PMID:24270147

Gotoh, Kensei; Nishimura, Naoko; Takeuchi, Suguru; Hattori, Fumihiko; Horiba, Kazuhiro; Isaji, Mai; Okai, Yu; Ohshima, Yasunori; Hosono, Haruki; Takemoto, Koji; Iwata, Yasushi; Nakane, Kazumasa; Funahashi, Keiji; Ozaki, Takao

2013-01-01

233

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

PubMed Central

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

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

2013-01-01

234

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

PubMed Central

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

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

2014-01-01

235

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

PubMed

In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid, sensitive, and inexpensive detection of nervous necrosis virus (NNV) in olive flounder, Paralichthys olivaceus, in Korea. A set of six specific primers was designed to target the RNA 2 gene encoding the coat protein of Korean NNV strains. The RT-LAMP reaction successfully detected NNV after 30 min at 65 degrees C. When the sensitivities among RT-LAMP, RT-PCR, and nested RTPCR were compared, the RT-LAMP was shown to be able to detect the RNA template at 2.58 × 10(-2) TCID50/ml, whereas the RT-PCR and nested RT-PCR were only able to detect the RNA template at 2.58 × 10(2) TCID50/ml and 2.58 TCID50/ml, respectively. Thus, the sensitivity of the RT-LAMP assay was higher than those of the RT-PCR assays. In the specificity test of the RT-LAMP, 2 genotypes of NNVs (SJNNV and RGNNV) were positive; however, no other fish viruses were positive with the primers, indicating that the RT-LAMP assay is only specific to NNV. A total of 102 olive flounder were collected from hatcheries between 2009 and 2011. The occurrence of NNV in olive flounder was determined to be 53.9% (55/ 102) by the RT-LAMP. On the other hand, the prevalence based on the nested RT-PCR and RT-PCR results was 33.8% (34/102) and 20.6% (21/102), respectively. This result indicates that the RT-LAMP assay developed in this study is suitable for early field diagnosis of NNV with high sensitivity. PMID:22580323

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

2012-07-01

236

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

PubMed

This study investigated loop-mediated isothermal amplification (LAMP) detection of Plasmodium falciparum and Plasmodium vivax in urine and saliva of malaria patients. From May to November 2011, 108 febrile patients referred to health centers in Sistan and Baluchestan Province of south-eastern Iran participated in the study. Saliva, urine, and blood samples were analyzed with nested PCR and LAMP targeting the species-specific nucleotide sequence of small subunit ribosomal RNA gene (18S rRNA) of P. falciparum and P. vivax and evaluated for diagnostic accuracy by comparison to blood nested PCR assay. When nested PCR of blood is used as standard, microscopy and nested PCR of saliva and urine samples showed sensitivity of 97.2%, 89.4% and 71% and specificity of 100%, 97.3% and 100%, respectively. LAMP sensitivity of blood, saliva, and urine was 95.8%, 47% and 29%, respectively, whereas LAMP specificity of these samples was 100%. Microscopy and nested PCR of saliva and LAMP of blood were comparable to nested PCR of blood (?=0.95, 0.83, and 0.94, respectively), but agreement for nested PCR of urine was moderate (?=0.64) and poor to fair for saliva LAMP and urine LAMP (?=0.38 and 0.23, respectively). LAMP assay showed low sensitivity for detection of Plasmodium DNA in human saliva and urine compared to results with blood and to nested PCR of blood, saliva, and urine. However, considering the advantages of LAMP technology and of saliva and urine sampling, further research into the method is worthwhile. LAMP protocol and precise preparation protocols need to be defined and optimized for template DNA of saliva and urine. PMID:24721227

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

2014-08-01

237

Comparison of an isothermal amplification and bioluminescence detection of DNA method and ISO 6579:2002 for the detection of Salmonella enterica serovars in retail meat samples.  

PubMed

The aim of the study was the comparative evaluation of an isothermal amplification and bioluminescence detection of DNA (IMBD) method and method ISO 6579:2002 for detection of Salmonella in retail meat products of unknown contamination status. A total of 200 meat samples were tested: 116 minced meat and meat preparations to be eaten cooked (52 chicken, 48 pork, and 16 beef samples) and 84 fresh meat samples (68 poultry and 16 pork). With one or both methods, 21 samples (10.5%) were positive for Salmonella enterica. Fifteen samples were positive with both methods (71.4% of all positive samples), two more samples (9.5%) were positive with the IMBD method only, and four samples (19.1%) were positive with the ISO method only. One ISO-positive sample was inhibited with the IMBD method. For the IMBD method, relative accuracy was 97.0% (95% confidence interval [CI], 93.6 to 98.9%), relative sensitivity was 78.9% (95% CI, 54.4 to 93.9%), and relative specificity was 98.9% (95% CI, 96.1 to 99.7%). Time to negative results was shorter with the IMBD method (20 to 24 h). Also, positive results were available in 20 to 24 h but should be confirmed using other methods (presumptive-positive results). Rapidity of response of the IMBD method gave us the opportunity to test the presumptive-positive samples by the most-probable-number (MPN) method, which was not performed for samples that were positive only with the ISO method because of likely microbial changes during the long storage period (5 to 7 days) at refrigeration temperature. Salmonella MPN values in naturally contaminated meat were low, at <0.3 to 2.1 MPN/g. PMID:23575129

Bonardi, Silvia; Alpigiani, Irene; Bacci, Cristina; Brindani, Franco; Pongolini, Stefano

2013-04-01

238

Most-Probable-Number Loop-Mediated Isothermal Amplification-Based Procedure Enhanced with K Antigen-Specific Immunomagnetic Separation for Quantifying tdh(+) Vibrio parahaemolyticus in Molluscan Shellfish.  

PubMed

Although thermostable direct hemolysin-producing (tdh(+)) Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis, the enumeration of tdh(+) V. parahaemolyticus remains challenging due to its low densities in the environment. In this study, we developed a most-probable-number (MPN)-based procedure designated A-IS(1)-LAMP, in which an immunomagnetic separation (IMS) technique targeting as many as 69 established K antigens and a loop-mediated isothermal amplification (LAMP) assay targeting the thermostable direct hemolysin (tdh) gene were applied in an MPN format. Our IMS employed PickPen, an eight-channel intrasolution magnetic particle separation device, which enabled a straightforward microtiter plate-based IMS procedure (designated as PickPen-IMS). The ability of the procedure to quantify a wide range of tdh(+) V. parahaemolyticus levels was evaluated by testing shellfish samples in Japan and southern Thailand, where shellfish products are known to contain relatively low and high levels of total V. parahaemolyticus, respectively. The Japanese and Thai shellfish samples showed, respectively, relatively low (< 3 to 11 MPN/10 g) and considerably higher (930 to 110,000 MPN/10 g) levels of tdh(+) V. parahaemolyticus, raising concern about the safety of Thai shellfish products sold to domestic consumers at local morning markets. LAMP showed similar or higher performance than conventional PCR in the detection and quantification of a wide range of tdh(+) V. parahaemolyticus levels in shellfish products. Whereas a positive effect of PickPen-IMS was not observed in MPN determination, PickPen-IMS was able to concentrate tdh(+) V. parahaemolyticus 32-fold on average from the Japanese shellfish samples at an individual tube level, suggesting a possibility of using PickPen-IMS as an optional tool for specific shellfish samples. The A-IS(1)-LAMP procedure can be used by any health authority in the world to measure the tdh(+) V. parahaemolyticus levels in shellfish products. PMID:24988012

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

2014-07-01

239

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

PubMed

Rapid and specific detection methods for bacterial agents in drinking water are important for disease prevention and responding to suspected contamination events. In this study, an isothermal Genome Exponential Amplification Reaction (GEAR) assay for Escherichia coli O157:H7 was designed specifically to recognize a 199-bp fragment of the lipopolysaccharide gene (rfbE) for rapid testing of water samples. The GEAR assay was found to be specific for E. coli O157:H7 using 10 isolates of E. coli O157:H7 and a panel of 86 bacterial controls. The GEAR assay was performed at a constant temperature of 65°C using SYTO 9 intercalating dye. Detection limits were determined to be 20 CFU for the GEAR assay. When SYTO 9 fluorescence was measured using a real-time PCR instrument, the assay had the same detection limit as when malachite green was added to the reaction mix and a characteristic blue color was visually observed in positive reactions. The study also found that 50 and 20 CFU of E. coli O157:H7 seeded into 100-liter of tap water could be detected by the GEAR assays after the sample was concentrated by hollow-fiber ultrafiltration (HFUF) and approximately 10% of HFUF concentrate was cultured using trypticase soy broth-novobiocin. When applied to 19 surface water samples collected from Tennessee and Kentucky, the GEAR assay and a published real-time PCR assay both detected E. coli O157:H7 in two of the samples. The results of this study indicate that the GEAR assay can be sensitive for rapid detection of E. coli O157:H7 in water samples using fluorometric instruments and visual endpoint determination. PMID:24424127

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

2014-03-01

240

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

PubMed

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

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

2013-03-15

241

Loop-mediated isothermal amplification for Rickettsia typhi (the causal agent of murine typhus): problems with diagnosis at the limit of detection.  

PubMed

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

Dittrich, Sabine; Castonguay-Vanier, Josée; Moore, Catrin E; Thongyoo, Narongchai; Newton, Paul N; Paris, Daniel H

2014-03-01

242

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

2013-06-11

243

Development and application of a loop-mediated isothermal amplification assay for rapid detection of Borrelia burgdorferi s. l. in ticks.  

PubMed

A loop-mediated isothermal amplification (LAMP) assay was developed to detect Borrelia burgdorferi s. l. in ticks, which is a pathogen that causes Lyme disease. Cross-reactions with Chlamydia psittaci, Mycoplasma mycoides subsp. capri and some tick-borne pathogens were excluded. Analytical sensitivity of LAMP showed its detection limit was from 0.02 to 0.2 pg of DNA in detection of the reference samples at 65°C for 40 min. The performance of LAMP was assessed by testing 110 samples from susceptible tick species and comparing the results with conventional and nested PCR tests previously described. The results demonstrated that LAMP was significantly more sensitive than the conventional PCR (32.7% versus 15.5%, P < 0.05) and slightly more sensitive, although not significantly so, than nested PCR (32.7% versus 26.4%, P > 0.05). The assay was used to analyse a total of 1052 ticks collected from eight provinces in China. The results showed that the infection rates of B. burgdorferi s. l. varied from 12.5% to 88.9% across the different geographical sites. Selected positive samples were subjected to sequencing and sequence analysis for conformation of the accuracy of the assay. Here we report a highly sensitive, specific and easy diagnostic assay based on LAMP technology. These data indicate that LAMP is a useful approach for detecting B. burgdorferi s. l. in field-collected ticks and has the potential as an alternative tool for the ecological and epidemiological surveillance of Lyme disease. PMID:22587441

Yang, J; Guan, G; Niu, Q; Liu, Z; Li, Y; Liu, J; Ma, M; Ren, Q; Liu, A; Luo, J; Yin, H

2013-06-01

244

Comparative Detection of Trypanosomal DNA by Loop-Mediated Isothermal Amplification and PCR from Flinders Technology Associates Cards Spotted with Patient Blood ?  

PubMed Central

We analyzed DNA eluted from FTA (Flinders Technology Associates) cards spotted with blood from human African trypanosomiasis (HAT) patients admitted at Lwala Hospital in eastern Uganda and Kaliua Health Centre in northwestern Tanzania. The aims were to evaluate loop-mediated isothermal amplification (LAMP) for detection of trypanosomal DNA in clinical samples and to characterize the infecting trypanosomes to the subspecies level. LAMP targeting the Trypanozoon conserved random inserted mobile element (RIME-LAMP) and that for the serum resistance-associated (SRA) gene (SRA-LAMP) were performed. For comparison, PCRs for the SRA gene specific for Trypanosoma brucei rhodesiense (SRA-PCR) and that to amplify the Trypanosoma brucei gambiense-specific surface glycoprotein (TgSGP-PCR) were done. Out of 128 samples analyzed, SRA-PCR was positive in 101 samples (78.9% sensitivity; 95% confidence interval [CI], 71.1 to 85.1%), SRA-LAMP was positive in 120 (93.8%; 95% CI, 88.2 to 96.8%), while RIME-LAMP revealed signals in 122 (95.3%; 95% CI, 90.2 to 97.8%). RIME-LAMP and SRA-LAMP were each significantly more sensitive than SRA-PCR (P values of 0.000 and 0.001, respectively; Fisher's exact test). There was poor agreement between RIME-LAMP and SRA-LAMP and the SRA-PCR, yielding kappa values of 0.31 and 0.40, respectively. Agreement between SRA-LAMP and RIME-LAMP was almost perfect (kappa value, 0.85; 95% CI, 0.64 to 1). All the 128 field samples were negative by TgSGP-PCR. Blood spots from three T. b. gambiense HAT cases from northwestern Uganda were positive by TgSGP-PCR and RIME-LAMP. PCR took five times longer to execute than LAMP. LAMP may be useful to monitor emerging HAT foci or to test travelers returning from countries where HAT is endemic. It should be evaluated in a case-control study to determine its utility as a HAT diagnostic.

Matovu, Enock; Kuepfer, Irene; Boobo, Alex; Kibona, Stafford; Burri, Christian

2010-01-01

245

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

2013-07-01

246

Comparison of loop-mediated isothermal amplification (LAMP) and nested-PCR assay targeting the RE and B1 gene for detection of Toxoplasma gondii in blood samples of children with leukaemia.  

PubMed

Toxoplasmosis diagnosis constitutes an important measure for disease prevention and control. In this paper, a newly described DNA amplification technique, loop-mediated isothermal amplification (LAMP), and nested-PCR targeting the repeated element (RE) and B1 gene, were compared to each other for the detection of Toxoplasma gondii DNA in blood samples of children with leukaemia. One hundred ten blood samples from these patients were analyzed by LAMP and nested-PCR. Out of 50 seropositive samples (IgM+, IgG+), positive results were obtained with 92% and 86% on RE, B1-LAMP and 82% and 68% on RE, B1-nested PCR analyses, respectively. Of the 50 seronegative samples, three, two and one samples were detected positive by RE-LAMP, B1-LAMP and RE-nested PCR assays, respectively, while none were detected positive by B1-nested PCR. None of the 10 IgM-, IgG+ samples was detected positive after testing LAMP and nested-PCR assays in duplicate. This is the first report of a study in which the LAMP method was applied with high sensitivity and efficacy for the diagnosis of T. gonii in blood samples of children with leukaemia. PMID:24792838

Fallahi, Shirzad; Seyyed Tabaei, Seyyed Javad; Pournia, Yadollah; Zebardast, Nozhat; Kazemi, Bahram

2014-07-01

247

Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. cubense tropical race 4 in soil.  

PubMed

Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 10(3) spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China. PMID:24376590

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

2013-01-01

248

Ultrasensitive fluorescence detection of nucleic acids using exonuclease III-induced cascade two-stage isothermal amplification-mediated zinc (II)-protoporphyrin IX/G-quadruplex supramolecular fluorescent nanotags.  

PubMed

A cascadic sensing system was developed for detection of DNA target at ultralow concentration by a combination of magnetic nanoparticles (MNPs) and exonuclease III (Exo III)-induced cascade two-stage isothermal amplification in the study. An ingeniously designed capture hairpin probe (CHP) that integrates target-binding and signal transduction sequences within one multifunctional design was assembled on MNPs. Upon sensing of the analyte nucleic acid, the hairpin probe on MNPs could be opened and stepwise removed by Exo III accompanied by the releasing of target DNA for the successive hybridization and cleavage process and the generation of bare signal transduction sequences of CHP as a new trigger for next circular reaction. The new DNA triggers initiate hybridizing with hairpin DNA probe that contains a partially "caged" G-quadruplex sequence (GHP), forming a duplex structure and liberating the active G-quadruplex structure. Then, Exo III digests the resulting duplex domain, leading to the recycling of new DNA trigger and simultaneously generating numerous ZnPPIX/G-quadruplex supramolecular complexes with the help of the zinc (II)-protoporphyrin IX (ZnPPIX), as an optical label for amplified fluorescence sensing event. Finally, numerous liberated cascade ZnPPIX/G-quadruplex supramolecular complexes give a remarkable fluorescence response. Because of two-stage autocatalytic recycling amplification and the specifically catalyzed formation of ZnPPIX/G-quadruplex supramolecular complexes, this newly designed protocol provides a high sensitivity with a detection limit of 0.75 fM, can discriminate mismatched DNA from perfectly matched target DNA, and gives low matrix effect due to using MNPs as the separation and amplification elements in the real samples. Therefore, it holds great potential for early diagnosis in gene-related diseases. PMID:24912035

Xue, Qingwang; Lv, Yanqin; Zhang, Yuanfu; Xu, Shulin; Li, Rui; Yue, Qiaoli; Li, Haibo; Wang, Lei; Gu, Xiaohong; Zhang, Shuqiu; Liu, Jifeng

2014-11-15

249

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

PubMed Central

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

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

2013-01-01

250

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

PubMed

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

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

2014-06-15

251

[Comparative evaluation of the isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) and Roche Amplicor PCR and culture for detecting Mycobacterium tuberculosis complex in sputum samples].  

PubMed

We compared the ability of the newly developed ICAN MTB Detection Kit (TaKaRa Bio Inc.), which uses the Isothermal and Chimeric primer-initiated Amplification of Nucleic acid (ICAN), with that of COBAS Amplicor PCR System (Roche Diagnostics) to directly detect Mycobacterium tuberculosis complex (MTB) in sputum samples. A total of 142 sputum samples from 120 patients were examined in this study. The results were compared with those of acid-fast staining and MGIT liquid culture system (BD) following identification by the probe test (DDH Mycobacteria Kit). A total of 68 specimens were MGIT positive for MTB. In addition, 62 specimens were positive by the combination of staining and MGIT assay for MTB. When compared with that for MGIT, the sensitivity of each assay system was 88.2% for ICAN and 92.6% for COBAS Amplicor, respectively. The specificity of each assay system was 65.7% for ICAN and 62.7% for COBAS Amplicor, respectively. Coincidence between ICAN and COBAS Amplicor assay results was 96.3% (130 of 135 samples). No significant difference was observed between the results of the two assay methods. It is concluded that although both nucleic acid amplification methods are sensitive and specific for the detection of MTB in the respiratory specimens, ICAN system appeared to be more rapid (within 3.5 h from the specimen collection) than Amplicor system. The ICAN system will be useful in clinical laboratories for the rapid detection of MTB without specially programmed thermo-cycler. PMID:14509225

Kurashima, Atsuyuki; Machida, Kazuko; Nagai, Hideaki; Kawabe, Yoshiko; Akagawa, Shinobu; Nagayama, Naohiro; Baba, Motoo; Suzuki, Junko; Masuda, Kimihiko; Tamura, Atsuhisa; Komatsu, Hikotarou; Yotsumoto, Hideki

2003-08-01

252

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

253

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

PubMed Central

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

2012-01-01

254

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

PubMed Central

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

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

2012-01-01

255

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

PubMed

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

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

2012-01-01

256

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

257

Application of loop-mediated isothermal amplification assays for direct identification of pure cultures of Aspergillus flavus, A. nomius, and A. caelatus and for their rapid detection in shelled Brazil nuts.  

PubMed

Brazil nuts have a high nutritional content and are a very important trade commodity for some Latin American countries. Aflatoxins are carcinogenic fungal secondary metabolites. In Brazil nuts they are produced predominantly by Aspergillus (A.) nomius and A. flavus. In the present study we applied and evaluated two sets of primers previously published for the specific detection of the two species using loop-mediated isothermal amplification (LAMP) technology. Moreover, a primer set specific for A. caelatus as a frequently occurring non-aflatoxigenic member of Aspergillus section Flavi in Brazil nuts was newly developed. LAMP assays were combined with a simplified DNA release method and used for rapid identification of pure cultures and rapid detection of A. nomius and A. flavus from samples of shelled Brazil nuts. An analysis of pure cultures of 68 isolates representing the major Aspergillus species occurring on Brazil nuts showed that the three LAMP assays had individual accuracies of 61.5%, 84.4%, and 93.3% for A. flavus, A. nomius, and A. caelatus, respectively when morphological identification was used as a reference. The detection limits for conidia added directly to the individual LAMP reactions were found to be 10? conidia per reaction with the primer set ID9 for A. nomius and 10? conidia per reaction with the primer set ID58 for A. flavus. Sensitivity was increased to 10¹ and 10² conidia per reaction for A. nomius and A. flavus, respectively, when sample preparation included a spore disruption step. The results of LAMP assays obtained during the analysis of 32 Brazil nut samples from different regions of Brazil and from different steps in the production process of the commodity were compared with results obtained from mycological analysis and aflatoxin analysis of corresponding samples. Compared with mycological analysis of the samples, the Negative Predictive Values of LAMP assays were 42.1% and 12.5% while the Positive Predictive Values were 61.5% and 66.7% for A. nomius and A. flavus, respectively. When LAMP results were compared with the presence of aflatoxins in corresponding samples, the Negative Predictive Values were 22.2% and 44.4% and the Positive Predictive Values were 52.2% and 78.3% for aflatoxins produced by A. nomius and A. flavus, respectively. The LAMP assays described in this study have been demonstrated to be a specific, sensitive and easy to use tool for the survey of Brazil nuts for contaminations with potential aflatoxin-producing A. nomius and A. flavus in low tech environments where resources may be limited. PMID:24361827

Luo, Jie; Taniwaki, Marta H; Iamanaka, Beatriz T; Vogel, Rudi F; Niessen, Ludwig

2014-02-17

258

Isothermal Confinement  

NASA Astrophysics Data System (ADS)

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

Ohkawa, Tihiro

259

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

260

Multiplexed protein profiling on microarrays by rolling-circle amplification  

Microsoft Academic Search

Fluorescent-sandwich immunoassays on microarrays hold appeal for proteomics studies, because equipment and antibodies are readily available, and assays are simple, scalable, and reproducible. The achievement of adequate sensitivity and specificity, however, requires a general method of immunoassay amplification. We describe coupling of isothermal rolling-circle amplification (RCA) to universal antibodies for this purpose. A total of 75 cytokines were measured simultaneously

Barry Schweitzer; Scott Roberts; Brian Grimwade; Weiping Shao; Minjuan Wang; Qin Fu; Quiping Shu; Isabelle Laroche; Zhimin Zhou; Velizar T. Tchernev; Jason Christiansen; Mark Velleca; Stephen F. Kingsmore

2002-01-01

261

Isothermal and Adiabatic Measurements.  

ERIC Educational Resources Information Center

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

McNairy, William W.

1996-01-01

262

Theory and Experiments for Isothermal and Non-Isothermal Sintering.  

National Technical Information Service (NTIS)

New formulations of thermodynamics for surfaces and for non-isothermal systems are used as a basis for analyzing the kinetics of isothermal and non-isothermal sintering. The analysis suggests: (1) rates of microstructural changes in crystalline aggregates...

A. W. Searcy D. Beruto

1987-01-01

263

Isothermal Bainite in AF1410.  

National Technical Information Service (NTIS)

The isothermal bainite transformation was identified in AF1410 by the use of thermomagnetic analysis methods. The formation of isothermal bainite in AF1410 has not been previously reported. Mechanical property test results indicate that isothermally proce...

G. N. Vigilante P. J. Cote T. Hickey S. K. Pan

1996-01-01

264

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

PubMed

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

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

2014-06-10

265

Non-Isothermal Kinetics.  

ERIC Educational Resources Information Center

Discusses the principle of nonisothermal kinetics and some of the factors involved in such reactions, especially when considering the reliability of the kinetic parameters, compared to those of isothermal conditions. (GA)

Brown, M. E.; Phillpotts, C. A. R.

1978-01-01

266

Isothermal Containment Module  

NASA Technical Reports Server (NTRS)

Isothermal Containment Modules are the temperature-controlling carrier that BioServe built to carry Commercial Generic Bioprocessing Apparatus (CGBA) and in the future, Space Automated Bioproduct Lab (SABL) to the International Space Station.

1999-01-01

267

Hybrid Chirped Pulse Amplification  

SciTech Connect

We present a novel chirped pulse amplification method which combines optical parametric amplification and laser amplification. We have demonstrated this hybrid CPA concept with a combination of beta-barium borate and Ti:sapphire. High-efficiency, multi-terawatt compatible amplification is achieved without gain narrowing and without electro-optic modulators using a simple commercial pump laser.

Jovanovic, I; Barty, C P J

2002-05-07

268

Rapid amplification/detection of nucleic acid targets utilizing a HDA/thin film biosensor.  

PubMed

Thin film biosensors exploit a flat, optically coated silicon-based surface whereupon formation of nucleic acid hybrids are enzymatically transduced in a molecular thin film that can be detected by the unaided human eye under white light. While the limit of sensitivity for detection of nucleic acid targets is at sub-attomole levels (60?000 copies) many clinical specimens containing bacterial pathogens have much lower levels of analyte present. Herein, we describe a platform, termed HDA/thin film biosensor, which performs helicase-dependant nucleic acid amplification on a thin film biosensor surface to improve the limit of sensitivity to 10 copies of the mecA gene present in methicillin-resistant strains of Staphylococcus. As double-stranded DNA is unwound by helicase it was either bound by solution-phase DNA primers to be copied by DNA polymerase or hybridized to surface immobilized probe on the thin film biosensor surface to be detected. Herein, we show that amplification reactions on the thin film biosensor are equivalent to in standard thin wall tubes, with detection at the limit of sensitivity of the assay occurring after 30 minutes of incubation time. Further we validate the approach by detecting the presence of the mecA gene in methicillin-resistant Staphylococcus aureus (MRSA) from positive blood culture aliquots with high specificity (signal/noise ratio of 105). PMID:24899216

Jenison, Robert; Jaeckel, Heidi; Klonoski, Joshua; Latorra, David; Wiens, Jacinta

2014-06-30

269

Isothermal Community College  

ERIC Educational Resources Information Center

Isothermal Community College (ICC) is located in Spindale, North Carolina. The college serves approximately 2,000 curriculum students every fall and spring semester and about 1,000 curriculum students in summer semesters. The Student Affairs department at ICC is divided into 10 functional areas. Over the last several years, student affairs staff…

Jones, Karen Kitchens

2009-01-01

270

Surface Temperature: Contouring Isotherms  

NSDL National Science Digital Library

This interactive feature shows how an isothermal map of surface temperature is drawn. Students can select an individual contour value and watch as a virtual 'pencil' correctly places the line with respect to temperature values on the map. The animation also permits the user to color the spaces between the contour lines on the map.

2011-02-22

271

Automated adsorption isotherm device.  

PubMed Central

A device is described for the automated determination of the water adsorption isotherms of biological materials. The vapor pressure and weight of the adsorbate are measured directly with appropriate transducers, and equilibrium is defined on the basis of constant pressure. The accuracy of the device, determined on two samples with well-known water binding properties, is +/- 5%. Automation is achieved by electronic control.

Wilkinson, A; Morowitz, H J; Lund, W

1976-01-01

272

Random-primed, Phi29 DNA polymerase-based whole genome amplification.  

PubMed

Whole-genome amplification by multiple displacement amplification (MDA) is a patented method to generate potentially unlimited genomic material when researchers are challenged with trace samples, or the amount of genomic DNA required for analysis exceeds the amount on hand. It is an isothermal reaction, using Phi29 DNA polymerase and random hexamer primers for unbiased amplification of linear DNA molecules, such as genomic DNA. The random-primed MDA reaction provides extensive amplification coverage of the genome, generates extremely long DNA products, and provides high DNA yields. This unit explains the reaction, and describes use of the commercial kits available. PMID:24510438

Nelson, John R

2014-01-01

273

Isothermal and non-isothermal pyrolysis kinetics of Kapton ® polyimide  

Microsoft Academic Search

The kinetics involved in the thermal decomposition of Kapton® polyimide 100HN under nitrogen atmosphere were studied by applying various fitting techniques to the isothermal and non-isothermal gravimetric data. The correlation of the reaction mechanism fitting, the analytical model fitting and the isoconversional method to these data was examined in relation to the kinetic parameters and the kinetic predictions. The mechanisms

Aik Chong Lua; Jincai Su

2006-01-01

274

A mechanism for ramified rolling circle amplification  

PubMed Central

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

2010-01-01

275

Model for Isothermal Dendritic Coarsening.  

National Technical Information Service (NTIS)

Isothermal coarsening of dendrites in the solid-liquid region was experimentally studied for Al-4.5% Cu and Al-3.75% Cu-0.11% Ti alloys. Solid-liquid interface area per unit volume, S sub r, was plotted versus coarsening time, t, for various temperatures....

J. J. Reeves T. Z. Kattamis

1971-01-01

276

Isothermal Dendritic Growth Experiment Video  

NASA Technical Reports Server (NTRS)

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

1997-01-01

277

Gravitomagnetic amplification in cosmology  

SciTech Connect

Magnetic fields interact with gravitational waves in various ways. We consider the coupling between the Weyl and the Maxwell fields in cosmology and study the effects of the former on the latter. The approach is fully analytical and the results are gauge invariant. We show that the nature and the outcome of the gravitomagnetic interaction depends on the electric properties of the cosmic medium. When the conductivity is high, gravitational waves reduce the standard (adiabatic) decay rate of the B field, leading to its superadiabatic amplification. In poorly conductive environments, on the other hand, Weyl-curvature distortions can result into the resonant amplification of large-scale cosmological magnetic fields. Driven by the gravitational waves, these B fields oscillate with an amplitude that is found to diverge when the wavelengths of the two sources coincide. We present technical and physical aspects of the gravitomagnetic interaction and discuss its potential implications.

Tsagas, Christos G. [Section of Astrophysics, Astronomy and Mechanics, Department of Physics, Aristotle University of Thessaloniki, Thessaloniki 54124 (Greece)

2010-02-15

278

Voltage Amplification using Plasma  

SciTech Connect

The purpose of this work is to present experimental results about voltage amplification using plasma produced by a simple neon lamp, series connected with a signal generator and discrete circuit elements. The main advantage of employing plasma as an amplifier is due to its ability to drive larger power and potentially to operate in a larger frequency range compared with traditional amplifiers. Our results show that both, the voltage gain and the frequency range where the gain is bigger than one, are related to the plasma density which may be adjusted by a proper control of electrical discharge conditions. The plasma produced into the neon lamp exhibits a diode characteristic that is the principal responsible by the nonlinear plasma response. The amplification occurs when the plasma shows a negative conductance. In this regime the lamp works as an active amplifier and voltage gain higher than 18 was obtained.

Farias, E. E.; Cavalcanti, G. H.; Santiago, M. A. M. [Av. Gal Milton Tavares de Souza - 24310 240 - Instituto de Fisica - Universidade Federal Fluminense (Brazil)

2006-12-04

279

Assembly of Multiple DNA Components through Target Binding toward Homogeneous, Isothermally Amplified, and Specific Detection of Proteins.  

PubMed

We describe a strategy of utilizing specific target binding to trigger assembly of three DNA components that are otherwise unable to spontaneously assemble with one another. This binding-induced DNA assembly forms a three-arm DNA junction, subsequently initiating nicking endonuclease-assisted isothermal fluorescence signal amplification. Real-time monitoring of fluorescence enables amplified detection of specific protein targets. The implementation of the strategy necessitates the simultaneous binding of a single target molecule with two affinity ligands each conjugated to a DNA motif. Simple alternation of affinity ligands enables different protein targets to induce the formation of the DNA junction and subsequent isothermal amplification. The use of the strategy allowed us to develop a sensitive assay for proteins with three appealing features: homogeneous analysis without the need for separation, isothermal amplification, and high specificity. Streptavidin was chosen as an initial target to establish and optimize the assay. Sensitivity of protein detection was improved by 1000-fold upon the application of isothermal amplification. A limit of detection of 10 pM was achieved for detection of prostate-specific antigen in buffer and diluted serum. The combination of its three appealing features makes the assay attractive for potential applications in molecular diagnosis, point-of-care testing, and on-site analysis. PMID:24977952

Deng, Bin; Chen, Junbo; Zhang, Hongquan

2014-07-15

280

Investigation of the molecular mechanism of ICAN, a novel gene amplification method.  

PubMed

Isothermal and Chimeric primer-initiated Amplification of Nucleic acids (ICAN) allows the amplification of target DNA under isothermal conditions at around 55 degrees C using only a pair of 5'-DNA-RNA-3' chimeric primers, thermostable RNaseH and a DNA polymerase with strand-displacing activity (H. Mukai et al. J. Biochemistry, in the preceding paper in this issue). Here we elucidated the mechanism of ICAN by analysing the nicking site of RNaseH, behaviour of chimeric primers and extension products. We found that the ICAN reaction was composed of two unique mechanisms, multi-priming and template-switching, that were responsible for the highly efficient amplifying capability of ICAN. The simultaneous occurrence of two types of reactions, one based on multi-priming and the other based on template-switching, is likely to drive the DNA amplification in ICAN. PMID:17720719

Uemori, Takashi; Mukai, Hiroyuki; Takeda, Osamu; Moriyama, Mariko; Sato, Yoshimi; Hokazono, Shigekazu; Takatsu, Nariaki; Asada, Kiyozo; Kato, Ikunoshin

2007-08-01

281

Sorption isotherms for amaranth grains  

Microsoft Academic Search

Compiling of EMC–ERH data for amaranth grains (Amaranthus cruentus L.) in the range of water activity from 0.029 to 0.979 and temperature from 25 to 90 °C was performed. Included data sets comprised experimental values of EMC–ERH that summarize 78 identified points for desorption, 53 for adsorption and 16 not discerned points that were considered for mean sorption.Five isotherm equations

A. M. Pagano; R. H. Mascheroni

2005-01-01

282

IDGE: Isothermal Dendritic Growth Experiment  

NASA Technical Reports Server (NTRS)

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

1994-01-01

283

Isotherms of cadmium sorption density  

SciTech Connect

To study Cd sorption, 16 soil samples of different chemical soil reactions were taken and analyzed for their physical and chemical properties. Adsorption of ethylene glycol monoethyl ether (EGME) and N{sub 2} were determined to establish the specific surface area of the soils. The sorption of Cd was modeled on a per-mass basis using the Langmuir and Freundlich equations as well as per-surface area basis. Traditional sorption isotherms reveal the relation between the amount of Cd sorbed and the Cd concentration in the soil solution only for the soil under study and can therefore not be applied, or can be applied only with restrictions, to other soils. To meet the aim of modeling Cd sorption and mobility for other soils or locations differing greatly in their properties, we attempted to establish a generalizing sorption isotherm for soils of entirely different composition of the solid phase. On the basis of characteristic values the 16 soil samples taken were grouped into three different reaction types (aluminum-hydroxide, silicate, and carbonate-buffer range). The generalizing Cd sorption density isotherms for these different soil groups introduced in the following provide a useful mathematical model for the quantity-intensity relation of Cd in soils that differ greatly in their specific surface area and their composition. 16 refs., 7 figs., 5 tabs.

Schulte, A. [Mulawarman Univ., East Kalimantan (Indonesia); Beese, F. [Institut fuer Bodenoekologie/GSF, OberschleiBheim (Germany)

1994-07-01

284

Marine Raman image amplification  

NASA Astrophysics Data System (ADS)

Lite Cycles has developed a new type of range-gated, LIDAR sensing element based on Raman image amplification in a solid-state optical crystal. Marine Raman Image Amplification (MARIA) is a feasible technology for producing high-resolution imagery in an underwater environment. MARIA is capable of amplifying low-level optical images with gains up to 106 with the addition of only quantum-limited noise. The high gains available from MARIA can compensate for low quantum efficiency detectors. The range-gate of MARIA is controlled by the pulsewidth of the amplifier pump laser and can be made as short as 30 - 100 cm, using pump pulses of 2 - 6.7 nsec FWHM. The use of MARIA in an imaging LIDAR system has been shown to result in higher SNR images throughout a broad range of incident light levels, in contrast to the increasing noise factor occurring with reduced gain in ICCDs. The imaging resolution of MARIA in the marine environment can be superior to images produced by a laser line scan or standard range-gated imaging system. MARIA is also superior in rejecting unwanted sunlight background, further increasing the SNR of images. MARIA has the potential of providing the best overall system resolution and SNR, making it ideal for the identification of mine-like objects, even in bright sunlight conditions.

Calmes, Lonnie K.; Murray, James T.; Austin, William L.; Powell, Richard C.

1999-10-01

285

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

286

Oncogene amplification in breast cancer.  

PubMed

To refine the analysis of gene amplification in breast cancer, the authors have developed sensitive methods that can be used to screen nucleic acid prepared from a variety of sources. In their analysis, Southern hybridization and DNA dot-blot analysis were used to screen 49 breast cancer DNAs for Myc, Neu, and Int-2 gene amplification. The analysis detected minimal one extra gene copy) as well as expanded (two or more extra gene copies) gene amplifications, and in addition, distinguished between gene amplification and aneuploidy as the cause of extra gene copies. These quantitative methods were adapted to patient specimens routinely available in the anatomic pathology laboratory, including fresh tumor tissue, tumor nuclei discarded during estrogen receptor analysis, and paraffin blocks. One minimal gene amplification was found in three cases of intraductal cancer. Of 25 cases of nonmetastatic invasive cancer, 28% had at least one extra Myc gene, whereas 24% had Neu, and 21% had Int-2 gene amplification. Of 21 cases of metastatic invasive cancer, 43% had Myc, 43% had Neu, and 40% had Int-2 gene amplification. Among the nonmetastatic cancers, 47% had one, 12% had two, and 4% had three amplified genes. Within the metastatic cancers, 48% had one, 28% had two, and 5% had three amplified genes. Our data suggest relationships between tumor progression and both incidence and size of Myc, Neu, and Int-2 gene amplification. PMID:1849359

Donovan-Peluso, M; Contento, A M; Tobon, H; Ripepi, B; Locker, J

1991-04-01

287

Oncogene amplification in breast cancer.  

PubMed Central

To refine the analysis of gene amplification in breast cancer, the authors have developed sensitive methods that can be used to screen nucleic acid prepared from a variety of sources. In their analysis, Southern hybridization and DNA dot-blot analysis were used to screen 49 breast cancer DNAs for Myc, Neu, and Int-2 gene amplification. The analysis detected minimal one extra gene copy) as well as expanded (two or more extra gene copies) gene amplifications, and in addition, distinguished between gene amplification and aneuploidy as the cause of extra gene copies. These quantitative methods were adapted to patient specimens routinely available in the anatomic pathology laboratory, including fresh tumor tissue, tumor nuclei discarded during estrogen receptor analysis, and paraffin blocks. One minimal gene amplification was found in three cases of intraductal cancer. Of 25 cases of nonmetastatic invasive cancer, 28% had at least one extra Myc gene, whereas 24% had Neu, and 21% had Int-2 gene amplification. Of 21 cases of metastatic invasive cancer, 43% had Myc, 43% had Neu, and 40% had Int-2 gene amplification. Among the nonmetastatic cancers, 47% had one, 12% had two, and 4% had three amplified genes. Within the metastatic cancers, 48% had one, 28% had two, and 5% had three amplified genes. Our data suggest relationships between tumor progression and both incidence and size of Myc, Neu, and Int-2 gene amplification. Images Figure 1 Figure 2

Donovan-Peluso, M.; Contento, A. M.; Tobon, H.; Ripepi, B.; Locker, J.

1991-01-01

288

Equilibria of rotating isothermal clouds  

SciTech Connect

The effect of rotation on the equilibrium structure of an axisymmetric isothermal gas cloud embedded in a stationary external medium is investigated by means of numerical simulations. The underlying assumptions, basic equations, input parameters, and numerical approach are explained, and the results are presented in extensive tables and graphs and characterized in detail. The critical central density and rotation energy, beyond which clouds become unstable to global contraction/expansion and ring formation, respectively, are found to be 800 times the boundary-surface density (BSD) and 0.44 times the gravitational energy. Stable rotating clouds are shown to have maximum mass 31 times that of nonrotating clouds, maximum mean rotation velocity 2.7 times the sound speed, and maximum mean density 6 times BSD. An expression for the maximum height of the boundary surface above the equatorial plane is derived. 41 references.

Kiguchi, M.; Narita, S.; Miyama, S.M.; Hayashi, C.

1987-06-01

289

Lunar ash flows - Isothermal approximation.  

NASA Technical Reports Server (NTRS)

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

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

1972-01-01

290

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

291

Detection of Nucleic Acid Targets Using Ramified Rolling Circle DNA Amplification: A Single Nucleotide Polymorphism Assay Model  

PubMed Central

Background Isothermal amplification methods provide alternatives to PCR that may be preferable for some nucleic acid target detection tasks. Among current isothermal target detection methods, ramified rolling circle amplification (RAM) of single-stranded DNA circles that are formed by ligation of linear DNA probes (C-probes or padlock probes) offers a unique target detection system by linked primers and a simple amplification system that is unconstrained by the target’s sequence context. Earlier implementations of RAM-based target detection were reported to be limited by background noise, due in part to unligated C-probe in the amplification reaction. We show here that a target-detection system using a biotinylated target-capture probe together with automated bead-handling reduces or eliminates background amplification noise. We demonstrate the system’s performance by detection of a single-nucleotide polymorphism in human genomic DNA. Methodology Target detection by RAM entails hybridization and ligation of a C-probe, followed by amplification and RAM signal detection. We evaluated RAM target detection in genomic DNA using recognition of a human Factor V gene single nucleotide polymorphism (G1691A) as a model. Locus-specific C-probes were annealed and ligated to genomic DNAs that represent the 3 possible genotypes at this locus, then ligated C-probes were amplified by real time RAM. The majority of the steps in the assay were performed with a magnetic bead-based chemistry on an automated platform. We show that the specificity of C-probe ligation permits accurate genotyping of this polymorphism. The assay as described here eliminates some of the background noise previously described for C-probe ligation, RAM amplification assays. Conclusion The methods and results presented here show that a combination of C-probe detection, automated sample processing, and isothermal RAM amplification provide a practical approach for detecting DNA targets in complex mixtures.

Smith, James H.; Beals, Thomas P.

2013-01-01

292

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

293

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

294

Non-isothermal Turbulence in Molecular Clouds  

Microsoft Academic Search

Chemical and dynamical simulations of molecular clouds generally assume that the gas is perfectly isothermal. However, it is not clear that this approximation is always appropriate; supersonically turbulent gas will inevitably deviate from isothermality to some degree due to intermittent shock heating, but the extent of these deviations and their effects on the dynamical evolution and observational properties of molecular

S. C. O. Glover; M.-M. Mac Low; M. D. Smith; G. Pavlovski; A. Rosen

2003-01-01

295

Isothermal Circumstellar Dust Shell Model for Teaching  

ERIC Educational Resources Information Center

We introduce a model of radiative transfer in circumstellar dust shells. By assuming that the shell is both isothermal and its thickness is small compared to its radius, the model is simple enough for students to grasp and yet still provides a quantitative description of the relevant physical features. The isothermal model can be used in a…

Robinson, G.; Towers, I. N.; Jovanoski, Z.

2009-01-01

296

CARBON ADSORPTION ISOTHERMS FOR TOXIC ORGANICS  

EPA Science Inventory

An experimental protocol for measuring the activated carbon adsorption isotherm was developed and applied to a wide range of organic compounds. Methods for treatment of the isotherm data and a standard format for presentation of results are shown. In the early phase of the study ...

297

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

298

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

299

The Isothermal Dendritic Growth Experiment  

NASA Technical Reports Server (NTRS)

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

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

1998-01-01

300

Rapid detection of porcine kobuvirus in feces by reverse transcription loop-mediated isothermal amplification  

PubMed Central

Background PKV is a new emerging pathogen detected in diarrhea pigs. At present, no more detection methods were reported except RT-PCR method. this study was to develop a fast diagnostic method based on the LAMP reaction for rapid detection of PKV nucleic acid in fecal samples. Findings Two pairs of primers were designed to amplify the conservative 3D gene of PKV genome. The PKV RT-LAMP method possessed well specificity and had 100 times higher sensitivity than common reverse transcription PCR (RT-PCR), which could detect up to 10 RNA copies of the target gene. Conclusions The results showed that the optimal reaction condition for RT-LAMP was achieved at 64°C for 50 min. Furthermore, the RT-LAMP procedure does not demand special equipment and is time-saving.

2014-01-01

301

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

302

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

2013-02-01

303

Characterization of Norovirus RNA replicase for in vitro amplification of RNA  

PubMed Central

Background The isothermal amplification of RNA in vitro has been used for the study of in vitro evolution of RNA. Although Q? replicase has been traditionally used as an enzyme for this purpose, we planned to use norovirus replicase (NV3Dpol) due to its structural simplicity in the scope of in vitro autonomous evolution of the protein. Characteristics of the enzyme NV3Dpolin vitro were re-evaluated in this context. Results NV3Dpol, synthesized by using a cell-free translation system, represented the activities which were reported in the previous several studies and the reports were not fully consistent each other. The efficiency of the initiation of replication was dependent on the 3’-terminal structure of single-stranded RNA template, and especially, NV3Dpol preferred a self-priming small stem-loop. In the non-self-priming and primer-independent replication reaction, the presence of -CCC residues at the 3’-terminus increased the initiation efficiency and we demonstrated the one-pot isothermal RNA (even dsRNA) amplification by 16-fold. NV3Dpol also showed a weak activity of elongation-reaction from a long primer. Based on these results, we present a scheme of the primer-independent isothermal amplification of RNA with NV3Dpolin vitro. Conclusions NV3Dpol can be used as an RNA replicase in in vitro RNA?+?protein evolution with the RNA of special terminal sequences.

2013-01-01

304

Chromosomal destabilization during gene amplification.  

PubMed Central

Acentric extrachromosomal elements, such as submicroscopic autonomously replicating circular molecules (episomes) and double minute chromosomes, are common early, and in some cases initial, intermediates of gene amplification in many drug-resistant and tumor cell lines. In order to gain a more complete understanding of the amplification process, we investigated the molecular mechanisms by which such extrachromosomal elements are generated and we traced the fate of these amplification intermediates over time. The model system consists of a Chinese hamster cell line (L46) created by gene transfer in which the initial amplification product was shown previously to be an unstable extrachromosomal element containing an inverted duplication spanning more than 160 kilobases (J. C. Ruiz and G. M. Wahl, Mol. Cell. Biol. 8:4302-4313, 1988). In this study, we show that these molecules were formed by a process involving chromosomal deletion. Fluorescence in situ hybridization was performed at multiple time points on cells with amplified sequences. These studies reveal that the extrachromosomal molecules rapidly integrate into chromosomes, often near or at telomeres, and once integrated, the amplified sequences are themselves unstable. These data provide a molecular and cytogenetic chronology for gene amplification in this model system; an early event involves deletion to generate extrachromosomal elements, and subsequent integration of these elements precipitates a cascade of chromosome instability. Images

Ruiz, J C; Wahl, G M

1990-01-01

305

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...

306

Theoretical isothermal equation of state of rhenium  

SciTech Connect

A first-principles computation of the zero-degree isotherm has been carried out for Re, employing a linear muffin-tin orbital electron-band theory technique. This computed isotherm departs significantly from the isotherm derived from shock-wave data, but is in reasonable agreement with those from empirical equations of states based on zero-pressure parameters. Consequently, it is suggested that the 200--250-GPa pressures achieved by Vohra, Duclos, and Ruoff in recent diamond-anvil-cell experiments are underestimated by about half a megabar.

Sikka, S.K.; Vijayakumar, V.

1988-11-15

307

Multichange Isothermal Mutagenesis: a new strategy for multiple site-directed mutations in plasmid DNA  

PubMed Central

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.

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

2014-01-01

308

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

309

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

310

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

311

Linear Non-Isothermal Viscoelastic Solids.  

National Technical Information Service (NTIS)

With the use of a suitable representation for the free energy, explicit forms of the constitutive relations, the dissipation function and related thermodynamical restrictions are derived for infinitesimal deformation of linear non-isothermal viscoelastic ...

R. M. Christensen P. M. Naghdi

1966-01-01

312

ON THE ISOTHERMALITY OF SOLAR PLASMAS  

SciTech Connect

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

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

2010-11-01

313

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

PubMed

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

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

2014-01-01

314

Microwave amplification with nanomechanical resonators.  

PubMed

The sensitive measurement of electrical signals is at the heart of modern technology. According to the principles of quantum mechanics, any detector or amplifier necessarily adds a certain amount of noise to the signal, equal to at least the noise added by quantum fluctuations. This quantum limit of added noise has nearly been reached in superconducting devices that take advantage of nonlinearities in Josephson junctions. Here we introduce the concept of the amplification of microwave signals using mechanical oscillation, which seems likely to enable quantum-limited operation. We drive a nanomechanical resonator with a radiation pressure force, and provide an experimental demonstration and an analytical description of how a signal input to a microwave cavity induces coherent stimulated emission and, consequently, signal amplification. This generic scheme, which is based on two linear oscillators, has the advantage of being conceptually and practically simpler than the Josephson junction devices. In our device, we achieve signal amplification of 25 decibels with the addition of 20 quanta of noise, which is consistent with the expected amount of added noise. The generality of the model allows for realization in other physical systems as well, and we anticipate that near-quantum-limited mechanical microwave amplification will soon be feasible in various applications involving integrated electrical circuits. PMID:22170682

Massel, F; Heikkilä, T T; Pirkkalainen, J-M; Cho, S U; Saloniemi, H; Hakonen, P J; Sillanpää, M A

2011-12-15

315

Modeling isothermal and non-isothermal flows in porous media  

NASA Astrophysics Data System (ADS)

A complete understanding of the physics of flow and heat transfer phenomena in porous media is vital for accurate simulation of flow processes in industrial applications. In one such application pertaining to liquid composite molding (LCM) for manufacturing polymer composites, the fiber preforms used in LCM as reinforcements are limited not only to the single-scale porous media in the form of random fiber-mats, but also include dual-scale porous media in the form of woven or stitched fiber-mats. The conventional flow physics is not able to model the resin filling process in LCM involving the dual-scale porous media. In this study, the flow in dual-scale porous media is studied in order to predict the permeability of these fiber mats. The effect of aspect ratio of the fiber preform on the accuracy and flow during permeability estimation in single- and dual-scale porous media is analyzed experimentally and numerically. Flow of liquid in a free channel bounded on one side by porous medium is studied next, and two well-known boundary conditions of stress continuity and stress jump at the interface of the two regions are evaluated numerically. A point-wise solution for Stokes flow through periodic and non periodic porous media (made of cylindrical particles) adjacent to the free channel is presented using the Imite element based CFD software COMSOL. The efficacy of the two interfacial conditions is evaluated after volume averaging the point-wise velocity using a long averaging volume, also called the representative elementary volume or REV, and then comparing such a volume-averaged velocity profile with the available analytical solution. The investigation is carried out for five different porosities at three different Reynolds numbers to cover a wide range of applications. The presence of randomly-placed cylinders during the creation of non-periodic porous media damps out spatial fluctuations in the averaged velocity observed in periodic porous media. The analytical solutions obtained after applying the stress-continuity and stress-jump boundary conditions are found to work well at low porosities, which is in contradiction with the results achieved earlier by other researchers. The traditional approach of using averaged equations in the regions of sharp gradients in porous media to describe flow and transport is theoretically untenable and perhaps inaccurate. A novel ensemble averaging method is being proposed to test the accuracy of the volume averaged or smoothed description of flows in porous media in the regions of sharp gradients. In the new method, the flow in a certain arrangement of particles (called a realization) is averaged using a small unit cell, much smaller than the REV. Then such an averaged flow variable is further averaged over a whole gamut of randomly-generated particle realizations. First the accuracy of the ensemble averaging method was tested by comparing the permeability of an artificially generated porous medium obtained by the proposed method against the permeability predicted by some established theoretical models of permeability. The proposed method was found to be quite accurate. Later the ensemble average method was applied to the open-channel porous-medium interface region characterized by a sharp gradient in the flow velocities. It was discovered that the volume averaged description of such flows, characterized by the use of the Brinkman equation along with the stress-continuity and stress-jump conditions, is quite accurate for a range of Reynolds numbers. The non-isothermal transport during flow in porous media is examined next. The main focus in this area of research is the thermal dispersion term found in the heat transfer equation for single- and dual-scale porous media. Most of the previous efforts on modeling the heat transfer phenomena in porous media were devoted to isotropic porous media. However, for the anisotropic porous media widely in many industrial applications, not much research on the dispersion tensor is available. A new combined experimental/numerical approach to estimating

Mohseni Languri, Ehsan

316

Isothermal and non-isothermal torrefaction characteristics and kinetics of microalga Scenedesmus obliquus CNW-N.  

PubMed

Isothermal and non-isothermal torrefaction characteristics and kinetics of microalga Scenedesmus obliquus (S. obliquus) CNW-N are studied using thermogravimetric analysis. The pyrolysis of S. obliquus CNW-N with increasing temperature is characterized by four-stage decomposition. Depending on the torrefaction temperature, light, mild, and severe torrefaction from the weight loss and the maximum decomposition rate of the microalga can be classified. Under the same average temperature and torrefaction duration, non-isothermal torrefaction gives more severe pretreatment than the isothermal one. Increasing the heating rate of non-isothermal torrefaction also intensifies the pretreatment severity. Therefore, microalgae can be torrefied via non-isothermal torrefaction in a shorter time under the same pretreatment extent. The atomic H/C ratio in the microalga decreases with increasing torrefaction severity, whereas the atomic O/C ratio rises. The analysis suggests that the activation energy of isothermal torrefaction is 57.52×10(3)Jmol(-1), while it is between 40.14×10(3) and 88.41×10(3)Jmol(-1) for non-isothermal torrefaction. PMID:24457308

Chen, Wei-Hsin; Wu, Zih-Ying; Chang, Jo-Shu

2014-03-01

317

Frequency domain optical parametric amplification  

PubMed Central

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

Schmidt, Bruno E.; Thire, Nicolas; Boivin, Maxime; Laramee, Antoine; Poitras, Francois; Lebrun, Guy; Ozaki, Tsuneyuki; Ibrahim, Heide; Legare, Francois

2014-01-01

318

Frequency domain optical parametric amplification.  

PubMed

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

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

2014-01-01

319

Frequency domain optical parametric amplification  

NASA Astrophysics Data System (ADS)

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

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

2014-05-01

320

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

PubMed Central

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 spanning primer pairs to control false positive results in RT-PCR. Using NASBA, mRNA of the intronless human interferon-beta gene was demonstrated with a sensitivity of 10 copies, whereas 100 ng genomic DNA gave a negative result.

Heim, A; Grumbach, I M; Zeuke, S; Top, B

1998-01-01

321

Higher order Godunov schemes for isothermal hydrodynamics  

NASA Technical Reports Server (NTRS)

In this paper we construct higher order Godunov schemes for isothermal flow. Isothermal hydrodynamics serves as a good representation for several systems of astrophysical interest. The schemes designed here have second-order accuracy in space and time and some are third-order accurate for advection. Moreover, several ingredients of these schemes are essential components of even higher order. The methods designed here have excellent ability to represent smooth flow yet capture shocks with high resolution. Several test problems are presented. The algorithms presented here are compared with other algorithms having a comparable formal order of accuracy.

Balsara, Dinshaw S.

1994-01-01

322

Enzymatic Amplification of DNA/RNA Hybrid Molecular Beacon Signaling in Nucleic Acid Detection  

PubMed Central

A rapid assay operable under isothermal or non-isothermal conditions is described wherein the sensitivity of a typical molecular beacon (MB) system is improved by utilizing thermostable RNase H to enzymatically cleave an MB comprised of a DNA stem and RNA loop (R/D-MB). Upon hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (~5.7x above background) due to an opening of the probe and concomitant reduction in the Förster resonance energy transfer efficiency. 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 (12.9x above background), resulting in a ~2–2.8 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 PCR reactions by measuring enhancement of donor fluorescence upon 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.

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

2012-01-01

323

Performance Characteristics of an Isothermal Freeze Valve  

SciTech Connect

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

Hailey, A.E.

2001-08-22

324

Radiative heat transfer in isothermal spherical media  

Microsoft Academic Search

Radiative heat transfer in emitting, absorbing, and scattering spherical media is analyzed. The medium is assumed to be gray, isothermal, and linear-anisotropically scattering. The medium is confined in the space between two gray concentric spheres, which diffusely emit, and specularly and diffusely reflect radiation. Approximate solutions of the equation of radiative transfer are obtained using the spherical harmonics method. Results

W. Li; T. W. Tong

1990-01-01

325

Apparatus to Measure Adiabatic and Isothermal Processes.  

ERIC Educational Resources Information Center

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

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

1996-01-01

326

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

327

Rapid Isothermal Processing (RIP) of Dielectrics.  

National Technical Information Service (NTIS)

Rapid isothermal processing (RIP) based on incoherent sources of light has emerged as a major semiconductor processing technique. In this paper, a review of our own and other works on the RIP of dielectrics is presented. The inadequacy of conventional fur...

R. Singh F. Radpour

1988-01-01

328

A nucleic acid sequence-based amplification (NASBA) system for Listeria monocytogenes and simple method for detection of amplimers  

Microsoft Academic Search

A NASBA system amplifying specific sequences of the Listeria monocytogenes hlyA gene and an immunoenzymatic assay for the detection of amplimers was developed. The immunoenzymatic assay utilized a simple microtiter plate format and an anti-RNA:DNA hybrid antibody for the detection of NASBA product (predominantly RNA) hybridized to an immobilized DNA probe. This highly sensitive isothermal amplification and detection system was

Burton W. Blais; Geoff Turner; Roy Sooknananz; Lawrence T. Malek; Lucille M. Phillippe

1996-01-01

329

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

PubMed

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

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

2014-04-01

330

Pyrolysis kinetics of coking coal mixed with biomass under non-isothermal and isothermal conditions.  

PubMed

To investigate the kinetic characteristics of coking coal mixed with biomass during pyrolysis, thermogravimetric (TG) and thermo-balance reactor (TBR) analyses were conducted under non-isothermal and isothermal condition. Yellow poplar as a biomass (B) was mixed with weak coking coal (WC) and hard coking coal (HC), respectively. The calculated activation energies of WC/B blends were higher than those of HC/B blends under non-isothermal and isothermal conditions. The coal/biomass blends show increased reactivity and decreased activation energy with increasing biomass blend ratio, regardless of the coking properties of the coal. The different char structures of the WC/B and HC/B blends were analyzed by BET and SEM. PMID:24472746

Jeong, Ha Myung; Seo, Myung Won; Jeong, Sang Mun; Na, Byung Ki; Yoon, Sang Jun; Lee, Jae Goo; Lee, Woon Jae

2014-03-01

331

Laser amplification of incoherent radiation  

NASA Technical Reports Server (NTRS)

The amplification of noise in a laser amplifier is treated theoretically. The model for the active medium and its description using density-matrix techniques, are taken from the theory of laser operation. The spectral behavior of the radiation in the nonlinear regime is studied and the formalism is written from the onset in the frequency domain. The statistics of the light are gradually modified by the nonlinear amplification process, and expressions are derived for the rate of change of fluctuations in intensity as a measure of statistical changes. In addition, the range of validity of Litvak's Gaussian-statistics approximation is discussed. In the homogeneous-broadening case, the evolution of initially broadband Gaussian radiation toward quasimonochromatic oscillations with laserlike statistics is explored in several numerical examples. The connections of this study with the time-domain work on self-pulsing in a ring-laser configuration, are established. Finally, spectral-narrowing and -rebroadening effects in Doppler-broadened media are discussed both analytically and with numerical examples. These examples show the distinct contribution of pulsations in the population ('Raman-type terms'), and saturation phenomena.

Menegozzi, L. N.; Lamb, W. E., Jr.

1978-01-01

332

Increasing amplification success of forensic DNA samples using multiple displacement amplification  

Microsoft Academic Search

Multiple displacement amplification (MDA) is capable of amplifying nanogram template amounts of DNA with high accuracy to\\u000a generate micrograms of representative product. Although MDA is able to amplify small template amounts (<100 pg), increased\\u000a levels of preferential amplification and allelic dropout are observed. The use of molecular crowders (polyethylene glycol\\u000a 400) can decrease the amplification bias and increase amplification success. We

Kaye N. Ballantyne; Roland A. H. van Oorschot; R. John Mitchell

2007-01-01

333

Signal amplification of glucosamine-6-phosphate based on ribozyme glmS.  

PubMed

Ribozyme glmS based isothermal amplification assay is developed for the colorimetric detection of glucosamine-6-phosphate (GlcN6P). Upon binding to the metabolite target GlcN6P, self-cleavage of glmS ribozyme is initiated to release RNA fragment that can trigger the cascade signal amplification to release large amount of G-quadruplex DNAzymes as reporter for colorimetric detection. Given the importance of GlcN6P for cell wall biosynthesis, the glmS riboswitch has become a new drug target for the development of antibiotics. This assay not only offers a convenient detection of GlcN6P with high specificity and sensitivity, but also provides a platform for high-throughput screening of antibiotics based on glmS riboswitches. PMID:25038539

Zhao, Yongyun; Chen, Haodong; Du, Feng; Yasmeen, Afshan; Dong, Juan; Cui, Xin; Tang, Zhuo

2014-12-15

334

Instrument-free nucleic acid amplification assays for global health settings  

PubMed Central

Many infectious diseases that affect global health are most accurately diagnosed through nucleic acid amplification and detection. However, existing nucleic acid amplification tests are too expensive and complex for most low-resource settings. The small numbers of centralized laboratories that exist in developing countries tend to be in urban areas and primarily cater to the affluent. In contrast, rural area health care facilities commonly have only basic equipment and health workers have limited training and little ability to maintain equipment and handle reagents.1 Reliable electric power is a common infrastructure shortfall. In this paper, we discuss a practical approach to the design and development of non-instrumented molecular diagnostic tests that exploit the benefits of isothermal amplification strategies. We identify modular instrument-free technologies for sample collection, sample preparation, amplification, heating, and detection. By appropriately selecting and integrating these instrument-free modules, we envision development of an easy to use, infrastructure independent diagnostic test that will enable increased use of highly accurate molecular diagnostics at the point of care in low-resource settings.

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

2014-01-01

335

Isothermal crystallization of tripalmitin in sesame oil  

Microsoft Academic Search

Crystallization of tripalmitin (TP) in sesame oil was investigated under isothermal conditions at a cooling rate similar to\\u000a the one achieved in industrial crystallizers (1 K\\/min). The results obtained indicated that, at TP concentrations <0.98%,\\u000a triacylglycerides of sesame oil developed mixed crystals with TP. However, at concentrations within the interval of 0.98 to\\u000a 3.44%, tripalmitin crystallized independently from sesame oil.

Elena Dibildox-Alvarado; Jorge F. Toro-Vazquez

1997-01-01

336

Isothermal crystallization kinetics of refined palm oil  

Microsoft Academic Search

The induction times for the crystallization, under isothermal conditions, of refined, bleached, and deodorized palm oil from\\u000a the melt were studied by viscometry. At temperatures below 295 K, the crystallization of palm oil was observed to occur in\\u000a a two-stage process. This two-stage process was caused by the fractionation of palm oil, most probably into the stearin and\\u000a olein fractions.

C. W. Chen; O. M. Lai; H. M. Ghazali; C. L. Chong

2002-01-01

337

On the Isothermality of Solar Plasmas  

NASA Technical Reports Server (NTRS)

Recent measurements have shown that the quiet unstructured solar corona observed at the solar limb is close to isothermal, at a temperature that does not appear to change over wide areas or with time. Some in dividual active loop structures have also been found to be nearly iso thermal both along their axis and across their cross-section. Even a complex active region observed at the solar limb has been found to be composed of three distinct isothermal plasmas. If confirmed, these r esults would pose formidable challenges to the current theoretical understanding of the thermal structure and heating of the solar corona. For example, no current theoretical model can explain the excess dens ities and lifetimes of many observed loops if the loops are in fact i sothermal. All of these measurements are based on the so-called emiss ion measure (EM) diagnostic technique that is applied to a set of opt ically thin lines under the assumption of isothermal plasma. It provi des simultaneous measurement of both the temperature and EM. However, no study has ever been carried out to quantify the uncertainties in the technique and to rigorously assess its ability to discriminate bet ween isothermal and multithermal plasmas. Such a study is the topic o f the present work. We define a formal measure of the uncertainty in the EM diagnostic technique that can easily be applied to real data. We here apply it to synthetic data based on a variety of assumed plas ma thermal distributions, and develop a method to quantitatively asse ss the degree of multithermality of a plasma.

Landi, E.; Klimchuk, J. A.

2010-01-01

338

MOISTURE EQUILIBRIUM ISOTHERMS FOR A HANDMADE KRAFT PAPER  

Microsoft Academic Search

Moisture equilibrium isotherms are very important in drying and storage processes analysis. So, the DEQ\\/UEM process separation group is building a data bank for cellulose, paper and textile isotherms and their fit with available literature equations. This work intends to obtain the experimental isotherms of a brazilian handmade kraft paper. The experimental equilibrium data were obtained by exposing the material

MOTTA LIMA; Guilherme D. MACHADO; Roberta M. LUCHEIS; Nehemias C. PEREIRA

339

On numerical classification of solution adsorption isotherms.  

PubMed

To numerically classify solution adsorption isotherms, a difference or deviation measure, DS(mc) (the relative difference between two sums of the adsorption maximum's characteristics of selectivity isotherms x 100), is derived. The measure is applicable to completely miscible binary solutions on solids. This quantity evaluates the difference between an adsorption system and the ideal adsorption system (ideal adsorbed and bulk phases, homogeneous surface, and equal molar area solution components) at the point of maximum adsorption. For model systems, DS(mc)s are calculated at several levels of surface heterogeneity (Gaussian distribution of surface energy) and for different signs of phase nonideality (regular solution phases) on a homogeneous surface and on a simple two-site-type heterogeneous surface. All heterogeneous surfaces have negative DS(mc) values, but nonideal phases have DS(mc)s with signs opposite to the sign of deviation from Raoult's law. DS(mc)s from both U- and S-shape isotherms are reported for 16 experimental systems consisting of hydrocarbon mixtures and both alcohol + hydrocarbon and alcohol + water solutions or acetone + carbon tetrachloride on several silica gels and a variety of carbons. PMID:15807590

Coltharp, Marcellus T

2005-04-12

340

Tsunami Amplification due to Focusing  

NASA Astrophysics Data System (ADS)

Tsunami runup measurements over the periphery of the Pacific Ocean after the devastating Great Japan tsunami of 11 March 2011 showed considerable variation in far-field and near-field impact. This variation of tsunami impact have been attributed to either directivity of the source or by local topographic effects. Directivity arguments alone, however, cannot explain the complexity of the radiated patterns in oceans with trenches and seamounts. Berry (2007, Proc. R. Soc. Lond. A 463, 3055-3071) discovered how such underwater features may concentrate tsunamis into cusped caustics and thus cause large local amplifications at specific focal points. Here, we examine focusing and local amplification, not by considering the effects of underwater diffractive lenses, but by considering the details of the dipole nature of the initial profile, and propose that certain regions of coastline are more at-risk, not simply because of directivity but because typical tsunami deformations create focal regions where abnormal tsunami wave height can be registered (Marchuk and Titov, 1989, Proc. IUGG/IOC International Tsunami Symposium, Novosibirsk, USSR). In this work, we present a new general analytical solution of the linear shallow-water wave equation for the propagation of a finite-crest-length source over a constant depth without any restriction on the initial profile. Unlike the analytical solution of Carrier and Yeh (2005, Comp. Mod. Eng. & Sci. 10(2), 113-121) which was restricted to initial conditions with Gaussian profiles and involved approximation, our solution is not only exact, but also general and allows the use of realistic initial waveform such as N-waves as defined by Tadepalli and Synolakis (1994, Proc. R. Soc. Lond. A 445, 99-112). We then verify our analytical solution for several typical wave profiles, both with the NOAA tsunami forecast model MOST (Titov and Synolakis, 1998, J. Waterw. Port Coast. Ocean Eng. 124(4), 157-171) which is validated and verified through (Synolakis et al., 2008, Pure Appl. Geophys. 165(11-12), 2197-2228), and with a Boussinesq model, to illustrate the role focusing can play for different initial conditions, and to show the robust nature of focusing with respect to dispersion. We also show how the focusing effect might have played a role in the 17 July 1998 Papua New Guinea and 17 July 2006 Java events, and also the 11 March 2011 Great Japan earthquake and tsunami. Our results strongly imply that focusing increases the shoreline amplification of the tsunami.; Schematic of focusing; initial displacement (upper left), wave evolution (upper right, lower left), maximum wave amplitude with focusing (lower right).

Moore, C. W.; Kanoglu, U.; Titov, V. V.; Aydin, B.; Spillane, M. C.; Synolakis, C. E.

2012-12-01

341

Mechanisms of Metal-Induced Centrosome Amplification  

PubMed Central

Exposure to toxic and carcinogenic metals is widespread; however, their mechanisms of action remain largely unknown. One potential mechanism for metal-induced carcinogenicity and toxicity is centrosome amplification. Here, we review the mechanisms for metal-induced centrosome amplification, including arsenic, chromium, mercury and nano-titanium dioxide.

Holmes, Amie L.; Wise, John Pierce

2014-01-01

342

Third Sound Amplification and Detailed Balance  

NASA Astrophysics Data System (ADS)

Condensation of atoms from the vapor into a third sound resonance is expected to be capable of acoustic amplification. This results from normal to superfluid conversion that coherently accommodates atoms into the third sound velocity field. Consideration of third sound in light of the equilibrium detailed balance between vapor particles and the superfluid film provides further evidence that acoustic amplification is attainable.

Eddinger, J. D.; Ellis, F. M.

2006-09-01

343

Synchronous Amplification of Subpicosecond Pulses.  

NASA Astrophysics Data System (ADS)

The amplification of subpicosecond optical pulses using ultrashort synchronized pump pulses (synchronous amplification) is investigated in this thesis. The research presented here includes the construction and assessment, both experimental and theoretical, of a unique high power organic dye amplifier system pumped by modelocked, frequency doubled, Nd:YAG laser pulses. Advantages of this system over previous amplifier systems pumped with longer Q-switched pulses include a higher pump energy to output energy conversion efficiency and significantly reduced amplified spontaneous emission (ASE) generation. The availability of high power, wavelength tunable, subpicosecond laser pulses has opened up a number of rapidly developing research areas in time resolved Physics, Chemistry, Biology and Quantum Optics. The design and operation of a three stage dye amplifier, which amplifies subpicosecond pulses to a peak power of 1 gigawatt, is described. Components of the system include a dye oscillator, which produces optical pulses at 605 nm as short as 70 femtoseconds, and two Nd:YAG laser systems, each of which is capable of preparing the ultrashort synchronized pump pulses used to drive the three stage dye amplifier. The synchronization between the pump pulse and the subpicosecond input pulse is found to be 5 ps. A section on dye amplifier design includes a discussion of staging, pumping geometry, spot size, dye solvent, alignment and timing. The experimental assessment of the system includes measurements of the gain, pulsewidth, ASE, amplitude fluctuations, beam quality and nonlinear index effects. In order to optimize the synchronously pumped amplifier system, the factors that limit the gain in a single amplifier stage are investigated experimentally. The effects of saturation, and three loss mechanisms: fluorescence, molecular reorientation and ASE, are presented. Measurements of the transient gain, using a pump-probe technique, are used to investigate the loss mechanisms. A theory that models the behavior of a dye amplifier system under ultrashort pulse excitation has been developed. Because no steady state gain assumption is included, the theory can predict the transient gain, and results are compared to experiment. The theory is also used to predict saturation behavior of the amplifier including pulse broadening. A theoretical comparison of amplifiers pumped with modelocked pump pulses and Q-switched pump pulses is also presented.

Kafka, James David

344

Pressure-composition-isotherms of palladium alloys  

SciTech Connect

About one year ago a summary report was submitted covering the previous three years of the contract. This earlier report should be consulted as a useful survey and evaluation of the research carried out by the authors. Because of difficulties during the current contract period arising from the anomalous nature of the melt-spun alloys received from LANL, it is not possible to contribute much beyond that given in last year's summary with regard to the overall picture of the behavior of Pd-rich alloys towards hydrogen and its isotopes. In this contract year deuterium was employed instead of hydrogen and instead of using cycled alloys, the alloys employed for each isotherm measurement were in their virgin condition. Because of the anomalous behavior of the melt-spun alloys, it was not feasible or worthwhile in some cases, e.g., when the alloy behaved anomalously, to carry out all of the originally proposed work. Nonetheless considering these obstacles, some useful data were obtained. For example, the obtaining of deuterium isotherms for the Pd-Rh alloys down to {minus}40 C using internally oxidized melt-spun alloys may prove to be useful.

Flanagan, T.B.

1996-11-01

345

Computer Modeling of Non-Isothermal Crystallization  

NASA Technical Reports Server (NTRS)

A realistic computer model for simulating isothermal and non-isothermal phase transformations proceeding by homogeneous and heterogeneous nucleation and interface-limited growth is presented. A new treatment for particle size effects on the crystallization kinetics is developed and is incorporated into the numerical model. Time-dependent nucleation rates, size-dependent growth rates, and surface crystallization are also included. Model predictions are compared with experimental measurements of DSC/DTA peak parameters for the crystallization of lithium disilicate glass as a function of particle size, Pt doping levels, and water content. The quantitative agreement that is demonstrated indicates that the numerical model can be used to extract key kinetic data from easily obtained calorimetric data. The model can also be used to probe nucleation and growth behavior in regimes that are otherwise inaccessible. Based on a fit to data, an earlier prediction that the time-dependent nucleation rate in a DSC/DTA scan can rise above the steady-state value at a temperature higher than the peak in the steady-state rate is demonstrated.

Kelton, K. F.; Narayan, K. Lakshmi; Levine, L. E.; Cull, T. C.; Ray, C. S.

1996-01-01

346

Numerical Model for Isothermal and Non-Isothermal Crystallization of Liquids and Glasses  

NASA Technical Reports Server (NTRS)

A new numerical model of isothermal and non-isothermal first order phase transformations, such as the crystallization of liquids and glasses, is presented. This model computes directly the volume fraction transformed, taking into account time-dependent nucleation rates and cluster-size-dependent growth velocities. The model is applied to the crystallization of lithium disilicate glass, using the appropriate kinetic and thermodynamic parameters. The model is used (1) to determine the validity of common methods for computing the volume fraction transformed as a function of time in isothermal experiments when a time-dependent nucleation rate is expected, (2) to simulate non-isothermal differential scanning calorimetry (DSC) studies of glass devitrification as a function of scan rate, and (3) to compute the effect of preannealing on the DSC peak parameters. A novel behavior of the nucleation rate with scan rate is predicted, arising because the relaxation of the cluster distribution cannot be described by a single relaxation time. Comparisons of the calculations with experimental data on this glass demonstrate the validity of the model.

Kelton, K. F.

1993-01-01

347

Tension amplification in branched macromolecules  

NASA Astrophysics Data System (ADS)

A molecule's topology can greatly affect the distribution of tension within its bonds. Pom-pom molecules consist of a short linear spacer linking two star polymers, each containing z long arms. The striking ability of this molecular architecture to magnify spacer tension in solution by several orders of magnitude, from the pN to the nN level is due to the steric repulsion between densely packed side chains. In fact, the tension can increase to values that significantly alter the molecule's chemical properties, or even initiate the scission of a carbon-carbon covalent bond in the spacer chain. We study the tension distribution in the spacer and in side branches using molecular dynamics simulations and scaling theory. The dependences of observables such as spacer tension and root-mean-square spacer length on the number of side chains, chemical spacer length, and length of side branches have been quantified. Scaling models are used to explain the interrelated phenomena of tension amplification and spacer elongation and to interpret the results of molecular dynamics simulations.

Bacanu, Alexandru; Brock, James; Panyukov, Sergey; Rubinstein, Michael

2012-02-01

348

Energy amplification in turbulent channels  

NASA Astrophysics Data System (ADS)

We study the temporal stability of the Orr-Sommerfeld and Squire equations in channels with turbulent mean velocity profiles and turbulent eddy viscosities. All the eigensolutions of this problem are damped, but initial perturbations with wavelengths ?x> ?z can grow temporarily before decaying. For each wavelength, the structure of the most amplified solution agrees with that of the most energetic POD eigenfunction obtained from the available direct numerical simulations (180 <=Re?<=1900). The transient growth has two local maxima at ?z^+ = 100 and ?z/h = 3, which coincide with the widths of the near-wall streaks and of the largest structures of the outer layer. The dynamics of both the near-wall and the outer solutions are similar. They start with a wall-normal v event which does not grow but which forces streamwise velocity fluctuations by stirring the mean shear (uv<0). The resulting u fluctuations grow significantly and last longer than the v ones, containing nearly all the kinetic energy at the instant of maximum amplification.

del Alamo, Juan C.; Jimenez, Javier; Zandonade, Paulo; Moser, Robert D.

2005-11-01

349

Surface Elastic-Wave Propagation and Amplification.  

National Technical Information Service (NTIS)

Surface elastic-wave propagation, transduction, and amplification (in a piezoelectric semiconductor) are discussed with emphasis on characteristics useful in electronic devices. Computed curves show the dependence on distance from the surface of the elast...

R. M. White

1967-01-01

350

Chirped Pulse Amplification: Present and Future.  

National Technical Information Service (NTIS)

Short pulses with ultrahigh peak powers have been generated in Nd: glass and Alexandrite using the Chirped Pulse Amplification (CPA) technique. This technique has been successful in producing picosecond terawatt pulses with a table-top laser system. In th...

P. Maine D. Strickland M. Pessot J. Squier P. Bado

1988-01-01

351

AMPLIFICATION OF RIBOSOMAL RNA SEQUENCES - Book Chapter  

EPA Science Inventory

This book chapter contains the following headings and subheadings: Introduction; Experimental Approach - Precautions, Template, Primers, Reaction Conditions, Enhancers, Post Amplification; Procedures - Template DNA, Basic PCR, Thermal Cycle Parameters, Enzyme Addition, Agarose Ge...

352

Amplification of Angular Rotations Using Weak Measurements  

NASA Astrophysics Data System (ADS)

We present a weak measurement protocol that permits a sensitive estimation of angular rotations based on the concept of weak-value amplification. The shift in the state of a pointer, in both angular position and the conjugate orbital angular momentum bases, is used to estimate angular rotations. This is done by an amplification of both the real and imaginary parts of the weak-value of a polarization operator that has been coupled to the pointer, which is a spatial mode, via a spin-orbit coupling. Our experiment demonstrates the first realization of weak-value amplification in the azimuthal degree of freedom. We have achieved effective amplification factors as large as 100, providing a sensitivity that is on par with more complicated methods that employ quantum states of light or extremely large values of orbital angular momentum.

Magaña-Loaiza, Omar S.; Mirhosseini, Mohammad; Rodenburg, Brandon; Boyd, Robert W.

2014-05-01

353

Self-Pumped Josephson Parametric Amplification.  

National Technical Information Service (NTIS)

Parametric gain has been demonstrated at 30 MHz using the non-linear inductance of Josephson point contact devices. SQUID type configurations were used to achieve degenerate mode, negative resistance amplification with self-pumping achieved via the intern...

A. H. Silver H. Kanter

1971-01-01

354

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

355

Amplification of chromosomal DNA in situ  

DOEpatents

Amplification of chromosomal DNA in situ to increase the amount of DNA associated with a chromosome or chromosome region is described. The amplification of chromosomal DNA in situ provides for the synthesis of Fluorescence in situ Hybridization (FISH) painting probes from single dissected chromosome fragments, the production of cDNA libraries from low copy mRNAs and improved in Comparative Genomic Hybridization (CGH) procedures.

Christian, Allen T. (Tracy, CA); Coleman, Matthew A. (Livermore, CA); Tucker, James D. (Livermore, CA)

2002-01-01

356

Broadband amplification of ultraviolet laser pulses  

Microsoft Academic Search

Broadband amplification of laser pulses at 400 nm is demonstrated with a maximum gain of 3550 using non-collinear optical parametric amplification. Without depleting the pump, the spectral gain was found to be almost flat along the available 17-nm-wide spectrum of the signal pulse. It is also shown that the group velocity difference between the signal and pump pulses eventually results in

K. Osvay; G. Kurdi; J. Klebniczki; M. Csatari; I. N. Ross; E. J. Divall; C. J. Hooker; A. J. Langley

2002-01-01

357

Isothermal crystallisation behaviour and kinetics of polyvinylalcohol\\/silica nanocomposite  

Microsoft Academic Search

In the present study, the isothermal crystallisation kinetics and behaviour of polyvinylalcohol\\/silica (PVA\\/SiO2) nanocomposite prepared with self assembly monolayer (SAM) technique are characterized by conducting differential scanning calorimetric (DSC) analysis. Several kinetics equations are employed to describe the isothermal crystallisation of PVA\\/SiO2 nanocomposite compared to the pure PVA. During the isothermal crystallisation, the peak crystallisation time (tp), half time of

Zheng Peng; Ling Xue Kong; Si-Dong Li

2005-01-01

358

Non-isothermal crystallization of polyvinylalcohol-co-ethylene  

Microsoft Academic Search

Non-isothermal crystallization of polyvinylalcohol-co-ethylene with different ethylene contents was studied. Several models were used to predict the crystallization behavior of these materials under non-isothermal conditions at a constant cooling rate. Kinetic parameters determined from isothermal date were employed. Experimental data were in accordance with model prediction at low cooling rate and relative degree of crystallization lower than 0.8, but it

V. A. Alvarez; P. M. Stefani; A. Vázquez

2005-01-01

359

Suppression of Gene Amplification in Human Cell Hybrids  

Microsoft Academic Search

Gene amplification, one example of genetic instability, is of prognostic and clinical importance in neoplasia. In tumorigenic cells, gene amplification occurs at a very high frequency, whereas in normal diploid fibroblasts the event is undetectable by the clonogenic assay. To investigate genetic control of gene amplification, amplification frequency was measured in hybrids of tumorigenic cells and normal diploid cells. The

Thea D. Tlsty; Alicia White; Janet Sanchez

1992-01-01

360

Isothermal aging of three polyurethane elastomers  

SciTech Connect

Two polyurethane systems, EN-7 and L-100, have a long history as encapsulants and coatings in Sandia programs. These materials contain significant amounts of toluene diisocyanate (TDI), a suspect human carcinogen. As part of efforts to reduce the use of hazardous materials in the workplace, PET-90A, a polyurethane with less than 0.1% free TDI, was identified as a candidate for new applications and as a replacement for the more hazardous polyurethanes in selected programs. This report documents the results of a two-year accelerated aging study of PET-90A, EN-7, and L-100 polyurethane elastomers to characterize the effect of 135{degrees}F isothermal aging on selected physical, electrical, mechanical and thermal properties. In general, there was very little change in properties over the two year period for the three elastomers. The largest changes occurred in EN-7, which is the polyurethane with the longest service history in Sandia applications.

Guess, T.R.

1996-05-01

361

The equilibria of rotating isothermal clouds  

NASA Astrophysics Data System (ADS)

The effect of rotation on the equilibrium structure of an axisymmetric isothermal gas cloud embedded in a stationary external medium is investigated by means of numerical simulations. The underlying assumptions, basic equations, input parameters, and numerical approach are explained, and the results are presented in extensive tables and graphs and characterized in detail. The critical central density and rotation energy, beyond which clouds become unstable to global contraction/expansion and ring formation, respectively, are found to be 800 times the boundary-surface density (BSD) and 0.44 times the gravitational energy. Stable rotating clouds are shown to have maximum mass 31 times that of nonrotating clouds, maximum mean rotation velocity 2.7 times the sound speed, and maximum mean density 6 times BSD. An expression for the maximum height of the boundary surface above the equatorial plane is derived.

Kiguchi, Masayoshi; Narita, Shinji; Miyama, Shoken M.; Hayashi, Chushiro

1987-06-01

362

Isothermal Dendritic Growth Experiment - SCN Dendrites  

NASA Technical Reports Server (NTRS)

The Isothermal Dendritic Growth Experiment (IDGE), flown on three Space Shuttle missions, is yielding new insights into virtually all industrially relevant metal and alloy forming operations. IDGE used transparent organic liquids that form dendrites (treelike structures) similar to the crystals that form inside metal alloys. Comparing Earth-based and space-based dentrite growth velocity, tip size and shape provid a better understanding of the fundamentals of dentritic growth, including gravity's effects. These shadowgraphic images show succinonitrile (SCN) dentrites growing in a melt (liquid). The space-grown crystals also have cleaner, better defined sidebranches. IDGE was developed by Rensselaer Polytechnic Institude (RPI) and NASA/ Glenn Research Center(GRC). Advanced follow-on experiments are being developed for flight on the International Space Station. Photo gredit: NASA/Glenn Research Center

1995-01-01

363

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

364

ANALYTICAL SOLUTIONS OF SINGULAR ISOTHERMAL QUADRUPOLE LENS  

SciTech Connect

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. [Key Laboratory for Research in Galaxies and Cosmology, Shanghai Astronomical Observatory, Chinese Academy of Sciences, 80 Nandan Road, Shanghai 200030 (China); Yang Xiaofeng, E-mail: chuzhe@shao.ac.cn, E-mail: linwp@shao.ac.cn [School of Astronomy and Space Science, Nanjing University, Nanjing 210093 (China)

2013-06-20

365

Circle-to-circle amplification on a digital microfluidic chip for amplified single molecule detection.  

PubMed

We demonstrate a novel digital microfluidic nucleic acid amplification concept which is based on padlock probe mediated DNA detection and isothermal circle-to-circle amplification (C2CA). This assay platform combines two digital approaches. First, digital microfluidic manipulation of droplets which serve as micro-reaction chambers and shuttling magnetic particles between these droplets facilitates the integration of complex solid phase multistep assays. We demonstrate an optimized novel particle extraction and transfer protocol for superparamagnetic particles on a digital microfluidic chip that allows for nearly 100% extraction efficiencies securing high assay performance. Second, the compartmentalization required for digital single molecule detection is solved by simple molecular biological means, circumventing the need for complex microfabrication procedures necessary for most, if not all, other digital nucleic acid detection methods. For that purpose, padlock probes are circularized in a strictly target dependent ligation reaction and amplified through two rounds of rolling circle amplification, including an intermediate digestion step. The reaction results in hundreds of 500 nm sized individually countable DNA nanospheres per detected target molecule. We demonstrate that integrated miniaturized digital microfluidic C2CA results in equally high numbers of C2CA products ?L(-1) as off-chip tube control experiments indicating high assay performance without signal loss. As low as 1 aM synthetic Pseudomonas aeruginosa DNA was detected with a linear dynamic range over 4 orders of magnitude up to 10 fM proving excellent suitability for infectious disease diagnostics. PMID:24934991

Kühnemund, Malte; Witters, Daan; Nilsson, Mats; Lammertyn, Jeroen

2014-08-21

366

Heat induces gene amplification in cancer cells  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer This study discovered that heat exposure (hyperthermia) results in gene amplification in cancer cells. Black-Right-Pointing-Pointer Hyperthermia induces DNA double strand breaks. Black-Right-Pointing-Pointer DNA double strand breaks are considered to be required for the initiation of gene amplification. Black-Right-Pointing-Pointer The underlying mechanism of heat-induced gene amplification is generation of DNA double strand breaks. -- Abstract: Background: Hyperthermia plays an important role in cancer therapy. However, as with radiation, it can cause DNA damage and therefore genetic instability. We studied whether hyperthermia can induce gene amplification in cancer cells and explored potential underlying molecular mechanisms. Materials and methods: (1) Hyperthermia: HCT116 colon cancer cells received water-submerged heating treatment at 42 or 44 Degree-Sign C for 30 min; (2) gene amplification assay using N-(phosphoacetyl)-L-aspartate (PALA) selection of cabamyl-P-synthetase, aspartate transcarbarmylase, dihydro-orotase (cad) gene amplified cells; (3) southern blotting for confirmation of increased cad gene copies in PALA-resistant cells; (4) {gamma}H2AX immunostaining to detect {gamma}H2AX foci as an indication for DNA double strand breaks. Results: (1) Heat exposure at 42 or 44 Degree-Sign C for 30 min induces gene amplification. The frequency of cad gene amplification increased by 2.8 and 6.5 folds respectively; (2) heat exposure at both 42 and 44 Degree-Sign C for 30 min induces DNA double strand breaks in HCT116 cells as shown by {gamma}H2AX immunostaining. Conclusion: This study shows that heat exposure can induce gene amplification in cancer cells, likely through the generation of DNA double strand breaks, which are believed to be required for the initiation of gene amplification. This process may be promoted by heat when cellular proteins that are responsible for checkpoints, DNA replication, DNA repair and telomere functions are denatured. To our knowledge, this is the first study to provide direct evidence of hyperthermia induced gene amplification.

Yan, Bin, E-mail: yanbin@mercyhealth.com [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States) [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Mercy Cancer Center, Mercy Medical Center-North Iowa, Mason City, IA 50401 (United States); Ouyang, Ruoyun [Department of Respiratory Medicine, The Second Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410011 (China)] [Department of Respiratory Medicine, The Second Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410011 (China); Huang, Chenghui [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States) [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Department of Oncology, The Third Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410013 (China); Liu, Franklin [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States)] [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States); Neill, Daniel [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States)] [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Li, Chuanyuan [Dermatology, Duke University Medical Center, Durham, NC 27710 (United States)] [Dermatology, Duke University Medical Center, Durham, NC 27710 (United States); Dewhirst, Mark [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States)] [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States)

2012-10-26

367

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

368

Evaluation of an ultrabroadband high-gain amplification technique for chirped pulse amplification facilities.  

PubMed

Recently, an amplification technique for ultrashort pulses was explored in detail in a theoretical paper by Ross et al. [Opt. Commun. 144, 125 (1997)]. The technique, based on nonlinear optics, is called optical parametric chirped pulse amplification. It has a number of features that, in principle, make it highly attractive. It primarily offers extremely large gains simultaneously with extremely large bandwidths. Additional attractions are virtually no spatial and temporal phase distortion of the amplified pulse, high efficiencies and a low thermal loading, reduced amplified spontaneous emission levels, small optical material lengths, and an inherent simplicity of implementation. We present an evaluation of the technique as a front end amplifier for the ultrashort pulse amplification chain of the Vulcan laser system. Such a device could replace some of the existing amplification in Nd:glass and additionally have a wider effect as a direct replacement of Ti:sapphire regenerative amplifiers on large-scale chirped pulse amplification scale facilities. PMID:18324304

Collier, J; Hernandez-Gomez, C; Ross, I N; Matousek, P; Danson, C N; Walczak, J

1999-12-20

369

Sorption Isotherms of Acetone on Various Building Materials  

Microsoft Academic Search

The physical modelling of Indoor Air Quality (IAQ) suf fers a lack of sorption data for the most common Volatile Organic Compounds (VOC) on building materials. This paper deals with an experimental facility that aimed to provide the sorption isotherms of gaseous contaminants on various materials. It was used to determine the sorp tion isotherms of acetone on chipboard, acrylic

Anne-Lise Tiffonnet; Patrice Blondeau; Francis Allard; Fariborz Haghighat

2002-01-01

370

Giant planet formation. A first classification of isothermal protoplanetary equilibria  

Microsoft Academic Search

We present a model for the equilibrium of solid planetary cores embedded in a gaseous nebula. From this model we are able to extract an idealized roadmap of all hydrostatic states of the isothermal protoplanets. The complete classification of the isothermal protoplanetary equilibria should improve the understanding of the general problem of giant planet formation, within the framework of the

B. Pecnik; G. Wuchterl

2005-01-01

371

Financial Statement Audit Report of Isothermal Community College.  

ERIC Educational Resources Information Center

This report presents the results of the Isothermal Community College financial statement audit for the fiscal year ending on June 30, 1998. Isothermal Community College is a component of the State of North Carolina, thus the authority to audit is granted by Article 5A of G.S. 147. The accounts and operations of the institution were subject to…

Campbell, Ralph

372

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

373

Remote fiber sensors and optical amplification  

NASA Astrophysics Data System (ADS)

This work discusses remote fiber sensors enabled by optical amplification. Continuous wave numerical modeling based on the propagation of pumps and signal lasers coupled to optical fibers explores Raman amplification schemes to predict the sensor's behavior. Experimental analyses report the results to a temperature remote optical sensor with 50 km distance between the central unit and the sensor head. An electrical interrogation scheme is used due to their low cost and good time response. Different architectures in remote sensor systems are evaluated, where diffraction gratings are the sensor element. A validation of calculated results is performed by experimental analyses and, as an application, the noise generated by Raman amplification in the remote sensors systems is simulated applying such numerical modeling. The analyses of sensors systems based on diffraction gratings requires optical broadband sources to interrogate the optical sensor unit, mainly in long period gratings that shows a characteristic rejection band. Therefore, the sensor distance is limited to a few kilometers due to the attenuation in optical fibers. Additional attenuation is introduced by the sensor element. Hence, to extend the distance in the optical sensor system, the optical amplification system is needed to compensate the losses in the optical fibers. The Raman amplification technology was selected mainly due to the flexibility in the gain bandwidth. The modeling can be applied to sensor systems that monitor sites located at long distances, or in places that the access is restricted due to harsh environment conditions in such cases conventional sensors are relatively fast deteriorated.

Pontes, M. J.; Coelho, Thiago V. N.; Carvalho, Joel P.; Santos, J. L.; Guerreiro, A.

2013-11-01

374

Loop-Mediated Isothermal Amplification Compared to Real-Time PCR and Enzyme Immunoassay for Toxigenic Clostridium difficile Detection  

PubMed Central

Clostridium difficile infection is the primary cause of health care-associated diarrhea. While most laboratories have been using rapid antigen tests for detecting C. difficile toxins, they have poor sensitivity; newer molecular methods offer rapid results with high test sensitivity and specificity. This study was designed to compare the performances of two molecular assays (Meridian illumigene and BD GeneOhm) and two antigen assays (Wampole Quik Chek Complete and TechLab Tox A/B II) to detect toxigenic C. difficile. Fecal specimens from hospitalized patients (n = 139) suspected of having C. difficile infection were tested by the four assays. Nine specimens were positive and 109 were negative by all four methods. After discrepant analysis by toxigenic culture (n = 21), the total numbers of stool specimens classified as positive and negative for toxigenic C. difficile were 21 (15%) and 118 (85%), respectively. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were as follows: GeneOhm (95.2%, 100%, 100%, and 99.2%), illumigene (95.2%, 96.6%, 83.3%, and 99.2%), Tox A/B II (52.4%, 97.5%, 78.6%, and 92.4%), and Quik Chek Complete (47.6%, 100%, 100%, and 91.9%). The illumigene assay performed comparably to the GeneOhm assay with a slight decrease in test specificity; the sensitivities of both far exceeded those of the antigen assays. The clinical characteristics of the concordant and discrepant study patients were similar, including stool consistency and frequency. In the era of rapid molecular-based tests for toxigenic C. difficile, toxin enzyme immunoassays (EIAs) should no longer be considered the standard of care.

Sural, Preethi; Loomis, Caroline R.; Pesta, Christine; Gonzalez-Krellwitz, Laura; Robinson-Dunn, Barbara; Riska, Paul

2012-01-01

375

Loop-mediated isothermal amplification compared to real-time PCR and enzyme immunoassay for toxigenic Clostridium difficile detection.  

PubMed

Clostridium difficile infection is the primary cause of health care-associated diarrhea. While most laboratories have been using rapid antigen tests for detecting C. difficile toxins, they have poor sensitivity; newer molecular methods offer rapid results with high test sensitivity and specificity. This study was designed to compare the performances of two molecular assays (Meridian illumigene and BD GeneOhm) and two antigen assays (Wampole Quik Chek Complete and TechLab Tox A/B II) to detect toxigenic C. difficile. Fecal specimens from hospitalized patients (n = 139) suspected of having C. difficile infection were tested by the four assays. Nine specimens were positive and 109 were negative by all four methods. After discrepant analysis by toxigenic culture (n = 21), the total numbers of stool specimens classified as positive and negative for toxigenic C. difficile were 21 (15%) and 118 (85%), respectively. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were as follows: GeneOhm (95.2%, 100%, 100%, and 99.2%), illumigene (95.2%, 96.6%, 83.3%, and 99.2%), Tox A/B II (52.4%, 97.5%, 78.6%, and 92.4%), and Quik Chek Complete (47.6%, 100%, 100%, and 91.9%). The illumigene assay performed comparably to the GeneOhm assay with a slight decrease in test specificity; the sensitivities of both far exceeded those of the antigen assays. The clinical characteristics of the concordant and discrepant study patients were similar, including stool consistency and frequency. In the era of rapid molecular-based tests for toxigenic C. difficile, toxin enzyme immunoassays (EIAs) should no longer be considered the standard of care. PMID:22189114

Boyanton, Bobby L; Sural, Preethi; Loomis, Caroline R; Pesta, Christine; Gonzalez-Krellwitz, Laura; Robinson-Dunn, Barbara; Riska, Paul

2012-03-01

376

Rapid and Real-Time Detection of Chikungunya Virus by Reverse Transcription Loop-Mediated Isothermal Amplification Assay  

Microsoft Academic Search

template and time of positivity value over a range of 2 108 to 2 102 copies was obtained. The feasibility of CHIKV RT-LAMP for clinical diagnosis was validated with patient serum samples from an ongoing epidemic in Southern India. Optimal assay conditions with zero background were established for the detection of low levels of CHIKV in acute-phase patient serum samples.

M. M. Parida; S. R. Santhosh; P. K. Dash; N. K. Tripathi; V. Lakshmi; N. Mamidi; A. Shrivastva; N. Gupta; P. Saxena; J. Pradeep Babu; P. V. Lakshmana Rao; Kouichi Morita

2007-01-01

377

Etiologic Diagnosis of Lower Respiratory Tract Bacterial Infections Using Sputum Samples and Quantitative Loop-Mediated Isothermal Amplification  

Microsoft Academic Search

Etiologic diagnoses of lower respiratory tract infections (LRTI) have been relying primarily on bacterial cultures that often fail to return useful results in time. Although DNA-based assays are more sensitive than bacterial cultures in detecting pathogens, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. Here we

Yu Kang; Rui Deng; Can Wang; Tao Deng; Peichao Peng; Xiaoxing Cheng; Guoqing Wang; Minping Qian; Huafang Gao; Bei Han; Yusheng Chen; Yinghui Hu; Rong Geng; Chengping Hu; Wei Zhang; Jingping Yang; Huanying Wan; Qin Yu; Liping Wei; Jiashu Li; Guizhen Tian; Qiuyue Wang; Ke Hu; Siqin Wang; Ruiqin Wang; Juan Du; Bei He; Jianjun Ma; Xiaoning Zhong; Lan Mu; Shaoxi Cai; Xiangdong Zhu; Wanli Xing; Jun Yu; Minghua Deng; Zhancheng Gao

2012-01-01

378

Centrosome Amplification Can Initiate Tumorigenesis in Flies  

PubMed Central

SUMMARY Centrosome amplification is a common feature of many cancer cells, and it has been previously proposed that centrosome amplification can drive genetic instability and so tumorigenesis. To test this hypothesis, we generated Drosophila lines that have extra centrosomes in ~60% of their somatic cells. Many cells with extra centrosomes initially form multipolar spindles, but these spindles ultimately become bipolar. This requires a delay in mitosis that is mediated by the spindle assembly checkpoint (SAC). As a result of this delay, there is no dramatic increase in genetic instability in flies with extra centrosomes, and these flies maintain a stable diploid genome over many generations. The asymmetric division of the larval neural stem cells, however, is compromised in the presence of extra centrosomes, and larval brain cells with extra centrosomes can generate metastatic tumors when transplanted into the abdomens of wild-type hosts. Thus, centrosome amplification can initiate tumorigenesis in flies.

Basto, Renata; Brunk, Kathrin; Vinadogrova, Tatiana; Peel, Nina; Franz, Anna; Khodjakov, Alexey; Raff, Jordan W.

2009-01-01

379

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

380

A "release" protocol for isothermal titration calorimetry.  

PubMed Central

Isothermal titration calorimetry (ITC) has become a standard method for investigating the binding of ligands to receptor molecules or the partitioning of solutes between water and lipid vesicles. Accordingly, solutes are mixed with membranes (or ligands with receptors), and the subsequent heats of incorporation (or binding) are measured. In this paper we derive a general formula for modeling ITC titration heats in both binding and partitioning systems that allows for the modeling of the classic incorporation or binding protocols, as well as of new protocols assessing the release of solute from previously solute-loaded vesicles (or the dissociation of ligand/receptor complexes) upon dilution. One major advantage of a simultaneous application of the incorporation/binding and release protocols is that it allows for the determination of whether a ligand is able to access the vesicle interior within the time scale of the ITC experiment. This information cannot be obtained from a classical partitioning experiment, but it must be known to determine the partition coefficient (or binding constant and stochiometry) and the transfer enthalpy. The approach is presented using the partitioning of the nonionic detergent C12EO7 to palmitoyloleoylphosphatidylcholine vesicles. The release protocol could also be advantageous in the case of receptors that are more stable in the ligand-saturated rather than the ligand-depleted state.

Heerklotz, H H; Binder, H; Epand, R M

1999-01-01

381

Isothermal thermogravimetric data acquisition analysis system  

NASA Technical Reports Server (NTRS)

The description of an Isothermal Thermogravimetric Analysis (TGA) Data Acquisition System is presented. The system consists of software and hardware to perform a wide variety of TGA experiments. The software is written in ANSI C using Borland's Turbo C++. The hardware consists of a 486/25 MHz machine with a Capital Equipment Corp. IEEE488 interface card. The interface is to a Hewlett Packard 3497A data acquisition system using two analog input cards and a digital actuator card. The system provides for 16 TGA rigs with weight and temperature measurements from each rig. Data collection is conducted in three phases. Acquisition is done at a rapid rate during initial startup, at a slower rate during extended data collection periods, and finally at a fast rate during shutdown. Parameters controlling the rate and duration of each phase are user programmable. Furnace control (raising and lowering) is also programmable. Provision is made for automatic restart in the event of power failure or other abnormal terminations. Initial trial runs were conducted to show system stability.

Cooper, Kenneth, Jr.

1991-01-01

382

Material Compatibility with Isothermal Pb-Li  

SciTech Connect

Eutectic Pb-Li is a leading candidate for current fusion blanket concepts as a coolant. However, there is very little data about the compatibility of most materials with Pb-Li above 500 C where the dissolution rate of many conventional alloys increases rapidly. Current work is beginning to assess Pb-Li compatibility from 500 to 800 C using isothermal capsule experiments. Aluminide coatings hold some promise in protecting conventional Fe-base alloys at 600-700 C. However, there is a significant initial Al loss that has not been clearly explained. Furthermore, the reaction product with coated materials is LiAlO{sub 2} rather than Al{sub 2}O{sub 3} at 600 and 700 C. Even when pre-oxidized to form {alpha}-Al{sub 2}O{sub 3}, an alumina layer on FeCrAl transformed to LiAlO{sub 2} at 700 and 800 C. At 500 C, the preformed oxide partially transformed from alumina and some Li was detected in the oxide layer.

Pint, Bruce A [ORNL; Walker, Larry R [ORNL; Unocic, Kinga A [ORNL

2012-01-01

383

Isothermal Dendritic Growth Experiment - PVA Dendrites  

NASA Technical Reports Server (NTRS)

The Isothermal Dendritic Growth Experiment (IDGE), flown on three Space Shuttle missions, is yielding new insights into virtually all industrially relevant metal and alloy forming operations. IDGE used transparent organic liquids that form dendrites (treelike structures) similar to those inside metal alloys. Comparing Earth-based and space-based dendrite growth velocity, tip size and shape provides a better understanding of the fundamentals of dentritic growth, including gravity's effects. Shalowgraphic images of pivalic acid (PVA) dendrites forming from the melt show the subtle but distinct effects of gravity-driven heat convection on dentritic growth. In orbit, the dendrite grows as its latent heat is liberated by heat conduction. This yields a blunt dendrite tip. On Earth, heat is carried away by both conduction and gravity-driven convection. This yields a sharper dendrite tip. In addition, under terrestrial conditions, the sidebranches growing in the direction of gravity are augmented as gravity helps carry heat out of the way of the growing sidebranches as opposed to microgravity conditions where no augmentation takes place. IDGE was developed by Rensselaer Polytechnic Institute and NASA/Glenn Research Center. Advanced follow-on experiments are being developed for flight on the International Space Station. Photo Credit: NASA/Glenn Research Center

1997-01-01

384

ISOTHERMAL AIR-INGRESS VALIDATION EXPERIMENTS  

SciTech Connect

Idaho National Laboratory has conducted airingress experiments as part of a campaign to validate computational fluid dynamics (CFD) calculations for very high-temperature gas-cooled reactor (VHTR) analysis. An isothermal test loop was designed to recreate exchange or stratified flow that occurs in the lower plenum of VHTR after a break in the primary loop allows helium to leak out and reactor building air to enter the reactor core. The experiment was designed to measure stratified flow in the inlet pipe connecting to the lower plenum of the General Atomics gas turbine–modular helium reactor (GT-MHR). Instead of helium and air, brine and sucrose were used as heavy fluids, and water was used as the lighter fluid to create, using scaling laws, the appropriate flow characteristics of the lower plenum immediately after depressurization. These results clearly indicate that stratified flow is established even for very small density differences. Corresponding CFD results were validated with the 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. The calculated current speed showed very good agreement with the experimental data, indicating that current CFD methods are suitable for simulating density gradient stratified flow phenomena in an air-ingress accident.

Chang H. Oh; Eung S. Kim

2013-01-01

385

Iodine isothermal migration behaviour in titanium nitride  

NASA Astrophysics Data System (ADS)

Titanium nitride is one of the inert matrixes proposed to surround the fuel in gas cooled fast reactor (GFR) systems. These reactors will operate at high temperature and refractory materials with a high chemical stability and good mechanical properties are required. Furthermore, a total retention of the most volatile fission products, such as I, Xe or Cs, by the inert matrix is needed during the in-pile process. The isothermal migration of iodine in TiN was studied by implanting 800 keV I ++ ions in sintered samples at an ion fluence of 5 × 10 15 cm -2. Thermal treatments were performed under secondary vacuum at temperatures ranging from 1200 to 1700 °C. Iodine concentration profiles were determined by 2.5 MeV ?-particle elastic backscattering. The migration of iodine seems to be correlated with point defects created by implanted ions near the surface. The Arrhenius plot corresponding to iodine detrapping is curved with possibly two straight-line regions which could indicate either the presence of two types of traps, or a strong dependence of trap's concentration on temperature above 1500 °C. The activation energies associated with each linear region of the Arrhenius plot were found to be: Ea = 2.4 ± 0.2 eV below 1500 °C and E=11.4±0.2 eV above 1500 °C. Nitrogen evaporation from TiN surface under secondary vacuum was proposed as a contributing factor to the enhanced mobility of iodine at high temperature.

Gavarini, S.; Jaffrezic, H.; Martin, P.; Peaucelle, C.; Toulhoat, N.; Cardinal, S.; Moncoffre, N.; Pichon, C.; Tribet, M.

2008-02-01

386

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

387

Signal amplification by unidirectional coupling of oscillators  

NASA Astrophysics Data System (ADS)

We report our investigation on the input signal amplification in unidirectionally coupled monostable Duffing oscillators in one- and two dimensions with first oscillator alone driven by a weak periodic signal. Applying a perturbation theory, we obtain a set of nonlinear equations for the response amplitude of the coupled oscillators. We identify the conditions for undamped signal propagation with enhanced amplitude through the coupled oscillators. When the number of oscillators increases the response amplitude approaches a limiting value. We determine this limiting value. Also, we analyse the signal amplification in the coupled oscillators in two dimensions with a fraction of oscillators chosen randomly for coupling and forcing.

Rajamani, S.; Rajasekar, S.

2013-07-01

388

On sensitivity amplification in intracellular signaling cascades.  

PubMed

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. PMID:18663279

Rácz, Eva; Slepchenko, Boris M

2008-01-01

389

ON SENSITIVITY AMPLIFICATION IN INTRACELLULAR SIGNALING CASCADES  

PubMed Central

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.

Racz, Eva; Slepchenko, Boris M.

2009-01-01

390

Optical amplification enhancement in photonic crystals  

SciTech Connect

Improving and controlling the efficiency of a gain medium is one of the most challenging problems of laser research. By measuring the gain length in an opal-based photonic crystal doped with laser dye, we demonstrate that optical amplification is more than twenty-fold enhanced along the {Gamma}-K symmetry directions of the face-centered-cubic photonic crystal. These results are theoretically explained by directional variations of the density of states, providing a quantitative connection between density of the states and light amplification.

Sapienza, R.; Leonetti, M.; Froufe-Perez, L. S.; Galisteo-Lopez, J. F.; Lopez, C. [Instituto de Ciencia de Materiales de Madrid (CSIC) and Unidad Asociada CSIC-UVigo, Cantoblanco, E-28049 Madrid (Spain); Conti, C. [Research Center INFM-CNR, c/o Universita di Roma Sapienza, I-00185 Roma (Italy)

2011-02-15

391

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

392

Amplification of entangled states of squeezed light  

SciTech Connect

Two amplification schemes are considered for entangled squeezed states of light, including an Einstein-Podolsky-Rosen entangled state of continuous variables (EPR pair): propagation in a nonlinear medium and reflection from a cavity. Both schemes make use of a parametric process that can be implemented in a periodic nonlinear medium. The existence of an integral of motion makes it possible to amplify an entangled state of light while preserving the initial entanglement. To analyze the cavity-based scheme, a master equation is derived for the density matrix of the field inside the cavity. The feasibility of amplification that preserves entanglement of an EPR pair is demonstrated for this scheme.

Gorbachev, V. N., E-mail: vn@vg3025.spb.edu; Trubilko, A. I. [St. Petersburg State University of Aerospace Instrumentation, Laboratory of Quantum Information and Computing (Russian Federation)], E-mail: tai@at3024.spb.edu

2007-08-15

393

Determining Kinetic Parameters for Isothermal Crystallization of Glasses  

NASA Technical Reports Server (NTRS)

Non-isothermal crystallization techniques are frequently used to determine the kinetic parameters for crystallization in glasses. These techniques are experimentally simple and quick compared to the isothermal techniques. However, the analytical models used for non-isothermal data analysis, originally developed for describing isothermal transformation kinetics, are fundamentally flawed. The present paper describes a technique for determining the kinetic parameters for isothermal crystallization in glasses, which eliminates most of the common problems that generally make the studies of isothermal crystallization laborious and time consuming. In this technique, the volume fraction of glass that is crystallized as a function of time during an isothermal hold was determined using differential thermal analysis (DTA). The crystallization parameters for the lithium-disilicate (Li2O.2SiO2) model glass were first determined and compared to the same parameters determined by other techniques to establish the accuracy and usefulness of the present technique. This technique was then used to describe the crystallization kinetics of a complex Ca-Sr-Zn-silicate glass developed for sealing solid oxide fuel cells.

Ray, C. S.; Zhang, T.; Reis, S. T.; Brow, R. K.

2006-01-01

394

In-situ Rapid Isothermal Processing (RIP) of InP Based Devices.  

National Technical Information Service (NTIS)

Rapid isothermal processing based on incoherent sources of light is emerging as a reduced thermal budget processing technique. As compared to a stand alone rapid isothermal annealing unit, the integration of deposition system and the rapid isothermal proc...

R. Singh R. P. Thakur A. Katz A. J. Nelson J. Narayan

1990-01-01

395

Adsorption isotherms on nicotinamide-imprinted polymer stationary phase.  

PubMed

The molecular-imprinted technique is applied for the preparation of a polymer selector by using methacrylic acid as functional monomer, ethyleneglycol dimethacrylate as the cross-linker, 2,2'-azobisisobutyronitrile as the initiator, and nicotinamide as the template. The adsorption isotherms of nicotinamide and nicotinic acid and the competitive adsorption isotherms of nicotinamide and nicotinic acid on the imprinted stationary phase are determined using rectangular pulse frontal analysis and static method. Aqueous solution is used as the mobile phase in frontal analysis. It is found that the adsorption data fit well to both Langmuir and Freundlich isotherm models. PMID:16176649

Li, Zhiwei; Yang, Gengliang; Liu, Shubin; Chen, Yi

2005-08-01

396

Detection of cochlear amplification and its activation.  

PubMed

The operation of the mammalian cochlea relies on a mechanical traveling wave that is actively boosted by electromechanical forces in sensory outer hair cells (OHCs). This active cochlear amplifier produces the impressive sensitivity and frequency resolution of mammalian hearing. The cochlear amplifier has inspired scientists since its discovery in the 1970s, and is still not well understood. To explore cochlear electromechanics at the sensory cell/tissue interface, sound-evoked intracochlear pressure and extracellular voltage were measured using a recently developed dual-sensor with a microelectrode attached to a micro-pressure sensor. The resulting coincident in vivo observations of OHC electrical activity, pressure at the basilar membrane and basilar membrane displacement gave direct evidence for power amplification in the cochlea. Moreover, the results showed a phase shift of voltage relative to mechanical responses at frequencies slightly below the peak, near the onset of amplification. Based on the voltage-force relationship of isolated OHCs, the shift would give rise to effective OHC pumping forces within the traveling wave peak. Thus, the shift activates the cochlear amplifier, serving to localize and thus sharpen the frequency region of amplification. These results are the most concrete evidence for cochlear power amplification to date and support OHC somatic forces as its source. PMID:23972858

Dong, Wei; Olson, Elizabeth S

2013-08-20

397

Photoinduced amplification of hypersound in superlattices  

NASA Astrophysics Data System (ADS)

The high-frequency acoustic-phonon scattering by electrons in superlattices in the presence of an intense radiation field is discussed. It is shown that in the intense field regime of the electromagnetic wave the hypersound attenuation coefficient may revert its sign and hypersound amplification can be obtained. An application is made for GaAs based superlattices.

Nunes, O. A. C.; Fonseca, A. L. A.; Agrello, D. A.

2001-09-01

398

HER2 Amplification in Gastroesophageal Adenocarcinoma  

PubMed Central

We assessed 103 resected gastroesophageal adenocarcinomas for HER2 amplification by fluorescence in situ hybridization (FISH) and 2 commercial immunohistochemical assays. Of 103, 30 (29%) were FISH-amplified. Both immunohistochemical assays had greater than 95% concordance with FISH. However, as a screening test for FISH amplification, the Ventana Medical Systems (Tucson, AZ) 4B5 antibody demonstrated superior sensitivity (87%) compared with the DAKO (Carpinteria, CA) A0485 (70%). Of the cases, 28 were immunohistochemically 3+ or immunohistochemically 2+/FISH-amplified with the 4B5 assay compared with only 22 cases with the A0485 assay, representing a large potential difference in patient eligibility for anti-HER2 therapy. Cases with low-level FISH amplification (HER2/CEP17, 2.2–4.0) express lower levels of HER2 protein compared with cases with high-level amplification (HER2/CEP17, ?4.0), raising the possibility of a differential response to anti-HER2 therapy. The H score and digital image analysis may have a limited role in improving HER2 test performance.

Radu, Oana M.; Foxwell, Tyler; Cieply, Kathleen; Navina, Sarah; Dacic, Sanja; Nason, Katie S.; Davison, Jon M.

2014-01-01

399

Optical amplifier-powered quantum optical amplification  

SciTech Connect

I show that an optical amplifier, when combined with photon subtraction, can be used for quantum state amplification, adding noise at a level below the standard minimum. The device could be used to significantly decrease the probability of incorrectly identifying coherent states chosen from a finite set.

Jeffers, John [Department of Physics, Scottish Universities Physics Alliance, University of Strathclyde, John Anderson Building, 107 Rottenrow, Glasgow G4 0NG (United Kingdom)

2011-05-15

400

Surface elastic-wave propagation and amplification  

Microsoft Academic Search

Surface elastic-wave propagation, transduction, and amplification (in a piezoelectric semiconductor) are discussed with emphasis on characteristics useful in electronic devices. Computed curves show the dependence on distance from the surface of the elastic and the electric fields associated with surface elastic-wave propagation in cadmium sulfide. The interaction impedance, relating the external electric field to power flow, is computed for propagation

RICHARD M. WHITE

1967-01-01

401

KASER: knowledge amplification by structured expert randomization  

Microsoft Academic Search

In this paper and attached video, we present a third-generation expert system named Knowledge Amplification by Structured Expert Randomization (KASER) for which a patent has been filed by the U.S. Navy's SPAWAR Systems Center, San Diego, CA (SSC SD). KASER is a creative expert system. It is capable of deductive, inductive, and mixed derivations. Its qualitative creativity is realized by

Stuart Harvey Rubin; S. N. Jayaram Murthy; Michael H. Smith; Ljiljana Trajkovic

2004-01-01

402

Ripple Signal Amplification: Measurement, Modelling and Mitigation  

Microsoft Academic Search

Use of high frequency ripple control signals for load control purposes has existed for many years. Although the level of the injected signal at the zone substation is generally small, amplification can occur when the network resonant frequency is close to the injected ripple signal frequency. Ripple signal level at the injection point is generally 5 - 7V in magnitude

Sean Elphick; Sarath Perera; Neil Browne