Nasba

Sooknanan, Roy; Malek, L T

doi:10.1038/nbt0695-563

Cited by 22 publications

(8 citation statements)

References 12 publications

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“…A variety of isothermal amplification methods have been developed, for example strand displacement amplification 2 3 , nucleic acid sequence based amplification 4 , self-sustained sequence replication 5 , rolling circle amplification 6 , loop mediated amplification 7 , and helicase dependent amplification 8 . While overcoming some of the technical and cost barriers associated with PCR and providing amplification of target sequences on par with PCR, these methods are still relatively complex protocols requiring the use of multiple enzymes and/or special reagents.…”

mentioning

confidence: 99%

Cross Priming Amplification: Mechanism and Optimization for Isothermal DNA Amplification

Zhong

et al. 2012

Sci Rep

181

148

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

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mentioning

confidence: 99%

Cross Priming Amplification: Mechanism and Optimization for Isothermal DNA Amplification

Zhong

et al. 2012

Sci Rep

181

148

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“…Several platforms of isothermal nucleic acid amplification have been invented and developed in the past 18 years. However, so far most of them, such as transcription-mediated amplification (TMA) [ 1 ], rolling cycle amplification (RCA) [ 2 ], loop-mediated isothermal amplification (LAMP) [ 3 ], nucleic acid sequence based amplification (NASBA) [ 4 ], strand displacement amplification (SDA) [ 5 ], the helicase-dependent amplification (HDA) [ 6 , 7 ] require 1 hour or more of amplification time to detect <100 copies of template DNA. The improvement on speed (e.g.…”

Section: Introductionmentioning

confidence: 99%

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

2011

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BackgroundIn the past decades the rapid growth of molecular diagnostics (based on either traditional PCR or isothermal amplification technologies) meet the demand for fast and accurate testing. Although isothermal amplification technologies have the advantages of low cost requirements for instruments, the further improvement on sensitivity, speed and robustness is a prerequisite for the applications in rapid pathogen detection, especially at point-of-care diagnostics. Here, we describe and explore several strategies to improve one of the isothermal technologies, helicase-dependent amplification (HDA).ResultsMultiple strategies were approached to improve the overall performance of the isothermal amplification: the restriction endonuclease-mediated DNA helicase homing, macromolecular crowding agents, and the optimization of reaction enzyme mix. The effect of combing all strategies was compared with that of the individual strategy. With all of above methods, we are able to detect 50 copies of Neisseria gonorrhoeae DNA in just 20 minutes of amplification using a nearly instrument-free detection platform (BESt™ cassette).ConclusionsThe strategies addressed in this proof-of-concept study are independent of expensive equipments, and are not limited to particular primers, targets or detection format. However, they make a large difference in assay performance. Some of them can be adjusted and applied to other formats of nucleic acid amplification. Furthermore, the strategies to improve the in vitro assays by maximally simulating the nature conditions may be useful in the general field of developing molecular assays. A new fast molecular assay for Neisseria gonorrhoeae has also been developed which has great potential to be used at point-of-care diagnostics.

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“…There are several platforms for isothermal amplification, including strand displacement amplification [SDA (8)], transcription-mediated amplification [TMA (9)], rolling cycle amplification [RCA (10)], loop-mediated amplification LAMP [11]), nucleic acid sequence-based amplification [NASBA (12)], and helicase-dependent amplification [HDA (1315)]. Most of the isothermal amplification technologies rely on a 5-3 exonuclease deficient DNA polymerase (SDA, RCA, LAMP) or an RNA polymerase (TMA, NASBA).…”

Section: Introductionmentioning

confidence: 99%

Development of Isothermal TaqMan Assays for Detection of Biothreat Organisms

Tong¹,

Tang²,

Kim³

et al. 2008

BioTechniques

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

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Nasba

Cited by 22 publications

References 12 publications

Cross Priming Amplification: Mechanism and Optimization for Isothermal DNA Amplification

Cross Priming Amplification: Mechanism and Optimization for Isothermal DNA Amplification

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

Development of Isothermal TaqMan Assays for Detection of Biothreat Organisms

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