Electrochemical detection of Francisella tularensis genomic DNA using solid-phase recombinase polymerase amplification

Río, Jonathan Sabaté del; Adly, Nouran; Acero-Sánchez, Josep Lluís; Henry, Olivier; O’Sullivan, Ciara K.

doi:10.1016/j.bios.2013.11.035

Cited by 64 publications

(43 citation statements)

References 17 publications

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“…In solution-phase, due to the unimpeded diffusion of primers and reaction reagents, amplification kinetics are favoured and the achieved limit of detection is subsequently usually better and amplification is achieved in a faster time than solid-phase. Nevertheless, solidphase and bridge amplification present some advantages, such as the potential for spatially resolved multiplexed amplification or the possibility to couple the amplification with diverse detection techniques including ring resonators [27e29], electrochemical [30e36] and colorimetric detection [5,6,30,33,37,38]. Several methods have been developed with solid phase amplification with performances usually inferior to that achieved with solution phase amplification [5,27e30,39] as primer accessibility is more restricted impeding amplification efficiency, and future work will need to focus on strategies to decrease amplification time.…”

Section: Solid Phase Rpamentioning

confidence: 99%

Recombinase polymerase amplification: Basics, applications and recent advances

Lobato

O’Sullivan

2018

TrAC Trends in Analytical Chemistry

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a b s t r a c tRecombinase polymerase amplification (RPA) is a highly sensitive and selective isothermal amplification technique, operating at 37e42 C, with minimal sample preparation and capable of amplifying as low as 1e10 DNA target copies in less than 20 min. It has been used to amplify diverse targets, including RNA, miRNA, ssDNA and dsDNA from a wide variety of organisms and samples. An ever increasing number of publications detailing the use of RPA are appearing and amplification has been carried out in solution phase, solid phase as well as in a bridge amplification format. Furthermore, RPA has been successfully integrated with different detection strategies, from end-point lateral flow strips to real-time fluorescent detection amongst others. This review focuses on the different methodologies and advances related to RPA technology, as well as highlighting some of the advantages and drawbacks of the technique.

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Section: Solid Phase Rpamentioning

confidence: 99%

Recombinase polymerase amplification: Basics, applications and recent advances

Lobato

O’Sullivan

2018

TrAC Trends in Analytical Chemistry

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727

522

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“…Therefore, analysis times and costs are reduced. The assays can potentially be further simplified and expedited by utilizing isothermal recombinase polymerase amplification and an amplicon detection method that does not involve gel electrophoresis (Del Río et al, 2014). The application of the O. sinensis -specific primer pair along with the five primer pairs targeting DNA from common adulterants should allow determining if a sample said to only contain O. sinensis actually also, or exclusively, contains adulterants added inadvertently or deliberately.…”

Section: Discussionmentioning

confidence: 99%

Detection of Ophiocordyceps sinensis and Its Common Adulterates Using Species-Specific Primers

et al. 2017

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Ophiocordyceps sinensis is a fungus that infects Hepialidae caterpillars, mummifying the larvae and producing characteristic fruiting bodies (stromata) that are processed into one of the most valued traditional Chinese medicines (TCM). The product commands a very high price due to a high demand but a very limited supply. Adulteration with other fungi is a common problem and there is a need to test preparation for the presence of the correct fungus. In the current study, a PCR-based approach for the identification of O. sinensis based on a segment of the internal transcribed spacer (ITS) region was developed. The segments is 146-bp in size and is likely to be amplified even in materials where processing led to DNA fragmentation. Primer development was based on the alignment of sequence data generated from a total of 89 samples of O. sinensis and potential adulterants as well as sequences date from 41 Ophiocordyceps species and 26 Cordyceps species available in GenBank. Tests with primer pair, DCF4/DCR4, demonstrated generation of an amplicon from DNA extracted from O. sinensis stromata, but not from extracts derived from adulterants. Species-specific primer pairs were also developed and tested for detection of the common adulterants, Cordyceps gunnii, Cordyceps cicadae, Cordyceps militaris, Cordyceps liangshanensis and Ophiocordyceps nutans. The collection of primers developed in the present study will be useful for the authentication of preparation claiming to only contain O. sinensis and for the detection of fungi used as adulterants in these preparations.

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“…Shin et al [69] have demonstrated one of the most sophisticated and sensitive approaches for isothermal PCR-like amplification of ds-DNA using a solid-phase immobilized primer. This was carried out using recombinase polymerase amplification (RPA) [70,71] which employs a uvsX recombinase enzyme of specific activity isolated from T4 phage. In this case the enzyme is intended to "substitute" the thermal denaturation step normally performed during PCR amplification.…”

Section: Isothermal Amplification On Microarraysmentioning

confidence: 99%