Influence of sequence mismatches on the specificity of recombinase polymerase amplification technology

Daher, Rana; Stewart, Gale; Boissinot, Maurice; Boudreau, Dominique K.; Bergeron, Michel G.

doi:10.1016/j.mcp.2014.11.005

Cited by 157 publications

(133 citation statements)

References 28 publications

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“…Interestingly, the GII.3 template was inconsistently amplified by the NOF5-NOR11-NOP1 primer-probe set, the amplified region of which contained base mismatches on the 3′ end of NOF5 and NOR11 target regions in addition to a large number of mismatches (Table 4). Such placement of mismatches was found by Daher et al 34. to be more inhibitory to amplification than internal or 5′ mismatches.…”

Section: Discussionmentioning

confidence: 66%

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Development of a Recombinase Polymerase Amplification Assay for Detection of Epidemic Human Noroviruses

Moore

Jaykus

2017

Sci Rep

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Human norovirus is a leading cause of viral gastroenteritis worldwide. Rapid detection could facilitate control, however widespread point-of-care testing is infrequently done due to the lack of robust and portable methods. Recombinase polymerase amplification (RPA) is a novel isothermal method which rapidly amplifies and detects nucleic acids using a simple device in near real-time. An RT-RPA assay targeting a recent epidemic human norovirus strain (GII.4 New Orleans) was developed and evaluated in this study. The assay successfully detected purified norovirus RNA from multiple patient outbreak isolates and had a limit of detection of 3.40 ± 0.20 log10 genomic copies (LGC), which is comparable to most other reported isothermal norovirus amplification methods. The assay also detected norovirus in directly boiled stool, and displayed better resistance to inhibitors than a commonly used RT-qPCR assay. The assay was specific, as it did not amplify genomes from 9 non-related enteric viruses and bacteria. The assay detected norovirus in some samples in as little as 6 min, and the entire detection process can be performed in less than 30 min. The reported RT-RPA method shows promise for sensitive point-of-care detection of epidemic human norovirus, and is the fastest human norovirus amplification method to date.

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Section: Discussionmentioning

confidence: 66%

“…In our report, GII.4 Sydney containing 6 total mismatches in the target region was readily amplified. The data of Daher et al 34,. which focused on using RPA for amplification of conserved genes for several bacteria, showed a similar tolerance to base pair mismatches.…”

Section: Discussionmentioning

confidence: 94%

Development of a Recombinase Polymerase Amplification Assay for Detection of Epidemic Human Noroviruses

Moore

Jaykus

2017

Sci Rep

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“…It has been reported that target specific RPA primers with mismatches at their 3' end can prevent amplification with RPA (Daher et al, 2015). Within the 174 sequences that amplified there were 16 (9.1%) that had a single mismatch within the first three bases at the 3’ end of the forward primer and one isolate, KF716479, had mismatches at both positions 1 and 3 and yet still amplified 100 copies of RNA.…”

Section: Resultsmentioning

confidence: 99%

“…In particular the group O isolates had significant mismatches to the assay primer/probe sequences with a total of 13–15 polymorphisms (6–7 of which were in the reverse primer) and yet were reproducibly detected at the 100 copy level. In contrast, the sequence analysis of the isolates that did not efficiently amplify had relatively fewer mismatches (range 5–6), most of which were not in the 3’ end of the primer binding sites (Daher et al, 2014). As others have observed, we did note that mismatches at the 3’ end of a primer prevented amplification.…”

Section: Discussionmentioning

confidence: 99%

Cross-subtype detection of HIV-1 using reverse transcription and recombinase polymerase amplification

Lillis

Lehman

Siverson

et al. 2016

Journal of Virological Methods

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A low complexity diagnostic test that rapidly and reliably detects HIV infection in infants at the point of care could facilitate early treatment, improving outcomes. However, many infant HIV diagnostics can only be performed in laboratory settings. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that can rapidly amplify proviral DNA from multiple subtypes of HIV-1 in under twenty minutes without complex equipment. In this study we added reverse transcription (RT) to RPA to allow detection of both HIV-1 RNA and DNA. We show that this RT-RPA HIV-1 assay has a limit of detection of 10 to 30 copies of an exact sequence matched DNA or RNA, respectively. In addition, at 100 copies of RNA or DNA, the assay detected 171 of 175 (97.7 %) sequence variants that represent all the major subtypes and recombinant forms of HIV-1 Groups M and O. This data suggests that the application of RT-RPA for the combined detection of HIV-1 viral RNA and proviral DNA may prove a highly sensitive tool for rapid and accurate diagnosis of infant HIV.

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“…FMDV serotype C, SAT1–3, SVDV, VESV and SVA were not included in the analytical specificity analysis, which is a shortcoming of this study. RPA is tolerant to 5–9 mismatches in primer and probe showing no influence on the performance of the assay [20, 28], and the maximum number of mismatches found within one sequence was four in some FMDV serotypes available in GenBank (e.g. accession numbers DQ533483, HQ412603, KU821590, JF749862, KJ820999, and KT968663).…”

Section: Discussionmentioning

confidence: 99%

Visual and equipment-free reverse transcription recombinase polymerase amplification method for rapid detection of foot-and-mouth disease virus

Liu

Wang

Zhang³

et al. 2018

BMC Vet Res

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BackgroundFoot-and-mouth disease (FMD), which is caused by foot-and-mouth disease virus (FMDV), is a highly contagious tansboundary disease of cloven-hoofed animals and causes devastating economic damages. Accurate, rapid and simple detection of FMDV is critical to containing an FMD outbreak. Recombinase polymerase amplification (RPA) has been explored for detection of diverse pathogens because of its accuracy, rapidness and simplicity. A visible and equipment-free reverse-transcription recombinase polymerase amplification assay combined with lateral flow strip (LFS RT-RPA) was developed to detect the FMDV using primers and LF probe specific for the 3D gene.ResultsThe FMDV LFS RT-RPA assay was performed successfully in a closed fist using body heat for 15 min, and the products were visible on the LFS inspected by the naked eyes within 2 min. The assay could detect FMDV serotypes O, A and Asia1, and there were no cross-reactions with vesicular stomatitis virus (VSV), encephalomyocarditis virus (EMCV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 (PCV2) and pseudorabies virus (PRV). The analytical sensitivity was 1.0 × 102 copies in vitro transcribed FMDV RNA per reaction, which was the same as a real-time RT-PCR. For the 55 samples, FMDV RNA positive rate was 45.5% (25/55) by LFS RT-RPA and 52.7% (29/55) by real-time RT-PCR. For the LFS RT-RPA assay, the positive and negative predicative values were 100% and 80%, respectively.ConclusionsThe performance of the LFS RT-RPA assay was comparable to real-time RT-PCR, while the LFS RT-RPA assay was much faster and easier to be performed. The developed FMDV LFS RT-RPA assay provides an attractive and promising tool for rapid and reliable detection of FMDV in under-equipped laboratory and at point-of-need facility, which is of great significance in FMD control in low resource settings.

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Influence of sequence mismatches on the specificity of recombinase polymerase amplification technology

Cited by 157 publications

References 28 publications

Development of a Recombinase Polymerase Amplification Assay for Detection of Epidemic Human Noroviruses

Development of a Recombinase Polymerase Amplification Assay for Detection of Epidemic Human Noroviruses

Cross-subtype detection of HIV-1 using reverse transcription and recombinase polymerase amplification

Visual and equipment-free reverse transcription recombinase polymerase amplification method for rapid detection of foot-and-mouth disease virus

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