[Letter to the editor] Combined FAM-Labeled Taqman Probe Detection and SYBR Green I Melting Curve Analysis in Multiprobe qPCR Genotyping Assays

Poucke, Mario Van; Zeveren, Alex Van; Peelman, Luc

doi:10.2144/000113808

Cited by 11 publications

(9 citation statements)

References 15 publications

(20 reference statements)

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“…The primer sets used for the LAMP assay were based on previously published primer sequences for P. falciparum (45), and Plasmodium genus (31). A LAMP assay that amplified a BCRA1 gene fragment served as a positive control (46). The primers were all purchased from Eurofins Genomics.…”

Section: Methodsmentioning

confidence: 99%

Paper-based microfluidics for DNA diagnostics of malaria in low resource underserved rural communities

Reboud

Garrett

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

276

254

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Rapid, low-cost, species-specific diagnosis, based upon DNA testing, is becoming important in the treatment of patients with infectious diseases. Here, we demonstrate an innovation that uses origami to enable multiplexed, sensitive assays that rival polymerase chain reactions (PCR) laboratory assays and provide high-quality, fast precision diagnostics for malaria. The paper-based microfluidic technology proposed here combines vertical flow sample-processing steps, including paper folding for whole-blood sample preparation, with an isothermal amplification and a lateral flow detection, incorporating a simple visualization system. Studies were performed in village schools in Uganda with individual diagnoses being completed in <50 min (faster than the standard laboratory-based PCR). The tests, which enabled the diagnosis of malaria species in patients from a finger prick of whole blood, were both highly sensitive and specific, detecting malaria in 98% of infected individuals in a double-blind first-in-human study. Our method was more sensitive than other field-based, benchmark techniques, including optical microscopy and industry standard rapid immunodiagnostic tests, both performed by experienced local healthcare teams (which detected malaria in 86% and 83% of cases, respectively). All assays were independently validated using a real-time double-blinded reference PCR assay. We not only demonstrate that advanced, low-cost DNA-based sensors can be implemented in underserved communities at the point of need but also highlight the challenges associated with developing and implementing new diagnostic technologies in the field, without access to laboratories or infrastructure.

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Section: Methodsmentioning

confidence: 99%

Paper-based microfluidics for DNA diagnostics of malaria in low resource underserved rural communities

Reboud

Garrett

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

276

254

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“…Recently, it was shown that two dyes with the same emission wavelengths could be combined 22 . Researchers used four different TaqMan probes labelled with FAM dye, phosphoramidite (HEX), Texas Red and Cy5 dyes combined with the intercalating dye SYBR Green I, which exhibits nearly identical excitation/emission spectra as FAM.…”

mentioning

confidence: 99%

Doubling Throughput of a Real-Time PCR

Ahrberg

Neužil

2015

Sci Rep

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The invention of polymerase chain reaction (PCR) in 1983 revolutionized many areas of science, due to its ability to multiply a number of copies of DNA sequences (known as amplicons). Here we report on a method to double the throughput of quantitative PCR which could be especially useful for PCR-based mass screening. We concurrently amplified two target genes using only single fluorescent dye. A FAM probe labelled olionucleotide was attached to a quencher for one amplicon while the second one was without a probe. The PCR was performed in the presence of the intercalating dye SYBR Green I. We collected the fluorescence amplitude at two points per PCR cycle, at the denaturation and extension steps. The signal at denaturation is related only to the amplicon with the FAM probe while the amplitude at the extension contained information from both amplicons. We thus detected two genes within the same well using a single fluorescent channel. Any commercial real-time PCR systems can use this method doubling the number of detected genes. The method can be used for absolute quantification of DNA using a known concentration of housekeeping gene at one fluorescent channel.

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“…The idea to integrate the TaqMan and melting analysis into a single assay is not entirely new [ 34 – 36 ]. In 2006, Lind and colleagues [ 34 ] were the first to present a system combining the FAM-labeled TaqMan probes with the BOXTO dye that allowed parallel signal processing in the green and yellow channels, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…In 2006, Lind and colleagues [ 34 ] were the first to present a system combining the FAM-labeled TaqMan probes with the BOXTO dye that allowed parallel signal processing in the green and yellow channels, respectively. The two remaining works relied on SG, ROX, and Cy5 [ 35 ] or FAM, HEX, TexasRed, and Cy5 [ 36 ] probe labels. Although proven fully functional, these techniques were designed for specific applications [ 35 , 36 ], and their potential for broader use has not yet been evaluated [ 34 ].…”

Section: Resultsmentioning

confidence: 99%

“…The two remaining works relied on SG, ROX, and Cy5 [ 35 ] or FAM, HEX, TexasRed, and Cy5 [ 36 ] probe labels. Although proven fully functional, these techniques were designed for specific applications [ 35 , 36 ], and their potential for broader use has not yet been evaluated [ 34 ]. Based on the studies comparing the performance of multiple DNA dyes in qPCR [ 10 , 11 ], we took advantages of the S82 DNA binding dye to integrate the FAM-TaqMan and melting analysis into a single assay.…”

Section: Resultsmentioning

confidence: 99%

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MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay

et al. 2016

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In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence.

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[Letter to the editor] Combined FAM-Labeled Taqman Probe Detection and SYBR Green I Melting Curve Analysis in Multiprobe qPCR Genotyping Assays

Cited by 11 publications

References 15 publications

Paper-based microfluidics for DNA diagnostics of malaria in low resource underserved rural communities

Paper-based microfluidics for DNA diagnostics of malaria in low resource underserved rural communities

Doubling Throughput of a Real-Time PCR

MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay

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