Doubling Throughput of a Real-Time PCR

Ahrberg, Christian D.; Neužil, Pavel

doi:10.1038/srep12595

Cited by 16 publications

(13 citation statements)

References 24 publications

(26 reference statements)

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“…Our qPCR assay had a detection limit of 10 attamoles (~300 molecules of 160 bp ds (double-stranded) DNA at 90 kD) of Gouldian finch control region DNA in a qPCR. This level of sensitivity is consistent with that reported for other qPCR assays (Ahrberg & Neužil 2015).…”

Section: Molecular Sensitivity Of the Gouldian Finch Dna Detection Assaysupporting

confidence: 91%

“…Molecular methods for eDNA analysis have been changing constantly for the past decade (Jarman et al 2018). The design of our qPCR assay for Gouldian finch eDNA involved multiple channels of fluorescent detection (Ahrberg & Neužil 2015) to provide a versatile test for the Gouldian finch and other finches. The group-specific approach for PCR amplification also lowers the chance of encountering a Gouldian finch with sequence variation in the PCR primer-binding sites leading to undetectable false negative outcomes.…”

Section: Discussionmentioning

confidence: 99%

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Development and validation of an environmental DNA test for the endangered Gouldian finch

Day¹,

Campbell²,

Fisher³

et al. 2019

Endang. Species. Res.

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Detecting animals by identifying their DNA in water is a valuable tool for locating and monitoring species that are difficult to detect through other survey techniques. We developed a test for detecting the endangered Gouldian finch Erythrura gouldiae, a small bird endemic to northern Australia. Only 1 previous study has reported an environmental DNA (eDNA) test that unequivocally identifies a bird species using the water bodies from which they drink. In controlled aviary trials with a pair of Gouldian finches, first detection in 200 ml of water occurred after as little as 6 h, but the detection rate was higher at 30 h. DNA persisted in water exposed to the sun for <12 h and in the shade for 12 h. In trials with 55 finches, persistence was up to 144 h. The eDNA test for finches and the Gouldian finch-specific test were positive for waterholes where Gouldian and other finch species were observed each morning over 3 d. Importantly, where no Gouldian finches were observed for up to 72 h prior to water sampling, the Gouldian test was negative. Where other species of finch but no Gouldian finch were observed and counted, the finch test was positive, but the Gouldian finch test was negative. This approach could be developed for broadscale monitoring of this endangered species, and potentially applied to a much broader range of terrestrial species that shed DNA into water bodies.

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Section: Molecular Sensitivity Of the Gouldian Finch Dna Detection Assaysupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Development and validation of an environmental DNA test for the endangered Gouldian finch

Day¹,

Campbell²,

Fisher³

et al. 2019

Endang. Species. Res.

View full text Add to dashboard Cite

show abstract

“…The testing time can be shortened either by system redesign [36] or using probe-based PCR and performing a single PCR cycle as fast as 10 s [32], thus having a total required time for the 40 cycle PCR protocol as short as ≈ 6 min. The system can be further expanded using multiplexing just by reprogramming and effectively doubling the throughput [36], reprogramming the PCR readout, or using multicolor LEDs with a dual bandpass filter.…”

Section: Discussionmentioning

confidence: 99%

IoT PCR for pandemic disease detection and its spread monitoring

Zhu

Podešva

Liu

et al. 2020

Sensors and Actuators B: Chemical

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A B S T R A C TDuring infectious disease outbreaks, the centers for disease control need to monitor particular areas. Considerable effort has been invested in the development of portable, user-friendly, and cost-effective systems for point-of-care (POC) diagnostics, which could also create an Internet of Things (IoT) for healthcare via a global network. However, at present IoT based on a functional POC instrument is not available. Here we show a fast, user-friendly, and affordable IoT system based on a miniaturized polymerase chain reaction device. We demonstrated the system's capability by amplification of complementary deoxyribonucleic acid (cDNA) of the dengue fever virus. The resulting data were then automatically uploaded via a Bluetooth interface to an Androidbased smartphone and then wirelessly sent to a global network, instantly making the test results available anywhere in the world. The IoT system presented here could become an essential tool for healthcare centers to tackle infectious disease outbreaks identified either by DNA or ribonucleic acid.

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“…The system throughput can be doubled using a single channel multiplexing method as demonstrated earlier. 27 …”

Section: Discussionmentioning

confidence: 99%

Handheld real-time PCR device

et al. 2016

Self Cite

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Here we report one of the smallest real-time polymerase chain reaction (PCR) system up to date with approximate size of 100 mm × 60 mm × 33 mm. The system is an autonomous unit requiring an external 12 V power supply. Four simultaneous reactions are performed in form of virtual reaction chambers (VRC) where a ≈ 200 nL sample is covered with mineral oil and placed on a glass cover slip. Fast, 40 cycle amplification of an amplicon from the H7N9 gene was used to demonstrate PCR performance. The standard curve slope was (−3.02 ± 0.16) cycles at threshold per decade (mean ± standard deviation) corresponding to an amplification efficiency of (0.91 ± 0.05) per cycle (mean ± standard deviation). The PCR device was capable of detecting a single deoxyribonucleic acid (DNA) copy. These results further suggest that our handheld PCR device may have broad, technologically-relevant applications extending to rapid detection of infectious diseases in small clinics.

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Doubling Throughput of a Real-Time PCR

Cited by 16 publications

References 24 publications

Development and validation of an environmental DNA test for the endangered Gouldian finch

Development and validation of an environmental DNA test for the endangered Gouldian finch

IoT PCR for pandemic disease detection and its spread monitoring

Handheld real-time PCR device

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