Microchip device with dry-stored reagents for Loop mediated isothermal amplification

Tupik, A. N.; Rudnitskaya, G. E.; Bulyanitsa, A. L.; Lukashenko, T. A.; Varlamov, D. A.; Evstrapov, A. A.

doi:10.1088/1742-6596/1124/3/031015

Cited by 3 publications

(1 citation statement)

References 10 publications

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“…LAMP is similar to polymerase chain reaction (PCR) in that it amplifies target DNA sequences. LAMP provides some advantages over PCR as a portable detection method because it is isothermal and does not require a bulky heat source, can be used on less pure DNA samples, and the reagents can be dried to a matrix to extend the shelf life without a freezer 11,12,13,14 .…”

Section: Introductionmentioning

confidence: 99%

Detection of mycotoxin-producing fungi in cereal crops using LAMP-based assays in a microfluidic device

Reynolds,

Wu,

Thamaratnothai

et al. 2023

Sensing for Agriculture and Food Quality and Safety XV

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The consumption of mycotoxins generated by fungi can have severe effects on the health of both humans and animals. These toxins can exist at dangerous levels in food products made from crops that have been infected with mycotoxinproducing fungi. Numerous methods have been developed for detecting mycotoxins in order to divert contaminated commodities from the food supply, but only allow for reactive, not preventive approaches. Furthermore, under favorable conditions toxin-producing fungi can continue to produce mycotoxins during storage and throughout the crop processing stages. By identifying mycotoxin-producing fungal species on crops or commodities, remediation such as fungicide application can be carried out, preventing the spread of infection and potential contamination of healthy crops, reducing waste of resources and ultimately improving food safety. Loop-mediated isothermal amplification (LAMP) has advantages for portable DNA detection due to its isothermal nature, resistance to matrix inhibitors, and the possibility of a long shelflife when reagents are dried onto a matrix. The developed microfluidic device allows for the homogenized wheat sample input after DNA extraction. The microfluidic device functions as a disposable cassette and can be heated by an independent, portable, isothermal heating device. The LAMP assay is combined with calcein for fluorescence detection. In this experiment, Fusarium graminearum, a trichothecene mycotoxin producer, was used as a proof-of-concept for the device with a LAMP assay targeting the gaoA gene, which codes for the enzyme galactose oxidase (GO), a unique enzyme produced by only a few other fungal species. The presence of Fusarium graminearum was detected in contaminated wheat samples utilizing the described methods, indicating the potential detection of mycotoxin-producing fungi. In the future, the device will be expanded to test for multiple mycotoxin-producing genes.

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Section: Introductionmentioning

confidence: 99%

Detection of mycotoxin-producing fungi in cereal crops using LAMP-based assays in a microfluidic device

Reynolds,

Wu,

Thamaratnothai

et al. 2023

Sensing for Agriculture and Food Quality and Safety XV

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All-in-one pumpless portable genetic analysis microsystem for rapid naked-eye detection

Choi

Song

Kim

et al. 2021

Sensors and Actuators B: Chemical

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Reagent integration and controlled release for multiplexed nucleic acid testing in disposable thermoplastic 2D microwell arrays

et al. 2021

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The seamless integration of reagents into microfluidic devices can serve to significantly reduce assay complexity and cost for disposable diagnostics. In this work, the integration of multiplexed reagents into thermoplastic 2D microwell arrays is demonstrated using a scalable pin spotting technique. Using a simple and low-cost narrow-bore capillary spotting pin, high resolution deposition of concentrated reagents within the arrays of enclosed nanoliter-scale wells is achieved. The pin spotting method is further employed to encapsulate the deposited reagents with a chemically modified wax layer that serves to prevent disruption of the dried assay components during sample introduction through a shared microchannel, while also enabling temperature-controlled release after sample filling is complete. This approach supports the arbitrary patterning and release of different reagents within individual wells without crosstalk for multiplexed analyses. The performance of the in-well spotting technique is characterized using on-chip rolling circle amplification to evaluate its potential for nucleic acid-based diagnostics.

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Microchip device with dry-stored reagents for Loop mediated isothermal amplification

Cited by 3 publications

References 10 publications

Detection of mycotoxin-producing fungi in cereal crops using LAMP-based assays in a microfluidic device

Detection of mycotoxin-producing fungi in cereal crops using LAMP-based assays in a microfluidic device

All-in-one pumpless portable genetic analysis microsystem for rapid naked-eye detection

Reagent integration and controlled release for multiplexed nucleic acid testing in disposable thermoplastic 2D microwell arrays

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