A rapid, instrument-free, sample-to-result nucleic acid amplification test

Abstract

The prototype demonstrated here is the first fully integrated sample-to-result diagnostic platform for performing nucleic acid amplification tests that requires no permanent instrument or manual sample processing. The multiplexable autonomous disposable nucleic acid amplification test (MAD NAAT) is based on two-dimensional paper networks, which enable sensitive chemical detection normally reserved for laboratories to be carried out anywhere by untrained users. All reagents are stored dry in the disposable test device and are rehydrated by stored buffer. The paper network is physically multiplexed to allow independent isothermal amplification of multiple targets; each amplification reaction is also chemically multiplexed with an internal amplification control. The total test time is less than one hour. The MAD NAAT prototype was used to characterize a set of human nasal swab specimens pre-screened for methicillin-resistant Staphylococcus aureus (MRSA) bacteria. With qPCR as the quantitative reference method, the lowest input copy number in the range where the MAD NAAT prototype consistently detected MRSA in these specimens was ∼5 × 10(3) genomic copies (∼600 genomic copies per biplexed amplification reaction).

A paperfluidic platform to detect Neisseria gonorrhoeae in clinical samples

“…When comparing to the 96% success rate of the pre-isolated NG DNA trials, the lowered success of NGIC amplification in NG-negative swab device trials suggests that both urethral and vaginal patient sample matrices introduce some inhibition to amplification. Indeed, this has been seen in other recent paperfluidic NAATs, which had a 62% success rate of Staphylococcus aureus detection using clinical nasal samples (Lafleur et al 2016). To improve our device success rate in future trials with NG negative patient samples, NGIC amplification will require further optimization, such as adjusting assay salt balance and ensuring complete washing for removal of the patient sample matrices.…”

“…Here, we expand the platform to NG and enhance clinical reliability by incorporating an internal amplification control to differentiate between negative test results and invalid tests, a feature that will inevitably be required for regulatory approval of POC NAATs (Johnson et al 2002; U.S. Food and Drug Administration 2011; Lafleur et al 2016). In our NG diagnostic, cells in patient urethral and vaginal samples are added to lysis buffer which is then washed through a porous polyethersulfone (PES) substrate to precipitate and concentrate sample DNA.…”

Point-of-Care Diagnostics: Recent Developments in a Connected Age

“…Multiple research groups are developing paper-based diagnostic tests to perform molecular diagnostic tests 280–283 . Diagnostics For All is a non-profit company developing paper-based diagnostic technologies from the Whitesides lab 201 .…”

On-chip conductometric detection of short DNA sequences via electro-hydrodynamic aggregation

“…Yager's papers. 29,31 The SDA mix was composed of 1× HRCA was then performed by adding 0.5 µl of HRCA Primer 1, 0.5 µl of HRCA Primer 2 (final concentrations 1 µM) and 5 U of Phi29 polymerase to the previous mix. Polymerisation was realised during 1 h at 35°C, followed by a facultative heat inactivation at 65°C during 2 minutes.…”