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

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).

7-aminobutynyl-8-aza-7-deazahypoxanthine as a quasi-universal nucleobase.

Several 7-(hydroxy, amino, methylureido, and guanidino)alkynyl-substituted 8-aza-7-deaza- hypoxanthine analogues were investigated as potential universal nucleobases. 7-Aminobutynyl-8-aza-7-deazahypoxanthine was found to be the most promising quasi-universal nucleobase with improved hybridization and polymerase chain reaction (PCR) enhancing properties as compared to commonly used hypoxanthine (the nucleobase of inosine). It demonstrated improved ambiguity for pairing with A, T, and C bases and its base pairing properties can be summarized as follows: X:C∼X:A∼X:T > X:G. The improvement in PCR performance directly correlated with primer's Tm. Primers containing multiple 7-aminobutynyl-8-aza-7-deazahypoxanthines were successfully used without noticeable inhibition of Taq polymerase activity provided the modifications are positioned more than two bases away from the 3' end.