Highlights • SAMRS primers support high levels of multiplexing by not interacting with each other. • Nicking enzyme signal amplification was used to improve molecular beacon sensitivity. • Addition of AEGIS nucleotides to the beacon stem eliminated unwanted stem invasion. • 50 Copies of MERS-coV were detected by using a nicking-AEGIS molecular beacon. This paper combines two advances to detect MERS-CoV, the causative agent of Middle East Respiratory Syndrome, that have emerged over the past few years from the new field of “synthetic biology”. Both are based on an older concept, where molecular beacons are used as the downstream detection of viral RNA in biological mixtures followed by reverse transcription PCR amplification. The first advance exploits the artificially expanded genetic information systems (AEGIS). AEGIS adds nucleotides to the four found in standard DNA and RNA (xNA); AEGIS nucleotides pair orthogonally to the A:T and G:C pairs. Placing AEGIS components in the stems of molecular beacons is shown to lower noise by preventing unwanted stem invasion by adventitious natural xNA. This should improve the signal-to-noise ratio of molecular beacons operating in complex biological mixtures. The second advance introduces a nicking enzyme that allows a single target molecule to activate more than one beacon, allowing “signal amplification”. Combining these technologies in primers with components of a self-avoiding molecular recognition system (SAMRS), we detect 50 copies of MERS-CoV RNA in a multiplexed respiratory virus panel by generating fluorescence signal visible to human eye and/or camera.
【저자키워드】 RT-PCR, MERS-CoV, Nicking molecular beacon, Synthetic biology nucleotides, 【초록키워드】 amplification, sensitivity, respiratory virus, Viral RNA, molecular, respiratory, reverse transcription PCR, nucleotide, Invasion, Middle East, Support, Older, enzyme, complex, copy, primer, genetic information, component, downstream, DNA and RNA, IMPROVE, shown, was used, detect, activate, eliminated, MERS-CoV RNA, Nicking, 【제목키워드】 molecular, Amplicon, Standard, Middle East, respiratory syndrome coronavirus, detect,