Real-time detection of amplification products through fluorescence quenching or energy transfer

Abstract

Molecular diagnostics of microbiological agents involves detection of microbial nucleic acids (i.e., DNA and/or RNA) and often requires the amplification of targeted sequences due to low abundances of the microbes or relevant molecular targets. Target amplification technology became available with the advent of polymerase chain reaction (PCR) in the 1980s, which eventually revolutionized the field of clinical diagnostics. While PCR remains the mainstay ever since, several alternative isothermal target amplification technologies have also become available and been used in practice [1, 2], including nucleic acid sequence-based amplification (NASBA) [3, 4], self-sustained sequence replication (3SR) [5, 6], transcription-mediated amplification (TMA) [7], strand displacement amplification (SDA) [8, 9], loop-mediated isothermal amplification (LAMP) [10, 11], recombinase polymerase amplification [12], helicase-dependent amplification (HDA) [13, 14], single primer isothermal amplification (SPIA) [15], isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) [16], isothermal chain amplification (ICA) [17], and Ionian Technologies amplification [18].