Nucleic acid probe with single fluorophore label bound to internal cytosine for use in loop mediated isothermal amplification

A method of detecting a target nucleic acid sequence in a sample, the method comprising:

• isothermally amplifying a target nucleic acid in the sample by loop-mediated isothermal amplification to provide an amplified nucleic acid, wherein the isothermal amplification incorporates a probe comprising an oligonucleotide sequence into the amplified nucleic acid, the oligonucleotide sequence being complementary to a region of a target nucleic acid sequence, wherein said oligonucleotide sequence has only one fluorophore label, which label is bound to an internal cytosine base that is not disposed in the first three bases from either the 5′ or the 3′ end, and wherein said probe comprising the oligonucleotide sequence does not have a 3′ end terminator;
• measuring fluorescence of the incorporated fluorophore label in the amplified nucleic acid during the isothermal nucleic acid amplification;
• and
• detecting the presence of the target nucleic acid, wherein an increase in fluorescence of the amplified nucleic acid during the isothermal nucleic acid amplification indicates the presence of the target nucleic acid in the sample.