Patent ID: 9222124
Filing Date: 2015-12-29
Classification: C12Q

Abstract:
1. A method for detecting and differentiating a plurality of pathogenic organisms having a copy number of less than 6000 in a clinical sample in a single reaction vessel, the clinical sample comprising a plurality of different target RNA templates and/or a plurality of different target DNA templates derived from the plurality of pathogenic organisms, each target DNA template comprising a first target segment and a second target segment, the combination of both the first target segment and the second target segment being specific for a particular target DNA template, wherein the first target segment and the second target segment are essentially adjacent to one another and wherein the first target segment is located 3′ from the second target segment, said method comprising the steps of: (a) a reverse transcription step in the single reaction vessel of the plurality of different target RNA templates into the plurality of different target DNA templates and/or an optional pre-amplification step of the plurality of different target DNA templates in the single reaction vessel; (b) bringing said plurality of different target DNA templates into contact with a plurality of different probe sets in the single reaction vessel, each probe set being specific for a particular target DNA template of the plurality of different target DNA templates and allowing the particular target DNA template to hybridise thereto, each probe set comprising: (c) forming a plurality of connected probe assemblies in the single reaction vessel, wherein the plurality of connected probe assemblies comprises the plurality of different probe sets and is formed by ligating said first nucleic acid probe and said second nucleic acid probe of each probe set; (d) amplifying the plurality of connected probe assemblies in the single reaction vessel to obtain a plurality of amplicons, wherein the plurality of connected probe assemblies are amplified by allowing the plurality of connected probe assemblies to contact with a plurality of nucleic acid primer pairs, each nucleic acid primer pair comprising: (e) detecting and differentiating the plurality of internally labelled amplicons in the single reaction vessel, wherein the plurality of internally labelled amplicons are detected and differentiated by performing a real-time melting curve analysis comprising: