| 主权项 |
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:
a first nucleic acid probe having a first target region hybridisable to the first target segment of said particular target DNA template and a first tag region, wherein the first tag region is located 5′ from the first target region and comprises a first tag sequence, anda second nucleic acid probe having a second target region hybridisable to the second target segment of said particular target DNA template and a second tag region, wherein the second tag region is located 3′ from the second target region and comprises a second tag sequence, and wherein at least one of the first nucleic acid probe or the second nucleic acid probe contains a detection sequence located 5′ from the second tag sequence or located 3′ from the first tag sequence; (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:
a primer 1 and a primer 2, wherein at least one of primer 1 or primer 2 comprises at least one internal donor or acceptor fluorescent label at or near its 3′ end, thereby providing a plurality of internally labelled amplicons upon amplification of said plurality of connected probe assemblies, wherein the internal donor or acceptor fluorescent label is incorporated in the first or second tag region and is essentially adjacent to the detection sequence; and (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:
(1) providing a plurality of labelled detection probes, each labelled detection probe being specific for a particular detection sequence of a particular internally labelled amplicon and comprising:
at least one fluorescent donor or acceptor label complementary to the internal donor or acceptor fluorescent label incorporated in the first or second tag region by said plurality of nucleic acid primer pairs, wherein a single pair of donor and acceptor labels is used in detecting a plurality of internally labelled amplicons, wherein each similarly labelled detection probe exhibits a different melting temperature upon hybridisation to the particular detection sequence of the particular internally labelled amplicon, such that the plurality of internally labelled amplicons is distinguishable; anda nucleic acid region specifically hybridisable to said particular detection sequence of the particular internally labelled amplicon,(2) allowing the plurality of internally labelled amplicons to hybridise with the plurality of labelled detection probes, and(3) monitoring hybridisation and the different melting temperatures of the plurality of labelled detection probes by measuring the fluorescence of the acceptor labels over a pre-selected increasing temperature range within different fluorescent detection channels, wherein:
said hybridisation of the plurality of labelled detection probes is indicative for the presence of the plurality of different pathogenic organisms in the clinical sample,the different melting temperatures in combination with the different fluorescent labels of the plurality of labelled detection probes allow detection and distinguishability of a plurality of different pathogenic organisms in the clinical sample, such that up to about 30 different pathogenic organisms in the clinical sample may be detected and distinguished; andsteps (a)-(e) are performed in a closed system. |
