Method of detection in vitro of a target substance in a sample comprising the labelling of said substance with a reporter gene and with the sequences necessary for the expression of said reporter gene in vitro

Abstract

The present invention relates to a method of detection in vitro of a target substance in a sample. The sample can comprise, among other things, a nucleic sequence or more generally any type of substance. In particular, the present invention provides a method of detection comprising (i) specifically labeling a substance with a reporter gene and any sequences necessary for the in vitro expression of the reporter gene; (ii) in vitro transcription and translation of the reporter gene; and (iii) in vitro detection of a reporter protein coded by the reporter gene.

1 ) Method of detection of a target substance in a sample, comprising: 
 a) specific labeling of said substance by a reporter gene and by sequences necessary for expression of said reporter gene in vitro;    b) transcription and translation in vitro of said reporter gene; and    c) The detection in vitro of the a reporter protein encoded by said reporter gene.    
     
     
         2 - 4 . (canceled)  
     
     
         5 ) Method of detection of a target nucleic acid sequence in a sample according to  claim 1 , comprising 
 a) The preparation, starting from the sample of a recombinant nucleic acid molecule comprising, in the 5′ to 3′ sense, an RNA polymerase promoter, a reporter gene possessing the sequences necessary for its expression and possibly an RNA polymerase terminator, the target nucleic acid sequence being localized between any two of these elements,    b) The transcription and the translation of the amplification products obtained at step (a),    c) The revelation of the activity of the protein encoded by the reporter gene, designated the reporter molecule, obtained in step (b) by any appropriate means.    
     
     
         6 ) Method of detection of a target nucleic acid sequence in a sample according to  claim 5 , characterized in that the preparation of the nucleic acid molecule of step (a) is carried out by amplification in vitro of the target nucleic acid sequence.  
     
     
         7 - 10 . (canceled)  
     
     
         11 ) A set of primers capable of being used at step (a) of the method according to  claim 6 , comprising: 
 A sense primer comprising at least one part homologous to the 5′ region of the target sequence,    An anti-sense primer, comprising at least one part homologous to the region 3′ of the target sequence,    said primers permitting after amplification of the target nucleic acid sequence, and after the step (b) the expression of a reporter gene.    
     
     
         12 ) A kit for the detection of a target nucleic acid sequence in a sample, characterized in that it comprises a set of primers according to  claim 11 , a mixture necessary for amplification, the triphosphate nucleotides, a DNA dependent RNA polymerase, a cellular translation extract, the mixtures necessary for transcription, for translation, and optionally for the revelation of the reporter molecule, and optionally one or several substances permitting revelation of the activity of a reporter molecule.  
     
     
         13 ) Method of detection in vitro of a target nucleic acid sequence in a sample according to  claim 6 , characterized in that the preparation of the nucleic acid molecule of step (a) is carried out by hybridization and ligation of a Padlock-type probe with the target sequence.  
     
     
         14 - 15 . (canceled)  
     
     
         16 ) A Padlock probe capable of being used in a method according to  claim 13 , characterized in that the sequences of said segments 3′ and 5′ of the Padlock probe are defined such as being able to hybridize in a complementary and joined manner to a target sequence.  
     
     
         17 ) A Padlock probe capable of being used in a method according to  claim 13 , characterized in that the sequences of said segments 3′ and 5′ of the Padlock probe are complementary to a target sequence having a nucleotide capable of being mutated so as to form with it a hybrid where said nucleotide is found at their junction when they are hybridized to the target sequence.  
     
     
         18 ) Method of detection in vitro of a target nucleic acid sequence in a sample according to  claim 13 , characterized in that after circularization, the probe is used as a matrix for its replication by rolling circle.  
     
     
         19 - 20 . (canceled)  
     
     
         21 ) A kit comprising: 
 a probe according to  claim 16 , a DNA dependent DNA polymerase, a nick-sealing ligase, the triphosphate nucleotides and the triphosphate deoxynucleotides, a primer for initiating replication, a primer permitting the synthesis of the second DNA strand, a DNA dependent RNA polymerase, the mixtures necessary for ligation, for replication, for transcription, for translation and for the revelation of the reporter molecule.    
     
     
         22 ) Method of detection in vitro of a target nucleic acid sequence in a sample according to  claim 6 , characterized in that the target nucleic acid sequence is isolated from a nucleic acid sample at step (a) by specific isothermic amplification, with the aid of a DNA dependent DNA polymerase and of two specific primers of the target sequence, wherein at least one is composed of a 3′ part which can be specifically hybridized to the target sequence and of a 5′ part comprising at least one inverted repeat sequence for forming at a suitable temperature a so called “hairpin” structure.  
     
     
         23 - 24 . (canceled)  
     
     
         25 ) A set of primers capable of being used in the method according to  claim 22 , characterized in that it comprises: 
 A sense primer comprising at least a part homologous to the 5′ region of the target sequence,    An anti-sense primer, comprising at least a part homologous to the 3′ region of the target sequence    said primers permitting after amplification of the target nucleic acid sequence, and after the step (b) the expression of a reporter gene, and at least one of said primers comprising a 5′ inverted repeat sequence.    
     
     
         26 . (canceled)  
     
     
         27 ) A set of primers according to  claim 25 , characterized in that at least one of the two primers contains a restriction site, wherein if the two possess a restriction site, the latter is identical or different.  
     
     
         28 ) A set of primers according to  claim 27 , characterized in that the restriction site present on at least one of the primers is situated in a loop of a hairpin, more precisely between two inverted repeat sequences of one of the primers.  
     
     
         29 ) A kit comprising: a set of primers according to  claim 25 , a amplification reaction mixture, optionally one or several restriction enzymes, a DNA dependent RNA polymerase, a DNA dependent DNA polymerase, the mixtures necessary for transcription, for translation, and for the revelation of the reporter molecule.  
     
     
         30 . (canceled)  
     
     
         31 ) A kit for the implementation of the method of detection of a target substance according to  claim 1 , comprising at least a reporter gene, the mixtures necessary for the transcription, for the translation and for the revelation of the reporter protein encoded by the reporter gene.  
     
     
         32 ) A substance labeled by a reporter gene and the elements necessary for the expression in vitro of said reporter gene capable of being obtained by the process for labeling a substance by step (a) according to  claim 1 .  
     
     
         33 ) A kit comprising: 
 a probe according to  claim 17 , a DNA dependent DNA polymerase, a nick-sealing ligase, the triphosphate nucleotides and the triphosphate deoxynucleotides, a primer for initiating replication, a primer permitting the synthesis of the second DNA strand, a DNA dependent RNA polymerase, the mixtures necessary for ligation, for replication, for transcription, for translation and for the revelation of the reporter molecule.    
     
     
         34 ) A kit comprising: 
 a set of primers according to  claim 27 , a amplification reaction mixture, optionally one or several restriction enzymes, a DNA dependent RNA polymerase, a DNA dependent DNA polymerase, the mixtures necessary for transcription, for translation, and for the revelation of the reporter molecule.    
     
     
         35 ) A kit comprising: 
 a set of primers according to  claim 28 , a amplification reaction mixture, optionally one or several restriction enzymes, a DNA dependent RNA polymerase, a DNA dependent DNA polymerase, the mixtures necessary for transcription, for translation, and for the revelation of the reporter molecule.