Method and rapid test for detection of specific nucleic acid sequences

Abstract

A universally usable method for specific detection of target nucleic acid sequences, which method can be performed very rapidly and also simply and furthermore which does not need any expensive instrumental systems. The method is intended to be suitable as a molecular genetic rapid test and to respect the requirements of diagnostic specificity assurance. In this regard it is important that only one specific amplification product be detected and that amplification artifacts can be unambiguously discriminated. A nucleic acid amplification kit suitable for performing this method.

1 . A method for assaying at least one specific nucleic acid sequence (target sequence) comprising:
 amplifying a nucleic acid sequence to be assayed with at least one primer, if necessary, followed by strand separation, and   hybridizing with at least one probe completely or partly complementary to the target sequence, and   detecting the hybridization reaction;   wherein   a) the amplification, and if necessary, the strand separation, and the hybridization take place in one reaction vessel, and   b) at least one primer is labeled with a molecule, and   c) the hybridization probe is provided with a label and it hybridizes to the strand of the target sequence that contains the labeled primer and   d) the detection of the hybridization reaction takes place on a solid phase outside the reaction vessel mentioned under a) and   e) the solid phase contains a binding site for the label either of the primer or of the probe and thereby the hybridization product is thereby bound to the solid phase and   f) the detection of the hybridization reaction takes place on a solid phase outside the reaction vessel mentioned under a) by the fact that
 the solid phase has a binding site that permits binding with the label of the primer or with the label of the hybridization probe, whereby the hybridization product is bound to the solid phase and detection of the bound hybridization product takes place by direct or indirect detection of the label that is still free or 
 the label of the primer or the label of the hybridization probe enters into binding with a detection molecule and then the free label enters into binding with a binding site of the solid phase, whereby the hybridization product is bound to the solid phase and detection of the hybridization product bound to the solid phase takes place via the detection molecule. 
   
     
     
         2 . The method according to  claim 1 , wherein the visualization or the measurement of the PCR hybridization result takes place by means of an optical device. 
     
     
         3 . The method according to  claim 1 , wherein in that the hybridization probe is protected against the 5′→3′ polymerase activity. 
     
     
         4 . The method according to  claim 3 , wherein the hybridization probe is protected against the 5′→3′ polymerase activity by labeling or by phosphorylation. 
     
     
         5 . The method according to  claim 1 , wherein the at least one primer is labeled with biotin. 
     
     
         6 . The method according to  claim 1 , wherein the hybridization probe is labeled with FITC (fluorescein isothiocyanate). 
     
     
         7 . The method according to  claim 1 , wherein there is used as the solid phase a test strip that contains a streptavidin site for coupling the biotin-labeled label and an FITC binding site for functional control of the test strip. 
     
     
         8 . The method according to  claim 7 , wherein the PCR mixture (amplification mixture) is mixed with a running buffer and applied on the test strip. 
     
     
         9 . The method according to  claim 7 , wherein gold particles coated with anti-FITC antibodies are located in the lower zone of the test strip, where the sample is applied, and in that the streptavidin binding site is located further along the test strip. 
     
     
         10 . The method according to  claim 1 , wherein an asymmetric PCR is performed instead of the standard PCR reaction. 
     
     
         11 . The method according to  claim 1 , wherein a reverse transcription takes place in the case of RNA assay before amplification. 
     
     
         12 . A test kit for performing the method according to  claim 1 , comprising:
 a reaction vessel for performing the amplification, the strand separation and the hybridization with the probe,   at least one primer labeled with a molecule,   at least one probe that is completely or partly complementary to the target sequence, that is protected against the 5′→3′ polymerase activity and/or is provided with a label, and that hybridizes to the strand of the target sequence that contains the labeled primer,   at least one solid phase, which contains a binding site for the label either of the primer or of the probe, and/or   PCR reagents known in themselves, such as PCR buffers, polymerases, dNTPs and
 if necessary further additives 
 as well as at least one running agent for detection of hybridization. 
   
     
     
         13 . The test kit according to  claim 12 , wherein the reaction vessel contains the primer, the probe and the PCR reagents known in themselves in solid form. 
     
     
         14 . The method of  claim 1 , which is a qualitative method for detecting the target nucleic acid. 
     
     
         15 . The method according to  claim 1  that is a rapid test in which detection takes less than one hour. 
     
     
         16 . The method according to  claim 1  that is a rapid test that comprises multiplex detection, wherein several primers and probes labeled by either identical or different molecules are employed. 
     
     
         17 . The method according to  claim 1 , wherein the target nucleic acid is from a virus or a bacterium. 
     
     
         18 . A method for food diagnosis, environmental analysis, or hospital hygiene comprising the method of  claim 1 . 
     
     
         19 . The method according to  claim 1 , wherein the target nucleic acid is from  Salmonella, Listeria, E. coli, Campylobacter, Shigella, Enterobacter , MRSA microbes or  Legionella.    
     
     
         20 . The method according to  claim 1 , wherein the target nucleic acid is from  Borrelia, Rickettsia, Erlichia, Babesia , or another tick-born pathogen. 
     
     
         21 . The method according to  claim 1 , wherein said target nucleic acid comprises a SNP, mutation or methylated sequence motif. 
     
     
         22 . A method for detecting a target nucleic acid comprising:
 conducting a polymerase chain reaction (PCR) on a sample suspected of containing the target nucleic acid in a PCR reaction mixture comprising:   two PCR primers suitable for amplifying the target nucleic acid, one of which is labeled at its 5′ end with a labeling molecule, and   a hybridization probe which is able to hybridize to the strand of the target nucleic acid containing the sequence of primer labeled at its 5′ end, but which has been chemically modified so that it is not elongated by a 5′→3′ polymerase used for the PCR;   denaturing the PCR reaction mixture at a temperature sufficient to separate the strands of a PCR amplification product generated by the PCR,   cooling the denatured PCR reaction mixture to a hybridization temperature of the hybridization probe for a time and under conditions sufficient for the probe to bind to the complementary strand of the nucleic acid amplification product, and   contacting the resulting mixture with a solid phase support comprising a nucleic acid complementary to the PCR probe;   wherein the occurrence of, or amount of, binding to the solid phase support compared to a control indicates the presence of the target nucleic acid in the sample.   
     
     
         23 . The method of  claim 22 , wherein contacting the resulting mixture with a solid phase support comprising a nucleic acid complementary to the PCR probe is conducted outside of a reaction vessel used to perform amplification and hybridization. 
     
     
         24 . The method of  claim 23 , wherein said solid phase is a lateral-flow test strip.