Method for improved specificity in probe based assays

Abstract

Disclosed are compositions and methods for analyzing a target sequence in a sample. Generally, the method includes use of at least one pair of probes (Probe A and Probe B). In one embodiment, Probe A hybridizes to wanted and unwanted nucleic acid in the sample and bears a fluorophore and Probe B hybridizes to unwanted nucleic acid in the sample and bears a quencher. Fluorescence signal from Probe A hybridizing to unwanted nucleic acid is quenched by any relatively close hybridization of Probe B hereby increasing the specificity for the presence, amount or absence of the wanted target sequence. In preferred embodiments, the method is referred to as Fluorescence In-Situ Hybridization (FISH). The invention has many useful applications including rapidly detecting a microbial target sequence in a clinical sample.

1 .- 21 . (canceled) 
     
     
         22 . A kit suitable for performing an assay which detects the presence, absence or amount of target sequence in a sample, wherein said kit comprises a Probe A comprised of a nucleotide sequence, which hybridizes to a target region of both wanted and unwanted DNA or RNA and is labeled with a fluorophore at the end which, upon hybridization is closest to the adjacent target region for Probe B; and a Probe B comprised of a nucleotide sequence which hybridizes to the target region of unwanted DNA or RNA adjacent to the target region of Probe A and is labeled with a quencher at the end which, upon hybridization is closest to Probe A. 
     
     
         23 . The kit of  claim 22 , wherein Probe A comprises the following nucleotide sequence: GCT-TCT-CGT-CCG-TTC (SEQ ID NO: 1) and is labeled with the fluorophore. 
     
     
         24 . The kit of  claim 22  or  23 , wherein Probe B comprises the following nucleotide sequence: ACT-TCA-AAG-GAG-CM (SEQ ID NO: 2) and is labeled with the quencher. 
     
     
         25 . The kit of  claim 22 , wherein Probe A consists essentially of the following nucleotide sequence: GCT-TCT-CGT-CCG-TTC (SEQ ID NO: 1) and is labeled with a fluorophore. 
     
     
         26 . The kit of  claim 22  or  23 , wherein Probe B consists essentially of the following nucleotide sequence: ACT-TCA-AAG-GAG-CAA (SEQ ID NO: 2) and is labeled with the quencher. 
     
     
         27 . The kit of  claim 22 , wherein Probe A has the following nucleotide sequence: GCT-TCT-CGT-CCG-TTC (SEQ ID NO: 1) and is labeled with the fluorophore. 
     
     
         28 . The kit of  claim 22  or  27 , wherein Probe B has the following nucleotide sequence: ACT-TCA-AAG-GAG-CAA (SEQ ID NO: 2) and is labeled with the quencher. 
     
     
         29 . The kit of  claim 22 , wherein Probe A has the following nucleotide sequence: GCT-TCT-CGT-CCG-TTC (SEQ ID NO: 1) and is labeled with a fluorophore; and Probe B has the following nucleotide sequence: ACT-TCA-AAG-GAG-CAA (SEQ ID NO: 2) and is labeled with the quencher. 
     
     
         30 . The kit of  claim 22 , wherein the kit is adapted for use in a fluorescence in-situ hybridization assay. 
     
     
         31 . The kit of  claim 22 , wherein the kit is adapted to detect organisms in food, beverages, water, pharmaceutical products, personal care products, dairy products or environmental samples. 
     
     
         32 . The kit of  claim 22 , wherein the kit is adapted to test raw materials, products or processes. 
     
     
         33 . The kit of  claim 22 , wherein the kit is adapted to examine clinical samples. 
     
     
         34 . The kit of  claim 33 , wherein the clinical samples are clinical specimens or equipment, fixtures and products used to treat humans or animals. 
     
     
         35 . The kit of  claim 22 , wherein the kit is adapted to detect a target sequence which is specific for a genetically based disease or is specific for a predisposition to a genetically based disease. 
     
     
         36 . The kit of  claims 22 , wherein the kit is adapted to detect a target sequence associated with a disease selected from the group consisting of 5-Thalassemia, sickle cell anemia, Factor-V Leiden, cystic fibrosis and cancer related targets such as p53, pIO, BRC-1 and BRC-2. 
     
     
         37 . The kit of  claim 22 , wherein the kit is adapted to detect a target sequence in a forensic technique. 
     
     
         38 . The kit of  claim 37 , wherein the forensic technique is at least one of prenatal screening, paternity testing, identity confirmation or crime investigation.