Non-radioactive hybridization assay and kit

Abstract

A non-radioactive hybridization assay and kit for the detection of genetic defects, microbial infections or viral infections, such as human papillomavirus. A test sample is treated with a base and incubated with nucleic acid probes, diluted in a neutralizing buffer, specific for target nucleic acids. The hybrids are captured onto a solid phase coated with an anti-hybrid antibody, unhybridized probe is eliminated, and the bound hybrid detected.

We claim:  
     
         1 . A non-radioactive hybridization assay for the detection of a target nucleic acid sequence in a biological sample the improvement comprising the steps of: 
 a) hydrolyzing the sample with a base;    b) hybridizing a nucleic acid sequence in the treated sample to a complementary nucleic acid probe to form a double-stranded hybrid;    c) capturing the hybrid onto a solid phase to which an anti-hybrid antibody or anti-hybrid fragment has been immobilized; and,    d) eliminating any non-hybridized probe; and    e) detecting the bound hybrid.    
     
     
         2 . The assay of  claim 1  wherein the non-hybridized probe is eliminated by digestion with an enzyme.  
     
     
         3 . The assay of  claim 1  wherein the target nucleic acid is DNA selected from the group consisting of, human papillomavirus DNA, hepatitis B DNA, and Chlamydia DNA.  
     
     
         4 . The assay of  claim 1  wherein the probe is an RNA sequence complementary to target DNA.  
     
     
         5 . The assay of  claim 1  wherein the double-stranded hybrid is an RNA/DNA hybrid.  
     
     
         6 . The assay of  claim 1  wherein the digesting enzyme is RNAase.  
     
     
         7 . The assay of  claim 1  wherein the concentration of probe is between 1 and 500 ng/ml.  
     
     
         8 . The assay of claim.  1  wherein the concentration of probe is between 20 and 200 ng/ml.  
     
     
         9 . The assay of  claim 1  wherein the concentration of probe is approximately 75 ng/ml.  
     
     
         10 . The assay of  claim 1  wherein the base is sodium hydroxide in a concentration of between 0.1 and 2.0 M, incubated with the sample at a temperature between 20 and 100° C. for a period of between 5 and 120 minutes.  
     
     
         11 . The assay of  claim 1  wherein the base is sodium hydroxide in a concentration of between 0.2 and 0.8 M, incubated with the sample at a temperature between 60 and 70° C. for a period of between 30 and 60 minutes.  
     
     
         12 . The assay of  claim 1  wherein the base is sodium hydroxide in a concentration of approximately 0.415 M, incubated with the sample at a temperature of approximately 65° C. for a period of approximately 45 minutes.  
     
     
         13 . The assay of  claim 6  wherein the RNAase is added to the sample in a concentration between 0.01 and 1 mg/ml and incubated with the sample at a temperature between 4 and 45° C. for a period of between 5 minutes and 24 hours.  
     
     
         14 . The assay of  claim 6  wherein the RNAase is added to the sample in a concentration between 0.05 and 0.5 mg/ml and incubated with the sample at a temperature between 20 and 30° C. for a period of between 10 and 60 minutes.  
     
     
         15 . The assay of  claim 6  wherein the RNAase is added to the sample in a concentration of approximately 0.2 mg/ml and incubated with the sample at room temperature for a period of approximately 30 minutes.  
     
     
         16 . The assay of  claim 1  further comprising diluting the probe in a buffer that restores the sample to a neutral pH.  
     
     
         17 . The assay of  claim 16  wherein the buffer comprises 2-[bis(2-Hydroxyethyl) amino] ethane sulfonic acid and sodium acetate.  
     
     
         18 . A kit for the detection of a target nucleic acid sequence for diagnosing genetic defects, microbial or viral infections in a biological sample comprising: 
 a) a sample transport medium for stabilization of the biological sample;    b) a base for treating the sample by nicking and degrading the target nucleic acid sequence therein;    c) a probe complementary to the treated target nucleic acid sequence for formation of a double-stranded nucleic acid hybrid;    d) a neutralizing probe diluent for diluting the probe and neutralizing the treated target nucleic acid sequence;    e) a solid phase to which an anti-hybrid antibody or an anti-hybrid antibody fragment has been immobilized, wherein the antibody is specific for a hybrid formed by hybridization of the probe and the target nucleic acid sequence;    f) means for eliminating any non-hybridized probe; and,    g) means for detecting the hybrid formed by hybridization of the probe and the target nucleic acid sequence.    
     
     
         19 . The kit of  claim 18  wherein the means for eliminating non-hybridized probe is an enzyme that digests non-hybridized probe.  
     
     
         20 . The kit of  claim 18  wherein the target nucleic acid is DNA selected from the group consisting of human papillomavirus DNA, hepatitis B virus DNA and Chlamydia DNA.  
     
     
         21 . The kit of  claim 18  wherein the probe is an RNA sequence complementary to target DNA.  
     
     
         22 . The kit of  claim 18  wherein the digesting enzyme is RNAase.  
     
     
         23 . The kit of  claim 18  wherein the base is sodium hydroxide in a concentration of between 0.1 and 2 M.  
     
     
         24 . The kit of  claim 18  wherein the digesting enzyme and detecting means are combined in a single reagent.  
     
     
         25 . A buffer for use in a hybridization assay comprising 2-[bis(2-Hydroxyethyl) amino] ethane sulfonic acid and sodium acetate wherein the pH of the buffer is between approximately 5 and 5.5.  
     
     
         26 . The buffer of  claim 25  further comprising an antimicrobial agent, a metal chelating agent, a detergent, and a hybridization accelerator.  
     
     
         27 . The buffer of  claim 25  further comprising a probe in a concentration between 1 and 500 ng/ml for hybridization to a target nucleic acid.