Universal Multi-Variant Detection System

Abstract

The present invention provides a method to diagnostically detect the variants of a given pathogen, such as HIV, hepatitis C, hepatitis B (HBV), Parvovirus B19, etc., with the use of a single detection probe.

1 . A kit for the detection of a target nucleic acid molecule in a sample, wherein the target nucleic acid molecule is known to have variant sequences, comprising:
 (i) a set of nose-to-nose primers comprising a forward primer and a reverse primer, each comprising a sequence of nucleotides and wherein the forward and reverse primers are characterized in that a nucleotide at the 3′ end of the reverse primer hybridizes with:
 (i) a nucleotide at the 5′ end of a forward primer extension product or; 
 (ii) a nucleotide separated from the nucleotide at the 5′ end of the forward primer extension product by a gap; 
   and a nucleotide at the 3′ end of the forward primer hybridizes with:
 (i) a nucleotide at the 5′ end of a reverse primer extension product or; 
 (ii) a nucleotide separated from the nucleotide at the 5′ end of the reverse primer extension product by the gap; 
   (ii) reagents for performing a primer extension chain reaction and;   (iii) a self-altering signal-generating probe which detects the presence of primer amplification products, wherein the probe comprises a first nucleic acid sequence attached to a reporter moiety capable of generating a detectable signal; a second nucleic acid sequence attached to an interactive moiety capable of altering the signal of the reporter moiety; and a probe sequence which connects the first and second nucleic acid sequences.   
     
     
         2 . The kit according to  claim 1 , wherein the target nucleic acid molecule is a virus selected from the group consisting of human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV). 
     
     
         3 . The kit according to  claim 1 , wherein the gap comprises a highly conserved region of the genome of the virus. 
     
     
         4 . The kit according to  claim 1 , wherein the gap comprises from about one to about five nucleotides. 
     
     
         5 . The kit according to  claim 1 , wherein the nucleic acid molecule which is complementary to the target nucleic acid molecule of step (d)(i) is provided separately as the cDNA of the target nucleic acid molecule. 
     
     
         6 . The kit according to  claim 1 , wherein the second nucleic acid sequence of the self-altering signal-generating probe is hybridized to the first nucleic acid sequence of the self-altering signal-generating probe and wherein the probe sequence comprises either:
 (a) the nucleotide sequence of a segment of the forward primer; or   (b) the nucleotide sequence of a segment of the reverse primer; and   wherein upon contacting the amplification products with the probe, the probe sequence of the probe hybridizes with the additional reverse primer amplification product or with the additional forward primer amplification product, and the first and second nucleic acid sequences become denatured, thereby generating a signal by the reporter moiety; and wherein the signal generated by the reporter moiety indicates the presence of the target molecule.   
     
     
         7 . The kit according to  claim 6 , wherein the probe sequence comprises either:
 (a) the nucleotide sequence of a segment of the forward primer, and not the nucleotide sequence of a segment which is complementary to the reverse primer; or   (b) the nucleotide sequence of a segment of the reverse primer, and not the nucleotide sequence of a segment which is complementary to the forward primer.   
     
     
         8 . The kit according to  claim 6 , wherein the probe sequence comprises either:
 (a) the nucleotide sequence of a segment of the forward primer and the nucleotide sequence of a segment which is complementary to the reverse primer; or   (b) the nucleotide sequence of a segment of the reverse primer and the nucleotide sequence of a segment which is complementary to the forward primer.   
     
     
         9 . The kit according to  claim 6 , wherein the level of the detectable signal generated by the probe is proportional to the quantity of the target nucleic acid molecule in the sample. 
     
     
         10 . The kit according to  claim 8 , wherein about sixty to about ninety-five percent of the probe sequence comprises either:
 (i) the nucleotide sequence of a segment of the forward primer; or   (ii) the nucleotide sequence of a segment of the reverse primer.   
     
     
         11 . The kit according to  claim 6 , wherein:
 if the probe sequence comprises the nucleotide sequence of a segment of the reverse primer, the molar ratio of the reverse primer to the forward primer is in the range from about 1:5 to about 1:20; or   if the probe sequence comprises the nucleotide sequence of a segment of the forward primer, the molar ratio of the forward primer to the reverse primer is in the range from about 1:5 to about 1:20.   
     
     
         12 . The kit according to  claim 6 , wherein the probe sequence comprises from about ten to about thirty nucleotide residues. 
     
     
         13 . The kit according to  claim 9 , wherein the probe sequence comprises from about eighteen to about twenty-four nucleotide residues. 
     
     
         14 . The kit according to  claim 6 , wherein the detectable signal is a luminescent signal. 
     
     
         15 . The kit according to  claim 14 , wherein the luminescent signal is a fluorescent signal or a chemiluminescent signal. 
     
     
         16 . The kit according to  claim 6 , wherein the reporter moiety is attached at the 5′ terminus or 3′ terminus of the self-altering signal-generating probe. 
     
     
         17 . The kit according to  claim 6 , wherein the interactive moiety is attached at the 5′ terminus or 3′ terminus of the self-altering signal-generating probe. 
     
     
         18 . The kit according to  claim 6 , wherein the reporter moiety is a fluorophore selected from the group consisting of a xanthene dye, a cyanine dye, a dansyl derivative, EDANS, coumarin, Lucifer yellow, BODIPY, Cy3, Cy5, Cy7, Texas red, erythrosine, naphthylamine, Oregon green, or combinations thereof. 
     
     
         19 . The kit according to  claim 6 , wherein the interactive moiety is a quencher or a fluorophore. 
     
     
         20 . The kit according to  claim 19 , wherein the quencher is DABCYL, anthroquinone, nitrothiazole, nitroimidazole or malachite green. 
     
     
         21 . The kit according to  claim 1 , wherein the amplification products are measured and quantitated by end-point analysis or by real-time analysis. 
     
     
         22 . The kit according to  claim 1 , wherein the amplification products are measured using a standard curve derived from a series of threshold cycle measurements.