Detection of nucleic acids by type-specific hybrid capture methods

Abstract

Target-specific hybrid capture (TSHC) provides a nucleic acid detection method that is not only rapid and sensitive, but is also highly specific and capable of discriminating highly homologous nucleic acid sequences. The method produces DNA/RNA hybrids which can be detected by a variety of methods.

1 . A nucleic acid probe having a nucleic acid sequence consisting of a sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 160. 
     
     
         2 . A nucleic acid probe set comprising:
 a) at least one capture sequence probe, wherein the capture sequence probe is 15 to 100 nucleotides in length and comprises at least one sequence that is at least 75% complementary to a first target region of a target nucleic acid, wherein the first target region is at least 6 nucleotides in length;   b) at least one signal sequence probe;   c) optionally, at least one bridge probe; and   d) optionally, at least one blocker probe at least 5 nucleotides in length, wherein the blocker probe is at least 75% complementary to at least a portion of the capture sequence probe,   
       wherein either the signal sequence probe or the bridge probe is capable of hybridizing under stringent hybridization conditions to a second target region of the target nucleic acid, wherein the first target region and the second target region do not overlap. 
     
     
         3 . The nucleic acid probe set of  claim 2 , wherein the first target sequence is from 20 to 40 nucleotides in length. 
     
     
         4 . The nucleic acid probe set of  claim 2 , wherein the target nucleic acid is HSV1 and the capture sequence probe comprises at least one sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 29. 
     
     
         5 . The nucleic acid probe set of  claim 4  comprising at least one blocker probe, wherein the blocker probe comprises a sequence selected from the group consisting of SEQ ID NO: 30 to SEQ ID NO: 49. 
     
     
         6 . The nucleic acid probe set of  claim 2 , wherein the target nucleic acid is HSV2 and the capture sequence probe comprises at least one sequence selected from the group consisting of SEQ ID NO: 50 to SEQ ID NO: 62. 
     
     
         7 . The nucleic acid probe set of  claim 6  comprising at least one blocker probe, wherein the blocker probe comprises a sequence selected from the group consisting of SEQ ID NO: 63 to SEQ ID NO: 74. 
     
     
         8 . The nucleic acid probe set of  claim 2 , wherein the target nucleic acid is an HPV16 nucleic acid and the capture sequence probe comprises at least one sequence selected from the group consisting of SEQ ID NO: 75 to SEQ ID NO: 78. 
     
     
         9 . The nucleic acid probe set of  claim 8  comprising at least one blocker probe, wherein the blocker probe comprises a sequence selected from the group consisting of SEQ ID NO: 122 to SEQ ID NO: 129. 
     
     
         10 . The nucleic acid probe set of  claim 2 , wherein the target nucleic acid is an HPV31 nucleic acid and the capture sequence probe comprises at least one sequence selected from the group consisting of SEQ ID NO: 79 to SEQ ID NO: 82. 
     
     
         11 . The nucleic acid probe set of  claim 10  comprising at least one blocker probe, wherein the blocker probe comprises a sequence selected from the group consisting of SEQ ID NO: 130 to SEQ ID NO: 137. 
     
     
         12 . The nucleic acid probe set of  claim 2 , wherein the target nucleic acid is an HPV18 nucleic acid and the capture sequence probe comprises at least one sequence selected from the group consisting of SEQ ID NO: 83 to SEQ ID NO: 95. 
     
     
         13 . The nucleic acid probe set of  claim 12  comprising at least one blocker probe, wherein the blocker probe comprises a sequence selected from the group consisting of SEQ ID NO: 112 to SEQ ID NO: 117, SEQ ID NO: 147, SEQ ID NO: 150, and SEQ ID NO: 151. 
     
     
         14 . The nucleic acid probe set of  claim 2 , wherein the target nucleic acid is an HPV45 nucleic acid and the capture sequence probe comprises at least one sequence selected from the group consisting of SEQ ID NO: 96 to SEQ ID NO: 109. 
     
     
         15 . The nucleic acid probe set of  claim 12  comprising at least one blocker probe, wherein the blocker probe comprises a sequence selected from the group consisting of SEQ ID NO: 110 to SEQ ID NO: 111, SEQ ID NO: 118 to SEQ ID NO: 121, and SEQ ID NO: 138 to SEQ ID NO: 149. 
     
     
         16 . The probe set of  claim 2 , wherein the capture sequence probe comprises complementary sequences to two or more distinct regions of the target nucleic acid. 
     
     
         17 . The probe set of  claim 2 , wherein the signal sequence probe is capable of hybridizing under stringent hybridization conditions to the second target region of the target nucleic acid. 
     
     
         18 . The probe set of  claim 2  comprising a bridge probe, wherein the bridge probe is capable of hybridizing under stringent hybridization conditions to the second target region of the target nucleic acid, and wherein the signal sequence probe is capable of hybridizing under stringent hybridization conditions to the bridge probe, but not to the target nucleic acid. 
     
     
         19 . The probe set of  claim 2 , wherein the blocker probe has a melting temperature at least 10 degrees lower than that of the capture sequence probe. 
     
     
         20 . The probe set of  claim 2 , wherein the first target region and the second target region are within 50,000 bases of each other. 
     
     
         21 . The probe set of  claim 20 , wherein the distance between first target region and the second target region is less than 3,000 bases. 
     
     
         22 . The probe set of  claim 20 , wherein the distance between first target region and the second target region is less than 1,000 bases. 
     
     
         23 . The probe set of  claim 2 , wherein the capture sequence probe comprises at least one sequence that is 100% complementary to a first target region of a target nucleic acid. 
     
     
         24 . The probe set of  claim 2 , wherein the capture sequence probe contains one or more modifications in the nucleic acid which allows specific capture of the probe onto a solid phase. 
     
     
         25 . The probe set of  claim 2 , wherein the signal sequence probe is unlabeled.