Compositions and methods for detecting
View Patent ↗Disclosed are nucleic acid oligomers, including amplification oligomers, detection probes, and capture probes, for detection of Plasmodium species nucleic acid in a sample. Also disclosed are methods of specific nucleic acid amplification and detection, including amplification and detection of target nucleic acid in real time, using the disclosed oligomers, as well as corresponding reaction mixtures and kits.
1 . A method for specifically detecting Plasmodium species nucleic acid in a sample, said method comprising:
(1) contacting a sample, said sample suspected of containing Plasmodium species nucleic acid, with at least two oligomers for amplifying a target region of a Plasmodium species target nucleic acid, wherein the at least two amplification oligomers comprise:
(a) at least one first amplification oligomer comprising a target-hybridizing sequence
(i) that is from about 14 to about 20 contiguous nucleotides in length, is contained in the sequence of SEQ ID NO: 162, and comprises the sequence of SEQ ID NO: 163; or
(ii) that is from about 14 to about 25 contiguous nucleotides in length, is contained in the sequence of SEQ ID NO: 166, and comprises the sequence of SEQ ID NO: 167 or SEQ ID NO: 168; and
(b) at least one second amplification oligomer comprising a target-hybridizing sequence that is from about 15 to about 33 contiguous nucleotides in length, is contained in SEQ ID NO: 169 and comprises the sequence of SEQ ID NO: 171, SEQ ID NO:172, or SEQ ID NO:173;
(2) performing an in vitro nucleic acid amplification reaction, wherein any Plasmodium target nucleic acid present in said sample is used as a template for generating an amplification product; and
(3) contacting the in vitro nucleic acid amplification with at least one detection probe oligomer configured to specifically hybridize to the amplification product, wherein the at least one detection probe oligomer comprises a detectable label; and
(4) detecting the presence or absence of the amplification product, thereby indicating the presence or absence of Plasmodium species target nucleic acid in said sample.
2 . The method of claim 1 , wherein the target-hybridizing sequence of the at least one first amplification oligomer
(A) comprises a sequence selected from the group consisting of SEQ ID NOs: 21, 23-25, 32, 33, 35, 54, and 55;
(B) is contained in the sequence of SEQ ID NO: 164 and comprises the sequence of SEQ ID NO: 165;
(C) comprises the sequence of SEQ ID NO:167;
(D) comprises a sequence selected from the group consisting of SEQ ID NOs: 28-31, 34, 40, 41, and 49-51;
(E) comprises the sequence of SEQ ID NO:168; or
(F) comprises the sequence selected from the group consisting of SEQ ID NOs: 38, 39, 43, 44, and 53.
3 . The method claim 1 , wherein the target-hybridizing sequence of the at least one second amplification oligomer
(A) comprises a sequence selected from the group consisting of SEQ ID NOs: 80-82 and 85-100; or
(B) is contained in SEQ ID NO:170, and comprises the sequence of SEQ ID NO: 171 or SEQ ID NO: 172.
4 . The method of claim 1 , wherein the second amplification oligomer is
(A) a promoter primer;
(B) a promoter provider further comprising a promoter sequence located 5′ to the target-hybridizing sequence;
(C) a promoter provider further comprising a promoter sequence located 5′ to the target-hybridizing sequence wherein the promoter sequence is a T7 promoter sequence; or
(D) comprises a sequence selected from the group consisting of SEQ ID NOs: 57-59 and 62-77.
5 . The method of claim 1 , wherein the target-hybridizing sequences of the first and second amplification oligomers, respectively, are
(A) SEQ ID NO:30 and SEQ ID NO:82;
(B) SEQ ID NO:33 and SEQ ID NO:82;
(C) SEQ ID NO: 49 and SEQ ID NO: 82;
(D) SEQ ID NO:21 and SEQ ID NO:89;
(E) SEQ ID NO:30 and SEQ ID NO:89;
(F) SEQ ID NO:33 and SEQ ID NO:89;
(G) SEQ ID NO:49 and SEQ ID NO:89;
(H) SEQ ID NO:21 and SEQ ID NO:92;
(I) SEQ ID NO:30 and SEQ ID NO:92;
(J) SEQ ID NO:21 and SEQ ID NO:94;
(K) SEQ ID NO: 34 and SEQ ID NO: 94;
(L) SEQ ID NO:53 and SEQ ID NO:94;
(M) SEQ ID NO: 21 and SEQ ID NO: 95;
(N) SEQ ID NO:34 and SEQ ID NO:95; or
(O) SEQ ID NO:53 and SEQ ID NO:95.
6 . The method of claim 1 , wherein the at least one first amplification oligomer comprises at least two first amplification oligomers, wherein the at least two amplification oligomers are selected from the group consisting of:
(A) an amplification oligomer comprising a target-hybridizing sequence comprising the sequence of SEQ ID NO:167, or an amplification oligomer comprising a target-hybridizing sequence comprising the sequence of SEQ ID NO:34; and
(B) an amplification oligomer comprising a target-hybridizing sequence comprising the sequence of SEQ ID NO:168, an amplification oligomer comprising a target-hybridizing sequence comprising the sequence of SEQ ID NO:53, an amplification oligomer comprising a target-hybridizing sequence that is contained in the sequence of SEQ ID NO:164 and comprises the sequence of SEQ ID NO: 165, or an amplification oligomer comprising a target-hybridizing sequence consisting of SEQ ID NO:21.
7 . The method of claim 1 , wherein the at least one second amplification oligomer comprises at least two second amplification oligomers wherein the at least two second amplification oligomers are selected from the group consisting of: an amplification oligomer comprising a target-hybridizing sequence comprising the sequence of SEQ ID NO:94 and an amplification oligomer comprising a target-hybridizing sequence comprising the sequence of SEQ ID NO:95.
8 . The method of claim 1 , further comprising purifying the target nucleic acid from other components in the sample before step (1), wherein the purifying step comprises contacting the sample with at least one capture probe oligomer comprising a target-hybridizing sequence covalently attached to a sequence or moiety that binds to an immobilized probe, wherein said target-hybridizing sequence is up to about 30 contiguous nucleotides in length and comprises a sequence selected from the group consisting of SEQ ID NOs: 11-15, 17, 19, and 20, including DNA equivalents and DNA/RNA chimerics thereof.
9 . The method of claim 1 , wherein the at least one detection probe oligomer comprises a target-hybridizing sequence, wherein the detection probe oligomer target-hybridizing sequence
(A) is about 13 to about 40 nucleotides in length, is contained in the sequence of SEQ ID NO:196 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof, and comprises a sequence selected from the group consisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, and SEQ ID NO:178, including complements, DNA equivalents, and DNA/RNA equivalents thereof; or
(B) comprises a sequence selected from the group consisting of SEQ ID NOs: 131, 132, 135, 140, 145, 147-157, and 159-161, including complements, DNA equivalents, and DNA/RNA chimerics thereof;
whereby the presence or absence of the amplification product is determined, thereby indicating the presence or absence of Plasmodium species in said sample.
10 . The method of claim 9 , wherein the detection probe oligomer target-hybridizing sequence
(A) is contained in the sequence of SEQ ID NO: 197 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof, and comprises the sequence of SEQ ID NO:174 or SEQ ID NO:175, including complements, DNA equivalents, and DNA/RNA chimerics thereof;
(B) comprises a sequence selected from the group consisting of SEQ ID NOs: 131, 132, 135, 140, 147-157, 160, and 161, including complements, DNA equivalents, and DNA/RNA chimerics thereof, or
(C) comprises a sequence selected from the group consisting of SEQ ID NO:177 and SEQ ID NO:178, including complements, DNA equivalents, and DNA/RNA chimerics thereof.
11 . The method of claim 9 , wherein the at least one detection probe oligomer comprises a first detection probe oligomer and a second detection probe oligomer, wherein
(A) the first detection probe oligomer comprises a target-hybridizing sequence that is contained in the sequence of SEQ ID NO: 197 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof and comprises the sequence of SEQ ID NO:175 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof; or comprises the target-hybridizing sequence of SEQ ID NO: 157 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof; and
(B) the second detection probe oligomer comprises a target-hybridizing sequence that is contained in the sequence of SEQ ID NO: 197 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof and comprises the sequence of SEQ ID NO: 176 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof, or comprises a target-hybridizing sequence comprising a sequence selected from the group consisting of SEQ ID NO: 148 and SEQ ID NO: 152, including complements, DNA equivalents, and DNA/RNA chimerics thereof.
12 . The method of claim 1 , wherein the amplification reaction at step (2) is an isothermal amplification reaction; wherein the isothermal amplification reaction can be a transcription-mediated amplification (TMA) reaction; and wherein the isothermal amplification reaction or the TMA can be is a real-time amplification reaction.
13 . A combination of oligomers for determining the presence or absence of Plasmodium species in a sample, said oligomer combination comprising:
(1) at least two amplification oligomers for amplifying a target region of Plasmodium species target nucleic acid, wherein the at least two amplification oligomers comprise:
(a) at least one first amplification oligomer comprising a target-hybridizing sequence
(i) that is from about 14 to about 20 contiguous nucleotides in length, is contained in the sequence of SEQ ID NO:162, and comprises the sequence of SEQ ID NO:163; or
(ii) that is from about 14 to about 25 contiguous nucleotides in length, is contained in the sequence of SEQ ID NO:166, and comprises the sequence of SEQ ID NO: 167 or SEQ ID NO:168; and
(b) at least one second amplification oligomer comprising a target-hybridizing sequence that is from about 15 to about 33 contiguous nucleotides in length, is contained in SEQ ID NO: 169 and comprises the sequence of SEQ ID NO: 171, SEQ ID NO: 172, or SEQ ID NO: 173; and
(2) at least one detection probe oligomer configured to specifically hybridize to an amplification product of the at least two amplification oligomers, wherein the at least one detection probe oligomer comprises a fluorescent label, a chemiluminescent label, or a fluorescent label and a non-fluorescent quencher.
14 . The combination of claim 13 , wherein the target-hybridizing sequence of the at least one first amplification oligomer:
(A) comprises a sequence selected from the group consisting of SEQ ID NOs: 21, 23-25, 32, 33, 35, 54, and 55;
(B) is contained in the sequence of SEQ ID NO:164, and comprises the sequence of SEQ ID NO:165;
(C) comprises the sequence of SEQ ID NO:167
(D) comprises a sequence selected from the group consisting of SEQ ID NOs: 28-31, 34, 40, 41, and 49-51;
(E) comprises the sequence of SEQ ID NO:168; or
(F) comprises a sequence selected from the group consisting of SEQ ID NOs: 38, 39, 43, 44, and 53.
15 . The combination of claim 13 , wherein the target-hybridizing sequence of the at least one second amplification oligomer
(A) comprises a sequence selected from the group consisting of SEQ ID NOs: 80-82 and 85-100; or
(B) is contained in SEQ ID NO: 170, comprises the sequence of SEQ ID NO:171 or SEQ ID NO:172.
16 . The combination of claim 13 , wherein the second amplification oligomer is
(A) a promoter primer;
(B) a promoter provider further comprising a promoter sequence located 5′ to the target-hybridizing sequence;
(C) a promoter provider further comprising a promoter sequence located 5′ to the target-hybridizing sequence wherein the promoter sequence is a T7 promoter sequence; or
(D) comprises a sequence selected from the group consisting of SEQ ID NOs: 57-59 and 62-77.
17 . The combination of claim 13 , wherein the target-hybridizing sequences of the first and second amplification oligomers, respectively, are
(A) SEQ ID NO:30 and SEQ ID NO:82;
(B) SEQ ID NO:33 and SEQ ID NO:82;
(C) SEQ ID NO:49 and SEQ ID NO:82;
(D) SEQ ID NO:21 and SEQ ID NO:89
(E) SEQ ID NO:30 and SEQ ID NO:89;
(F) SEQ ID NO:33 and SEQ ID NO:89;
(G) SEQ ID NO:49 and SEQ ID NO:89;
(H) SEQ ID NO:21 and SEQ ID NO:92;
(I) SEQ ID NO:30 and SEQ ID NO:92;
(J) SEQ ID NO:21 and SEQ ID NO:94;
(K) SEQ ID NO:34 and SEQ ID NO:94;
(L) SEQ ID NO:53 and SEQ ID NO:94;
(M) SEQ ID NO:21 and SEQ ID NO:95;
(N) SEQ ID NO:34 and SEQ ID NO:95; or
(O) SEQ ID NO:53 and SEQ ID NO:95.
18 . The combination of claim 13 , wherein the at least one first amplification oligomer comprises at least two first amplification oligomers, wherein the at least two amplification oligomers are selected from the group consisting of:
(A) an amplification oligomer comprising a target-hybridizing sequence comprising the sequence of SEQ ID NO:167, or an amplification oligomer comprising a target-hybridizing sequence comprising the sequence of SEQ ID NO:34; and
(B) an amplification oligomer comprising a target-hybridizing sequence comprising the sequence of SEQ ID NO:168, an amplification oligomer comprising a target-hybridizing sequence comprising the sequence of SEQ ID NO:53, an amplification oligomer comprising a target-hybridizing sequence that is contained in the sequence of SEQ ID NO:164 and comprises the sequence of SEQ ID NO:165, or an amplification oligomer comprising a target-hybridizing sequence comprising the sequence of SEQ ID NO:21.
19 . The combination of claim 13 , wherein the at least one second amplification oligomer comprises at least two second amplification oligomers wherein the at least two second amplification oligomers are selected from the group consisting of: an amplification oligomer comprising a target-hybridizing sequence comprising the sequence of SEQ ID NO:94 and an amplification oligomer comprising a target-hybridizing sequence comprising the sequence of SEQ ID NO:95.
20 . The combination of claim 13 , further comprising at least one capture probe oligomer wherein the capture probe oligomer comprises a target-hybridizing sequence covalently attached to a sequence or moiety that binds to an immobilized probe, wherein said target-hybridizing sequence is up to about 30 contiguous nucleotides in length and comprises a sequence selected from the group consisting of SEQ ID NOs: 11-15, 17, 19, and 20, including DNA equivalents and DNA/RNA chimerics thereof.
21 . The combination of claim 13 , wherein the at least one detection probe oligomer comprises a target-hybridizing sequence, wherein the detection probe oligomer target-hybridizing sequence
(A) is about 13 to about 40 nucleotides in length, is contained in the sequence of SEQ ID NO:196 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof, and comprises a sequence selected from the group consisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, and SEQ ID NO:178, including complements, DNA equivalents, and DNA/RNA equivalents thereof, or
(B) comprises a sequence selected from the group consisting of SEQ ID NOs: 131, 132, 135, 140, 145, 147-157, and 159-161, including complements, DNA equivalents, and DNA/RNA chimerics thereof.
22 . The combination of claim 21 , wherein the detection probe oligomer target-hybridizing sequence
(A) is contained in the sequence of SEQ ID NO: 197 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof, and comprises a sequence selected from the group consisting of SEQ ID NO: 174 and SEQ ID NO:175, including complements, DNA equivalents, and DNA/RNA equivalents thereof;
(B) comprises a sequence selected from the group consisting of SEQ ID NOs: 131, 132, 135, 140, 147-157, 160, and 161, including complements, DNA equivalents, and DNA/RNA equivalents thereof; or
(C) comprises a sequence selected from the group consisting of SEQ ID NO:177 and SEQ ID NO:178, including complements, DNA equivalents, and DNA/RNA chimerics thereof.
23 . The combination of claim 21 , wherein the at least one detection probe oligomer comprises a first detection probe oligomer and a second detection probe oligomer, wherein
(A) the first detection probe oligomer comprises a target-hybridizing sequence that is contained in the sequence of SEQ ID NO: 197 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof and comprises the sequence of SEQ ID NO: 175 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof; or comprises the target-hybridizing sequence of SEQ ID NO:157 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof, and
(B) the second detection probe oligomer comprises a target-hybridizing sequence that is contained in the sequence of SEQ ID NO: 197 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof and comprises the sequence of SEQ ID NO: 176 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof, or comprises a target-hybridizing sequence comprising a sequence selected from the group consisting of SEQ ID NO: 148 and SEQ ID NO: 152, including complements, DNA equivalents, and DNA/RNA equivalents thereof.
24 . The combination of claim 13 , wherein the at least two oligomers are provided in a kit or a reaction mixture.