Compositions and methods for detecting PLASMODIUM species nucleic acids

Abstract

Nucleic acid oligomers, including amplification oligomers, detection probes, and capture probes, are disclosed for detecting Plasmodium species nucleic acids in a sample. Specific nucleic acid amplification and detection methods, including real-time amplification and detection of target nucleic acids using the disclosed oligomers, and corresponding reaction mixtures and kits are also disclosed.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 782,945, filed December 20, 2018, which is incorporated by reference in its entirety.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The said ASCII copy, created on October 28, 2019, is entitled "GDS_0110PC_20191028_Seq_Listing_ST25" and is 56,087 bytes in size. [Background technology]

[0003] Malaria is a serious disease caused by intraerythrocytic parasites of the genus Plasmodium. The parasites are transmitted by the bite of infected female Anopheles mosquitoes. There are five species of parasites that cause malaria in humans: P. falciparum, P. knowlesi, P. malariae, P. ovale, and P. vivax. According to the World Health Organization (WHO), there were 216 million cases of malaria worldwide in 2016, including 445,000 deaths.

[0004] The United States Food and Drug Administration (USFDA) has implemented strict blood screening guidelines to accept or defer blood donors who have traveled to malaria-endemic areas. Travelers are deferred for one year after travel to endemic areas, or three years if the donor is a former resident of an endemic area. Those diagnosed with malaria are deferred for three years after completing treatment and becoming symptom-free. There are no approved tests available in the United States to screen blood donations for Plasmodium. This requires careful screening of prospective donors by questionnaire.

[0005] In endemic countries, WHO recommends testing by thick smear or using a sensitive enzyme immunoassay. In non-endemic countries, WHO recommends that donors be deferred for 6 months from the last potential exposure, in combination with malaria antibody testing using a sensitive enzyme immunoassay. If there is no evidence of malaria antibodies, the donor may be returned. There is a risk that these methods are not effective in detecting low levels of parasitemia where transmission may occur.

[0006] Transfusion-transmitted malaria (TTM) has been documented in the United States. Between 1911 and 2015, 46 cases of TTM were reported, with the most recent case reported in 2018. To reduce the number of TTM and deferred donors, specific highly sensitive nucleic acid tests (NATs) for the detection of Plasmodium species in samples are needed. Summary of the Invention

[0007] In one aspect, the invention provides a method for specifically detecting Plasmodium species nucleic acid in a sample. The method generally comprises: (1) contacting a 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, (2) performing an in vitro nucleic acid amplification reaction in which any Plasmodium target nucleic acid present in the sample is used as a template to generate an amplification product, and (3) detecting the presence or absence of the amplification product, thereby indicating the presence or absence of the Plasmodium species target nucleic acid in the sample. In some embodiments, the at least two amplification oligomers include (a) an amplification oligomer (i) that comprises a target hybridizing sequence that is about 14 to about 20 contiguous nucleotides in length, that is contained in the sequence of SEQ ID NO:162 and that includes the sequence of SEQ ID NO:163, or (ii) that is about 14 to about 25 contiguous nucleotides in length, that is contained in the sequence of SEQ ID NO:166 and that includes SEQ ID NO:167 or SEQ ID NO:168, and (b) an amplification oligomer that comprises a target hybridizing sequence that is about 15 to about 33 contiguous nucleotides in length, that is contained in SEQ ID NO:169 and that includes the sequence of SEQ ID NO:171, SEQ ID NO:172, or SEQ ID NO:173. In other embodiments, the at least two amplification oligomers include (a') an amplification oligomer containing a target hybridizing sequence contained in the sequence of SEQ ID NO:185 and including the sequence of SEQ ID NO:37, SEQ ID NO:46, or SEQ ID NO:187, and (b') an amplification oligomer containing a target hybridizing sequence contained in the sequence of SEQ ID NO:188 and including the sequence of SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO:182.

[0008] In some embodiments of the above methods, where the at least two amplification oligomers comprise amplification oligomers (a)(i) and (b), the target hybridizing sequence of (a)(i) is selected from SEQ ID NOs: 21, 23-25, 32, 33, 35, 54, and 55. In other embodiments, the target hybridizing sequence of (a)(i) is contained within the sequence of SEQ ID NO: 164, including the sequence of SEQ ID NO: 165. In some such variations, the target hybridizing sequence of (a)(i) is selected from SEQ ID NOs: 21, 23-25, 32, 33, and 35.

[0009] In some embodiments of the above methods, where the at least two amplification oligomers comprise amplification oligomers (a)(ii) and (b), the target hybridizing sequence of (a)(ii) comprises the sequence of SEQ ID NO: 167. In some such variations, the target hybridizing sequence of (a)(ii) is selected from SEQ ID NOs: 28-31, 34, 40, 41, and 49-51. In other embodiments, the target hybridizing sequence of (a)(ii) comprises the sequence of SEQ ID NO: 168. In some such variations, the target hybridizing sequence of (a)(ii) is selected from SEQ ID NOs: 38, 39, 43, 44, and 53.

[0010] In some embodiments of the above method, where the at least two amplification oligomers include amplification oligomers (a) and (b), the target hybridization sequence of (b) is selected from SEQ ID NOs: 80-82 and 85-100. In other embodiments, the target hybridization sequence of (b) is contained in the sequence of SEQ ID NO: 170 and includes the sequence of SEQ ID NO: 171 or SEQ ID NO: 172. In some variations where the target hybridization sequence of (b) is contained in the sequence of SEQ ID NO: 170 and includes the sequence of SEQ ID NO: 171, the target hybridization sequence of (b) is selected from SEQ ID NOs: 81, 82, 85, 87-90, 94, and 96-98. In some variations where the target hybridization sequence of (b) is contained in the sequence of SEQ ID NO: 170 and includes the sequence of SEQ ID NO: 172, the target hybridization sequence of (b) is selected from SEQ ID NOs: 80, 82, 85, and 87-100.

[0011] In some embodiments of the above methods, where at least two amplification oligomers comprise amplification oligomers (a') and (b'), the target hybridizing sequence of (a') is selected from SEQ ID NO: 37, 46, 183, and 184. In other embodiments, the target hybridizing sequence of (a') is contained in the sequence of SEQ ID NO: 186. In some such variations, the target hybridizing sequence of (a') is SEQ ID NO: 183 or SEQ ID NO: 184. In certain embodiments, where at least two amplification oligomers comprise amplification oligomers (a') and (b'), the target hybridizing sequence of (b') is selected from SEQ ID NO: 83, 84, and 182. In more specific variations, the target hybridizing sequence of (a') is SEQ ID NO: 183 or SEQ ID NO: 184, and the target hybridizing sequence of (b') is SEQ ID NO: 182.

[0012] In some embodiments of the above method, the amplification oligomer of (b) or (b') is a promoter primer or promoter provider, respectively, further comprising a promoter sequence located 5' of the target hybridizing sequence of (b) or (b'). A particularly suitable promoter sequence is, for example, a T7 promoter sequence, such as SEQ ID NO: 179. In certain variations in which the amplification oligomer of (b) comprises a promoter sequence, the amplification oligomer of (b) comprises a sequence selected from SEQ ID NOs: 57-59 and 62-77. In certain variations in which the amplification oligomer of (b') comprises a promoter sequence, the amplification oligomer of (b') comprises a sequence selected from SEQ ID NOs: 60, 61, and 181.

[0013] Particularly preferred pairs of amplification oligomer target hybridizing sequences of (a) and (b) are, respectively, (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, and (O) SEQ ID NO:53 and SEQ ID NO:95. In some such embodiments, the amplification oligomer of (b) is a promoter primer or a promoter provider that further comprises a promoter sequence (e.g., a T7 promoter sequence such as SEQ ID NO: 179) located 5' to the target hybridizing sequence of (b).

[0014] In some embodiments of the above methods, in which the at least two amplification oligomers comprise amplification oligomers of (a) and (b), the at least two amplification oligomers comprise a first and a second amplification oligomer as in (a). In some such variations, the at least two amplification oligomers comprise a first and a second amplification oligomer as in (a)(ii). Particularly preferred first and second amplification oligomers of (a)(ii) comprise a first amplification oligomer that comprises a target hybridizing sequence comprising the sequence of SEQ ID NO: 167 (e.g., the target hybridizing sequence of SEQ ID NO: 34) and a second amplification oligomer that comprises a target hybridizing sequence comprising the sequence of SEQ ID NO: 168 (e.g., the target hybridizing sequence of SEQ ID NO: 53).

[0015] In other embodiments of the above methods, in which the at least two amplification oligomers comprise amplification oligomers of (a) and (b), the at least two amplification oligomers comprise an amplification oligomer as in (a)(i) and an amplification oligomer as in (a)(ii). In some such embodiments, the amplification oligomer as in (a)(i) is contained in the sequence of SEQ ID NO: 164 and comprises a target hybridizing sequence that comprises the sequence of SEQ ID NO: 165 (e.g., the hybridizing sequence of target SEQ ID NO: 21), and the amplification oligomer as in (a)(ii) comprises a target hybridizing sequence that comprises the sequence of SEQ ID NO: 167 (e.g., the target hybridizing sequence of SEQ ID NO: 34).

[0016] In some embodiments of the above method, in which the at least two amplification oligomers include amplification oligomers of (a) and (b), the at least two amplification oligomers include a first and a second amplification oligomer of (b). In some such embodiments, each of the first and second amplification oligomers of (b) is contained in SEQ ID NO: 170 and includes a target hybridization sequence that includes a sequence of SEQ ID NO: 171 or SEQ ID NO: 172 (e.g., a first amplification oligomer that includes a target hybridization sequence of SEQ ID NO: 94, and a second amplification oligomer that includes a target hybridization sequence of SEQ ID NO: 95). In some variations, each of the first and second amplification oligomers of (b) is a promoter primer or a promoter provider that further includes a promoter sequence located 5' of the target hybridization sequence of (b). A particularly suitable promoter sequence is, for example, a T7 promoter sequence, such as SEQ ID NO: 179. In a particular variation in which the first and second amplification oligomers of (b) each contain a promoter sequence, the first amplification oligomer of (b) contains the sequence of SEQ ID NO: 71 and the second amplification oligomer of (b) contains the sequence of SEQ ID NO: 72.

[0017] In certain embodiments of the method for detecting Plasmodium species nucleic acid in a sample as described above, the method further comprises purifying the target nucleic acid from other components in the sample prior to step (1). In some such variations, the purification 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 the immobilized probe, the target hybridizing sequence being configured to specifically hybridize to the Plasmodium species target nucleic acid. Particularly suitable capture probe oligomer target hybridizing sequences are up to about 30 contiguous nucleotides in length and include sequences selected from SEQ ID NOs: 11-15, 17, 19, and 20 (including DNA equivalents and DNA / RNA chimeras thereof). In more particular variations, the capture probe oligomer target hybridizing sequences are selected from SEQ ID NOs: 11-15, 17, 19, and 20 (including DNA equivalents and DNA / RNA chimeras thereof). In some embodiments, the purification step comprises contacting the sample with at least two capture probe oligomers (e.g., at least two capture probe oligomers as described above). In some such variations, the at least two capture probe oligomers comprise a first capture probe oligomer that includes a target hybridizing sequence of SEQ ID NO:19 and a second capture probe oligomer that includes a target hybridizing sequence of SEQ ID NO:20.

[0018] In some embodiments of the method for detecting Plasmodium species nucleic acid in a sample as described above, the detecting step (3) comprises contacting the in vitro nucleic acid amplification reaction with at least one detection probe oligomer comprising a target hybridizing sequence configured to specifically hybridize to an amplification product under conditions in which the presence or absence of the amplification product is determined, thereby indicating the presence or absence of the Plasmodium species in the sample. In some such embodiments in which the at least two amplification oligomers comprise amplification oligomers of (a) and (b), the detection probe oligomer target hybridizing sequence is about 13 to about 40 nucleotides in length and comprises (i) a sequence of SEQ ID NO: 196 or its complement, or a DNA equivalent or DNA / RNA chimera thereof, and (ii) a sequence selected from SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO: 178 (including complements thereof, DNA equivalents, and DNA / RNA equivalents). Suitable detection probe oligomer target hybridizing sequences include SEQ ID NOs: 131, 132, 135, 140, 145, 147-157, and 159-161 (including complements thereof, DNA equivalents, and DNA / RNA chimeras). In certain embodiments, the detection probe oligomer target hybridizing sequence is (i) contained in the sequence of SEQ ID NO: 197 or its complement, or its DNA equivalent or DNA / RNA chimera, and (ii) comprises the sequence of SEQ ID NO: 174 or SEQ ID NO: 175 (including complements thereof, DNA equivalents, and DNA / RNA chimeras). In some such variations including the sequence of SEQ ID NO: 174 or its complement, or its DNA equivalent or DNA / RNA chimera, the detection probe oligomer target hybridizing sequence is selected from SEQ ID NOs: 148-155 and 159 (including complements thereof, DNA equivalents, and DNA / RNA chimeras). In other such variations including SEQ ID NO: 175 or its complement, or its DNA equivalent or DNA / RNA chimera, the detection probe oligomer target hybridizing sequence is selected from SEQ ID NOs: 147, 156, 157, 160, and 161 (including their complements, DNA equivalents, and DNA / RNA chimeras).In certain embodiments, the detection probe oligomer target hybridizing sequence comprises (i) a sequence contained in SEQ ID NO: 196 or its complement, or a DNA equivalent or DNA / RNA chimera thereof, (ii) a sequence selected from SEQ ID NO: 177 and SEQ ID NO: 178 (including complements thereof, DNA equivalents, and DNA / RNA chimeras). In some such variations, the detection probe oligomer target hybridizing sequence is selected from SEQ ID NO: 131, 132, 135, 140, 147, 156, 157, 160, and 161 (including complements thereof, DNA equivalents, and DNA / RNA chimeras).

[0019] In other embodiments of the above method, where the detecting step (3) comprises contacting an in vitro nucleic acid amplification reaction with at least one detection probe oligomer, and where the at least two amplification oligomers comprise the amplification oligomers of (a') and (b'), the detection probe oligomer target hybridizing sequence is at least about 13 nucleotides in length and (i) is contained in the sequence of SEQ ID NO: 189 or its complement, or its DNA equivalent or DNA / RNA chimera, and (ii) comprises a sequence selected from SEQ ID NO: 190 and SEQ ID NO: 191 (including complements thereof, DNA equivalents, and DNA / RNA equivalents). In some such variations, the detection probe oligomer target hybridizing sequence is selected from SEQ ID NOs: 125-130 and 143 (including complements thereof, DNA equivalents, and DNA / RNA chimeras).

[0020] In certain variations of the method for detecting Plasmodium species nucleic acid in a sample as described above, where the detecting step (3) comprises contacting an in vitro nucleic acid amplification reaction with at least one detection probe oligomer, and where the at least two amplification oligomers comprise amplification oligomers of (a) and (b), the amplification oligomer target hybridizing sequence of (a), the amplification oligomer target hybridizing sequence of (b), and the detection probe oligomer target hybridizing sequence are, respectively, (A) SEQ ID NO: 30, SEQ ID NO: 82, and SEQ ID NO: 151, or a phase thereof. (B) SEQ ID NO:30, SEQ ID NO:82, and SEQ ID NO:157 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:157 or its complements; (C) SEQ ID NO:33, SEQ ID NO:82, and SEQ ID NO:155 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:155 or its complements; (D) SEQ ID NO:49, SEQ ID NO:82, and SEQ ID NO:150 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:150 or its complements. or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:49, SEQ ID NO:82, and SEQ ID NO:155 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:155 or its complement; (E) SEQ ID NO:49, SEQ ID NO:82, and SEQ ID NO:155 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:155 or its complement; (F) SEQ ID NO:21, SEQ ID NO:89, and SEQ ID NO:148 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:148 or its complement; (G) SEQ ID NO:21, SEQ ID NO:89, and SEQ ID NO:152 or their complements, or the DNA equivalent of SEQ ID NO:152 or its complements. or a DNA / RNA chimera, (H) SEQ ID NO:30, SEQ ID NO:89, and SEQ ID NO:148 or their complements, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:148 or its complement, (I) SEQ ID NO:30, SEQ ID NO:89, and SEQ ID NO:152 or their complements, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:152 or its complement, (I) SEQ ID NO:33, SEQ ID NO:89, and SEQ ID NO:158 or their complements, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:158 or its complement,(K) SEQ ID NO: 49, SEQ ID NO: 89, and SEQ ID NO: 150 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO: 150 or its complement; (L) SEQ ID NO: 21, SEQ ID NO: 92, and SEQ ID NO: 148 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO: 148 or its complement; (M) SEQ ID NO: 21, SEQ ID NO: 92, and SEQ ID NO: 152 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO: 152 or its complement; (N) SEQ ID NO: 30 , SEQ ID NO:92, and SEQ ID NO:148 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:148 or its complement; (O) SEQ ID NO:30, SEQ ID NO:92, and SEQ ID NO:152 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:152 or its complement; (P) SEQ ID NO:21, SEQ ID NO:94, and SEQ ID NO:148 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:148 or its complement; (Q) SEQ ID NO:21, SEQ ID NO:94, and and SEQ ID NO:152 or its complement, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:152 or its complement; (R) SEQ ID NO:34, SEQ ID NO:94, and SEQ ID NO:148 or its complement, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:148 or its complement; (S) SEQ ID NO:34, SEQ ID NO:94, and SEQ ID NO:152 or its complement, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:152 or its complement; (T) SEQ ID NO:34, SEQ ID NO:94, and SEQ ID NO:157. or a complement thereof, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:157 or its complement; (U) SEQ ID NO:53, SEQ ID NO:94, and SEQ ID NO:148 or a complement thereof, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:148 or its complement; (V) SEQ ID NO:53, SEQ ID NO:94, and SEQ ID NO:152 or a complement thereof, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:152 or its complement; (W) SEQ ID NO:53, SEQ ID NO:94, and SEQ ID NO:157 or a complement thereof;or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:157 or its complement; (X) SEQ ID NO:21, SEQ ID NO:95, and SEQ ID NO:148 or its complement, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:148 or its complement; (Y) SEQ ID NO:21, SEQ ID NO:95, and SEQ ID NO:152 or its complement, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:152 or its complement; (Z) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO:148 or its complement, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:148 or its complement; (AA) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO:152 or its complement, or the DNA equivalent of SEQ ID NO:152 or its complement. (AB) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO:157 or its complement, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:157 or its complement; (AC) SEQ ID NO:53, SEQ ID NO:95, and SEQ ID NO:148 or its complement, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:148 or its complement; (AD) SEQ ID NO:53, SEQ ID NO:95, and SEQ ID NO:152 or its complement, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:152 or its complement; or (AE) SEQ ID NO:53, SEQ ID NO:95, and SEQ ID NO:157 or its complement, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:157 or its complement.

[0021] In certain variations of the method for detecting Plasmodium species nucleic acid in a sample as described above, where the detecting step (3) comprises contacting an in vitro nucleic acid amplification reaction with at least one detection probe oligomer, and where the at least two amplification oligomers comprise amplification oligomers of (a') and (b'), the amplification oligomer target hybridizing sequence of (a'), the amplification oligomer target hybridizing sequence of (b'), and the detection probe oligomer target hybridizing sequence are, respectively, (A) SEQ ID NO:183, SEQ ID NO:182, and SEQ ID NO:126 or a complement thereof, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:126 or a complement thereof, (B) SEQ ID NO:183, SEQ ID NO:182, and SEQ ID NO:127 or a complement thereof, or SEQ ID NO:127 or a complement thereof, (C) SEQ ID NO:183, SEQ ID NO:182, and SEQ ID NO:128 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:128 or its complement; (D) SEQ ID NO:183, SEQ ID NO:182, and SEQ ID NO:143 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:143 or its complement; (E) SEQ ID NO:183, SEQ ID NO:182, and SEQ ID NO:129 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:129 or its complement; or (F) SEQ ID NO:184, SEQ ID NO:182, and SEQ ID NO:126 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:126 or its complement.

[0022] In some embodiments of the above methods utilizing at least one detection probe oligomer, the detection probe oligomer comprises a 2' methoxy modification of at least one of the nucleotide residue members of the detection probe oligomer nucleotide sequence.

[0023] In some embodiments of the above method utilizing at least one detection probe oligomer, the detection probe oligomer further comprises a detectable label, such as, for example, a fluorescent or chemiluminescent label. Particularly suitable chemiluminescent labels are chemiluminescent acridinium ester (AE) compounds linked between two nucleobases of the detection probe oligomer. In some embodiments including a detectably labeled probe oligomer, the detectable label is a fluorescent label and the detection probe oligomer further comprises a non-fluorescent quencher.

[0024] In some embodiments of the above methods, the detecting step (3) occurs during the amplifying step (2). In some such embodiments, the method utilizes a detection probe oligomer that includes a fluorescent label and a quencher (e.g., a molecular torch, a molecular beacon, or a TaqMan detection probe).

[0025] In some embodiments of the above methods utilizing at least one detection probe oligomer, the detection probe further comprises a non-target hybridizing sequence. In certain variations, the detection probe oligomer comprising the non-target hybridizing sequence is a hairpin detection probe, such as, for example, a molecular beacon or a molecular torch.

[0026] In certain embodiments, the method for detecting Plasmodium species nucleic acid in a sample as described above utilizes at least two detection probe oligomers. In some such embodiments, the at least two detection probe oligomers include a first and a second detection probe oligomer, (A) the first detection probe oligomer includes a target hybridization sequence that includes (i) the sequence of SEQ ID NO: 197 or its complement, or its DNA equivalent or DNA / RNA chimera, and (ii) the sequence of SEQ ID NO: 175 or its complement, or its DNA equivalent or DNA / RNA chimera, and (B) the second detection probe oligomer includes a target hybridization sequence that includes (i) the sequence of SEQ ID NO: 197 or its complement, or its DNA equivalent or DNA / RNA chimera, and (ii) the sequence of SEQ ID NO: 176 or its complement, or its DNA equivalent or DNA / RNA chimera. In a more specific variation, the first detection probe oligomer comprises a target hybridizing sequence of SEQ ID NO:157 or its complement, or a DNA equivalent or DNA / RNA chimera thereof, and the second detection probe oligomer comprises a target hybridizing sequence selected from SEQ ID NO:148 and SEQ ID NO:152 (including their complements, DNA equivalents, and DNA / RNA chimeras thereof).

[0027] In certain variations of the methods for detecting Plasmodium species nucleic acid in a sample as described above, the in vitro nucleic acid amplification reaction in step (2) is an isothermal amplification reaction (eg, a transcription-mediated amplification (TMA) reaction).

[0028] In a particular variant of the method for detecting Plasmodium species nucleic acid in a sample as described above, the amplification reaction is a real-time amplification reaction.

[0029] In some embodiments of the method for detecting Plasmodium species nucleic acid in a sample as described above, the sample is a clinical sample. In some embodiments, the sample is a blood sample, such as a red blood cell sample (e.g., a lysed blood cell sample or a lysed red blood cell sample).

[0030] In another aspect, the invention provides a combination of at least two oligomers for determining the presence or absence of a Plasmodium species in a sample. The combination of oligomers generally includes at least two oligomers for amplifying a target region of a Plasmodium species target nucleic acid. In some embodiments, the at least two amplification oligomers include (a) an amplification oligomer that includes a target hybridizing sequence that is about 14 to about 20 contiguous nucleotides in length, contained in the sequence of SEQ ID NO: 162 and includes the sequence of SEQ ID NO: 163, or (ii) is about 14 to about 25 contiguous nucleotides in length, contained in the sequence of SEQ ID NO: 166 and includes SEQ ID NO: 167 or SEQ ID NO: 168, and (b) an amplification oligomer that includes a target hybridizing sequence that is about 15 to about 33 contiguous nucleotides in length, contained in SEQ ID NO: 169 and includes the sequence of SEQ ID NO: 171, SEQ ID NO: 172, or SEQ ID NO: 173. In other embodiments, the at least two amplification oligomers include (a') an amplification oligomer containing a target hybridizing sequence contained in the sequence of SEQ ID NO:185 and including the sequence of SEQ ID NO:37, SEQ ID NO:46, or SEQ ID NO:187, and (b') an amplification oligomer containing a target hybridizing sequence contained in the sequence of SEQ ID NO:188 and including the sequence of SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO:182.

[0031] In some embodiments of the above oligomer combinations, where at least two amplification oligomers comprise amplification oligomers (a)(i) and (b), the target hybridizing sequence of (a)(i) is selected from SEQ ID NOs: 21, 23-25, 32, 33, 35, 54, and 55. In other embodiments, the target hybridizing sequence of (a)(i) is contained within the sequence of SEQ ID NO: 164, including the sequence of SEQ ID NO: 165. In some such variations, the target hybridizing sequence of (a)(i) is selected from SEQ ID NOs: 21, 23-25, 32, 33, and 35.

[0032] In some embodiments of the above oligomer combinations, where at least two amplification oligomers comprise amplification oligomers (a)(ii) and (b), the target hybridizing sequence of (a)(ii) comprises the sequence of SEQ ID NO: 167. In some such variations, the target hybridizing sequence of (a)(ii) is selected from SEQ ID NOs: 28-31, 34, 40, 41, and 49-51. In other embodiments, the target hybridizing sequence of (a)(ii) comprises the sequence of SEQ ID NO: 168. In some such variations, the target hybridizing sequence of (a)(ii) is selected from SEQ ID NOs: 38, 39, 43, 44, and 53.

[0033] In some embodiments of the above oligomer combinations, where at least two amplification oligomers comprise amplification oligomers (a) and (b), the target hybridizing sequence of (b) is selected from SEQ ID NOs: 80-82 and 85-100. In other embodiments, the target hybridizing sequence of (b) is contained in the sequence of SEQ ID NO: 170 and comprises the sequence of SEQ ID NO: 171 or SEQ ID NO: 172. In some variations where the target hybridizing sequence of (b) is contained in the sequence of SEQ ID NO: 170 and comprises the sequence of SEQ ID NO: 171, the target hybridizing sequence of (b) is selected from SEQ ID NOs: 81, 82, 85, 87-90, 94, and 96-98. In some variations where the target hybridizing sequence of (b) is contained in the sequence of SEQ ID NO: 170 and comprises the sequence of SEQ ID NO: 172, the target hybridizing sequence of (b) is selected from SEQ ID NOs: 80, 82, 85, and 87-100.

[0034] In some embodiments of the above oligomer combinations, where at least two amplification oligomers comprise amplification oligomers (a') and (b'), the target hybridizing sequence of (a') is selected from SEQ ID NO: 37, 46, 183, and 184. In other embodiments, the target hybridizing sequence of (a') is contained in the sequence of SEQ ID NO: 186. In some such variations, the target hybridizing sequence of (a') is SEQ ID NO: 183 or SEQ ID NO: 184. In certain embodiments, where at least two amplification oligomers comprise amplification oligomers (a') and (b'), the target hybridizing sequence of (b') is selected from SEQ ID NO: 83, 84, and 182. In more specific variations, the target hybridizing sequence of (a') is SEQ ID NO: 183 or SEQ ID NO: 184, and the target hybridizing sequence of (b') is SEQ ID NO: 182.

[0035] In some embodiments of the above oligomer combinations, the amplification oligomer (b) or (b') is a promoter primer or promoter provider, respectively, further comprising a promoter sequence located 5' of the target hybridizing sequence of (b) or (b'). A particularly suitable promoter sequence is, for example, a T7 promoter sequence, such as SEQ ID NO: 179. In certain variations in which the amplification oligomer (b) comprises a promoter sequence, the amplification oligomer (b) comprises a sequence selected from SEQ ID NOs: 57-59 and 62-77. In certain variations in which the amplification oligomer (b') comprises a promoter sequence, the amplification oligomer (b') comprises a sequence selected from SEQ ID NOs: 60, 61, and 181.

[0036] Particularly preferred pairs of amplification oligomer target hybridizing sequences of (a) and (b) are, respectively, (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, and (O) SEQ ID NO:53 and SEQ ID NO:95. In some such embodiments, the amplification oligomer of (b) is a promoter primer or a promoter provider that further comprises a promoter sequence (e.g., a T7 promoter sequence such as SEQ ID NO: 179) located 5' to the target hybridizing sequence of (b).

[0037] In some embodiments of the above oligomer combinations, where at least two amplification oligomers comprise amplification oligomers of (a) and (b), the at least two amplification oligomers comprise a first and a second amplification oligomer as in (a). In some such variations, the at least two amplification oligomers comprise a first and a second amplification oligomer as in (a)(ii). Particularly preferred first and second amplification oligomers of (a)(ii) comprise a first amplification oligomer comprising a target hybridizing sequence comprising the sequence of SEQ ID NO: 167 (e.g., the target hybridizing sequence of SEQ ID NO: 34) and a second amplification oligomer comprising a target hybridizing sequence comprising the sequence of SEQ ID NO: 168 (e.g., the target hybridizing sequence of SEQ ID NO: 53).

[0038] In other embodiments of the above oligomer combinations, where at least two amplification oligomers comprise amplification oligomers of (a) and (b), the at least two amplification oligomers comprise an amplification oligomer as in (a)(i) and an amplification oligomer as in (a)(ii). In some such embodiments, the amplification oligomer as in (a)(i) is contained in the sequence of SEQ ID NO: 164 and comprises a target hybridizing sequence comprising the sequence of SEQ ID NO: 165 (e.g., the hybridizing sequence of target SEQ ID NO: 21), and the amplification oligomer as in (a)(ii) comprises a target hybridizing sequence comprising the sequence of SEQ ID NO: 167 (e.g., the target hybridizing sequence of SEQ ID NO: 34).

[0039] In some embodiments of the above oligomer combinations, where at least two amplification oligomers include amplification oligomers of (a) and (b), the at least two amplification oligomers include a first and a second amplification oligomer of (b). In some such embodiments, each of the first and second amplification oligomers of (b) is contained in SEQ ID NO: 170 and includes a target hybridization sequence that includes a sequence of SEQ ID NO: 171 or SEQ ID NO: 172 (e.g., a first amplification oligomer that includes a target hybridization sequence of SEQ ID NO: 94, and a second amplification oligomer that includes a target hybridization sequence of SEQ ID NO: 95). In some variations, each of the first and second amplification oligomers of (b) is a promoter primer or promoter provider that further includes a promoter sequence located 5' of the target hybridization sequence of (b). A particularly suitable promoter sequence is, for example, a T7 promoter sequence, such as SEQ ID NO: 179. In a particular variation in which the first and second amplification oligomers of (b) each contain a promoter sequence, the first amplification oligomer of (b) contains the sequence of SEQ ID NO: 71 and the second amplification oligomer of (b) contains the sequence of SEQ ID NO: 72.

[0040] In certain embodiments of the oligomer combination for detecting Plasmodium species nucleic acid in a sample as described above, the oligomer combination further comprises at least one capture probe oligomer comprising a target hybridizing sequence covalently attached to a sequence or moiety that binds to the immobilized probe, the target hybridizing sequence being configured to specifically hybridize to the Plasmodium species target nucleic acid. Particularly preferred capture probe oligomer target hybridizing sequences are up to about 30 contiguous nucleotides in length and include sequences selected from SEQ ID NOs: 11-15, 17, 19, and 20 (including DNA equivalents and DNA / RNA chimeras thereof). In more specific variations, the capture probe oligomer target hybridizing sequences are selected from SEQ ID NOs: 11-15, 17, 19, and 20 (including DNA equivalents and DNA / RNA chimeras thereof). In some embodiments, the oligomer combination comprises at least two capture probe oligomers (e.g., at least two capture probe oligomers as described above). In some such variations, the at least two capture probe oligomers include a first capture probe oligomer that includes a target hybridizing sequence of SEQ ID NO:19 and a second capture probe oligomer that includes a target hybridizing sequence of SEQ ID NO:20.

[0041] In some embodiments of the oligomer combination for detecting Plasmodium species nucleic acid in a sample as described above, the oligomer combination further comprises at least one detection probe oligomer comprising a target hybridizing sequence configured to specifically hybridize to a Plasmodium species amplicon amplifiable by at least two amplification oligomers. In some such embodiments, where the at least two amplification oligomers comprise amplification oligomers (a) and (b), the detection probe oligomer target hybridizing sequence is about 13 to about 40 nucleotides in length and comprises (i) a sequence of SEQ ID NO: 196 or its complement, or a DNA equivalent or a DNA / RNA chimera thereof, or (ii) a sequence selected from SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO: 178 (including complements thereof, DNA equivalents, and DNA / RNA equivalents). Suitable detection probe oligomer target hybridizing sequences include SEQ ID NOs: 131, 132, 135, 140, 145, 147-157, and 159-161 (including complements thereof, DNA equivalents, and DNA / RNA chimeras). In certain embodiments, the detection probe oligomer target hybridizing sequence is (i) contained in the sequence of SEQ ID NO: 197 or its complement, or its DNA equivalent or DNA / RNA chimera, and (ii) comprises the sequence of SEQ ID NO: 174 or SEQ ID NO: 175 (including complements thereof, DNA equivalents, and DNA / RNA chimeras). In some such variations including the sequence of SEQ ID NO: 174 or its complement, or its DNA equivalent or DNA / RNA chimera, the detection probe oligomer target hybridizing sequence is selected from SEQ ID NOs: 148-155 and 159 (including complements thereof, DNA equivalents, and DNA / RNA chimeras). In other such variations including SEQ ID NO: 175 or its complement, or its DNA equivalent or DNA / RNA chimera, the detection probe oligomer target hybridizing sequence is selected from SEQ ID NOs: 147, 156, 157, 160, and 161 (including their complements, DNA equivalents, and DNA / RNA chimeras).In certain embodiments, the detection probe oligomer target hybridizing sequence comprises (i) a sequence contained in SEQ ID NO: 196 or its complement, or a DNA equivalent or DNA / RNA chimera thereof, (ii) a sequence selected from SEQ ID NO: 177 and SEQ ID NO: 178 (including complements thereof, DNA equivalents, and DNA / RNA chimeras). In some such variations, the detection probe oligomer target hybridizing sequence is selected from SEQ ID NO: 131, 132, 135, 140, 147, 156, 157, 160, and 161 (including complements thereof, DNA equivalents, and DNA / RNA chimeras).

[0042] In other embodiments of the above oligomer combinations further comprising at least one detection probe oligomer and at least two amplification oligomers comprising amplification oligomers (a') and (b'), the detection probe oligomer target hybridizing sequence is at least about 13 nucleotides in length and (i) is contained in the sequence of SEQ ID NO: 189 or its complement, or its DNA equivalent or DNA / RNA chimera, and (ii) comprises a sequence selected from SEQ ID NO: 190 and SEQ ID NO: 191 (including complements, DNA equivalents, and DNA / RNA equivalents thereof). In some such variations, the detection probe oligomer target hybridizing sequence is selected from SEQ ID NOs: 125-130 and 143 (including complements, DNA equivalents, and DNA / RNA chimeras thereof).

[0043] In certain variations of the above oligomer combinations further comprising at least one detection probe oligomer, wherein at least two amplification oligomers comprise amplification oligomers of (a) and (b), the amplification oligomer target hybridizing sequence of (a), the amplification oligomer target hybridizing sequence of (b), and the detection probe oligomer target hybridizing sequence are, respectively, (A) SEQ ID NO:30, SEQ ID NO:82, and SEQ ID NO:151 or a complement thereof, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:151 or a complement thereof; (B) SEQ ID NO:30, SEQ ID NO:82, and SEQ ID NO:157 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO:157 or its complement; (C) SEQ ID NO:33, SEQ ID NO:82, and SEQ ID NO:155 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO:155 or its complements; (D) SEQ ID NO:49, SEQ ID NO:82, and SEQ ID NO:150 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO:150 or its complements; (E) SEQ ID NO:49, SEQ ID NO: (F) SEQ ID NO:21, SEQ ID NO:89, and SEQ ID NO:148 or its complement, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:148 or its complement; (G) SEQ ID NO:21, SEQ ID NO:89, and SEQ ID NO:152 or its complement, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:152 or its complement; (H) SEQ ID NO:30, SEQ ID NO:89, and SEQ ID NO:1 48 or its complement, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:148 or its complement, (I) SEQ ID NO:30, SEQ ID NO:89, and SEQ ID NO:152 or its complement, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:152 or its complement, (I) SEQ ID NO:33, SEQ ID NO:89, and SEQ ID NO:158 or its complement, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:158 or its complement, (K) SEQ ID NO:49, SEQ ID NO:89, and SEQ ID NO:150 or its complement,or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:150 or its complement; (L) SEQ ID NO:21, SEQ ID NO:92, and SEQ ID NO:148 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:148 or its complement; (M) SEQ ID NO:21, SEQ ID NO:92, and SEQ ID NO:152 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:152 or its complement; (N) SEQ ID NO:30, SEQ ID NO:92, and SEQ ID NO:148 or its complement, or SEQ ID NO:148 or (O) SEQ ID NO:30, SEQ ID NO:92, and SEQ ID NO:152 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:152 or its complement; (P) SEQ ID NO:21, SEQ ID NO:94, and SEQ ID NO:148 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:148 or its complement; (Q) SEQ ID NO:21, SEQ ID NO:94, and SEQ ID NO:152 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:152 or its complements. (R) SEQ ID NO:34, SEQ ID NO:94, and SEQ ID NO:148 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:148 or its complement; (S) SEQ ID NO:34, SEQ ID NO:94, and SEQ ID NO:152 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:152 or its complement; (T) SEQ ID NO:34, SEQ ID NO:94, and SEQ ID NO:157 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:157 or its complement; (U) ) SEQ ID NO:53, SEQ ID NO:94, and SEQ ID NO:148 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:148 or its complement, (V) SEQ ID NO:53, SEQ ID NO:94, and SEQ ID NO:152 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:152 or its complement, (W) SEQ ID NO:53, SEQ ID NO:94, and SEQ ID NO:157 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:157 or its complement, (X) SEQ ID NO:21, SEQ ID NO:95,and SEQ ID NO:148 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:148 or its complement; (Y) SEQ ID NO:21, SEQ ID NO:95, and SEQ ID NO:152 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:152 or its complement; (Z) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO:148 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:148 or its complement; (AA) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO:152 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:152 or its complement; (AB) SEQ ID NO:34, sequence No. 95, and SEQ ID NO: 157 or its complement, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO: 157 or its complement; (AC) SEQ ID NO: 53, SEQ ID NO: 95, and SEQ ID NO: 148 or its complement, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO: 148 or its complement; (AD) SEQ ID NO: 53, SEQ ID NO: 95, and SEQ ID NO: 152 or its complement, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO: 152 or its complement; or (AE) SEQ ID NO: 53, SEQ ID NO: 95, and SEQ ID NO: 157 or its complement, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO: 157 or its complement.

[0044] In certain variations of the above oligomer combinations further comprising at least one detection probe oligomer, wherein at least two amplification oligomers comprise amplification oligomers of (a') and (b'), the amplification oligomer target hybridizing sequence of (a'), the amplification oligomer target hybridizing sequence of (b'), and the detection probe oligomer target hybridizing sequence are, respectively: (A) SEQ ID NO:183, SEQ ID NO:182, and SEQ ID NO:126 or a complement thereof, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:126 or a complement thereof; (B) SEQ ID NO:183, SEQ ID NO:182, and SEQ ID NO:127 or a complement thereof, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO:127 or a complement thereof. (C) SEQ ID NO:183, SEQ ID NO:182, and SEQ ID NO:128 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:128 or its complement; (D) SEQ ID NO:183, SEQ ID NO:182, and SEQ ID NO:143 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:143 or its complement; (E) SEQ ID NO:183, SEQ ID NO:182, and SEQ ID NO:129 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:129 or its complement; or (F) SEQ ID NO:184, SEQ ID NO:182, and SEQ ID NO:126 or their complements, or the DNA equivalent or DNA / RNA chimera of SEQ ID NO:126 or its complement.

[0045] In some embodiments of the above oligomer combinations further comprising at least one detection probe oligomer, the detection probe oligomer comprises a 2' methoxy modification of at least one of the nucleotide residue members of the detection probe oligomer nucleotide sequence.

[0046] In some embodiments of the above oligomer combinations further comprising at least one detection probe oligomer, the detection probe oligomer further comprises a detectable label, such as, for example, a fluorescent or chemiluminescent label.A particularly suitable chemiluminescent label is a chemiluminescent acridinium ester (AE) compound linked between two nucleobases of the detection probe oligomer.In some embodiments comprising a detectably labeled probe oligomer, the detectable label is a fluorescent label, and the detection probe oligomer further comprises a non-fluorescent quencher, and particularly suitable detection probe oligomers comprise a fluorescent label and a quencher, including molecular torches, molecular beacons, and TaqMan detection probes.

[0047] In some embodiments of the above oligomer combinations further comprising at least one detection probe oligomer, the detection probe further comprises a non-target hybridizing sequence. In certain variations, the detection probe oligomer comprising the non-target hybridizing sequence is a hairpin detection probe, such as, for example, a molecular beacon or a molecular torch.

[0048] In certain embodiments, the oligomer combination for detecting Plasmodium species nucleic acid in a sample as described above comprises at least two detection probe oligomers. In some such embodiments, the at least two detection probe oligomers comprise a first and a second detection probe oligomer, (A) the first detection probe oligomer comprises a target hybridization sequence comprising (i) the sequence of SEQ ID NO: 197 or its complement, or its DNA equivalent or DNA / RNA chimera, (ii) the sequence of SEQ ID NO: 175 or its complement, or its DNA equivalent or DNA / RNA chimera, and (B) the second detection probe oligomer comprises a target hybridization sequence comprising (i) the sequence of SEQ ID NO: 197 or its complement, or its DNA equivalent or DNA / RNA chimera, and (ii) the sequence of SEQ ID NO: 176 or its complement, or its DNA equivalent or DNA / RNA chimera. In a more specific variation, the first detection probe oligomer comprises a target hybridizing sequence of SEQ ID NO:157 or its complement, or a DNA equivalent or DNA / RNA chimera thereof, and the second detection probe oligomer comprises a target hybridizing sequence selected from SEQ ID NO:148 and SEQ ID NO:152 (including their complements, DNA equivalents, and DNA / RNA chimeras thereof).

[0049] In another aspect, the present invention provides the use of a combination of at least two oligomers as described above for specifically amplifying Plasmodium species nucleic acid in a sample.

[0050] In another aspect, the invention provides detection probe oligomers for specifically detecting a Plasmodium species target nucleic acid in a sample. In some embodiments, the detection probe oligomer comprises a target hybridizing sequence that is about 13 to about 40 nucleotides in length and is configured to specifically hybridize to a target sequence contained within a Plasmodium species target region amplifiable by a combination of oligomers comprising a first and a second Plasmodium-specific amplification oligomer, wherein (a) the first amplification oligomer comprises a target hybridizing sequence that is (i) about 14 to about 20 contiguous nucleotides in length and contained in the sequence of SEQ ID NO: 162 and comprises the sequence of SEQ ID NO: 163, or (ii) about 14 to about 25 contiguous nucleotides in length and 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) the second amplification oligomer comprises a target hybridizing sequence that is about 15 to about 33 contiguous nucleotides in length and contained in SEQ ID NO: 169 and comprises the sequence of SEQ ID NO: 171, SEQ ID NO: 172, or SEQ ID NO: 173. In some such embodiments, the detection probe oligomer target hybridizing sequence (i) comprises the sequence of SEQ ID NO: 196 or its complement, or a DNA equivalent or DNA / RNA chimera thereof, and (ii) comprises a sequence selected from SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO: 178 (including complements, DNA equivalents, and DNA / RNA chimeras thereof). Suitable detection probe oligomer target hybridizing sequences include SEQ ID NOs: 131, 132, 135, 140, 145, 147-157, and 159-161 (including complements, DNA equivalents, and DNA / RNA chimeras thereof). In certain embodiments, the detection probe oligomer target hybridizing sequence (i) comprises the sequence of SEQ ID NO: 197 or its complement, or a DNA equivalent or DNA / RNA chimera thereof, and (ii) comprises the sequence of SEQ ID NO: 174 or SEQ ID NO: 175 (including complements, DNA equivalents, and DNA / RNA chimeras thereof).In some such variations, including the sequence of SEQ ID NO: 174 or its complement, or its DNA equivalent or DNA / RNA chimera, the detection probe oligomer target hybridizing sequence is selected from SEQ ID NOs: 148-155 and 159, including their complements, DNA equivalents, and DNA / RNA chimeras. In other such variations, including SEQ ID NO: 175 or its complement, or its DNA equivalent or DNA / RNA chimera, the detection probe oligomer target hybridizing sequence is selected from SEQ ID NOs: 147, 156, 157, 160, and 161, including their complements, DNA equivalents, and DNA / RNA chimeras. In certain embodiments, the detection probe oligomer target hybridizing sequence (i) is contained in the sequence of SEQ ID NO: 196 or its complement, or its DNA equivalent or DNA / RNA chimera, and (ii) comprises a sequence selected from SEQ ID NO: 177 and SEQ ID NO: 178, including their complements, DNA equivalents, and DNA / RNA chimeras. In some such variations, the detection probe oligomer target hybridizing sequences are selected from SEQ ID NOs: 131, 132, 135, 140, 147, 156, 157, 160, and 161 (including complements, DNA equivalents, and DNA / RNA chimeras thereof).

[0051] In other embodiments of detection probe oligomers for specifically detecting a Plasmodium species target nucleic acid in a sample, the detection probe oligomer comprises a target hybridizing sequence that is at least about 13 nucleotides in length and is configured to specifically hybridize to a target sequence contained within a Plasmodium species target region amplifiable by a combination of oligomers comprising a first and a second Plasmodium-specific amplification oligomer, where (a) the first amplification oligomer comprises a target hybridizing sequence contained in the sequence of SEQ ID NO:185 and comprises the sequence of SEQ ID NO:37, SEQ ID NO:46, or SEQ ID NO:187, and (b) the second amplification oligomer comprises a target hybridizing sequence contained in SEQ ID NO:188 and comprises the sequence of SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO:182. In some such embodiments, the detection probe oligomer target hybridizing sequence comprises (i) a sequence contained in SEQ ID NO: 189 or its complement, or a DNA equivalent or DNA / RNA chimera thereof, or (ii) a sequence selected from SEQ ID NO: 190 and SEQ ID NO: 191 (including complements, DNA equivalents, and DNA / RNA equivalents thereof). In more specific variations, the detection probe oligomer target hybridizing sequence is selected from SEQ ID NOs: 125-130 and 143 (including complements, DNA equivalents, and DNA / RNA chimeras thereof).

[0052] In some embodiments of the above detection probe oligomers, the detection probe oligomer comprises a 2' methoxy modification of at least one of the nucleotide residue members of the detection probe oligomer nucleotide sequence.

[0053] In some embodiments of the above detection probe oligomer, the detection probe oligomer further comprises a detectable label, such as, for example, a fluorescent or chemiluminescent label.A particularly suitable chemiluminescent label is a chemiluminescent acridinium ester (AE) compound bound between two nucleic acid bases of the detection probe oligomer.In some embodiments that comprise a detectable label, the detectable label is a fluorescent label, and the detection probe oligomer further comprises a non-fluorescent quencher, and particularly suitable detection probe oligomers comprise a fluorescent label and a quencher, including molecular torch, molecular beacon, and TaqMan detection probe.

[0054] In some embodiments of the above detection probe oligomers, the detection probe further comprises a non-target hybridizing sequence. In certain variations, the detection probe oligomer that comprises a non-target hybridizing sequence is a hairpin detection probe, such as, for example, a molecular beacon or a molecular torch.

[0055] In another aspect, the present invention provides a combination of at least two oligomers for detecting Plasmodium species target nucleic acid in a sample, the combination of oligomers comprising at least two detection probe oligomers as described above. In some embodiments, the at least two detection probe oligomers comprise: (A) a first detection probe oligomer comprising (i) a target hybridization sequence comprising the sequence of SEQ ID NO: 197 or its complement, or its DNA equivalent or DNA / RNA chimera, and (ii) a target hybridization sequence comprising the sequence of SEQ ID NO: 175 or its complement, or its DNA equivalent or DNA / RNA chimera; and (B) a second detection probe oligomer comprising (i) a target hybridization sequence comprising the sequence of SEQ ID NO: 197 or its complement, or its DNA equivalent or DNA / RNA chimera, and (ii) a target hybridization sequence comprising the sequence of SEQ ID NO: 176 or its complement, or its DNA equivalent or DNA / RNA chimera. In a more specific variation, the first detection probe oligomer comprises a target hybridizing sequence of SEQ ID NO:157 or its complement, or a DNA equivalent or DNA / RNA chimera thereof, and the second detection probe oligomer comprises a target hybridizing sequence selected from SEQ ID NO:148 and SEQ ID NO:152 (including their complements, DNA equivalents, and DNA / RNA chimeras thereof).

[0056] In another aspect, the present invention provides the use of a detection probe oligomer or combination of oligomers described above for specifically detecting Plasmodium species nucleic acid in a sample.

[0057] In another aspect, the invention provides capture probe oligomers for specifically isolating Plasmodium species nucleic acids from a sample. In some embodiments, the capture probe oligomer comprises a target hybridizing sequence covalently attached to a sequence or moiety that binds to an immobilized probe, the target hybridizing sequence being up to about 30 contiguous nucleotides in length and comprising a sequence selected from SEQ ID NOs: 11-15, 17, 19, and 20 (including DNA equivalents and DNA / RNA chimeras thereof). In more particular variations, the capture probe oligomer target hybridizing sequence is selected from SEQ ID NOs: 11-15, 17, 19, and 20 (including DNA equivalents and DNA / RNA chimeras thereof).

[0058] In another aspect, the present invention provides a combination of at least two oligomers for specifically isolating Plasmodium species nucleic acids from a sample, the combination of oligomers comprising at least two capture probe oligomers as described above. In some embodiments, the at least two capture probe oligomers comprise a first capture probe oligomer comprising a target hybridizing sequence of SEQ ID NO: 19, or a DNA equivalent or a DNA / RNA chimera thereof, and a second capture probe oligomer comprising a hybridizing sequence of SEQ ID NO: 20, or a DNA equivalent or a DNA / RNA chimera thereof.

[0059] In another aspect, the invention provides the use of a capture probe oligomer or combination of oligomers described above for specifically capturing Plasmodium species nucleic acids from a sample.

[0060] In another aspect, the present invention provides a kit comprising a combination of at least two oligomers as described above.

[0061] In another aspect, the present invention provides a reaction mixture comprising a combination of at least two oligomers as described above.

[0062] These and other aspects of the present invention will become evident upon reference to the following detailed description of the invention and the accompanying drawings.

[0063] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art related to the methods and compositions described. As used herein, the following terms and phrases have the meanings ascribed to them unless otherwise specified.

[0064] The terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0065] A "sample" includes any specimen that may contain or is suspected of containing a Plasmodium species or a component thereof (such as a nucleic acid or a fragment of a Plasmodium nucleic acid). A sample may be an isolated sample. A sample includes a "biological sample", which includes any tissue or material from a living or dead human that may contain a Plasmodium parasite or a component thereof (e.g., a target nucleic acid derived therefrom), including, for example, blood, peripheral blood, and red blood cells. The use of other sample types that may contain a Plasmodium parasite or a component thereof (e.g., a target nucleic acid derived therefrom), such as plasma, serum, lymph nodes, gastrointestinal tissue, feces, urine, semen, or other bodily fluids or materials, is also contemplated. A biological sample may be treated to physically or mechanically disrupt tissue or cell structures, thus releasing intracellular components into solutions that may further contain enzymes, buffers, salts, detergents, etc., that are used to prepare the biological sample for analysis using standard methods. For example, the sample may be treated with a cell lysis reagent, such as the lysis reagents described in U.S. Pat. No. 10,093,989 or PCT Publication No. WO2017 / 189746, each of which is incorporated herein by reference. Samples may also include treated samples, such as those obtained from passing a sample over or through a filtration device, or after centrifugation, or by attachment to a medium, matrix, or support.

[0066] "Nucleic acid" refers to a polymeric compound containing two or more covalently linked nucleosides or nucleoside analogs with nitrogen-containing heterocyclic bases, or base analogs, where the nucleosides are linked together by phosphodiester bonds or other linkages to form a polynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNA polymers, or oligonucleotides, and analogs thereof. The nucleic acid "backbone" can be composed of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid linkages (as in "peptide nucleic acid" or PNA, see PCT Publication No. WO95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar moiety of the nucleic acid can be either ribose or deoxyribose, or similar compounds with known substitutions, such as 2' methoxy and 2' halide substitutions (e.g., 2'-F). Nitrogenous bases include the conventional bases (A, G, C, T, U), their analogs (e.g., inosine, 5-methylisocytosine, isoguanine, The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992, BioTechniques (2007) 43:617-24), which include derivatives of purine or pyrimidine bases (e.g., N-methyldeoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases having substituents at the 5- or 6-positions, purine bases having modified or replaced substituents at the 2-, 6-, and / or 8-positions, such as 2-amino-6-methylaminopurine, 0-methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine, and 0-alkyl-pyrimidines, as well as pyrazolo compounds, such as unsubstituted or 3-substituted pyrazolo[3,4-d]pyrimidines, U.S. Pat. Nos. 5,378,825, 6,949,367, and PCT Publication No. WO 93 / 13121). Nucleic acids may contain "non-basic" residues, in which the backbone does not contain one or more nitrogenous bases (US Pat. No. 5,585,481).Nucleic acids may contain only conventional sugars, bases, and linkages found in RNA and DNA, or may contain conventional components and substitutions (e.g., conventional bases linked by a 2' methoxy backbone, or nucleic acids containing a mixture of conventional bases and one or more base analogs). Nucleic acids may include "locked nucleic acids" (LNAs), in which one or more nucleotide monomers have bicyclic furanose units locked into an RNA-mimicking sugar conformation, which enhances hybridization affinity to complementary sequences of single-stranded RNA (ssRNA), single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA) (Biochemistry (2004) 43:13233-41). Nucleic acids may contain modified bases that change the function or behavior of the nucleic acid, for example, the addition of a 3' terminal dideoxyribonucleotide that blocks additional nucleotides from being added to the nucleic acid. Synthetic methods for making nucleic acids in vitro are well known in the art.

[0067] As used herein, the term "polynucleotide" refers to a nucleic acid strand. Throughout this application, nucleic acids are referred to from the 5' to the 3' end. Standard nucleic acids, such as DNA and RNA, are typically synthesized "5' to 3'", i.e., by the addition of nucleotides to the 3' end of a growing nucleic acid.

[0068] As used herein, a "nucleotide" is a subunit of a nucleic acid consisting of a phosphate group, a five-carbon sugar, and a nitrogenous base. The five-carbon sugar found in RNA is ribose. In DNA, the five-carbon sugar is 2'-deoxyribose. The term also includes analogs of such subunits, such as a methoxy group at the 2' position of ribose (2'-0-Me).

[0069] As used herein, a "nucleic acid-based detection assay" is an assay for detecting a target sequence within a target nucleic acid and utilizing another oligonucleotide that specifically hybridizes to the target sequence.

[0070] In certain embodiments, nucleic acid-based detection assay is an "amplification-based assay", i.e., an assay that utilizes one or more steps to amplify nucleic acid target sequence.Various amplification methods for use in detection assay are known in the art, some of which are further summarized herein.For clarity, amplification-based assay may include one or more steps that do not amplify target sequence, such as the steps used in non-amplification-based assay methods (e.g., hybridization assay or cleavage-based assay).

[0071] In other embodiments, the nucleic acid-based detection assay is a "non-amplification-based assay", i.e., an assay that does not rely on any step to amplify the nucleic acid target sequence. For clarity, a nucleic acid-based detection assay that includes a reaction for primer extension in the absence of a corresponding downstream amplification oligomer (e.g., primer extension by reverse transcriptase to generate an RNA:DNA duplex, followed by RNase digestion of the RNA to obtain a single-stranded cDNA complementary to the RNA target, but does not generate a copy of the cDNA) is understood to be a non-amplification-based assay.

[0072] An exemplary non-amplification-based assay is "cleavage-based assay", which is an assay that relies on the specific cleavage of the linear double-stranded break structure formed by the specific hybridization of overlapping oligonucleotides to target nucleic acid by flap endonuclease.In these assays, the probe oligonucleotide containing non-target hybridizing flap region is cleaved by flap endonuclease in an overlap-dependent manner to release the cleavage product, which is then detected.The principle of cleavage-based assay is well known in the art, and exemplary assays are described in, for example, Nat.Biotechnol.(1999)17:292-296, Mol.Diagn.(1999)4:135-144, J.Clin.Microbiol.(2006)44:3443-3447, and U.S. Patent Nos. 5,846,717, 6,706,471, and 5,614,402. Cleavage-based assays include, for example, the commercially available Invader® assay (Hologic, Inc., Madison, Wis.).

[0073] As used herein, "target nucleic acid" refers to the nucleic acid that contains the target sequence to be detected. Target nucleic acid may be DNA or RNA as described herein, and may be either single-stranded or double-stranded. Target nucleic acid may also contain other sequences other than the target sequence.

[0074] By "isolated" it is meant that the sample containing the target nucleic acid is removed from its natural environment, although the term does not imply any degree of purification.

[0075] As used herein, the term "target sequence" refers to a specific nucleotide sequence of a target nucleic acid to be detected. "Target sequence" includes a complexing sequence with which an oligonucleotide (e.g., a probe oligonucleotide, a priming oligonucleotide, and / or a promoter oligonucleotide) forms a complex during a detection process (e.g., an amplification-based detection assay such as TMA or PCR, or a non-amplification-based detection assay such as a cleavage-based assay). If the target nucleic acid is originally single-stranded, the term "target sequence" also refers to a sequence complementary to the "target sequence" as present in the target nucleic acid. If the target nucleic acid is originally double-stranded, the term "target sequence" refers to both the sense (+) strand and the antisense (-) strand. In selecting a target sequence, those skilled in the art will understand that a "unique" sequence should be selected to distinguish unrelated or closely related target nucleic acids.

[0076] "Target hybridizing sequence" is used herein to refer to the portion of an oligomer that is configured to hybridize with a target nucleic acid sequence. Preferably, the target hybridizing sequence is configured to specifically hybridize with a target nucleic acid sequence. Target hybridizing sequences may be, but are not necessarily, 100% complementary to the portion of the target sequence to which they are configured to hybridize. Target hybridizing sequences may also include nucleotide residues that are inserted, deleted, and / or substituted relative to the target sequence. Less than 100% complementarity of the target hybridizing sequence to the target sequence may occur when the target nucleic acid is multiple strains within a species, such as in the case of oligomers configured to hybridize to various strains of Plasmodium. It is understood that there are other reasons to configure a target hybridizing sequence to have less than 100% complementarity to the target nucleic acid.

[0077] The term "targeting a sequence" as used herein with respect to a region of a Plasmodium species nucleic acid refers to the process by which an oligonucleotide hybridizes to a target sequence in a manner that allows for detection as described herein. In one embodiment, the oligonucleotide is complementary to the target Plasmodium species nucleic acid sequence and does not contain mismatches. In another embodiment, the oligonucleotide is complementary but contains 1, 2, 3, 4, or 5 mismatches with the target Plasmodium species nucleic acid sequence. Preferably, the oligonucleotide that hybridizes to the target nucleic acid sequence contains at least 10 to as many as 50 nucleotides that are complementary to the target sequence. At least 10 to 50 is understood to be an inclusive range such that 10, 50, and each integer therebetween are included. Preferably, the oligomer specifically hybridizes to the target sequence.

[0078] The term "configured to" refers to the actual arrangement of the polynucleotide sequence composition of a referenced oligonucleotide target hybridizing sequence. For example, an oligonucleotide configured to specifically hybridize to a target sequence has a polynucleotide sequence that specifically hybridizes to the referenced sequence under stringent hybridization conditions.

[0079] As used herein, the term "configured to specifically hybridize to" means that the target hybridizing region of the oligonucleotide is designed to have a polynucleotide sequence that can target the sequence of the referenced Plasmodium species target region. Such oligonucleotides are not limited to targeting only that sequence, but rather are useful as compositions, in kits, or in methods for targeting Plasmodium species target nucleic acids. The oligonucleotides are designed to function as components of an assay for the detection of Plasmodium species from a sample, and thus are designed to target Plasmodium species in the presence of other nucleic acids commonly found in test samples. "Specifically hybridizing to" does not mean exclusively hybridizing, as some small level of hybridization to non-target nucleic acids may occur, as is understood in the art. Rather, "specifically hybridizing to" means that the oligonucleotide is configured to function in the assay to primarily hybridize the target, such that accurate detection of the target nucleic acid in the sample can be determined. The term "configured to" refers to the actual arrangement of the polynucleotide sequence configuration of the oligonucleotide target hybridizing sequence.

[0080] The term "fragment" as used herein with respect to a Plasmodium species target nucleic acid refers to a portion of a contiguous nucleic acid.

[0081] As used herein, the term "region" refers to a portion of a nucleic acid, which portion is smaller than the entire nucleic acid.For example, if the nucleic acid in reference is an oligonucleotide promoter primer, the term "region" can be used to refer to the smaller promoter portion of the entire oligonucleotide.As a non-limiting example, if the nucleic acid in reference is an amplicon, the term region can be used to refer to the smaller nucleotide sequence that is specified for hybridization by the target hybridization sequence of the probe.

[0082] The interchangeable terms "oligomer," "oligo," and "oligonucleotide" generally refer to nucleic acids having less than 1,000 nucleotides (nt) residues (including polymers in the range having a lower limit of about 5 nt residues and an upper limit of about 500-900 nt residues). In some embodiments, oligonucleotides are in a size range having a lower limit of about 12-15 nt and an upper limit of about 50-600 nt, while other embodiments are in a range having a lower limit of about 15-20 nt and an upper limit of about 22-100 nt. Oligonucleotides may be purified from naturally occurring sources or may be synthesized using any of a variety of well-known enzymatic or chemical methods. The term oligonucleotide does not indicate any particular function for the reagent, but rather is used generically to encompass all such reagents described herein. Oligonucleotides may serve a variety of different functions. For example, it can function as a primer if it is specific for and capable of hybridizing to a complementary strand and capable of being further extended in the presence of a nucleic acid polymerase; it can function as a primer if it contains a sequence recognized by an RNA polymerase and allows for transcription; it can provide a promoter (e.g., a T7 primer); it can function to detect a target nucleic acid if it is capable of hybridizing to a target nucleic acid, or an amplicon thereof, and further provides a detectable moiety (e.g., an acridinium-ester compound).

[0083] As used herein, an oligonucleotide can "substantially correspond" to a designated reference nucleic acid sequence, meaning that the oligonucleotide is sufficiently similar to the reference nucleic acid sequence such that the oligonucleotide has similar hybridization properties to the reference nucleic acid sequence in that it hybridizes to the same target nucleic acid sequence under stringent hybridization conditions. Those skilled in the art will understand that a "substantially corresponding oligonucleotide" can be different from the reference sequence and still hybridize to the same target nucleic acid sequence. It is also understood that a first nucleic acid that corresponds to a second nucleic acid includes its RNA or DNA, including its complement, unless the context clearly indicates otherwise. This variation from the nucleic acid can be described in terms of the percentage of identical bases in the sequence, or the percentage of perfectly complementary bases between the probe or primer and its target sequence. Thus, in certain embodiments, an oligonucleotide "substantially corresponds" to a reference nucleic acid sequence if these percentages of base identity or complementarity are between 100% and about 80%. In a preferred embodiment, the percentage is between 100% and about 85%. In more preferred embodiments, this percentage is 100% to about 90%. In other preferred embodiments, this percentage is 100% to about 95%. Similarly, a region of a nucleic acid or amplified nucleic acid may be referred to herein as corresponding to a reference nucleic acid sequence. Those skilled in the art will understand the various modifications to hybridization conditions that may be required at various percentages of complementarity to allow hybridization to a specific target sequence without causing unacceptable levels of non-specific hybridization.

[0084] Exemplary sequences of Plasmodium species target nucleic acids are shown in Table 19 below. Specifically, SEQ ID NOs: 180 and 192-195 are reference sequences corresponding to the ribosomal RNA sequences of Plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi, Plasmodium ovale, and Plasmodium malariae, respectively. When a target region of Plasmodium species is described herein as "corresponding" to a defined region of SEQ ID NO: 180, it is understood that such reference includes any one or more homologous regions of SEQ ID NOs: 192-195. It is also understood that such reference to a region "corresponding" to a defined region of SEQ ID NO: 180 includes any one or more homologous regions of naturally occurring variants of SEQ ID NOs: 180 and 192-195 that may be present in a sample.

[0085] An "amplification oligomer" is an oligomer that has at least its 3' end complementary to a target nucleic acid, hybridizes to the target nucleic acid or its complement, and participates in a nucleic acid amplification reaction. One example of an amplification oligomer is a "primer" that hybridizes to a target nucleic acid and contains a 3' OH end that is extended by a polymerase in the amplification process. Another example of an amplification oligomer is an oligomer that is not extended by a polymerase (e.g., because it has a 3' blocked end), but participates in or facilitates amplification. For example, the 5' region of an amplification oligonucleotide may contain a promoter sequence (which may be referred to as a "promoter primer" or "promoter provider") that is non-complementary to the target nucleic acid. Those skilled in the art will understand that an amplification oligomer that functions as a primer may be modified to include a 5' promoter sequence and thus function as a promoter primer. The promoter primer is further modified by incorporating a 3' blocked end, which can provide an upstream promoter sequence that hybridizes to the target nucleic acid and acts to initiate transcription, but does not provide a primer for oligo extension. Such modified oligos are referred to herein as "promoter provider" oligomers. Size ranges for amplification oligonucleotides include those that are about 10 to about 70 nt in length (not including any promoter sequence or polyA tail) and contain at least about 10 contiguous bases, or as many as at least 12 contiguous bases that are complementary to a region of the target nucleic acid sequence (or its complementary strand). The contiguous bases are at least 80%, or at least 90%, or fully complementary to the target sequence to which the amplification oligomer binds. Amplification oligomers may optionally contain modified nucleotides or analogs, or additional nucleotides that participate in the amplification reaction but are not complementary to or contained in the target nucleic acid or template sequence. When referring to a range for the length of an oligonucleotide, amplicon, or other nucleic acid, it is understood that the range includes all integers (e.g., a length of 19 to 25 contiguous nucleotides includes 19, 20, 21, 22, 23, 24, and 25).

[0086] As used herein, a "promoter" is a specific nucleic acid sequence that binds to a nucleic acid and is recognized by a DNA-dependent RNA polymerase (a "transcriptase") as a signal to initiate transcription of RNA at a specific site.

[0087] As used herein, a "promoter provider" or "provider" refers to an oligonucleotide that includes a first and a second region and is modified to prevent the initiation of DNA synthesis from its 3' end. The "first region" of the promoter provider oligonucleotide includes a base sequence that hybridizes to a DNA template, and the hybridizing sequence is located 3' to the promoter region, but is not necessarily adjacent to it. The hybridizing portion of the promoter oligonucleotide is typically at least 10 nucleotides in length and can extend up to 50 or more nucleotides in length. The "second region" includes a promoter sequence for an RNA polymerase. The promoter oligonucleotide is engineered so that it cannot be extended by an RNA or DNA-dependent DNA polymerase, e.g., reverse transcriptase, and preferably includes a blocking portion at its 3' end as described above. As referred to herein, a "T7 provider" is a blocked promoter provider oligonucleotide that provides an oligonucleotide sequence recognized by T7 RNA polymerase.

[0088] "Amplification" refers to any known procedure for obtaining multiple copies of a target nucleic acid sequence or its complement or a fragment thereof. The multiple copies may be referred to as amplicons or amplification products. Known amplification methods include both thermal cycling and isothermal amplification methods. In some embodiments, isothermal amplification methods are preferred. Replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and transcription-mediated or transcription-associated amplification are non-limiting examples of nucleic acid amplification methods. Replicase-mediated amplification uses self-replicating RNA molecules and replicases such as QB replicase (e.g., U.S. Pat. No. 4,786,600). PCR amplification uses DNA polymerase, primer pairs, and thermal cycling to synthesize multiple copies of two complementary strands of dsDNA or from cDNA (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159). LCR amplification uses four or more different oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation (e.g., U.S. Pat. Nos. 5,427,930 and 5,516,663). SDA uses a primer containing a recognition site for a restriction endonuclease and an endonuclease that nicks one strand of a semi-modified DNA duplex containing the target sequence, thereby causing amplification in a series of primer extension and strand displacement steps (e.g., U.S. Pat. Nos. 5,422,252, 5,547,861, and 5,648,211). A preferred embodiment uses an amplification method suitable for amplifying an RNA target nucleic acid, such as transcription-mediated amplification (TMA) or NASBA, but it will be apparent to one skilled in the art that the oligomers disclosed herein can be readily used as primers in other amplification methods.

[0089] "Transcription-associated amplification," also referred to herein as "transcription-mediated amplification" (TMA), refers to nucleic acid amplification that uses an RNA polymerase to produce multiple RNA transcripts from a nucleic acid template. These methods generally employ an RNA polymerase, a DNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, and template-complementary oligonucleotides that contain a promoter sequence and may optionally contain one or more other oligonucleotides. The TMA method is an embodiment of the amplification method used to amplify and detect Plasmodium target sequences, as described herein. Variations of transcription-associated amplification are well known in the art, as previously disclosed in detail (e.g., U.S. Patent Nos. 4,868,105, 5,124,246, 5,130,238, 5,437,990, 5,554,516, and 7,374,885, and PCT Publication Nos. WO 88 / 01302, WO 88 / 10315, and WO 95 / 03430).Those skilled in the art will understand that the disclosed compositions can be used in amplification methods based on the extension of oligomer sequences by polymerase.

[0090] As used herein, the term "real-time TMA" refers to transcription-mediated amplification ("TMA") of a target nucleic acid monitored by a real-time detection means.

[0091] The term "amplicon," which is used interchangeably with "amplification product," refers to a nucleic acid molecule generated during an amplification procedure that is complementary or homologous to a sequence contained within a target sequence. These terms can be used to refer to a single-stranded amplification product, a double-stranded amplification product, or one of the strands of a double-stranded amplification product.

[0092] "Probe", "detection probe", "detection oligonucleotide", and "detection probe oligomer" are used interchangeably herein to refer to a nucleic acid oligomer that specifically hybridizes to a target sequence in a nucleic acid or amplified nucleic acid under conditions that promote hybridization to allow detection of the target sequence or amplified nucleic acid. Detection can be either direct (e.g., a probe directly hybridized to its target sequence) or indirect (e.g., a probe linked to its target via an intermediate molecular structure). Probes can be DNA, RNA, analogs thereof, or combinations thereof, and they can be labeled or unlabeled. The "target sequence" of a probe generally refers to a smaller nucleic acid sequence within a larger nucleic acid sequence that specifically hybridizes to at least a portion of the probe oligomer by standard base pairing. The probes may contain target-specific sequences and other sequences that contribute to the three-dimensional conformation of the probe (e.g., U.S. Patent Nos. 5,118,801, 5,312,728, 6,849,412, 6,835,542, 6,534,274, and 6,361,945, and U.S. Publication No. 2006 / 0068417). In a preferred embodiment, the detection probes contain a 2' methoxy backbone, which may result in a higher signal.

[0093] The term "TaqMan® probe" refers to a detection oligonucleotide that typically contains a fluorescent dye on the 5' base and a non-fluorescent quencher dye (quencher) on the 3' base. When illuminated, the excited fluorescent dye, rather than fluorescing, transfers energy to a nearby quencher dye molecule, resulting in a non-fluorescent substrate. During amplification, the exonuclease activity of the polymerase cleaves the TaqMan probe, separating the fluorophore from the quencher, thereby allowing an unquenched signal to be emitted from the fluorophore as an indication of amplification.

[0094] As used herein, "label" refers to a moiety or compound directly or indirectly attached to the probe to be detected or to the probe that produces a detectable signal. Direct labeling can occur through a bond or interaction that links the label to the probe, including covalent or non-covalent interactions, such as hydrogen bonds, hydrophobic and ionic interactions, or the formation of chelate or coordination complexes. Indirect labeling can occur through the use of a bridging moiety or "linker," such as a binding pair member, an antibody, or an additional oligomer, that can be either directly or indirectly labeled and amplify the detectable signal. Labels include any detectable moiety, such as a radionuclide, a ligand (e.g., biotin, avidin), an enzyme or enzyme substrate, a reactive group, or a chromophore (e.g., a dye, particle, or bead that gives a detectable color), a light-emitting compound (e.g., a bioluminescent, phosphorescent, or chemiluminescent label), or a fluorophore. A label can be detectable in a homogeneous assay in which the bound labeled probe in the mixture exhibits a different detectable change, such as instability or different degradation characteristics, than the unbound labeled probe. A "homogeneous detectable label" can be detected without physically removing the unbound form from the bound form of the label or labeled probe (e.g., U.S. Pat. Nos. 5,283,174, 5,656,207, and 5,658,737). Labels include chemiluminescent compounds, such as acridinium ester ("AE") compounds, including standard AE and derivatives (e.g., U.S. Pat. Nos. 5,656,207, 5,658,737, and 5,639,604). The synthesis and methods of attaching labels to nucleic acids and detecting the labels are well known (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) Chapter 10; U.S. Pat. Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333).More than one label, and more than one type of label, may be present on a particular probe, or detection may use a mixture of probes, where each probe is labeled with a compound that produces a detectable signal (e.g., U.S. Pat. Nos. 6,180,340 and 6,350,579).

[0095] As used herein, the structure referred to as "molecular torch" is designed to contain separate regions of self-complementarity ("closing domains") that are connected by binding regions ("target binding domains") and hybridize to each other under a given hybridization assay condition. All or part of the nucleotide sequence that contains the closing domain can also function as a target binding domain. Thus, the closing domain can include target binding sequences, non-target binding sequences, and combinations thereof.

[0096] As used herein, structures called "molecular beacons" are designed to contain target binding sequences flanked at both their 5' and 3' ends by sequences that are complementary to each other and hybridize to each other under defined hybridization assay conditions. The flanking complementary regions may be referred to as "switch sequences."

[0097] "Capture probe", "capture oligonucleotide", "target capture oligonucleotide", and "capture probe oligomer" are used interchangeably herein to refer to a nucleic acid oligomer that specifically hybridizes to a target sequence in a target nucleic acid by standard base pairing and binds to a binding partner on an immobilized probe to capture the target nucleic acid to a support. One example of a capture oligomer includes an oligonucleotide that includes two binding regions: a target hybridization sequence and an immobilized probe binding region. As a variation of this example, the two regions can be on two different oligomers that are linked together by one or more linkers. In another embodiment of a capture oligomer, the target hybridization sequence is a sequence that includes random or nonrandom polyGU, polyGT, or polyU sequences for nonspecifically binding to the target nucleic acid and linking the target nucleic acid to the immobilized probe on the support (see, for example, PCT Publication No. WO 2008 / 016988). The immobilized probe binding region can be a nucleic acid sequence called a tail. The tail comprises a substantially homopolymeric tail of about 10-40 nucleotides (e.g., A10-A40) or about 17-33 nt (e.g., T3A14-T3A30) that binds to a complementary immobilized sequence attached to a support particle or support matrix. Thus, non-limiting examples of preferred nucleic acid tails include, in some embodiments, T 0~4 A 10~40 Another example of a capture oligomer contains two regions, a target hybridizing sequence and a binding pair member that is not a nucleic acid sequence.

[0098] As used herein, "immobilized oligonucleotide", "immobilized probe", or "immobilized nucleic acid" refers to a nucleic acid binding partner that directly or indirectly binds a capture oligomer to a support. The immobilized probe bound to a support facilitates separation of the capture probe-bound target from unbound material in a sample. One embodiment of the immobilized probe is an oligomer bound to a support that facilitates separation of the bound target sequence from unbound material in a sample. The support may be made of nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene, silane, polypropylene, metal, or other compositions, and may include known materials such as matrices and particles free in solution, one embodiment of which is a magnetically attractable particle. The support may be monodisperse magnetic spheres (e.g., uniform size +5%) to which the immobilized probe is directly (via covalent bond, chelation, or ionic interaction) or indirectly (via one or more linkers) bound, and the binding or interaction between the probe and the support is stable during hybridization conditions.

[0099] explanation The present invention generally relates to methods and compositions for determining the presence or absence of the protozoan parasite Plasmodium spp. in a sample, such as a blood sample. Suitably, the methods and compositions described herein can detect the presence or absence of Plasmodium falciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale, and / or Plasmodium vivax. In some embodiments, the present invention provides a method for the detection of Plasmodium spp. in a sample, the method comprising performing an amplification-based detection of a target nucleic acid from a Plasmodium spp. The present invention further provides compositions (including reaction mixtures) and kits comprising oligomer combinations for detecting Plasmodium spp. in a sample, including Plasmodium falciparum, and / or Plasmodium knowlesi, and / or Plasmodium malariae, and / or Plasmodium ovale, and / or Plasmodium vivax. The oligomer combinations generally comprise at least two amplification oligomers for detecting Plasmodium species (including Plasmodium falciparum, and / or Plasmodium knowlesi, and / or Plasmodium malariae, and / or Plasmodium ovale, and / or Plasmodium vivax) in a sample, and may further comprise one or more additional oligomers described herein for performing amplification-based detection of Plasmodium species (including Plasmodium falciparum, and / or Plasmodium knowlesi, and / or Plasmodium malariae, and / or Plasmodium ovale, and / or Plasmodium vivax), e.g., capture probes and / or detection probes.

[0100] Methods for detecting the presence or absence of Plasmodium species in a sample from a subject generally include performing a nucleic acid-based detection assay for the specific detection of Plasmodium species nucleic acid in the sample. The nucleic acid-based detection assay generally utilizes an oligonucleotide that specifically hybridizes to the target nucleic acid of the Plasmodium species with minimal cross-reactivity with other nucleic acids suspected to be in the sample. In some variations, the oligonucleotide or combination of oligonucleotides for nucleic acid-based detection of Plasmodium species has minimal cross-reactivity with Babesia species (e.g., B. microti) nucleic acid.

[0101] In a particular aspect of the present invention, a combination of at least two oligomers is provided for determining the presence or absence of Plasmodium species in a sample. Typically, the combination of oligomers comprises at least a first and a second amplification oligomer for amplifying a Plasmodium species target region corresponding to the region of SEQ ID NO: 180. In such an embodiment, at least one amplification oligomer comprises a target hybridization sequence in a sense orientation ("sense THS") and at least one amplification oligomer comprises a target hybridization sequence in an antisense orientation ("antisense THS"), the sense THS and antisense THS of the amplification oligomer are each configured to specifically hybridize to the Plasmodium species target sequence corresponding to the sequence contained in SEQ ID NO: 180, and the target hybridization sequence is selected such that the Plasmodium sequence targeted by the antisense THS is located downstream of the Plasmodium sequence targeted by the sense THS (i.e., the at least two amplification oligomers are located adjacent to the target region that they are amplified).

[0102] In some embodiments, the Plasmodium species target region corresponds to a region of SEQ ID NO: 180 from about nucleotide position 844 or about nucleotide position 910 to about nucleotide position 1038, about nucleotide position 1051, about nucleotide position 1060, or about nucleotide position 1077. In other embodiments, the Plasmodium species target region corresponds to a region of SEQ ID NO: 180 from about nucleotide position 1153, about nucleotide position 1169, or about nucleotide position 1182 to about nucleotide position 1327, about nucleotide position 1354, or about nucleotide position 1382.

[0103] In some embodiments, the composition comprises amplification oligomers that include a Plasmodium-specific target hybridizing sequence that substantially corresponds to or is identical to a sequence set forth in any one of SEQ ID NOs: 21-56, 80-102, and 182-184. In such variations, the combination of oligomers includes at least one amplification oligomer that includes a Plasmodium-specific target hybridizing sequence of opposite polarity (sense versus antisense or vice versa) to the target hybridizing sequence of the oligomer, such that at least two amplification oligomers flank the target region to be amplified.

[0104] In some embodiments, the composition comprises (1) at least one amplification oligomer comprising a Plasmodium specific target hybridizing region substantially corresponding to at least one sense oligomer sequence shown in Table 1 below, and (2) at least one amplification oligomer comprising a Plasmodium specific target hybridizing region substantially corresponding to at least one antisense oligomer sequence shown in Table 1. In certain variations, the sense and / or antisense target hybridizing sequence(s) of the amplification oligomer combination comprises or consists of the sense and / or antisense sequence(s) selected from Table 1. [Table 1-1]

Table 1-2

[0105] In some embodiments, the oligomer combination includes (a) an amplification oligomer that comprises a target hybridizing sequence that is about 14 to about 20 contiguous nucleotides in length, contained in the sequence of SEQ ID NO:162 and includes the sequence of SEQ ID NO:163, or (ii) a target hybridizing sequence that is about 14 to about 25 contiguous nucleotides in length, contained in the sequence of SEQ ID NO:166 and includes SEQ ID NO:167 or SEQ ID NO:168, and (b) an amplification oligomer that comprises a target hybridizing sequence that is about 15 to about 33 contiguous nucleotides in length, contained in SEQ ID NO:169 and includes the sequence of SEQ ID NO:171, SEQ ID NO:172, or SEQ ID NO:173. In some such embodiments, the oligomer combination comprises an amplification oligomer of (a)(i), wherein the target hybridizing sequence is selected from SEQ ID NOs: 21, 23-25, 32, 33, 35, 54, and 55, or the target hybridizing sequence is contained within the sequence of SEQ ID NO: 164 and comprises the sequence of SEQ ID NO: 165 (e.g., a target hybridizing sequence selected from SEQ ID NOs: 21, 23-25, 32, 33, and 35). In yet other embodiments, the oligomer combination comprises an amplification oligomer of (a)(ii), wherein the target hybridizing sequence comprises the sequence of SEQ ID NO: 167 (e.g., a target hybridizing sequence selected from SEQ ID NOs: 28-31, 34, 40, 41, and 49-51), or the target hybridizing sequence comprises the sequence of SEQ ID NO: 168 (e.g., a target hybridizing sequence selected from SEQ ID NOs: 38, 39, 43, 44, and 53). In certain embodiments of the above oligomer combinations, the target hybridizing sequence of (b) is selected from SEQ ID NOs: 80 to 82 and 85 to 100. In other embodiments, the target hybridizing sequence of (b) is contained in the sequence of SEQ ID NO: 170 and includes the sequence of SEQ ID NO: 171 (e.g., a target hybridizing sequence selected from SEQ ID NOs: 81, 82, 85, 87 to 90, 94, and 96 to 98), or is contained in the sequence of SEQ ID NO: 170 and includes the sequence of SEQ ID NO: 172 (e.g., a target hybridizing sequence selected from SEQ ID NOs: 80, 82, 85, and 87 to 100).

[0106] In some embodiments, the combination of oligomers includes (a') an amplification oligomer that includes a target hybridizing sequence contained in the sequence of SEQ ID NO: 185 and includes a sequence of SEQ ID NO: 37, SEQ ID NO: 46, or SEQ ID NO: 187, and (b') an amplification oligomer that includes a target hybridizing sequence contained in the sequence of SEQ ID NO: 188 and includes a sequence of SEQ ID NO: 83, SEQ ID NO: 84, or SEQ ID NO: 182. In some embodiments, the amplification oligomer of (a') includes a target hybridizing sequence selected from SEQ ID NO: 37, 46, 183, and 184, or a target hybridizing sequence contained in the sequence of SEQ ID NO: 186 (e.g., a target hybridizing sequence of SEQ ID NO: 183 or SEQ ID NO: 184). In certain embodiments, the amplification oligomer of (b') includes a target hybridizing sequence selected from SEQ ID NO: 83, 84, and 182.

[0107] In certain embodiments, the amplification oligomers described herein are promoter primers or promoter providers that further comprise a promoter sequence located 5' of the target hybridizing sequence and are non-complementary to the Plasmodium species target nucleic acid. For example, in some embodiments of the oligomer combinations described herein, the amplification oligomer of (b) or (b') above is a promoter primer that further comprises a 5' promoter sequence. In certain embodiments, the promoter sequence is a T7 RNA polymerase promoter sequence, such as, for example, a T7 promoter sequence having the sequence set forth in SEQ ID NO: 179. In certain variations, the amplification oligomer is a promoter primer having the sequence set forth, selected from SEQ ID NOs: 57-77 and 181.

[0108] Table 2 shows particularly preferred combinations of amplification oligomer target hybridizing sequences ("Amp 1" and "Amp 2") for detection of Plasmodium species target nucleic acids. [Table 2]

[0109] In some embodiments, the oligomer combination includes at least two sense amplification oligomers and / or at least two antisense amplification oligomers that flank a Plasmodium species target region. For example, the oligomer combination can include (a) at least two amplification oligomers (e.g., two or three amplification oligomers), each of which is about 14 to about 25 contiguous nucleotides in length, is contained in SEQ ID NO: 166, and includes a target hybridizing sequence that includes SEQ ID NO: 167 or SEQ ID NO: 16, and / or (b) at least two amplification oligomers (e.g., two or three amplification oligomers), each of which is about 15 to about 33 contiguous nucleotides in length, is contained in SEQ ID NO: 169, and includes a target hybridizing sequence that includes SEQ ID NO: 171, SEQ ID NO: 172, or SEQ ID NO: 173. In some such variations, the combination of oligomers includes (a) a first amplification oligomer that includes a target hybridizing sequence comprising the sequence of SEQ ID NO: 167 (e.g., the target hybridizing sequence of SEQ ID NO: 34) and (b) a second amplification oligomer that includes a target hybridizing sequence comprising the sequence of SEQ ID NO: 168 (e.g., the target hybridizing sequence of SEQ ID NO: 53). In other embodiments comprising at least two sense amplification oligomers and / or at least two antisense amplification oligomers, the combination of oligomers comprises (a) (i) an amplification oligomer that is about 14 to about 20 contiguous nucleotides in length, is contained in the sequence of SEQ ID NO:162 and comprises a target hybridizing sequence that comprises the sequence of SEQ ID NO:163, and (ii) an amplification oligomer that is about 14 to about 25 contiguous nucleotides in length, is contained in the sequence of SEQ ID NO:166 and comprises a target hybridizing sequence that comprises SEQ ID NO:167 or SEQ ID NO:168, and / or (b) at least two amplification oligomers (e.g., two or three amplification oligomers), each of which is about 15 to about 33 contiguous nucleotides in length, is contained in SEQ ID NO:169 and comprises a target hybridizing sequence that comprises SEQ ID NO:171, SEQ ID NO:172, or SEQ ID NO:173.In some such variations, the oligomer combination comprises (a)(i) an amplification oligomer that is contained in SEQ ID NO: 164 and includes a target hybridizing sequence that includes a sequence of SEQ ID NO: 165 (e.g., a target hybridizing sequence of SEQ ID NO: 21), and (a)(ii) an amplification oligomer that includes a target hybridizing sequence that includes a sequence of SEQ ID NO: 167 (e.g., a target hybridizing sequence of SEQ ID NO: 34). In some embodiments of the above oligomer combinations that include at least two amplification oligomers of (b), the oligomer combination comprises a first and a second amplification oligomer of (b), each of which includes a target hybridizing sequence that is contained in SEQ ID NO: 170 and includes a sequence of SEQ ID NO: 171 or SEQ ID NO: 172 (e.g., a first amplification oligomer that includes a target hybridizing sequence of SEQ ID NO: 94, and a second amplification oligomer that includes a target hybridizing sequence of SEQ ID NO: 95).

[0110] In some embodiments, the oligomer combinations described herein further comprise at least one capture probe oligomer comprising a target hybridizing sequence configured to specifically hybridize to a Plasmodium species target nucleic acid. In some such embodiments, the capture probe oligomer comprises a target hybridizing sequence that is a sequence substantially corresponding to a sequence contained in the complement of SEQ ID NO: 180. In some embodiments, the capture probe oligomer target hybridizing sequence is covalently linked to a sequence or moiety that binds to the immobilized probe. Suitable capture probe oligomer target hybridizing sequences include sequences that are up to about 30 contiguous nucleotides in length and include sequences that substantially correspond to sequences selected from SEQ ID NOs: 11-15, 17, 19, and 20 (e.g., target hybridizing sequences that comprise or consist of sequences selected from SEQ ID NOs: 11-15, 17, 19, and 20, including DNA equivalents and DNA / RNA chimeras thereof). In some embodiments, the capture probe oligomer comprises or consists of a sequence selected from SEQ ID NOs: 1-5, 7, 9, and 10. In some embodiments, the oligomer combination comprises at least two capture probe oligomers (e.g., at least two capture probe oligomers described above). A first capture probe oligomer comprising a target hybridizing sequence of SEQ ID NO: 19 (e.g., a capture probe oligomer comprising a sequence of SEQ ID NO: 9) and a second capture probe oligomer comprising a target hybridizing sequence of SEQ ID NO: 20 (e.g., a capture probe oligomer comprising a sequence of SEQ ID NO: 10) are particularly suitable for use together in the oligomer combinations described herein.

[0111] In certain variations, the oligomer combinations described herein further comprise at least one detection probe oligomer configured to specifically hybridize to a Plasmodium species target sequence that is amplifiable using at least two amplification oligomers targeting the Plasmodium species target region. In some embodiments where the Plasmodium species target region corresponds to a region of SEQ ID NO: 180 from about nucleotide position 844 or about nucleotide position 910 to about nucleotide position 1038, about nucleotide position 1051, about nucleotide position 1060, or about nucleotide position 1077, the oligomer combination comprises a detection probe oligomer that specifically hybridizes to a target region corresponding to a region of SEQ ID NO: 180 from about nucleotide position 951 to about nucleotide position 998, or a complete complement thereof. For example, a detection probe oligomer may be about 13 to about 40 nucleotides in length and include a target hybridizing sequence that (i) is contained in the sequence of SEQ ID NO: 196 or its complement, and (ii) includes a sequence selected from SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO: 178 (including complements thereof). In a more specific variation, the detection probe oligomer target hybridizing sequence (i) is contained in the sequence of SEQ ID NO: 197 or its complement, and (ii) includes the sequence of SEQ ID NO: 174 or SEQ ID NO: 175 (including complements thereof). In another variation, the detection probe oligomer target hybridizing sequence (i) is contained in the sequence of SEQ ID NO: 196 or its complement, and (ii) includes a sequence selected from SEQ ID NO: 177 and SEQ ID NO: 178 (including complements thereof). Particularly preferred detection probe oligomer target hybridizing sequences include SEQ ID NOs: 131, 132, 135, 140, 145, 147-157, and 159-161 (including complements thereof). Suitable detection probes further include DNA equivalents and DNA / RNA chimeras of any of the above.

[0112] In some embodiments where the Plasmodium species target region corresponds to a region of SEQ ID NO: 180 from about nucleotide position 1153, about nucleotide position 1169, or about nucleotide position 1182 to about nucleotide position 1327, about nucleotide position 1354, or about nucleotide position 1382, the oligomer combination comprises a detection probe oligomer that specifically hybridizes to a target region corresponding to a region of SEQ ID NO: 180 from about nucleotide position 1210 to about nucleotide position 1233, or a complete complement thereof. For example, the detection probe oligomer may comprise a target hybridizing sequence that is at least about 13 nucleotides in length and (i) is contained in the sequence of SEQ ID NO: 189, or its complement, and (ii) comprises a sequence selected from SEQ ID NO: 190 and SEQ ID NO: 191, including complements thereof. Particularly suitable detection probe oligomer target hybridizing sequences include SEQ ID NOs: 125-130 and 143, including complements thereof. Suitable detection probes further include DNA equivalents and DNA / RNA chimeras of any of the above.

[0113] Table 3 shows exemplary combinations of detection probe target hybridizing sequences and first and second amplification oligomer target hybridizing sequences ("Amp 1" and "Amp 2") for detection of Plasmodium species target nucleic acids. [Table 3]

[0114] In some variations, the oligomer combination comprises at least two detection probe oligomers (e.g., at least two specific detection probes described herein). For example, the Plasmodium species target region corresponds to a region of SEQ ID NO: 180 from about nucleotide position 844 or about nucleotide position 910 to about nucleotide position 1038, about nucleotide position 1051, about nucleotide position 1060, or about nucleotide position 1077, and the oligomer combination may comprise: (A) a first detection probe oligomer (i) comprising a target hybridizing sequence comprising a sequence of SEQ ID NO: 197 or its complement, and (ii) a target hybridizing sequence comprising a sequence of SEQ ID NO: 175 or its complement, and (B) a second detection probe oligomer (i) comprising a sequence of SEQ ID NO: 197 or its complement, and (ii) a target hybridizing sequence comprising a sequence of SEQ ID NO: 176 or its complement (including the DNA equivalent or DNA / RNA chimera described above). A particularly preferred combination of first and second detection probe oligomers includes a first detection probe oligomer that comprises the target hybridizing sequence of SEQ ID NO:157 or its complement, or a DNA equivalent or DNA / RNA chimera thereof, and a second detection probe oligomer that comprises a target hybridizing sequence selected from SEQ ID NO:148 and SEQ ID NO:152 (including its complement, DNA equivalent, and DNA / RNA chimera).

[0115] The detection probe oligomer may contain a 2'-methoxy backbone at one or more linkages in the nucleic acid backbone. In some embodiments, at least one detection probe oligomer is provided in the amplicon detection reaction mixture.

[0116] Typically, the detection probe oligomer according to the present invention further comprises a label. Particularly suitable labels include compounds that emit detectable light signals, such as fluorophores or luminescent (e.g., chemiluminescent) compounds that can be detected in a homogeneous mixture. Two or more labels and two or more types of labels may be present on a particular probe, or detection may rely on using a mixture of probes, each of which is labeled with a compound that generates a detectable signal (see, for example, U.S. Patent Nos. 6,180,340 and 6,350,579, each of which is incorporated herein by reference). Labels may be attached to probes by various means, including covalent binding, chelation, and ionic interactions, but preferably, the labels are covalently attached. For example, in some embodiments, the detection probe has an attached chemiluminescent label, such as, for example, an acridinium ester (AE) compound (see, e.g., U.S. Pat. Nos. 5,185,439, 5,639,604, 5,585,481, and 5,656,744, each of which is incorporated herein by reference), which in a typical variation is attached to the probe by a non-nucleotidic linker (see, e.g., U.S. Pat. Nos. 5,585,481, 5,656,744, and 5,639,604, especially column 10, line 6 to column 11, line 3, and Example 8, each of which is incorporated herein by reference). In other embodiments, the detection probe includes both a fluorescent label and a quencher, a combination that is particularly useful in fluorescence resonance energy transfer (FRET) assays. Particular variations of such detection probes include, for example, TaqMan detection probes (Roche Molecular Diagnostics), as well as "molecular beacons" (see, e.g., Tyagi et al., Nature Biotechnol. 16:49-53, 1998; U.S. Pat. Nos. 5,118,801 and 5,312,728, each of which is incorporated herein by reference).

[0117] The detection probe oligomer according to the present invention may further comprise a non-target hybridizing sequence. Particular embodiments of such detection probes include probes that form conformations that are held by intramolecular hybridization, such as conformations that are commonly referred to as hairpins. Particularly suitable hairpin probes include "molecular torches" (see, for example, U.S. Pat. Nos. 6,849,412, 6,835,542, 6,534,274, and 6,361,945, each of which is incorporated herein by reference) and "molecular beacons" (see, for example, Tyagi et al., supra, U.S. Pat. Nos. 5,118,801 and 5,312,728, supra). Methods for using such hairpin probes are well known in the art.

[0118] In yet other embodiments, the detection probe is a linear oligomer that does not substantially form a conformation held together by intramolecular bonds. In a particular variation, the linear detection probe oligomer comprises a chemiluminescent compound as a label, preferably an acridinium ester (AE) compound.

[0119] Also provided by the present invention are detection probe oligomers, capture probe oligomers, and combinations thereof as described herein.

[0120] In another aspect, the present invention provides a method for detecting the presence or absence of Plasmodium species in a sample from a subject. Such a method generally includes carrying out a nucleic acid-based detection assay for specific detection in a sample of Plasmodium species nucleic acid. The nucleic acid-based detection assay for specific detection of Plasmodium species may use any one or more of the Plasmodium species-specific oligomers described herein (e.g., a combination of oligomers described herein comprising at least two amplification oligomers, or a detection probe or combination of detection probes described herein). A positive signal from the nucleic acid-based detection assay according to the present invention indicates the presence of one or more of Plasmodium falciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale, and / or Plasmodium vivax in the sample.

[0121] In some embodiments of the methods involving the use of a nucleic acid-based detection assay, an amplification-based assay is used to detect Plasmodium species. Such amplification-based assay methods generally include performing nucleic acid amplification of a Plasmodium species target region and detecting the amplification product (e.g., by specifically hybridizing the amplification product to a nucleic acid detection probe that provides a signal indicative of the presence of Plasmodium species in the sample). The amplification step includes contacting the sample with one or more amplification oligomers specific for a target sequence in the Plasmodium species target nucleic acid to produce an amplification product if Plasmodium species nucleic acid is present in the sample. In certain embodiments, a combination of at least two amplification oligomers described herein is used in the amplification step. Amplification uses at least one nucleic acid polymerase and an amplification oligomer to synthesize additional copies of the target sequence or its complement by producing copies from a template strand (e.g., by using the template strand to extend a sequence from a primer). Suitable amplification methods include, for example, replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and transcription-mediated or transcription-associated amplification (TMA). Such amplification methods are well known in the art (see, for example, the discussion of amplification methods in the Definitions section above) and are readily used in accordance with the methods of the present disclosure.

[0122] Detection of the amplification products can be achieved by various methods to detect signals specifically associated with the amplified target sequence. Nucleic acids can be associated with a surface that results in a physical change, such as a detectable electrical change. Amplified nucleic acids can be detected by concentrating them in or on a matrix and detecting the nucleic acids or their associated dyes (e.g., intercalating agents such as ethidium bromide or SYBR Green), or by detecting an increase in dyes associated with the nucleic acids in the solution phase. Other detection methods include contacting the amplification products with at least one detection probe configured to specifically hybridize to a sequence in the amplification product and detecting the presence of a probe:product complex, or may use a hybridization step by using a complex of probes that can amplify a detectable signal associated with the amplification product (e.g., U.S. Patent Nos. 5,424,413, 5,451,503, and 5,849,481). Directly or indirectly labeled probes that specifically associate with the amplification products provide a detectable signal that indicates the presence of the target nucleic acid in the sample. In some embodiments, methods utilizing an amplification-based assay for the detection of Plasmodium species utilize one or more detection probe oligomers described herein for detection of the amplification product.

[0123] The detection probe that hybridizes to the complementary amplified sequence can be a DNA or RNA oligomer, or an oligomer containing a combination of DNA and RNA nucleotides, or an oligomer synthesized with a modified backbone, for example, an oligomer containing one or more 2'-methoxy substituted ribonucleotides. The probe used to detect the amplified sequence can be unlabeled and indirectly detected (e.g., by binding of another binding partner to a moiety on the probe), or can be labeled with a variety of detectable labels. In some embodiments of the method for detecting Plasmodium species, such as certain embodiments using transcription-mediated amplification (TMA), the detection probe is a linear chemiluminescent labeled probe, such as, for example, a linear acridinium ester (AE) labeled probe. The detecting step can also provide additional information about the amplified sequence, such as, for example, all or a portion of its nucleic acid base sequence. Detection can be performed after the amplification reaction is completed, or can be performed simultaneously with the amplification of the target region, for example, in real time. In one embodiment, the detecting step allows for homogeneous detection, e.g., detection of hybridized probes without removing unhybridized probes from the mixture (see, e.g., U.S. Pat. Nos. 5,639,604 and 5,283,174).

[0124] In embodiments that detect amplification products near or at the end of the amplification step, linear detection probes can be used to provide a signal indicating the hybridization of the probe to the amplification product. One example of such detection uses a luminescent label probe that hybridizes to the target nucleic acid. The luminescent label is then hydrolyzed from the unhybridized probe. Detection is performed by chemiluminescence using a luminometer (see, for example, International Patent Application Publication No. WO89 / 002476). In other embodiments that use real-time detection, the detection probe can be, for example, a hairpin probe such as a molecular beacon, a molecular torch, or a hybridization switch probe that is labeled with a reporter moiety that is detected when the probe binds to the amplification product. Such a probe can include a target hybridizing sequence and a non-target hybridizing sequence. Various forms of such probes have been previously described (see, e.g., U.S. Pat. Nos. 5,118,801, 5,312,728, 5,925,517, 6,150,097, 6,849,412, 6,835,542, 6,534,274, and 6,361,945, as well as U.S. Patent Application Publication Nos. 20060068417A1 and 20060194240A1).

[0125] Some amplification methods using TMA amplification include the following steps. Briefly, a target nucleic acid containing a sequence to be amplified is provided as a single-stranded nucleic acid (e.g., ssRNA or ssDNA). Those skilled in the art will understand that conventional melting of double-stranded nucleic acids (e.g., dsDNA) can be used to provide a single-stranded target nucleic acid. A promoter primer specifically binds to the target nucleic acid at its target sequence, and a reverse transcriptase (RT) extends the 3' end of the promoter primer using the target strand as a template to create a cDNA copy of the target sequence strand, resulting in an RNA:DNA duplex. RNase digests the RNA strand of the RNA:DNA duplex, and a second primer specifically binds to its target sequence located on the cDNA strand downstream of the promoter primer end. RT then synthesizes a new DNA strand by extending the 3' end of the second primer using the first cDNA as a template to create a dsDNA containing a functional promoter sequence. RNA polymerase specific to the promoter sequence then initiates transcription to produce RNA transcripts that are approximately 100-1000 amplified copies ("amplicons") of the initial target strand of the reaction. Amplification continues when a second primer specifically binds to its target sequence in each of the amplicons, and RT creates DNA copies from the amplicon RNA template to produce an RNA:DNA duplex. RNase in the reaction mixture digests the amplicon RNA from the RNA:DNA duplex, and the promoter primer specifically binds to its complementary sequence in the newly synthesized DNA. RT extends the 3' end of the promoter primer to create dsDNA containing a functional promoter to which RNA polymerase binds and transcribes additional amplicons that are complementary to the target strand. The autocatalytic cycle of creating more amplicon copies is repeated over the course of the reaction, resulting in approximately one billion-fold amplification of the target nucleic acid present in the sample. The amplification products can be detected in real time during amplification or at the end of the amplification reaction by using probes that specifically bind to the target sequence contained in the amplification products. Detection of a signal arising from the bound probe indicates the presence of the target nucleic acid in the sample.

[0126] In some embodiments, the method utilizes a "reverse" TMA reaction. In such variations, the first or "forward" amplification oligomer is a priming oligonucleotide that hybridizes to the target nucleic acid near the 3' end of the target region. Reverse transcriptase (RT) synthesizes a cDNA strand by extending the 3' end of the primer using the target nucleic acid as a template. The second or "reverse" amplification oligomer is a promoter primer or promoter provider that has a target hybridizing sequence configured to hybridize to the target sequence contained within the synthesized cDNA strand. If the second amplification oligomer is a promoter primer, the RT extends the 3' end of the promoter primer using the cDNA strand as a template to create a second cDNA copy of the target sequence strand, thereby creating a dsDNA that includes a functional promoter sequence. Amplification then continues essentially as described above with initiation of transcription from the promoter sequence utilizing an RNA polymerase. Alternatively, when the second amplification oligomer is a promoter provider, a terminating oligonucleotide that hybridizes to a target sequence near the 5' end of the target region is typically utilized to terminate the extension of the priming oligomer at the 3' end of the terminating oligonucleotide, thereby providing a defined 3' end to the first cDNA strand synthesized by extension from the priming oligomer. The target hybridizing sequence of the promoter provider then hybridizes to the defined 3' end of the first cDNA strand, and the 3' end of the cDNA strand is extended to add a sequence complementary to the promoter sequence of the promoter provider, resulting in the formation of a double-stranded promoter sequence. The first cDNA strand is then used as a template to transcribe multiple RNA transcripts complementary to the first cDNA strand that do not include the promoter portion, using an RNA polymerase that recognizes and initiates transcription from the double-stranded promoter. Each of these RNA transcripts is available to serve as a template for further amplification from the first priming amplification oligomer.

[0127] In some embodiments of the method involving the use of nucleic acid-based detection assays, non-amplification-based assays are used to detect Plasmodium species. In some such embodiments, the non-amplification-based assay is a hybridization assay that involves hybridization of a specific detection probe to a target nucleic acid. Methods for carrying out polynucleotide hybridization assays have been well developed in the art. The procedure and conditions of the hybridization assay vary depending on the application and are selected according to known general binding methods, including, for example, those mentioned in Maniatis et al, Molecular Cloning: A Laboratory Manual (3rd ed. Cold Spring Harbor, NY, 2002), and Berger and Kimmel, Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc., San Diego, Calif., 1987). In general, the probe and sample are mixed under conditions that allow specific nucleic acid hybridization, and then the specific hybridization of the probe to its respective target is detected. Nucleic acid hybridization can be adapted to various assay formats. One suitable format is the sandwich assay format, which is particularly suitable for hybridization under non-denaturing conditions. The main component of the sandwich-type assay is a solid support, which has an immobilized nucleic acid probe adsorbed or covalently attached thereto, which is unlabeled and complementary to a portion of the DNA sequence. The target nucleic acid is hybridized to the immobilized probe, and a second labeled detection probe, which is complementary to a second different region of the same DNA strand to which the immobilized unlabeled nucleic acid probe is hybridized, is hybridized to the [target nucleic acid]:[immobilized probe] duplex to detect the target nucleic acid. Another exemplary format utilizes electrochemical detection of the target nucleic acid hybridized to an unlabeled detection probe immobilized on an electrode surface suitable as a signaling agent.See, e.g., Drummond et al., Nat. Biotechnol. 21:1192, 2003; Gooding, Electroanalysis 14:1149, 2002; Wang, Anal. Chim. Acta 469:63, 2002; Cagnin et al., Sensors 9:3122, 2009; Katz and Willner, Electroanalysis 15:913, 2003; Daniels and Pourmand, Electroanalysis 19:1239, 2007.

[0128] In certain embodiments of methods for detecting Plasmodium species that involve a hybridization assay, the hybridization assay utilizes one or more detection probe oligomers described herein.

[0129] In some embodiments, the non-amplification-based assay for detecting Plasmodium species is a cleavage-based assay, in which a probe oligonucleotide containing a non-target hybridizing flap region is cleaved by a flap endonuclease in an overlap-dependent manner to release a cleavage product, which is then detected. Exemplary cleavage-based assay reagents are described, for example, in Lyamichev et al. Nat. Biotechnol. 17: 292-296, 1999), Ryan et al. (Mol. Diagn. 4: 135-144, 1999), and Allawi et al. (J. Clin. Microbiol. 44: 3443-3447, 2006).

[0130] Suitable conditions for the flap endonuclease reaction are known or can be readily determined using methods known in the art (see, eg, Kaiser et al., J. Biol. Chem. 274:2138-721394, 1999). Exemplary flap endonucleases that may be used in the methods include Thermus aquaticus DNA polymerase I, Thermus thermophilus DNA polymerase I, mammalian FEN-1, Archaeoglobus fulgidus FEN-1, Methanococcus jannaschii FEN-1, Pyrococcus fiiriosus FEN-1, Methanobacterium thermoautotrophicum FEN-1, Thermus thermophilus FEN-1, CLEAVASE® (Hologic, Inc., Madison, WI), S. cerevisiae RTH1, S. cerevisiae RAD27, Schizosaccharomyces pombe rad2, bacteriophage T5 5'-3' exonuclease, Pyrococcus horikoshii Included are FEN-1, human endonuclease 1, calf thymus 5'-3' exonuclease (including its homologs in eubacteria, eukaryotes, and archaea), e.g., members of the class II family of structure-specific enzymes, and enzymatically active mutants or variants thereof.Descriptions of flap endonucleases can be found, for example, in Lyamichev et al., Science 260:778-783, 1993; Eis et al., Nat. Biotechnol. 19:673-676, 2001; Shen et al., Trends in Bio. Sci. 23:171-173, 1998; Kaiser et al., J. Biol. Chem. 274:21387-21394, 1999; Ma et al., J. Biol. Chem. 275:24693-24700, 2000; Allawi et al., J. Mol. Biol. 328:537-554, 2003; Sharma et al., J. Biol. Chem. 278:23487-23496, 2003; and Feng et al. al., Nat. Struct. Mol. Biol. 11:450-456, 2004.

[0131] In certain variations, the cleavage-based assay detects RNA target nucleic acids of Plasmodium species, where the cleavage-based assay utilizes a flap endonuclease capable of cleavage and an RNA:DNA linear duplex structure. In some alternative embodiments, the cleavage-based assay detects DNA target nucleic acids of Plasmodium species, where the cleavage-based assay utilizes a flap endonuclease capable of cleavage and a DNA:DNA linear duplex structure. Exemplary flap endonucleases capable of cleaving RNA:DNA duplexes include the polymerase-deficient 5' nucleases of the Thermus genus, as well as certain CLEAVASE® enzymes (Hologic, Inc., Madison, WI), such as CLEAVASE® BN (a BstX-Notl deletion of Taq polymerase, see U.S. Pat. No. 5,614,402), CLEAVASE® II (an "AG" mutant of full-length Taq polymerase, see U.S. Pat. No. 5,614,402), CLEAVASE® VII (a synthesis-deficient mutation of full-length Thermus thermophilus polymerase), CLEAVASE® IX (a polymerase-deficient mutant of Tth DNA polymerase), and CLEAVASE® XII (a polymerase-deficient mutant of taq DNA polymerase and Tth Exemplary flap endonucleases capable of cleaving DNA:DNA duplexes include the flap endonucleases described above, as well as CLEAVASE® 2.0 (Archaeoglobus fulgidus FEN-1), CLEAVASE® 2.1 (Archaeoglobus fulgidus FEN-1 with six histidines on the C-terminus), CLEAVASE® 3.0 (Archaeoglobus veneficus FEN-1), and CLEAVASE® 3.1 (Archaeoglobus veneficus FEN-1 with six histidines on the C-terminus).

[0132] In some embodiments, the cleavage-based assay detects an RNA target nucleic acid of a Plasmodium species, and the assay includes a step for synthesizing a DNA complement of the RNA target region, the cDNA strands of which are then hybridized to overlapping first and second probe oligonucleotides to form a linear double-stranded break structure for cleavage by a flap endonuclease. Reaction conditions for synthesizing cDNA from an RNA template using an RNA-dependent DNA polymerase (reverse transcriptase) are well known in the art.

[0133] In certain embodiments utilizing a nucleic acid-based detection assay, the method further comprises purifying the Plasmodium species target nucleic acid from other components in the sample. Such purification may include methods of separating and / or concentrating organisms contained in the sample from other sample components. In certain embodiments, purifying the target nucleic acid comprises capturing the target nucleic acid to specifically or non-specifically separate the target nucleic acid from other sample components. Non-specific target capture methods may involve selective precipitation of the nucleic acid from a substantially aqueous mixture, attachment of the nucleic acid to a support that is washed to remove other sample components, or other means of physically separating the nucleic acid from a mixture containing Plasmodium species nucleic acid and other sample components. In some embodiments, purification comprises lysing a sample of cells, such as, for example, blood cells (e.g., red blood cells), and purifying any Plasmodium species target nucleic acid from the lysed cell sample. Exemplary lysis reagents and methods for use in accordance with the present invention are described in U.S. Pat. No. 10,093,989 and PCT Publication No. WO 2017 / 189746, each of which is incorporated herein by reference.

[0134] In some embodiments, the target nucleic acid of a Plasmodium species is separated from other sample components by hybridizing the target nucleic acid to a capture probe oligomer. The capture probe oligomer comprises a target hybridizing sequence configured to specifically or non-specifically hybridize to the target nucleic acid to form a [target nucleic acid]:[capture probe] complex that is separated from other sample components. Capture probes comprising target hybridizing sequences suitable for non-specific capture of target nucleic acids are described, for example, in PCT Publication No. WO 2008 / 016988. In some particular variations, the Plasmodium species-specific capture probe comprises a target hybridizing sequence(s) configured to specifically hybridize to a Plasmodium species target nucleic acid (e.g., a target hybridizing sequence that comprises or consists of a sequence selected from SEQ ID NOs:11-15, 17, 19, and 20, including DNA equivalents and DNA / RNA chimeras thereof) that is up to about 30 contiguous nucleotides in length and that substantially corresponds to a sequence selected from SEQ ID NOs:11-15, 17, 19, and 20. In a preferred variation, the capture probe binds the [target nucleic acid]:[capture probe] complex to the immobilized probe to form a [target nucleic acid]:[capture probe]:[immobilized probe] complex that is separated from the sample and, if necessary, washed to remove non-target sample components (see, e.g., U.S. Patent Nos. 6,110,678, 6,280,952, and 6,534,273). In such variations, the capture probe oligomer further comprises a sequence or moiety that binds the capture probe, with its bound target sequence, to an immobilized probe attached to a solid support, thereby enabling the hybridized target nucleic acid to be separated from other sample components.

[0135] In more specific embodiments, the capture probe oligomer includes a tail portion (e.g., a 3' tail) that is not complementary to the target nucleic acid, but functions as a portion that specifically hybridizes to a sequence on the immobilized probe, thereby allowing the target nucleic acid to be separated from other sample components, as previously described in U.S. Patent No. 6,110,678. Any sequence may be used in the tail region, which is generally about 5-50 nt in length, with preferred embodiments including a substantially homopolymeric tail of about 10-40 nt (e.g., A10-A40), more preferably about 14-33 nt (e.g., A14-A30 or T3A14-T3A30), which binds to a complementary immobilized sequence (e.g., polyT) attached to a solid support, e.g., a matrix or particle. In some such embodiments comprising a target hybridizing sequence(s) configured to specifically hybridize to a Plasmodium species target nucleic acid, the Plasmodium species-specific capture probe comprises or consists of a nucleotide sequence selected from SEQ ID NOs: 1-5, 7, 9, and 10.

[0136] Target capture typically occurs in a liquid phase mixture containing one or more capture probe oligomers that hybridize to the target nucleic acid under hybridization conditions, usually at a temperature higher than the Tm of the [tail sequence]:[immobilized probe sequence] duplex. For embodiments that include a capture probe tail, the [target nucleic acid]:[capture probe] complex is captured by adjusting the hybridization conditions such that the capture probe tail hybridizes to the immobilized probe, and then the entire complex on the solid support is separated from other sample components. The support with the attached [immobilized probe]:[capture probe]:[target nucleic acid] can be washed one or more times to further remove other sample components. A preferred embodiment uses particulate solid supports, such as paramagnetic beads, whereby the particles with the attached [target nucleic acid]:[capture probe]:[immobilized probe] complexes can be suspended in a wash solution and retrieved from the wash solution, preferably using magnetic attraction. In embodiments of the method that include the use of an amplification-based detection assay, in order to limit the number of handling steps, the target nucleic acid may be amplified by simply mixing the target nucleic acid in a complex on the support with an amplification oligomer and proceeding with the amplification step.

[0137] According to the present disclosure, detecting the presence or absence of Plasmodium species may be performed separately (e.g., in separate reaction vessels) or together with another assay as a multiplex reaction system. Thus, in some embodiments, the methods described herein utilize a multiplex reaction, where the reaction mixture contains reagents for assaying multiple (e.g., at least two, three, four, or more) different target sequences in parallel. In these cases, the reaction mixture may contain multiple different target-specific oligonucleotides for performing the detection assay. For example, in methods utilizing an amplification-based detection assay, the multiplex reaction may contain multiple sets (e.g., multiple pairs) of amplification oligomers (e.g., multiple pairs of PCR primers or multiple pairs of TMA amplification oligomers (e.g., multiple pairs of promoter primers and non-promoter primers for TMAs, or multiple pairs of promoter provider and non-promoter primers)). In other embodiments utilizing a cleavage-based detection assay, the multiplex reaction may include multiple probe oligonucleotides with different flaps, multiple different overlapping probe oligonucleotides, and multiple different FRET cassettes for detecting different flaps once cleaved.

[0138] The oligomer combination described herein can be in the form of a reaction mixture or kit that includes the oligomer. The reaction mixture or kit can further include a number of optional components, such as, for example, a capture probe nucleic acid or an array of capture probe nucleic acids. For amplification reaction mixtures, the reaction mixture typically includes other reagents suitable for performing in vitro amplification, such as buffers, salt solutions, suitable nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, and UTP), and / or enzymes (e.g., reverse transcriptase and / or RNA polymerase), and typically includes test sample components that may or may not include Plasmodium species target nucleic acid. The kit that includes the oligomer combination for amplification of Plasmodium species also includes other reagents suitable for performing in vitro amplification, such as buffers, salt solutions, suitable nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, and UTP), and / or enzymes (e.g., reverse transcriptase and / or RNA polymerase). For oligomer combinations (e.g., reaction mixtures or kits) that include a detection probe along with a combination of amplification oligomers that target a common target nucleic acid, the selection of amplification oligomers and detection probe oligomers are linked by a common target region (i.e., the combination includes a probe that binds to a sequence amplifiable by the combination of amplification oligomers).

[0139] Compositions, methods, reaction mixtures, systems, kits, and the like for the detection of Plasmodium nucleic acids are further illustrated by the following non-limiting examples. EXAMPLES

[0140] "Parasite transport solution" generally refers to a solution formulated to preserve a sample and, in some cases, to at least partially dissolve one or more cell types in the sample. One exemplary parasite transport solution includes 15 mM monobasic sodium phosphate, 15 mM dibasic sodium phosphate, 1 mM EDTA, 1 mM EGTA, and 110 mM lithium lauryl sulfate (LLS), pH 6.7. Another exemplary parasite transport solution includes an aqueous solution of 100 mM TRIS, 30 mM magnesium chloride, and 6% (v / v) LLS, pH 7.5. A further exemplary parasite transport solution includes an aqueous solution of 14 mM sodium bicarbonate, 250 mM ammonium chloride, 5% (v / v) LLS, and 0.1 mM EDTA, pH 7.4. Other formulations of parasite transport solution may work well as well.

[0141] "Target capture reagent" generally refers to a solution containing multiple components that facilitate the capture of nucleic acids from a solution. One exemplary target capture reagent is 250 mM HEPES, 310 mM lithium hydroxide, 1.88 M lithium chloride, 100 mM EDTA, pH 6.4, and dT 14 The oligomers are covalently attached to magnetic particles (1 micron SERA-MAG™ MG-CM particles, GE Healthcare Lifesciences) at 250 μg / mL. Another exemplary target capture reagent contains 790 mM HEPES, 453 mM lithium hydroxide, 10% w / v LLS, 230 mM succinic acid, 0.03% w / v Foam Ban MS-575, and dT 14 It contains 0.0125% w / v magnetic particles (1 micron SERA-MAG^TM MG-CM particles, GE Healthcare Lifesciences) to which oligomers are covalently attached. Other formulations of target capture reagent may work equally well.

[0142] "Washing solution" generally refers to a solution containing 10 mM HEPES, 150 mM sodium chloride, 6.5 mM sodium hydroxide, 1 mM EDTA, 0.3% (v / v) ethanol, 0.02% (w / v) methylparaben, 0.01% (w / v) propylparaben, and 0.1% (w / v) sodium lauryl sulfate, pH 7.5.

[0143] "Probe reagent" generally refers to a solution containing one or more labeled detection probes. One exemplary probe reagent is a solution composed of about 75 to about 100 mM lithium succinate, 2% (w / v) LLS, 15 mM mercaptoethanesulfonate, 1.2 M lithium chloride, 20 mM EDTA, and 3% (v / v) ethanol, pH 4.7. Another exemplary probe reagent is a solution composed of about 75 to about 100 mM succinic acid, 3.5% (w / v) LLS, 75 mM lithium hydroxide, 15 mM aldrithiol-2, 1.0 M lithium chloride, 1 mM EDTA, and 3.0% (v / v) ethanol, pH 4.1 to 4.3. Other formulations may work well as well.

[0144] "Amplification reagents" generally refer to a concentrated mixture of reaction components to facilitate an amplification reaction. Amplification reagents include a number of different reagents at various concentrations depending on factors such as the type of amplification (PCR, TMA, etc.), the target nucleic acid (GC content), etc. One exemplary amplification reagent contains 47.6 mM Na-HEPES, 12.5 mM N-acetyl-L-cysteine, 2.5% TRITON™ X-100, 54.8 mM KCl, 23 mM MgCl, 3 mM NaOH, 0.35 mM of each dNTP (dATP, dCTP, dGTP, dTTP), 7.06 mM rATP, 1.35 mM rCTP, 1.35 mM UTP, 8.85 mM rGTP, 0.26 mM NaEDTA, 5% v / v glycerol, 2.9% trehalose, 0.225% ethanol, 0.075% methylparaben, 0.015% propylparaben, and 0.002% phenol red, pH 7.5-7.6. Another exemplary amplification reagent contains 19.1 mM Trizma base, 7.5 mM Trizma hydrochloride, 23.3 mM KCl, 21.5 mM MgCl2, 1 mM of each dNTP (dATP, dCTP, dGTP, dTTP), 6.5 mM rATP, 4.0 mM rCTP, 4.0 mM UTP, 6.5 mM rGTP, 3.33% v / v glycerol, 0.05 mM zinc acetate, 6 ppm Pro Clin 300 preservative, pH 8.25-8.45. Other formulations of amplification reagent may work equally well. Primers may be added to the amplification reagent or may be added to the amplification reaction separate from the amplification reagent. The enzymes in the amplification reagent include Moloney murine leukemia virus reverse transcriptase (MMLV-RT) and bacteriophage T7 RNA polymerase, with the units functionally defined as follows: 1 U of MMLV-RT will incorporate 1 nmol of dTTP in 10 min at 37°C using 200-400 micromoles of oligo-dT primed poly(A) as a template, and 1 U of T7 RNA polymerase will incorporate 1 nmol of ATP into RNA in 1 h at 37°C using a DNA template containing a T7 promoter.

[0145] "Hybridization reagent" generally refers to a solution comprised of the following reagents with concentrations ranging from about 75-100 mM succinic acid, 2%-3.5% (w / v) LLS, 75-100 mM lithium hydroxide, 14-16 mM aldrithiol-2, 1.0-1.2 M lithium chloride, 20-1000 mM EDTA, and 2.0-4.0% (v / v) ethanol, pH 4 to 5. Other formulations of hybridization reagent may work equally well.

[0146] "Selection Reagent" generally refers to a solution containing 600 mM boric acid, 182.5 mM sodium hydroxide, 1% (v / v) octoxynol (TRITON® X-100), pH 8.5.

[0147] "Detection Reagent" includes "Detection Reagent I," which generally refers to a solution containing 1 mM nitric acid and 32 mM hydrogen peroxide, and "Detection Reagent II," which generally refers to a solution of 1.5 M sodium hydroxide.

[0148] Example 1 Primer screening was performed using transcription-mediated amplification (TMA) on a manual Procleix Enhanced Semi-automated System (eSAS) using Plasmodium falciparum in vitro transcripts (IVT). The assay rack consisted of 10 rows of 10 tube units (TTU). Seventy-five microliters (75 μL) of amplification reagent and 5 picomoles of each T7 promoter provider oligonucleotide and non-T7 primer oligonucleotide were added to the appropriate tubes on the rack, whereby each combination of amplification oligomers was tested with three replicates of P. falciparum IVT at 30 and 10 copies per reaction, and two replicates of B. microti IVT at 1,000,000 copies per reaction, where applicable. B. microti was included in the initial screen as a cross-reactive specimen due to the conserved region between Babesia and Plasmodium. It is necessary to determine that the amplification and detection system is specific to Plasmodium. To achieve the target copy number per reaction, 10 μL of P. falciparum IVT at 3 c / μL or 1 c / μL diluted in buffer was spiked into the appropriate tube, and 10 μL of B. microti IVT at 100,000 c / μL diluted in buffer was spiked into the appropriate tube. Various combinations of primers were tested. This setup allows for 10 primer combinations to be tested per rack. Once the primer combinations and IVT were spiked, 200 μL of oil was added to each tube, the rack was covered with a sealing card, and vortexed for a minimum of 20 seconds.

[0149] The racks were then incubated in a 60±1°C water bath for 10±1 min, followed by 9-20 min in a 41.5±1°C water bath. With the racks still in the water bath, the sealing cards were removed and 25 μL of commercial Procleix Ultrio Plus enzyme reagent (Grifols Diagnostic Solutions Inc.) was added to each reaction tube, which was then re-covered with a sealing card. The racks were gently shaken to mix, then re-covered with a sealing card and incubated in a 41.5±1°C water bath for an additional 60±5 min.

[0150] After incubation was complete, the rack was transferred to the Hybridization Protection Assay (HPA) area where the sealing card was removed. 100 μL of probe reagent, consisting of acridinium-ester (AE)-labeled probe, was added to the hybridization reagent at the desired total concentration of at least 2.5e6 relative light units (RLU) per reaction. The probe reagent was then added to the appropriate reaction tubes. The tubes were covered with sealing cards and the rack was vortexed for a minimum of 20 seconds, after which the rack was incubated in a 61 ± 2 °C water bath for 15 ± 1 minutes.

[0151] The rack was removed from the water bath, the sealing cards were removed, and 250 μL of commercially available Procleix Ultrio Plus selection reagent (Grifols Diagnostic Solutions Inc.) was added to each tube. The tubes were covered with sealing cards, vortexed for a minimum of 20 seconds, and then returned to the 61±2°C water bath and incubated for 10±1 minutes. After incubation, the rack was allowed to cool in the 23±4°C water bath for a minimum of 10 minutes.

[0152] For detection, the TTUs were removed from the rack and loaded into the automated reader instrument for subsequent light-off using commercially available Procleix Auto Detect 1 and 2 reagents (Grifols Diagnostic Solutions Inc.), and the results were exported for analysis of the signal in relative light units (RLU).

[0153] The AE-labeled probes screened in this example are shown below in Table 4. Probes were screened with amplification oligomer pairs in four experimental groups: group 1, groups 2a and 2b, and group 3. [Table 4]

[0154] Group 1. Probes 1, 3, 4-6, 12, and 14-16 were each tested with SEQ ID NO:59 (T7 promoter provider oligomer) and SEQ ID NO:30 (non-T7 oligomer). The results of this probe screen are shown in Table 5 below. The candidates showed no cross-reactivity with B. microti IVT at 1e6 copies per reaction. In addition, probes with the same nucleotide sequence but different 2MeAE linker sites (see, e.g., probes 1 and 3, also see probes 4, 5, and 6) performed well regardless of their different labels. [Table 5]

[0155] Groups 2a and 2b. In group 2a, the following primer / probe combinations were tested: probes 8 and 20-22, each paired with SEQ ID NO:59 and SEQ ID NO:33 (T7 / NT7), probes 7 and 20-22, each paired with SEQ ID NO:59 and SEQ ID NO:52 (T7 / NT7), and probes 7 and 8, each paired with SEQ ID NO:59 and SEQ ID NO:49 (T7 / NT7). In group 2b, the following primer / probe combinations were tested: probes 20-22, each paired with SEQ ID NO:66 and SEQ ID NO:33 (T7 / NT7), probes 7 and 20-22, each paired with SEQ ID NO:66 and SEQ ID NO:52 (T7 / NT7), and probe 7, each paired with SEQ ID NO:66 and SEQ ID NO:49 (T7 / NT7).

[0156] The results of this probe screen are shown in Tables 6 and 7 below. Systems containing the non-T7 oligomer of SEQ ID NO:52 did not amplify Plasmodium. Systems with the T7 / NT7 oligomer pair of SEQ ID NO:59 / SEQ ID NO:33 paired with any of probes 20-22 did not detect Plasmodium, but these probes do detect Plasmodium when used with the T7 / NT7 oligomer pair of SEQ ID NO:66 / SEQ ID NO:33. [Table 6] [Table 7]

[0157] Group 3. Redesign of the non-T7 primer of SEQ ID NO:33 was tested. Each of probes 7 and 8 was paired with each of SEQ ID NO:66 / SEQ ID NO:25 and SEQ ID NO:66 / SEQ ID NO:35 (T7 / NT7). Additionally, probe 20 was paired with SEQ ID NO:66 / SEQ ID NO:33 (T7 / NT7).

[0158] The results of this probe screen are shown below in Table 8. The T7 / NT7 oligomer pair SEQ ID NO:66 / SEQ ID NO:33 performed well with probe 20. Redesigns of non-T7 oligomers of SEQ ID NO:33 (SEQ ID NO:25 and SEQ ID NO:35) induced false positives with probes 7 and 8. [Table 8]

[0159] Example 2 This example describes materials and methods for screening candidate amplification systems using TMA on a fully automated Procleix Panther system (Grifols Diagnostic Solutions Inc.).

[0160] Specimens included P. falciparum, P. knowlesi, P. malariae, P. ovale, and / or P. vivax IVT diluted in buffer. Specimens may also include B. microti IVT as a cross-reactive specimen. It is necessary to determine that the amplification and detection system is specific for Plasmodium. An assay calibrator containing a P. falciparum IVT panel at 500c / mL was included to determine the analyte cutoff for the run. The assay software uses the analyte cutoff to determine whether a sample is reactive or non-reactive. Samples with a signal to cutoff ratio of 1 or greater are considered reactive, while samples with a signal to cutoff ratio of less than 1 are non-reactive. The assay reagents used included: target capture reagent (TCR) comprised of at least one target capture oligomer (TCO), amplification reagent comprising at least one T7 promoter provider and at least one non-T7 primer, probe reagent comprised of at least one AE-labeled probe, Ultrio Plus enzyme reagent, and selection reagent.

[0161] Example 3: Screening of Plasmodium target capture probes Candidate target capture probes (TCOs) were screened using samples containing 500 c / mL P. falciparum IVT prepared in 3 mL of parasite transport medium (PTM, 100 mM TRIS, 30 mM magnesium chloride, and 6% (v / v) LLS, pH 7.5) with 1 mL of whole blood. TMA reactions were performed on a fully automated Procleix Panther system essentially as described in Example 2. TCOs of SEQ ID NOs: 1-5, 7, 9, and 10 were tested using T7 oligomers of SEQ ID NOs: 66 and 69 (5 pmol / reaction each), non-T7 oligomers of SEQ ID NOs: 21 and 30 (5 pmol / reaction each), and detection probes of SEQ ID NOs: 148 and 152 (1.9e6 RLU / reaction each).

[0162] The results are shown below in Tables 9 and 10. Assay performance was best with TCO SEQ ID NO: 9 and SEQ ID NO: 10. SEQ ID NO: 3 was suboptimal in this assay. [Table 9] [Table 10]

[0163] Example 4: Analytical Sensitivity - LoD in RNA copies / mL The limit of detection (LoD) of the candidate amplification systems was evaluated by probit analysis using in vitro synthesized transcripts of P. falciparum, P. knowlesi, P. malariae, P. ovale, and P. vivax. TMA reactions were performed on a fully automated Procleix Panther system, essentially as described in Example 2. The IVTs of each species were serially diluted in buffer to 100, 30, 10, 3, 1, and 0 copies / mL, and 32 replicates were tested at each level per species. Specimens were tested using TMA on a fully automated Procleix Panther system (Grifols Diagnostic Solutions Inc.). An assay calibrator containing a P. falciparum IVT panel at 500 c / mL was included to determine the analyte cutoff for the run. The assay software uses the analyte cutoff to determine whether a sample is reactive or non-reactive. Samples with a signal-to-cutoff ratio of 1 or greater are considered reactive, while samples with a signal-to-cutoff ratio of less than 1 are non-reactive. For this assay, the following oligomers were used: T7 oligomers of SEQ ID NO:66 and SEQ ID NO:69 (5 pmol / reaction each), non-T7 oligomers of SEQ ID NO:21 and SEQ ID NO:30 (5 pmol / reaction each), detection probes of SEQ ID NO:148 and SEQ ID NO:152 (1.9e6 RLU / reaction each), and TCOs of SEQ ID NO:9 and SEQ ID NO:10.

[0164] The results are shown in Table 11. Similar LoD values ​​were observed for the five species tested. The 95% LoD ranged from 9.4 to 14.8 copies / mL. [Table 11]

[0165] Example 5: Analytical sensitivity - LoD of RNA parasites / mL The limit of detection (LoD) of the candidate amplification systems was assessed by probit analysis using cultured parasite-infected cells. Specifically, conditions were tested using lysed negative whole blood specimens and diluted cultured P. falciparum-infected red blood cells. Culture samples were received with known percent parasitemia and RBC counts determined by fluorescence-activated cell sorting (FACS) to estimate concentration (parasites per mL value). Based on estimated parasitemia per mL, samples were diluted with normal negative human whole blood to an estimated 6, 4, 2, 1, and 0.5 parasitemia per mL. Diluted Plasmodium-infected whole blood was lysed at a ratio of 0.9 mL of whole blood in 2.7 mL of parasite transport medium (PTM, 100 mM TRIS, 30 mM magnesium chloride, and 6% (v / v) LLS, pH 7.5). Specimens were tested using TMA on a fully automated Procleix Panther system (Grifols Diagnostic Solutions Inc.). An assay calibrator containing 500c / mL P. falciparum IVT panel was included to determine the analyte cutoff for the run. The assay software uses the analyte cutoff to determine whether a sample is reactive or non-reactive. Samples with a signal to cutoff ratio of 1 or greater are considered reactive, while samples with a signal to cutoff ratio less than 1 are non-reactive. The assay reagents used included: target capture reagent (TCR) containing TCOs of SEQ ID NO:9 and SEQ ID NO:10, amplification reagent containing T7 oligomers of SEQ ID NO:66 and SEQ ID NO:69 (5 pmol / reaction each), non-T7 oligomers of SEQ ID NO:21 and SEQ ID NO:30 (5 pmol / reaction each), probe reagent containing AE-labeled detection probes of SEQ ID NO:148 and SEQ ID NO:152 (1.9e6 RLU / reaction each), Ultrio Plus enzyme reagent, and selection reagent.

[0166] The results are shown in Table 12. The 95% LoD was 2.14 parasites / mL. Each of the 480 replicates of the internal control buffer, PTM, and negative lysates were evaluated with no false positives (0 / 1,440). [Table 12]

[0167] Example 6: Interference and cross-reactivity with Babesia Interference and cross-reactivity with Babesia microti (a homologous protozoan) was evaluated using in vitro synthesized transcripts. Tests were performed in TMA reactions carried out on a fully automated Procleix Panther system, essentially as described in Example 2, using P. falciparum IVT dilutions of 100 to 1c / mL, with and without the addition of 1e6c / mL B. microti IVT. The following oligomers were used: T7 oligomers of SEQ ID NO: 71 and SEQ ID NO: 72 (5 pmol / reaction each), non-T7 oligomers of SEQ ID NO: 34 and SEQ ID NO: 53 (5 pmol / reaction each), AE-labeled detection probes of SEQ ID NO: 148 and SEQ ID NO: 157 (1.9e6 RLU / reaction each), and TCOs of SEQ ID NO: 9 and SEQ ID NO: 10.

[0168] The results are shown below in Tables 13 and 14. Comparable reactivity and analyte RLUs were observed for P. falciparum IVT with and without the presence of B. microti IVT. [Table 13] [Table 14]

[0169] Example 7: Evaluation of Redundant Probes Redundancy probe combinations were evaluated using in vitro synthesized transcripts. Tests were performed in TMA reactions carried out on a fully automated Procleix Panther system, substantially as described in Example 2. The following oligomers were used: (i) T7 oligomers of SEQ ID NO: 71 and SEQ ID NO: 72 (5 pmol each / reaction), (ii) non-T7 oligomers of SEQ ID NO: 34 and SEQ ID NO: 53 (5 pmol each / reaction), (iii) AE-labeled detection probes of SEQ ID NO: 148 ((4,5 2MeAE linker) and SEQ ID NO: 157 (1.27e6 RLU / reaction each), with SEQ ID NO: 148 (5,6 2MeAE linker) or SEQ ID NO: 148 (6,7 2MeAE linker), and (iv) TCOs of SEQ ID NO: 9 and SEQ ID NO: 10.

[0170] The results are shown in Table 15. "C1" refers to the probe combination containing SEQ ID NO: 148 (5,6 2MeAE linker). "C2" refers to the probe combination containing SEQ ID NO: 148 (6,7 2MeAE linker). Equivalent reactivity was observed for all Plasmodium species at 30 and 10 c / mL using the redundant probe combination. Consistent with the results of Example 1, probes with the same nucleotide sequence (SEQ ID NO: 148) but different 2MeAE linker sites performed well regardless of their different labels. [Table 15]

[0171] Example 8: Analytical sensitivity - LoD of RNA parasites / mL The limit of detection (LoD) of the candidate amplification systems was evaluated by probit analysis using cultured P. falciparum infected erythrocytes. Testing was performed in TMA reactions carried out on a fully automated Procleix Panther system, substantially as described in Example 5. The following oligomers were used: (i) T7 oligomers of SEQ ID NO: 71 and SEQ ID NO: 72 (5 pmol each / reaction), (ii) non-T7 oligomers of SEQ ID NO: 34 and SEQ ID NO: 53 (5 pmol each / reaction), (iii) AE-labeled detection probes of SEQ ID NO: 148 (4,5 2MeAE linker), SEQ ID NO: 157, and SEQ ID NO: 148 (5,6 2MeAE linker) (1.17e6, 7.92e5, and 2.03e6 RLU / reaction, respectively), and (iv) TCOs of SEQ ID NO: 9 and SEQ ID NO: 10.

[0172] The results are shown in Table 16. The 95% LoD was 2.31 parasites / mL. There were no false positives in the negative specimens tested. These results are comparable to the system evaluated in Example 5, where the 95% LoD was 2.14 parasites / mL. [Table 16]

[0173] Example 9 Primer screening was performed using a manual two-phase real-time TMA assay procedure. In the target capture step, 400 μL of target capture reagent (TCR), containing at least one target capture oligo (TCO) and one T7 promoter provider, was added to a 2 mL deep-well 96-well plate (Thermo Scientific catalog number 95040450), followed by the addition of 500 μL of specimen. Specimens consisted of Plasmodium species in vitro transcripts (IVT) diluted in buffer for Plasmodium detection, and Babesia species IVT diluted in buffer for Babesia detection. Specimens could also include B. microti IVT or P. falciparum IVT as cross-reactive specimens for the opposite detection system due to conserved regions between Babesia and Plasmodium. It is necessary to determine that the amplification and detection system is specific for the analyte system. The plate was covered with a sealing card, loaded onto a Torrey Pines plate incubator, and covered with a lid. Incubation steps in the Torrey Pines incubator included 7 min at 80°C, followed by 17 min at 62°C and 15–25 min at 25°C, respectively.

[0174] After the target capture incubation step, the plate was loaded with deep-well comb tips (Thermo Scientific Catalog No. 97002534) and placed on a Kingfisher 96 instrument (Thermo Scientific Type 710 REF 5400500) fitted with a deep-well magnet. In addition, the Kingfisher instrument was loaded with a wash plate consisting of a 2 mL 96-well plate (Nunc Deep Well Plate Catalog No. 278752) prepared with 500 μL of commercial Procleix wash buffer reagent (Grifols Diagnostic Solutions Inc.), and a second wash plate consisting of a 200 μL 96-well plate (Thermo Scientific Catalog No. 97002540) containing 200 μL of wash buffer. For the deep-well wash step, the plate containing the hybridized TCR-sample mixture was mixed for 5 minutes, after which the magnetic beads were collected for 20 counts and eluted into the plate containing 500 μL of wash buffer for 20 seconds. After mixing 500 μL of the wash plate for 1 min, the magnetic beads were collected for 10 counts and eluted into the wash plate containing 200 μL of wash buffer for 20 s.

[0175] The second wash plate containing the mixture of hybridized magnetic beads and wash buffer was removed from the Kingfisher instrument and transferred to a second Kingfisher 96 instrument (Thermo Scientific Type 710 REF 5400500) loaded with a small PCR tip comb (Thermo Scientific 97002514) and fitted with a PCR magnet. The Kingfisher instrument was loaded with a 96-well PCR plate (Axygen catalogue no. PCR-96-HS-C) containing 30 μL of amplification reagent (without phenol red) containing at least one non-T7 primer (amplification plate). To transfer the hybridized magnetic beads to the amplification plate, the wash plate was mixed for 5 minutes, after which the magnetic beads were collected for 30 counts and eluted for 30 seconds into the amplification plate. The wash plate was mixed again for 1 minute, after which the magnetic beads were collected for 30 counts and eluted for 30 seconds into the amplification plate to complete the transfer.

[0176] The amplification plate was covered with a sealing card and loaded onto a Stratagene instrument (Mx3005P Multiplex Quantitative PCR System) and incubated at 43°C for 5 minutes. The plate was transferred to a heat block set at 42°C, the cover was removed, 10 μL of commercial Ultrio Plus enzyme reagent (Grifols Diagnostic Solutions Inc.) was added, and the sealing card was re-covered. The plate was mixed on the heat block at 1400 RMP for 1 minute, re-loaded onto the Stratagene instrument, and incubated at 43°C for 5 minutes. The plate was transferred back to the heat block, the cover was removed, and 15 μL of promoter reagent (amplification reagent without phenol red) was added, containing a mixture of at least one T7 promoter provider and at least one fluorescently labeled molecular torch or beacon (5'-hexochloro-fluorescein (HEX) for Plasmodium, or 5'-fluorescein (FAM) for Babesia) and sealed with a clear adhesive plate cover. The plate was mixed on a heat block at 1400 RMP for 1 minute and reloaded onto the Stratagene for the read protocol, which consisted of incubation at 43° C. and reading every 30 seconds for 120 or 150 cycles.

[0177] Raw data exported from the Stratagene instrument was analyzed using an in-house software tool. Fluorescence curves were analyzed using a threshold of 1,000 relative fluorescence units (RFU). The time (TTime) at which a specimen met or exceeded the threshold was determined by the software. Specimens with a TTime were considered reactive for Plasmodium in the HEX channel or Babesia in the FAM channel. A lower TTime indicated better performance of the system tested. Specimens without a TTime or below the threshold were considered non-reactive.

[0178] In this example, combinations of molecular torch or beacon probe and amplification oligomer pairs were tested in three groups: groups 1, 2, and 3. The oligomer combinations tested are shown in Table 17 below. [Table 17]

[0179] Results for Group 1. Probes of SEQ ID NO: 104-107 and 121 successfully detected Plasmodium with fluorescence curves that produced an average TTime of 30.69 minutes or less at 10 c / mL. For probe of SEQ ID NO: 108, no fluorescence curve with TTime was produced, and it was concluded that Plasmodium could not be detected.

[0180] Group 2 Results. Amplification oligomers of SEQ ID NO: 181 and 184 in combination with the torch probe of SEQ ID NO: 104 successfully detected Plasmodium as demonstrated by a fluorescence curve producing a TTime of 20.89 minutes or less at 10 c / mL, and showed no cross-reactivity with Babesia IVT as demonstrated by the absence of a fluorescence curve with a TTime.

[0181] Group 2 Results. The T7 amplification oligomer of SEQ ID NO:59 and the non-T7 amplification oligomers of SEQ ID NO:51, 31, and 50 in combination with the torch probe of SEQ ID NO:123 each successfully detected Plasmodium as demonstrated by fluorescence curves that produced TTimes of 22.82 minutes or less.

[0182] Example 10 Candidate amplification systems were tested using real-time TMA on a fully automated Panther system equipped with a real-time fluorometer (Hologic Inc.). Specimens consisted of Plasmodium species IVT diluted in buffer for Plasmodium detection, and Babesia species IVT diluted in buffer for Babesia detection. Specimens could also include B. microti IVT or P. falciparum IVT as cross-reactive specimens. It is necessary to determine that the amplification and detection system is specific to the analyte system. The assay reagents used included: TCR composed of at least one TCO and one T7 promoter provider, amplification reagent composed of at least one non-T7 primer, promoter reagent (phenol red-free amplification reagent) composed of at least one T7 promoter provider and at least one fluorescently labeled molecular torch or beacon (5'-hexochloro-fluorescein (HEX) for Plasmodium, or 5'-fluorescein (FAM) for Babesia), and Ultrio Plus enzyme reagent.

[0183] The Panther RT-Dev Tool (Hologic Inc.) was used for analysis of raw data exported from the Panther system. Fluorescence curves were analyzed using a threshold of 1,000 relative fluorescence units (RFU). The time (TTime) at which a specimen met or exceeded the threshold was determined by the software. Specimens with a TTime were considered reactive for Plasmodium in the HEX channel or Babesia in the FAM channel. A lower TTime indicated better performance of the tested system. Specimens without a TTime or below the threshold were considered non-reactive.

[0184] The oligomers tested in this example were the T7 oligomer of SEQ ID NO:59, the non-T7 oligomer of SEQ ID NO:30, and the Torch probe of SEQ ID NO:123. Results demonstrated detection of 8 of 8 replicates tested at Plasmodium IVT at 30 c / mL and 3 of 8 replicates tested at 10 c / mL. Fluorescence curves produced a TTime of 33.18 minutes or less for the replicates detected at 10 c / mL.

[0185] Example 11: Detection of Plasmodium in clinical specimens Candidate amplification systems were tested using real-time TMA on a fully automated Panther system fitted with a real-time fluorometer (Hologic Inc.). Specimens consisted of positive controls of B. microti and P. falciparum IVT diluted to 300 c / mL in buffer, and a negative control consisting of negative buffer. Clinical specimens consisted of whole blood and plasma specimens of P. falciparum and P. ovale. Whole blood specimens were prepared by manually adding 100 μL to 3 mL of lysis buffer (14 mM sodium bicarbonate, 250 mM ammonium chloride, 5% (v / v) LLS, and 0.1 mM EDTA, pH 7.4). Plasma specimens were prepared by manually adding 100 μL to 3 mL of processed human plasma. Specimens were tested on the Panther system. The assay reagents used included the following: TCR, composed of at least one TCO and one T7 promoter provider, amplification reagent, composed of at least one non-T7 primer, promoter reagent (phenol red-free amplification reagent), composed of at least one T7 promoter provider and at least one fluorescently labeled molecular torch or beacon (5'-hexochloro-fluorescein (HEX) for Plasmodium, or 5'-fluorescein (FAM) for Babesia), and enzyme reagent.

[0186] The Panther RT-Dev Tool (Hologic Inc.) was used for analysis of raw data exported from the Panther system. Fluorescence curves were analyzed using a threshold of 1,000 relative fluorescence units (RFU). The time (TTime) at which a specimen met or exceeded the threshold was determined by the software. Specimens with a TTime were considered reactive for Plasmodium in the HEX channel or Babesia in the FAM channel. A lower TTime indicated better performance of the tested system. Specimens without a TTime or below the threshold were considered non-reactive.

[0187] The oligomers tested in this example were the T7 oligomer of SEQ ID NO:59, the non-T7 oligomer of SEQ ID NO:30, and the torch probe of SEQ ID NO:123. The results are shown in Table 18. Real-time TMA results with the tested oligomers showed 100% concordance with the PCR results. Based on a standard curve generated using in vitro Plasmodium infected red blood cells, the samples tested have between 7.14E6 and 2.14E8 parasites / mL in whole blood. No cross-reactivity was observed with Babesia. [Table 18]

[0188] Example 12 Candidate amplification systems were tested using real-time TMA on a fully automated Panther system equipped with a real-time fluorometer (Hologic Inc.). Specimens consisted of five strains of Plasmodium-infected erythrocytes: S 05 F Benin I, US 05 F Santa Lucia, US 08 F Nigeria XII, US05 F FC27 / A3, and US05 F PH1. Infected erythrocytes were provided with an estimated parasite / mL value. Each strain was serially diluted in normal negative human whole blood to an estimate of 10 parasites / mL. Diluted Plasmodium-infected whole blood was manually lysed in a ratio of 1 mL of whole blood in 3 mL of lysis buffer (14 mM sodium bicarbonate, 250 mM ammonium chloride, 5% (v / v) LLS, and 0.1 mM EDTA, pH 7.4). Specimens were tested on the Panther system. The assay reagents used included the following: TCR, composed of at least one TCO and one T7 promoter provider, amplification reagent, composed of at least one non-T7 primer, promoter reagent (phenol red-free amplification reagent), composed of at least one T7 promoter provider and at least one fluorescently labeled molecular torch or beacon (5'-hexochloro-fluorescein (HEX) for Plasmodium, or 5'-fluorescein (FAM) for Babesia), and enzyme reagent.

[0189] The Panther RT-Dev Tool (Hologic Inc.) was used for analysis of raw data exported from the Panther system. Fluorescence curves were analyzed using a threshold of 1,000 relative fluorescence units (RFU). The time (TTime) at which a specimen met or exceeded the threshold was determined by the software. Specimens with a TTime were considered reactive for Plasmodium in the HEX channel or Babesia in the FAM channel. A lower TTime indicated better performance of the tested system. Specimens without a TTime or below the threshold were considered non-reactive.

[0190] The oligomers tested in this example were a T7 oligomer of SEQ ID NO: 59, a non-T7 oligomer of SEQ ID NO: 30, and a Torch probe of SEQ ID NO: 123. Results showed that all five strains of Plasmodium infected erythrocytes were detected in six of six replicates tested at 10 parasites / mL, with fluorescence curves producing TTimes ranging from 14.29 to 17.17 minutes. [Table 19-1] [Table 19-2] [Table 19-3] [Table 19-4] [Table 19-5] [Table 19-6] [Table 19-7]

[0191] Embodiment Embodiment 1. A method for specifically detecting Plasmodium species nucleic acid in a sample, comprising: (1) contacting a 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) (i) is about 14 to about 20 contiguous nucleotides in length and is contained within the sequence of SEQ ID NO: 162 and includes the sequence of SEQ ID NO: 163; or (ii) an amplification oligomer comprising a target hybridizing sequence that is about 14 to about 25 contiguous nucleotides in length, contained within the sequence of SEQ ID NO: 166, and including the sequence of SEQ ID NO: 167 or SEQ ID NO: 168; and (b) contacting an amplification oligomer comprising a target hybridizing sequence that is about 15 to about 33 contiguous nucleotides in length and is contained in SEQ ID NO: 169, and includes a sequence of SEQ ID NO: 171, SEQ ID NO: 172, or SEQ ID NO: 173; (2) performing an in vitro nucleic acid amplification reaction, in which any Plasmodium target nucleic acid present in said sample is used as a template to generate an amplification product; (3) detecting the presence or absence of an amplification product, thereby indicating the presence or absence of a Plasmodium species target nucleic acid in said sample.

[0192] Embodiment 2. The method of embodiment 1, wherein the at least two amplification oligomers comprise an amplification oligomer of (a)(i).

[0193] The method of embodiment 2, wherein the target hybridizing sequence of embodiment 3 (a)(i) is selected from the group consisting of SEQ ID NOs: 21, 23-25, 32, 33, 35, 54, and 55.

[0194] Embodiment 4. The method of embodiment 2, wherein the target hybridizing sequence of (a)(i) is contained within the sequence of SEQ ID NO: 164 and includes the sequence of SEQ ID NO: 165.

[0195] Embodiment 5. The method of embodiment 4, wherein the target hybridizing sequence of (a)(i) is selected from the group consisting of SEQ ID NOs: 21, 23-25, 32, 33, and 35.

[0196] Embodiment 6. The method of embodiment 1, wherein the at least two amplification oligomers comprise an amplification oligomer of (a)(ii).

[0197] Embodiment 7. The method of embodiment 6, wherein the target hybridizing sequence of (a)(ii) comprises the sequence of SEQ ID NO: 167.

[0198] Embodiment 8. The method of embodiment 7, wherein the target hybridizing sequence of (a)(ii) is selected from the group consisting of SEQ ID NOs: 28-31, 34, 40, 41, and 49-51.

[0199] Embodiment 9. The method of embodiment 6, wherein the target hybridizing sequence of (a)(ii) comprises the sequence of SEQ ID NO: 168.

[0200] Embodiment 10. The method of embodiment 9, wherein the target hybridizing sequence of (a)(ii) is selected from the group consisting of SEQ ID NOs: 38, 39, 43, 44, and 53.

[0201] The method according to any one of the preceding embodiments, wherein the target hybridizing sequence of embodiment 11 (b) is selected from the group consisting of SEQ ID NOs: 80-82 and 85-100.

[0202] The method of any one of embodiments 1 to 10, wherein the target hybridizing sequence of embodiment 12 (b) is contained in SEQ ID NO: 170, and includes the sequence of SEQ ID NO: 171 or SEQ ID NO: 172.

[0203] The method of embodiment 12, wherein the target hybridizing sequence of embodiment 13 (b) comprises the sequence of SEQ ID NO: 171.

[0204] The method of embodiment 13, wherein the target hybridizing sequence of embodiment 14 (b) is selected from the group consisting of SEQ ID NOs: 81, 82, 85, 87-90, 94, and 96-98.

[0205] The method of embodiment 12, wherein the target hybridizing sequence of embodiment 15 (b) comprises the sequence of SEQ ID NO: 172.

[0206] The method of embodiment 15, wherein the target hybridizing sequence of embodiment 16 (b) is selected from the group consisting of SEQ ID NOs: 80, 82, 85, and 87-100.

[0207] Embodiment 17. The method of any one of embodiments 1 to 16, wherein the amplification oligomer of (b) is a promoter primer or a promoter provider, further comprising a promoter sequence located 5' of the target hybridizing sequence of (b).

[0208] Embodiment 18 The method of embodiment 17, wherein the promoter sequence is a T7 promoter sequence.

[0209] Embodiment 19. The method of embodiment 18, wherein the T7 promoter sequence is SEQ ID NO: 179.

[0210] The method of embodiment 19, wherein the amplification oligomer of embodiment 20 (b) comprises a sequence selected from the group consisting of SEQ ID NOs: 57-59 and 62-77.

[0211] Embodiment 21. The target hybridizing sequences of (a) and (b) each comprise: (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.

[0212] Embodiment 22. The method of embodiment 21, wherein the amplification oligomer of (b) is a promoter primer or a promoter provider, further comprising a promoter sequence located 5' of the target hybridizing sequence of (b).

[0213] Embodiment 23 The method of embodiment 22, wherein the promoter sequence is a T7 promoter sequence.

[0214] Embodiment 24 The method of embodiment 23, wherein the T7 promoter sequence is SEQ ID NO: 179.

[0215] Embodiment 25. The method of embodiment 1, wherein the at least two amplification oligomers comprise a first and a second amplification oligomer as in (a).

[0216] Embodiment 26 The method of embodiment 25, wherein the at least two amplification oligomers comprise a first and a second amplification oligomer as in (a)(ii).

[0217] EMBODIMENT 27. a first amplification oligomer as in (a)(ii) comprising a target hybridizing sequence comprising the sequence of SEQ ID NO: 167; 27. The method of embodiment 26, wherein the second amplification oligomer as in (a)(ii) comprises a target hybridizing sequence comprising the sequence of SEQ ID NO: 168.

[0218] Embodiment 28. The method of embodiment 27, wherein the first amplification oligomer comprises the target hybridizing sequence of SEQ ID NO:34 and the second amplification oligomer comprises the target hybridizing sequence of SEQ ID NO:53.

[0219] Embodiment 29. The method of embodiment 25, wherein the at least two amplification oligomers comprise an amplification oligomer as in (a)(i) and an amplification oligomer as in (a)(ii).

[0220] EMBODIMENT 30. (a) an amplification oligomer as in (i) comprising a target hybridizing sequence contained in the sequence of SEQ ID NO: 164 and including the sequence of SEQ ID NO: 165; 30. The method of embodiment 29, wherein the amplification oligomer as in (a)(ii) comprises the sequence of SEQ ID NO: 167.

[0221] Embodiment 31. The method of embodiment 30, wherein the amplification oligomer as in (a)(i) comprises a target hybridizing sequence of SEQ ID NO:21 and the amplification oligomer as in (a)(ii) comprises a target hybridizing sequence of SEQ ID NO:34.

[0222] Embodiment 32. The method of any one of embodiments 1 and 25 to 31, wherein the at least two amplification oligomers comprise a first and a second amplification oligomer of (b).

[0223] The method of embodiment 32, wherein each of the first and second amplification oligomers of embodiment 33 (b) comprises a target hybridizing sequence contained in the sequence of SEQ ID NO: 170 and including the sequence of SEQ ID NO: 171 or SEQ ID NO: 172.

[0224] Embodiment 34. The method of embodiment 33, wherein the first amplification oligomer as in (b) comprises a target hybridizing sequence of SEQ ID NO: 94, and the second amplification oligomer as in (b) comprises a target hybridizing sequence of SEQ ID NO: 95.

[0225] Embodiment 35. The method of any one of embodiments 32 to 34, wherein each of the amplification oligomers of (b) is a promoter primer or a promoter provider, further comprising a promoter sequence located 5' of the target hybridizing sequence of (b).

[0226] Embodiment 36 The method of embodiment 35, wherein the promoter sequence is a T7 promoter sequence.

[0227] Embodiment 37 The method of embodiment 36, wherein the T7 promoter sequence is SEQ ID NO: 179.

[0228] Embodiment 38. The method according to any one of embodiments 1 to 37, further comprising purifying the target nucleic acid from other components in the sample prior to step (1).

[0229] Embodiment 39. The method of embodiment 38, wherein the purification step comprises contacting the sample with at least one capture probe oligomer comprising a target hybridizing sequence covalently linked to a sequence or moiety that binds to the immobilized probe, said target hybridizing sequence being up to about 30 contiguous nucleotides in length and comprising a sequence selected from the group consisting of SEQ ID NOs: 11-15, 17, 19, and 20 (including DNA equivalents and DNA / RNA chimeras thereof).

[0230] Embodiment 40. The method of embodiment 39, wherein the capture probe oligomer target hybridizing sequence is selected from the group consisting of SEQ ID NOs: 11-15, 17, 19, and 20 (including DNA equivalents and DNA / RNA chimeras thereof).

[0231] Embodiment 41 The method of embodiment 39, wherein the purifying step comprises contacting the sample with at least two capture probe oligomers.

[0232] Embodiment 42. At least two capture probe oligomers are a first capture probe oligomer comprising a target hybridizing sequence of SEQ ID NO: 19; and a second capture probe oligomer comprising the target hybridizing sequence of SEQ ID NO:20.

[0233] Embodiment 43. The method of any one of embodiments 1 to 42, wherein the detecting step (3) comprises contacting the in vitro nucleic acid amplification reaction with at least one detection probe oligomer comprising a target hybridizing sequence configured to specifically hybridize to the amplification product under conditions in which the presence or absence of the amplification product is determined, thereby indicating the presence or absence of the Plasmodium species in said sample.

[0234] Embodiment 44. The method of embodiment 43, wherein the detection probe oligomer target hybridizing sequence is about 13 to about 40 nucleotides in length and (i) is contained in the sequence of SEQ ID NO: 196 or its complement, or its DNA equivalent or DNA / RNA chimera; and (ii) 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 their complements, DNA equivalents, and DNA / RNA equivalents).

[0235] Embodiment 45. The method of embodiment 44, wherein the detection probe oligomer target hybridizing sequence is 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 chimeras thereof).

[0236] Embodiment 46. The method of embodiment 44, wherein the detection probe oligomer target hybridizing sequence is (i) contained in the sequence of SEQ ID NO: 197 or its complement, or its DNA equivalent or DNA / RNA chimera, and (ii) comprises the sequence of SEQ ID NO: 174 or SEQ ID NO: 175 (including its complement, DNA equivalent, and DNA / RNA chimera).

[0237] Embodiment 47. The method of embodiment 46, wherein the detection probe oligomer target hybridizing sequence comprises (ii) SEQ ID NO: 174 or a complement thereof, or a DNA equivalent or a DNA / RNA chimera thereof.

[0238] Embodiment 48. The method of embodiment 47, wherein the detection probe oligomer target hybridizing sequence is selected from the group consisting of SEQ ID NOs: 148-155 and 159 (including complements thereof, DNA equivalents, and DNA / RNA chimeras).

[0239] Embodiment 49. The method of embodiment 46, wherein the detection probe oligomer target hybridizing sequence comprises (ii) SEQ ID NO: 175 or a complement thereof, or a DNA equivalent or a DNA / RNA chimera thereof.

[0240] Embodiment 50. The method of embodiment 49, wherein the detection probe oligomer target hybridizing sequence is selected from the group consisting of SEQ ID NOs: 147, 156, 157, 160, and 161 (including complements, DNA equivalents, and DNA / RNA chimeras thereof).

[0241] Embodiment 51. The method of embodiment 44, wherein the detection probe oligomer target hybridizing sequence comprises (ii) a sequence selected from the group consisting of SEQ ID NO: 177 and SEQ ID NO: 178 (including complements thereof, DNA equivalents, and DNA / RNA chimeras).

[0242] Embodiment 52. The method of embodiment 51, wherein the detection probe oligomer target hybridizing sequence is selected from the group consisting of SEQ ID NOs: 131, 132, 135, 140, 147, 156, 157, 160, and 161 (including complements, DNA equivalents, and DNA / RNA chimeras thereof).

[0243] Embodiment 53 The method of any one of embodiments 43 to 52, wherein the detection probe oligomer comprises a 2' methoxy modification of at least one of the nucleotide residue members of the detection probe oligomer nucleotide sequence.

[0244] Embodiment 54. The amplification oligomer target hybridizing sequence of (a), the amplification oligomer target hybridizing sequence of (b), and the detection probe oligomer target hybridizing sequence each comprise: (A) SEQ ID NO: 30, SEQ ID NO: 82, and SEQ ID NO: 151 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO: 151 or its complement; (B) SEQ ID NO: 30, SEQ ID NO: 82, and SEQ ID NO: 157 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 157 or its complements; (C) SEQ ID NO: 33, SEQ ID NO: 82, and SEQ ID NO: 155 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 155 or its complements; (D) SEQ ID NO: 49, SEQ ID NO: 82, and SEQ ID NO: 150 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 150 or its complements; (E) SEQ ID NO: 49, SEQ ID NO: 82, and SEQ ID NO: 155 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 155 or their complements; (F) SEQ ID NO:21, SEQ ID NO:89, and SEQ ID NO:148 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO:148 or its complements; (G) SEQ ID NO:21, SEQ ID NO:89, and SEQ ID NO:152 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO:152 or its complements; (H) SEQ ID NO: 30, SEQ ID NO: 89, and SEQ ID NO: 148 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 148 or its complements; (I) SEQ ID NO: 30, SEQ ID NO: 89, and SEQ ID NO: 152 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO: 152 or its complement; (I) SEQ ID NO: 33, SEQ ID NO: 89, and SEQ ID NO: 158 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 158 or its complements; (K) SEQ ID NO: 49, SEQ ID NO: 89, and SEQ ID NO: 150 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 150 or its complements; (L) SEQ ID NO: 21, SEQ ID NO: 92, and SEQ ID NO: 148 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 148 or its complements; (M) SEQ ID NO: 21, SEQ ID NO: 92, and SEQ ID NO: 152 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 152 or its complements; (N) SEQ ID NO: 30, SEQ ID NO: 92, and SEQ ID NO: 148 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO: 148 or its complement; (O) SEQ ID NO: 30, SEQ ID NO: 92, and SEQ ID NO: 152 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 152 or their complements; (P) SEQ ID NO: 21, SEQ ID NO: 94, and SEQ ID NO: 148 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 148 or its complements; (Q) SEQ ID NO: 21, SEQ ID NO: 94, and SEQ ID NO: 152 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 152 or its complements; (R) SEQ ID NO: 34, SEQ ID NO: 94, and SEQ ID NO: 148 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 148 or its complements; (S) SEQ ID NO: 34, SEQ ID NO: 94, and SEQ ID NO: 152 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO: 152 or its complement; (T) SEQ ID NO: 34, SEQ ID NO: 94, and SEQ ID NO: 157 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO: 157 or its complement; (U) SEQ ID NO: 53, SEQ ID NO: 94, and SEQ ID NO: 148 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 148 or its complements; (V) SEQ ID NO: 53, SEQ ID NO: 94, and SEQ ID NO: 152 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO: 152 or its complement; (W) SEQ ID NO: 53, SEQ ID NO: 94, and SEQ ID NO: 157 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO: 157 or its complement; (X) SEQ ID NO: 21, SEQ ID NO: 95, and SEQ ID NO: 148 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 148 or its complements; (Y) SEQ ID NO: 21, SEQ ID NO: 95, and SEQ ID NO: 152 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 152 or its complements; (Z) SEQ ID NO: 34, SEQ ID NO: 95, and SEQ ID NO: 148 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 148 or its complements; (AA) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO:152 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO:152 or its complements; (AB) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO:157 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO:157 or its complements; (AC) SEQ ID NO:53, SEQ ID NO:95, and SEQ ID NO:148 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO:148 or its complements; (AD) SEQ ID NO:53, SEQ ID NO:95, and SEQ ID NO:152 or their complements, or the DNA equivalents or DNA / RNA chimeras of SEQ ID NO:152 or their complements; or The method of any one of embodiments 43 to 53, wherein the nucleic acid sequence is selected from the group consisting of SEQ ID NO: 53, SEQ ID NO: 95, and SEQ ID NO: 157 or its complement, or a DNA equivalent or a DNA / RNA chimera of SEQ ID NO: 157 or its complement.

[0245] Embodiment 55. The method of any one of embodiments 43 to 54, wherein the detection probe oligomer comprises a detectable label.

[0246] Embodiment 56 The method of embodiment 55, wherein the detectable label is a chemiluminescent label or a fluorescent label.

[0247] Embodiment 57. The method of embodiment 56, wherein the detectable label is a chemiluminescent acridinium ester (AE) compound linked between the two nucleobases of the detection probe oligomer.

[0248] Embodiment 58. The method of any one of embodiments 1 to 57, wherein the detecting step (3) occurs during the amplifying step (2).

[0249] Embodiment 59. The method of embodiment 56 or 58, wherein the detection probe comprises a fluorescent label and a quencher.

[0250] Embodiment 60. The method of embodiment 59, wherein the detection probe is selected from the group consisting of a molecular torch, a molecular beacon, and a TaqMan detection probe.

[0251] Embodiment 61 The method of any one of embodiments 43 to 60, wherein the detection probe further comprises a non-target hybridizing sequence.

[0252] Embodiment 62. The method of embodiment 61, wherein the detection probe is a molecular torch or a molecular beacon.

[0253] Embodiment 63 The method of embodiment 43, wherein the at least one detection probe oligomer comprises at least two detection probe oligomers.

[0254] Embodiment 64. The at least two detection probe oligomers include a first and a second detection probe oligomer; (A) a first detection probe oligomer comprises a target hybridizing sequence that comprises (i) the sequence of SEQ ID NO: 197 or its complement, or a DNA equivalent or DNA / RNA chimera thereof, and (ii) the sequence of SEQ ID NO: 175 or its complement, or a DNA equivalent or DNA / RNA chimera thereof; (B) The method of embodiment 44, wherein the second detection probe oligomer comprises a target hybridizing sequence that comprises (i) the sequence of SEQ ID NO: 197 or its complement, or a DNA equivalent or DNA / RNA chimera thereof, and (ii) the sequence of SEQ ID NO: 176 or its complement, or a DNA equivalent or DNA / RNA chimera thereof.

[0255] 65. the first detection probe oligomer comprises the target hybridizing sequence of SEQ ID NO: 157 or its complement, or a DNA equivalent or a DNA / RNA chimera thereof; 65. The method of embodiment 64, wherein the second detection probe oligomer comprises a target hybridizing sequence selected from the group consisting of SEQ ID NO: 148 and SEQ ID NO: 152 (including complements thereof, DNA equivalents, and DNA / RNA chimeras).

[0256] Embodiment 66. The method according to any one of embodiments 1 to 65, wherein the amplification reaction in step (2) is an isothermal amplification reaction.

[0257] Embodiment 67. The method of embodiment 66, wherein the amplification reaction is a transcription-mediated amplification (TMA) reaction.

[0258] Embodiment 68. The method according to any one of embodiments 1 to 67, wherein the amplification reaction is a real-time amplification reaction.

[0259] Embodiment 69. The method of any one of embodiments 1 to 68, wherein the sample is a clinical sample.

[0260] Embodiment 70. The method of any one of embodiments 1 to 69, wherein the sample is a blood sample.

[0261] Embodiment 71. The method of embodiment 70, wherein the sample is a lysed blood cell sample, and optionally the sample is a lysed red blood cell sample.

[0262] Embodiment 72. The method of embodiment 70, wherein the sample is a red blood cell sample.

[0263] Embodiment 73. A combination of at least two oligomers for determining the presence or absence of Plasmodium species in a sample, said combination of oligomers comprising at least two oligomers for amplifying a target region of a Plasmodium species target nucleic acid, wherein the at least two amplification oligomers are: (a) (i) is about 14 to about 20 contiguous nucleotides in length and is contained within the sequence of SEQ ID NO: 162 and includes the sequence of SEQ ID NO: 163; or (ii) an amplification oligomer having a length of about 14 to about 25 contiguous nucleotides, the amplification oligomer comprising a target hybridizing sequence contained in the sequence of SEQ ID NO: 166, the target hybridizing sequence comprising the sequence of SEQ ID NO: 167 or SEQ ID NO: 168; (b) an amplification oligomer comprising a target hybridizing sequence having a length of about 15 to about 33 contiguous nucleotides, the target hybridizing sequence being contained in SEQ ID NO: 169 and comprising the sequence of SEQ ID NO: 171, SEQ ID NO: 172, or SEQ ID NO: 173.

[0264] Embodiment 74. The combination of oligomers according to embodiment 73, wherein at least two amplification oligomers comprise an amplification oligomer of (a)(i).

[0265] Embodiment 75. The combination of oligomers described in embodiment 74, wherein the target hybridizing sequences of (a)(i) are selected from the group consisting of SEQ ID NOs: 21, 23-25, 32, 33, 35, 54, and 55.

[0266] Embodiment 76. A combination of oligomers according to embodiment 74, wherein the target hybridizing sequence of (a)(i) is contained in the sequence of SEQ ID NO: 164 and comprises the sequence of SEQ ID NO: 165.

[0267] Embodiment 77. The combination of oligomers described in embodiment 76, wherein the target hybridizing sequences of (a)(i) are selected from the group consisting of SEQ ID NOs: 21, 23-25, 32, 33, and 35.

[0268] Embodiment 78. The combination of oligomers according to embodiment 73, wherein at least two amplification oligomers comprise an amplification oligomer of (a)(ii).

[0269] Embodiment 79. A combination of oligomers according to embodiment 78, wherein the target hybridizing sequence of (a)(ii) comprises the sequence of SEQ ID NO: 167.

[0270] Embodiment 80. The combination of oligomers according to embodiment 79, wherein the target hybridizing sequences of (a)(ii) are selected from the group consisting of SEQ ID NOs: 28-31, 34, 40, 41, and 49-51.

[0271] Embodiment 81. The combination of oligomers described in embodiment 78, wherein the target hybridizing sequence of (a)(ii) comprises the sequence of SEQ ID NO: 168.

[0272] Embodiment 82. The combination of oligomers according to embodiment 81, wherein the target hybridizing sequences of (a)(ii) are selected from the group consisting of SEQ ID NOs: 38, 39, 43, 44, and 53.

[0273] A combination of oligomers according to any one of embodiments 73 to 82, wherein the target hybridizing sequence of embodiment 83 (b) is selected from the group consisting of SEQ ID NOs: 80 to 82 and 85 to 100.

[0274] Embodiment 84. A combination of oligomers according to any one of embodiments 73 to 82, wherein the target hybridizing sequence of (b) is contained in SEQ ID NO: 170 and includes the sequence of SEQ ID NO: 171 or SEQ ID NO: 172.

[0275] Embodiment 85. A combination of oligomers according to embodiment 84, wherein the target hybridizing sequence of (b) comprises the sequence of SEQ ID NO: 171.

[0276] The combination of oligomers according to embodiment 85, wherein the target hybridizing sequence of embodiment 86 (b) is selected from the group consisting of SEQ ID NOs: 81, 82, 85, 87-90, 94, and 96-98.

[0277] Embodiment 87. A combination of oligomers according to embodiment 84, wherein the target hybridizing sequence of (b) comprises the sequence of SEQ ID NO: 172.

[0278] The combination of oligomers according to embodiment 87, wherein the target hybridizing sequence of embodiment 88 (b) is selected from the group consisting of SEQ ID NOs: 80, 82, 85, and 87-100.

[0279] Embodiment 89. A combination of oligomers according to any one of embodiments 1 to 88, wherein the amplification oligomer of (b) is a promoter primer or a promoter provider, further comprising a promoter sequence located 5' of the target hybridizing sequence of (b).

[0280] Embodiment 90. The combination of oligomers according to embodiment 89, wherein the promoter sequence is a T7 promoter sequence.

[0281] Embodiment 91. A combination of oligomers according to embodiment 90, wherein the T7 promoter sequence is SEQ ID NO: 179.

[0282] A combination of oligomers according to embodiment 91, wherein the amplification oligomer of embodiment 92 (b) comprises a sequence selected from the group consisting of SEQ ID NOs: 57-59 and 62-77.

[0283] Embodiment 93. The target hybridizing sequences of (a) and (b) each comprise: (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) A combination of oligomers according to embodiment 73, which are SEQ ID NO: 53 and SEQ ID NO: 95.

[0284] Embodiment 94. The combination of oligomers according to embodiment 93, wherein the amplification oligomer of (b) is a promoter primer or a promoter provider, further comprising a promoter sequence located 5' of the target hybridizing sequence of (b).

[0285] Embodiment 95. The combination of oligomers according to embodiment 94, wherein the promoter sequence is a T7 promoter sequence.

[0286] Embodiment 96. A combination of oligomers according to embodiment 95, wherein the T7 promoter sequence is SEQ ID NO: 179.

[0287] Embodiment 97. The combination of oligomers according to embodiment 73, wherein the at least two amplification oligomers comprise a first and a second amplification oligomer as in (a).

[0288] Embodiment 98. The combination of oligomers according to embodiment 97, wherein the at least two amplification oligomers comprise a first and a second amplification oligomer as in (a)(ii).

[0289] EMBODIMENT 99. a first amplification oligomer as in (a)(ii) comprising a target hybridizing sequence comprising the sequence of SEQ ID NO: 167; The combination of oligomers described in embodiment 98, wherein the second amplification oligomer as in (a)(ii) comprises a target hybridizing sequence comprising the sequence of SEQ ID NO: 168.

[0290] Embodiment 100. The combination of oligomers according to embodiment 99, wherein the first amplification oligomer comprises the target hybridizing sequence of SEQ ID NO:34 and the second amplification oligomer comprises the target hybridizing sequence of SEQ ID NO:53.

[0291] Embodiment 101. The combination of oligomers according to embodiment 97, wherein the at least two amplification oligomers comprise an amplification oligomer as in (a)(i) and an amplification oligomer as in (a)(ii).

[0292] EMBODIMENT 102. (a) an amplification oligomer as in (i) comprising a target hybridizing sequence contained in the sequence of SEQ ID NO: 164 and including the sequence of SEQ ID NO: 165; 102. The combination of oligomers described in embodiment 101, wherein the amplification oligomer as in (a)(ii) comprises the sequence of SEQ ID NO: 167.

[0293] Embodiment 103. A combination of oligomers according to embodiment 102, wherein the amplification oligomer as in (a)(i) comprises a target hybridizing sequence of SEQ ID NO:21 and the amplification oligomer as in (a)(ii) comprises a target hybridizing sequence of SEQ ID NO:34.

[0294] Embodiment 104. A combination of oligomers according to any one of embodiments 73 and 97 to 103, wherein the at least two amplification oligomers comprise a first and a second amplification oligomer of (b).

[0295] Embodiment 105. A combination of oligomers according to embodiment 104, wherein each of the first and second amplification oligomers of (b) is contained in SEQ ID NO: 170 and comprises a target hybridizing sequence comprising the sequence of SEQ ID NO: 171 or SEQ ID NO: 172.

[0296] Embodiment 106. A combination of oligomers according to embodiment 105, wherein the first amplification oligomer as in (b) comprises a target hybridizing sequence of SEQ ID NO: 94, and the second amplification oligomer as in (b) comprises a target hybridizing sequence of SEQ ID NO: 95.

[0297] Embodiment 107. The combination of oligomers according to embodiment 93, wherein each of the amplification oligomers of (b) is a promoter primer or a promoter provider, further comprising a promoter sequence located 5' of the target hybridizing sequence of (b).

[0298] Embodiment 108. A combination of oligomers according to embodiment 107, wherein the promoter sequence is a T7 promoter sequence.

[0299] Embodiment 109. A combination of oligomers according to embodiment 108, wherein the T7 promoter sequence is SEQ ID NO: 179.

[0300] Embodiment 110. A combination of oligomers according to any one of embodiments 1 to 109, further comprising at least one capture probe oligomer comprising a target hybridizing sequence covalently linked to a sequence or moiety that binds to the immobilized probe, said target hybridizing sequence being up to about 30 contiguous nucleotides in length and comprising a sequence selected from the group consisting of SEQ ID NOs: 11 to 15, 17, 19, and 20 (including DNA equivalents and DNA / RNA chimeras thereof).

[0301] Embodiment 111. The combination of oligomers according to embodiment 110, wherein the capture probe oligomer target hybridizing sequences are selected from the group consisting of SEQ ID NOs: 11-15, 17, 19, and 20 (including DNA equivalents and DNA / RNA chimeras thereof).

[0302] Embodiment 112. The oligomer combination of embodiment 110, wherein the oligomer combination comprises at least two capture probe oligomers.

[0303] Embodiment 113. At least two capture probe oligomers are a first capture probe oligomer comprising a target hybridizing sequence of SEQ ID NO: 19, or its DNA equivalent or DNA / RNA chimera; and a second capture probe oligomer comprising the target hybridizing sequence of SEQ ID NO: 20, or a DNA equivalent or a DNA / RNA chimera thereof.

[0304] Embodiment 114. A combination of oligomers according to any one of embodiments 1 to 113, further comprising at least one detection probe oligomer comprising a target hybridizing sequence configured to specifically hybridize to a Plasmodium species amplicon amplifiable by at least two amplification oligomers.

[0305] Embodiment 115. The combination of oligomers described in embodiment 114, wherein the detection probe oligomer target hybridizing sequence is about 13 to about 40 nucleotides in length and comprises (i) the sequence of SEQ ID NO: 196 or its complement, or its DNA equivalent or DNA / RNA chimera, and (ii) 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 their complements, DNA equivalents, and DNA / RNA equivalents).

[0306] Embodiment 116. The combination of oligomers according to embodiment 115, wherein the detection probe oligomer target hybridizing sequences are 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 equivalents thereof).

[0307] Embodiment 117. The combination of oligomers according to embodiment 115, wherein the detection probe oligomer target hybridizing sequence comprises (i) a sequence of SEQ ID NO: 197 or its complement, or a DNA equivalent or a DNA / RNA chimera thereof, and (ii) a sequence selected from the group consisting of SEQ ID NO: 174 and SEQ ID NO: 175 (including their complements, DNA equivalents, and DNA / RNA equivalents).

[0308] Embodiment 118. A combination of oligomers according to embodiment 117, wherein the detection probe oligomer target hybridizing sequence comprises (ii) the sequence of SEQ ID NO: 174 or its complement, or a DNA equivalent or a DNA / RNA chimera thereof.

[0309] Embodiment 119. The combination of oligomers according to embodiment 118, wherein the detection probe oligomer target hybridizing sequences are selected from the group consisting of SEQ ID NOs: 148-155 and 159 (including complements, DNA equivalents, and DNA / RNA equivalents thereof).

[0310] Embodiment 120. A combination of oligomers according to embodiment 117, wherein the detection probe oligomer target hybridizing sequence comprises (ii) the sequence of SEQ ID NO: 175 or its complement, or a DNA equivalent or a DNA / RNA chimera thereof.

[0311] Embodiment 121. The combination of oligomers according to embodiment 120, wherein the detection probe oligomer target hybridizing sequences are selected from the group consisting of SEQ ID NOs: 147, 156, 157, 160, and 161 (including their complements, DNA equivalents, and DNA / RNA equivalents).

[0312] Embodiment 122. The combination of oligomers according to embodiment 115, wherein the detection probe oligomer target hybridizing sequence comprises (ii) a sequence selected from the group consisting of SEQ ID NO: 177 and SEQ ID NO: 178 (including their complements, DNA equivalents, and DNA / RNA equivalents).

[0313] Embodiment 123. The combination of oligomers according to embodiment 122, wherein the detection probe oligomer target hybridizing sequences are selected from the group consisting of SEQ ID NOs: 131, 132, 135, 140, 147, 156, 157, 160, and 161 (including complements, DNA equivalents, and DNA / RNA equivalents thereof).

[0314] Embodiment 124. A combination of oligomers according to any one of embodiments 114 to 123, wherein the detection probe oligomer comprises a 2' methoxy modification of at least one of the nucleotide residue members of the detection probe oligomer nucleotide sequence.

[0315] Embodiment 125. The amplification oligomer target hybridizing sequence of (a), the amplification oligomer target hybridizing sequence of (b), and the detection probe oligomer target hybridizing sequence each comprise: (A) SEQ ID NO: 30, SEQ ID NO: 82, and SEQ ID NO: 151 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO: 151 or its complement; (B) SEQ ID NO: 30, SEQ ID NO: 82, and SEQ ID NO: 157 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 157 or its complements; (C) SEQ ID NO: 33, SEQ ID NO: 82, and SEQ ID NO: 155 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 155 or its complements; (D) SEQ ID NO: 49, SEQ ID NO: 82, and SEQ ID NO: 150 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 150 or its complements; (E) SEQ ID NO: 49, SEQ ID NO: 82, and SEQ ID NO: 155 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 155 or their complements; (F) SEQ ID NO:21, SEQ ID NO:89, and SEQ ID NO:148 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO:148 or its complements; (G) SEQ ID NO:21, SEQ ID NO:89, and SEQ ID NO:152 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO:152 or its complements; (H) SEQ ID NO: 30, SEQ ID NO: 89, and SEQ ID NO: 148 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 148 or its complements; (I) SEQ ID NO: 30, SEQ ID NO: 89, and SEQ ID NO: 152 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO: 152 or its complement; (I) SEQ ID NO: 33, SEQ ID NO: 89, and SEQ ID NO: 158 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 158 or its complements; (K) SEQ ID NO: 49, SEQ ID NO: 89, and SEQ ID NO: 150 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 150 or its complements; (L) SEQ ID NO: 21, SEQ ID NO: 92, and SEQ ID NO: 148 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 148 or its complements; (M) SEQ ID NO: 21, SEQ ID NO: 92, and SEQ ID NO: 152 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 152 or its complements; (N) SEQ ID NO: 30, SEQ ID NO: 92, and SEQ ID NO: 148 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO: 148 or its complement; (O) SEQ ID NO: 30, SEQ ID NO: 92, and SEQ ID NO: 152 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 152 or their complements; (P) SEQ ID NO: 21, SEQ ID NO: 94, and SEQ ID NO: 148 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 148 or its complements; (Q) SEQ ID NO: 21, SEQ ID NO: 94, and SEQ ID NO: 152 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 152 or its complements; (R) SEQ ID NO: 34, SEQ ID NO: 94, and SEQ ID NO: 148 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 148 or its complements; (S) SEQ ID NO: 34, SEQ ID NO: 94, and SEQ ID NO: 152 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO: 152 or its complement; (T) SEQ ID NO: 34, SEQ ID NO: 94, and SEQ ID NO: 157 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO: 157 or its complement; (U) SEQ ID NO: 53, SEQ ID NO: 94, and SEQ ID NO: 148 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 148 or its complements; (V) SEQ ID NO: 53, SEQ ID NO: 94, and SEQ ID NO: 152 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO: 152 or its complement; (W) SEQ ID NO: 53, SEQ ID NO: 94, and SEQ ID NO: 157 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO: 157 or its complement; (X) SEQ ID NO: 21, SEQ ID NO: 95, and SEQ ID NO: 148 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 148 or its complements; (Y) SEQ ID NO: 21, SEQ ID NO: 95, and SEQ ID NO: 152 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 152 or its complements; (Z) SEQ ID NO: 34, SEQ ID NO: 95, and SEQ ID NO: 148 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 148 or its complements; (AA) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO:152 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO:152 or its complements; (AB) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO:157 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO:157 or its complements; (AC) SEQ ID NO:53, SEQ ID NO:95, and SEQ ID NO:148 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO:148 or its complements; (AD) SEQ ID NO:53, SEQ ID NO:95, and SEQ ID NO:152 or their complements, or the DNA equivalents or DNA / RNA chimeras of SEQ ID NO:152 or their complements; or (AE) A combination of oligomers according to embodiments 114 to 124, which is SEQ ID NO: 53, SEQ ID NO: 95, and SEQ ID NO: 157 or its complement, or a DNA equivalent or a DNA / RNA chimera of SEQ ID NO: 157 or its complement.

[0316] Embodiment 126. A combination of oligomers according to any one of embodiments 114 to 125, wherein the detection probe oligomer comprises a detectable label.

[0317] Embodiment 127. The oligomeric combination according to embodiment 126, wherein the detectable label is a chemiluminescent or fluorescent label.

[0318] Embodiment 128. The oligomeric combination according to embodiment 127, wherein the detectable label is a chemiluminescent acridinium ester (AE) compound linked between the two nucleobases of the detection probe oligomer.

[0319] Embodiment 129. The oligomer combination according to embodiment 127, wherein the detection probe comprises a fluorescent label and a quencher.

[0320] Embodiment 130. The combination of oligomers according to embodiment 129, wherein the detection probe is selected from the group consisting of molecular torches, molecular beacons, and TaqMan detection probes.

[0321] Embodiment 131. A combination of oligomers according to any one of embodiments 114 to 130, wherein the detection probe further comprises a non-target hybridizing sequence.

[0322] Embodiment 132. The oligomeric combination according to embodiment 131, wherein the detection probe is a molecular torch or a molecular beacon.

[0323] Embodiment 133. The combination of oligomers according to embodiment 114, wherein the at least two detection probe oligomers comprise a first and a second detection probe oligomer.

[0324] Embodiment 134. The at least two detection probe oligomers include a first and a second detection probe oligomer; (A) a first detection probe oligomer comprises a target hybridizing sequence that comprises (i) the sequence of SEQ ID NO: 197 or its complement, or a DNA equivalent or DNA / RNA chimera thereof, and (ii) the sequence of SEQ ID NO: 175 or its complement, or a DNA equivalent or DNA / RNA chimera thereof; (B) The combination of oligomers described in embodiment 115, wherein the second detection probe oligomer comprises a target hybridizing sequence that includes (i) the sequence of SEQ ID NO: 197 or its complement, or a DNA equivalent or DNA / RNA chimera thereof, and (ii) the sequence of SEQ ID NO: 176 or its complement, or a DNA equivalent or DNA / RNA chimera thereof.

[0325] EMBODIMENT 135. the first detection probe oligomer comprises the target hybridizing sequence of SEQ ID NO: 157 or its complement, or a DNA equivalent or a DNA / RNA chimera thereof; The combination of oligomers described in embodiment 134, wherein the second detection probe oligomer comprises a target hybridizing sequence selected from the group consisting of SEQ ID NO: 148 and SEQ ID NO: 152 (including complements thereof, DNA equivalents, and DNA / RNA equivalents).

[0326] Embodiment 136. A detection probe oligomer for specifically detecting a Plasmodium species target nucleic acid in a sample, the detection probe oligomer being about 13 to about 40 nucleotides in length and configured to specifically hybridize to a target sequence contained within a Plasmodium species target region amplifiable by a combination of oligomers comprising a first and a second Plasmodium-specific amplification oligomer; (a) a first amplification oligomer comprises a target hybridizing sequence that is (i) about 14 to about 20 contiguous nucleotides in length, is contained within the sequence of SEQ ID NO: 162, and comprises the sequence of SEQ ID NO: 163, or (ii) about 14 to about 25 contiguous nucleotides in length, is contained within the sequence of SEQ ID NO: 166, and comprises the sequence of SEQ ID NO: 167 or SEQ ID NO: 168; (b) a detection probe oligomer, wherein the second amplification oligomer is about 15 to about 33 contiguous nucleotides in length and comprises a target hybridizing sequence contained in SEQ ID NO:169, comprising the sequence of SEQ ID NO:171, SEQ ID NO:172, or SEQ ID NO:173.

[0327] Embodiment 137. The detection probe oligomer according to embodiment 136, wherein the detection probe oligomer target hybridizing sequence is (i) contained in the sequence of SEQ ID NO: 196 or its complement, or its DNA equivalent or DNA / RNA chimera, and (ii) 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 their complements, DNA equivalents, and DNA / RNA equivalents).

[0328] Embodiment 138. The detection probe oligomer according to embodiment 137, wherein the detection probe oligomer target hybridizing sequence is selected from the group consisting of SEQ ID NOs: 131, 132, 135, 140, 145, 147-157, and 159-161 (including complements thereof, DNA equivalents, and DNA / RNA chimeras).

[0329] Embodiment 139. A detection probe oligomer according to embodiment 137, wherein the detection probe oligomer target hybridizing sequence is (i) contained in the sequence of SEQ ID NO: 197 or its complement, or its DNA equivalent or DNA / RNA chimera, and (ii) comprises the sequence of SEQ ID NO: 174 or SEQ ID NO: 175 (including its complement, DNA equivalent, and DNA / RNA chimera).

[0330] Embodiment 140. A detection probe oligomer according to embodiment 139, wherein the detection probe oligomer target hybridizing sequence comprises (ii) the sequence of SEQ ID NO: 174 or its complement, or a DNA equivalent or a DNA / RNA chimera thereof.

[0331] Embodiment 141. The detection probe oligomer of embodiment 140, wherein the detection probe oligomer target hybridizing sequence is selected from the group consisting of SEQ ID NOs: 148-155 and 159 (including complements thereof, DNA equivalents, and DNA / RNA chimeras).

[0332] Embodiment 142. A detection probe oligomer according to embodiment 139, wherein the detection probe oligomer target hybridizing sequence comprises (ii) the sequence of SEQ ID NO: 175 or its complement, or a DNA equivalent or a DNA / RNA chimera thereof.

[0333] Embodiment 143. The detection probe oligomer according to embodiment 142, wherein the detection probe oligomer target hybridizing sequence is selected from the group consisting of SEQ ID NOs: 147, 156, 157, 160, and 161 (including complements thereof, DNA equivalents, and DNA / RNA chimeras).

[0334] Embodiment 144. The detection probe oligomer of embodiment 137, wherein the detection probe oligomer target hybridizing sequence comprises (ii) a sequence selected from the group consisting of SEQ ID NO: 177 and SEQ ID NO: 178 (including complements thereof, DNA equivalents, and DNA / RNA chimeras).

[0335] Embodiment 145. The detection probe oligomer according to embodiment 144, wherein the detection probe oligomer target hybridizing sequence is selected from the group consisting of SEQ ID NOs: 131, 132, 135, 140, 147, 156, 157, 160, and 161 (including complements thereof, DNA equivalents, and DNA / RNA chimeras).

[0336] Embodiment 146. A detection probe oligomer according to any one of embodiments 136 to 145, wherein the detection probe oligomer comprises a 2' methoxy modification of at least one of the nucleotide residue members of the detection probe oligomer nucleotide sequence.

[0337] Embodiment 147. A detection probe oligomer according to any one of embodiments 136 to 146, wherein the detection probe oligomer comprises a label.

[0338] Embodiment 148. The detection probe oligomer according to embodiment 147, wherein the label is a chemiluminescent or fluorescent label.

[0339] Embodiment 149. The detection probe oligomer according to embodiment 148, wherein the label is a chemiluminescent acridinium ester (AE) compound linked between the two nucleobases of the detection probe oligomer.

[0340] Embodiment 150. The detection probe oligomer of embodiment 148, wherein the detection probe comprises a fluorescent label and a quencher.

[0341] Embodiment 152. The detection probe oligomer of embodiment 150, wherein the detection probe is selected from the group consisting of a molecular torch, a molecular beacon, and a TaqMan detection probe.

[0342] Embodiment 153. A detection probe oligomer according to any one of embodiments 136 to 152, wherein the detection probe further comprises a non-target hybridizing sequence.

[0343] Embodiment 154. The detection probe oligomer according to embodiment 153, wherein the detection probe is a molecular torch or a molecular beacon.

[0344] Embodiment 155. A combination of at least two oligomers for detecting a Plasmodium species target nucleic acid in a sample, comprising at least two detection probe oligomers according to embodiment 136.

[0345] Embodiment 156. The at least two detection probe oligomers include a first and a second detection probe oligomer; (A) a first detection probe oligomer comprises a target hybridizing sequence that comprises (i) the sequence of SEQ ID NO: 197 or its complement, or a DNA equivalent or DNA / RNA chimera thereof, and (ii) the sequence of SEQ ID NO: 175 or its complement, or a DNA equivalent or DNA / RNA chimera thereof; (B) The combination of oligomers described in embodiment 155, wherein the second detection probe oligomer comprises a target hybridizing sequence that includes (i) the sequence of SEQ ID NO: 197 or its complement, or a DNA equivalent or DNA / RNA chimera thereof, and (ii) the sequence of SEQ ID NO: 176 or its complement, or a DNA equivalent or DNA / RNA chimera thereof.

[0346] 157. the first detection probe oligomer comprises the target hybridizing sequence of SEQ ID NO: 157 or its complement, or a DNA equivalent or a DNA / RNA chimera thereof; The combination of oligomers described in embodiment 156, wherein the second detection probe oligomer comprises a target hybridizing sequence selected from the group consisting of SEQ ID NO: 148 and SEQ ID NO: 152 (including complements thereof, DNA equivalents, and DNA / RNA chimeras).

[0347] Embodiment 158. A capture probe oligomer for specifically isolating Plasmodium species nucleic acids from a sample, said capture probe oligomer comprising a target hybridizing sequence covalently linked to a sequence or moiety that binds to an immobilized probe, said target hybridizing sequence being up to about 30 contiguous nucleotides in length and comprising a sequence selected from the group consisting of SEQ ID NOs: 11-15, 17, 19, and 20 (including DNA equivalents and DNA / RNA chimeras thereof).

[0348] Embodiment 159. The capture probe oligomer of embodiment 158, wherein the capture probe oligomer target hybridizing sequence is selected from the group consisting of SEQ ID NOs: 11-15, 17, 19, and 20 (including DNA equivalents and DNA / RNA chimeras thereof).

[0349] Embodiment 160. A combination of at least two oligomers for specifically isolating Plasmodium species nucleic acids from a sample, comprising at least two capture probe oligomers according to embodiment 158.

[0350] Embodiment 161. At least two capture probe oligomers are a first capture probe oligomer comprising a target hybridizing sequence of SEQ ID NO: 19, or its DNA equivalent or DNA / RNA chimera; and a second capture probe oligomer comprising the target hybridizing sequence of SEQ ID NO: 20, or a DNA equivalent or a DNA / RNA chimera thereof.

[0351] Embodiment 162. A kit comprising a combination of at least two oligomers according to any one of embodiments 73 to 135, 155 to 157, 160, and 161.

[0352] Embodiment 163. A reaction mixture comprising a combination of at least two oligomers according to any one of embodiments 73-135, 155-157, 160, and 161.

[0353] Embodiment 164. Use of a combination of at least two oligomers according to any one of embodiments 73 to 135 for specifically amplifying Plasmodium species nucleic acids in a sample.

[0354] Embodiment 165. Use of a detection probe oligomer or a combination of oligomers according to any one of embodiments 136 to 157 for specifically detecting Plasmodium species nucleic acids in a sample.

[0355] Embodiment 166. Use of a capture probe oligomer or a combination of oligomers according to any one of embodiments 158 to 161 for specifically capturing Plasmodium species nucleic acids from a sample.

[0356] Embodiment 167. A method for specifically detecting Plasmodium species nucleic acids in a sample, comprising: (1) contacting a 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) an amplification oligomer comprising a target hybridizing sequence contained in the sequence of SEQ ID NO: 185, and comprising the sequence of SEQ ID NO: 37, SEQ ID NO: 46, or SEQ ID NO: 187; and (b) contacting an amplification oligomer comprising a target hybridizing sequence contained in SEQ ID NO: 188, the target hybridizing sequence comprising a sequence of SEQ ID NO: 83, SEQ ID NO: 84, or SEQ ID NO: 182; (2) performing an in vitro nucleic acid amplification reaction, in which any Plasmodium target nucleic acid present in said sample is used as a template to generate an amplification product; (3) detecting the presence or absence of an amplification product, thereby indicating the presence or absence of a Plasmodium species target nucleic acid in said sample.

[0357] Embodiment 168. The method of embodiment 167, wherein the target hybridizing sequence of (a) is selected from the group consisting of SEQ ID NOs: 37, 46, 183, and 184.

[0358] Embodiment 169. The method of embodiment 168, wherein the target hybridizing sequence of (a) is contained in the sequence of SEQ ID NO: 186.

[0359] Embodiment 170. The method of embodiment 169, wherein the target hybridizing sequence of (a) is SEQ ID NO: 183 or SEQ ID NO: 184.

[0360] The method of any one of embodiments 167 to 170, wherein the target hybridizing sequence of embodiment 171 (b) is selected from the group consisting of SEQ ID NOs: 83, 84, and 182.

[0361] Embodiment 172. The method of embodiment 167, wherein the target hybridizing sequence of (a) is SEQ ID NO: 183 or SEQ ID NO: 184, and the target hybridizing sequence of (b) is SEQ ID NO: 182.

[0362] Embodiment 173. The method according to any one of embodiments 167 to 172, wherein the detecting step (3) comprises contacting the in vitro nucleic acid amplification reaction with at least one detection probe oligomer comprising a target hybridizing sequence configured to specifically hybridize to the amplification product under conditions in which the presence or absence of the amplification product is determined, thereby indicating the presence or absence of the Plasmodium species in said sample.

[0363] Embodiment 174. The method of embodiment 173, wherein the detection probe oligomer target hybridizing sequence is at least about 13 nucleotides in length and (i) is contained in the sequence of SEQ ID NO: 189 or its complement, or its DNA equivalent or DNA / RNA chimera; and (ii) comprises a sequence selected from the group consisting of SEQ ID NO: 190 and SEQ ID NO: 191 (including their complements, DNA equivalents, and DNA / RNA equivalents).

[0364] Embodiment 175. The method of embodiment 174, wherein the detection probe oligomer target hybridizing sequence is selected from the group consisting of SEQ ID NOs: 125-130 and 143 (including complements thereof, DNA equivalents, and DNA / RNA chimeras).

[0365] Embodiment 176. The amplification oligomer target hybridizing sequence of (a), the amplification oligomer target hybridizing sequence of (b), and the detection probe oligomer target hybridizing sequence each comprise: (A) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 126 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 126 or its complements; (B) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 127 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 127 or its complements; (C) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 128 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 128 or its complements; (D) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 143 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 143 or its complements; (E) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 129 or their complements, or the DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 129 or their complements, or (F) The method of embodiment 173, wherein the sequence is SEQ ID NO: 184, SEQ ID NO: 182, and SEQ ID NO: 126 or a complement thereof, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO: 126 or a complement thereof.

[0366] Embodiment 177. A combination of at least two oligomers for determining the presence or absence of Plasmodium species in a sample, said combination of oligomers comprising at least two oligomers for amplifying a target region of a Plasmodium species target nucleic acid, wherein the at least two amplification oligomers are: (a) an amplification oligomer comprising a target hybridizing sequence contained in the sequence of SEQ ID NO: 185, and comprising the sequence of SEQ ID NO: 37, SEQ ID NO: 46, or SEQ ID NO: 187; (b) an amplification oligomer contained in SEQ ID NO:188, the amplification oligomer comprising a target hybridizing sequence comprising the sequence of SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO:182.

[0367] Embodiment 178. A combination of oligomers according to embodiment 177, wherein the target hybridizing sequences of (a) are selected from the group consisting of SEQ ID NOs: 37, 46, 183, and 184.

[0368] Embodiment 179. A combination of oligomers according to embodiment 178, wherein the target hybridizing sequence of (a) is contained in the sequence of SEQ ID NO: 186.

[0369] Embodiment 180. The combination of oligomers according to embodiment 179, wherein the target hybridizing sequence of (a) is SEQ ID NO: 183 or SEQ ID NO: 184.

[0370] Embodiment 181. A combination of oligomers according to any one of embodiments 177 to 180, wherein the target hybridizing sequence of (b) is selected from the group consisting of SEQ ID NOs: 83, 84, and 182.

[0371] Embodiment 182. A combination of oligomers according to embodiment 177, wherein the target hybridizing sequence of (a) is SEQ ID NO: 183 or SEQ ID NO: 184, and the target hybridizing sequence of (b) is SEQ ID NO: 182.

[0372] Embodiment 183. A combination of oligomers according to any one of embodiments 177 to 182, further comprising at least one detection probe oligomer comprising a target hybridizing sequence configured to specifically hybridize to a Plasmodium species amplicon amplifiable by at least two amplification oligomers.

[0373] Embodiment 184. The combination of oligomers described in embodiment 183, wherein the detection probe oligomer target hybridizing sequence is at least about 13 nucleotides in length and comprises (i) a sequence of SEQ ID NO: 189 or its complement, or a DNA equivalent or DNA / RNA chimera thereof, and (ii) a sequence selected from the group consisting of SEQ ID NO: 190 and SEQ ID NO: 191 (including their complements, DNA equivalents, and DNA / RNA equivalents).

[0374] Embodiment 185. The combination of oligomers according to embodiment 174, wherein the detection probe oligomer target hybridizing sequences are selected from the group consisting of SEQ ID NOs: 125-130 and 143 (including complements thereof, DNA equivalents, and DNA / RNA chimeras).

[0375] Embodiment 186. The amplification oligomer target hybridizing sequence of (a), the amplification oligomer target hybridizing sequence of (b), and the detection probe oligomer target hybridizing sequence each comprise: (A) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 126 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 126 or its complements; (B) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 127 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 127 or its complements; (C) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 128 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 128 or its complements; (D) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 143 or their complements, or DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 143 or its complements; (E) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 129 or their complements, or the DNA equivalents or DNA / RNA chimeras of SEQ ID NO: 129 or their complements, or (F) A combination of oligomers according to embodiment 173, which is SEQ ID NO: 184, SEQ ID NO: 182, and SEQ ID NO: 126 or its complement, or a DNA equivalent or DNA / RNA chimera of SEQ ID NO: 126 or its complement.

[0376] Embodiment 187. A detection probe oligomer for specifically detecting a Plasmodium species target nucleic acid in a sample, said detection probe oligomer being at least about 13 nucleotides in length and configured to specifically hybridize to a target sequence contained within a Plasmodium species target region amplifiable by a combination of oligomers comprising a first and a second Plasmodium-specific amplification oligomer; (a) a first amplification oligomer is contained in the sequence of SEQ ID NO: 185 and comprises a target hybridizing sequence comprising the sequence of SEQ ID NO: 37, SEQ ID NO: 46, or SEQ ID NO: 187; (b) a detection probe oligomer, wherein the second amplification oligomer is contained in SEQ ID NO:188 and comprises a target hybridizing sequence comprising the sequence of SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO:182.

[0377] Embodiment 188. A kit comprising a combination of at least two oligomers according to any one of embodiments 177 to 186.

[0378] Embodiment 189. A reaction mixture comprising a combination of at least two oligomers according to any one of embodiments 177 to 186.

[0379] Embodiment 190. Use of a combination of at least two oligomers according to any one of embodiments 177 to 186 for specifically amplifying Plasmodium species nucleic acids in a sample.

[0380] Embodiment 191. Use of a detection probe oligomer or a combination of oligomers according to any one of embodiments 177 to 187 for specifically detecting Plasmodium species nucleic acids in a sample.

[0381] From the foregoing, it will be understood that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

1. 1. A method for specifically detecting Plasmodium species nucleic acid in a sample, comprising: (1) contacting a sample suspected of containing a Plasmodium species nucleic acid with at least two amplification oligomers for amplifying a target region of a Plasmodium species target nucleic acid, the at least two amplification oligomers comprising: (a) (i) is 14 to 20 contiguous nucleotides in length and is contained within the sequence of SEQ ID NO: 162 and includes the sequence of SEQ ID NO: 163; or (ii) at least one first amplification oligomer comprising a target hybridizing sequence that is 14-25 contiguous nucleotides in length, contained within the sequence of SEQ ID NO:166, and including the sequence of SEQ ID NO:167 or SEQ ID NO:168; and (b) contacting at least one second amplification oligomer comprising a target hybridizing sequence that is 15-33 contiguous nucleotides in length and is contained in SEQ ID NO:169, and includes 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, in which any Plasmodium target nucleic acid present in the sample is used as a template to generate an amplification product; (3) detecting the presence or absence of said amplification product, thereby indicating the presence or absence of a Plasmodium sp. target nucleic acid in said sample.

2. the target hybridizing sequence of the at least one first amplification oligomer comprises: (A) selected from the group consisting of SEQ ID NOs: 21, 23-25, 32, 33, 35, 54, and 55; (B) contained in the sequence of SEQ ID NO: 164 and including the sequence of SEQ ID NO: 165; (C) comprising the sequence of SEQ ID NO: 167; (D) selected from the group consisting of SEQ ID NOs: 28-31, 34, 40, 41, and 49-51; (E) comprising the sequence of SEQ ID NO: 168; or (F) the method of claim 1, selected from the group consisting of SEQ ID NOs: 38, 39, 43, 44, and 53.

3. the target-hybridizing sequence of the at least one second amplification oligomer comprises: (A) selected from the group consisting of SEQ ID NOs: 80-82 and 85-100; or (B) The method of claim 1 or 2, comprising the sequence of SEQ ID NO: 170, including the sequence of SEQ ID NO: 171 or SEQ ID NO:

172.

4. The second amplification oligomer comprises: (A) a promoter primer, (B) a promoter provider comprising a promoter sequence located 5' to the target hybridizing sequence; (C) a promoter provider comprising a promoter sequence located 5' of the target hybridizing sequence, the promoter sequence being a T7 promoter sequence; or (D) a sequence selected from the group consisting of SEQ ID NOs: 57-59 and 62-77.

5. the target-hybridizing sequences of the first and second amplification oligomers each comprise: (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. The method of claim 1,

6. The at least one first amplification oligomer comprises at least two first amplification oligomers, the at least two first amplification oligomers comprising: (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 of 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 of SEQ ID NO: 53, an amplification oligomer comprising a target hybridizing sequence contained in the sequence of SEQ ID NO: 164 and comprising the sequence of SEQ ID NO: 165, or an amplification oligomer comprising the target hybridizing sequence of SEQ ID NO:

21.

7. The at least one second amplification oligomer comprises at least two second amplification oligomers, the at least two second amplification oligomers comprising: The method of claim 1 or 6, wherein the amplification oligomer comprises the target hybridizing sequence of SEQ ID NO:94, and the amplification oligomer comprises the target hybridizing sequence of SEQ ID NO:

95.

8. The method of claim 1, further comprising a purification step for purifying the target nucleic acid from other components in the sample, the purification step comprising contacting the sample with at least one capture probe oligomer comprising a target hybridizing sequence covalently attached to a moiety that binds to an immobilized probe, the target hybridizing sequence being up to 30 contiguous nucleotides in length and comprising a sequence selected from the group consisting of SEQ ID NOs: 11-15, 17, 19, and 20, the capture probe oligomer comprising a DNA sequence, an RNA sequence, or a chimeric DNA / RNA sequence, and the purification step is performed prior to contacting the sample with the at least two amplification oligomers.

9. The method of claim 8, wherein detecting the presence or absence of the amplification product comprises contacting the in vitro nucleic acid amplification reaction with at least one detection probe oligomer; the detection probe oligomer comprises a detection probe target hybridizing sequence, the detection probe oligomer target hybridizing sequence comprising: (A) is 13 to 40 nucleotides in length and is contained in the sequence of SEQ ID NO: 196 or its complement, 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, or its complement; or (B) SEQ ID NOs: 131, 132, 135, 140, 145, 147-157, and 159-161, or their complements; the detection probe oligomer comprises a DNA sequence, an RNA sequence, or a chimeric DNA / RNA sequence; A method according to any one of claims 1 to 8, thereby indicating the presence or absence of Plasmodium species in said sample.

10. the detection probe oligomer target hybridizing sequence being (A) contained in the sequence of SEQ ID NO: 197 or its complement and comprising the sequence of SEQ ID NO: 174 or SEQ ID NO: 175 or its complement; (B) selected from the group consisting of SEQ ID NOs: 131, 132, 135, 140, 147-157, 160, and 161, or their complements; or (C) a sequence selected from the group consisting of SEQ ID NOs: 177 and 178 or complements thereof.

11. the at least one detection probe oligomer comprises a first detection probe oligomer and a second detection probe oligomer; (A) the first detection probe oligomer comprises a target hybridizing sequence contained in the sequence of SEQ ID NO: 197 or its complement, and including the sequence of SEQ ID NO: 175 or its complement, or comprises the target hybridizing sequence of SEQ ID NO: 157 or its complement, the first detection probe oligomer comprises a DNA sequence, an RNA sequence, or a chimeric DNA / RNA sequence; and (B) The method of claim 10, wherein the second detection probe oligomer comprises a target hybridizing sequence contained in the sequence of SEQ ID NO: 197 or its complement and including the sequence of SEQ ID NO: 176 or its complement, or comprises a target hybridizing sequence selected from the group consisting of SEQ ID NO: 148 and SEQ ID NO: 152, and wherein the second detection probe oligomer comprises a DNA sequence, an RNA sequence, or a chimeric DNA / RNA sequence.

12. A method according to any one of claims 1 to 11, wherein the in vitro nucleic acid amplification reaction is an isothermal amplification reaction, a transcription-mediated amplification (TMA) reaction, a real-time amplification reaction, or a real-time TMA reaction.

13. 1. A combination of at least two oligomers for determining the presence or absence of a Plasmodium species in a sample, said combination comprising at least two amplification oligomers for amplifying a target region of a Plasmodium species target nucleic acid, said at least two amplification oligomers comprising: (a) (i) is 14 to 20 contiguous nucleotides in length and is contained within the sequence of SEQ ID NO: 162 and includes the sequence of SEQ ID NO: 163; or (ii) at least one first amplification oligomer comprising a target hybridizing sequence that is 14 to 25 contiguous nucleotides in length, contained within the sequence of SEQ ID NO: 166, and including the sequence of SEQ ID NO: 167 or SEQ ID NO: 168; (b) at least one second amplification oligomer comprising a target hybridizing sequence that is 15-33 contiguous nucleotides in length, contained in SEQ ID NO:169, and includes the sequence of SEQ ID NO:171, SEQ ID NO:172, or SEQ ID NO:

173.

14. the target hybridizing sequence of the at least one first amplification oligomer comprises: (A) selected from the group consisting of SEQ ID NOs: 21, 23-25, 32, 33, 35, 54, and 55; (B) contained in the sequence of SEQ ID NO: 164 and including the sequence of SEQ ID NO: 165; (C) comprising the sequence of SEQ ID NO: 167; (D) selected from the group consisting of SEQ ID NOs: 28-31, 34, 40, 41, and 49-51; (E) comprising the sequence of SEQ ID NO: 168; or (F) the combination described in claim 13, selected from the group consisting of SEQ ID NOs: 38, 39, 43, 44, and 53.

15. the target-hybridizing sequence of the at least one second amplification oligomer comprises: (A) selected from the group consisting of SEQ ID NOs: 80-82 and 85-100; or (B) The combination described in claim 13 or 14, which is contained in sequence number 170 and includes the sequence of sequence number 171 or sequence number 172.

16. The second amplification oligomer comprises: (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' of the target hybridizing sequence, the promoter sequence being a T7 promoter sequence; or (D) a sequence selected from the group consisting of SEQ ID NOs: 57-59 and 62-77.

17. the target-hybridizing sequences of the first and second amplification oligomers each comprise: (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. The combination of claim 16.

18. The at least one first amplification oligomer comprises at least two first amplification oligomers, the at least two first amplification oligomers comprising: (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 that is SEQ ID NO: 34; and (B) The combination of claim 13, which is an amplification oligomer comprising a target hybridizing sequence comprising the sequence of SEQ ID NO: 168, an amplification oligomer comprising a target hybridizing sequence that is SEQ ID NO: 53, an amplification oligomer comprising a target hybridizing sequence contained in SEQ ID NO: 164 and comprising the sequence of SEQ ID NO: 165, or an amplification oligomer comprising a target hybridizing sequence comprising SEQ ID NO:

21.

19. The at least one second amplification oligomer comprises at least two second amplification oligomers, the at least two second amplification oligomers comprising:

20. The combination of claim 13 or 18, selected from the group consisting of an amplification oligomer comprising the target hybridizing sequence of SEQ ID NO:94 and an amplification oligomer comprising the target hybridizing sequence of SEQ ID NO:

95.

20. 20. The combination of any one of claims 13 to 19, further comprising at least one capture probe oligomer, said capture probe oligomer comprising a capture probe oligomer target hybridizing sequence covalently attached to a moiety that binds to an immobilized probe, said capture probe oligomer target hybridizing sequence being up to 30 contiguous nucleotides in length and comprising a sequence selected from the group consisting of SEQ ID NOs: 11-15, 17, 19, and 20, and said capture probe oligomer comprising a DNA sequence, an RNA sequence, or a chimeric DNA / RNA sequence.

21. The method further comprises the steps of: at least one detection probe oligomer, said detection probe oligomer comprising a detection probe target hybridizing sequence, said detection probe oligomer target hybridizing sequence comprising: (A) is 13 to 40 nucleotides in length and is contained in the sequence of SEQ ID NO: 196 or its complement, 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 or its complement; or (B) SEQ ID NOs: 131, 132, 135, 140, 145, 147-157, and 159-161 or complements thereof, and the detection probe oligomer comprises a DNA sequence, an RNA sequence, or a chimeric DNA / RNA sequence.

22. the detection probe oligomer target hybridizing sequence being (A) comprising a sequence selected from the group consisting of SEQ ID NO: 174 and SEQ ID NO: 175, or a complement thereof, contained in the sequence of SEQ ID NO: 197, or a complement thereof; (B) selected from the group consisting of SEQ ID NOs: 131, 132, 135, 140, 147-157, 160, and 161, or their complements; or (C) the combination of claim 21, comprising a sequence selected from the group consisting of SEQ ID NOs: 177 and 178 or complements thereof.

23. the at least one detection probe oligomer comprises a first detection probe oligomer and a second detection probe oligomer; (A) the first detection probe oligomer comprises a target hybridizing sequence contained in the sequence of SEQ ID NO: 197 or its complement, and including the sequence of SEQ ID NO: 175 or its complement, or comprises the target hybridizing sequence of SEQ ID NO: 157 or its complement, the first detection probe oligomer comprises a DNA sequence, an RNA sequence, or a chimeric DNA / RNA sequence; and (B) The combination of claim 21, wherein the second detection probe oligomer comprises a target hybridizing sequence contained in the sequence of SEQ ID NO: 197 or its complement and including the sequence of SEQ ID NO: 176 or its complement, or comprises a target hybridizing sequence selected from the group consisting of SEQ ID NO: 148 and SEQ ID NO: 152 or its complement, and wherein the second detection probe oligomer comprises a DNA sequence, an RNA sequence, or a chimeric DNA / RNA sequence.

24. The combination according to any one of claims 13 to 23, wherein the at least two oligomers are provided in a kit or reaction mixture.

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