WO2010080616A1 - Molecular assay for diagnosis of malaria - Google Patents

Molecular assay for diagnosis of malaria Download PDF

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WO2010080616A1
WO2010080616A1 PCT/US2009/068725 US2009068725W WO2010080616A1 WO 2010080616 A1 WO2010080616 A1 WO 2010080616A1 US 2009068725 W US2009068725 W US 2009068725W WO 2010080616 A1 WO2010080616 A1 WO 2010080616A1
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nucleic acid
acid sequence
seq id
set
primer
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PCT/US2009/068725
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French (fr)
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Mark A. Hayden
Thomas G. Laffler
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Abbott Laboratories
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    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6893Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
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    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869Methods for sequencing
    • C12Q1/6872Methods for sequencing involving mass spectrometry
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    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/158Expression markers
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    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/16Primer sets for multiplex assays

Abstract

The invention is directed to compositions, methods and kits for diagnosing malaria using primers that amplify target sequences in Plasmodium falciparum, P. malariae, P. vivax, and P. ovale. The amplified target sequences are then analyzed by any number of mass spectrometric techniques, which data are queried against a database of base composition signatures of Plasmodia sp.

Description

MOLECULAR ASSAY FOR DIAGNOSIS OF MALARIA

FIELD OF THE INVENTION

[0001] The present invention is generally directed to the diagnosis of malaria; specifically, directed to detecting the causative agents of malaria using molecular tools.

GOVERNMENT SUPPORT [0002] Not applicable.

COMPACT DISC FOR SEQUENCE LISTINGS AND TABLES [0003] Not applicable.

BACKGROUND OF THE INVENTION

[0004] Malaria

[0005] Although eliminated in the United States by 1951, malaria is still a risk for over 2 billion people (Snow et al., 2005). People at risk for the disease live in tropical or subtropical regions. The World Health Organization (WHO) launched a malaria eradication program in 1955 (1999), but abandoned the cause in 1972 in view of waning political will and the emergence of chloroquine -resistant Plasmodia parasites (Brito, 2001). Interestingly, WHO never attempted to eradicate malaria from Africa, a continent severely plagued with malaria transmission. Epidemics have broken out as recently as 1987-1988 in Madagascar (Roberts et al., 2000); children continue to perish at alarming rates — in the millions, mostly in Africa from infection by the unicellular parasite (Snow et al., 2001).

[0006] Four species of Plasmodium cause malaria in humans: P. falciparum, P. vivax, P. ovale and P. malariae. P. falciparum is the most widespread and dangerous of the malaria parasites and causes most of the severe forms of the disease and the majority of the deaths. A fifth species, P. knowlesi, can infect a limited number of people, such as those of Malaysia (Singh et al., 2004). P. vivax is less deadly than P. falciparum, but is highly disabling (Greenwood et al., 2008). Both P. vivax and P. ovale can remain dormant for months, hiding in the liver. P. malariae can persist asymptomatically in the blood stream for decades.

[0007] The life cycle of the parasite is complex. (See (Greenwood et al., 2008) for a brief overview). Female Anopheles mosquitos, inoculate victims when feasting on a blood meal. If the inoculation is in the human dermis, elongated motile sporozites enter blood vessels and make their way to infect the liver through liver macrophages and hepatocytes. After about a week, infected hepatocytes rupture to release mersomes, which are aggregates of merozoites. These invade red blood cells — erythrocytes. Within the red blood cells the parasites multiply asexually, periodically being released from the red blood cells to invade fresh red blood cells. These amplification cycles correspond to the classical descriptions of waves of fever.

[0008] Diagnosis in the field is especially difficult. Malaria presents clinically in different forms, depending on the patient's genetic make-up, age, complicating co-infections (such as with Human Immunodeficiency Virus (HIV)), etc. Furthermore, many of the symptoms are not unique to malaria infection, which can confuse diagnosis. Laboratory diagnoses, however, can remove uncertainty. Typically, a blood sample is analyzed under the microscope for the unicellular parasite, but this test is a simple yes-no examination, depends on visually detectable levels of the parasite, and does not always indicate the Plasmodium species that is infecting the patient. Other diagnostic tests incorporate immunochromatographic capture procedures that use conjugated monoclonal antibodies which bind to antigens presented by the parasites, such as HRP-2 from P. falciparum and parasite-specific lactate dehydrogenase or Plasmodium aldolase from the parasite glycolytic pathway found in all species (Moody, 2002). Reagents to carry out these sophisticated assays are susceptible to deterioration in those environments that malaria is most often found (Jorgensen et al., 2006).

[0009] Thus traditional methods for detection and identification of microorganisms lack the speed and sensitivity for quick diagnosis in the field. Molecular recognition systems that can be used for rapid identification can improve response time and thus avert or reduce mortality or complications arising from malaria by providing quick and targeted treatment. This aspect becomes even more important as Plasmodium biology is further understood and treatments are developed that are specifically targeted to the different species of the parasite.

[0010] Nucleic acid-based molecular diagnostics

[0011] Molecular diagnostics have been championed for identifying pathogens. Polymerase chain reaction (PCR)-based diagnostics, wherein target polynucleotide sequences are amplified in vitro and then detected, have been successfully developed for a wide variety of pathogens.

[0012] The principal shortcomings of applying PCR assays to the clinical setting include the inability to eliminate background DNA contamination, interference with the PCR amplification by competing substrates, and limited capacity to discern speciation, antibiotic resistance and pathogen subtype. Despite significant progress, contamination remains problematic, and methods directed towards eliminating exogenous sources of DNA often also result in significant diminution in assay sensitivity. Although simple DNA sequencing can be performed to identify and characterize PCR products, sequencing and the subsequent analysis can be laborious and time-consuming.

[0013] Mass spectrometric techniques, such as high resolution electrospray ionization- Fourier transform-ion cyclotron resonance mass spectrometry (ESI-FT-ICR MS), can be used for quick PCR product detection and characterization. Accurate measurement of the exact mass combined with knowledge of the number of at least one nucleotide allows for calculating the total base composition for PCR duplex products of approximately 100 base pairs (Muddiman and Smith, 1998). For example, Aaserud et al demonstrated that accurate mass measurements obtained by high-performance mass spectrometry can be used to derive base compositions from double-stranded synthetic DNA constructs using the mathematical constraints imposed by the complementary nature of the two strands (Aaserud et al., 1996). Muddiman et al. developed an algorithm that allowed for deriving unambiguous base compositions from the exact mass measurements of the complementary single-stranded oligonucleotides (Muddiman et al., 1997). Wunschel et al showed that PCR products amplified from templates differing by a single nucleotide can be resolved and identified using ESI-FTICR at the 89-bp level in PCR product amplified from a 16/23S rDNA interspace region from Bacillus cereus (Wunschel et al., 1998). Electrospray ionization-Fourier transform-ion cyclotron resistance (ESI-FT-ICR) MS can be used to determine the mass of double-stranded, 500 base-pair PCR products via the average molecular mass (Hurst et al., 1996). The use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for characterizing PCR products has also been exploited (Muddiman et al., 1999). f

[0014] Examples of mass spectrometric analysis of polynucleotides include:

[0015] U.S. Pat. No. 5,965,363 discloses methods for screening nucleic acids for polymorphisms by analyzing amplified target nucleic acids using mass spectrometric techniques and procedures for improving mass resolution and mass accuracy of these methods.

[0016] WO 99/14375 describes methods, PCR primers and kits for use in analyzing preselected DNA tandem nucleotide repeat alleles by mass spectrometry. [0017] WO 98/12355 discloses methods of determining the mass of a target nucleic acid by mass spectrometric analysis, by cleaving the target nucleic acid to reduce its length, making the target single- stranded and using MS to determine the mass of the single-stranded shortened target. Also disclosed are methods of preparing a double-stranded target nucleic acid for MS analysis comprising amplification of the target nucleic acid, binding one of the strands to a solid support, releasing the second strand and then releasing the first strand which is then analyzed by MS. Kits for target nucleic acid preparation are also provided.

[0018] PCT WO97/33000 discloses methods for detecting mutations in a target nucleic acid by non-randomly fragmenting the target into a set of single-stranded nonrandom length fragments and determining their masses by MS.

[0019] U.S. Pat. No. 5,605,798 describes a fast and highly accurate mass spectrometer-based process for detecting the presence of a particular nucleic acid in a biological sample for diagnostic purposes.

[0020] WO 98/21066 describes processes for determining the sequence of a particular target nucleic acid by mass spectrometry. Processes for detecting a target nucleic acid present in a biological sample by PCR amplification and mass spectrometry detection are disclosed, as are methods for detecting a target nucleic acid in a sample by amplifying the target with primers that contain restriction sites and tags, extending and cleaving the amplified nucleic acid, and detecting the presence of extended product, wherein the presence of a DNA fragment of a mass different from wild-type is indicative of a mutation. Methods of sequencing a nucleic acid via mass spectrometry methods are also described.

[0021] WO 97/37041, WO 99/31278 and U.S. Pat. No. 5,547,835 describe methods of sequencing nucleic acids using mass spectrometry. U.S. Pat. Nos. 5,622,824, 5,872,003 and 5,691,141 describe methods, systems and kits for exonuclease-mediated mass spectrometric sequencing.

[0022] U.S. Patent Nos. 7,217,510, 7,108,974, 7,255,992, 7,226,739 and US 2004/0219517 describe methods and compositions for identifying one or more bioagents that uses at least one pair of oligonucleotide primers, wherein one pair hybridizes to two distinct conserved regions of a nucleic acid encoding a pathogen ribosomal RNA, wherein the two distinct conserved regions flank a variable nucleic acid region that when amplified creates a base composition "signature" that is characteristic of the bioagents. The base composition signature, the exact base composition determined from the molecular mass of the amplified product, is determined by first determining the molecular mass of the amplification product by mass spectrometry, after which the base composition is determined from the molecular mass. The bioagent is determined by matching the base composition signature to those stored in a database.

SUMMARY OF THE INVENTION

[0023] In a first aspect, the invention is directed to methods of identifying the presence or absence of a causative agent of malaria in a test sample, comprising: providing a test sample; forming a reaction mixture comprising a primer pair set selected from the group consisting of set A, B and C, wherein: set A comprises a forward primer comprising a nucleic acid sequence of SEQ ID NO: 1, and a reverse primer comprising a nucleic acid sequence of SEQ ID NO:2 set B comprises a forward primer comprising a nucleic acid sequence of SEQ ID NO:3, and a reverse primer comprising a nucleic acid sequence of SEQ ID NO:4; and set C comprises a forward primer comprising a nucleic acid sequence of SEQ ID NO:5, and a reverse primer comprising a nucleic acid sequence of SEQ ID NO:6, subjecting the mixture to amplification conditions to generate an amplification product; determining the molecular mass of the amplification product; and comparing the molecular mass of the amplification product to calculated or measured molecular masses of target sequences in a database to identify the presence or absence of the causative agent of malaria.

[0024] In a second aspect, the invention is directed to methods of identifying the presence or absence of a causative agent of malaria in a test sample, comprising: providing a test sample; forming a reaction mixture comprising a primer pair set selected from the group consisting of set A, B and C, wherein: set A comprises a forward primer comprising a nucleic acid sequence of SEQ ID NO: 1, and a reverse primer comprising a nucleic acid sequence of SEQ ID NO:2 set B comprises a forward primer comprising a nucleic acid sequence of SEQ ID NO:3, and a reverse primer comprising a nucleic acid sequence of SEQ ID NO:4; and set C comprises a forward primer comprising a nucleic acid sequence of SEQ ID NO:5, and a reverse primer comprising a nucleic acid sequence of SEQ ID NO:6, subjecting the mixture to amplification conditions to generate an amplification product; determining the base composition of the amplification product; and comparing the base composition of the amplification product to calculated or measured base compositions of target sequences in a database to identify the presence or absence of a causative agent of malaria.

[0025] In either of the methods of the first and second aspects, identifying the target sequence does not comprise sequencing of the amplification product, and the mass spectrometry can be Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) or time of flight mass spectrometry (TOF-MS), such as electrospray ionization time of flight mass spectrometry (ESI-TOF). Additionally, in either of the first and second aspects, the primer set can comprise at least one nucleotide analog, wherein the nucleotide analog is, for example, inosine, uridine, 2,6- diaminopurine, propyne C, and propyne T, and the reaction mixture comprises at least two primer sets. The amplification products of both aspects can further comprise incorporating a molecular mass-modifying tag, such as an isotope of carbon, for example, 13C.

[0026] In a third aspect, the invention is directed to methods of identifying the presence or absence of a causative agent of malaria in a test sample, comprising: providing a test sample; forming a reaction mixture comprising: a primer pair set selected from the group consisting of set A, B and C, wherein: set A comprises a forward primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO: 1, and a reverse primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO:2; set B comprises a forward primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO:3, and a reverse primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO:4; and set C comprises a forward primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO:5, and a reverse primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO: 6, subjecting the mixture to amplification conditions to generate an amplification product; determining the molecular mass of the amplification product; and comparing the molecular mass of the amplification product to calculated or measured molecular masses of target sequences in a database to identify the presence or absence of a causative agent of malaria.

[0027] In a fourth aspect, the invention is directed to methods of identifying the presence or absence of a causative agent of malaria in a test sample, comprising: providing a test sample; forming a reaction mixture comprising: a primer pair set selected from the group consisting of set A, B and C, wherein: set A comprises a forward primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO: 1, and a reverse primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO:2 set B comprises a forward primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO:3, and a reverse primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO:4; and set C comprises a forward primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO:5, and a reverse primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO: 6, subjecting the mixture to amplification conditions to generate an amplification product; determining the base composition of the amplification product; and comparing the base composition of the amplification product to calculated or measured base compositions of target sequences in a database to identify the presence or absence of a causative agent of malaria.

[0028] In either of the methods of the third and fourth aspects, identifying the target sequence does not comprise sequencing of the amplification product, and the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) or time of flight mass spectrometry (TOF-MS), such as electrospray ionization time of flight mass spectrometry. Additionally, in either of the first and second aspects, the primer set can comprise at least one nucleotide analog, wherein the nucleotide analog is, for example, inosine, uridine, 2,6- diaminopurine, propyne C, and propyne T, and the reaction mixture comprises at least two primer sets. The amplification products of both aspects can further comprise incorporating a molecular mass-modifying tag [0029] In a fifth aspect, the invention is directed to kits, comprising a primer pair set selected from the group consisting of set A, B and C, wherein: set A comprises a forward primer comprising a nucleic acid sequence of SEQ ID NO: 1, and a reverse primer comprising a nucleic acid sequence of SEQ ID NO:2 set B comprises a forward primer comprising a nucleic acid sequence of SEQ ID NO:3, and a reverse primer comprising a nucleic acid sequence of SEQ ID NO:4; and set C comprises a forward primer comprising a nucleic acid sequence of SEQ ID NO:5, and a reverse primer comprising a nucleic acid sequence of SEQ ID NO:6; and amplification reagents.

BRIEF DESCRIPTION OF THE DRAWING

[0030] FIG. 1 shows an alignment of 18S ribosomal RNA sequences and surrounding variable regions of Plasmodium falciparum, P. malariae, P. ovale, and P. vivαx (SEQ ID NOs:7-10).

DETAILED DESCRIPTION

[0031] Modern typical molecular assays for malaria typically use distinct molecular probes to identify the species of Plasmodium DNA present in test specimens. The present invention allows Plasmodium species to be determined based on amplicon base composition alone, thereby eliminating the need for species-specific molecular probes and labels. The invention thus simplifies detection in that fewer assay reagents are required. Other advantages include the ability to detect multiple malaria causative agents simultaneously, reduced false positive and negative results due to the ability to screen multiple causative agents simultaneously, and speed. [0032] The invention accomplishes its significant advantages in part by exploiting spectrometric technologies. For example, ElectroSpray Injection Time-of-Flight Mass Spectrometry (ESI-MS) can be used to determine the exact base composition of amplicons generated by target amplification technologies such as the polymerase chain reaction (PCR). The disclosed invention exploits primer pairs (sets A, B, and C) directed to highly conserved regions of Plasmodium genomes, such as the genes encoding 18S ribosomal RNA, that can be used to detect the four species of genus Plasmodium: falciparum, malariae, vivax and ovale that typically cause malaria in humans. The primers of the invention can be used to amplify DNA from any of these four species. Base composition analysis by ESI-MS can then be used to identify which of the four species is (or are) present in a test sample.

[0033] In one embodiment, the invention uses the novel primer pair of SEQ ID NOs: 1 and 2 (Set A). In another embodiment, the invention uses the novel primer pair of SEQ ID NOs:3 and 4 (Set B). In another embodiment, the invention uses the primer pair of SEQ ID NOs:5 and 6 (Set C). Rougemont et al. (Rougemont et al., 2004) have published only in part the primer pair Set C nucleic acid sequences as part of a primer pair-probe combination. These primer pair sets, which are complementary to conserved 18S ribosomal RNA sequences surrounding variable regions in the four Plasmodium genomes, can be used singly or in combination as desired. The primer sequences for sets A, B and C are shown in Table 1. FIG. 1 shows the alignment of the 18S ribosomal sequences from Plasmodium falciparum (SEQ ID NO: 7), Plasmodium malariae (SEQ ID NO:8), Plasmodium ovale (SEQ ID NO:9) and Plasmodium vivax (SEQ ID NO: 10). Tables 2, 3, and 4 show the target sequences when the primer pairs are used to amplify sequences from the four Plasmodium species.

[0034] In one embodiment, Sets A, B, and C are used singly to amplify target sequences. In other embodiments, the sets are used in pairs, e.g., A with B, B with C, and A with C, in the same reaction mix. In yet a third embodiment, Sets A, B, and C are used simultaneously in a reaction mix. Of course, the sets can be used sequentially, if desired, or in parallel samples simultaneously

TABLE 1

Primer pair sets and sequences

Primer Sequence SEQ ID NO

A (Forward) cggctcatta aaacagttat 1

A (Reverse) tagctacagc ttttccgta 2

B (Forward) cctaccgatt gaaagatatg a 3

B (Reverse) cggaaacctt gttacgac 4

C (Forward) gttaagggag tgaagacgat caga 5

C (Reverse) aacccaaaga ctttgatttc tcatag 6

TABLE 2 Sequences amplified by primer set A (SEQ ID NOs: 1 and 2)

Plasmodium sp. Sequence SEQ ID NO:

P. falciparum cggctcatta aaacagttat aatctacttg atgtttttaa tataaggata actacggaaa 60 11 atctgtagct a 71 P. malariae cggctcatta aaacagttat agtctacttg acattttttt tataaggata actacggaaa 60 14 agctgtagct a 71 P. ovale cggctcatta aaacagttat aatctacttg aaatttctac cttacaagga taactacgga 60 17 aaagctgtag eta 73 P. vivax cggctcatta aaacagttat aatctacttg acattttttt ctataaggat aactacggaa 60 20 aagctgtagc ta 72

TABLE 3 Sequences amplified by primer set B (SEQ ID N0s:3 and 4)

Plasmodium sp. Sequence SEQ ID NO:

P. falciparum cctaccgatt gaaagatatg ataaattgtt tggatatgaa ttaaaataat agaagtcgta 60 12 acaaggtttc eg 72

P. malariae cctaccgatt gaaagatatg atgaattgtt tggacaagaa aaaaggtttt tattcttttt 60 15 tctggaaaaa tcgtaaatcc tatcttttaa aggaaggaga aagtcgtaac aaggtttccg 120

P. ovale cctaccgatt gaaagatatg atgaattgtt tggacaagaa aagaaagaat ttatattctt 60 18 ttttttctgg aaaaaccgta aatcctatct tttaaaggaa ggagaagtcg taacaaggtt 120 tccg 124

P. vivax cctaccgatc gaaagatatg atgaattgtt tggacaagaa gaaaggggat tatatcttct 60 21 tttttctgga aaaaccgtaa atcctgtctt ttaaaggaag gagaagtcgt aacaaggttt 120 ccg 123

TABLE 4 Sequences amplified by primer set C (SEQ ID N0s:5 and 6)

Plasmodium sp. Sequence SEQ ID NO:

P. falciparum gttaagggag tgaagacgat cagataccgt cgtaatctta accataaact ataccgacta 60 13 ggtgttggat gaatataaaa aatatataaa tatgtagcat ttcttaggga atgttgattt 120 tatattagaa ttgcttcctt cagtacctta tgagaaatca aagtctttgg gtt 173

P. malariae gttaagggag tgaagacgat cagataccgt cgtaatctta accataaact atgccgacta 60 16 ggtgttggat gatagtgtaa aaaataaaag agacattctt atatatgagt gtttctttta 120 gatagcttcc ttcagtacct tatgagaaat caaagtcttt gggtt 165

P. ovale gttaagggag tgaagacgat cagataccgt cgtaatctta accataaact atgccgacta 60 19 ggttttggat gaaagatttt taaataagaa aattcctttc ggggaaattt cttagattgc 120 ttccttcagt accttatgag aaatcaaagt ctttgggtt 159

P. vivax gttaagggag tgaaaacgat cagataccgt cgtaatctta accataaact ataccgacta 60 22 ggttttggat gaaagttaaa caaataagga tagtctcttc ggggatagtc cttagatttc 120 ttccttcagt accctatgag aaatcaaagt ctttgggtt 159

[0035] In another embodiment, the primer sets are subjected to amplification conditions, wherein the first cycle comprises incubating the reaction mix with at least one nucleic acid polymerase, such as a DNA polymerase, at 94 0C for 10 seconds, followed by 55-60 0C for 20 seconds, and then 72 0C for 20 seconds. The cycle can be repeated multiple times, such as for 35 cycles. A final cycle can be added, wherein the reaction mix is held at 40 0C. These conditions can be adjusted as necessary, and easily, by one of skill in the art. Amplification conditions are discussed further below.

[0036] After amplification, the reaction mix is subjected to spectrometric analysis, such as ESI- MS. The sample is injected into a spectrometer, the molecular mass or corresponding "base composition signature" (BCS) of any amplification product is then determined and matched against a database of molecular masses or BCS's. A BCS is the exact base composition determined from the molecular mass of a bioagent identifying amplicon. BCS's provide a useful index of a specific gene in a specific organism. A BCS differs from nucleic acid sequence in that the signature does not order the bases, but instead represents the nucleic acid base composition of the nucleic acid (e.g., A, G, C, T). The present method thus provides rapid throughput and does not require nucleic acid sequencing of the amplified target sequence for detection and identification. Furthermore, time-consuming separation technologies, such as gel electrophoresis, coupled with detection of the separated sequences, whether from simple gel staining or hybridization with a probe comprising a detectable label, is avoided. In the methods of the invention, all target species of Plasmodium can be detected in a sample with a simple detection step and database interrogation.

[0037] In one embodiment, samples are obtained from a subject, which can be a mammal, such as a human. The sample is typically blood, but can be any other tissues that can harbor causative agents of malaria, such as liver.

[0038] DEFINITIONS

[0039] "Specifically hybridize" refers to the ability of a nucleic acid to bind detectably and specifically to a second nucleic acid. Polynucleotides specifically hybridize with target nucleic acid strands under hybridization and wash conditions that minimize appreciable amounts of detectable binding by non-specific nucleic acids.

[0040] "Target sequence" or "target nucleic acid sequence" means a nucleic acid sequence of Plasmodium or complements thereof, that is amplified, detected, or both using one or more of the polynucleotide primer sets of SEQ ID NOs: 1 and 2, SEQ ID NOs:3 and 4, and SEQ ID NOs:5 and 6. Additionally, while the term target sequence sometimes refers to a double stranded nucleic acid sequence; a target sequence can also be single- stranded. In cases where the target is double- stranded, polynucleotide primer sequences of the present invention preferably amplify both strands of the target sequence. A target sequence can be selected that is more or less specific for a particular organism. For example, the target sequence can be specific to an entire genus, to more than one genus, to a species or subspecies, serogroup, auxotype, serotype, strain, isolate or other subset of organisms.

[0041] "Test sample" means a sample taken from an organism, including mosquitoes, or a biological fluid, wherein the sample may contain a Plasmodium target sequence. A test sample can be taken from any source, for example, tissue, blood, saliva, sputa, mucus, sweat, urine, urethral swabs, cervical swabs, urogenital or anal swabs, conjunctival swabs, ocular lens fluid, cerebral spinal fluid, etc. A test sample can be used (i) directly as obtained from the source; or (ii) following a pre -treatment to modify the character of the sample. Thus, a test sample can be pre- treated prior to use by, for example, preparing plasma or serum from blood, disrupting cells or viral particles, preparing liquids from solid materials, diluting viscous fluids, filtering liquids, adding reagents, purifying nucleic acids, etc. Typically, test samples contain blood or liver tissue when analyzing for malaria causative agents.

[0042] "Subjects" include a mammal, a bird, or a reptile. The subject can be a cow, horse, dog, cat, or a primate. The biological entity can also be a human. The biological entity may be living or dead.

[0043] A "polynucleotide" is a nucleic acid polymer of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), modified RNA or DNA, or RNA or DNA mimetics (such as PNAs), and derivatives thereof, and homologues thereof. Thus, polynucleotides include polymers composed of naturally occurring nucleobases, sugars and covalent inter-nucleoside (backbone) linkages as well as polymers having non-naturally-occurring portions that function similarly. Such modified or substituted nucleic acid polymers are well known in the art and for the purposes of the present invention, are referred to as "analogues." Oligonucleotides are generally short polynucleotides from about 10 to up to about 160 or 200 nucleotides.

[0044] "Plasmodium variant polynucleotide" or "Plasmodium variant nucleic acid sequence" means a polynucleotide having at least about 60% nucleic acid sequence identity, more preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with the nucleic acid sequence of SEQ ID NOs: 1-6. Variants do not encompass the native nucleotide sequence. [0045] Ordinarily, Plasmodium variant polynucleotides are at least about 8 nucleotides in length, often at least about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 35, 40, 45, 50, 55, 60 nucleotides in length, or even about 75-200 nucleotides in length, or more.

[0046] "Percent (%) nucleic acid sequence identity" with respect to nucleic acid sequences is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the sequence of interest, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining % nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST- 2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0047] When nucleotide sequences are aligned, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) can be calculated as follows:

% nucleic acid sequence identity = W/Z* 100 where

W is the number of nucleotides scored as identical matches by the sequence alignment program's or algorithm's alignment of C and D and

Z is the total number of nucleotides in D.

[0048] When the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.

[0049] "Consisting essentially of a polynucleotide having a % sequence identity" means that the polynucleotide does not substantially differ in length, but may differ substantially in sequence. Thus, a polynucleotide "A" consisting essentially of a polynucleotide having at least 80% sequence identity to a known sequence "B" of 100 nucleotides means that polynucleotide "A" is about 100 nts long, but up to 20 nts can vary from the "B" sequence. The polynucleotide sequence in question can be longer or shorter due to modification of the termini, such as, for example, the addition of 1-15 nucleotides to produce specific types of probes, primers and other molecular tools, etc., such as the case of when substantially non-identical sequences are added to create intended secondary structures. Such non-identical nucleotides are not considered in the calculation of sequence identity when the sequence is modified by "consisting essentially of."

[0050] The specificity of single stranded DNA to hybridize complementary fragments is determined by the stringency of the reaction conditions. Hybridization stringency increases as the propensity to form DNA duplexes decreases. In nucleic acid hybridization reactions, the stringency can be chosen to favor specific hybridizations (high stringency). Less-specific hybridizations (low stringency) can be used to identify related, but not exact, DNA molecules (homologous, but not identical) or segments.

[0051] DNA duplexes are stabilized by: (1) the number of complementary base pairs, (2) the type of base pairs, (3) salt concentration (ionic strength) of the reaction mixture, (4) the temperature of the reaction, and (5) the presence of certain organic solvents, such as formamide, which decrease DNA duplex stability. A common approach is to vary the temperature: higher relative temperatures result in more stringent reaction conditions. Ausubel et al. provide an excellent explanation of stringency of hybridization reactions (Ausubel et al., 1987).

[0052] Hybridization under "stringent conditions" means hybridization protocols in which nucleotide sequences at least 60% homologous to each other remain hybridized.

[0053] Polynucleotides can include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane. In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (van der Krol et al., 1988) or intercalating agents (Zon, 1988). The oligonucleotide can be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.

[0054] Useful polynucleotide analogues include polymers having modified backbones or non- natural inter-nucleoside linkages. Modified backbones include those retaining a phosphorus atom in the backbone, such as phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, as well as those no longer having a phosphorus atom, such as backbones formed by short chain alkyl or cycloalkyl inter-nucleoside linkages, mixed heteroatom and alkyl or cycloalkyl inter-nucleoside linkages, or one or more short chain heteroatomic or heterocyclic inter-nucleoside linkages. Modified nucleic acid polymers (analogues) can contain one or more modified sugar moieties. [0055] Analogs that are RNA or DNA mimetics, in which both the sugar and the inter- nucleoside linkage of the nucleotide units are replaced with novel groups, are also useful. In these mimetics, the base units are maintained for hybridization with the target sequence. An example of such a mimetic, which has been shown to have excellent hybridization properties, is a peptide nucleic acid (PNA) (Buchardt et al., 1992; Nielsen et al., 1991). Another example is a locked nucleic acids (LNA) where the 2' and 4'glycosidic carbons are linked by a 2'-O- methylene bridge.

[0056] The realm of nucleotides includes derivatives wherein the nucleic acid molecule has been covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring nucleotide.

[0057] The polynucleotides of the present invention thus comprise primers that specifically hybridize to target sequences, for example the nucleic acid molecules having any one of the nucleic acid sequences of SEQ ID NOs: 1-6, including analogues and/or derivatives of the nucleic acid sequences, and homologs thereof. The polynucleotides of the invention can be used as primers to amplify or detect Plasmodium sp. polynucleotides.

[0058] The polynucleotides of SEQ ID NOs: 1 -6 can be prepared by conventional techniques, such as solid-phase synthesis using commercially available equipment, such as that available from Applied Biosystems USA Inc. (Foster City, CA; USA), DuPont, (Wilmington, DE; USA), or Milligen (Bedford, MA; USA). Modified polynucleotides, such as phosphorothioates and alkylated derivatives, can also be readily prepared by similar methods known in the art (Fino, 1995; Mattingly, 1995; Ruth, 1990).

[0059] PRACTICING THE INVENTION

[0060] The invention includes methods of detecting Plasmodium nucleic acids wherein a test sample is collected; amplification reagents and Plasmodium-speciύc primers, such as those of SEQ ID Nos: 1-6, are added; the sample subjected to amplification; the amplified nucleic acid (amplicons), if any, is analyzed using mass spectrometry; and the resulting data used to interrogate a database.

[0061] Amplification of Plasmodium nucleic acids [0062] The polynucleotides of SEQ ID NOs: 1 -6 can be used as primers to amplify Plasmodium polynucleotides in a sample. The polynucleotides are used as primers, wherein the primer pairs are Set A: SEQ ID NOs: 1 and 2; Set B: SEQ ID NOs:3 and 4; and Set C: SEQ ID NOs:5 and 6.

[0063] The amplification method generally comprises (a) a reaction mixture comprising nucleic acid amplification reagents, at least one primer set of the present invention, and a test sample suspected of containing at least one target sequence; and (b) subjecting the mixture to amplification conditions to generate at least one copy of a nucleic acid sequence complementary to the target sequence if the target sequence is present.

[0064] Step (b) of the above method can be repeated any suitable number of times prior to, for example, a detection step; e.g., by thermal cycling the reaction mixture between 10 and 100 times (or more), typically between about 20 and about 60 times, more typically between about 25 and about 45 times.

[0065] Nucleic acid amplification reagents include enzymes having polymerase activity, enzyme co-factors, such as magnesium or manganese; salts; nicotinamide adenine dinucleotide (NAD); and deoxynucleotide triphosphates (dNTPs), (dATP, dGTP, dCTP and dTTP).

[0066] Amplification conditions are those that promote annealing and extension of one or more nucleic acid sequences. Such annealing is dependent in a rather predictable manner on several parameters, including temperature, ionic strength, sequence length, complementarity, and G:C content of the sequences. For example, lowering the temperature in the environment of complementary nucleic acid sequences promotes annealing. Typically, diagnostic applications use hybridization temperatures that are about 2 0C to 18 0C (e.g., approximately 10 0C) below the melting temperature, Tm. Ionic strength also impacts Tm. Typical salt concentrations depend on the nature and valency of the cation but are readily understood by those skilled in the art. Similarly, high G:C content and increased sequence length stabilize duplex formation.

[0067] Finally, the hybridization temperature is selected close to or at the Tm of the primers. Thus, obtaining suitable hybridization conditions for a particular primer set is within the ordinary skill of the PCR arts.

[0068] Amplification procedures are well-known in the art and include the polymerase chain reaction (PCR), transcription-mediated amplification (TMA), rolling circle amplification, nucleic acid sequence based amplification (NASBA), ligase chain reaction and strand displacement amplification (SDA). One skilled in the art understands that for use in certain amplification techniques, the primers may need to be modified; for example, SDA primers usually comprise additional nucleotides near the 5' ends that constitute a recognition site for a restriction endonuclease. For NASBA, the primers can include additional nucleotides near the 5' end that constitute an RNA polymerase promoter. Polynucleotides thus modified are considered to be within the scope of the present invention.

[0069] The present invention includes the use of the polynucleotides of SEQ ID NOs:l-6 in methods to specifically amplify target nucleic acid sequences in a test sample in a single vessel format.

[0070] Chemical Modification of Primers

[0071] Primers can be chemically modified, for example, to improve the efficiency of hybridization. For example, because variation (due to codon wobble in the 3rd position) in conserved regions among species often occurs in the third position of a DNA triplet, the primers of SEQ ID NOs: 1 -6 can be modified such that the nucleotide corresponding to this position is a "universal base" that can bind to more than one nucleotide. For example, inosine (I) binds to U, C or A; guanine (G) binds to U or C, and uridine (U) binds to A or G. Other examples of universal bases include nitroindoles such as 5-nitroindole or 3-nitropyrrole (Loakes et al., 1995), the degenerate nucleotides dP or dK (Hill et al.), an acyclic nucleoside analog containing 5- nitroindazole (Van Aerschot et al., 1995) or the purine analog l-(2-deoxy-β-D-ribofuranosyl)- imidazole-4-carboxamide (SaIa et al., 1996).

[0072] In another embodiment, to compensate for the somewhat weaker binding by the "wobble" base, the oligonucleotide primers can be designed such that the first and second positions of each triplet are occupied by nucleotide analogs which bind with greater affinity than the unmodified nucleotide. Examples of these analogs include 2,6-diaminopurine, which binds to thymine; propyne T, which binds to adenine; and propyne C and phenoxazines, including G- clamp, which bind to G. Propynylated pyrimidines are described in U.S. Pat. Nos. 5,645,985, 5,830,653 and 5,484,908. Phenoxazines are described in U.S. Pat. Nos. 5,502,177, 5,763,588, and 6,005,096. G-clamps are described in U.S. Pat. Nos. 6,007,992 and 6,028,183.

[0073] Controls

[0074] Various controls can be instituted in the methods of the invention to assure, for example, that amplification conditions are optimal. An internal standard can be included in the reaction. Such internal standards generally comprise a control target nucleic acid sequence. The internal standard can optionally further include an additional pair of primers. The primary sequence of these control primers can be unrelated to the polynucleotides of the present invention and specific for the control target nucleic acid sequence.

[0075] In the context of the present invention, a control target nucleic acid sequence is a nucleic acid sequence that:

(a) can be amplified either by a primer or primer pair being used in a particular reaction or by distinct control primers; and

(b) is detected by mass spectrometric techniques.

[0076] Mass Spectrometric Characterization ofAmplicons

[0077] Mass spectrometry (MS)-based detection and characterizing PCR products has several distinct advantages. MS is intrinsically a parallel detection scheme without the need for radioactive or fluorescent labels, since every amplification product is identified by its molecular mass. Less than femtomole quantities of material are required. An accurate assessment of the molecular mass of a sample can be quickly obtained. Intact molecular ions can be generated from amplification products using one of a variety of ionization techniques to convert the sample to gas phase. These ionization methods include electrospray ionization (ES), matrix-assisted laser desorption ionization (MALDI) and fast atom bombardment (FAB). For example, MALDI of nucleic acids, along with examples of matrices for use in MALDI of nucleic acids, are described in WO 98/54751.

[0078] Upon ionization, several peaks are observed from one sample due to the formation of ions with different charges. Averaging the multiple readings of molecular mass obtained from a single mass spectrum affords an estimate of molecular mass of the amplicon. Electrospray ionization mass spectrometry (ESI-MS) is particularly useful for very high molecular weight polymers such as proteins and nucleic acids having molecular weights greater than 10 kDa, since it yields a distribution of multiply-charged molecules of the sample without causing a significant amount of fragmentation.

[0079] Suitable mass detectors for the present invention include Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF, and triple quadrupole.

[0080] In general, useful mass spectrometric techniques that can be used in the present invention include tandem mass spectrometry, infrared multiphoton dissociation and pyro lytic gas chromatography mass spectrometry (PGC-MS). [0081] The accurate measurement of molecular mass for large DNAs is limited by the adduction of cations from the PCR reaction to each strand, resolution of the isotopic peaks from natural abundance 13C and 15N isotopes, and assignment of the charge state for any ion. The cations are removed by in-line dialysis using a flow-through chip that brings the solution containing the PCR products into contact with a solution containing ammonium acetate in the presence of an electric field gradient orthogonal to the flow. The latter two problems can be addressed by operating with a resolving power of > 100,000 and by incorporating isotopically-depleted nucleotide triphosphates into the DNA. The resolving power of the instrument is also a consideration. At a resolving power of 10,000, the modeled signal from the [M-14H+]14" charge state of an 84-mer PCR product is poorly characterized and assignment of the charge state or exact mass is impossible. At a resolving power of 33,000, the peaks from the individual isotopic components are visible. At a resolving power of 100,000, the isotopic peaks are resolved to the baseline and assignment of the charge state for the ion is straightforward. The [13C, 15N] -depleted triphosphates are obtained, for example, by growing microorganisms on depleted media and harvesting the nucleotides (Batey et al, 1992). [0082] Tandem mass spectrometry techniques can provide more definitive information pertaining to molecular identity or sequence. Tandem MS involves the coupled use of two or more stages of mass analysis where both the separation and detection steps are based on mass spectrometry. The first stage is used to select an ion or component of a sample from which further structural information is to be obtained. The selected ion is then fragmented using, e.g., blackbody irradiation, infrared multiphoton dissociation, or collisional activation. For example, ions generated by electrospray ionization (ESI) can be fragmented using IR multiphoton dissociation. This activation leads to dissociation of glycosidic bonds and the phosphate backbone, producing two series of fragment ions, called the w-series (having an intact 3 ' terminus and a 5 ' phosphate following internal cleavage) and the a-Base series (having an intact 5' terminus and a 3' furan). [0083] The second stage of mass analysis is then used to detect and measure the mass of these resulting fragments of product ions. Such ion selection followed by fragmentation routines can be performed multiple times so as to essentially completely dissect the molecular sequence of a sample. [0084] PCR amplicons when analyzed by ESI-TOF mass spectrometry give a pair of masses, one for each strand of the double-stranded DNA amplicon. In some cases, the molecular mass of one strand alone provides enough information to unambiguously identify a given Plasmodium sp.. In other cases, however, determining information from both strands is preferred.

[0085] The molecular mass of a single strand can also be consistent with more than on BCS. This can also be true for the complementary strand. These ambiguities are resolved when the added constraint of complementarity is applied. Thus a strand with a BCS of A28T24G29C25 is paired with its complement A24T28G25C29. Typically when sets of possible BCS solutions for the two strands of an amplicon are compared, usually only one pair of strands are complements of each other. That pair represents a unique solution for an amplicon's BCS; the other potential solutions are discarded because they are non- complementary.

[0086] For example, an amplicon is analyzed by ESI-TOF mass spectrometry that gives two masses: a first mass of 32,889.45 Da for one strand, and a second mass of 33,071.46 Da for the second. Assuming an average mass for the DNA bases are as follows:

A = 313.0576 amu G = 328.0526 amu C = 289.0464 amu; and T = 304.0461 amu.

Each strand has 5 possible solutions, each solution resulting in the measured mass. The calculated possible solutions for the first and second strands are:

First Strand (32.889.45 Da) Second strand (33.071.46 Da)

A24G27C27 T24 A25G26C30 T25

A28G31C27 T24 A24G25C29T28

A26G30C25 T25 A25G25C30 T26

A28G29C25 T24 A24G27C31 T28

A25G30C26T25 A24G27C27 T24

Inspecting these possible solutions, there is only one solution where the first strand is the complement of the second strand when the constraint of complementarity is applied; that is, for every A in the first strand, there is a T in the second; for every G in the first strand, there is a C in the second, and so on. Thus, the only solution is:

First strand: A28G29C25T24 Second strand: T28C29G25A24

[0087] Mass-modifying "tags" can also be used. A nucleotide analog or "tag" is incorporated during amplification (e.g., a 5-(trifluoromethyl) deoxythymidine triphosphate) that has a different molecular weight than the unmodified base so as to improve distinction of masses. Such tags are described in, for example, WO97/33000. This further limits the number of possible base compositions consistent with any mass. For example, 5-(trifluoromethyl)deoxythymidine triphosphate can be used in place of dTTP in a separate nucleic acid amplification reaction. Measurement of the mass shift between a conventional amplification product and the tagged product is used to quantitate the number of thymidine nucleotides in each of the single strands. Because the strands are complementary, the number of adenosine nucleotides in each strand is also determined. [0088] In another amplification reaction, the number of G and C residues in each strand is determined using, for example, the cytidine analog 5-methylcytosine (5-meC) or propyne C. The combination of the A/T reaction and G/C reaction, followed by molecular weight determination, provides a unique base composition. This method is summarized in Table 5.

TABLE 5

Mass tag Double strand Single strand Total mass Base info Base info (other Total base Total base comp. sequence sequence (this strand) (this strand) strand) comp. (top) (bottom)

T*mass T*ACGT*ACGT* T*ACGT*ACGT* 3x 3T 3A

( T* -T ) = x AT*GCAT*GCA

AT*GCAT*GCA 2x 2T 2A 3T 3A 2A 2T

C*mass TAC*GTAC*GT TAC*GTAC*GT 2y 2T 2G 2C 2G

(C* -C ) =y ATGC*ATGC*A 2G 2C

ATGC*ATGC*A 2y 2C 2G

[0089] In Table 5, the mass tag phosphorothioate A (A*) was used to distinguish a Bacillus anthracis cluster. The B. anthracis (A14G9C14T9) had an average MW of 14072.26, and the B. anthracis (AiA* 13GgCi4Tg) had an average molecular weight of 14281.11 and the phosphorothioate A had an average molecular weight of +16.06 as determined by ESI-TOF MS.

[0090] In another example, assume the measured molecular masses of each strand are 30,000.115 Da and 31,000.115 Da respectively, and the measured number of dT and dA residues are (30,28) and (28,30). If the molecular mass is accurate to 100 ppm, there are 7 possible combinations of dG+dC possible for each strand. However, if the measured molecular mass is accurate to 10 ppm, there are only 2 combinations of dG+dC, and at 1 ppm accuracy there is only one possible base composition for each strand.

[0091] Base Composition Signatures as Indices of Identifying Amplicons and Database

Interrogation

[0092] Conversion of molecular mass data to a base composition signature is useful for certain analyses. A "base composition signature" (BCS) is the exact base composition determined from the molecular mass of an amplicon. The BCS can provide an index of a specific gene in a specific organism.

[0093] Base compositions, like sequences, vary slightly from isolate to isolate within species. It is possible to manage this diversity by building "base composition probability clouds" around the composition constraints for each Plasmodium species. This permits identification of the species of Plasmodium in a fashion similar to sequence analysis. A "pseudo four-dimensional plot" can be used to visualize the concept of base composition probability clouds. See, for example, U.S.

Patent Nos. 7,217,510, 7,108,974, 7,255,992, 7,226,739 and US 2004/0219517.

[0094] The BCS's collected from mass spectrometric analysis can be used to query a database that contains, for example, the information from the sequences of SEQ ID NOs: 11-22, including

G+C content, molecular mass, and of course, the BCS's of SEQ ID NOs: 11-22, etc. From this interrogation, the species of Plasmodium can be identified from the amplified target sequence.

See, for example, U.S. Patent Nos. 7,217,510, 7,108,974, 7,255,992, 7,226,739 and US

2004/0219517. [0095] Databases

[0096] The invention in part exploits 18S ribosomal RNA sequences, wherein the polynucleotides of the invention, SEQ ID NOs: 1-6, are designed to hybridize to conserved 18S ribosomal RNA sequences that flank variable regions. Ribosomal RNA (rRNA) gene sequences are useful BCS's because rRNA genes contain sequences that are extraordinarily conserved across bacterial domains interspersed with regions of high variability that are more specific to each species. Variable regions flanked by conserved sequences, such as those flanked by primer sets A, B, and C, can be used to build a database of BCS's. The strategy involves creating a structure-based alignment of sequences of the 18S rRNA subunits. For example, the SILVA rRNA database project is part of the ARB software package and provides comprehensive, quality checked and regularly updated databases of aligned small (16S/18S, SSU) and large subunit (23S/28S, LSU) ribosomal RNA (rRNA) sequences for all three domains of life (Bacteria, Archaea and Eukarya) (Ludwig, W. et al., 2004). Databases can also be assembled by surveying a number of Plasmodium sp. isolated from the field, using the primer sets A, B, and C. In another embodiment, databases combine data from known sequences, such as those from the SILVA rRNA database, and that data collected from the field.

[0097] Databases useful for the invention contain known Plasmodium sp. molecular masses and BCS's of the targeted sequences (as defined by the primer sets A, B, and C) and, optionally, BCS's and masses from homologous regions from benign background organisms. The latter is used to estimate and subtract the signature produced by the background organisms. Optionally, a maximum likelihood detection of known background organisms is implemented using matched filters and a running-sum estimate of the noise covariance. Background signal strengths are estimated and used along with the matched filters to form signatures which are then subtracted. The maximum likelihood process is applied to this "cleaned up" data in a similar manner employing matched filters for the organisms and a running-sum estimate of the noise-covariance for the cleaned up data.

[0098] An example of data that could be compiled into a simple database is shown in Table 6, which shows the BCS's, G+C content and molecular mass, as derived from the amplified sequences from the primer sets A, B, and C (resulting in the sequences of SEQ ID NOs: 11-22; note that the amplified sequences include the primer sequences themselves).

TABLE 6

Data from Amplified Target Sequences SEQ ID NO: BCS (%v) G+C content (%) Average Molecular Mass A G C

11 36.6 33.8 14.1 15.5 29.6 22433.691

12 38.9 30.6 11.1 19.4 30.6 22860.977

13 34.1 33.5 12.7 19.7 32.4 55906.604

14 33.8 33.8 15.5 16.9 32.4 21967.337

15 35.0 32.5 12.5 20.0 32.5 37733.661

16 32.7 32.1 13.9 21.2 35.2 52354.197

17 37.0 28.8 19.2 15.1 34.2 22991.050

18 36.3 31.5 12.9 19.4 32.3 39630.944

19 31.4 32.7 15.1 20.8 35.8 50427.925

20 34.7 33.3 16.7 15.3 31.9 22385.641

21 33.3 29.3 14.6 22.8 37.4 39360.723

22 32.1 30.2 17.0 20.8 37.7 50391.902 bounded to the nearest 0.1.

[0099] Kits

[00100] The polynucleotides of SEQ ID NOs: 1 -6 can be included as part of kits that allow for the detection of Plasmodium nucleic acids. Such kits comprise one or more of the polynucleotides of the invention. In one embodiment, the polynucleotides are provided in the kits in combinations for use as primers to specifically amplify Plasmodium nucleic acids in a test sample.

[00101] Kits for the detection of Plasmodium nucleic acids can also include a control target nucleic acid. Kits can also include control primers, which specifically amplify a sequence of the control target nucleic acid sequence.

[00102] Kits can also include amplification reagents, reaction components and/or reaction vessels. One or more of the polynucleotides can be modified as previously discussed. One or more of the components of the kit may be lyophilized, and the kit can further include reagents suitable for reconstituting the lyophilized products. The kit can additionally contain instructions for use.

[00103] In an additional embodiment, the kits further contain computer-readable media that contains a database that allows for the identification of BCS's. Optionally, the computer-readable media can contain software that allows for data collection and/or database interrogation. Kits can also be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic -readable medium, such as a floppy disc, CD- ROM, DVD-ROM, Zip disc, videotape, audiotape, etc. [00104] When a kit is supplied, the different components of the composition can be packaged in separate containers and admixed immediately before use. Such packaging of the components separately can permit long-term storage of the active components. For example, one or more of the particles having polynucleotides attached thereto, the substrate, and the nucleic acid enzyme are supplied in separate containers.

[00105] The reagents included in the kits can be supplied in containers of any sort such that the different components are preserved and are not adsorbed or altered by the materials of the container. For example, sealed glass ampoules can contain one of more of the reagents or buffers that have been packaged under a neutral, non-reacting gas, such as nitrogen. Ampoules can consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, etc.; ceramic, metal or any other material typically used to hold similar reagents. Other examples of suitable containers include simple bottles that can be fabricated from similar substances as ampoules, and envelopes, that can have foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, etc.

[00106] EXAMPLES

[00107] The following examples are for illustrative purposes only and should not be interpreted as limitations of the claimed invention. There are a variety of alternative techniques and procedures available to those of skill in the art which would similarly permit one to successfully perform the intended invention.

[00108] Example 1. Primer design

[00109] In this example, the A and B primer sets suitable for use polymerase chain reactions and other polynucleotide amplification protocols were designed to produce amplification products that were suitable for mass spectrometric analysis to allow identification of four species of

Plasmodium: malariae, falciparum, ovale and vivax. Primers were designed using conserved 18S ribosomal RNA sequences surrounding variable regions.

[00110] The primers were designed using OLIGO 6 software (Molecular Biology Insights, Inc.;

Cascade, CO), using the following design parameters:

200 nM primer concentration

50 mM monovalent cation

0.7 mM free divalent cation [00111] Example 2 Demonstration of primer efficacy to amplify target sequence (Prophetic) [00112] Two primer sets, A (SEQ ID NOs: 1 and 2) and B (SEQ ID N0s:3 and 4), as shown in Table 1 and reproduced in Table 7, are tested for their ability to amplify the target sequence and for the amplified sequence to be detected. The primers themselves can further incorporate a nucleotide analog, such as inosine, uridine, 2,6-diaminopurine, propyne C or propyne T.

TABLE 7 Primer pair sets and sequences

Primer Sequence SEQ Length, Tm, 0C Amplicon

ID NO nt length, bp

A (Forward) cggctcatta aaacagttat 1 20 61 -70

A (Reverse) tagctacagc ttttccgta 2 19 60

B (Forward) cctaccgatt gaaagatatg a 3 21 63 -120

B (Reverse) cggaaacctt gttacgac 4 18 63 (-70 for falciparum)

[00113] The conditions for the PCR are:

94 0C I x 10 sec. 35 cycles

55-60 0C 1 x 20 sec.

72 0C I x 20 sec. 4 0C hold 1 cycle

Alternatively, a "hot start" polymerase can be used in order to avoid false priming during the initial rounds of PCR. Adjustments to the PCR cycling parameters would include a heat activation step prior to the standard PCR cycling.

94 0C 5-10 min. 1 cycle

94 0C I x 10 sec. 35 cycles

55-60 0C 1 x 20 sec.

72 0C I x 20 sec. 4 0C hold 1 cycle [00114] The predicted amplification products are subjected to mass spectrometric analysis, their BCS determined and coupled with database interrogation, wherein the database contains the BCS information from the regions targeted by the primer sets A (SEQ ID NOs: 1 and T), B (SEQ ID NOs:3 and 4), and C (SEQ ID NOs:5 and 6), including the mass and/or BCS of each target sequence.

[00115] Example 3 Mass Spectrometry (Prophetic)

[00116] Fourier transform ion cyclotron resonance (FTICR) mass spectrometry Instrumentation: The FT-ICR instrument is based on a 7 tesla actively shielded superconducting magnet and modified Bruker Daltonics Apex II 7Oe ion optics (Bruker Daltonics; Billerica; MA) and vacuum chamber. The spectrometer is interfaced to a LEAP PAL autosampler (LEAP Technologies; Carrboro, NC) and a custom fluidics control system for high throughput screening applications. Samples are analyzed directly from 96-well or 384-well microtiter plates at a rate of about 1 sample/minute. The Bruker data- acquisition platform is supplemented with a lab-built ancillary data station that controls the autosampler and contains an arbitrary waveform generator capable of generating complex rf-excite waveforms (frequency sweeps, filtered noise, stored waveform inverse Fourier transform (SWIFT), etc.) for tandem MS experiments. Typical performance characteristics include mass resolving power in excess of 100,000 (FWHM), low ppm mass measurement errors, and an operable m/z range between 50 and 5000 m/z. [00117] Modified ESI Source: In sample-limited analyses, analyte solutions are delivered at 150 nL/minute to a 30 mm i.d. fused-silica ESI emitter mounted on a 3-D micromanipulator. The ESI ion optics consists of a heated metal capillary, an rf-only hexapole, a skimmer cone, and an auxiliary gate electrode. The 6.2 cm rf-only hexapole is comprised of 1 mm diameter rods and is operated at a voltage of 380 Vpp at a frequency of 5 MHz. An electro-mechanical shutter can be used to prevent the electrospray plume from entering the inlet capillary unless triggered to the "open" position via a TTL pulse from the data station. When in the "closed" position, a stable electrospray plume is maintained between the ESI emitter and the face of the shutter. The back face of the shutter arm contains an elastomeric seal that can be positioned to form a vacuum seal with the inlet capillary. When the seal is removed, a 1 mm gap between the shutter blade and the capillary inlet allows constant pressure in the external ion reservoir regardless of whether the shutter is in the open or closed position. When the shutter is triggered, a "time slice" of ions is allowed to enter the inlet capillary and is subsequently accumulated in the external ion reservoir. The rapid response time of the ion shutter (<25 ms) provides reproducible, user defined intervals during which ions can be injected into and accumulated in the external ion reservoir. [00118] Apparatus for Infrared Multiphoton Dissociation: A 25-watt CW CO2 laser operating at 10.6 μm is interfaced to the spectrometer to enable infrared multiphoton dissociation (IRMPD) for tandem MS applications. An aluminum optical bench is positioned approximately 1.5 m from the actively shielded superconducting magnet such that the laser beam is aligned with the central axis of the magnet. Using standard infrared-compatible mirrors and kinematic mirror mounts, the unfocused 3 mm laser beam is aligned to traverse directly through the 3.5 mm holes in the trapping electrodes of the FT-ICR trapped ion cell and longitudinally traverse the hexapole region of the external ion guide finally impinging on the skimmer cone. This scheme allows infrared multiphoton dissociation (IRMPD) to be conducted in an mlz selective manner in the trapped ion cell (e.g. following a SWIFT isolation of the species of interest), or in a broadband mode in the high pressure region of the external ion reservoir where collisions with neutral molecules stabilize IRMPD-generated metastable fragment ions resulting in increased fragment ion yield and sequence coverage.

[00119] Example 4 Assaying for the presence of Plasmodium sp.from a test sample (Prophetic) [00120] A sample from an organism or subject suspected of carrying Plasmodium sp. organisms is processed using well-known methods and is assayed using primer set A, B, and/or C using PCR using standard methods, such as those shown in Example 2. The amplified products are assayed by mass spectrometry using the set-up described in Example 3. [00121] If necessary, nucleic acid is isolated from the samples, for example, by cell lysis, centrifugation and ethanol precipitation or any other technique well known in the art. [00122] Mass measurement accuracy can be assayed using an internal mass standard in the ESI- MS study of PCR products. A mass standard, such as a 20-mer phosphorothioate oligonucleotide added to a solution containing a primer set A, B, and/or C PCR product(s) from Plasmodium sp. [00123] The predicted amplification products are subjected to mass spectrometric analysis coupled with database interrogation, wherein the database contains the information from the regions targeted by the primer sets A (SEQ ID NOs: 1 and T), B (SEQ ID NOs:3 and 4), and C (SEQ ID NOs:5 and 6), including the mass and/or BCS of each target sequence (such as the data provided in Table 5).

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49.

Claims

We claim:
1. A method of identifying the presence or absence of a causative agent of malaria in a test sample, comprising: providing a test sample; forming a reaction mixture comprising: a primer pair set selected from the group consisting of set A, B and C, wherein: set A comprises a forward primer comprising a nucleic acid sequence of SEQ ID NO: 1, and a reverse primer comprising a nucleic acid sequence of SEQ ID NO:2 set B comprises a forward primer comprising a nucleic acid sequence of SEQ ID NO:3, and a reverse primer comprising a nucleic acid sequence of SEQ ID NO:4; and set C comprises a forward primer comprising a nucleic acid sequence of SEQ ID NO: 5, and a reverse primer comprising a nucleic acid sequence of SEQ ID NO: 6, subjecting the mixture to amplification conditions to generate an amplification product; determining the molecular mass of the amplification product; and comparing the molecular mass of the amplification product to calculated or measured molecular masses of target sequences in a database to identify the presence or absence of the causative agent of malaria.
2. A method of identifying the presence or absence of a causative agent of malaria in a test sample, comprising: providing a test sample; forming a reaction mixture comprising: a primer pair set selected from the group consisting of set A, B and C, wherein: set A comprises a forward primer comprising a nucleic acid sequence of SEQ ID NO: 1, and a reverse primer comprising a nucleic acid sequence of SEQ ID NO:2 set B comprises a forward primer comprising a nucleic acid sequence of SEQ ID NO:3, and a reverse primer comprising a nucleic acid sequence of SEQ ID NO:4; and set C comprises a forward primer comprising a nucleic acid sequence of SEQ ID NO: 5, and a reverse primer comprising a nucleic acid sequence of SEQ ID NO: 6, subjecting the mixture to amplification conditions to generate an amplification product; determining the base composition of the amplification product; and comparing the base composition of the amplification product to calculated or measured base compositions of target sequences in a database to identify the presence or absence of a causative agent of malaria.
3. The method of claim 1 or 2, wherein the identifying the target sequence does not comprise sequencing of the amplification product.
4. The method of claim 1 or 2, wherein the reaction mixture comprises at least two primer pair sets.
5. The method of claim 1 or 2 wherein the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), time of flight mass spectrometry (TOF-MS), or electrospray ionization time of flight spectroscopy.
6. The method of claim 1 or 2, wherein the primer set comprises at least one nucleotide analog.
7. The method of claim 6, wherein the nucleotide analog is selected from the group consisting of inosine, uridine, 2,6-diaminopurine, propyne C, and propyne T.
8. The method of claim 1 or 2, wherein a molecular mass-modifying tag is incorporated into the amplification product.
9. A method of identifying the presence or absence of a causative agent of malaria in a test sample, comprising: providing a test sample; forming a reaction mixture comprising: a primer pair set selected from the group consisting of set A, B and C, wherein: set A comprises a forward primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO : 1 , and a reverse primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO:2 set B comprises a forward primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO:3, and a reverse primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO:4; and set C comprises a forward primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO:5, and a reverse primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO:6, subjecting the mixture to amplification conditions to generate an amplification product; determining the molecular mass of the amplification product; and comparing the molecular mass of the amplification product to calculated or measured molecular masses of target sequences in a database to identify the presence or absence of a causative agent of malaria.
10. A method of identifying the presence or absence of a causative agent of malaria in a test sample, comprising: providing a test sample; forming a reaction mixture comprising: a primer pair set selected from the group consisting of set A, B and C, wherein: set A comprises a forward primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO: 1 , and a reverse primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO:2 set B comprises a forward primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO:3, and a reverse primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO:4; and set C comprises a forward primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO:5, and a reverse primer comprising a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO:6, subjecting the mixture to amplification conditions to generate an amplification product; determining the base composition of the amplification product; and comparing the base composition of the amplification product to calculated or measured base compositions of target sequences in a database to identify the presence or absence of a causative agent of malaria.
11. The method of claim 9 or 10, wherein the identifying the target sequence does not comprise sequencing of the amplification product.
12. The method of claim 9 or 10, wherein the reaction mixture comprises at least two primer pair sets.
13. The method of claim 9 or 10, wherein the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), time of flight mass spectrometry (TOF-MS), or electrospray ionization time of flight spectroscopy.
14. The method of claim 9 or 10, wherein the primer set comprises at least one nucleotide analog.
15. The method of claim 14, wherein the nucleotide analog is selected from the group consisting of inosine, uridine, 2,6-diaminopurine, propyne C, and propyne T.
16. The method of claim 9 or 10, wherein a molecular mass-modifying tag is incorporated into the amplification product.
17. A kit, comprising a primer pair set selected from the group consisting of set A, B and C, wherein: set A comprises a forward primer comprising a nucleic acid sequence of SEQ ID NO: 1, and a reverse primer comprising a nucleic acid sequence of SEQ ID NO:2 set B comprises a forward primer comprising a nucleic acid sequence of SEQ ID NO:3, and a reverse primer comprising a nucleic acid sequence of SEQ ID NO:4; and set C comprises a forward primer comprising a nucleic acid sequence of SEQ ID NO: 5, and a reverse primer comprising a nucleic acid sequence of SEQ ID NO: 6, amplification reagents; and a means to access a database comprising calculated or measured base compositions or molecular masses of target sequences in a database to identify the presence or absence of a causative agent of malaria.
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