# Patent

## Title
Nucleic acid probes and methods for detecting plasmodium parasites

## Abstract

This invention relates to novel nucleic acid-based probes and methods for detecting Plasmodium parasites in biological samples as well as detecting different Plasmodium parasites selectively from one another.

## Background
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 11, 2010, is named 01532009.txt, and is 1,353 bytes in size.
BACKGROUND OF THE INVENTION
Plasmodium is a genus of parasitic protozoa. Infection with this genus is known as malaria. The parasite always has two hosts in its life cycle: a mosquito vector and a vertebrate host. At least ten species infect humans including P. falciparum, P. vivax, P. malariae, and P. ovale. Malaria is an infectious disease that is widespread in tropical and subtropical regions. Malaria represents a threat to survival of men, women and children. It infects between 300 and 500 million people every year and causes between one and three million deaths annually, mostly among young children in Sub-Saharan Africa.
Malaria is one of the most common infectious diseases and an enormous public-health problem. The disease is caused by protozoan parasites of the genus Plasmodium. The most serious forms of the disease are caused by Plasmodium falciparum and Plasmodium vivax, but other related species (Plasmodium ovale and Plasmodium malariae) can also infect humans. Determining the infectious species can help determine the course of treatment for the patient.
The preferred and most reliable diagnosis of malaria is microscopic examination of blood films because each of the four major parasite species has distinguishing characteristics. Two sorts of blood film are traditionally used. Thin films are similar to usual blood films and allow species identification because the parasite's appearance is best preserved in this preparation. Thick films allow the microscopist to screen a larger volume of blood and are about eleven times more sensitive than the thin film, so picking up low levels of infection is easier on the thick film, but the appearance of the parasite is much more distorted and therefore distinguishing between the different species can be much more difficult.
From the thick film, an experienced microscopist can detect parasite levels (or parasitemia) down to as low as 0.0000001% of red blood cells. However, microscopic diagnosis can be difficult because the early trophozoites (“ring form”) of all four species look identical and it is never possible to diagnose species on the basis of a single ring form; species identification is always based on several trophozoites.
In areas where microscopy is not available, there are antigen detection tests that require only a drop of blood. OptiMAL-IT® (TCS Bio Sciences, Buckingham, England) will reliably detect falciparum down to 0.01% parasitemia and non-falciparum down to 0.1%. Paracheck-Pf® (Orchard Biomedical Systems, India) will detect parasitemias down to 0.002% but will not distinguish between falciparum and non-falciparum malaria. Parasite nucleic acids may also be detected using polymerase chain reaction. This technique is more accurate than microscopy. However, it is expensive and requires a specialized laboratory. Moreover, levels of parasitemia are not necessarily correlative with the progression of disease, particularly when the parasite is able to adhere to blood vessel walls. Limited molecular methods are available in some clinical laboratories and rapid real-time assays, for example, QT-NASBA (real-time quantitative nucleic acid sequence-based amplification) based on the polymerase chain reaction) but are only now being developed. Therefore, sensitive, low-tech diagnosis tools need to be developed for in order to detect low levels of parasitaemia in the field.
What is need are reagents and methods for the rapid and accurate detection and discrimination of various malaria causing Plasmodium species.
SUMMARY OF THE INVENTION
This invention relates to nucleic acid probes that detect and discriminate between different species of Plasmodium parasites in, for example, hybridization assays. Accordingly, in a first aspect, the invention features nucleic acid fragments to be used as probes for detecting Plasmodium in a hybridization assay. The invention also includes probes (DNA, RNA and PNA) that can discriminate between P. falciparum, P. vivax, P. malariae and P. ovale. In the context of the present invention the term “discriminates between” (or similar terms) means that the probe binds to nucleic acid (e.g., RNA, DNA, rRNA [ribosomal RNA] or rDNA [ribosomal DNA]) from one species more favorably than the other 3 species.
The probes of the present invention are able to detect specific species of Plasmodium in a sample without cross-reacting with other species of Plasmodium to any significant extent. In this regard, cross-reactivity between the probes of the present invention in the detection of various Plasmodium species is less than about 20%, less than about 15%, less than about 10%, less than about 5% or less than about 2%.
In a another aspect, the invention features a nucleic acid fragment containing a sequence selected from at least five, preferably, at least ten, more preferably at least thirteen or most preferably at least fifteen consecutive nucleotides or the entire sequence of one or more of probes designated SEQ ID NO: 1 through SEQ ID NO.: 4 or the partial or full-length complementary sequences thereof.
In a another aspect, the invention features a method for detecting the presence of Plasmodium in a sample. In this method, a sample is contacted with a nucleic acid fragment containing a sequence selected from, preferably, at least five, at least ten, more preferably at least thirteen or most preferably at least fifteen consecutive nucleotides or the entire sequence of a PV1, PF4, PM1 or PO1, probe selected from or the partial or full-length complementary sequence thereof (or any combination thereof); under conditions that permit the nucleic acid fragment to hybridize to Plasmodium nucleic acid. Detection of the nucleic acid fragment bound to the Plasmodium nucleic acid in the sample is used as an indication of the presence of Plasmodium in the sample. Detection with probes of the present invention that discriminate between the four species of Plasmodium listed above indicates the presence of that species of Plasmodium. 
In specific aspects, the present invention contemplates a method for detecting the presence of Plasmodium in a sample, said method comprising the steps of: contacting said sample with a probe for detecting Plasmodium in a hybridization assay, wherein said probe discriminates between Plasmodium species, under conditions that permit said probe to hybridize to Plasmodium nucleic acid; and detecting said probe bound to said Plasmodium nucleic acid in said sample as an indication of the presence of Plasmodium in said sample.
In the preceding embodiment, the nucleic acid fragment comprises a sequence that is selected from at least five consecutive nucleotides of probe PV1, PF4, PM1 or PO1 the full-length complementary sequence thereof.
The present invention also contemplates a nucleic acid fragment comprising a sequence selected from at least ten (10), at least thirteen (13) or at least fifteen (15) consecutive nucleotides of a probe selected from a group consisting of PV1, PF4, PM1, PO1 or the full-length complementary sequence thereof.
The present invention also contemplates a nucleic acid fragment comprising the complete sequence selected of a probe selected from a group consisting of PV1, PF4, PM1, PO1 or the full-length complementary sequence thereof.
In specific aspects, the present invention contemplates a method of selecting nucleic acid probes that discriminate between the species P. falciparum, P. vivax, P. malariae and P. ovale, the method comprising: preparing a nucleic acid fragment or polypeptide nucleic acid (PNA) corresponding to, or complementary to, a sequence of at least five nucleotides of nucleic acid from P. falciparum, P. vivax, P. malariae and P. ovale; comparing the ability of the probe to detect one or more of the Plasmodium species in a hybridization assay; and selecting the probe or probes that detect one, two or three species of Plasmodium but not all four species of Plasmodium. 
The present invention also contemplates a method for detecting and differentiating between P. falciparum, P. Vivax, P. malariae and P. ovale in a sample, said method comprising: providing: i) a sample from, for example, a subject suspected of having malaria and ii) probes comprising nucleic acid suitable for detecting and differentiating between P. falciparum, P. Vivax, P. malariae and P. ovale; contacting said sample with said probes under conditions suitable for hybridization of said probes to targets; and determining the presence of P. falciparum, P. Vivax, P. malariae and P. ovale, if any, in the sample.
In any of the preceding embodiments, the probe or probes used may be of the entire nucleotide sequence as disclosed herein or the complementary strand thereof as well as the complementary sequence of the at least five (5), at least ten (10), at least thirteen (13) or at least fifteen (15) contiguous nucleotides of the probes.


DETAILED DESCRIPTION OF THE INVENTION
The invention features nucleic acid probes for detecting Plasmodium parasites (e.g., P. falciaprum, P. vivax, P. malariae and P. ovale) in, for example, hybridization assays. The probes of the invention may be used in methods for detecting the presence of Plasmodium in a biological sample. In these methods, a probe of the invention is contacted with a biological sample or specimen (e.g., whole blood, cerebrospinal fluid (CSF), a tissue sample or any other biological sample suspected of having the presence of Plasmodium) in a hybridization assay and detection of the probe bound to the nucleic acid in the sample is used as an indication of the presence of Plasmodium in the sample.
Probes included in the invention may be identified by:
A (1) preparing a nucleic acid fragment (DNA, RNA) or polypeptide nucleic acid (PNA) (i.e., a probe) corresponding to or complementary to, a sequence of at least five, at least ten, at least thirteen or at least fifteen nucleotides of nucleic acid from P. falciparum and (2) comparing the ability of the probe to detect all the Plasmodium species in a hybridization assay. Probes that hybridize to P. falciparum more favorably than to other three species are included in the invention.
B (1) preparing a nucleic acid fragment (DNA, RNA) or PNA (i.e., a probe) corresponding to, or complementary to, a sequence of at least five, at least ten, at least thirteen or at least fifteen nucleotides from P. vivax and (2) comparing the ability of the probe to detect all the Plasmodium species in a hybridization assay. Probes that hybridize to P. vivax more favorably than to other three species are included in the invention.
C (1) preparing a nucleic acid fragment (DNA, RNA) or PNA (i.e., a probe) corresponding to, or complementary to, a sequence of at least five, at least ten, at least thirteen or at least fifteen nucleotides from P. malariae and (2) comparing the ability of the probe to detect all the Plasmodium species in a hybridization assay. Probes that hybridize to P. malariae more favorably than to other three species are included in the invention.
D (1) preparing a nucleic acid fragment (DNA, RNA) or PNA (i.e., a probe) corresponding to, or complementary to, a sequence of at least five, at least ten, at least thirteen or at least fifteen nucleotides from P. ovale and (2) comparing the ability of the probe to detect all the Plasmodium species in a hybridization assay. Probes that hybridize to P. ovale more favorably than to other three species are included in the invention.
P. falciaprum, P. vivax, P. malariae and P. ovale nucleic acid may be obtained from biological samples (such as whole blood, bone marrow, CSF) from infected individuals, using standard nucleic acid isolation methods in the art. P. falciaprum (as well as P. vivax, P. malariae and P. ovale) can also be obtained from culture. For example, DNA encoding Plasmodium ribosomal RNA may be obtained by PCR amplification of DNA prepared from a whole blood sample of an infected patient using the methods and primers described herein and known in the art.
Any Plasmodium sequence (e.g., a sequence encoding 58, 5.8S, 18S, or 28S ribosomal RNA) may be selected as a candidate sequence for the identification of probes. Preferred sequences are those that diverge from analogous sequences in non-human Plasmodium or other protozoan parasites like, for example, Babesia or Thileria, as determined by phylogenetic comparison. The nucleic acid probes of the invention are at least 5, at least 10, at least 13 or at least 15 nucleotides in length and may contain deoxyribonucleotides (DNA probes), ribonucleotides (RNA probes), peptide nucleic acid (PNA probes) or combinations or modifications thereof. The probes may be single stranded or double stranded and may be prepared by any of a number of standard methods in the art. For example, the probes may be made by chemical synthesis, restriction endonuclease digestion of a vector (e.g., a plasmid containing a sequence corresponding to the probe), polymerase chain reaction (PCR) amplification, or in vitro transcription of a vector containing a sequence corresponding to the probe (see, e.g., Ausubel, et al., Current Protocols in Molecular Biology, Greene Publishing, New York, N.Y., 1994, incorporated herein by reference). The probes may be labeled during or after synthesis. For example, labeled nucleotides containing, e.g., radioisotopes (e.g., p32, S35, or H3), biotin or digoxigenin may be incorporated into the probe during synthesis. Probes containing biotin are detected by the use of a secondary reagent such as avidin or streptavidin, which contains a detectable label such as a fluorochrome (e.g., fluorescein or rhodamine) or an enzyme (e.g., alkaline phosphatase or horseradish peroxidase). Similarly, probes containing digoxigenin may be detected by using a labeled antidigoxigenin antibody. Probes may also be labeled after synthesis by, e.g., nick translation or the use of T4 RNA ligase, poly(A) polymerase, terminal transferase or T4 polynucleotide kinase, in standard methods (see, e.g., Ausubel, et al., supra). The probes may also contain modified nucleotides in order to increase the stability of the probe. For example, ribonucleotides containing 2′-0-alkyl groups on the ribose group may be used. The probes may also contain modifications that facilitate capture of the probe onto a solid support. For example, poly-dA or poly-deaza-guanosine tails may be added to the 3′ ends of the probes, using terminal transferase, in order to facilitate probe binding to a solid support, e.g., poly-dT or poly-dC labeled magnetic particles. The probes may be purified prior to use, using standard methods such as denaturing polyacrylamide gel electrophoresis, high performance liquid chromatography or gel filtration chromatography (see, e.g., Ausubel, et al., supra). The probes of the invention may be used in any standard hybridization assay to detect the presence of Plasmodium in a sample. For example, Southern blot, dot blot, in situ hybridization, real-time hybridization detection by biosensors or dual probe, sandwich-type hybridization assays may be used (see, e.g., U.S. patent application Ser. No. 07/826,657 [now U.S. Pat. No. 5,519,127] and U.S. Pat. No. 5,629,156 [International Publication Number WO 94/10335], all of which are incorporated herein by reference). Alternatively, the probes may be used as primers in a polymerase chain reaction assay (see, e.g., Ausubel, et al., supra). Biological samples that may be analyzed using the probes and methods of the invention include whole blood, CSF, bone marrow and tissue samples from, e.g., the spleen. Nucleic acid is extracted from the sample by standard methods (except in the case of in situ hybridization, where the cells are kept intact) and is analyzed using the probes in the assays listed above. A single probe or combinations of probes may be used in the assay. The hybridization conditions used with the probes (e.g., in the methods of the invention) fall within the range of, for example, about 30 to about 50% formamide at about 25° C. to about 42° C. or mixtures of GuSCN and formamide at about 25° C. to about 37° C. As is “known by one skilled in the art,” selection of hybridization conditions depends on the length and nucleotide content (i.e., GC compared to AT) of the probe. Accordingly, hybridization conditions may be adjusted to accommodate these factors. In addition, the use of different salts (e.g., guanidine thiocyanate or guanidine hydrochoride compared with NaCl) and denaturing agents (e.g., NP-40, sodium dodecyl sulfate) may require adjustment of the salt concentration and the temperature, as can readily be determined by one skilled in the art.
Non-limiting examples of hybridization conditions that may be used in the present invention are as follows. In Southern blot analysis, the following hybridization conditions may be used: 30% to 50% formamide in 2×SSC at about 42° C. After hybridization, the filters are washed using standard methods. For example, three 15 minute post-hybridization washes at about 25° C. in 2×SSC to 0.1×SSC and 0.1% SDS may be carried out in order to remove unbound probes. For RNA blots hybridizations in about 30% formamide at room temperature overnight were performed. Excess probes were removed by washing three 15 min washes in 2×SSC with 0.1% SDS.
The term “hybridization” refers to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the Tm of the formed hybrid, and the G-C ratio within the nucleic acids. A single molecule that contains pairing of complementary nucleic acids within its structure is said to be “self-hybridized.”
It is well known that numerous equivalent conditions may be employed to comprise suitable hybridization conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions. In addition, the art knows conditions that promote hybridization under conditions of high stringency (e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.).
For in situ hybridization, the following conditions may be used as described in U.S. Pat. No. 6,165,723 and U.S. patent application Ser. No. 11/494,430 (which are herein incorporated by reference): GuSCN (1.5 to 3.5 M depending on the probe sequence) between room temperature and about 37° C. or mixtures of GuSCN and formamide between room temperature and about 37° C. for 30 minutes to one hour, followed by washes in SSC (2× to 0.1×) and 0.1% SDS.
Fluorescent markers (dyes) suitable for use in the present invention include, but are not limited to, for example, Alexa 488, rhodamin, Texas red, fluorescein, Oregon Green, Tokyo Green as well as others known to those of ordinary skill in the field at the time of the invention.
EXEMPLIFICATION
Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever.
Hybridization of Probes PF4, PV1, PM1 and PO1 to P. falciaprum, P. vivax, P. ovale and P. malariae Samples, Respectively.
Dot-blot analysis data is shown in Table 1. Dot-blot protocol and analysis is known in the art. See, for example, Brown, T., Dot and Slot Blotting DNA, Curr. Protoc. Mol. Biol., 2001 May; Chapter 2: Unit2.9B and references therein. In the present experiments, approximately 0.1 ug of a plasmid containing nucleotide sequences encoding the respective 18S rRNA subunits was used per spot. The blots were hybridized with dig-labeled probes under the hybridization conditions described above.
In another embodiment, fluorescent in situ hybridization (FISH) may be used to detect the probe(s) of the present invention. FISH is well known to those of skill in the art. For example, see, Volpi, and Bridger, FISH glossary: An Overview of the Fluorescence in situ Hybridization Technique, Biotechniques, 2008 October; 45(4):385-6, 388, 390 passim, and references therein.
In the present experiment, a dot blot assay was employed. In the case of RNA, RNA was synthesized and 0.1 ug in 6×SSC was spotted on nitrocellulose membrane. Hybridization with the dig-labeled probes (digoxigenin-labeled probes) was performed overnight at room temperature in formamide. This method was used to compare hybridization signals between the different organisms and probes.
Plasmodium Species 18S rRNA-Specific Probes
Plasmodium species specific probes of the invention include probes which have the sequences of a probe selected from PV1, PF4, PM1 and PO1 or the full-length complementary sequence thereof.









P. vivax Specific Probe







[SEQ ID NO.: 1]










PV1
5′-AGCAAAATGCGCACAAAGTCGATACGAAGTATC-3′



 









P. falciparum Specific Probe







[SEQ ID NO.: 2]










PF4
5′-TTACAAAACCAAAAATTGGCCTTGCATTGTTATTT-3′



 









P. malariae Specific Probe







[SEQ ID NO.: 3]










PM1
5′-GAAACACTCATATATAAGAATGTCTC-3′



 









P. ovale Specific Probe







[SEQ ID NO.: 4]










PO1
5′-AATTTCCCCGAAAGGAATTTTC-3′

















TABLE 1





Probes
P. falciparum
P. vivax
P. malariae
P. ovale







PV1
Negative
Positive
Negative
Negative


PF4
Positive
Negative
Negative
Negative


PM1
Negative
Negative
Positive
Negative


PO1
Negative
Negative
Negative
Positive









From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

## Claims

1. A method for detecting the presence of Plasmodium falciparum in a sample, said method comprising the steps of:
a) contacting said sample with a nucleic acid probe suitable for detecting Plasmodium falciparum in a hybridization assay, the probe consisting of SEQ ID NO: 2 or the full-length complement of SEQ ID NO: 2, and optionally a label, under conditions that permit said probe to hybridize to Plasmodium falciparum nucleic acid; and
b) detecting said probe bound to said Plasmodium falciparum nucleic acid as an indication of the presence of Plasmodium falciparum in said sample.

2. The method of claim 1, wherein said nucleic acid probe is the full-length complement of SEQ ID NO: 2.

3. The method of claim 1 wherein said detecting comprises detecting probe bound to said Plasmodium falciparum nucleic acid by Fluorescent in Situ Hybridization (FISH).

4. The method of claim 1 wherein said detecting comprises detecting probe bound to said Plasmodium falciparum nucleic acid by a Dot Blot assay.