WO2013159293A1

WO2013159293A1 - Method, composition and kit for high throughput detection of genus plasmodium

Info

Publication number: WO2013159293A1
Application number: PCT/CN2012/074669
Authority: WO
Inventors: Zhi Zheng; Zhibin Cheng
Original assignee: Institute of Basic Medical Sciences of AMMS; Institute of Basic Medical Sciences of CAMS and PUMC
Current assignee: Institute of Basic Medical Sciences of AMMS; Institute of Basic Medical Sciences of CAMS and PUMC
Priority date: 2012-04-25
Filing date: 2012-04-25
Publication date: 2013-10-31
Anticipated expiration: 2014-10-25
Also published as: CN104284986B; CN104284986A

Description

METHOD, COMPOSITION AND KIT FOR HIGH THROUGHPUT DETECTION OF GENUS

PLASMODIUM

Technical field of the invention

The present invention relates to a method for high throughput and sensitive detection of Genus Plasmodium. The present invention also relates to the composition and kit thereof.

Background of the invention

Malaria remains as one of the leading infectious diseases in the world. In recent years, however, its decline of transmission in many countries makes it possible to consider elimination of the disease (WHO. World malaria report; 2009). As countries approach malaria elimination, disease monitoring, evaluation, and surveillance activities will need to shift from measuring morbidity and mortality to detecting infections and measuring transmission. This calls for new diagnostic tools with higher sensitivity for detecting asymptomatic infections and higher throughput for mass screening and surveillance (A research agenda for malaria eradication: diagnoses and diagnostics. PLoS Med 2011;8:el000396). For example, with the increasing spread of the disease to non-endemic areas as a result of global traveling and migrations, there is an urgent need for large scale, active malaria screening in at-risk populations that may or may not show clinical symptoms, as subclinically-infected individuals can be significant sources of infection (Harris I, Sharrock WW, Bain LM, et al. A large proportion of asymptomatic Plasmodium infections with low and sub-microscopic parasite densities in the low transmission setting of Temotu Province, Solomon Islands: challenges for malaria diagnostics in an elimination setting. Malar J 2010;9:254). The parasites responsible for malaria are Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi. The most serious and fatal type of malaria is caused by P. falciparum, while P. vivax-caused malaria is more prone to relapse (Galinski MR, Barnwell JW. Plasmodium vivax: who cares? Malar J 2008;7 Suppl 1 :S9). Overall, P. falciparum and P. vivax are the most prevalent parasites for malaria, and countries with low, unstable transmission are encouraged to proceed to malaria elimination by focusing on both falciparum elimination and vivax elimination (WHO. World malaria report; 2010).

Current diagnostic methods lack the sensitivity or throughput needed for monitoring and controlling malaria transmission. The most-widely used microscopic examination cannot detect Plasmodia infection lower than 50 parasites/μΐ (Moody A. Rapid diagnostic tests for malaria parasites. Clinical Microbiology Reviews 2002; 15:66), and can miss a substantial proportion of infections in surveys of endemic populations, especially in areas with low transmission of infection (Okell LC, Ghani AC, Lyons E, Drakeley CJ. Submicroscopic infection in Plasmodium falciparum-endemic populations: a systematic review and meta-analysis. J Infect Dis 2009; 200: 1509-17). Recently developed molecular methods, such as PCR (Snounou G, Viriyakosol S, Jarra W, Thaithong S, Brown K . Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections. Mol Biochem Parasitol 1993; 58:283-92), quantitative-PCR (Rougemont M, Van Saanen M, Sahli R, Hinrikson HP, Bille J, Jaton K. Detection of four Plasmodium species in blood from humans by 18S rRNA gene subunit-based and species-specific real-time PCR assays. J Clin Microbiol 2004;42:5636-43), Nucleic Acid Sequence-Based Amplification (NASBA) (Mens PF, Schoone GJ, Kager PA, Schallig HD. Detection and identification of human Plasmodium species with real-time quantitative nucleic acid sequence-based amplification. Malar J 2006;5:80) and Loop Mediated Isothermal Amplification (LAMP) (Lucchi NW, Demas A, Narayanan J, et al. Real-Time Fluorescence Loop Mediated Isothermal Amplification for the Diagnosis of Malaria. PLoS One 2010;5) are more sensitive and call for less malaria expertise, yet their dependence on DNA RNA extraction can strongly influence the performance of the diagnosis. Furthermore, such dependence also hindered their use in high throughout patient screening. The protein-based Rapid Diagnostic Tests (RDTs) are easy, sensitive and useful for malaria screening, but they are more reliable for detecting P. falciparum infection and some cannot differentiate between active infection and past malaria experience, reducing their effectiveness as alternatives to microscopic diagnosis.

Summary of the invention

The present invention provides an easy, sensitive and reliable method suitable for large-scale detection of Genus Plasmodium. We adopted our previously developed sandwich RNA hybridization assay (Zheng Z, Luo Y, McMaster GK. Sensitive and quantitative measurement of gene expression directly from a small amount of whole blood. Clin Chem 2006;52: 1294-302; and US patent application US2007/016015) to detect the presence of genus Plasmodium in a sample. All of the contents of the references are incorporated herein.

In detail, the present provides a method for detection of genus Plasmodium in a sample, comprising :

(a) providing a test sample suspected of containing target genus

Plasmodium;

(b) optionally, releasing the polynucleic acids from the test samples of (a);

(c) contacting the said test sample or the released polynucleic acids with probe mixture containing one or more capture extender (CE) probes and one or more label extender (LE) probes;

wherein the CE probe consists of nucleotides hybridizing to a region of the 18S ribosomal RNA of genus Plasmodium and the nucleotides hybridizing to nucleic acids covalently or non-covalently attached to a solid support;

and wherein the LE probes consists of nucleotides hybridizing to a region of 18S ribosomal RNA of genus Plasmodium and the nucleotides hybridizing to a detectable molecule;

for a time and under conditions sufficient for hybridization to take place; and

(d) contacting the said test sample with a detectable molecule after the said step (c), for a time and under conditions sufficient for hybridization to take place;

(e) detecting any hybridization which has taken place in step (c); wherein the presence or absence of hybridization is indicative of the presence or absence of said genus Plasmodium in said test sample. In another aspect of the invention, the present provides a method for detection of genus Plasmodium in a sample, comprising :

(a) providing a test sample suspected of containing target genus

Plasmodium;

(b) optionally, releasing the polynucleic acids from the test samples of (a);

(c) contacting the said test sample or the released polynucleic acids with probe mixture containing one or more CE probes, one or more LE probes and at least one blocking probe;

wherein the CE probe consists of nucleotides hybridizing to the region of the 18S ribosomal RNA of genus Plasmodium and the nucleotides hybridizing to nucleic acids covalently or non-covalently attached to a solid support;

and wherein the LE probes consists of nucleotides hybridizing to the region of 18S ribosomal RNA of genus Plasmodium and the nucleotides hybridizing to a detectable molecule;

and wherein the blocking probe consists of nucleotides hybridizing to 18S ribosomal RNA of genus Plasmodium to reduce or eliminate non-specific hybridization of other probes to the sequence, and to enhance the hybridization of adjacent probes by stacking effect.

for a time and under conditions sufficient for hybridization to take place; and

(e) detecting any hybridization which has taken place in step (c); wherein the presence or absence of hybridization is indicative of the presence or absence of said genus Plasmodium in said test sample.

In a preferable embodiment, the amount of CE probe is from 1 wt% to 99 wt% by total weight of the composition; the amount of LE probe is from 1 wt% to 99 wt% total weight of the composition; the amount of blocking probe is from 1 wt% to 99 wt% total weight of the composition.

In a preferable embodiment, the said blocking probe has a sequence as shown in SEQ ID NO:22.

In a preferable embodiment, the said genus Plasmodium is selected from the group consisting of R falciparum, R vivax, R malariae, R ovale and R knowlesi.

In another embodiment, the test sample is a blood sample including plasma, serum or blood clot, cultured erythrocytes, or blood sample dried on a filter paper.

In a preferable embodiment, the test sample is treated with proteinase K. Preferably, the test sample is treated with proteinase K at 50^°C to 60^°C for 1 hour. More preferably, the test sample is treated with proteinase K at 56 ^°C for 1 hour.

In a preferable embodiment, the said one or more CE probes are bound to a solid support. More preferably, the solid support is a planar solid support or a bead. Most preferably, the support is a 96-well plate. In a preferable embodiment, the detectable molecule is labeled with an enzyme or a fluorescent label.

In a preferable embodiment, the nucleotides hybridizing to the region of 18S ribosomal R A of genus Plasmodium have at least 80% identity, more preferably, at least 85%, more preferably 90%, most preferably 95% identity to the nucleotides as shown in SEQ ID NOs 23 to 43.

In other embodiments, the at least one or more CE probes are selected from the group consisting of SEQ ID NOs 1 to 7; and the at least one or more LE probe is selected from the group consisting of SEQ ID NOs 8 to 21.

In another aspect, the present invention provides a method for detection of genus Plasmodium in a sample, comprising:

(a) providing a test sample suspected of containing target genus

Plasmodium;

(b) optionally, releasing the polynucleic acids from the test samples of (a);

(c) contacting the said test sample or the released polynucleic acids with probes mixture containing all of the probes represented as SEQ ID NOs 1 to 22; for a time and under conditions sufficient for hybridization to take place; and

In another aspect, the present invention provides a composition for detection of genus Plasmodium in a sample, comprising:

(a) one or more CE probes; wherein the said CE probe consists of nucleotides hybridizing to a region of the 18S ribosomal RNA of genus Plasmodium and the nucleotides hybridizing to nucleic acids covalently non-covalently attached to a solid support; and

(b) one or more LE probes; and wherein the LE probe consists of nucleotides hybridizing to a region of 18S ribosomal RNA of genus Plasmodium and the nucleotides hybridizing to a detectable molecule.

(a) one or more CE probes; wherein the said CE probe consists of nucleotides hybridizing to the region of the 18S ribosomal RNA of genus Plasmodium and the nucleotides hybridizing to nucleic acids covalently non-covalently attached to a solid support;

(b) one or more LE probes; and wherein the LE probe consists of nucleotides hybridizing to the region of 18S ribosomal RNA of genus Plasmodium and the nucleotides hybridizing to a detectable molecule; and (c) at least one blocking consisting of nucleotides hybridizing tol8S ribosomal RNA of genus Plasmodium to reduce or eliminate non-specific hybridization of other probes to the sequence, and to enhance the hybridization of adjacent probes by stacking effect.

In a preferable embodiment, the nucleotides hybridizing to the region of 18S ribosomal RNA of genus Plasmodium have at least 80% identity, more preferably, at least 85%, more preferably 90%, most preferably 95% identity to the nucleotides as shown in SEQ ID NOs 23 to 43.

In a preferable embodiment, the said probe is selected from the group consisting of SEQ ID NOs 1 to 7; and the LE probe is selected from the group consisting of SEQ ID NOs 8-21.

In another respect, the present invention provides a composition for detection of genus Plasmodium in a sample, comprising all of the probes as shown in SEQ ID NOs 1 to 23.

In another respect, the present invention provides a kit for detection of genus Plasmodium in sample, comprising

(a) a composition comprising:

(i) one or more CE probes; wherein the said CE probe consists of nucleotides hybridizing to a region of the 18S ribosomal RNA of genus Plasmodium and the nucleotides hybridizing to nucleic acids covalently non-covalently attached to a solid support; and

(ii) one or more LE probes; and wherein the LE probe consists of nucleotides hybridizing to a region of 18S ribosomal RNA of genus Plasmodium and the nucleotides hybridizing to a detectable molecule;

(b) a solid support;

(c) a detectable molecule.

(a) a composition comprising:

(iii) at least one blocking consisting of nucleotides hybridizing tol 8S ribosomal RNA of genus Plasmodium to reduce or eliminate non-specific hybridization of other probes to the sequence, and to enhance the hybridization of adjacent probes by stacking effect. (b) a solid support;

(c) a detectable molecule.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a molecule" includes a plurality of such molecules, and the like.

The terms "polynucleic acid", "polynucleotide", "nucleotide" and "nucleic acid" are interchangeably used herein and encompass any physical string of monomer units that can be corresponded to a string of nucleotides, including a polymer of nucleotides (e.g., a typical DNA or RNA polymer), peptide nucleic acids (PNAs), modified oligonucleotides (e.g., oligonucleotides comprising nucleotides that are not typical to biological RNA or DNA, such as 2'-0-methylated oligonucleotides), and the like.

The term "capture extender probe" or "CE probe" is a polynucleotide that is capable of hybridizing to 18S ribosomal RNA of genus Plasmodium and is capable of hybridizing to nucleic acids covalently or non-covalently attached to a solid support. The capture extender probe (CE probe) typically has a first polynucleotide portion, which is complementary to 18S ribosomal RNA of genus Plasmodium, and a second polynucleotide portion, which is complementary to a polynucleotide covalently or non-covalently attached to a solid support. The first and the second polynucleotide are typically not complementary to each other. The CE probe is preferably single-stranded.

The term "label extender probe" or "LE probe" is a polynucleotide that is capable of hybridizing to 18S ribosomal RNA of genus Plasmodium and is capable of hybridizing to a detectable molecule. The label extender probe (LE probe) typically has a first polynucleotide portion, which is complementary to 18S ribosomal RNA of genus Plasmodium, and a second polynucleotide portion, which is complementary to a detectable molecule. The first and the second polynucleotide are typically not complementary to each other. The LE probe is preferably single-stranded.

The term "detectable molecule" comprises one or more polynucleotides that collectively comprise a label and a polynucleotide sequence M-l, which is capable of hybridizing to at least one LE probe. The label provides a signal, directly or indirectly. Polynucleotide sequence M-l is typically complementary to the second polynucleotide in the LE probe.

The terms "hybridization" and "hybridizing" are used herein to refer to denote the paring of complementary nucleotide sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid. Defining appropriate hybridization conditions is within the skill of the art.

Brief Description of the Drawings

Figure 1 indicates the correlation of assay signal with cultured P. falciparum in human erythrocytes. P. falciparum in human erythrocyte culture was determined as described in Method for detection of Genus Plasmodium. The limit of detection was determined as the minimal amount of cultured Plasmodia added to the erythrocytes that gave a net signal above 3 times the SD of the background erythrocyte control. Each dilution is prepared using culture medium. Triplicate samples were used in the assay and the data is representative of three independent runs. Averaged limit of detection was 0.04 parasite/μΐ blood. RLU: relative light unit

Figure 2 indicates the correlation between assay signals and parasitemia of poorly stored blood samples. Stored blood samples from 59 patients, which underwent multiple freeze-and-thaw cycles after collection due to limiting resources, were assayed (20μ1 each) and the net signals were plotted against parasitemia determined microscopically at admission. Each sample was assayed in duplicate and the averaged signal was used. Signal intensities beyond lxlO⁷ RLU were considered approaching detection saturation of the photon detector.

Detailed Descriptions of the Invention

Examples

Sample collection

Veinous heparin blood samples were collected from 202 patients with fever of unknown origin in Kachine, Myanmar, and in Yunnan, China. Among them, 59 were collected in 2008, and 143 was collected in 201 1. Control blood samples (n=13) used for analytical evaluation were taken from healthy subjects in Beijing, China. All samples were frozen and stored at -80 °C before use. Samples were collected with written informed consent. Ethical approval was granted by the Institutional Review Board (IRB) of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. Due to limited resources, the 59 blood samples have suffered several freeze-and-thaw cycles before the test.

Method for detection of Genus Plasmodium

1. Microscopy

At time of admission, 2 slides, each containing a thick and a thin blood film, were collected from each patient and dyed with 2% Giemsa Stain solution for 30 minutes. Slides were then examined independently by two professional microscopists for Plasmodium parasites, and were pronounced negative only when a minimum of 500 fields had been carefully examined by each microscopist for the absence of parasites. The parasite density was counted if the slide was positive.

2. RDT

RDT tests in this study were done with CARESTART™ (Accessbio, Monmouth Junction, NJ) according to the manufacture's protocol. The kit is among the list of RDT procurement recommendations issued by WHO .

3. Real time qPCR

DNA was extracted from 200 μΐ of thawed blood with QIAamp DNA Blood Mini Kit (QIAGEN), according to the manufacturer's instructions. The genus Plasmodium 18s screening primer and probe sequences and real time qPCR condition were adopted from previously published work. If the fluorescent signal did not increase within 40 cycles (Ct 40), the sample was considered negative. At least 1 positive control and 1 negative control were included to each experiment. Each sample was tested in duplicate.

4. Sandwich RNA hybridization assay

22 oligonucleotide probes (having the sequences indicated in SEQ ID Nos: 1 to 22) targeting at several highly conserved regions in 18S ribosomal RNA of the genus Plasmodium, including P. falciparum (GeneBank accession number M19172.1), P. vivax (U03079.1), P. malariae (AF488000.1), P. ovale (L48987.1) and P. knowlesi(L07560.1)

Probe Name SEQ ID NO Sequences

CE1 SEQ ID NO 1 atcaaaagctgataggtcagaaacTTTTTctcttggaaagaaagt

CE2 SEQ ID NO 2 ccatgttaggccaataccctaacTTTTTctcttggaaagaaagt

CE3 SEQ ID NO 3 cttgtcactacctctcttctttagaatTTTTTctcttggaaagaaagt

CE4 SEQ ID NO 4 aattggccttgcattgttatttTTTTTctcttggaaagaaagt

CE5 SEQ ID NO 5 actcccttaactttcgttcttgatTTTTTctcttggaaagaaagt

CE6 SEQ ID NO 6 cacctagtcggcatagtttatggtTTTTTctcttggaaagaaagt

CE7 SEQ ID NO 7 gcctttcggcggaggaaTTTTTctcttggaaagaaagt

LEI SEQ ID NO 8 tttattacgtgttacttctttgttataattTTTTTgaagttaccgtttt

LE2 SEQ ID NO 9 tcgattgatacacactaaataaaataaaTTTTTctgagtcaaagcat

LE3 SEQ ID NO 10 accattccaattacaaaaccaaaTTTTTgaagttaccgtttt

LE4 SEQ ID NO 11 tctgggaaggttttaaattcccTTTTTctgagtcaaagcat

LE5 SEQ ID NO 12 gtattcaaacacagtaaatgctttaactTTTTTgaagttaccgtttt

LE6 SEQ ID NO 13 tgttcaattttgttattccatgctataTTTTTctgagtcaaagcat

LE7 SEQ ID NO 14 ctcctattaatcgtaactaagccaaTTTTTgaagttaccgtttt

LE8 SEQ ID NO 15 gaatacgaatgtccccaagctaTTTTTctgagtcaaagcat

LE9 SEQ ID NO 16 atctaagaatttcacctctgacatctTTTTTgaagttaccgtttt

LE10 SEQ ID NO 17 cgcagttgttcgtctccagaaTTTTTctgagtcaaagcat

LE1 1 SEQ ID NO 18 tcacgatatatattgataaagattacctacTTTTTgaagttaccgtttt LE12 SEQ ID NO: 19 ttaataattgcaataatctatccccaTTTTTctgagtcaaagcat

LE13 SEQ ID NO:20 tactaggcattcctcgttcaagaTTTTTgaagttaccgtttt

LE14 SEQ ID NO:21 agcacaatctgatgaatcatgctTTTTTctgagtcaaagcat

Blocking probe SEQ ID NO:22 tacgacggtatctgatcgtcttc

For each assay, 20 μΐ of fresh or thawed blood or erythrocyte culture of P. falciparum was lysed with 50 μΐ of Lysis Mixture (Panomics/Affymetrx), 28 μΐ of water and 2 μΐ of 50 μg/ ml proteinase K at 60 °C for 1 h with vigorous shaking. The lysate were mixed with probes and hybridized tothe probe set comprising the above 22 oligonucleotide probes, wherein the amounts of CE, LE and BE probes are 150, 600 and 300 fmol, respectively. The mixture was incubated at 58 °C overnight without shaking. After the unbound probes were washed off, captured targets were sequentially hybridized, with washing in between, with Preamplifier, Amplifier, Label Probe and Substrate as described in the Quantigene Assay Kit (Panomics/Affymetrix). The resulting chemiluminescence was quantified in a Modulus plate reader (Turner Biosciences) as previously described Zheng Z et ah, 2006 (Zheng Z, Luo Y, McMaster GK. Sensitive and quantitative measurement of gene expression directly performed from a small amount of whole blood. Clin Chem 2006;52: 1294-302) For malaria diagnosis, the background signal from blank control was subtracted from sample signal to obtain the net signal, and samples with net signal above the detection threshold (3xSD of blank control) was diagnosed as positive, while below as negative. Each sample was tested in duplicate.

Example 1

We investigated the detection limit and the quantification capability of the RNA hybridization assay we developed. We tested a 3-fold serial dilution of fresh human erythrocyte-cultured P. falciparum. The assay gave a detection limit of about 0.04 parasite/μΐ, with signals above the threshold proportional to parasite numbers (R2=0.999) (Figure 1), indicating that our assay is highly sensitive, quantitative and able to detect low parasitemia in intact samples. All samples collected from health volunteers (n=13) are negative, showing that the method is highly specific.

Example 2

We tested our assay with clinical blood samples from 202 febrile patients with undetermined cause. Among these samples, 66 were determined by microscopy as positive (P. falciparum (n=27) and P. vivax (n=39)), with parasitemia ranging from 320 parasites/μΐ to 6x l0⁵ parasites/ μΐ, while the remaining 136 samples were microscopy-negative. Our assay identified all 66 microscopy-positive samples. There was no apparent difference between detecting P. falciparum infection and P.vivax infection. Although an overnight incubation was required, the overall hands-on time of the assay was less than 2 hours as the samples were assayed in parallel in 96-well plates. The blood lysates for this assay can be stored at -20°C with stability for at least 6 months (data not shown). These results suggest the assay is well sufficient for simultaneous qualitative detection of malaria infection in a large number of samples.

Despite broad agreement between microscopy and our assay (66 positive and 131 negative), there were, as may be expected, 5 microscopy negative samples which showed positive in our assay. We decided to further investigate microscopic-negative samples with RDT and real-time qPCR, and compared the results (Table 1 and Table 2). We tested all 136 microscopy-negative and 7 microscopic positive samples with RDT. The two methods have good agreement for 134 negative samples and 4 positive samples. However, 3 microscopy positive and 2 microscopy negative samples have opposite result in RDT. All these 5 samples were later proved to be positive both by our assay and by real time qPCR.

Table 1 Results of microscopy, real time qPCR, RDT and R A hybridization assay

P.f=plasmodium falciparum, P.v=plasmodium vivax

Table 2 Results of the microscopy false-negative samples

There are in total 8 disagreements out of 202 samples between the results of microscopy, RDT and our assay. We tested all these 8 samples, along with other 36 randomly chosen samples, by real time qPCR. For these 8 samples with discrepancy, both qPCR (including DNA extraction) and our assay were repeated twice for conformation. For 43 samples out of 44, the result is consistent with diagnosis of our assay (20 positive, 23 negative), the remaining one sample is positive in our assay but is negative in real time qPCR and other two methods. For this one, our assay gave rather weak signal, but above detection threshold, indicating the rather low parasite load; the Ct value of the qPCR assay for this sample is 41, close to the threshold cycle number of 40 (Table 2). Doubling the DNA input in the qPCR assay did not improve the Ct value of this sample.

Using microscopy as the gold standard, our assay has a sensitivity of 100% (66/66); using real time qPCR as the gold standard, our assay has a sensitivity of 100% (20/20). There was no sample that was negative by our assay but was positive by other assays tested.

Example 3

To test for the assay's robustness and to determine if the assay is capable of diagnosing samples of sub-optimal quality, we examined the assay results of the 59 malaria samples that were poorly stored and likely partially degraded. These samples were microscopy-positive, and included both P. falciparum samples and P. vivax samples that underwent repeated freeze -thaw during collection and storage. Signals of all 59 patient samples were above detection threshold, and a majority of them approached detection saturation (Figure 2), despite the fact that the samples may have suffered from R A degradation due to several freeze-and-thaw cycles and prolonged storage. The assay had a mean intra-assay CV of 5%.

Reference

1. WHO. World malaria report; 2009.

2. A research agenda for malaria eradication: diagnoses and diagnostics. PLoS Med 201 1;8:el000396.

3. Harris I, Sharrock WW, Bain LM, et al. A large proportion of asymptomatic Plasmodium infections with low and sub-microscopic parasite densities in the low transmission setting of Temotu Province, Solomon Islands: challenges for malaria diagnostics in an elimination setting. Malar J 2010;9:254.

4. Galinski MR, Barnwell JW. Plasmodium vivax: who cares? Malar J 2008;7 Suppl 1 :S9.

5. WHO. World malaria report; 2010.

6. Moody A. Rapid diagnostic tests for malaria parasites. Clinical Microbiology Reviews 2002; 15:66.

7. Okell LC, Ghani AC, Lyons E, Drakeley CJ. Submicroscopic infection in Plasmodium falciparum-endemic populations: a systematic review and meta-analysis. J Infect Dis 2009; 200: 1509-17.

8. Snounou G, Viriyakosol S, Jarra W, Thaithong S, Brown K . Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections. Mol Biochem Parasitol 1993; 58:283-92.

9. Rougemont M, Van Saanen M, Sahli R, Hinrikson HP, Bille J, Jaton K. Detection of four Plasmodium species in blood from humans by 18S rRNA gene subunit-based and species-specific real-time PCR assays. J Clin Microbiol 2004;42:5636-43.

10. Mens PF, Schoone GJ, Kager PA, Schallig HD. Detection and identification of human Plasmodium species with real-time quantitative nucleic acid sequence-based amplification. Malar J 2006;5:80.

11. Lucchi NW, Demas A, Narayanan J, et al. Real-Time Fluorescence Loop Mediated Isothermal Amplification for the Diagnosis of Malaria. PLoS One 2010;5:-.

12. Zheng Z, Luo Y, McMaster GK. Sensitive and quantitative measurement of gene expression directly from a small amount of whole blood. Clin Chem 2006;52: 1294-302.

13. Information note on recommended selection criteria for malaria rapid diagnostic tests (RDTs). 2010. (Accessed October 4, 2011 , at http://www.who.int/malaria/diagnosis_treatment/diagnosis/RDT_selection_criteria.pdf.)

14. Bushnell S, Budde J, Catino T, et al. ProbeDesigner for the design of probesets for branched DNA (bDNA) signal amplification assays. Bioinformatics 1999;15:348-55.

15. Rapid diagnostic tests for malaria—Haiti, 2010. MMWR Morb Mortal Wkly Rep 2010;59: 1372-3.

16. Gilles H. Diagnostic methods in malaria. In:Gilles HM, Warrell DA, eds. Essential malariology. 3rd Ed. London: P Edward Arnold 1993:78.

17. Yang W, Maqsodi B, Ma YQ, et al. Direct quantification of gene expression in homogenates of formalin-fixed, paraffin-embedded tissues. Biotechniques 2006;40:481-6.

18. Cox-Singh J, Davis TME, Lee KS, et al. Plasmodium knowlesi malaria in humans is widely distributed and potentially life threatening. Clinical Infectious Diseases 2008;46: 165-71.

Claims

What is claimed is:

1. A method for detection of genus Plasmodium in a sample, comprising the steps of

(a) providing a test sample suspected of containing target genus Plasmodium;

(b) optionally, releasing the polynucleic acids from the test samples of the step

(a);

(c) contacting the said test sample or the released polynucleic acids with probe mixture containing one or more CE probes and one or more LE probes for a time and under conditions sufficient for hybridization to take place, wherein the CE probe consists of nucleotides hybridizing to a region of the 18S ribosomal RNA of genus Plasmodium and the nucleotides hybridizing to nucleic acids covalently non-covalently attached to a solid support; and the LE probes consists of nucleotides hybridizing to a region of 18S ribosomal RNA of genus Plasmodium and the nucleotides hybridizing to a detectable molecule,

(d) contacting the said test sample with a detectable molecule after the said step (c), for a time and under conditions sufficient for hybridization to take place; and

(e) detecting any hybridization which has taken place in the step (c); wherein the presence or absence of hybridization is indicative of the presence or absence of said genus Plasmodium in said test sample.

2. The method according to claim 1, wherein the method further comprises the steps of contacting the said test sample with at least one blocking probe consisting of nucleotides hybridizing to 18S ribosomal RNA of genus Plasmodium to reduce or eliminate non-specific hybridization of other probes to the sequence, and to enhance the hybridization of adjacent probes by stacking effect.

3. The method according to claim 2, wherein the said blocking probe has a sequence as shown in SEQ ID NO:22.

4. The method according to any one of claims 1 to 3, wherein the said genus Plasmodium is selected from the group consisting of P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi.

5. The method according to any one of claims 1 to 4, wherein the said test sample is a blood sample, cultured erythrocytes, or blood sample dried on a filter paper.

6. The method according to any one of claims 1 to 5, wherein the said one or more CE probes are bound to the said solid support.

7. The method according to claim 6, wherein the said support is a planar solid support or a bead.

8. The method according to claim 7, wherein the said support is a 96-well plate.

9. The method according to any one of claims 1 to 8, wherein the said detectable molecule is labeled with an enzyme or a fluorescent label.

10. The method according to any one of claims 1 to 9, wherein the nucleotides hybridizing to the region of 18S ribosomal RNA of genus Plasmodium are selected from the nucleotides having at least 80% identity to the nucleotides as shown in SEQ ID NOs 23 to 43.

11. The method according to any one of claims 1 to 10, wherein the said CE probe is selected from the group consisting of SEQ ID NOs 1 to 7; and the said LE probe is selected from the group consisting of SEQ ID NOs 8 to 21.

12. A method for detection of genus Plasmodium in a sample, comprising the steps of

(a) providing a test sample suspected of containing target genus Plasmodium;

(a);

(c) contacting the said test sample or the released polynucleic acids with probes mixture containing at least one probe selected from the group consisting of the probes represented as SEQ ID NOs 1 to 22 for a time and under conditions sufficient for hybridization to take place;

(d) contacting the said test sample with a detectable molecule after the step (c), for a time and under conditions sufficient for hybridization to take place; and

13. A composition for detection of genus Plasmodium in a sample, comprising :

(a) one or more CE probes; wherein the said CE probe consists of nucleotides hybridizing to the region of the 18S ribosomal RNA of genus Plasmodium and the nucleotides hybridizing to nucleic acids covalently non-covalently attached to a solid support; and

(b) one or more LE probes; and wherein the LE probe consists of nucleotides hybridizing to the region of 18S ribosomal RNA of genus Plasmodium and the nucleotides hybridizing to a detectable molecule;

wherein the said CE probes and LE probes are in effective amounts sufficiently for hybridization to take place.

14. The composition according to claim 13, further optionally comprising a blocking probe in effective amounts sufficiently for hybridizing to 18S ribosomal RNA of genus Plasmodium to reduce or eliminate non-specific hybridization of other probes to the sequence, and to enhance the hybridization of adjacent probes by stacking effect.

15. The composition of claim 14, wherein the amount of CE probe is from 1 wt% to 99 wt% by total weight of the composition; the amount of LE probe is from 1 wt% to 99 wt% total weight of the composition; the amount of blocking probe is from 1 wt% to 99 wt% total weight of the composition.

16. The composition according to claim 13 or 14, wherein the nucleotides hybridizing to the region of 18S ribosomal RNA of genus Plasmodium are selected from the nucleotides having at least 80% identity to the nucleotides as shown in SEQ ID NOs 23 to 43.

17. The composition according to any one of claims 13 to 15, wherein the said CE probe is selected from the group consisting of SEQ ID NOs 1 to 7; and the said LE probe is selected from the group consisting of SEQ ID NOs 8-21.

18. The composition according to any one of claims 14 to 16, wherein the said blocking probe has a sequence as shown in SEQ ID NO:22.

19. A composition for detection of genus Plasmodium in a sample, comprising all of the probes as shown in SEQ ID NOs 1 to 22.

20. A kit for detection of genus Plasmodium in sample, comprising a) a composition according to any one of claims 13 to 18; b) a solid support; c) a detectable molecule and d) an instruction.