Compositions and methods for detecting malaria

Abstract

A method for rapidly detecting the presence or absence of malaria parasites (including Plasmodium) in biological or non-biological samples is described. The method can include performing an amplifying step, a hybridizing step, and a detecting step. Furthermore, the assay can be a multiplex assay for simultaneously amplifying and detecting multiple Plasmodium targets, which offers advantages over singleplex assays. Additionally, oligonucleotide primers and probes targeting malaria parasites (including Plasmodium) are provided, as well as kits designed for the detection of Plasmodium species, including, but not limited to, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium simulans. Kits, reaction mixtures, and oligonucleotides (e.g., primers and probes) for amplifying and detecting malaria parasites (including Plasmodium) are also described.

Description

[Technical Field]

[0001] Cross-reference of related applications This patent application claims priority to U.S. Provisional Patent Application No. 63 / 121,338, filed on 4 December 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] This disclosure relates to the field of in vitro diagnostics. In this field, the present invention relates to the amplification and detection of target nucleic acids that may be present in a sample, in particular to the amplification, detection and quantification of target nucleic acids, including sequence mutations and / or individual mutations of the malaria parasite, using primers and probes. The present invention further provides reaction mixtures and kits containing primers and probes for the amplification and detection of malaria. [Background technology]

[0003] Malaria is a mosquito-borne infectious disease that affects humans and other animals. It is caused by the parasitic single-celled microorganism Plasmodium, which is transmitted to humans after being bitten by an infected female Anopheles mosquito. Initial symptoms of malaria include fever, headache, and chills, which can be treated with available antimalarial drugs. If left untreated, it can develop into severe malaria with symptoms including anemia, cerebral malaria, and respiratory distress, and can ultimately be fatal.

[0004] According to a 2018 World Health Organization (WHO) report, approximately 3.2 billion people are at risk of malaria in 87 countries, primarily located in tropical and subtropical regions. There are approximately 219 million annual cases of malaria, 92% from Africa, 5% from Southeast Asia, and 2% from Eastern Mediterranean countries. The United States sees approximately 1,700 cases per year. Malaria causes 435,000 deaths annually, 266,000 of which are children under five years old. Efforts to eradicate and prevent malaria are underway, including insecticide-treated nets, increased access to healthcare for patients, drug development, vaccines, and genetically modified mosquitoes (GMOs). While the United States is not considered a malaria-endemic country, global migration to and from the US could lead to an increase in imported malaria cases.

[0005] There are at least five species of malaria parasites known to infect humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. Clinical symptoms can vary depending on the species of malaria parasite. Plasmodium falciparum is the most deadly species, known to cause rapid cell division in infected red blood cells, leading to anemia and occlusion of blood vessels to the brain. The life cycle of Plasmodium falciparum is complex, with the parasite alternating between sexual reproduction in mosquitoes and asexual reproduction in human hosts. This life cycle is shown in Figure 1, obtained from the CDC website (https: / / www.cdc.gov / malaria). Symptoms are caused by the parasite during the blood stage and may appear 7 to 30 days after a mosquito bite. Plasmodium vivax (P. vivax) and Plasmodium ovale (P. ovale) remain dormant in the liver and can reactivate after several months or even years.

[0006] Regarding blood supply and donation, significant challenges already exist in the southern and eastern United States to maintain an adequate blood supply, which is severely impacted by malaria shortages, and there are further problems with blood supply and donation. For example, although the United States is considered a non-endemic country, global travel is increasing imported cases of malaria, which could affect the blood supply worldwide. Current strategies for malaria employed to maintain the safety of the blood supply include (1) selective testing and (2) donor deferral, and the United States follows the donor deferral policy. Typically, donors are often deferred based on their history, place of residence, and travel, based on their responses to screening questionnaires. In the United States, an estimated 191,000 donors are temporarily deferred (for 1-3 years), with the majority of deferrals due to travel to endemic areas. Furthermore, when blood donations are deferred, only about a quarter of temporarily deferred donors actually return for their next donation after the deferral period ends, thus negatively impacting the blood supply. Donor deferral negatively impacts the blood supply by excluding donations based on questionnaire responses, regardless of whether or not the donor has malaria. Selective testing, on the other hand, reduces the impact on blood supply inventory compared to donor deferral. Selective testing of blood donations is also based on questionnaire responses, but a selective testing policy allows for shortening the deferral period or allowing donors to rejoin depending on their malaria status. In countries with a selective testing policy, testing is performed using enzyme immunoassay. At least three countries—France, the UK, and Australia—conduct malaria testing in blood donations. In France, out of 3,000,000 blood donations per year, approximately 180,000 were tested, and of those 180,000 tested donations, approximately 3,300 (or 1.8%) were positive.

[0007] Several diagnostic and screening methods exist for malaria. Microscopic examination of Giemsa-stained blood smears allows for species identification, is inexpensive, and remains the gold standard. However, microscopic examination cannot perform high-throughput screening / detection. Rapid diagnostic tests (RDTs) using immunochromatographic detection methods for malaria antigens are rapid and inexpensive, but have low sensitivity (200-5,000 p / μL) and still require confirmation by microscopy. Enzyme immunoassay (EIA) for detecting anti-malarial parasite antibodies in serum is widely used, but also has low sensitivity. In contrast, nucleic acid testing (NAT) is suitable for high-throughput screening / detection and has high sensitivity (<1 p / μL). Therefore, due to its high sensitivity and high processing capacity, nucleic acid testing is the best method for detecting and screening malaria in blood samples. However, currently, there are no in vitro diagnostic nucleic acid tests available for screening blood donations. Therefore, there is a need in this field for rapid, reliable, specific, and highly sensitive methods for detecting and quantifying the presence of malaria in biological samples such as blood.

[0008] In the field of molecular diagnostics, nucleic acid amplification and detection are of considerable importance. Such methods can be used to detect any number of viruses and microorganisms such as bacteria. The most prominent and widely used amplification technique is polymerase chain reaction (PCR). Other amplification techniques include ligase chain reaction, polymerase-ligase chain reaction, Gap-LCR, repair chain reaction, 3SR, NASBA, strand substitution amplification (SDA), transcription-mediated amplification (TMA), and Qβ amplification. Automated systems for PCR-based analysis often utilize real-time detection of product amplification during the PCR process in the same reaction vessel. A key aspect of such methods is the use of modified oligonucleotides with reporter groups or labels.

[0009] The safety of blood donation is threatened by the lack of reliable, rapid, inexpensive, and highly sensitive methods for detecting malaria. Therefore, there is a need in the art for rapid, reliable, specific, and highly sensitive methods for detecting and quantifying the presence of malaria in biological samples such as blood. This disclosure relates to polymerase chain reaction (PCR)-based assays for detecting and screening malaria parasite species (Platypleura falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium knowlesi, and Plasmodium malariae) in blood samples and offerings. [Overview of the project] [Problems that the invention aims to solve]

[0010] The safety of blood donation is threatened by the lack of reliable, rapid, inexpensive, and highly sensitive methods for detecting malaria. Therefore, there is a need in the art for rapid, reliable, specific, and highly sensitive methods for detecting and quantifying the presence of malaria in biological samples such as blood. This disclosure relates to polymerase chain reaction (PCR)-based assays for detecting and screening malaria parasite species (Platypleura falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium knowlesi, and Plasmodium malariae) in blood samples and offerings. [Means for solving the problem]

[0011] This disclosure relates to a method for the rapid detection of the presence or absence of malaria parasites (such as Plasmodium) in biological or non-biological samples, for example, for the multiple detection and quantification of malaria parasites (such as Plasmodium) by real-time polymerase chain reaction (PCR) in a single test tube or container. Disclosed herein is a method for detecting malaria parasites (such as Plasmodium) comprising performing at least one cycling step, which may include an amplification step and a hybridization step. Furthermore, oligonucleotide primers, oligonucleotide probes, and kits designed for the detection of malaria parasites (such as Plasmodium) in a single test tube or container are provided.

[0012] One embodiment relates to a method for detecting one or more malaria parasite species in a sample, comprising: (a) an amplification step, which includes contacting the sample with a set of one or more primers to produce an amplification product, if target nucleic acids of one or more malaria parasite species are present in the sample; (b) a hybridization step, which includes contacting one or more probes with the amplification product, if target nucleic acids of one or more malaria parasite species are present in the sample; and (c) a step to detect the presence or absence of the amplification product, wherein the presence of the amplification product indicates the presence of one or more malaria parasite species in the sample, and the absence of the amplification product indicates the absence of one or more malaria parasite species in the sample; and the method comprising one or more primer sets and one or more probes, which comprises (1) a set of primers comprising primers comprising nucleic acid sequences of SEQ ID NOs. 34, 35, and 36 or their complements; a probe comprising the nucleic acid sequence of SEQ ID NO. 4 or its complement; and / or (2) a set of primers comprising primers comprising nucleic acid sequences of SEQ ID NOs. 56 and 57 or their complements, and a probe comprising the nucleic acid sequence of SEQ ID NO. 58 or its complement. In another embodiment, the set of one or more primers and one or more probes includes (1) a set of primers containing the nucleic acid sequences of SEQ ID NOs. 34, 35, and 36 or their complements, and a probe containing the nucleic acid sequence of SEQ ID NO. 4 or its complement; and (2) a set of primers containing the nucleic acid sequences of SEQ ID NOs. 56 and 57 or their complements, and a probe containing the nucleic acid sequence of SEQ ID NO. 58 or its complement. In another embodiment, one or more malaria parasite species belong to the genus Plasmodium. In another embodiment, one or more malaria parasite species belonging to the genus Plasmodium are Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and / or Plasmodium malariae. In one embodiment, the sample is a biological sample. In another embodiment, the biological sample is whole blood, respiratory specimen, urine, fecal specimen, blood specimen, plasma, skin swab, nasal swab, wound swab, blood culture, skin or soft tissue infection. In other embodiments, the biological sample is whole blood.In another embodiment, one or more probes are labeled. In another embodiment, one or more probes are labeled with a donor fluorescent portion and a corresponding acceptor portion. Step (c) further includes detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent portion and the acceptor portion of one or more probes, the presence or absence of which indicates the presence or absence of one or more malaria parasite species in the sample.

[0013] Another aspect is a method for detecting a first target nucleic acid and / or a second target nucleic acid of one or more malaria parasite species in a sample, comprising: (a) if the first target nucleic acid and / or the second target nucleic acid of one or more malaria parasite species are present in the sample, an amplification step comprising contacting the sample with one or more sets of primers to produce an amplification product; (b) if the first target nucleic acid and / or the second target nucleic acid of one or more malaria parasite species are present in the sample, a hybridization step comprising contacting one or more probes with the amplification product; and (c) a step of detecting the presence or absence of the amplification product, wherein the presence of the amplification product indicates the presence of one or more malaria parasite species in the sample, and the amplification product The method comprises the step of indicating the absence of one or more malaria parasite species in a sample; and a set of one or more primers and one or more probes comprising: (1) a set of primers and a probe for a first target nucleic acid of one or more malaria parasite species (the set of primers comprising primers containing the nucleic acid sequences of SEQ ID NOs. 34, 35, and 36 or their complements, and the probe comprising the nucleic acid sequence of SEQ ID NOs. 4 or its complement); and (2) a set of primers and a probe for a second target nucleic acid of one or more malaria parasite species (the set of primers comprising primers containing the nucleic acid sequences of SEQ ID NOs. 56 and 57 or their complements, and the probe comprising the nucleic acid sequence of SEQ ID NOs. 58 or its complement).Another embodiment relates to a method for detecting one or more malaria parasite species in a sample, comprising: (a) an amplification step, which includes contacting the sample with a set of one or more primers to produce an amplification product, if target nucleic acids of one or more malaria parasite species are present in the sample; (b) a hybridization step, which includes contacting one or more probes with the amplification product, if target nucleic acids of one or more malaria parasite species are present in the sample; and (c) a step for detecting the presence or absence of the amplification product, wherein the presence of the amplification product indicates the presence of one or more malaria parasite species in the sample, and the absence of the amplification product indicates the absence of one or more malaria parasite species in the sample; and a method comprising a set of one or more primers, the set of primers comprising primers comprising the nucleic acid sequences of SEQ ID NOs. 34, 35, and 36 or their complements, and a probe comprising the nucleic acid sequence of SEQ ID NO. 4 or its complement. Another embodiment relates to a method for detecting one or more malaria parasite species in a sample, comprising: (a) an amplification step, comprising contacting the sample with a set of one or more primers to produce an amplification product, if target nucleic acids of one or more malaria parasite species are present in the sample; (b) a hybridization step, comprising contacting one or more probes with the amplification product, if target nucleic acids of one or more malaria parasite species are present in the sample; and (c) a step of detecting the presence or absence of the amplification product, wherein the presence of the amplification product indicates the presence of one or more malaria parasite species in the sample, and the absence of the amplification product indicates the absence of one or more malaria parasite species in the sample; and a method comprising one or more sets of primers and one or more primers comprising a set of primers comprising primers comprising the nucleic acid sequences of SEQ ID NOs. 56 and 57 or complements thereof, and a probe comprising the nucleic acid sequence of SEQ ID NO. 58 or a complement thereof. In the relevant embodiment, the one or more malaria parasite species belong to the genus Plasmodium. In another embodiment, one or more malaria parasite species belonging to the genus Plasmodium are Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium quartanum, and / or Plasmodium malariae. In one embodiment, the sample is a biological sample.In another embodiment, the biological sample is whole blood, respiratory specimen, urine, fecal specimen, blood specimen, plasma, skin swab, nasal swab, wound swab, blood culture, or skin or soft tissue infection. In yet another embodiment, the biological sample is whole blood. In yet another embodiment, one or more probes are labeled. In yet another embodiment, the one or more probes are labeled with a donor fluorescence portion and a corresponding acceptor portion.

[0014] Another embodiment is a kit for detecting one or more malaria parasite species that may be present in a sample, comprising a DNA polymerase, a nucleotide monomer (e.g., nucleoside triphosphate), and an amplification reagent comprising one or more sets of primers and one or more probes, wherein the set of primers and one or more probes comprises (1) a set of primers comprising a primer containing the nucleic acid sequences of SEQ ID NOs. 34, 35, and 36 or their complements, and a probe comprising the nucleic acid sequence of SEQ ID NOs. 4 or its complement; and / or (2) a set of primers comprising a primer containing the nucleic acid sequences of SEQ ID NOs. 56 and 57 or their complements, and a probe comprising the nucleic acid sequence of SEQ ID NOs. 58 or its complement. In another embodiment, the set of primers and one or more probes comprises (1) a set of primers comprising a primer containing the nucleic acid sequences of SEQ ID NOs. 34, 35, and 36 or their complements, and a probe comprising the nucleic acid sequence of SEQ ID NOs. 4 or its complement; and (2) a set of primers comprising a primer containing the nucleic acid sequences of SEQ ID NOs. 56 and 57 or their complements, and a probe comprising the nucleic acid sequence of SEQ ID NOs. 58 or its complement. In another embodiment, one or more malaria parasite species belong to the genus Plasmodium. In another embodiment, one or more malaria parasite species belonging to the genus Plasmodium are Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium quartanum, and / or Plasmodium malariae. In one embodiment, the sample is a biological sample. In another embodiment, the biological sample is whole blood, respiratory specimen, urine, fecal specimen, blood specimen, plasma, skin swab, nasal swab, wound swab, blood culture, skin or soft tissue infection. In another embodiment, the biological sample is whole blood. In another embodiment, one or more probes are labeled. In another embodiment, the one or more probes are labeled with a donor fluorescence portion and a corresponding acceptor portion.Another embodiment relates to a kit for detecting a first target nucleic acid and / or a second target nucleic acid of one or more malaria parasite species that may be present in a sample, comprising a DNA polymerase, a nucleotide monomer (e.g., nucleoside triphosphate), and an amplification reagent comprising one or more sets of primers and one or more probes, wherein the one or more sets of primers and one or more probes comprise: (1) a set of primers and a probe for a first target nucleic acid of one or more malaria parasite species (the set of primers comprises primers containing the nucleic acid sequences of SEQ ID NOs. 34, 35, and 36 or their complements, and the probe comprises the nucleic acid sequence of SEQ ID NOs. 4 or its complement); and (2) a set of primers and a probe for a second target nucleic acid of one or more malaria parasite species (the set of primers comprises primers containing the nucleic acid sequences of SEQ ID NOs. 56 and 57 or their complements, and the probe comprises the nucleic acid sequence of SEQ ID NOs. 58 or its complement). Another embodiment relates to a kit for detecting one or more malaria parasite species that may be present in a sample, comprising a DNA polymerase, a nucleotide monomer (e.g., nucleoside triphosphate), and an amplification reagent comprising one or more sets of primers and one or more probes, wherein the set of primers and one or more probes comprises a set of primers comprising the nucleic acid sequences of SEQ ID NOs. 34, 35, and 36 or their complements, and a probe comprising the nucleic acid sequence of SEQ ID NOs. 4 or its complement. Another embodiment relates to a kit for detecting one or more malaria parasite species that may be present in a sample, comprising a DNA polymerase, a nucleotide monomer (e.g., nucleoside triphosphate), and an amplification reagent comprising one or more sets of primers and one or more probes, wherein the set of primers and one or more probes comprises a set of primers comprising the nucleic acid sequences of SEQ ID NOs. 56 and 57 or their complements, and a probe comprising the nucleic acid sequence of SEQ ID NOs. 58 or its complement. In another embodiment, the one or more malaria parasite species belong to the genus Plasmodium.In another embodiment, one or more malaria parasite species belonging to the genus Plasmodium are Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium quartanum, and / or Plasmodium malariae. In one embodiment, the sample is a biological sample. In another embodiment, the biological sample is whole blood, respiratory specimen, urine, fecal specimen, blood specimen, plasma, skin swab, nasal swab, wound swab, blood culture, skin or soft tissue infection. In yet another embodiment, the biological sample is whole blood. In yet another embodiment, one or more probes are labeled. In yet another embodiment, the one or more probes are labeled with a donor fluorescence portion and a corresponding acceptor portion.

[0015] In one embodiment, a method for detecting malaria parasites in a sample, comprising: (b) an amplification step including contacting the sample with a set of one or more primers to produce an amplification product if the target nucleic acid of malaria parasites is present in the sample; (c) a hybridization step including contacting one or more probes with the amplification product if the target nucleic acid of malaria parasites is present in the sample; and (d) a step for detecting the presence or absence of the amplification product, wherein the presence of the amplification product indicates the presence of malaria parasites in the sample, and the absence of the amplification product indicates the absence of malaria parasites in the sample; A method is provided in which the set of primers and the one or more probes include (1) a set of primers including a first primer containing the nucleic acid sequence of SEQ ID NO: 34 or its complement, a second primer containing the nucleic acid sequence of SEQ ID NO: 36 or its complement, and a probe containing the nucleic acid sequence of SEQ ID NO: 4 or its complement; and / or (2) a set of primers including a first primer containing the nucleic acid sequence of SEQ ID NO: 22 or its complement, a second primer containing the nucleic acid sequence of SEQ ID NO: 27 or its complement, and a probe containing the nucleic acid sequence of SEQ ID NO: 25 or its complement. In one embodiment, the malaria parasite belongs to the genus Plasmodium. In another embodiment, the malaria parasite belonging to the genus Plasmodium is Plasmodium falciparum. In one embodiment, the sample is a biological sample such as whole blood, respiratory specimen, urine, fecal specimen, blood specimen, plasma, skin swab, nasal swab, wound swab, blood culture, or skin or soft tissue infection. In one embodiment, the biological sample is whole blood. In another embodiment, one or more probes are labeled. In another embodiment, one or more probes are labeled with a donor fluorescent portion and a corresponding acceptor portion. In another embodiment, step (c) further includes detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent portion and the acceptor portion of one or more probes, the presence or absence of fluorescence indicating the presence or absence of malaria parasites in the sample.

[0016] In one embodiment, a method for detecting a first target nucleic acid and a second target nucleic acid of a malaria parasite in a sample, comprising: (a) an amplification step, if the first target nucleic acid and / or the second target nucleic acid of a malaria parasite are present in the sample, including contacting the sample with a set of one or more primers to produce an amplification product; (b) a hybridization step, if the first target nucleic acid and / or the second target nucleic acid of a malaria parasite are present in the sample, including contacting one or more probes with the amplification product; and (c) a step for detecting the presence or absence of the amplification product, wherein the presence of the amplification product indicates the presence of a malaria parasite in the sample, and the absence of the amplification product indicates the absence of a malaria parasite in the sample; a set of one or more primers and one or more A method is provided in which the probe comprises (1) a set of primers and a probe for a first target nucleic acid of the malaria parasite (the set of primers comprises a first primer comprising the nucleic acid sequence of SEQ ID NO: 34 or its complement, and a second primer comprising the nucleic acid sequence of SEQ ID NO: 36 or its complement, and the probe comprising the nucleic acid sequence of SEQ ID NO: 4 or its complement), and / or (2) a set of primers and a probe for a second target nucleic acid of the malaria parasite (the set of primers comprises a first primer comprising the nucleic acid sequence of SEQ ID NO: 22 or its complement, and a second primer comprising the nucleic acid sequence of SEQ ID NO: 27 or its complement, and the probe comprising the nucleic acid sequence of SEQ ID NO: 25 or its complement).Another aspect of the present invention relates to a method for detecting malaria parasites in a sample, comprising: (a) an amplification step, if the target nucleic acid of malaria parasites is present in the sample, including contacting the sample with a set of one or more primers to produce an amplification product; (b) a hybridization step, if the target nucleic acid of malaria parasites is present in the sample, including contacting the amplification product with one or more probes; and (c) a step of detecting the presence or absence of the amplification product, wherein the presence of the amplification product indicates the presence of malaria parasites in the sample, and the absence of the amplification product indicates the absence of malaria parasites in the sample; and the method comprising a set of one or more primers and one or more probes, including a set of primers including a first primer containing the nucleic acid sequence of SEQ ID NO: 34 or its complement, and a second primer containing the nucleic acid sequence of SEQ ID NO: 36 or its complement; and a probe containing the nucleic acid sequence of SEQ ID NO: 4 or its complement. Another aspect of the present invention relates to a method for detecting malaria parasites in a sample, comprising: (a) performing an amplification step, which includes contacting the sample with a set of one or more primers to produce an amplification product, if the target nucleic acid of malaria parasites is present in the sample; (b) performing a hybridization step, which includes contacting the amplification product with one or more probes, if the target nucleic acid of malaria parasites is present in the sample; and (c) detecting the presence or absence of the amplification product, wherein the presence of the amplification product indicates the presence of malaria parasites in the sample, and the absence of the amplification product indicates the absence of malaria parasites in the sample; and the method comprising a set of one or more primers and one or more probes, which includes a set of primers comprising a first primer comprising the nucleic acid sequence of SEQ ID NO: 22 or its complement, and a second primer comprising the nucleic acid sequence of SEQ ID NO: 27 or its complement; and a probe comprising the nucleic acid sequence of SEQ ID NO: 25 or its complement. In a relevant embodiment, the malaria parasite belongs to the genus Plasmodium. In another embodiment, the malaria parasite belonging to the genus Plasmodium is Plasmodium falciparum. In one embodiment, the sample is a biological sample.In related embodiments, the sample is a biological sample such as whole blood, respiratory specimen, urine, fecal specimen, blood specimen, plasma, skin swab, nasal swab, wound swab, blood culture, skin or soft tissue infection. In other embodiments, the biological sample is whole blood. In one embodiment, one or more probes are labeled. In another embodiment, one or more probes are labeled with a donor fluorophore and a corresponding acceptor moiety.

[0017] In one aspect, a kit for detecting Plasmodium that may be present in a sample, comprising a DNA polymerase, nucleotide monomers (e.g., nucleoside triphosphates), and an amplification reagent comprising a set of one or more primers and one or more probes, wherein the set of one or more primers and one or more probes comprises: (1) a set of primers comprising a first primer comprising the nucleic acid sequence of SEQ ID NO: 34 or its complement, and a second primer comprising the nucleic acid sequence of SEQ ID NO: 36 or its complement; and a probe comprising the nucleic acid sequence of SEQ ID NO: 4 or its complement; and / or (2) a set of primers comprising a first primer comprising the nucleic acid sequence of SEQ ID NO: 22 or its complement, and a second primer comprising the nucleic acid sequence of SEQ ID NO: 27 or its complement; and a probe comprising the nucleic acid sequence of SEQ ID NO: 25 or its complement. In related embodiments, the Plasmodium belongs to the genus Plasmodium. In another embodiment, the Plasmodium belonging to the genus Plasmodium is Plasmodium falciparum. In one embodiment, the sample is a biological sample. In another embodiment, the biological sample is such that the biological sample is whole blood, respiratory specimen, urine, fecal specimen, blood specimen, plasma, skin swab, nasal swab, wound swab, blood culture, skin or soft tissue infection. In other embodiments, the biological sample is whole blood. In one embodiment, one or more probes are labeled. In another embodiment, one or more probes are labeled with a donor fluorophore and a corresponding acceptor moiety.

[0018] In one embodiment, a kit is provided for detecting a first target nucleic acid and / or a second target nucleic acid of a malaria parasite that may be present in a sample, comprising a DNA polymerase, a nucleotide monomer (e.g., nucleoside triphosphate), and an amplification reagent comprising one or more sets of primers and one or more probes, wherein the one or more sets of primers and one or more probes comprise (1) a set of primers and a probe for a first target nucleic acid of a malaria parasite (the set of primers comprises a first primer comprising the nucleic acid sequence of sequence number 34 or its complement, and a second primer comprising the nucleic acid sequence of sequence number 36 or its complement, and the probe comprising the nucleic acid sequence of sequence number 4 or its complement), and / or a set of primers and a probe for a second target nucleic acid of a malaria parasite (the set of primers comprises a first primer comprising the nucleic acid sequence of sequence number 22 or its complement, and a second primer comprising the nucleic acid sequence of sequence number 27 or its complement, and the probe comprising the nucleic acid sequence of sequence number 25 or its complement). Another embodiment relates to a kit for detecting malaria parasites that may be present in a sample, comprising a DNA polymerase, a nucleotide monomer (e.g., nucleoside triphosphate), and an amplification reagent comprising one or more sets of primers and one or more probes, wherein the set of primers and one or more probes comprises a first primer comprising the nucleic acid sequence of SEQ ID NO: 34 or its complement, a second primer comprising the nucleic acid sequence of SEQ ID NO: 36 or its complement, and the probe comprising the nucleic acid sequence of SEQ ID NO: 4 or its complement. Another embodiment relates to a kit for detecting malaria parasites that may be present in a sample, comprising a DNA polymerase, a nucleotide monomer (e.g., nucleoside triphosphate), and an amplification reagent comprising one or more sets of primers and one or more probes, wherein the set of primers and one or more probes comprises a first primer comprising the nucleic acid sequence of SEQ ID NO: 22 or its complement, a second primer comprising the nucleic acid sequence of SEQ ID NO: 27 or its complement, and a probe comprising the nucleic acid sequence of SEQ ID NO: 25 or its complement.In a related embodiment, the malaria parasite belongs to the genus Plasmodium. In another embodiment, the malaria parasite belonging to the genus Plasmodium is Plasmodium falciparum. In one embodiment, the sample is a biological sample. In another embodiment, the biological sample is whole blood, respiratory specimen, urine, fecal specimen, blood specimen, plasma, skin swab, nasal swab, wound swab, blood culture, skin or soft tissue infection. In one embodiment, the biological sample is whole blood. In another embodiment, one or more probes are labeled. In another embodiment, one or more probes are labeled with a donor fluorescence moiety and a corresponding acceptor moiety.

[0019] Disclosures in other aspects also provide oligonucleotides comprising or consisting of nucleotide sequences selected from SEQ ID NOs: 1-58 or their complements, and having 100 or fewer nucleotides. As used herein, the present disclosure provides oligonucleotides comprising a nucleic acid having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90% or 95%, etc.) with one of SEQ ID NOs: 1-58 or its complement, and having 100 or fewer nucleotides. Generally, these oligonucleotides can be primer nucleic acids, probe nucleic acids, etc. In certain aspects, the oligonucleotide has 40 or fewer nucleotides (e.g., 35 or fewer nucleotides, 30 or fewer nucleotides, 25 or fewer nucleotides, 20 or fewer nucleotides, 15 or fewer nucleotides, etc.). In some aspects, the oligonucleotide comprises at least one modified nucleotide, e.g., to change nucleic acid hybridization stability as compared to unmodified nucleotides. The oligonucleotide optionally comprises at least one label and / or optionally at least one quencher moiety. In some aspects, the oligonucleotide comprises at least one conservatively modified mutation. A "conservatively modified mutation" or simply "conservative mutation" of a particular nucleic acid sequence refers to a nucleic acid encoding the same or essentially the same amino acid sequence, or, if the nucleic acid does not encode an amino acid sequence, an essentially the same sequence. One of ordinary skill in the art will recognize that individual substitutions, deletions or additions that change, add or delete a single nucleotide or a low percentage of nucleotides (typically less than 5%, more typically less than 4%, 2% or 1%) in the encoded sequence are "conservatively modified mutations" that result in an amino acid deletion, an amino acid addition, or a substitution of an amino acid with a chemically similar amino acid.

[0020] In one embodiment, amplification may be performed using a polymerase enzyme having nuclease activity from 5' to 3'. Thus, the donor fluorescent moiety and acceptor moiety, e.g., the quencher, may be within 5 to 20 nucleotides (e.g., 7 or 10 nucleotides) of each other along the length of the probe. In another embodiment, the probe includes a nucleic acid sequence that allows for the formation of a secondary structure. The formation of such a secondary structure may result in spatial proximity between the first fluorescent moiety and the second fluorescent moiety. According to this method, the second fluorescent moiety on the probe may be the quencher.

[0021] This disclosure also provides methods for detecting the presence or absence of malaria parasites (such as Plasmodium) or malaria parasite (such as Plasmodium) nucleic acids in biological samples from individuals. These methods may be used to detect the presence or absence of malaria parasite (such as Plasmodium) nucleic acids in plasma for use in blood screening and diagnostic tests. Furthermore, the same tests may be used by those skilled in the art to evaluate urine and other types of samples to detect and / or quantify malaria parasite (such as Plasmodium) nucleic acids. Such methods generally involve performing at least one cycling step, which includes an amplification step and a dye-binding step. Typically, the amplification step involves contacting the sample with a plurality of pairs of oligonucleotide primers that produce one or more amplification products if nucleic acid molecules are present in the sample, and the dye-binding step involves contacting the amplification product with a double-stranded DNA-binding dye. Such methods also include detecting the presence or absence of binding of the double-stranded DNA-binding dye to the amplification product, where the presence of binding indicates the presence of malaria parasite (such as Plasmodium) nucleic acids in the sample, and the absence of binding indicates the absence of malaria parasite nucleic acids in the sample. A typical double-stranded DNA binding dye is ethidium bromide. Other nucleic acid binding dyes include DAPI, Hoechst dyes, PicoGreen®, RiboGreen®, OliGreen®, and cyanine dyes, such as YO-YO® and SYBR® Green. Furthermore, such a method may also include measuring the melting temperature between the amplification product and the double-stranded DNA binding dye, which confirms the presence or absence of malaria parasite (including plasmodium) nucleic acids.

[0022] In a further embodiment, a kit is provided for detecting and / or quantifying one or more nucleic acids of malaria parasites (such as Plasmodium). The kit may comprise one or more sets of oligonucleotide primers specific for amplification of a gene target; and one or more detectable oligonucleotide probes specific for detection of the amplification product.

[0023] In one embodiment, the kit may include a donor and corresponding acceptor moiety, for example, a probe already labeled with another fluorescent moiety or dark quencher, or a fluorophore moiety for labeling the probe. The kit may also further include a nucleoside triphosphate, a nucleic acid polymerase, and buffers necessary for the function of the nucleic acid polymerase. The kit may also include an accompanying leaflet, as well as instructions for using the primers, probe, and fluorescent dye moiety to detect the presence or absence of malaria parasite (e.g., Plasmodium) nucleic acids in a sample.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the invention pertains. Methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of the subject matter, but preferred methods and materials are described below. In addition, materials, methods, and examples are illustrative and not intended to be limiting. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. In case of any conflict, this specification, including definitions, shall prevail.

[0025] Details of one or more embodiments of the present invention are shown in the accompanying drawings and the following description. Other features, purposes and advantages of the present invention will become apparent from the drawings and the modes for carrying out the invention, as well as from the claims.

[0026] A patent or application file must include at least one drawing made in color. A copy of the published patent or patent application containing the color drawing will be provided by the Patent Office upon request and payment of the required fee. [Brief explanation of the drawing]

[0027] [Figure 1]Figure 1 shows a diagram of a complex life cycle plasmodium in which the parasite alternates between sexual reproduction in mosquitoes and asexual reproduction in human hosts (adapted from the CDC website (https: / / www.cdc.gov / malaria)).

[0028] [Figure 2A] Figure 2A shows data for the 18S-1 target (using primers with nucleic acid sequences of SEQ ID NOs: 1 and 2, and a probe with the nucleic acid sequence of SEQ ID NO: 3).

[0029] [Figure 2B] Figure 2B shows data for the 18S-3 target (using primers with nucleic acid sequences of SEQ ID NOs. 5 and 6, and a probe with the nucleic acid sequence of SEQ ID NO. 7).

[0030] [Figure 2C] Figure 2C shows data for the 18S-4 target (using primers with nucleic acid sequences of SEQ ID NOs. 8 and 9, and a probe with the nucleic acid sequence of SEQ ID NO. 10).

[0031] [Figure 2D] Figure 2D shows data for the MT-1 target (using primers with nucleic acid sequences SEQ ID NOs. 16 and 17, and a probe with the nucleic acid sequence SEQ ID NO. 18).

[0032] [Figure 2E] Figure 2E shows data for MT-2 targeting (using primers with nucleic acid sequences of SEQ ID NOs. 11 and 12, and a probe with the nucleic acid sequence of SEQ ID NO. 15).

[0033] [Figure 2F] Figure 2F shows data for the R125 target (using primers with nucleic acid sequences 21 and 22, and a probe with the nucleic acid sequence 23).

[0034] [Figure 3]Figure 3 shows data from Example 1 for the eluate at a 1:105 dilution level.

[0035] [Figure 4] Figure 4 shows the real-time PCR amplification curves of a plasmodium assay using Plasmodium falciparum cultures against an 18S-1 target using the redesigned probe (SEQ ID NO: 4) as described in Example 1. The curve using the original 18S-1 probe (SEQ ID NO: 3) is shown in blue, and the curve using the redesigned 18S-1 probe (SEQ ID NO: 4) is shown in red. All primers used have the nucleic acid sequences of SEQ ID NOs: 1 and 2.

[0036] [Figure 5] Figure 5 shows the results of ddPCR of Plasmodium falciparum cultures. The results show the copy number (per μl) of a 1:105 culture dilution of Plasmodium falciparum culture. For the 18S-1 target, primers with the nucleic acid sequences of SEQ ID NOs. 1 and 2 and probes with the nucleic acid sequence of SEQ ID NOs. 37 were used. For the 18S-3 target, primers with the nucleic acid sequences of SEQ ID NOs. 5 and 6 and probes with the nucleic acid sequence of SEQ ID NOs. 38 were used. For the 18S-4 target, primers with the nucleic acid sequences of SEQ ID NOs. 8 and 9 and probes with the nucleic acid sequence of SEQ ID NOs. 39 were used. For the MT-1 target, primers with the nucleic acid sequences of SEQ ID NOs. 16 and 17 and probes with the nucleic acid sequence of SEQ ID NOs. 42 were used. For the MT-2 target, primers with the nucleic acid sequences of SEQ ID NOs. 11 and 12 and probes with the nucleic acid sequence of SEQ ID NOs. 41 were used. For the R125 target, primers with the nucleic acid sequences of SEQ ID NOs. 21 and 22 and probes with the nucleic acid sequence of SEQ ID NOs. 40 were used.

[0037] [Figure 6A] Figure 6A shows data from multiplex assays using primers and probes against 18S-1 and 18S-4 targets.

[0038] [Figure 6B] Figure 6B shows real-time PCR amplification curves from multiplex assays using primers and probes for 18S-1 and 18S-4 targets. For the 18S-1 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs: 1 and 2, and a probe with the nucleic acid sequence of SEQ ID NO: 4. For the 18S-4 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs: 8 and 9, and a probe with the nucleic acid sequence of SEQ ID NO: 10.

[0039] [Figure 7A] Figure 7A shows data from multiplex assays using primers and probes targeting 18S-1 and R125, with Plasmodium falciparum cultures at four different dilution levels (1:104, 1:105, 1:106, 1:107) in a background of 500 ng of whole blood genomic DNA.

[0040] [Figure 7B] Figure 7B shows real-time PCR amplification curves from multiplex assays using primers and probes for the 18S-1 and R125 targets. For the 18S-1 target, this multiplex assay used primers with the nucleic acid sequences of SEQ ID NOs. 1 and 2, and a probe with the nucleic acid sequence of SEQ ID NO. 4. For the R125 target, this multiplex assay used primers with the nucleic acid sequences of SEQ ID NOs. 21 and 22, and a probe with the nucleic acid sequence of SEQ ID NO. 25.

[0041] [Figure 8]Figure 8 shows data from multiplex assays using primers and probes against 18S-1 and R125 targets, using in vitro transcripts of 18S-1 and R125 targets at five different levels (105, 104, 103, 102, and 10 copies) in a background of 500 ng of whole blood genomic DNA. For the 18S-1 target, this multiplex assay used primers with the nucleic acid sequences of SEQ ID NOs: 1 and 2, and a probe with the nucleic acid sequence of SEQ ID NO: 4. For the R125 target, this multiplex assay used primers with the nucleic acid sequences of SEQ ID NOs: 21 and 22, and a probe with the nucleic acid sequence of SEQ ID NO: 25.

[0042] [Figure 9] Figure 9 shows real-time PCR amplification curves from a singleplex assay using primers (SEQ ID NOs. 22 and 26) and probe (SEQ ID NOs. 25) to amplify and detect an in vitro transcript (103 copies) of the R125 target in a background of 500 ng of whole blood genomic DNA / RNA. The forward primer has the nucleic acid sequence of SEQ ID NOs. 26, the reverse primer has the nucleic acid sequence of SEQ ID NOs. 22, and the probe has the nucleic acid sequence of SEQ ID NOs. 25.

[0043] [Figure 10A] Figure 10A shows data from multiplex assays using primers and probes targeting 18S-1 and R125, with Plasmodium falciparum from Plasmodium falciparum cultures at four different dilution levels (1:104, 1:105, 1:106, 1:107) in a background of whole blood nucleic acids extracted with a cobas6800 instrument.

[0044] [Figure 10B]Figure 10B shows real-time PCR amplification curves from multiplex assays using primers and probes for 18S-1 and R125 targets. For the 18S-1 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs. 34 and 36, and a probe with the nucleic acid sequence of SEQ ID NO. 4. For the R125 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs. 27 and 22, and a probe with the nucleic acid sequence of SEQ ID NO. 25.

[0045] [Figure 11] Figure 11 shows real-time PCR amplification curves from multiplex assays using primers and probes for 18S-1 and 18S-3 targets, using Plasmodium falciparum from Plasmodium falciparum cultures at two different dilution levels (1:104 and 1:105) against a background of whole blood nucleic acids extracted with a cobas6800 instrument. For the 18S-1 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs. 34-36 and a probe with the nucleic acid sequence of SEQ ID NO. 4. For the 18S-3 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs. 56 and 57 and a probe with the nucleic acid sequence of SEQ ID NO. 58.

[0046] [Figure 12]Figure 12 shows real-time PCR amplification curves from multiplex assays using primers and probes for 18S-1 and 18S-3 targets, using Plasmodium vivax from Plasmodium vivax cultures at two different dilution levels (1:103 and 1:104) against a background of whole blood nucleic acids extracted with a cobas6800 instrument. For the 18S-1 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs. 34-36 and a probe with the nucleic acid sequence of SEQ ID NO. 4. For the 18S-3 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs. 56 and 57 and a probe with the nucleic acid sequence of SEQ ID NO. 58.

[0047] [Figure 13] Figure 13 shows real-time PCR amplification curves from multiplex assays using primers and probes against 18S-1 and 18S-3 targets, using a DNA plasmid containing 1000 copies of the 18S rRNA sequence of the Plasmodium malariae per PCR reaction level. For the 18S-1 target, this multiplex assay used primers with nucleic acid sequences SEQ ID NOs. 34-36 and a probe with the nucleic acid sequence SEQ ID NO. 4. For the 18S-3 target, this multiplex assay used primers with nucleic acid sequences SEQ ID NOs. 56 and 57 and a probe with the nucleic acid sequence SEQ ID NO. 58.

[0048] [Figure 14]Figure 14 shows real-time PCR amplification curves from multiplex assays using primers and probes against 18S-1 and 18S-3 targets, using a DNA plasmid containing 1000 copies of Plasmodium malariae 18S rRNA sequence per PCR reaction level. For the 18S-1 target, this multiplex assay used primers with nucleic acid sequences SEQ ID NOs. 34-36 and a probe with the nucleic acid sequence SEQ ID NO. 4. For the 18S-3 target, this multiplex assay used primers with nucleic acid sequences SEQ ID NOs. 56 and 57 and a probe with the nucleic acid sequence SEQ ID NO. 58.

[0049] [Figure 15] Figure 15 shows real-time PCR amplification curves from multiplex assays using primers and probes against 18S-1 and 18S-3 targets, using a DNA plasmid containing 1000 copies of the malaria parasite 18S rRNA sequence per PCR reaction level. For the 18S-1 target, this multiplex assay used primers with nucleic acid sequences SEQ ID NOs. 34-36 and a probe with the nucleic acid sequence SEQ ID NO. 4. For the 18S-3 target, this multiplex assay used primers with nucleic acid sequences SEQ ID NOs. 56 and 57 and a probe with the nucleic acid sequence SEQ ID NO. 58. [Modes for carrying out the invention]

[0050] The diagnosis of malaria parasite (Plasmodium, etc.) infection by nucleic acid amplification provides a method for rapid, accurate, reliable, specific, and sensitive detection and / or quantification of malaria parasite (Plasmodium, etc.) infection. Real-time PCR assays for detecting and / or quantifying malaria parasite nucleic acids, including DNA and / or RNA, in non-biological or biological samples are described herein. Primers and probes for detecting and / or quantifying malaria parasite (Plasmodium, etc.) are provided, as are manufactured products or kits containing such primers and probes. The increased specificity and sensitivity of real-time PCR for the detection of malaria parasite (Plasmodium, etc.) compared to other methods, along with improved features of real-time PCR including sample containment, real-time detection and quantification of amplification products, make the implementation of this technology for the routine diagnosis of malaria parasite (Plasmodium, etc.) infection in clinical laboratories feasible. Furthermore, this technology can be used for blood screening and prognostic diagnosis. This malaria parasite (Plasmodium, etc.) detection assay can also be multiplexed in parallel with other assays for the detection of other nucleic acids, such as other bacteria and / or viruses.

[0051] This disclosure includes, for example, oligonucleotide primers and fluorescently labeled hydrolysis probes that hybridize to the genome of malaria parasites (such as Plasmodium) for the specific identification of malaria parasites (such as Plasmodium) using TaqMan® amplification and detection techniques.

[0052] The disclosed method may include performing at least one cycling step, which involves amplifying one or more portions of a nucleic acid molecular gene target from a sample using one or more primer pairs. As used herein, “plasmodium primers” refer to oligonucleotide primers that specifically anneal to nucleic acid sequences found in the genome of malaria parasites (such as Plasmodium) and initiate DNA synthesis therefrom under appropriate conditions to produce their respective amplification products. Examples of nucleic acid sequences found in Plasmodium species and genomes include nucleic acids within mitochondrial DNA target regions (MT-1 and MT-2), RNA repeat sequences R125, and 18S ribosomal RNA, among other targets. Each of the plasmodium primers discussed anneals to a target such that at least a portion of each amplification product contains the nucleic acid sequence corresponding to the target. One or more amplification products are produced on the condition that one or more nucleic acids are present in the sample, and therefore the presence of one or more amplification products indicates the presence of Plasmodium in the sample. The amplification products should contain nucleic acid sequences complementary to one or more detectable probes for Plasmodium. As used herein, “plasmodium probe” refers to an oligonucleotide probe that specifically anneals to nucleic acid sequences found in the plasmodium genome. Each cycling step comprises an amplification step, a hybridization step, and a detection step, in which the sample is brought into contact with one or more detectable plasmodium probes to detect the presence or absence of plasmodium in the sample.

[0053] As used herein, the term “amplify” refers to the process of synthesizing a nucleic acid molecule complementary to one or both strands of a template nucleic acid molecule (e.g., a nucleic acid molecule from a plasmodium genome). Amplifying a nucleic acid molecule typically involves denaturing the template nucleic acid, annealing the primers to the template nucleic acid at a temperature below the primer melting temperature, and enzymatically extending from the primers to produce an amplification product. Amplification typically requires the presence of deoxyribonucleoside triphosphate, a DNA polymerase enzyme (e.g., Platinum® Taq), and appropriate buffers and / or cofactors for optimal activity of the polymerase enzyme (e.g., MgCl2 and / or KCl).

[0054] As used herein, the term “primer” is known to those skilled in the art and refers to oligomeric compounds, primarily oligonucleotides, but also to modified oligonucleotides that can “prime” DNA synthesis by template-dependent DNA polymerases, namely, for example, the 3' end of an oligonucleotide provides a free 3'-OH group to which a “nucleotide” can be further bound by a template-dependent DNA polymerase that establishes a 3'-to-5' phosphodiester bond, thereby using a deoxynucleoside triphosphate and releasing pyrophosphate.

[0055] The term "hybridize" refers to the annealing of one or more probes to an amplification product. "Hybridization conditions" typically include a temperature lower than the melting temperature of the probes but that avoids nonspecific hybridization of the probes.

[0056] The term "5'-to-3' nuclease activity" typically refers to the activity of nucleic acid polymerases involved in nucleic acid chain synthesis, where nucleotides are removed from the 5' end of the nucleic acid chain.

[0057] The term "thermally stable polymerase" refers to a polymerase enzyme that is thermally stable, meaning that it catalyzes the formation of primer extension products complementary to the template and does not irreversibly denature when exposed to high temperatures for the time required to cause denaturation of the double-stranded template nucleic acid. Generally, synthesis begins at the 3' end of each primer and proceeds along the template strand in the 5' to 3' direction. Thermally stable polymerases have been isolated from, for example, Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nevertheless, if the enzyme is replenished as needed, non-thermally unstable polymerases can also be used in PCR assays.

[0058] The term "complementary nucleic acid" refers to a nucleic acid that is the same length as a given nucleic acid and is precisely complementary to it.

[0059] When used in relation to nucleic acids, the terms "extension" or "lengthening" refer to the incorporation of additional nucleotides (or other similar molecules) into the nucleic acid. For example, nucleic acids can be arbitrarily lengthened by biocatalysts that incorporate nucleotides, or by polymerases that typically add nucleotides to the 3' end of nucleic acids.

[0060] In the context of two or more nucleic acid sequences, the term “identical” or “identity” percentage refers to two or more sequences or subsequences that have identical or a specific percentage of identical nucleotides when compared and aligned for the greatest match, for example, using one of the sequence comparison algorithms available to those skilled in the art or by visual inspection. An exemplary algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST program, for example, Altschul et al. (1990) "Basic local alignment search tool" J.Mol.Biol.215:403-410, Gish et al. (1993) "Identification of protein coding regions by database similarity search" Nature Genet.3:266-272, Madden et al. (1996) "Applications of network BLAST server" Meth.Enzymol.266:131-141, Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs" Nucleic Acids Res.25:3389-3402, and Zhang et al. (1997) "PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation" Genome Disclosed in Res.7:649-656, these are incorporated herein by reference.

[0061] In the context of oligonucleotides, "modified nucleotide" refers to a change in which at least one nucleotide in an oligonucleotide sequence is replaced by a different nucleotide that provides the oligonucleotide with desired properties. Exemplary modified nucleotides that may be substituted in the oligonucleotides described herein include, for example, t-butylbenzyl, C5-methyl-dC, C5-ethyl-dC, C5-methyl-dU, C5-ethyl-dU, 2,6-diaminopurine, C5-propynyl-dC, C5-propynyl-dU, C7-propynyl-dA, C7-propynyl-dG, C5-propargylamino-dC, C5-propargylamino-dU, C Examples include 7-propargylamino-dA, C7-propargylamino-dG, 7-deaza-2-deoxyxanthosine, pyrazolopyrimidine analogs, pseudo-dU, nitropyrrole, nitroindole, 2'-0-methylribo-U, 2'-0-methylribo-C, N4-ethyl-dC, N6-methyl-dA, 5-propynyl-dU, 5-propynyl-dC, and 7-deaza-deoxyguanosine (deaza-G(u-deaza)). Many other modified nucleotides that can be substituted in oligonucleotides are mentioned herein or are known in the art. In certain embodiments, the modified nucleotide substitution modifies the melting temperature (Tm) of the oligonucleotide compared to the melting temperature of the corresponding unmodified oligonucleotide. Further, certain modified nucleotide substitutions can, in some embodiments, reduce nonspecific nucleic acid amplification (e.g., minimizing primer dimer formation, etc.) and increase the yield of the intended target amplicon. Examples of these types of nucleic acid modifications are described, for example, in U.S. Patent No. 6,001,611, which is incorporated herein by reference. Other modifying nucleotide substitutions may alter the stability of oligonucleotides or provide other desirable characteristics.

[0062] Detection / quantification of target nucleic acids of malaria parasites (including Plasmodium). This disclosure provides a method for detecting malaria parasites (including Plasmodium) by, for example, amplifying a portion of the nucleic acid sequence of Plasmodium. Specifically, embodiments of this disclosure provide primers and probes for amplifying, detecting, and / or quantifying Plasmodium nucleic acid molecular targets.

[0063] Primers and probes for amplifying and detecting / quantifying plasmodium for the detection and / or quantification of malaria parasites (including plasmodium) are provided. Plasmodium nucleic acids other than those exemplified herein may also be used to detect plasmodium in a sample. For example, functional variants can be evaluated for specificity and / or sensitivity by those skilled in the art using routine methods. Representative functional variants may include, for example, one or more deletions, insertions, and / or substitutions in the plasmodium nucleic acids disclosed herein.

[0064] More specifically, each embodiment of an oligonucleotide comprises a nucleic acid having a sequence selected from SEQ ID NOs: 1 to 58, a substantially identical variant thereof having, for example, 80%, 90%, or 95% sequence identity with one of SEQ ID NOs: 1 to 58, or complementary and variant forms of SEQ ID NOs: 1 to 58.

[0065] [Table 1-1] [Table 1-2] [Table 1-3]

[0066] In one embodiment, the above set of plasmodium primers and probes are used to provide detection of malaria parasites (including plasmodium) in a biological sample suspected of containing plasmodium (Table 1). The set of primers and probes may include or consist of primers and probes specific to the nucleic acid sequences of malaria parasites (including plasmodium), which include or consist of the nucleic acid sequences of SEQ ID NOs. 1 to 58. In another embodiment, the primers and probes for a plasmodium target include or consist of a functionally active variant of any of the primers and probes of SEQ ID NOs. 1 to 58.

[0067] Functionally active variants of any of the primers and / or probes of SEQ ID NOs. 1-58 can be identified by using the primers and / or probes in the disclosed method. The functionally active variants of any of the primers and / or probes of SEQ ID NOs. 1-58 relate to primers and / or probes that, compared to the respective sequences of SEQ ID NOs. 1-58, provide similar or higher specificity and sensitivity in the described method or kit.

[0068] Variants can be altered from sequences 1-58 by adding, deleting, or substituting one or more nucleotides, for example, at the 5' and / or 3' ends of each sequence of sequence 1-58. As detailed above, primers and / or probes may be chemically modified, i.e., primers and / or probes may contain modified nucleotides or non-nucleotide compounds. Thus, the probe (or primer) is a modified oligonucleotide. A “modified nucleotide” (or “nucleotide analog”) differs from a natural “nucleotide” by several modifications, but still consists of a base or base-like compound, a pentofuranosyl sugar or pentofuranosyl sugar-like compound, a phosphate moiety or phosphate-like moiety, or a combination thereof. For example, a “modified nucleotide” can be obtained by attaching a “label” to the base portion of a “nucleotide.” The natural base in a “nucleotide” may be replaced, for example, with 7-deazapurine, which also yields a “modified nucleotide.” The terms “modified nucleotide” and “nucleotide analog” are used interchangeably in this application. "Modified nucleosides" (or "nucleoside analogs") differ from natural nucleosides through some form of modification, as outlined above for "modified nucleotides" (or "nucleotide analogs").

[0069] Oligonucleotides, including modified oligonucleotides and oligonucleotide analogs, that amplify nucleic acid molecules encoding plasmodium targets, such as nucleic acids encoding plasmodium alternative regions, can be designed using computer programs such as OLIGO (Molecular Biology Insights Inc., Cascade, Colorado). Key features in designing oligonucleotides to be used as amplification primers include, but are not limited to, appropriately sized amplification products to facilitate detection (e.g., by electrophoresis), similar melting temperatures for members of a pair of primers, and the length of each primer (i.e., primers must be long enough to anneal with sequence specificity and initiate synthesis, but not so long as to reduce accuracy during oligonucleotide synthesis). Typically, oligonucleotide primers are 8 to 50 nucleotides long (e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides long). Disclosed primers for the detection and amplification of malaria parasites (including Plasmodium) include SEQ ID NOs: 1, 2, 5, 6, 8, 9, 11-14, 16, 17, 21, 22, 26-29, 31-36, 43, 56, and 57.

[0070] In addition to a set of primers, this method may use one or more probes to detect the presence or absence of plasmodium. The term “probe” refers to a synthetically or biologically produced nucleic acid (DNA or RNA) that, by design or selection, contains a specific nucleotide sequence that enables it to hybridize specifically (i.e., preferentially) to a plasmodium (target) nucleic acid, in this case to the plasmodium (target) nucleic acid, under a defined stringency. A “probe” may be referred to as a “detection probe,” meaning that it detects the target nucleic acid.

[0071] In some embodiments, the plasmodium probes described may be labeled with at least one fluorescent label. In one embodiment, the plasmodium probe may be labeled with a donor fluorescent moiety, e.g., a fluorescent dye, and a corresponding acceptor moiety, e.g., a quencher. In one embodiment, the probe includes or comprises a fluorescent moiety, and the nucleic acid sequence includes or comprises SEQ ID NOs: 3, 4, 7, 10, 15, 18-20, 23-25, 30, 37-42, and 58. For example, probes disclosed for the detection of malaria parasites (including plasmodium) via hybridization / annealing to amplicons include SEQ ID NOs: 3, 4, 7, 10, 15, 18-20, 23-25, 30, 37-42, and 58.

[0072] The design of oligonucleotides used as probes can be carried out in a manner similar to that of primers. Multiple embodiments may use a single probe or a pair of probes for detection of the amplification product. Depending on the embodiment, the probes used may include at least one label and / or at least one quencher moiety. Similar to primers, probes typically have similar melting temperatures, and the length of each probe must be sufficient for sequence-specific hybridization to occur, but not so long as to degrade fidelity during synthesis. Oligonucleotide probes are generally 15 to 40 nucleotides long (e.g., 15, 16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, or 40).

[0073] The construct may include a vector, each containing one of the plasmodium primer and probe nucleic acid molecules (e.g., SEQ ID NOs: 1-58). The construct can be used, for example, as a control template nucleic acid molecule. Suitable vectors for use are commercially available and / or manufactured by recombinant nucleic acid techniques common in the art. Plasmodium nucleic acid molecules can be obtained, for example, by chemical synthesis, direct cloning from plasmodium, or nucleic acid amplification.

[0074] A construct suitable for use in this method typically includes a plasmodium nucleic acid molecule and / or primers / probes for amplification and / or detection of plasmodium (e.g., nucleic acid molecules containing one or more sequences of SEQ ID NOs: 1-58), as well as a sequence encoding a selection marker (e.g., an antibiotic resistance gene) for selecting the desired construct and / or transformant, and an origin of replication. The selection of a vector system usually depends on several factors, including but not limited to host cell selection, replication efficiency, selectivity, inducibility, and ease of recovery.

[0075] Constructs containing plasmodium nucleic acid molecules and / or primers / probes for amplification and / or detection of plasmodium can be grown in host cells. As used herein, the term host cell means prokaryotes and eukaryotes, including, for example, yeast, plant, and animal cells. Prokaryotic hosts may include Escherichia coli (E. coli), Salmonella typhimurium, Serratia marcescens, and Bacillus subtilis. Eukaryotic hosts include yeasts such as S. cerevisiae, S. pombe, and Pichia pastoris, mammalian cells such as COS cells or Chinese hamster ovary (CHO) cells, insect cells, and plant cells such as Arabidopsis thaliana and Nicotiana tabacum. The construct can be introduced into host cells using any technique commonly known to those skilled in the art. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and virus-mediated nucleic acid transfer are common methods for introducing nucleic acids into host cells. Furthermore, naked DNA can be delivered directly to cells (see, for example, U.S. Patents 5,580,859 and 5,589,466).

[0076] Polymerase chain reaction (PCR) U.S. Patents 4,683,202, 4,683,195, 4,800,159, and 4,965,188 disclose conventional PCR techniques. PCR typically uses two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA). Useful primers in some embodiments include oligonucleotides that can act as starting points for nucleic acid synthesis within the described plasmodium nucleic acid sequences (e.g., SEQ ID NOs: 1, 2, 5, 6, 8, 9, 11-14, 16, 17, 21, 22, 26-29, 31-36, and 43). Primers can be purified from restriction digests by conventional methods or produced synthetically. While single-stranded primers are preferred for maximum amplification efficiency, double-stranded primers may also be used. Double-stranded primers are first denatured, i.e., processed to separate the strands. One method of denaturing double-stranded nucleic acids is by heating.

[0077] If the template nucleic acid is double-stranded, it is necessary to separate the two strands before it can be used as a template in PCR. Strand separation can be achieved by any suitable denaturation method, including physical, chemical, or enzymatic means. One method for separating nucleic acid strands involves heating until the nucleic acid is predominantly denatured (e.g., denaturation of 50%, 60%, 70%, 80%, 90%, or more than 95%). The heating conditions required to denaturate the template nucleic acid depend, for example, the buffer salt concentration, as well as the length and nucleotide composition of the nucleic acid being denatured, but are typically in the range of about 90°C to about 105°C, depending on the reaction characteristics such as temperature and nucleic acid length. Denaturation typically takes place for about 30 seconds to 4 minutes (e.g., 1 minute to 2 minutes 30 seconds, or 1.5 minutes).

[0078] If the double-stranded template nucleic acid is denatured by heat, the reaction mixture is cooled to a temperature that promotes annealing of each primer to its target sequence. The annealing temperature is typically around 35°C to 65°C (e.g., around 40°C to 60°C, or around 45°C to 50°C). The annealing time can be around 10 seconds to 1 minute (e.g., around 20 seconds to 50 seconds, or around 30 seconds to 40 seconds). The reaction mixture is then adjusted to a temperature that promotes or optimizes polymerase activity, i.e., a temperature sufficient for extension to occur from the annealed primers to produce a product complementary to the template nucleic acid. The temperature must be sufficient to synthesize extension products from each primer annealed to the nucleic acid template, but should not be so high as to denature the extension products from their complementary templates (for example, the temperature for extension is generally in the range of about 40°C to 80°C (e.g., about 50°C to 70°C, about 60°C)). The extension time can be about 10 seconds to 5 minutes (e.g., about 30 seconds to 4 minutes, about 1 minute to 3 minutes, about 1 minute 30 seconds to 2 minutes).

[0079] The genomes of retroviruses or RNA viruses are composed of ribonucleic acid, i.e., RNA. In such cases, the template nucleic acid, RNA, must first be transcribed into complementary DNA (cDNA) via the action of the enzyme reverse transcriptase. Reverse transcriptase can direct the synthesis of the first strand of cDNA using the RNA template and short primers complementary to the 3' end of the RNA, and this can then be used directly as a template for polymerase chain reaction.

[0080] PCR assays may use plasmodium nucleic acids and / or primers / probes to amplify and / or detect plasmodium, such as RNA or DNA (cDNA). The template nucleic acid does not need to be purified; it may be a trace fraction of a complex mixture, such as plasmodium nucleic acids found in human cells. Plasmodium nucleic acid molecules and / or primers / probes to amplify and / or detect plasmodium can be extracted from biological samples by routine techniques, as described in Diagnostic Molecular Microbiology: Principles and Applications (Persing et al., ed., 1993, American Society for Microbiology, Washington DC). Nucleic acids can be obtained from any number of sources, such as plasmids, or from natural sources including bacteria, yeast, viruses, organelles, or higher organisms such as plants or animals.

[0081] Oligonucleotide primers (e.g., SEQ ID NOs: 1, 2, 5, 6, 8, 9, 11-14, 16, 17, 21, 22, 26-29, 31-36, and 43) are combined with the PCR reagent under reaction conditions that induce primer extension. For example, a chain extension reaction typically includes 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 15 mM MgCl2, 0.001% (w / v) gelatin, 0.5-1.0 μg of denatured template DNA, 50 pmol of each oligonucleotide primer, 2.5 U of Taq polymerase, and 10% DMSO. The reaction product usually contains 150-320 μM each of dATP, dCTP, dTTP, dGTP, or one or more of their analogues.

[0082] The newly synthesized chains form double-stranded molecules that can be used in subsequent steps of the reaction. The steps of chain separation, annealing, and extension can be repeated as many times as necessary to produce the desired amount of amplified product corresponding to the target nucleic acid molecule of the malaria parasite (including Plasmodium). Limiting factors of the reaction are the amount of primers, thermostable enzymes, and nucleoside triphosphates present during the reaction. The cycling steps (i.e., denaturation, annealing, and extension) are preferably repeated at least once. For use in detection, the number of cycling steps depends, for example, on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps will be required to amplify the target sequence sufficiently for detection. Generally, the cycling steps are repeated at least about 20 times, but may be repeated 40, 60, or 100 times.

[0083] Fluorescence resonance energy transfer (FRET) FRET technology (see, for example, U.S. Patents 4,996,143, 5,565,322, 5,849,489, and 6,162,603) is based on the concept that when a donor fluorescence moiety and a corresponding acceptor fluorescence moiety are placed within a certain distance of each other, energy transfer occurs between the two fluorescence moieties, which can be visualized or otherwise detected and / or quantified. Typically, the donor, when excited by irradiation with light of a suitable wavelength, transfers energy to the acceptor. Typically, the acceptor re-emits the transferred energy in the form of irradiation with light of a different wavelength. In certain systems, non-fluorescent energy can be transferred between the donor and acceptor moieties via a biomolecule containing a substantially non-fluorescent donor moiety (see, for example, U.S. Patent 7,741,467).

[0084] In one example, an oligonucleotide probe may contain a donor fluorescent moiety or dye (e.g., HEX or FAM) and a corresponding quencher (e.g., BlackHole Quencher® (BHQ) (BHQ-2, etc.)) which may or may not be fluorescent and dissipates energy transferred in a form other than light. If the probe is intact, energy transfer typically occurs between the donor and acceptor moieties so that fluorescence emission from the donor moiety is quenched by the acceptor moiety. During the extension step of the polymerase chain reaction, the probe bound to the amplified product is cleaved by the 5'-to-3' nuclease activity of, for example, Taq polymerase, so that fluorescence emission from the donor moiety is no longer quenched. Exemplary probes for this purpose are described, for example, in U.S. Patents 5,210,015, 5,994,056, and 6,171,785. Commonly used donor-acceptor pairs include the FAM-TAMRA pair. Commonly used quenchers are DABCYL and TAMRA. Commonly used dark quenchers include BlackHole Quencher® (BHQ) (BHQ2, etc.) (Biosearch Technologies, Inc., Novato, California), Iowa Black® (Integrated DNA Tech., Inc., Coralville, Iowa), and BlackBerry® Quencher 650 (BBQ-650) (Berry & Assoc, Dexter, Michigan).

[0085] In another example, two oligonucleotide probes, each containing a fluorescent moiety, may hybridize to an amplification product at a specific location determined by the complementarity of the oligonucleotide probes to the plasmodium-targeted nucleic acid sequence. When the oligonucleotide probes hybridize to the amplification product nucleic acid at the appropriate location, a FRET signal is generated. The hybridization temperature can range from approximately 35°C to approximately 65°C for approximately 10 seconds to approximately 1 minute.

[0086] Fluorescence analysis can be performed using, for example, a photon-counting epifluorescence microscope system (equipped with appropriate dichroic mirrors and filters for monitoring fluorescence emission over a specific range), a photon-counting photomultiplier tube system, or a fluorophotometer. Excitation to initiate energy transfer or to enable direct detection of the phosphor can be performed using an argon ion laser, a high-intensity mercury (Hg) arc lamp, a xenon lamp, a fiber optic light source, or other high-intensity light sources appropriately filtered for excitation over a desired range.

[0087] As used herein with respect to the donor portion and the corresponding acceptor portion, “corresponding” refers to an acceptor fluorescent portion or dark quencher having an absorbance spectrum that overlaps with the emission spectrum of the donor fluorescent portion. The maximum wavelength of the emission spectrum of the acceptor fluorescent portion must be at least 100 nm greater than the maximum wavelength of the excitation spectrum of the donor fluorescent portion. This allows for efficient non-radiative energy transfer between them.

[0088] The fluorescent donor and corresponding acceptor portions are generally selected for (a) highly efficient Foerster energy transfer, (b) a large final Stokes shift (>100 nm), (c) a shift of emission to the red portion of the visible spectrum (>600 nm) as much as possible, and (d) a shift of emission to wavelengths higher than the Raman water fluorescence emission produced by excitation at the donor excitation wavelength. For example, the donor fluorescent portion may be selected to have its maximum excitation wavelength near the laser line (e.g., helium-cadmium 442 nm or argon 488 nm), a high extinction coefficient, a high quantum yield, and good overlap of its fluorescence emission with the excitation spectrum of the corresponding acceptor fluorescent portion. The corresponding acceptor fluorescent portion may be selected to have a high extinction coefficient, a high quantum yield, good overlap of its excitation with the emission of the donor fluorescent portion, and emission in the red portion of the visible spectrum (>600 nm).

[0089] Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET technology include fluorescein, Lucifer Yellow, β-phycoerythrin, 9-acridine isothiocyanate, Lucifer Yellow VS, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, succinyl-1-pyrene butyrate, and derivatives of 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid. Typical acceptor fluorescence moieties include LC Red 640, LC Red 705, Cy5, Cy5.5, lysamine rhodamine B sulfonyl chloride, tetramethylrhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriamine pentaacetate, or other chelates of lanthanide ions (e.g., europium or terbium), depending on the donor fluorescence moiety used. Donor and acceptor fluorescence moieties can be obtained, for example, from Molecular Probes (Junction City, Oregon) or Sigma Chemical Co. (St. Louis, Missouri).

[0090] The donor and acceptor fluorescent moieties can be bound to a suitable probe oligonucleotide via linker arms. The length of each linker arm is important because it affects the distance between the donor and acceptor fluorescent moieties. The length of the linker arm may be the distance in angstroms (Å) from the nucleotide base to the fluorescent moiety. Generally, the linker arms are approximately 10 Å to 25 Å. The linker arms may be of the type described in International Publication 84 / 03285. International Publication 84 / 03285 also discloses methods for binding the linker arms to specific nucleotide bases and methods for binding the fluorescent moieties to the linker arms.

[0091] Acceptor fluorescent moieties such as LC Red 640 can be combined with oligonucleotides containing aminolinkers (e.g., C6-aminophosphoramidites available from ABI (Foster City, California) or Glen Research (Sterling, Virginia)) to produce, for example, LC Red 640-labeled oligonucleotides. Common linkers used to conjugate donor fluorescent moieties such as fluorescein to oligonucleotides include thiourea linkers (from FITC, e.g., fluorescein-CPG from Glen Research or ChemGene (Ashland, Massachusetts)), amide linkers (fluorescein-NHS-ester derived, such as CX-fluorescein-CPG from BioGenex (San Ramon, California)), or 3'-amino-CPGs that require fluorescein-NHS-ester conjugation after oligonucleotide synthesis.

[0092] Detection of plasmodium amplification products (amplicons) This disclosure provides a method for detecting the presence or absence of malaria parasites (including plasmodium) in biological or non-biological samples. The provided method avoids the problems of sample contamination, false negatives, and false positives. The method comprises performing at least one cycling step, which includes amplifying a portion of plasmodium-targeted nucleic acid molecules from a sample using one or more pairs of plasmodium primers, and a FRET detection step. Multiple cycling steps are preferably performed in a thermocycler. The method may be carried out using plasmodium primers and probes to detect the presence of plasmodium, and the detection of plasmodium indicates the presence of plasmodium in the sample.

[0093] As described herein, the amplification product may be detected using a labeled hybridization probe utilizing FRET technology. One FRET format utilizes TaqMan® technology to detect the presence or absence of the amplification product, and therefore the presence or absence of malaria parasites (including Plasmodium). TaqMan® technology utilizes a single-strand hybridization probe labeled with, for example, one fluorescent moiety or dye (e.g., HEX or FAM) and one quencher (e.g., BHQ-2), which may or may not be fluorescent. When the first fluorescent moiety is excited with light of an appropriate wavelength, the absorbed energy is transferred to a second fluorescent moiety or dark quencher according to the FRET principle. The second fluorescent moiety is generally a quencher molecule. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target DNA (i.e., the amplification product) and is subsequently degraded during the extension step by, for example, the 5'-to-3' nuclease activity of Taq polymerase. As a result, the fluorescent and quencher regions are spatially separated from each other. Consequently, when the first fluorescent region is excited in the absence of the quencher, fluorescence emission from the first fluorescent region can be detected. For example, the ABI PRISM® 7700 sequence detection system (Applied Biosystems) is suitable for performing the methods described herein to detect the presence or absence of malaria parasites (including Plasmodium) in a sample using TaqMan® technology.

[0094] Molecular beacons, combined with FRET, can also be used to detect the presence of amplification products using real-time PCR. Molecular beacon technology uses hybridization probes labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is typically a quencher, and the fluorescent label is usually located at each end of the probe. Molecular beacon technology uses probe oligonucleotides that have sequences (e.g., hairpins) that allow for secondary structure formation. As a result of secondary structure formation within the probe, both fluorescent sites are spatially close when the probe is in solution. After hybridization to the target nucleic acid (i.e., amplification product), the secondary structure of the probe is disrupted, and the fluorescent moieties separate from each other, thereby allowing detection of emission of the first fluorescent moiety after excitation with light of the appropriate wavelength.

[0095] Another common form of FRET technology utilizes two hybridization probes. Each probe can be labeled with a different fluorescent moiety and is generally designed to hybridize in close proximity to each other within a target DNA molecule (e.g., an amplification product). The donor fluorescent moiety, e.g., fluorescein, is excited at 470 nm by the light source of a LightCycler® instrument. During FRET, fluorescein transfers its energy to an acceptor fluorescent moiety, e.g., LightCycler®-Red 640 (LC Red 640) or LightCycler®-Red 705 (LC Red 705). The acceptor fluorescent moiety then emits longer wavelength light, which is detected by the optical detection system of the LightCycler® instrument. Efficient FRET can only occur if the fluorescent moieties are in direct, local proximity and the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety. The intensity of the emitted signal can be correlated with the number of original target DNA molecules (e.g., the number of plasmodium genomes). When amplification of the plasmodium target nucleic acid occurs and amplification products are produced, the hybridizing process yields a detectable signal based on FRET between members of the probe pair.

[0096] Generally, the presence of FRET indicates the presence of plasmodium in the sample, and the absence of FRET indicates the absence of plasmodium in the sample. However, inadequate sample collection, delayed transport, inappropriate transport conditions, or the use of specific collection swabs (calcium alginate or aluminum shafts) are all conditions that can affect the success and / or accuracy of the test results.

[0097] Typical biological samples that can be used in carrying out this method include, but are not limited to, whole blood, respiratory specimens, urine, fecal specimens, blood specimens, plasma, skin swabs, nasal swabs, wound swabs, blood cultures, and skin and soft tissue infections. Methods for collecting and storing biological samples are known to those skilled in the art. Biological samples can be processed (e.g., by nucleic acid extraction methods and / or kits known in the art) to release plasmodium nucleic acids, or in some cases, the biological sample can be brought into direct contact with PCR reaction components and appropriate oligonucleotides. In some examples, the biological sample is whole blood. When whole blood is typically collected, it is often collected in a container containing an anticoagulant such as heparin, citrate, or EDTA, which allows the whole blood to be stored at a suitable temperature. However, under such conditions, a considerable amount of nucleic acid in whole blood undergoes degradation. Therefore, it may be advantageous to collect the blood in reagents that dissolve, denature, and stabilize the whole blood components containing nucleic acids, such as nucleic acid stabilization solutions. In such cases, nucleic acids can be better preserved and stabilized for subsequent isolation and analysis by nucleic acid tests such as PCR. Such nucleic acid stabilizing solutions are well known in the art and include, but are not limited to, cobas PCR medium containing 4.2 M guanidinium salt (GuHCl) and 50 mM Tris at pH 7.5.

[0098] The sample may be collected by any method or apparatus designed to properly hold and store the sample before analysis. Such methods and apparatus are well known in the art. If the sample is a biological sample such as whole blood, the method or apparatus may include a blood collection tube. Such blood collection tubes are well known in the art and may include, for example, blood collection tubes. In many cases, it may be advantageous to use a blood collection tube, in which case the blood collection tube is under pressure in a space intended for sample collection, such as a blood tube with a vacuum chamber, such as a Vacutainer blood collection tube. Such blood collection tubes with a vacuum chamber, such as a Vacutainer blood collection tube, are well known in the art. It may be even more advantageous to collect the blood, with or without a vacuum chamber, into a blood collection tube containing a solution that dissolves, denatures, and stabilizes whole blood components containing nucleic acids, such as a nucleic acid stabilizing solution, so that the aspirated whole blood comes into immediate contact with the nucleic acid stabilizing solution in the blood collection tube.

[0099] Melting curve analysis is an additional step that can be included in cycle profiling. Melting curve analysis is based on the fact that DNA melts at a characteristic temperature called the melting temperature (Tm), which is defined as the temperature at which half of the DNA double strand separates into single strands. The melting temperature of DNA depends primarily on its nucleotide composition. Therefore, DNA molecules rich in G and C nucleotides have a higher Tm than DNA molecules rich in A and T nucleotides. The melting temperature of a probe can be determined by detecting the temperature at which the signal is lost. Similarly, the annealing temperature of a probe can be determined by detecting the temperature at which the signal is produced. The melting temperature of a plasmodium probe from a plasmodium amplification product can confirm the presence or absence of plasmodium in the sample.

[0100] Between each thermocycler run, a control sample may also be cycled. A positive control sample may amplify a target nucleic acid control template (other than the amplified product of the described target gene) using, for example, control primers and probes. A positive control sample may also amplify a plasmid construct containing, for example, the target nucleic acid molecule. Such a plasmid control may be amplified internally (e.g., in the sample) or in a separate sample run alongside the patient sample, using the same primers and probes used to detect the intended target. Such a control is an indicator of the success or failure of amplification, hybridization, and / or the FRET reaction. Each thermocycler run may also include a negative control lacking, for example, the target template DNA. The negative control can measure contamination. This ensures that the system and reagents do not produce false-positive signals. Thus, the control reaction can easily determine, for example, the ability of the primers to anneal and initiate extension by sequence specificity, as well as the ability of the probes to hybridize by sequence specificity and for FRET to occur.

[0101] In one embodiment, the method includes a step to avoid contamination. For example, an enzymatic method utilizing uracil-DNA glycosylase is described in U.S. Patents No. 5,035,996, No. 5,683,896, and No. 5,945,313 for reducing or eliminating contamination between one thermocycler operation and the next.

[0102] This method can be carried out using conventional PCR methods combined with FRET technology. In one embodiment, a LightCycler® instrument is used. The following patent applications: International Patent Publications 97 / 46707, 97 / 46714, and 97 / 46712 describe real-time PCR used with LightCycler® technology.

[0103] LightCycler® can be operated using a PC workstation and can utilize the Windows NT operating system. Signals from the sample are obtained as the machine sequentially positions capillaries on the optical unit. The software can display the fluorescence signal in real time immediately after each measurement. Fluorescence acquisition time is 10–100 milliseconds (msec). After each cycling step, a quantitative display of fluorescence versus cycle count can be continuously updated for all samples. The generated data can be saved for further analysis.

[0104] As an alternative to FRET, double-stranded DNA-binding dyes, such as fluorescent DNA-binding dyes (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)), can be used to detect amplification products. Upon interaction with double-stranded nucleic acids, these fluorescent DNA-binding dyes emit a fluorescent signal after being excited by light of a suitable wavelength. Double-stranded DNA-binding dyes, such as nucleic acid intercalating dyes, can also be used. When using double-stranded DNA-binding dyes, melting curve analysis is typically performed to confirm the presence of amplification products.

[0105] Those skilled in the art will understand that other nucleic acid or signal amplification methods may also be used. Examples of such methods, but are not limited to, branched DNA signal amplification, loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), autonomous sequence replication (3SR), strand displacement amplification (SDA), or smart amplification process version 2 (SMAP2).

[0106] Embodiments of this disclosure are not limited to the configuration of one or more commercially available devices.

[0107] Manufactured products / kits Embodiments of this disclosure further provide manufactured products or kits for detecting malaria parasites (including Plasmodium). The manufactured products may include primers and probes used to detect Plasmodium gene targets, along with appropriate packaging materials. Representative primers and probes for Plasmodium detection may hybridize to Plasmodium target nucleic acid molecules. Furthermore, the kits may also include appropriately packaged reagents and materials necessary for DNA immobilization, hybridization, and detection, such as solid supports, buffers, enzymes, and DNA standards. Methods for designing primers and probes are disclosed herein, and representative examples of primers and probes for amplifying and hybridizing Plasmodium target nucleic acid molecules are provided.

[0108] The manufactured product may also include one or more fluorescent moieties for labeling probes, or may label probes supplied with the kit. For example, the manufactured product may include donor and / or acceptor fluorescent moieties for labeling plasmodium probes. Examples of suitable FRET donor and corresponding acceptor fluorescent moieties are provided above.

[0109] The product may also include a package insert or packaging label with instructions for detecting plasmodium in a sample using plasmodium primers and probes. The product may further include reagents (e.g., buffers, polymerase enzymes, cofactors, or agents to prevent contamination) for carrying out the methods disclosed herein. Such reagents may be specific to one of the commercially available instruments described herein.

[0110] Embodiments of the present disclosure also provide a set of primers and one or more detectable probes for detecting malaria parasites (including Plasmodium) in a sample. [Examples]

[0111] Embodiments of this disclosure are further described in the following examples, which do not limit the scope of the invention as described in the claims.

[0112] The following embodiments and drawings are provided to aid in understanding the subject matter, and the true scope of that subject matter is set forth in the appended claims. It is understood that modifications may be made to the procedures described without departing from the spirit of the invention.

[0113] In this study, fully automated sample preparation (nucleic acid extraction and purification) was followed by PCR amplification and detection. The system used was the cobas® 6800 / 8800 system, consisting of a sample supply module, transfer module, processing module, and analysis module.

[0114] Selective amplification of the target nucleic acid was achieved by using specific forward and reverse primers selected from highly conserved regions of the target nucleic acid. A thermally stable DNA polymerase enzyme was used for both reverse transcription and amplification. The master mix contained deoxyuridine triphosphate (dUTP) instead of deoxythymidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplified product or amplicon). Any amplicons contaminating from previous PCR runs were destroyed by the AmpErase enzyme (uracil-N-glycosylase) present in the PCR mix by heating in the first thermal cycling step. However, the AmpErase enzyme was inactivated once exposed to temperatures above 55°C, so the newly formed amplicons were not destroyed.

[0115] The cobas® Plasmodium Master Mix contained detection probes specific to plasmodium and control nucleic acids. Each specific plasmodium and control detection probe was labeled with one of two unique fluorescent dyes acting as a reporter. Each probe also had a second dye acting as a quencher. The reporter dyes were measured at a defined wavelength, thus enabling the detection and identification of amplified plasmodium targets and controls. The fluorescence signal of the complete probe was suppressed by the quencher dye. During the PCR amplification process, hybridization of the probes to a specific single-strand DNA template led to cleavage by the 5'-to-3' nuclease activity of DNA polymerase, separating the reporter and quencher dyes and producing a fluorescence signal. With each PCR cycle, the amount of cleaved probe increased, and the cumulative signal of the reporter dye increased simultaneously. Since the two specific reporter dyes were measured at defined wavelengths, simultaneous detection and identification of amplified plasmodium targets and controls was possible.

[0116] Primers and probes for the Plasmodium assay were designed by seeding the primers and probes along the genome to the most conserved regions based on alignment. The primers and probes were then combined into an assay, and the assay was scored based on inclusiveness and exclusivity in the in silico evaluation. In addition to genome conservation, genome range (highly dependent on which sequences are publicly available) was also included in the assay scoring. Target regions of the Plasmodium genome were mitochondrial DNA targets (MT-1 and MT-2), the RNA repeat sequence R125, and 18S ribosomal RNA. The disclosed malaria parasite assay is designed to be a panmalaria assay capable of detecting the following Plasmodium species: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium malariae. This assay excludes target sequences that share homology with closely related species (e.g., parasites and bacteria) and humans, as well as environmental DNA that may be present in various assay reagents. The malaria assay is designed to detect Plasmodium species in a sample, such as a biological sample, such as whole blood. The disclosed malaria assay detects Plasmodium species in approximately 1.1 ml of whole blood. In some situations, the whole blood sample is in a tube / container, such as a vacuum tube / container, which also contains reagents / solutions that dissolve, denature, and stabilize the whole blood components containing nucleic acids, e.g., nucleic acid stabilizing solution, so that the whole blood being collected comes into immediate contact with the nucleic acid stabilizing solution in the blood collection container. The disclosed primer pairs having the nucleic acid sequences of SEQ ID NOs. 16 and 17, and the disclosed probes having the nucleic acid sequences of SEQ ID NOs. 18-20, detect and / or amplify the mitochondrial DNA target MT-1. The disclosed primers having the nucleic acid sequences of SEQ ID NOs. 11-14, and the disclosed probe having the nucleic acid sequence of SEQ ID NOs. 15, detect and / or amplify the mitochondrial DNA target MT-2. The disclosed primer pairs having the nucleic acid sequences of SEQ ID NOs. 21, 22 and 26, and the disclosed probes having the nucleic acid sequences of SEQ ID NOs. 23-25, detect and / or amplify the RNA repeat sequence R125.The disclosed primer pair having the nucleic acid sequences of SEQ ID NOs. 1 and 2, and the disclosed probe having the nucleic acid sequence of SEQ ID NOs. 3 and / or 4, detect and / or amplify the 18S ribosomal RNA target 18S-1. The disclosed primer pair having the nucleic acid sequences of SEQ ID NOs. 5 and 6, and the disclosed probe having the nucleic acid sequence of SEQ ID NOs. 7, detect and / or amplify the 18S ribosomal RNA target 18S-3. The disclosed primer pair having the nucleic acid sequences of SEQ ID NOs. 8 and 9, and the disclosed probe having the nucleic acid sequence of SEQ ID NOs. 10, detect and / or amplify the 18S ribosomal RNA target 18S-4.

[0117] Example 1: Amplification and detection of Plasmodium falciparum by real-time PCR Plasmodium nucleic acid assays were performed using Plasmodium falciparum cultures from ATCC (catalog no. 30930), and all targets (18S rRNA genes (including 18S-1, 18S-3, and 18S-4), mitochondrial genes (MT-1 and MT-2), and R-125) were tested in singleplex format. Six different dilution levels of Plasmodium falciparum were used: no dilution, 1:10, and 1:10. 2 , 1:10 3 , 1:10 4 , and 1:10 5 Plasmodium assays were performed. The reagents used included the cobas® 6800 / 8800 General Purpose PCR Master Mix, which has a profile and conditions for use with cobas® 6800 / 8800, and TaqMan® amplification and detection techniques. The final concentrations of oligonucleotides in the master mix were 0.3 μM for primers and 0.2 μM for probes. The cobas® 6800 / 8800 PCR profiles used are shown in Table 2 below: [Table 2]

[0118] In the test against the 18S-1 target, primers with nucleic acid sequences of SEQ ID NOs. 1 and 2, and a probe with the nucleic acid sequence of SEQ ID NO. 3 were used, and the results are shown in Figure 2A. In the test against the 18S-3 target, primers with nucleic acid sequences of SEQ ID NOs. 5 and 6, and a probe with the nucleic acid sequence of SEQ ID NO. 7 were used, and the results are shown in Figure 2B. In the test against the 18S-4 target, primers with nucleic acid sequences of SEQ ID NOs. 8 and 9, and a probe with the nucleic acid sequence of SEQ ID NO. 10 were used, and the results are shown in Figure 2C. In the study against the MT-1 target, primers with nucleic acid sequences of SEQ ID NOs. 16 and 17, and a probe with the nucleic acid sequence of SEQ ID NO. 18 were used, and the results are shown in Figure 2D. In the test against the MT-2 target, primers with nucleic acid sequences of SEQ ID NOs. 11 and 12, and a probe with the nucleic acid sequence of SEQ ID NO. 15 were used, and the results are shown in Figure 2E. In the test against the R125 target, primers with nucleic acid sequences of SEQ ID NOs. 21 and 22, and a probe with the nucleic acid sequence of SEQ ID NO. 23 were used, and the results are shown in Figure 2F. Figures 2A-2F and 3 show that all targets were detected at all dilution levels tested. Figure 3 is 1:10 5 The data for the eluate at different dilution levels is summarized below.

[0119] To improve the RFI signal of the 18S-1 target, the probe was redesigned. m This increased the signal. To that end, further testing of the 18S-1 target was performed using primers with the nucleic acid sequences of SEQ ID NOs. 1 and 2, and a probe with the nucleic acid sequence of SEQ ID NO. 4. The results of the original 18S-1 probe (SEQ ID NO. 3) and the redesigned 18S-1 probe (SEQ ID NO. 4) were compared, showing that the redesigned 18S-1 probe improved the RFI signal, as shown in Figure 4.

[0120] Therefore, these results demonstrate that the primers and probes for the plasmodium assay can efficiently and specifically amplify and detect the presence of Plasmodium falciparum in a real-time PCR assay.

[0121] Example 2: Droplet digital PCR (ddPCR) for copy number determination of Plasmodium falciparum cultures Using a droplet digital PCR (ddPCR) assay, in vitro transcripts, DNA minigenes, and stock target copy numbers of Plasmodium falciparum cultures were determined using Plasmodium falciparum cultures from ATCC (catalog number 30930). In the ddPCR assay, all targets (18S rRNA genes (including 18S-1, 18S-3, and 18S-4), mitochondrial genes (MT-1 and MT-2), and R-125) were tested in singleplex form. For the 18S-1 target, primers with the nucleic acid sequences of SEQ ID NOs. 1 and 2 and probes with the nucleic acid sequence of SEQ ID NOs. 37 were used. For the 18S-3 target, primers with the nucleic acid sequences of SEQ ID NOs. 5 and 6 and probes with the nucleic acid sequence of SEQ ID NOs. 38 were used. For the 18S-4 target, primers with the nucleic acid sequences of SEQ ID NOs. 8 and 9 and probes with the nucleic acid sequence of SEQ ID NOs. 39 were used. For the counter-test, primers with nucleic acid sequences of SEQ ID NOs. 16 and 17 and probes with the nucleic acid sequence of SEQ ID NO. 42 were used. For the MT-2 target test, primers with nucleic acid sequences of SEQ ID NOs. 11 and 12 and probes with the nucleic acid sequence of SEQ ID NO. 41 were used. For the R125 target test, primers with nucleic acid sequences of SEQ ID NOs. 21 and 22 and probes with the nucleic acid sequence of SEQ ID NO. 40 were used. Similar to Example 1, the reagent used was the cobas® 6800 / 8800 generic PCR Master Mix, which has a profile and conditions for use with cobas® 6800 / 8800 and uses TaqMan® amplification and detection techniques. The final concentrations of oligonucleotides in the master mix were 0.3 μM for the primers and 0.2 μM for the probes. The cobas® 6800 / 8800 PCR profiles used are shown in Table 2 above.

[0122] The ddPCR results of the Plasmodium falciparum cultures showed that the RNA targets were far more abundant than the DNA targets, as shown in Fig. 5. The results show the copy numbers (per μl) of the 1:10 5 culture dilutions, indicating higher copy numbers for the 18S-1, 18S-4, and R125 targets compared to the 18S-3, MT-1, and MT-2 targets.

[0123] Therefore, these tests showed that the 18S-1, 18S-4, and R125 targets had high copy numbers in the Plasmodium falciparum cultures.

[0124] Example 3: Multiplex Amplification and Detection of 18S-1 and 18S-4 Targets in Plasmodium falciparum Cultures by Real-Time PCR Since 18S-1 and 18S-4 were the most highly expressed targets in Plasmodium falciparum from a Plasmodium falciparum culture obtained from ATCC (Catalog No. 30930) (see Example 2, Fig. 5), a multiplex assay was designed and developed to simultaneously amplify and detect the 18S-1 and 18S-4 targets (i.e., dual) within the same sample. Specifically, the 18S-1 and 18S-4 targets were tested as a multiplex to determine whether the two 18S targets improved sensitivity. Generally, detecting multiple copy targets in parallel can improve sensitivity, and covering multiple targets reduces the risk that any singleplex assay will fail. This multiplex assay was performed at four different dilution levels (1:10 4 、1:10 5 、1:10 6 、1:10 7The tests were performed using the following reagents: cobas(registered trademark) 6800 / 8800 general-purpose PCR Master Mix, which has a profile and conditions for use with cobas(registered trademark) 6800 / 8800, and TaqMan(registered trademark) amplification and detection technology. The final concentrations of oligonucleotides in the master mix were 0.3 μM for primers and 0.2 μM for probes. The cobas(registered trademark) 6800 / 8800 PCR profiles used are shown in Table 2 above. For the 18S-1 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs. 1 and 2, and a probe with the nucleic acid sequence of SEQ ID NO. 4. For the 18S-4 target, this multiplex analysis used primers with nucleic acid sequences of SEQ ID NOs. 8 and 9, and a probe with the nucleic acid sequence of SEQ ID NO. 10. The 18S-1 and 18S-4 targets were run through the FAM channel, and the internal control (GIC) was run through the Cy5.5 channel. The data are shown in Figure 6A, and the results are shown in Figure 6B. Specifically, Figures 6A and 6B show that while the Ct values ​​for 18S-1 and 18S-4 are comparable, the RFI for 18S-1 is higher than that for 18S-4. These results demonstrate that the multiplex assay effectively and simultaneously amplifies and detects the plasmodium 18S-1 and 18S-4 targets. In a study on the impact of whole blood background on the multiplex assay, no issues of nonspecific interactions and / or significant PCR inhibition were observed (data not shown).

[0125] These studies demonstrate that multiplex assays can successfully and effectively amplify and detect plasmodium 18S-1 and 18S-4 targets simultaneously.

[0126] Example 4: Multiplex amplification and detection of 18S-1 and R125 targets in Plasmodium falciparum cultures by real-time PCR We designed and developed a multiplex assay for simultaneously amplifying (i.e., doubly) the 18S-1 and R125 targets within the same sample. Generally, detecting multiple copy targets in parallel allows for improved sensitivity and reduces the risk of any singleplex assay failing by covering multiple targets. For example, if a sequence variant is present, e.g., in the 18S sequence, the risk of not being able to detect the 18S sequence variant with an existing primer / probe set is mitigated by the presence of another set of primers / probes to detect a second distinct target. This is the greatest advantage of a multiplex assay over a singleplex assay. In this example, we used four different dilution levels of Plasmodium falciparum (1:10) in a background of 500 ng of whole blood genomic DNA. 4 , 1:10 5 , 1:10 6 , 1:10 7This multiplex assay was tested using the following reagents: cobas(registered) 6800 / 8800 general-purpose PCR Master Mix, which has a profile and conditions for use with cobas(registered) 6800 / 8800, and TaqMan(registered) amplification and detection technology. The final concentrations of oligonucleotides in the master mix were 0.3 μM for primers and 0.2 μM for probes. The cobas(registered) 6800 / 8800 PCR profiles used are shown in Table 2 above. For the 18S-1 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs. 1 and 2, and a probe with the nucleic acid sequence of SEQ ID NO. 4. For the R125 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs. 21 and 22, and a probe with the nucleic acid sequence of SEQ ID NO. 25. The 18S-1 and R125 targets were run through the FAM channel, and the internal control (GIC) was run through the Cy5.5 channel. The data are shown in Figure 7A, and the results are shown in Figure 7B. The data show that the Ct values ​​for 18S-1 and R125 are similar, but the RFI for 18S-1 is higher than that for R125. These results demonstrate that the multiplex assay effectively and simultaneously amplifies and detects the plasmodium 18S-1 and R125 targets. In a study on the effect of whole blood background on the multiplex assay, no issues of nonspecific interactions and / or significant PCR inhibition were observed (data not shown).

[0127] These tests demonstrate that multiplex assays can successfully and effectively amplify and detect plasmodium 18S-1 and R125 targets simultaneously.

[0128] Example 5: Multiplex amplification and detection of 18S-1 and R125 targets in in vitro transcripts in whole blood Designed and developed to simultaneously (i.e., dually) amplify and detect the 18S-1 and R125 targets within the same sample, the multiplex assay described in Example 4 above was tested on in vitro transcripts of the 18S-1 and R125 targets, and then added to DNA / RNA extracted from whole blood. As previously mentioned, in general, detecting multiple copy targets in parallel allows for improved sensitivity and reduces the risk of any singleplex assay failing by covering multiple targets. For example, if a sequence variant is present, for example, in the 18S sequence, the risk of not being able to detect the 18S sequence variant with an existing primer / probe set is mitigated by the presence of another set of primers / probes that detect a second distinct target. This is the greatest advantage of a multiplex assay over a singleplex assay. In this example, this multiplex assay was tested against a background of 500 ng of whole blood genomic DNA / RNA for 18S-1 and R125:10 5 , 10 4 , 10 3 , 10 2The assay was performed using 10 copies of in vitro transcripts at five different levels. The copy number of in vitro transcript stocks of 18S-1 and R125 transcripts was quantified by ddPCR. The reagents used included the cobas (for registration) 6800 / 8800 general-purpose PCR Master Mix, which has a profile and conditions for use with cobas® 6800 / 8800, and the TaqMan® amplification and detection technology. The final concentrations of oligonucleotides in the master mix were 0.3 μM for primers and 0.2 μM for probes. The cobas® 6800 / 8800 PCR profiles used are shown in Table 2 above. For the 18S-1 target, this multiplex assay used primers with the nucleic acid sequences of SEQ ID NOs. 1 and 2, and a probe with the nucleic acid sequence of SEQ ID NO. 4. For the R125 target, this multiplex assay used primers with the nucleic acid sequences of SEQ ID NOs. 21 and 22, and a probe with the nucleic acid sequence of SEQ ID NO. 25. The 18S-1 and R125 targets were flowed through the FAM channel, and the internal control (GIC) was flowed through the Cy5.5 channel. Multiplex assays were also performed separately as singleplex assays for comparison. The data are shown in Figure 8, which demonstrates that both the multiplex and singleplex assays were sensitive to 10 copies of input. In a study on the effect of whole blood background on the multiplex assay, no nonspecific interactions and / or significant PCR inhibition issues were observed (data not shown).

[0129] These tests demonstrate that multiplex assays can successfully and effectively amplify and detect plasmodium 18S-1 and R125 targets simultaneously.

[0130] Example 6: Amplification and detection of the R125 target in in vitro transcripts in whole blood In singleplex assays for the R125 target, a newly redesigned forward primer is used with an in vitro transcript of the R125 target (10 3The samples were tested against a copy of the R125 transcript and then added to DNA / RNA extracted from whole blood. The forward primer had the nucleic acid sequence of SEQ ID NO: 26, the reverse primer had the nucleic acid sequence of SEQ ID NO: 22, and the probe had the nucleic acid sequence of SEQ ID NO: 25. The copy number of the R125 transcript in the in vitro transcript stock was quantified by ddPCR as described above. The R125 target was flowed through a FAM channel, and the internal control (GIC) was flowed through a Cy5.5 channel. The results are shown in Figure 9, demonstrating that the redesigned oligonucleotide set (SEQ ID NOs: 22, 25, and 26) can amplify and detect R125 targets derived from malaria parasites, including Plasmodium malariae.

[0131] These tests demonstrate that the singleplex assay can successfully and effectively amplify and detect the R125 target of plasmodium.

[0132] Example 7: Multiplex amplification and detection of 18S-1 and R125 targets in Plasmodium falciparum cultures by real-time PCR We designed and developed a multiplex assay for simultaneously amplifying (i.e., doubly) the 18S-1 target and R125 target within the same sample. Generally, detecting multiple copy targets in parallel allows for improved sensitivity and reduces the risk of any singleplex assay failing by covering multiple targets. For example, if a sequence variant is present, e.g., in the 18S sequence, the risk of not being able to detect the 18S sequence variant with an existing primer / probe set is mitigated by the presence of another primer / probe set that detects a second distinct target. This is the greatest advantage of a multiplex assay over a singleplex assay. In this embodiment, this multiplex assay was performed on four different dilution levels of Plasmodium falciparum (1:10) in a background of whole blood nucleic acids extracted with a cobas® 6800 / 8800 instrument. 4 , 1:10 5 , 1:10 6 , 1:10 7The assay was performed using the following reagents: cobas(registered trademark) 6800 / 8800 general-purpose PCR Master Mix, which has a profile and conditions for use with cobas(registered trademark) 6800 / 8800, and TaqMan(registered trademark) amplification and detection technology. The final concentrations of oligonucleotides in the master mix were 0.3 μM for primers and 0.2 μM for probes. The cobas(registered trademark) 6800 / 8800 PCR profiles used are shown in Table 2 above. For the 18S-1 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs. 34 and 36, and a probe with the nucleic acid sequence of SEQ ID NO. 4. For the R125 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs. 22 and 27, and a probe with the nucleic acid sequence of SEQ ID NO. 25. The 18S-1 and R125 targets were run through the FAM channel, and the internal control (GIC) was run through the Cy5.5 channel. The data are shown in Figure 10A, and the results are shown in Figure 10B. The data show that while the Ct values ​​for 18S-1 and R125 are comparable, the RFI for 18S-1 is higher than that for R125. These results demonstrate that the multiplex assay effectively and simultaneously amplifies and detects the plasmodium 18S-1 and R125 targets. A study on the impact of whole blood background on the multiplex assay did not reveal any issues with nonspecific interactions and / or significant PCR inhibition (data not shown).

[0133] These tests demonstrate that multiplex assays can successfully and effectively amplify and detect plasmodium 18S-1 and R125 targets simultaneously.

[0134] Example 8: Multiplex amplification and detection of 18S-1 and 18S-3 targets in Plasmodium falciparum cultures by real-time PCR A multiplex assay was designed and developed to simultaneously amplify and detect two targets (i.e., dually) in the 18S rRNA region (18S-1 and 18S-3) within the same sample. Generally, detecting multiple copy targets in parallel allows for improved sensitivity and reduces the risk of any singleplex assay failing by covering multiple targets. For example, if a sequence variant is present, e.g., in the 18S sequence, the risk of not being able to detect the 18S sequence variant with an existing primer / probe set is mitigated by the presence of another set of primers / probes to detect a second distinct target. This is the greatest advantage of a multiplex assay over a singleplex assay. In this embodiment, this multiplex assay was performed on two different dilution levels of Plasmodium falciparum (1:10) in a background of whole blood nucleic acids extracted with a cobas® 6800 / 8800 instrument. 4 and 1:10 5The assay was performed using the following reagents: cobas(registered trademark) 6800 / 8800 general-purpose PCR Master Mix, which has a profile and conditions for use with cobas(registered trademark) 6800 / 8800, and TaqMan(registered trademark) amplification and detection technology. The final concentrations of oligonucleotides in the master mix were 0.3 μM for primers and 0.2 μM for probes. The cobas(registered trademark) 6800 / 8800 PCR profiles used are shown in Table 2 above. For the 18S-1 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs. 34-36 and probes with the nucleic acid sequence of SEQ ID NO. 4. For the 18S-3 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs. 56 and 57 and probes with the nucleic acid sequence of SEQ ID NO. 58. The 18S-1 and 18S-3 targets were run through the FAM channel, and the internal control (GIC) was run through the Cy5.5 channel. The results of this 18S-1 and 18S-3 multiplex malaria assay are shown in Figure 11. These results demonstrate that the multiplex assay effectively and simultaneously amplifies and detects the 18S-1 and 18S-3 targets of Plasmodium. In a study on the effect of whole blood background on the multiplex assay, no issues of nonspecific interactions and / or significant PCR inhibition were observed (data not shown).

[0135] These tests demonstrate that multiplex assays can successfully and effectively amplify and detect plasmodium 18S rRNA (targeting 18S-1 and 18S-3) regions simultaneously.

[0136] Example 9: Multiplex amplification and detection of 18S-1 and 18S-3 targets in Plasmodium vivax culture by real-time PCR A multiplex assay was designed and developed to simultaneously amplify and detect two targets (i.e., dually) in the 18S rRNA region (18S-1 and 18S-3) within the same sample. Generally, detecting multiple copy targets in parallel allows for improved sensitivity and reduces the risk of any singleplex assay failing by covering multiple targets. For example, if a sequence variant is present, e.g., in the 18S sequence, the risk of not being able to detect the 18S sequence variant with an existing primer / probe set is mitigated by the presence of another set of primers / probes to detect a second distinct target. This is the greatest advantage of a multiplex assay over a singleplex assay. In this embodiment, this multiplex assay was performed on four different dilution levels of Plasmodium vivax (1:10) in a background of whole blood nucleic acids extracted with a Cobas® 6800 / 8800 instrument. 3 and 1:10 4The assay was performed using the following reagents: cobas(registered trademark) 6800 / 8800 general-purpose PCR Master Mix, which has a profile and conditions for use with cobas(registered trademark) 6800 / 8800, and TaqMan(registered trademark) amplification and detection technology. The final concentrations of oligonucleotides in the master mix were 0.3 μM for primers and 0.2 μM for probes. The cobas(registered trademark) 6800 / 8800 PCR profiles used are shown in Table 2 above. For the 18S-1 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs. 34-36 and probes with the nucleic acid sequence of SEQ ID NO. 4. For the 18S-3 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs. 56 and 57 and probes with the nucleic acid sequence of SEQ ID NO. 58. The 18S-1 and 18S-3 targets were run through the FAM channel, and the internal control (GIC) was run through the Cy5.5 channel. The results of this 18S-1 and 18S-3 multiplex malaria assay are shown in Figure 12. These results demonstrate that the multiplex assay effectively and simultaneously amplifies and detects the 18S-1 and 18S-3 targets of Plasmodium. In a study on the impact of whole blood background on the multiplex assay, no nonspecific interactions and / or significant PCR inhibition issues were observed (data not shown).

[0137] These tests demonstrate that multiplex assays can successfully and effectively amplify and detect plasmodium 18S rRNA (targeting 18S-1 and 18S-3) regions simultaneously.

[0138] Example 10: Multiplex amplification and detection of 18S-1 and 18S-3 targets in the 18S rRNA sequence of the malaria parasite by real-time PCR A multiplex assay was designed and developed to simultaneously amplify and detect two targets (i.e., dually) in the 18S rRNA region (18S-1 and 18S-3) within the same sample. Generally, detecting multiple copy targets in parallel allows for improved sensitivity and reduces the risk of any singleplex assay failing by covering multiple targets. For example, if a sequence variant is present, e.g., in the 18S sequence, the risk of not being able to detect the 18S sequence variant with an existing primer / probe set is mitigated by the presence of another set of primers / probes to detect a second distinct target. This is the greatest advantage of a multiplex assay over a singleplex assay. In this example, the multiplex assay was tested on a DNA plasmid containing the 18S rRNA sequence of the malaria parasite at 1,000 copies per PCR reaction level. The reagents used included the cobas (for registration) 6800 / 8800 general-purpose PCR Master Mix, which has a profile and conditions for use with cobas® 6800 / 8800, and the TaqMan® amplification and detection technology. The final concentrations of oligonucleotides in the master mix were 0.3 μM for primers and 0.2 μM for probes. The cobas® 6800 / 8800 PCR profiles used are shown in Table 2 above. For the 18S-1 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs. 34-36 and a probe with the nucleic acid sequence of SEQ ID NO. 4. For the 18S-3 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs. 56 and 57 and a probe with the nucleic acid sequence of SEQ ID NO. 58. The 18S-1 and 18S-3 targets were run through the FAM channel, and the internal control (GIC) was run through the Cy5.5 channel. The results of the 18S-1 and 18S-3 multiplex malaria assays are shown in Figure 13.These results demonstrate that multiplex assays effectively and simultaneously amplify and detect 18S-1 and 18S-3 targets in DNA plasmids containing the 18S rRNA sequence of the malaria parasite.

[0139] These tests demonstrate that multiplex assays can successfully and effectively amplify and detect plasmodium 18S rRNA (targeting 18S-1 and 18S-3) regions simultaneously.

[0140] Example 11: Multiplex amplification and detection of 18S-1 and 18S-3 targets in Plasmodium vivax 18S rRNA sequences by real-time PCR A multiplex assay was designed and developed to simultaneously amplify and detect two targets (i.e., dually) in the 18S rRNA regions (18S-1 and 18S-3) within the same sample. Generally, detecting multiple copy targets in parallel allows for improved sensitivity and reduces the risk of any singleplex assay failing by covering multiple targets. For example, if a sequence variant is present, e.g., in the 18S sequence, the risk of not being able to detect the 18S sequence variant with an existing primer / probe set is mitigated by the presence of another set of primers / probes to detect a second distinct target. This is the greatest advantage of a multiplex assay over a singleplex assay. In this example, the multiplex assay was tested on a DNA plasmid containing the Plasmodium malariae 18S rRNA sequence at 1,000 copies per PCR reaction level. The reagents used included the cobas (for registration) 6800 / 8800 general-purpose PCR Master Mix, which has a profile and conditions for use with cobas® 6800 / 8800, and the TaqMan® amplification and detection technology. The final concentrations of oligonucleotides in the master mix were 0.3 μM for primers and 0.2 μM for probes. The cobas® 6800 / 8800 PCR profiles used are shown in Table 2 above. For the 18S-1 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs. 34-36 and a probe with the nucleic acid sequence of SEQ ID NO. 4. For the 18S-3 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs. 56 and 57 and a probe with the nucleic acid sequence of SEQ ID NO. 58. The 18S-1 and 18S-3 targets were run through the FAM channel, and the internal control (GIC) was run through the Cy5.5 channel. The results of the 18S-1 and 18S-3 multiplex malaria assays are shown in Figure 14.These results demonstrate that the multiplex assay effectively and simultaneously amplifies and detects the 18S-1 and 18S-3 targets of DNA plasmids containing the Plasmodium malariae 18S rRNA sequence.

[0141] These tests demonstrate that multiplex assays can successfully and effectively amplify and detect plasmodium 18S rRNA (targeting 18S-1 and 18S-3) regions simultaneously.

[0142] Example 12: Multiplex amplification and detection of 18S-1 and 18S-3 targets in the 18S rRNA sequence of Plasmodium ovale by real-time PCR A multiplex assay was designed and developed to simultaneously amplify and detect two targets (i.e., dually) in the 18S rRNA regions (18S-1 and 18S-3) within the same sample. Generally, detecting multiple copy targets in parallel allows for improved sensitivity and reduces the risk of any singleplex assay failing by covering multiple targets. For example, if a sequence variant is present, e.g., in the 18S sequence, the risk of not being able to detect the 18S sequence variant with an existing primer / probe set is mitigated by the presence of another set of primers / probes to detect a second distinct target. This is the greatest advantage of a multiplex assay over a singleplex assay. In this example, this multiplex assay was tested on a DNA plasmid containing the malaria parasite oophorus 18S rRNA sequence at 1,000 copies per PCR reaction level. The reagents used included the cobas (for registration) 6800 / 8800 general-purpose PCR Master Mix, which has a profile and conditions for use with cobas® 6800 / 8800, and the TaqMan® amplification and detection technology. The final concentrations of oligonucleotides in the master mix were 0.3 μM for primers and 0.2 μM for probes. The cobas® 6800 / 8800 PCR profiles used are shown in Table 2 above. For the 18S-1 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs. 34-36 and a probe with the nucleic acid sequence of SEQ ID NO. 4. For the 18S-3 target, this multiplex assay used primers with nucleic acid sequences of SEQ ID NOs. 56 and 57 and a probe with the nucleic acid sequence of SEQ ID NO. 58. The 18S-1 and 18S-3 targets were run through the FAM channel, and the internal control (GIC) was run through the Cy5.5 channel. The results of the 18S-1 and 18S-3 multiplex malaria assays are shown in Figure 15.These results demonstrate that multiplex assays effectively and simultaneously amplify and detect the 18S-1 and 18S-3 targets of DNA plasmids containing the 18S rRNA sequence of the malaria parasite oophorus.

[0143] These tests demonstrate that multiplex assays can successfully and effectively amplify and detect the 18S rRNA (targeting 18S-1 and 18S-3) regions of Plasmodium simultaneously. In particular, Examples 8-12 demonstrate that multiplex assays targeting 18S-1 and 18S-3 can specifically and efficiently amplify and detect Plasmodium parasites (including Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium quartanis) using oligonucleotides targeting 18S-1 with primers having the nucleic acid sequences of SEQ ID NOs. 34-36 and probes having the nucleic acid sequence of SEQ ID NOs. 4, and oligonucleotides targeting 18S-3 with primers having the nucleic acid sequences of SEQ ID NOs. 56 and 57 and probes having the nucleic acid sequence of SEQ ID NOs. 58.

[0144] Therefore, in summary, these results demonstrate that the oligonucleotide set of SEQ ID NOs: 1-58 specifically and efficiently amplifies and detects malaria parasites (including Plasmodium (Platypleura malariae), Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium quartanum)) in whole blood.

[0145] While the aforementioned invention has been described in some detail to clarify and understand it, it will be apparent to those skilled in the art that various modifications of form and detail can be made without departing from the true scope of the invention. For example, all the techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in whole for all purposes to the same extent as each individual publication, patent, patent application, and / or other document is individually indicated to be incorporated by reference for all purposes.

Claims

1. A method for detecting one or more malaria parasite species in a sample, wherein the method is (a) A step of carrying out an amplification step, which includes contacting the sample with a set of one or more oligonucleotide primers to produce an amplification product if the target nucleic acids of one or more malaria parasite species are present in the sample; (b) If one or more target nucleic acids of malaria parasite species are present in the sample, a step of performing a hybridization step including contacting one or more oligonucleotide probes with the amplification product; and (c) A step of detecting the presence or absence of the amplification product, wherein the presence of the amplification product indicates the presence of one or more malaria parasite species in the sample, and the absence of the amplification product indicates the absence of one or more malaria parasite species in the sample, The set of one or more oligonucleotide primers and the one or more oligonucleotide probes (1) A set of oligonucleotide primers including oligonucleotide primers containing the nucleic acid sequences of SEQ ID NOs: 34, 35, and 36, and an oligonucleotide probe containing the nucleic acid sequence of SEQ ID NO: 4 or its complement; and / or (2) A method comprising a set of oligonucleotide primers including oligonucleotide primers containing the nucleic acid sequences of SEQ ID NOs. 56 and 57, and an oligonucleotide probe containing the nucleic acid sequence of SEQ ID NO. 58 or its complement.

2. The set of one or more oligonucleotide primers and the one or more oligonucleotide probes (1) A set of oligonucleotide primers including oligonucleotide primers containing the nucleic acid sequences of SEQ ID NOs: 34, 35, and 36, and an oligonucleotide probe containing the nucleic acid sequence of SEQ ID NO: 4 or its complement; and (3) The method according to claim 1, comprising a set of oligonucleotide primers including oligonucleotide primers containing the nucleic acid sequences of SEQ ID NOs. 56 and 57, and an oligonucleotide probe containing the nucleic acid sequence of SEQ ID NO. 58 or its complement.

3. The method according to claim 1 or 2, wherein one or more malaria parasite species belong to the genus Plasmodium.

4. The method according to claim 3, wherein the one or more malaria parasite species belonging to the genus Plasmodium is Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium quartan, and / or Plasmodium malariae.

5. The method according to any one of claims 1 to 4, wherein the sample is a biological sample.

6. The method according to claim 5, wherein the biological sample is whole blood, respiratory specimen, urine, fecal specimen, blood specimen, plasma, skin swab, nasal swab, wound swab, blood culture, skin or soft tissue infection.

7. The method according to claim 6, wherein the biological sample is whole blood.

8. The method according to any one of claims 1 to 7, wherein one or more oligonucleotide probes are labeled.

9. The method according to claim 8, wherein one or more oligonucleotide probes are labeled with a donor fluorescent portion and a corresponding acceptor portion.

10. The method according to claim 9, further comprising step (c) detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent portion and the acceptor portion of the one or more oligonucleotide probes, wherein the presence or absence of fluorescence indicates the presence or absence of the one or more malaria parasite species in the sample.

11. A kit for detecting one or more malaria parasite species that may be present in a sample, wherein the kit comprises a DNA polymerase, a nucleotide monomer, and an amplification reagent comprising a set of one or more oligonucleotide primers and one or more oligonucleotide probes, the set of one or more oligonucleotide primers and the one or more oligonucleotide probes (1) A set of oligonucleotide primers including oligonucleotide primers containing the nucleic acid sequences of SEQ ID NOs: 34, 35, and 36, and an oligonucleotide probe containing the nucleic acid sequence of SEQ ID NO: 4 or its complement; and / or (2) A kit comprising a set of oligonucleotide primers, including oligonucleotide primers containing the nucleic acid sequences of SEQ ID NOs. 56 and 57, and an oligonucleotide probe containing the nucleic acid sequence of SEQ ID NO. 58 or its complement.

12. The set of one or more oligonucleotide primers and the one or more oligonucleotide probes (1) A set of oligonucleotide primers including oligonucleotide primers containing the nucleic acid sequences of SEQ ID NOs: 34, 35, and 36, and an oligonucleotide probe containing the nucleic acid sequence of SEQ ID NO: 4 or its complement; and (2) The kit according to claim 11, comprising a set of oligonucleotide primers including oligonucleotide primers containing the nucleic acid sequences of SEQ ID NOs. 56 and 57, and an oligonucleotide probe containing the nucleic acid sequence of SEQ ID NO. 58 or its complement.

13. The kit according to any one of claims 11 to 12, wherein one or more oligonucleotide probes are labeled.

14. The kit according to claim 13, wherein one or more oligonucleotide probes are labeled with a donor fluorescent portion and a corresponding acceptor portion.

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