Application of multi-species facultative detection reagent in preparation of kit for detecting dual parasites

Abstract

The invention provides application of a multi-species facultative detection reagent in preparation of a kit for detecting dual parasites. According to the multi-species facultative detection reagent, high-sensitivity detection of parasites of two genus, namely, plasmodium and babesia, can be realized by a double-system multiple qPCR detection method.

Description

Technical Field

[0001] The present invention belongs to the field of biological technology; more specifically, the present invention relates to the application of multi-species facultative detection reagents in the preparation of a kit for detecting dual-genera parasites. Background Art

[0002] Among the protozoa, the Sporozoa of the Apicomplexa all lead a parasitic life. Common species that have been reported to be able to parasitize in human red blood cells and cause disease in the host include Plasmodium of the Plasmodiidae family of the order Eucoccidiidae, including P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi, and Babesia microti, B. divergens, B. duncani and B. venatorum of the Babesiidae genus of the order Piroplasmida.

[0003] Malaria, caused by infection with Plasmodium, is one of the three major diseases that seriously endanger human health. In recent years, hundreds of millions of malaria cases have been reported worldwide each year, most of which are distributed in Africa. There are basically no local primary malaria cases in China, but in the context of global economic and trade integration, imported malaria cases and deaths still occur every year, seriously damaging people's lives and health; if cases are not discovered and diagnosed in time and prevented and controlled in time, or if treatment and prevention and control are not appropriate, there is a possibility of re-transmission of imported cases, threatening the existing malaria prevention and control results.

[0004] Commonly used detection methods for malaria diagnosis include rapid diagnostic kits (RDT), microscopy and nucleic acid testing. In the past, only RDT and microscopy were used at the grassroots and clinical levels. Nucleic acid testing is usually only used in case review in the laboratories of provincial or national centers for disease control and prevention (CDC). In recent years, with the promotion and application of fluorescent quantitative PCR (qPCR) technology and equipment in clinical and disease control units at all levels, the foundation has been laid for the application of Plasmodium nucleic acid detection technology in a wider range.

[0005] At present, the malaria nucleic acid detection systems or kits used in clinical or disease control agencies often only include the identification of Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale, and most of them do not include the identification of Plasmodium knowlesi. Although Plasmodium knowlesi is usually transmitted among monkeys by Anopheles mosquitoes, it can also infect humans. In recent years, the epidemic of Plasmodium knowlesi has been on the rise. Since 2011, the World Health Organization has listed Plasmodium knowlesi as the fifth malaria parasite that can infect humans in its annual World Malaria Report. The development cycle of Plasmodium knowlesi in a red blood cell is about 24 hours, which is shorter than the other four human malaria parasites. If not diagnosed and treated in time, there is a risk of developing severe illness or even death. Therefore, it is necessary to develop a detection kit that includes Plasmodium knowlesi. However, there is more than one strain of Plasmodium knowlesi reported, which is a major challenge to accurately detect the worm.

[0006] At the same time, people are generally susceptible to Babesia. The asexual reproduction period of Babesia can destroy human red blood cells, causing patients to experience chills, fever, sweating, fatigue, nausea, loss of appetite, muscle pain, joint pain, headache, abdominal pain, anemia or hemoglobinuria, etc. Severe patients may experience high fever (body temperature can reach 40°C), severe anemia, hemoglobinuria, soy sauce-colored urine, jaundice, respiratory distress, renal failure or coma, and even death. In recent years, there has been an upward trend in reports of residents infected with Babesia pathogens, the main species of which include Babesia volei, Babesia hunteri and Babesia divergentis.

[0007] Like Plasmodium, Babesia also mainly parasitizes human red blood cells in the human body. Its morphological characteristics are similar to those of the common Plasmodium ring body, and it is difficult to distinguish them by microscopic examination. At the same time, there are strains of Plasmodium that are positive in microscopy and negative in RDT that infect humans. In this case, the application of nucleic acid methods can be relatively more accurate. In 2017, a standard method (sequencing method) for Babesia nucleic acid detection was released, and the test range can theoretically include all Babesia that infects humans. However, this system does not belong to the same system as the qPCR method used to detect Plasmodium, and is not very convenient to use.

[0008] Although the detection of multiple species at one time is of great clinical significance, it is not easy to find a streamlined detection reagent with ideal sensitivity, given the complexity of the genomes of different species of parasites, the interference of homologous regions of other species' genomes, and the uncertainty of the detection experimental system. For example, the inventors established a qPCR based on the 18S rDNA of the asexual stage of Plasmodium to detect five species of Plasmodium infecting humans in a previous study, and evaluated its detection effect, but it requires different primer pairs, which is not suitable for simultaneous detection of multiple parasite species, and it is impossible to achieve specific detection of species other than Plasmodium using the same set of detection reagents with its characteristics.

[0009] In summary, there is a need in the art to simultaneously detect multiple genera of microorganisms in order to understand the presence of as many individual parasites as possible in a single sample. Summary of the invention

[0010] The object of the present invention is to provide the use of a multi-species facultative detection reagent in preparing a kit for detecting dual-genera parasites.

[0011] In a first aspect of the present invention, a multi-species facultative detection reagent is provided for use in preparing a kit for detecting parasites, wherein the multi-species facultative detection reagent comprises: universal primers: SEQ ID NOs: 1 to 3; probes: SEQ ID NOs: 4 to SEQ ID NOs: 11. The parasites include: Plasmodium parasites and Babesia parasites.

[0012] In one or more embodiments, the multi-species amphoteric detection reagent forms a dual system, including a probe method qPCR reaction system and a dye method qPCR reaction system.

[0013] In one or more embodiments, the probe method qPCR reaction system includes: universal primers SEQ ID NO: 1 to 3 and probes SEQ ID NO: 4 to SEQ ID NO: 11.

[0014] In one or more embodiments, the dye method qPCR reaction system includes: universal primers SEQ ID NO: 1-2.

[0015] In one or more embodiments, the Plasmodium parasites include: P. falciparum, P. knowlesi, P. malariae, P. ovale, P. vivax, P. cynomolgi, P. simium, P. brasilianum; preferably, the P. ovale includes: P. ovale Curtisi subspecies or P. ovale Wallikeri subspecies; preferably, the P. malariae includes: P. malariae strain 1 or P. malariae strain 2; preferably, the P. knowlesi includes: P. knowlesi strain 1 or P. knowlesi strain 2.

[0016] In one or more embodiments, the Babesia parasite includes: B. crassa; B. crassa-like; B. divergens; B. duncani; B. gibsoni; B. microti; B. odocoilei; B. sp; B. venatorum; B. vogeli.

[0017] In one or more embodiments, detection is performed on the 18S rDNA sequence of Plasmodium.

[0018] In one or more embodiments, the Plasmodium falciparum gene sequence includes: a Plasmodium falciparum A type 18S rDNA sequence or a Plasmodium falciparum S type 18S rDNA sequence.

[0019] In one or more embodiments, the Plasmodium vivax gene sequence includes: a Plasmodium vivax A type 18S rDNA sequence or a Plasmodium vivax S type 18S rDNA sequence.

[0020] In one or more embodiments, the Plasmodium malariae gene sequence comprises: a type A 18S rDNA sequence of Plasmodium malariae strain 1 or a type A 18S rDNA sequence of Plasmodium malariae strain 2.

[0021] In one or more embodiments, the Plasmodium malariae gene sequence comprises: an A or S type 18S rDNA sequence of Plasmodium knowlesi strain 1 or an A type 18S rDNA sequence of Plasmodium knowlesi strain 2.

[0022] In one or more embodiments, the Plasmodium ovale gene sequence comprises: a type A 18S rDNA sequence of Plasmodium ovale subspecies Curtisi or Plasmodium ovale subspecies Wallikeri.

[0023] In one or more embodiments, the probe-based qPCR reaction system detects Plasmodium falciparum, Plasmodium vivax and Plasmodium ovale.

[0024] In one or more embodiments, the dye-based qPCR reaction system detects Plasmodium and Babesia parasites; preferably, after qPCR amplification, the parasite species can be identified based on an amplification curve, a melting curve or sequencing.

[0025] In one or more embodiments, the detection reagent is highly non-specific for human DNA.

[0026] In another aspect of the present invention, a kit for detecting parasites is provided, the kit comprising: a container or a package, and a multi-species facultative detection reagent contained in (sub-packed in) the container or the package; wherein the multi-species facultative detection reagent comprises: universal primers: SEQ ID NOs: 1 to 3; probes: SEQ ID NOs: 4 to SEQ ID NOs: 11. wherein the parasites include: Plasmodium parasites and Babesia parasites.

[0027] In one or more embodiments, the multi-species facultative detection reagents form a double set, including a probe method reaction set and a dye method reaction set.

[0028] In one or more embodiments, the probe method reaction kit includes: universal primers SEQ ID NOs: 1 to 3 and probes SEQ ID NOs: 4 to 11.

[0029] In one or more embodiments, the dye method reaction kit includes: universal primers SEQ ID NOs: 1-2.

[0030] In one or more embodiments, the end of the probe carries a fluorescent group and a corresponding quenching group; preferably, the fluorescent group / quenching group includes: HEX / BHQ1, CY5.5 / BHQ3, CY5 / BHQ3, FAM / BHQ1, Texas RED / BHQ2.

[0031] In one or more embodiments, in the probe method reaction kit, the primer dosage (molar ratio) is: SEQ ID NO:1:SEQ ID NO:2:SEQ ID NO:3 is (8-12):(8-12):(3-5); preferably (9-11):(9-11):(3.5-4.5); for example 10:10:4.

[0032] In one or more embodiments, in the probe method reaction kit, the probe dosage (molar ratio): SEQID NO:4:SEQ ID NO:5:SEQ ID NO:6:SEQ ID NO:7:SEQ ID NO:8:SEQ ID NO:9:SEQ IDNO:10:SEQ ID NO:11 is (3-5):(3-5):(3-5):(2.5-3.5):(2.5-3.5):(3-5):(0.8-1.2):(0.8-1.2); preferably (3.5-4.5):(3.5-4.5):(3.5-4.5):(2.8-3.1):(2.8-3.2):(3.5-4.5):(0.9-1.1):(0.9-1.1); for example 4:4:4:3:3:4:1:1.

[0033] In one or more embodiments, in the dye method reaction kit, the primer dosage (molar ratio) is: SEQ ID NO:1:SEQ ID NO:2 is (0.7-1.3):(0.7-1.3); preferably (0.8-1.2):(0.8-1.2); more preferably (0.9-1.1):(0.9-1.1); for example 1:1.

[0034] In one or more embodiments, in the probe method reaction kit, each universal primer or probe is placed in an independent container or package, or is mixed in the container or package.

[0035] In one or more embodiments, during use, the primers and probes may be added separately, or a premix containing the same substances and amounts may be added.

[0036] In one or more embodiments, in the dye method reaction kit, each universal primer is placed in an independent container or package, or is mixed in the container or package.

[0037] In one or more embodiments, the kit further comprises one or more reagents selected from the following group: PCR reaction buffer solution, DNA extraction reagent, DNA polymerase (such as Taq enzyme), RNase-free deionized sterile water, and dNTPs.

[0038] In one or more embodiments, the kit further includes a positive control.

[0039] In one or more embodiments, the positive control includes: a positive control carrying a standard sequence segment (within 500 bp) of the amplified products of each Plasmodium and Babesia.

[0040] In one or more embodiments, the kit further includes a quality control product.

[0041] In one or more embodiments, the kit further includes a standard.

[0042] In one or more embodiments, the kit further includes: instructions for the Tm value of the standard dye method.

[0043] In one or more embodiments, the kit further includes instructions for use describing a method for simultaneously identifying multiple parasites.

[0044] In another aspect of the present invention, there is provided a use of any of the above-described kits for specifically detecting a variety of parasites.

[0045] In another aspect of the present invention, a method for specifically detecting parasites is provided, wherein the parasites include Plasmodium parasites and Babesia parasites; the method includes using a multi-species facultative detection reagent for detection, wherein the multi-species facultative detection reagent includes: universal primers SEQ ID NOs: 1 to 3, and probes SEQ ID NOs: 4 to SEQ ID NOs: 11; during detection, the multi-species facultative detection reagents form a detection system, including a probe method qPCR reaction system and / or a dye method qPCR reaction system (preferably including a double system); the probe method qPCR reaction system includes: universal primers SEQ ID NOs: 1 to 3 and probes SEQ ID NOs: 4 to SEQ ID NOs: 11; the dye method qPCR reaction system includes: universal primers SEQ ID NOs: 1 to 2.

[0046] In one or more embodiments, the detection system includes a probe method qPCR reaction system and a dye method qPCR reaction system; wherein the detection system is used to detect (a) Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium knowlesi, Plasmodium brasiliensis, Babesia scoparia, Babesia duncanii, Babesia microti or Babesia veseri at the genus and species level, and (b) Plasmodium howler monkey, Plasmodium cynomolgus, Babesia divergentis, Babesia ornithischia, Babesia ornithischia, Babesia scoparia, Babesia gibsonii or Babesia weseri at the genus level; preferably, after the genus level detection in (b), the insect species are further subdivided and identified by sequencing.

[0047] In one or more embodiments, the method for specifically detecting parasites is a non-diagnostic method, for example, used in the laboratory to classify (subdivide) parasites that are not parasitic on an organism host, or used in the determination of auxiliary sample parameters that are not for the purpose of diagnosis.

[0048] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 , Plasmodium falciparum, Plasmodium vivax and Plasmodium ovale species specificity assay results (the concentration of each target fragment plasmid DNA was 5×10 8 No non-specific results were found among the target insect species.

[0050] Figure 2 , Plasmodium, and Babesia probe specificity determination results (the concentration of each target fragment plasmid DNA was 5×10 8 copies / μL), and no nonspecific results were shown between different genera.

[0051] Figure 3 , Plasmodium falciparum and Plasmodium vivax low concentration 18S rDNA plasmid amplification curve (dye method, concentration gradient of 10 0 ~10 2 copies / μL).

[0052] Figure 4 Amplification curves of low-concentration 18S rDNA plasmids of Babesia microti and Babesia oriota (dye method, concentration gradient of 10 0 ~10 2 copies / μL).

[0053] Figure 5 , Plasmodium falciparum A-type and S-type 18S rDNA plasmid 2:3 ratio of the low-concentration 18S rDNA amplification curve (probe method, concentration gradient of 10 0 ~10 2 copies / μL).

[0054] Figure 6 , amplification curve of low concentration 18S rDNA plasmid of Plasmodium vivax (probe method, concentration gradient of 10 0 ~10 2 copies / μL).

[0055] Figure 7 , Amplification curve of low concentration 18S rDNA plasmid of Microtus elegans (probe method, concentration gradient of 10 1 ~10 3 copies / μL).

[0056] Figure 8 , amplification curve of low concentration 18S rDNA plasmid of Babesia orioides (probe method, concentration gradient of 10 1 ~10 3 copies / μL).

[0057] Fig. 9, concentration gradient is 10 1 ~10 7 Amplification curve of Plasmodium vivax 18S rDNA fragment copies / μL (dye method).

[0058] Fig.10 , concentration gradient is 10 1 ~10 7 Amplification curve of Babesia microti 18S rDNA fragment in copies / μL (dye method).

[0059] Fig.11 , concentration gradient is 10 0 ~10 7 Amplification curve of Plasmodium vivax 18S rDNA fragment at copies / μL (probe method).

[0060] Fig.12 , concentration gradient is 10 1 ~10 8 Amplification curve of 18S rDNA fragment of Babesia truncatum at copies / μL (probe method). DETAILED DESCRIPTION

[0061] In the field of species identification, PCR technology is widely used. However, when it is necessary to implement the simplest detection possible for some species with complex genomes and diverse species, various certain or uncertain complex factors need to be considered and dealt with. How to accurately and without omission detect such species without false positives and false negatives is a problem that needs to be solved in the field.

[0062] In order to achieve the goal of accurately detecting multiple genera of parasites at one time with as few detection reagents as possible, the inventors have conducted extensive and in-depth research and experiments, and on this basis, disclosed a group of multi-species facultative detection reagents, which can achieve high-sensitivity detection of parasites of the two genera of Plasmodium and Babesia through a dual-system (dual-tube) multiplex qPCR detection method.

[0063] Reagents and kits

[0064] The inventors have obtained a high-sensitivity detection reagent for broad-spectrum detection of parasites of the Plasmodium and Babesia genera by comparing and screening a variety of Plasmodium, Babesia and other species at the genera level, and have established a PCR method for detecting Plasmodium and Babesia species to achieve the purpose of accurate identification. The technical solution of the present invention uses a simplified detection reagent, uses universal primers, covers a variety of parasites but has controllable costs, and has a convenient detection process.

[0065] In a preferred embodiment of the present invention, a kit for specifically identifying Plasmodium and Babesia species is provided, comprising: universal primers: SEQ ID NOs: 1 to 3; and probes: SEQ ID NOs: 4 to 11.

[0066] In a preferred embodiment of the present invention, the multi-species facultative detection reagent forms a double set, including a probe method reaction set and a dye method reaction set; the probe method reaction set includes: universal primers SEQ ID NO: 1 to 3 and probes SEQ ID NO: 4 to SEQ ID NO: 11; the dye method reaction set includes: universal primers SEQ ID NO: 1 to 2.

[0067] The kit may also include standards and quality control products, such as plasmids carrying standard sequence segments (within 500 bp) of amplified products of various Plasmodium species and Babesia species, as positive controls or Tm values ​​of the standard dye method.

[0068] The primers and / or probes of the present invention may also be labeled with radioactive isotopes, biotin, enzymes, fluorescein or other chemiluminescent substances.

[0069] In addition, the kit may also contain other reagents for PCR reaction or upstream and downstream reactions, including but not limited to:

[0070] (A) various reagents for PCR reaction, such as but not limited to: Taq enzyme, PCR buffer, dNTP, DNA polymerase, etc.; or

[0071] (B) various reagents required for DNA extraction (i.e., preparation of PCR reaction templates), such as but not limited to: phenol, chloroform, isoamyl alcohol, NaCl, etc.; or

[0072] (C) Kit for extracting DNA.

[0073] In addition, the kit may also contain instructions and / or standard operating procedures for identifying Plasmodium and Babesia species.

[0074] The kit of the present invention can achieve the purpose of rapid and sensitive detection of Plasmodium and Babesia species with ideal accuracy.

[0075] Detection methods and applications

[0076] Based on the specific primers and probes suitable for identifying Plasmodium and Babesia species provided by the present invention, the present invention also provides a method for specifically detecting Plasmodium and Babesia species, the method comprising: using the DNA of the sample to be tested as a template, performing the following steps (a) and (b): (a) a probe method qPCR reaction system and a dye method qPCR reaction system; the probe method qPCR reaction system comprises: universal primers SEQ ID NOs: 1 to 3 and probes SEQ ID NOs: 4 to SEQ ID NOs: 11; the dye method qPCR reaction system comprises: universal primers SEQ ID NOs: 1 to 2; wherein, if specific amplification occurs, it indicates that the sample to be tested contains Plasmodium or Babesia species, and the genera and species are distinguished according to the fluorescence presented by the probe and the Tm value obtained by the dye method.

[0077] In a preferred embodiment of the present invention, each sample is amplified and detected using a Taqman probe and a TB green double-tube multiplex PCR system. Using the detection reagent of the present invention, the clinically important Plasmodium falciparum, Plasmodium vivax and Plasmodium ovale can be directly identified by genus and species, and other species can be identified by genus. After TB green qPCR amplification, the species can be identified based on the amplification curve, melting curve and sequencing.

[0078] The technical solution of the present invention only requires one pair of amplification primers, thereby ensuring the sensitivity of the system. For the PCR system for simultaneously detecting multiple insect species, the probe method can be used in the following ways: universal primer multiple probes, multiple primer universal probes, and multiple primer multiple probes; the dye method can be used in the following ways: universal primer diversified Tm values, multiple primer diversified Tm values. The more insects that need to be identified, the more difficult the system design is. As far as PCR amplification is concerned, a single pair of primers is less interfered with than multiple primer amplifications, that is, the amplification effect is better, especially when detecting low-density parasite infections, the sensitivity is higher.

[0079] The technical solution of the present invention introduces a mutant base to improve the specificity in a complex detection system. Specifically, a mutant base (from T to A) is introduced at a position close to the 3' end of the downstream primer, so that the homotypic mismatch between the primer and the target sequence at this site has a relatively small effect on the amplification of the target fragment. The inventor's analysis shows that human 18S rDNA also has a corresponding segment and a homotypic mismatch at the second position of the 3' end, then the impact of its composition is relatively large, and the non-specific enhancement of the human sequence, that is, the introduction of the mutant base improves the specificity of the detection reagent of the present invention.

[0080] The technical solution of the present invention reasonably matches species and genus probes / primers to ensure the direct detection of key parasites. Malaria is a Class B infectious disease, which has a relatively serious impact on human health, especially Plasmodium falciparum, which may lead to the death of patients if not treated in time. The top three malaria types in terms of the number of clinically reported cases are Plasmodium falciparum, Plasmodium vivax and Plasmodium ovale. The present invention selects suitable detection reagents through extensive screening and analysis, thereby realizing detection of these three malarial parasites by combining primers with species probes; for Plasmodium malariae and Plasmodium knowlesi, which have relatively few reported cases, they are distinguished and identified by the genus detection results of the probe method and the Tm value in the melting curve of the dye method. Both the accuracy of the detection and the actual clinical situation are taken into account.

[0081] The malarial parasite genome contains both A and S type 18S rDNA, wherein the 18S rDNA sequences of two types of Plasmodium falciparum, Plasmodium vivax and Plasmodium knowlesi are completely consistent with the sequences of universal primers (when no mutant base is introduced), so they can all be amplified. When the probe of Plasmodium is designed with the A type sequence as a template, the S type sequence cannot be detected. After comparison and analysis, the copy number ratios of A type and S type 18S rDNA in the three malarial parasite genomes are respectively 2:3 for Plasmodium falciparum, 1:3 for Plasmodium vivax and 1:1 for Plasmodium knowlesi. The copy number of A type 18S rDNA in the Plasmodium falciparum genomic DNA is less than that of S type. Although in theory, the copy number of 2 types of sequences can be amplified and detected within the range of 1:10, considering a more ideal detection result, the present invention provides a type 2 Plasmodium falciparum species-specific probe in the probe system.

[0082] Considering that after the introduction of the mutant base, the downstream primer and the 18S rDNA sequence of Plasmodium falciparum A type have two mismatched bases at the 3rd and 9th positions at the 3' end, the inventors also added a small amount of downstream degenerate primers specific to Plasmodium falciparum A type in the probe amplification system, which effectively ensures its detection. In a preferred embodiment, the inventors designed the type 2 probe to use the same luminescent group and quenching group, which actually increased the number of target fragments and further improved the sensitivity of detecting the insect species.

[0083] The content of Plasmodium A type 18S rRNA in the erythrocytic stage is hundreds to thousands of times that of 18S rDNA. The primers and probes of the present invention can detect Plasmodium A type 18S rDNA, which has the great advantage of developing and applying a more sensitive one-step reverse transcription detection system.

[0084] For the detection of Babesia, the probe system of the present invention not only finds probes that are conservative for most common Babesia, but also determines a more specific combined probe for Babesia volei, thus ensuring the accuracy and efficiency of its detection.

[0085] The technical solution of the present invention uses a universal primer and a dual-system detection mode, with a wide detection range and more reliable results. The detection mode, on the one hand, ensures that as many insect species as possible can be amplified and detected, and on the other hand, important parasites can be directly detected, and the PCR products of the dye method can be directly sequenced, eliminating the step of re-amplification after the probe method is negative, saving steps and time. In addition, the two systems are used for detection, and the results are mutually verified, which is more reliable.

[0086] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, Science Press, or according to the conditions recommended by the manufacturer.

[0087] Example 1. Design of detection reagents

[0088] Plasmodium has great complexity in different genera and species, and Babesia also has many closely related species and complexity. The inventors have done a lot of work to screen primers and probes using a variety of Plasmodium and Babesia as target species for analysis and research.

[0089] 1. Primer Design

[0090] The inventors collected 18S rRNA gene sequences of various malarial parasites that infect humans and compared them with a series of human malarial parasite detection systems. After analysis and preliminary experimental research, the focus was on the V4 region. The sequences of the malarial parasite V4 region were analyzed and compared. This region contains both a high-variability region (corresponding to the length of about 145bp of Plasmodium falciparum A-type 18S rDNA (PFA)) and a relatively conservative region (corresponding to the length of about 151bp of PFA). The bases in the high-variability regions between different species vary widely, with sequence consistency of 35-88%, except for PK and PV, which are greater than 80%, while the rest are less than 66%. The sequence consistency of the relatively conservative region is 66-99%, except for POS and PFS, the others are greater than 87%, and the similarity of the A-type 18S rDNA sequences of each species is greater than 94%. Analysis of 13 Babesia 18S rDNA sequences showed that the 18S rDNA sequence of Babesia also has the same characteristics in the region corresponding to the V4 region of Plasmodium 18S rDNA, with a sequence identity of 26-87%. However, the region corresponding to the relatively conservative region of V4 of Plasmodium 18S rDNA is a conserved region of Babesia. This region has a difference of less than 20% with Plasmodium. It can be seen that the sequence differences between different species are complex and difficult to design.

[0091] In order to obtain a streamlined detection reagent that can cover the overall detection of the Plasmodium "genus", specifically identify different Plasmodium "species", take into account the specific detection of Babesia parasites, do not cross-react with human DNA, and achieve high-sensitivity detection in experiments, the inventors conducted extensive and in-depth sequence analysis and experimental analysis.

[0092] Furthermore, in order to improve the specificity of detecting Plasmodium and Babesia, according to the influence of different base mutations at different sites of the 3' end of the primer on the amplification effect, the inventors introduced a mutant base at the third position of the 3' end of the primer, replacing T with A, to further reduce the possibility of amplifying human genome 18S rDNA by the amplification system.

[0093] Through sequence analysis and experimental analysis, the inventors designed the upstream and downstream primer sequences as follows:

[0094] Upstream primer - UP: 5'-GTTAAAACGCTCGTAGTTGA-3' (SEQ ID NO: 1);

[0095] Downstream primer - through LW: 5'-GTTATTCCATGCTGTAGTA A TC-3' (SEQ ID NO: 2);

[0096] Primers TongUP and TongLW can be used as universal primers for multiple insect species in two genera.

[0097] The inventor's analysis shows that in addition to the A-type 18S rDNA, there are two different 18S rDNA types, S and / or O-type, in the genomes of different malarial parasites, in which the primer sequence (except the mutation site) is completely consistent with the S-type 18S rDNA sequence of Plasmodium falciparum, Plasmodium vivax and Plasmodium knowlesi. In these three malarial parasite genomes, the ratios of the A-type and S-type 18S rDNA sequence copies are 2:3, 3:1 and 1:1, respectively. According to the priority order during PCR amplification, that is, the DNA template with a large number of copies is usually amplified preferentially, which may affect the amplification effect and the sensitivity of the detection. For this reason, the inventor added a downstream primer (degenerate primer) with A at the 9th position of the 3' end, so that some primers are completely consistent with the 18S rDNA of Plasmodium falciparum (except the mutation site):

[0098] Downstream primer - LF: 5'-GTTATTCCATGCTATAGTAATC-3' (SEQ ID NO: 3).

[0099] 2. Probe Design

[0100] In the present invention, the probe system is 5-fold, including Plasmodium probe (PG), Babesia probe (BG; degenerate probes BG1 and BG2), Plasmodium falciparum type A and type S probes (PFA and PFS), Plasmodium vivax species probe (PV) and Plasmodium ovale degenerate probes (POC and POW), and the 5' luminescent group and the 3' end quenching group are shown in Table 1.

[0101] Table 1. Probe sequences and modification groups

[0102]

[0103] 3. Reaction System

[0104] A single detection was performed with dual systems in parallel, and the reaction components in the probe method and dye method systems are shown in Tables 2 and 3. The inventors simultaneously considered the copying conditions and frequencies of the sequence segments corresponding to different amplification products in the genome, and optimized the usage of primers and probes.

[0105] Table 2. Amounts of components in the probe-based qPCR haploid reaction system

[0106]

[0107]

[0108] Table 3. Amounts of each component in the dye-based qPCR haploid reaction system

[0109]

[0110] 4. Reaction conditions

[0111] The reaction conditions of the system are: 95℃3min; 95℃8s, 55℃20s, 45 cycles; 95℃10s; 65℃~95℃, 0.5℃ / time, 0.5s each time. The baseline threshold automatically set by the instrument is used. After the reaction is completed, the amplification curve and melting curve of each PCR reaction of the dye method are checked. The baseline threshold is set to 50, and the Ct values ​​of all reactions and the Tm values ​​of the dye method qPCR are collected.

[0112] During the erythrocytic stage of malarial parasites, the blood contains a large amount of type A 18S rRNA sequences, which have a copy number hundreds or even tens of thousands of times that of 18S rDNA. Therefore, when a one-step reverse transcription kit is used for amplification, a reverse transcription amplification step can be added before the amplification conditions. The specific situation is determined based on all reagents.

[0113] 5. Result determination

[0114] 1. Validity determination

[0115] (1) Probe method: The positive control of the reaction system showed the corresponding genus and species standard amplification curve (i.e., S-shaped), while the negative control and blank control showed no amplification, i.e., there was no S-shaped amplification curve, and the qPCR amplification was effective.

[0116] (2) Dye method: In addition to meeting the requirements of the probe method, the positive control must also have a single peak in the melting curve and a Tm value consistent with the expected size (within ±0.5°C) (reference values ​​for Tm of each pair of primers are shown in Appendix B); the negative control and blank control must have no specific amplification, that is, no S-shaped amplification curve, or the Ct value of the amplification curve is >35, or the Tm value differs from the positive control by more than ±0.5°C, and qPCR amplification is valid.

[0117] (3) Specific amplification occurred in the positive control and negative control, but not in the blank control, indicating that DNA extraction was invalid.

[0118] 2. Determination of test results

[0119] Determine the test results based on the premise that qPCR amplification is effective

[0120] (1) When both the genus and species of Plasmodium are positive and the results are consistent, the test result can be directly judged as positive, and the species can be determined based on the species probe indication or Tm value. Usually, the template is unified, the ΔCt value between the genus and species probes is less than 3, and the ΔCt value between the probe method and the fuel method is less than 6. For species with the same Tm value that cannot be distinguished by combining the probe qPCR results, the dye method qPCR product can be sequenced; when the test results of both systems are negative, the sample test result is judged to be negative.

[0121] (2) If the Plasmodium probe is positive, the Babesia probe is negative, and the probe for any of the Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale species is positive, the test result can be determined to be positive for the corresponding species among the three Plasmodium species, and the dye method test result can be used for further reference verification.

[0122] (3) If the Plasmodium probe is positive and the other probes are negative, but the dye-based qPCR test shows amplification, the sample is determined to be positive for Plasmodium. The specific species can be determined based on the corresponding Tm value of the dye-based test (if the species is identified to the genus, the species can also be determined by sequencing the dye-based qPCR product).

[0123] (4) If the Babesia probe is positive, the other probes are negative, and the dye-based qPCR amplification is detected, the sample is determined to be positive for Babesia, and the specific insect species is determined based on its corresponding Tm value (if it is distinguished to the genus, the insect species can also be determined by sequencing the dye-based qPCR product).

[0124] 6. Testing

[0125] The positive samples involved in the test included DNA plasmids containing 18S rDNA fragments of Plasmodium or Babesia. Using the 18S rDNA of the A stage of five Plasmodium species as templates, a primer pair (rPLU1F: 5'-TCAAAGATTAAGCCATGCAAGTGA-3' (SEQ ID NO: 12) and rPLU1R: 5'-CTAGGCATTCCTCGTTCAAGATT-3' (SEQ ID NO: 13)) was used to PCR amplify a DNA fragment of about 1.8 kb. After sequencing and identification, the fragments were connected to the Puc19 plasmid to construct plasmids.

[0126] The plasmids of other positive samples were prepared by chemical synthesis based on the sequences published on the public platform. Plasmid DNA was extracted, the copy concentration of the target fragment was calculated, and the plasmid concentration was adjusted to 5×10 9 copies / μl, and perform 10-fold serial dilutions for later use.

[0127] The malaria-negative samples involved in the test come from whole blood DNA from healthy people.

[0128] Targeted malarial parasites: PFA: Plasmodium falciparum A type; PFS: Plasmodium falciparum S type; PK: Plasmodium knowlesi; PM: Plasmodium malariae; POC: Plasmodium ovale Curtisi subspecies; POW: Plasmodium ovale Wallikeri subspecies; PVA: Plasmodium vivax A type; PVS: Plasmodium vivax S type; PB: Plasmodium brasilianum; PC: Plasmodium cynomolgi; PS: Plasmodium simium, etc., see Table 4.

[0129] The targeted Babesia are: BC: Babesia crassa; BCL: Babesia crassa-like; BDI: Babesia divergens; BDU: Babesia duncani; BG: Babesia gibsoni; BM: Babesia microti; BOD: Babesia odocoilei; BSP: an unnamed Babesia; BV: Babesia venatorum; BVO: Babesia vogeli.

[0130] The detection capability of the dye-based qPCR system for various species of Plasmodium and Babesia using primer pairs Tong UP and Tong LW is shown in Table 4.

[0131] Table 4

[0132]

[0133] The results show that the detection reagent of the present invention can be used to detect various malarial parasites (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium knowlesi, Plasmodium brasiliensis) and Babesia (Babesia spp., Babesia duncani, Babesia voles, Babesia B.sp) at the genus and species level. It can detect various malarial parasites (Plasmodium spp., Plasmodium cynomolgus) and Babesia (Babesia divergentis, Babesia ornithis, Babesia ornithis, Babesia spp., Babesia spp., Babesia gibsoni, Babesia weseri) at the genus level.

[0134] Example 2: Comparison of universal primers before and after mutation (cross-reactivity with human DNA)

[0135] The upstream and downstream primers (upstream primer-Tong UP, downstream primer-Tong LW) obtained in Example 1 as universal primers are basically consistent with the Plasmodium 18S rDNA sequence (only one base in the middle of the downstream primer is different from PFA), and have individual base differences with some Babesia (10 / 14). Experimental analysis shows that good detection of Plasmodium and Babesia can be achieved.

[0136] When detecting a target template with a low concentration, one of the commonly used methods is to concentrate the initial template or increase the amount of template DNA in the amplification system in order to achieve the purpose of detection. However, when using conventional methods to extract Plasmodium or Babesia genomic DNA, it is impossible to separate Plasmodium or Babesia DNA from human genomic DNA. Therefore, the amount of human 18rDNA will also increase in the added or concentrated template DNA. In order to ensure the amplification effect when detecting actual samples, the higher the non-specificity of the primers to human genomic DNA, the better.

[0137] The comparison results of the universal primers (upstream primer-Tong UP and downstream primer-Tong LW) screened in Example 1 with the corresponding sequences of human 18S rDNA are as follows (yellow indicates mismatched bases):

[0138] Upstream primer: 5'-GTTAAAA C GCTCGTAGTTG A -3' (SEQ ID NO: 14);

[0139] Human 18S rDNA sequence: 5'-GTTAAAA A GCTCGTAGTTG G -3' (SEQ ID NO: 15);

[0140] Downstream primer: 5'-G TTATTCC AT GCTG TA GTA AT C-3' (SEQ ID NO: 16);

[0141] Human 18S rDNA sequence: 5'- A TTATTCC TA GCTG CG GTA TC C-3' (SEQ ID NO: 17).

[0142] When the DNA of the test sample is extracted, in the last step of DNA elution, if the volume of the added elution solution is the same as the sample volume, the extracted DNA solution is called the original concentration DNA template; if the elution solution is halved, it is called the concentrated DNA template. The test results show that when the T at the 3rd position of the 3' end of the downstream primer is changed to A, the amplification of various target fragments is minimal (ΔCt value ≤ 2). For human 18S rDNA, a heterotypic mutation at the 3' end is generated, coupled with the original homotypic mismatch at the 2nd position. After the introduction of the mutant base, the Ct value of the downstream primer for human 18S rDNA amplification can increase by 13.16, which is unexpected. In addition, the 1st position and other positions of the 3' end of the upstream primer have mismatches, and the non-specificity of the universal primer to human 18S rDNA is greatly improved.

[0143] 2 or 5ul of the original concentration DNA template and concentrated DNA template of 4 Plasmodium RDT and microscopic negative human blood samples were taken for DNA amplification (total volume 20uL), and the detection was carried out according to the method of Example 1. The results of amplifying 4 human blood DNA using original primers and mutant primers are compared in Table 5.

[0144] Table 5

[0145]

[0146]

[0147] As can be seen from the table, when the original primers were used, at least 3 of the 4 samples (46.7%, 7 / 15, Tm value 86.5-87.0℃) amplified human DNA during the qPCR reaction, and the Ct values ​​of amplification were mostly between 32-33℃ (91.7%, 11 / 12), and random non-specific amplification occurred 5 times (33.3%, 5 / 15) (i.e., there was a fluorescence value, but no peak in the melting curve). When the mutant primers were used, 7 of the 4 samples had no non-specific amplification (53.8%, 7 / 13), and 6 had random non-specific amplification (46.2%, 6 / 13); in the random non-specific amplification, the Ct value was close to the maximum positive critical value of 35 (34.88) (7.7%, 1 / 13) or significantly greater than the maximum positive critical value of 35 (92.3%, 12 / 13).

[0148] Therefore, mutation of the universal primer greatly reduces cross-reaction with human DNA, with remarkable effect.

[0149] Example 3: Primer and probe specificity test

[0150] The samples were respectively containing plasmid DNA templates of Plasmodium falciparum, Plasmodium vivax, Babesia microti and Babesia oriota, and the probe qPCR method of Example 1 was used for detection. The probe system of the present invention was based on the concentration of 5×10 8 Whether non-specific amplification curves occur in the non-target fragments with copies / μL is the criterion for determining whether the probe is specific.

[0151] The results showed that all probes had specific amplification curves for the target fragments and no amplification curves for the non-target fragments (see Figure 1 , Figure 2 ).

[0152] The dye method system does not conduct specificity evaluation, but can comprehensively detect a variety of Plasmodium and Babesia in addition to human genomic DNA.

[0153] Example 4: Sensitivity (minimum detection limit) test of primers and probes

[0154] In the system of the present invention, there are 4 types of base variation of 18S rDNA sequence alignment primer sequences of important and common species of Plasmodium and Babesia. Plasmodium falciparum, Plasmodium vivax, Babesia microti and Babesia oriolus were selected as representatives of each type to carry out system sensitivity determination.

[0155] 1. Minimum detection limit of dye-based qPCR system

[0156] The samples were respectively containing 18S rDNA plasmids of Plasmodium falciparum type A, Plasmodium vivax, Babesia microti, and Babesia oriota. The original concentration of each sample was 5×10 0 , 5×10 1 and 5×10 2 The copies / μL were detected using the qPCR dye method of Example 1 with 3 concentration gradients and 2 replicates for each concentration.

[0157] The minimum detection limit of the dye-based qPCR system for a certain sample is determined by the lowest concentration in the tested concentration gradient, with two replicates having amplification curves and the corresponding melting curve Tm value being consistent with that at a higher concentration (difference within ±0.5).

[0158] exist Figure 3 The green curve is the melting curve of the amplified Plasmodium falciparum 18S rDNA plasmid template. Among the three concentration gradients, only the lowest concentration of 5×10 0 The Tm value of one reaction with 10 copies / μL (red circled curve) is inconsistent with other samples, indicating nonspecific amplification. The purple curve is the melting curve of the amplified Plasmodium vivax plasmid DNA template, and the Tm values ​​of reactions at all concentrations remain consistent, indicating no nonspecific amplification.

[0159] exist Figure 4 The green curve is the melting curve of the amplified Babesia vole plasmid DNA template. Among the three concentration gradients, only the lowest concentration of 5×10 0 The purple curve is the melting curve of the amplified Babesia orionis plasmid DNA template. The difference between the Tm values ​​of each concentration reaction is 0.5℃, which means that they are consistent and no nonspecific amplification occurs.

[0160] From the above results, it can be seen that when the original concentration of plasmid DNA is ≥5×10 1 All samples and replicates were specifically amplified when the original concentration of plasmid DNA was 5×10 0 When the original concentration is 5×10 0 ~5×10 1 Copies / μL, that is, the final concentration in the system reaction can reach as low as 0.5 copies / μL, and it is most stable when ≥5 copies / μL. When the concentration is ≥5 copies / μL, the average Ct value of all samples is less than 33 (≤33), and the fluorescence signal value is no different from other samples with higher concentrations, that is, each sample can be accurately detected.

[0161] 2. Minimum detection limit of probe-based qPCR system

[0162] The samples contained 18S rDNA plasmids of Babesia molitori, 18S rDNA plasmids of Babesia oriorum, a mixture of 18S rDNA plasmids of Plasmodium falciparum type A and type S in a ratio of 2:3, and 18S rDNA plasmids of Plasmodium vivax. The original concentrations of Babesia molitori and Babesia oriorum were 5×10 1 copies / μL, 5×10 2 copies / μL and 5×10 3 There were 3 concentration gradients of copies / μL, and 5×10 copies / μL were selected for the samples of Plasmodium falciparum and Plasmodium vivax, respectively. 0 copies / μL, 5×10 1 copies / μL and 5×10 2 There were 3 concentration gradients of copies / μL, with 2 replicates for each concentration, and the detection was carried out using the qPCR probe method of Example 1.

[0163] The minimum detection limit of the probe-based qPCR system for a certain sample is determined by the lowest concentration in the tested concentration gradient and the presence of amplification curves for both replicates.

[0164] like Figure 5 , is the amplification curve of three concentration gradients of the mixture of Plasmodium falciparum A type and S type 18S rDNA plasmids at a ratio of 2:3. When the concentration gradient is ≥5×10 1 When the number of copies / μL was 1.04, the Ct values ​​of the sample species and genus amplification curves were ≤38, and the fluorescence signal values ​​were above 400.

[0165] like Figure 6 , is the amplification curve of the 18S rDNA fragment plasmid DNA template of Plasmodium vivax. 1 Except for 300 copies / μL, all other concentrations had amplification curves, and the Ct values ​​of the amplification curves were ≤38, and the fluorescence signal values ​​were above 400.

[0166] like Figure 7 , is the amplification curve of the 18S rDNA plasmid of Babesia volei. 1 Except for the two reactions with 0.1 copies / μL, the Ct values ​​of the amplification curves of other concentrations were ≤38, and the fluorescence signal values ​​were above 400.

[0167] like Figure 8 , is a test for the three concentration gradients of Babesia oriota 18S rDNA plasmid, except for the lowest concentration of 5×10 1 Except for one reaction with 10 copies / μL, the Ct values ​​of the amplification curves of other concentrations were ≤38, and the fluorescence signal values ​​were above 400.

[0168] From the above results, it can be seen that when the probe method qPCR is used to amplify the target template, the minimum detection limit of the system for Plasmodium is 5×10 1 The original template concentration was 10 copies / μL, which means that all the tested samples were accurately detected. The minimum detection limit of the system for Babesia was 5×10 1 ~5×10 2 The original template concentration is ≥5×10 2 copies / μL, that is, when the final concentration of the template in the system is ≥25 copies / μL, all tested samples are accurately detected.

[0169] Based on the test results of the dye method and the probe method, the minimum detection limit of this system for samples is 5×10 0 ~5×10 1 The original template concentration was 100 copies / μL. Only when the Ct value of the dye-based qPCR of the same sample was ≥32, or the Ct value of the probe-based qPCR was >38, or the fluorescence signal was lower than 400, the sample should be concentrated or the template amount should be increased before retesting.

[0170] Example 5: Examples of amplification efficiency of the system and Ct values ​​of samples of different concentrations

[0171] The samples were mixed with 18S rDNA plasmids of Babesia voles, 18S rDNA plasmids of Babesia oriorum, 18S rDNA plasmids of Plasmodium falciparum type A and type S in a ratio of 2:3 (the true copy number ratio in the genome of Plasmodium falciparum), and 18S rDNA plasmids of Plasmodium vivax. 5×10 0 ~10 7 There are 8 concentration gradients in total, and the probe method selects 5×10 1 ~10 7 There were 8 concentration gradients of copies / μL, and 2 replicates for each concentration. The detection was carried out using the dye method and probe method qPCR method of Example 1, respectively.

[0172] Collect the Ct values ​​of all reactions, and draw the standard curve of the amplification system with the lg value of each sample concentration as the horizontal axis and the corresponding Ct value as the vertical axis. Calculate the amplification efficiency (E) of the system for each sample based on the slope (K) of the curve and the following formula.

[0173] E=-1+10 1 / -K

[0174] An amplification system with an amplification efficiency (E) value between 90% and 110% is an ideal system.

[0175] The amplification efficiency of the system for samples with a concentration of 5×10 2 and 5×10 5The amplification Ct values ​​of each sample in copies / μL are shown in Table 6.

[0176] Table 6. Amplification efficiency of the system for each sample and amplification Ct values ​​for samples of different concentrations

[0177]

[0178] As can be seen from Table 6, the amplification efficiency of this system is basically in the range of 90-110% ( Figure 9-12 ), which meets the requirements of an ideal PCR system. In particular, for dye-based qPCR, the Ct value when the concentration copy is in the single digit is also close to the ideal state, that is, the PCR-Ct value of 1 copy template is 35 ( Figure 9-10 The amplification efficiency when using the probe method to detect Plasmodium falciparum samples is also high enough ( Fig.11 ).

[0179] For the same sample, there is a certain difference in the Ct value between the dye method and the probe method, and the difference is basically between 5 and 6, so the critical Ct value for positive judgment is higher for the probe method than for the dye method. This is related to the fact that the dye method qPCR system has fewer components and is simpler.

[0180] In the present invention, dye-based qPCR and probe-based qPCR are combined to detect samples, which is equivalent to increasing the number of detections, reducing the chance that the target DNA fragment cannot be amplified at low density to a certain extent, and improving the sensitivity of detection.

[0181] In the present invention, the determination of the detection result is verified from multiple angles through genus probes, species probes, amplification curves and melting curves, thereby increasing the accuracy and specificity of the result.

[0182] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Application of multi-species facultative detection reagents in the preparation of kits for detecting parasites; in, The multi-species facultative detection reagent comprises: Universal primers: SEQ ID NO: 1-3; Probe: SEQ ID NO: 4 to SEQ ID NO: 11; The parasites include Plasmodium parasites and Babesia parasites.

2. The use according to claim 1, characterized in that The multi-species facultative detection reagent forms a dual system, including a probe method qPCR reaction system and a dye method qPCR reaction system; The probe method qPCR reaction system includes: universal primers SEQ ID NO: 1 to 3 and probes SEQ ID NO: 4 to SEQ ID NO: 11; The dye method qPCR reaction system includes: universal primers SEQ ID NO: 1-2.

3. The use according to claim 1, characterized in that The Plasmodium parasites include: P. falciparum, P. knowlesi, P. malariae, P. ovale, P. vivax, P. cynomolgi, P. simium, P. brasilianum; preferably, the P. ovale includes: P. ovale Curtisi subspecies or P. ovale Wallikeri subspecies; preferably, the P. malariae includes: P. malariae strain 1 or P. malariae strain 2; preferably, the P. knowlesi includes: P. knowlesi strain 1 or P. knowlesi strain 2; The Babesia parasites include: Babesia crassa (B.crassa); Babesia crassa-like (B.crassa-like); Babesia divergens (B.divergens); Babesia duncani (B.duncani); Babesia gibsoni (B.gibsoni); Babesia microti (B.microti); Babesia odocoilei (B.odocoilei); Babesia B.sp; Babesia venatorum (B.venatorum); Babesia vogeli (B.vogeli).

4. The use according to claim 3, characterized in that The probe-based qPCR reaction system detects Plasmodium falciparum, Plasmodium vivax and Plasmodium ovale; The dye-based qPCR reaction system detects Plasmodium and Babesia parasites; preferably, after qPCR amplification, the parasite species can be identified based on an amplification curve, a melting curve or sequencing.

5. The use according to claim 1, characterized in that The detection reagent is highly non-specific for human DNA.

6. A kit for detecting parasites, characterized in that: The kit includes: a container or a package, and a multi-species facultative detection reagent contained in the container or the package; wherein the multi-species facultative detection reagent includes: Universal primers: SEQ ID NO: 1-3; Probe: SEQ ID NO: 4 to SEQ ID NO: 11; The parasites include Plasmodium parasites and Babesia parasites.

7. The kit according to claim 6, characterized in that The multi-species facultative detection reagents form a double set, including a probe method reaction set and a dye method reaction set; The probe method reaction kit includes: universal primers SEQ ID NO: 1 to 3 and probes SEQ ID NO: 4 to SEQ ID NO: 11; The dye method reaction kit includes: universal primers SEQ ID NOs: 1-2.

8. The kit according to claim 6, characterized in that The end of the probe carries a fluorescent group and a corresponding quenching group; preferably, the fluorescent group / quenching group includes: HEX / BHQ1, CY5.5 / BHQ3, CY5 / BHQ3, FAM / BHQ1, Texas RED / BHQ2; or In the probe method reaction kit, the amount of primers used is: SEQ ID NO:1:SEQ ID NO:2:SEQ ID NO:3 is (8-12):(8-12):(3-5); preferably (9-11):(9-11):(3.5-4.5); for example 10:10:4; or In the probe method reaction kit, the amount of the probe: SEQ ID NO:4:SEQ ID NO:5:SEQ ID NO:6:SEQ ID NO:7:SEQ ID NO:8:SEQ ID NO:9:SEQ ID NO:10:SEQ ID NO:11 is (3-5):(3-5):(3-5):(2.5-3.5):(2.5-3.5):(3-5):(0.8-1.2):(0.8-1.2); preferably (3.5-4.5):(3.5-4.5):(3.5-4.5):(2.8-3.1):(2.8-3.2):(3.5-4.5):(0.9-1.1):(0.9-1.1); for example 4:4:4:3:3:4:1:1; or In the dye method reaction kit, the primer dosage is: SEQ ID NO:1:SEQ ID NO:2 is (0.7-1.3):(0.7-1.3); preferably (0.8-1.2):(0.8-1.2); more preferably (0.9-1.1):(0.9-1.1); for example 1:

1.

9. A method for specifically detecting parasites, wherein the parasites include Plasmodium parasites and Babesia parasites; the method comprises detecting using a multi-species facultative detection reagent, wherein the multi-species facultative detection reagent comprises: Universal primers SEQ ID NOs: 1 to 3, probes SEQ ID NOs: 4 to 11; During detection, the multi-species facultative detection reagent forms a detection system, including a probe method qPCR reaction system and / or a dye method qPCR reaction system; the probe method qPCR reaction system includes: universal primers SEQ ID NO: 1 to 3 and probes SEQ ID NO: 4 to SEQ ID NO: 11; the dye method qPCR reaction system includes: universal primers SEQ ID NO: 1 to 2.

10. The method according to claim 9, characterized in that The detection system includes a probe method qPCR reaction system and a dye method qPCR reaction system; wherein the detection system is used to detect (a) Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium knowlesi, Plasmodium brasiliensis, Babesia scoparia, Babesia duncanii, Babesia microti or Babesia veseri at the genus and species level, and (b) Plasmodium howler monkey, Plasmodium cynomolgus, Babesia divergentis, Babesia ornithischia, Babesia ornithischia, Babesia scoparia, Babesia gibsonii or Babesia weseri at the genus level; preferably, after the genus level detection in (b), the insect species can also be subdivided and identified by sequencing.

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