Primer combination and kit for simultaneous detection of three bloodborne parasites

Abstract

This invention provides a method for simultaneous detection of three bloodborne parasites based on RAA-LFS and its applications. First, target genes and primers for specifically amplifying the genomic DNA of Toxoplasma gondii, Babesia, and Plasmodium are screened, identifying primer and probe combinations specifically for amplifying the 18S rRNA-1 gene of Toxoplasma gondii, the 18S rRNA-2 gene of Babesia, and the 18S rRNA-3 gene of Plasmodium. Using RAA isothermal amplification technology, rapid multiplex detection is achieved in a single reaction system. Combined with lateral flow chromatography test strips for intuitive result interpretation, detection efficiency is greatly improved, and results are obtained in a short time. The RAA-LFS detection method provided by this invention has significant advantages in applications such as epidemic prevention and control or blood screening.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a primer combination and kit for the simultaneous detection of three bloodborne parasites. Background Technology

[0002] Bloodborne parasitic infections (such as Toxoplasma gondii, Babesia, and Plasmodium) are significant infectious diseases threatening global public health. Because these three parasites are characterized by latent infection, mixed infections, and overlapping clinical symptoms, establishing highly sensitive and specific rapid detection technologies is crucial for early diagnosis and precise treatment.

[0003] Current mainstream parasite detection technologies still have significant technical limitations. Microscopic morphological detection, as the traditional gold standard, suffers from insufficient sensitivity (e.g., the detection limit for malaria thin-film microscopy is only 50-200 parasites / μL), complex and time-consuming procedures, and is highly dependent on the experience of the testing personnel. Immunological detection methods (such as ELISA and immunochromatographic strips) improve ease of use, but face problems such as antibody cross-reactivity and low sensitivity during the window period (e.g., the window period for Toxoplasma gondii IgM detection is 1-2 weeks after infection), failing to meet the needs of early infection detection. Molecular biological methods (such as PCR technology) significantly improve sensitivity by detecting pathogen nucleic acids, but still have obvious application limitations: for example, they require complex temperature control equipment for thermal cycling and need to be operated by specialized technicians, which limits their widespread adoption in primary healthcare institutions.

[0004] RAA (Recombinase-mediated Isothermal Amplification) is an isothermal nucleic acid amplification technique developed in recent years. Unlike traditional PCR, RAA does not rely on complex temperature control equipment but performs nucleic acid amplification under room temperature or isothermal conditions, typically between 37°C and 42°C. Its principle is based on the formation of a complex by the binding of recombinase and primers, scanning homologous regions of the target DNA, and achieving efficient nucleic acid amplification without thermal cycling. However, current RAA technology is mainly used for the detection of single pathogens and cannot simultaneously detect multiple parasites or pathogens. This presents a limitation for scenarios such as bloodborne parasites where multiple pathogens need to be detected simultaneously.

[0005] Therefore, it is urgent to provide a method for simultaneously detecting multiple bloodborne parasites, improving detection efficiency and sensitivity, for the early diagnosis and treatment of parasites. Summary of the Invention

[0006] The purpose of this invention is to provide a primer combination and kit for the simultaneous detection of three bloodborne parasites based on RAA-LFS. It provides primer and probe combinations for specifically amplifying the genomic DNA of the target parasites, optimizes reaction conditions, and combines the results with lateral flow chromatography test strips for visualization, thereby achieving efficient and rapid detection and improving the sensitivity and accuracy of the detection results.

[0007] Existing RAA isothermal amplification technology has certain bottlenecks and limitations in the simultaneous detection of multiple parasites or pathogens. Therefore, this invention innovatively combines RAA isothermal amplification technology with lateral flow chromatography test strips to achieve the simultaneous detection of three bloodborne parasites: Toxoplasma gondii, Babesia, and Plasmodium. Multiple sets of primers and probes are designed to amplify the conserved genes of the three parasites in order to screen for the best target genes.

[0008] To achieve the above objectives, the present invention employs the following technical solution:

[0009] On one hand, this invention provides a primer and probe combination for the simultaneous detection of three bloodborne parasites based on RAA-LFS, comprising:

[0010] (1) Forward primers having sequences as shown in SEQ ID NO.3, SEQ ID NO.8, and SEQ ID NO.15;

[0011] (2) The reverse primer has the sequences shown in SEQ ID NO.4, SEQ ID NO.11, and SEQ ID NO.18;

[0012] (3) The probe has the sequence shown in SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21, with the 5' end labeled with any one of Digoxin, FAM, or TAMRA, and the 3' end modified with a C3-spacer group.

[0013] Since different primers have a certain impact on the effect and sensitivity of isothermal amplification, in some embodiments, the present invention designed several sets of primers and probes for the conserved regions of Toxoplasma gondii 18S rRNA-1 gene and 529bp, Babesia 18S rRNA-2 gene and cytb-2, and Plasmodium 18S rRNA-3 gene and cytb-3. Specifically, for the 18S rRNA-1 gene of *Toxoplasma gondii*, the forward primer has the sequence shown in SEQ ID NO.1~SEQ ID NO.3, the reverse primer has the sequence shown in SEQ ID NO.4~SEQ ID NO.6, and the probe has the sequence shown in SEQ ID NO.19; for the 18S rRNA-2 gene of *Babesia*, the forward primer has the sequence shown in SEQ ID NO.7~SEQ ID NO.9, the reverse primer has the sequence shown in SEQ ID NO.10~SEQ ID NO.12, and the probe has the sequence shown in SEQ ID NO.20; for the 18S rRNA-3 gene of *Plasmodium*, the forward primer has the sequence shown in SEQ ID NO.13~SEQ ID NO.15, the reverse primer has the sequence shown in SEQ ID NO.16~SEQ ID NO.18, and the probe has the sequence shown in SEQ ID NO.21.

[0014] In some embodiments, the forward primer designed for the 529bp Toxoplasma gondii has the sequence shown in SEQ ID NO. 22~SEQ ID NO. 24, the reverse primer has the sequence shown in SEQ ID NO. 25~SEQ ID NO. 27, and the probe has the sequence shown in SEQ ID NO. 40; the forward primer designed for cytb-2 of Babesia has the sequence shown in SEQ ID NO. 28~SEQ ID NO. 30, the reverse primer has the sequence shown in SEQ ID NO. 31~SEQ ID NO. 33, and the probe has the sequence shown in SEQ ID NO. 41; the forward primer designed for cytb-3 of Plasmodium has the sequence shown in SEQ ID NO. 34~SEQ ID NO. 36, the reverse primer has the sequence shown in SEQ ID NO. 37~SEQ ID NO. 39, and the probe has the sequence shown in SEQ ID NO. 42.

[0015] Furthermore, the primer and probe combination specifically amplifies the 18S rRNA-1 gene of Toxoplasma gondii, the 18S rRNA-2 gene of Babesia, and the 18S rRNA-3 gene of Plasmodium.

[0016] In some embodiments, this invention utilizes fluorescent RAA isothermal amplification technology to analyze and screen primer and probe combinations for conserved genes 18S rRNA-1, 18S rRNA-2, 18S rRNA-3, 529bp, cytb-2, and cytb-3 in three parasites. The results show that, for *Toxoplasma gondii*, primer pair F3R1 exhibited the highest fluorescence intensity for amplifying the 18S rRNA-1 gene, and primer pair F4R6 exhibited the highest fluorescence intensity for amplifying the 529bp gene; for *Babesia*, primer pair F2R2 exhibited the highest fluorescence intensity for amplifying the 18S rRNA-2 gene, and primer pair F4R5 exhibited the highest fluorescence intensity for amplifying the cytb-2 gene; for *Plasmodium*, primer pair F3R3 exhibited the highest fluorescence intensity for amplifying the 18S rRNA-3 gene, and primer pair F5R6 exhibited the highest fluorescence intensity for amplifying the cytb-3 gene. Therefore, these primer pairs can be used as alternative primers for further screening. Subsequent negative screening experiments revealed that primer F4R5 for amplifying the cytb-2 gene and primer F5R6 for amplifying the cytb-3 gene would produce false positive results, so they were excluded.

[0017] In some embodiments, the present invention further combines the primer pairs selected above for the 18S rRNA-1 gene and 529bp of *Toxoplasma gondii*, the 18S rRNA-2 gene of *Babesia*, and the 18S rRNA-3 gene of *Plasmodium* to simultaneously detect the DNA of the three parasites, in order to screen for the optimal gene and primer combination for achieving multiplex amplification detection. The results show that only by using a mixture of primer pair F3R1 for amplifying the *Toxoplasma gondii* 18S rRNA-1 gene, primer pair F2R2 for amplifying the *Babesia* 18S rRNA-2 gene, and primer pair F3R3 for amplifying the *Plasmodium* 18S rRNA-3 gene as the primer mixture can the specific simultaneous detection of the three blood-borne parasites be achieved.

[0018] Furthermore, the forward primer for specifically amplifying the 18S rRNA-1 gene of *Toxoplasma gondii* has the sequence shown in SEQ ID NO. 3, the reverse primer has the sequence shown in SEQ ID NO. 4, and the probe has the sequence shown in SEQ ID NO. 19; the forward primer for specifically amplifying the 18S rRNA-2 gene of *Babesia* has the sequence shown in SEQ ID NO. 8, the reverse primer has the sequence shown in SEQ ID NO. 11, and the probe has the sequence shown in SEQ ID NO. 20; the forward primer for specifically amplifying the 18S rRNA-3 gene of *Plasmodium* has the sequence shown in SEQ ID NO. 15, the reverse primer has the sequence shown in SEQ ID NO. 18, and the probe has the sequence shown in SEQ ID NO. 21.

[0019] On the other hand, the present invention provides a kit for simultaneous detection of three bloodborne parasites based on RAA-LFS, including the primer and probe combination as described above.

[0020] In some embodiments, the present invention uses a single genomic DNA, a pairwise mixture, or a mixture of all three as RAA amplification templates to verify the feasibility of the RAA-LFS detection method. The results show that when a single genomic DNA template is used for RAA amplification, one C line and one T line appear on the test strip; when two genomic DNAs are mixed as templates for amplification, one C line and two T lines appear on the test strip; and when three genomic DNAs are mixed for amplification, one C line and three T lines appear on the test strip. This indicates that the RAA-LFS method can achieve specific detection of Toxoplasma gondii, Babesia, and Plasmodium, and also has the capability for multiplex detection.

[0021] In some embodiments, the present invention also uses genomic DNA from various parasites such as Trypanosoma, Bacillus cereus, Echinococcosis, and Tapeworm as templates to verify the specificity of the RAA-LFS detection method. The results show that only Toxoplasma gondii, Babesia, and Plasmodium are positive, while the results for the other pathogens are negative, indicating that the RAA-LFS detection method has good specificity.

[0022] In some embodiments, the present invention uses 10-fold serial dilutions of Toxoplasma gondii, Babesia, and Plasmodium malariae as templates to verify the sensitivity of the RAA-LFS detection method. The results show that, compared with nested PCR, RAA-LFS can achieve a lower limit of detection, indicating that RAA-LFS has higher sensitivity.

[0023] Compared to traditional isothermal amplification techniques or fluorescent PCR methods, the RAA-LFS rapid multiplex detection method provided by this invention can simultaneously detect three bloodborne parasitic infections—malaria, toxoplasmosis, and babesiosis—in a single reaction system. The detection process is simple and efficient, requiring no complex instruments or specialized personnel. Results are interpreted intuitively and clearly, effectively solving the time-consuming and labor-intensive problems of existing technologies. This innovative technology provides an efficient and convenient solution for the rapid screening and diagnosis of bloodborne parasitic infections.

[0024] On another note, this invention provides a method for simultaneously detecting three bloodborne parasites based on RAA-LFS, using the primer and probe combinations or kits described above for amplification.

[0025] Furthermore, the method includes the following steps:

[0026] (1) Genomic DNA extraction and amplification from samples;

[0027] (2) Test strip detection.

[0028] In another aspect, the present invention provides the use of a gene combination for preparing a reagent for simultaneously detecting three bloodborne parasites, the gene combination including the 18S rRNA-1 gene of Toxoplasma gondii, the 18S rRNA-2 gene of Babesia, and the 18S rRNA-3 gene of Plasmodium malariae.

[0029] Furthermore, the 18S rRNA-1 gene has the sequence shown in SEQ ID NO.43, the 18S rRNA-2 gene has the sequence shown in SEQ ID NO.44, and the 18S rRNA-3 gene has the sequence shown in SEQ ID NO.45.

[0030] In another aspect, the present invention provides the use of the primer and probe combination described above for preparing reagents to improve the detection sensitivity of bloodborne parasites, including Toxoplasma gondii, Babesia, and Plasmodium.

[0031] Furthermore, the primer and probe combination is used to detect the 18S rRNA-1 gene of Toxoplasma gondii, the 18S rRNA-2 gene of Babesia, and the 18S rRNA-3 gene of Plasmodium falciparum. The 18S rRNA-1 gene has the sequence shown in SEQ ID NO.43, the 18S rRNA-2 gene has the sequence shown in SEQ ID NO.44, and the 18S rRNA-3 gene has the sequence shown in SEQ ID NO.45.

[0032] The present invention has the following beneficial effects:

[0033] 1. This invention provides primer and probe combinations for specifically amplifying the 18S rRNA-1 gene of Toxoplasma gondii, the 18S rRNA-2 gene of Babesia, and the 18S rRNA-3 gene of Plasmodium. Using RAA isothermal amplification technology, it enables the simultaneous detection of three bloodborne parasitic infections—malaria, toxoplasmosis, and babesiosis—in a single reaction system, improving detection accuracy and sensitivity, and making the detection process simple and efficient.

[0034] 2. This invention provides a rapid multiplex detection method based on RAA-LFS, which can quickly complete multiplex detection through a single reaction system, eliminating the need for separate detection of multiple pathogens and greatly improving detection efficiency; the detection results can be intuitively judged through a visual detection method (lateral flow chromatography test strip), which greatly simplifies the operation process and greatly improves accessibility and practicality. Attached Figure Description

[0035] Figure 1 The diagram shows the RAA primer screening results for Toxoplasma gondii, Babesia, and Plasmodium. Figures A and D show the fluorescent RAA amplification results for the Toxoplasma gondii 18S rRNA-1 and 529bp genes, respectively; Figures B and E show the fluorescent RAA amplification results for the Babesia 18S rRNA-2 and cytb-2 genes, respectively; and Figures C and F show the fluorescent RAA amplification results for the Plasmodium 18S rRNA-3 and cytb-3 genes, respectively.

[0036] Figure 2 Figure 1 shows the negative screening results for primers for Toxoplasma gondii, Babesia, and Plasmodium. Figure 2 shows the screening results for Toxoplasma gondii primers, with F3R1 being the 18S rRNA-1 primer and F4R6 being the 529bp primer. Figure 3 shows the screening results for Babesia primers, with F2R2 being the 18S rRNA-2 primer and F4R5 being the cytb-2 primer. Figure 4 shows the screening results for Plasmodium primers, with F3R3 being the 18S rRNA-3 primer and F5R6 being the cytb-3 primer. Figures NC represent the negative controls for the corresponding primers.

[0037] Figure 3 The images show the results of multiplex amplification of different target genes. In the images, number 1 is the amplification image of method 1, number 2 is the negative control of method 1, number 3 is the amplification image of method 2, and number 4 is the negative control of method 2.

[0038] Figure 4 This is a feasibility validation diagram for RAA-LFS of Toxoplasma gondii, Babesia, and Plasmodium. In this diagram, number 1 represents the genome templates of Toxoplasma gondii, Babesia, and Plasmodium; number 2 represents the genome templates of Babesia and Plasmodium; number 3 represents the genome templates of Toxoplasma gondii and Plasmodium; number 4 represents the genome templates of Toxoplasma gondii and Babesia; number 5 represents the genome template of Plasmodium; number 6 represents the genome template of Babesia; number 7 represents the genome template of Toxoplasma gondii; and number 8 represents the negative control.

[0039] Figure 5 Figure 1 shows the optimization results of single primer and probe concentrations for the RAA-LFS detection method. In Figure A, 1-4 represent Toxoplasma gondii primer concentrations of 400 nM, 300 nM, 200 nM, and 100 nM, and probe concentrations of 120 nM, 90 nM, 60 nM, and 30 nM, respectively, with N representing the negative control. In Figure B, 1-4 represent Babesia worm primer concentrations of 400 nM, 300 nM, 200 nM, and 100 nM, and probe concentrations of 120 nM, 90 nM, 60 nM, and 30 nM, respectively, with N representing the negative control. In Figure C, 1-4 represent Plasmodium malariae primer concentrations of 400 nM, 300 nM, 200 nM, and 100 nM, and probe concentrations of 120 nM, 90 nM, 60 nM, and 30 nM, respectively, with N representing the negative control.

[0040] Figure 6 The figure shows the optimization results of multiplex primer and probe concentrations for the RAA-LFS detection method. In the figure, number 1 represents Toxoplasma gondii primer and probe concentrations of 200 nM and 60 nM, Babesia aegypti primer and probe concentrations of 300 nM and 90 nM, and Plasmodium aegypti primer and probe concentrations of 300 nM and 90 nM; number 2 represents Toxoplasma gondii primer and probe concentrations of 200 nM and 60 nM, Babesia aegypti primer and probe concentrations of 300 nM and 90 nM, and Plasmodium aegypti primer and probe concentrations of 400 nM and 120 nM; number 3 represents Toxoplasma gondii primer and probe concentrations of 200 nM and 60 nM, Babesia aegypti primer and probe concentrations of 400 nM and 120 nM, and Plasmodium aegypti primer and probe concentrations of 300 nM and 90 nM; number 4 represents Toxoplasma gondii primer and probe concentrations of 200 nM and 60 nM. nM, Babesia primer and probe concentrations were 400 nM and 120 nM, respectively; Plasmodium primer and probe concentrations were 400 nM and 120 nM, respectively.

[0041] Figure 7 The figure shows the optimization results of the reaction temperature for the RAA-LFS detection method. The numbers 1-8 represent the results at reaction temperatures of 25℃, 30℃, 35℃, 37℃, 39℃, 41℃, 43℃, and 45℃, respectively.

[0042] Figure 8 The figure shows the optimized reaction time of the RAA-LFS detection method. The numbers 1-8 represent the results with reaction times of 5 min, 8 min, 10 min, 12 min, 15 min, 18 min, 20 min, and 25 min, respectively.

[0043] Figure 9The figure shows the results of the specificity analysis of the RAA-LFS detection method. In the figure, number 1 is the genome template of Toxoplasma gondii, Babesia and Plasmodium; number 2 is the genome template of Leishmania; number 3 is the genome template of Bulbocystis hominis; number 4 is the genome template of Trypanosoma japonicum; number 5 is the genome template of Echinococcus hydatids; number 6 is the genome template of Taenia solium; and number 7 is the negative control.

[0044] Figure 10 The graph shows the sensitivity analysis of the RAA-LFS detection method. In the graph, numbers 1-6 represent the mixed template concentrations of Toxoplasma gondii, Babesia, and Plasmodium genomes at 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg, respectively. Number 7 represents the negative control.

[0045] Figure 11 Figure 1 shows the sensitivity analysis of nested PCR for Toxoplasma gondii, Babesia, and Plasmodium. Figure A shows the nested PCR results for Toxoplasma gondii; Figure B shows the nested PCR results for Babesia; and Figure C shows the nested PCR results for Plasmodium. Numbers 1-6 represent the mixed template concentrations of Toxoplasma gondii, Babesia, and Plasmodium genomes at 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg, respectively. Number 7 represents the negative control. Detailed Implementation

[0046] To make the above-mentioned objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0047] Unless otherwise specified, all raw materials used in the examples are commercially available.

[0048] Example 1: Design and screening of RAA isothermal fluorescence detection primer and probe sets

[0049] I. Screening of primer and probe combinations for amplifying target genes

[0050] The sequences of 18S rRNA-1 (GenBank: L37415.1, sequence as shown in SEQ ID NO.43) and 529bp (GenBank: AF146527, sequence as shown in SEQ ID NO.46) of *Toxoplasma gondii* were retrieved from the NCBI and PlasmoDB databases; the sequences of 18S rRNA-2 (GenBank: M93660, sequence as shown in SEQ ID NO.44) and cytb-2 (GenBank: MT114078.1, sequence as shown in SEQ ID NO.47) of *Babesia microsporum* were retrieved; and conserved portions of the 18S rRNA-3 gene sequences of four *Plasmodium* species were retrieved (sequences as shown in SEQ ID NO.43). Sequence No. 45 contains conserved portions of the cytb-3 gene sequences of four Plasmodium species (as shown in SEQ ID NO. 48), including *Plasmodium falciparum* (GenBank: M19173.1), *Plasmodium vivax* (GenBank: U07367.1), *Plasmodium ovale* (GenBank: KF018659.1), and *Plasmodium malariae* (GenBank: AF487999.1); and conserved portions of the cytb-3 gene sequences of these four Plasmodium species (as shown in SEQ ID NO. 48), including *Plasmodium falciparum* (PF3D7_MIT02300), *Plasmodium vivax* (PVP01_MIT03400), *Plasmodium ovale* (PocGH01_MIT000020), and *Plasmodium malariae* (PmUG01_MIT001100). After multiple sequence alignment using SnapGene software, conserved sequences of two conserved genes from *Toxoplasma gondii*, *Babesia*, and *Plasmodium* were ultimately selected. RAA-specific primers were designed using PrimerPremier 6 software, and corresponding probes were designed based on conserved sequences, as shown in Tables 1-4.

[0051] Table 1. Primer sequences for the 18S rRNA-1 gene of Toxoplasma gondii, the 18S rRNA-2 gene of Babesia, and the 18S rRNA-3 gene of Plasmodium.

[0052]

[0053] Table 2. Probe sequences for 18S rRNA-1, 18S rRNA-2, and 18S rRNA-3.

[0054]

[0055] Table 3 Primer sequences for Toxoplasma gondii (529 bp), Babesia cytb-2, and Plasmodium cytb-3.

[0056]

[0057] Table 4. Probe sequences of 529bp, cytb-2, and cytb-3

[0058]

[0059] The 5' ends of the Toxoplasma gondii, Babesia, and Plasmodium probes were labeled with Digoxin, FAM, and TAMRA, respectively; the internal components contained a base nucleotide analog replacing the nucleotide THF; and the 3' end had a polymerase extension blocking group C3-spacer. Furthermore, the 5' ends of the downstream primers were all labeled with biotin.

[0060] All primers were synthesized at Sangon Biotech. Wild-type samples of *Plasmodium falciparum*, *Babesia microsporum*, and *Toxoplasma gondii* were preserved in our laboratory. Fluorescent RAA amplification was performed using whole-genome DNA from *Toxoplasma gondii*, *Babesia*, and *Plasmodium* as templates. Primer pairs that amplified the target gene, exhibited the strongest fluorescence value, and showed no amplification in the negative control were selected to ensure the specificity and amplification efficiency of the RAA primers. These primers were then used as candidate primers for further optimization and application in subsequent experiments. The fluorescent RAA amplification detection steps are as follows:

[0061] (1) Genomic DNA extraction: DNA was extracted from all samples according to the instructions of the DNeasy® Blood & Tissue Kit (purchased from QIAGEN, Germany). The samples were eluted with 100 μl of TE solution and stored at -20°C for later use.

[0062] (2) RAA amplification was performed using RAA nucleic acid amplification reagent (basic type, purchased from Hangzhou Zhongce Biotechnology Co., Ltd.). The reaction system included: 25 μl of A Buffer, 2 μl each of upstream and downstream primers (2 μM), 13.5 μl of ddH2O, 2.5 μl of B Buffer, 4 μl of DNA sample, and 1 μl of SYBR Green I dye, for a total volume of 50 μl. The mixture was then placed in a fluorescent PCR instrument and reacted at 39℃ for 15 min.

[0063] (3) After the reaction, the amplification of each group of primers was analyzed using a real-time quantitative PCR instrument.

[0064] like Figure 1As shown, for Toxoplasma gondii, primer pair F3R1 showed the highest fluorescence intensity among primer combinations amplifying the 18S rRNA-1 gene, and primer pair F4R6 showed the highest fluorescence intensity among primer combinations amplifying the 529bp gene. For Babesia, primer pair F2R2 showed the highest fluorescence intensity among primer combinations amplifying the 18S rRNA-2 gene, and primer pair F4R5 showed the highest fluorescence intensity among primer combinations amplifying the cytb-2 gene. For Plasmodium, primer pair F3R3 showed the highest fluorescence intensity among primer combinations amplifying the 18S rRNA-3 gene, and primer pair F5R6 showed the highest fluorescence intensity among primer combinations amplifying the cytb-3 gene. Therefore, the primer pairs selected above were used as candidate primers for each group.

[0065] II. Further screening of candidate primer and probe combinations

[0066] Subsequently, based on the primer screening results of fluorescent RAA, in order to further screen for the best primers specifically amplifying the three parasites, this embodiment used fluorescent RAA to conduct further negative screening experiments on the above candidate primers to verify their specificity and exclude non-specific amplification of non-target genes. The specific method is as follows:

[0067] (1) Genomic DNA extraction: DNA was extracted from all samples according to the instructions of the DNeasy® Blood & Tissue Kit. The samples were eluted with 100 μl of TE solution and stored at -20°C for later use.

[0068] (2) RAA amplification was performed using RAA nucleic acid amplification reagent (basic type, purchased from Hangzhou Zhongce Biotechnology Co., Ltd.). The reaction system included: 25 μl of A Buffer, 2 μl each of upstream and downstream primers (2 μM), 13.5 μl of ddH2O, 2.5 μl of B Buffer, 4 μl of DNA sample, and 1 μl of SYBR Green I dye, for a total volume of 50 μl. The mixture was then placed in a fluorescent PCR instrument and reacted at 39℃ for 15 min before observing the fluorescence intensity.

[0069] The results are as follows Figure 2As shown, for Toxoplasma gondii, the fluorescence intensity of primer pairs F3R1 and F4R6 in the negative control group was low, indicating that both primer pairs could achieve specific amplification. For Babesia, the fluorescence intensity of primer pair F3R2 amplifying the 18S rRNA-2 gene in the negative control group was low, while the fluorescence intensity of primer pair F4R5 amplifying the cytb-2 gene in the negative control group was high, indicating the possibility of false positive amplification, which should be excluded. For Plasmodium falciparum, the fluorescence intensity of primer pair F3R3 amplifying the 18S rRNA-3 gene in the negative control group was low, while the fluorescence intensity of primer pair F5R6 amplifying the cytb-3 gene in the negative control group was high, indicating the possibility of false positive amplification, which should also be excluded.

[0070] Therefore, through negative screening, the optimal primer combinations for specifically amplifying the 18S rRNA genes of each parasite were finally determined. For the 18S rRNA-1 gene of Toxoplasma gondii, the primer pairs selected were Tox-F3 and Tox-R1; for the 539bp gene of Toxoplasma gondii, the primer pairs selected were Tox-F4 and Tox-R6; for the 18S rRNA-2 gene of Babesia, the primer pairs selected were Bm-F2 and Bm-R2; and for the 18S rRNA-3 gene of Plasmodium malariae, the primer pairs selected were P-F3 and P-R3.

[0071] III. Screening for Optimal Primer and Probe Combinations for Multiplex Amplification Detection

[0072] After the above screening process, the genes that can specifically amplify Toxoplasma gondii genomic DNA include 18S rRNA-1 and 529bp. In order to further screen the optimal gene and primer combination for simultaneous detection of Toxoplasma gondii, Babesia and Plasmodium, this invention further combines the primers and uses RAA isothermal amplification combined with lateral flow chromatography strip (RAA-LFS) technology for multiplex amplification.

[0073] Genomic DNA was extracted using the same method as above, with a mixed DNA of Toxoplasma gondii, Babesia, and Plasmodium as a template. The RAA amplification reaction system is shown in Table 5, where the primer mixture was prepared in a 1:1:1 volume ratio, and the mixing methods included the following two:

[0074] Method 1: Tox-F3R1, Bm-F2R2, P-F3R3

[0075] Method 2: Tox-F4R6, Bm-F2R2, P-F3R3

[0076] The reaction was then carried out at 39℃ for 15 min. 10 μl of the reaction solution was mixed with 190 μl of PBS, and 80 μl of the mixture was added to the well of the test strip. The solution was allowed to stand at room temperature for 5-15 min before the results were determined.

[0077] Table 5. RAA isothermal amplification reaction system

[0078]

[0079] Test strip results as follows Figure 3 The results showed that the primer mixture using method one, when used for RAA amplification, produced one C line and three T lines, while the negative control only showed a C line. However, the primer mixture using method two produced a false positive band in the negative control. This indicates that the combination of 529bp with 18S rRNA-2 and 18S rRNA-3 cannot be used to simultaneously detect three parasites. For Toxoplasma gondii, the 18S rRNA-1 gene should be selected, and the primer combination of Tox-F3R1, Bm-F2R2, and P-F3R3 is necessary to simultaneously detect Toxoplasma gondii, Babesia, and Plasmodium.

[0080] Example 2: Verification of the feasibility of the RAA-LFS method

[0081] To verify that the three primer and probe combinations screened in Example 1, used for RAA isothermal amplification combined with lateral flow chromatography strips (RAA-LFS), can simultaneously identify Toxoplasma gondii, Babesia, and Plasmodium, enabling rapid multiplex detection, this invention further mixes DNA templates of Toxoplasma gondii, Babesia, and Plasmodium in different combinations, and then uses RAA-LFS for detection to verify whether the system can simultaneously amplify multiple parasites. The mixing methods for the DNA templates of the three parasites are as follows:

[0082] 1. Individual detection: DNA from Toxoplasma gondii, Babesia, and Plasmodium was used as a template, respectively;

[0083] 2. Pairwise mixing: using the mixed DNA of Toxoplasma gondii and Babesia as a template, or the mixed DNA of Babesia and Plasmodium as a template, or the mixed DNA of Toxoplasma gondii and Plasmodium as a template.

[0084] 3. Mixed DNA: Using a mixed DNA of Toxoplasma gondii, Babesia, and Plasmodium as a template.

[0085] Genomic DNA extraction was performed using the same method as in Example 1. The RAA amplification reaction system is shown in Table 6, where the primer mixture consisted of Tox-F3R1, Bm-F2R2, and P-F3R3 in a 1:1:1 volume ratio. The mixture was then incubated at 39°C for 15 min. 10 μl of the reaction solution was mixed with 190 μl of PBS, and 80 μl was added to the well of the test strip. After standing at room temperature for 5-15 min, the results were determined.

[0086] The results are as follows Figure 4As shown, a single parasite DNA template results in one C line and one T line on the test strip; a mixture of two parasite DNA templates results in one C line and two T lines on the test strip; and a mixture of three parasite DNA templates results in one C line and three T lines on the test strip. This demonstrates that the RAA-LFS method can specifically detect Toxoplasma gondii, Babesia, and Plasmodium, and also has the capability for multiplex detection.

[0087] Example 3: Optimization of RAA-LFS Method Conditions

[0088] To further optimize the conditions of the RAA amplification reaction and screen the optimal conditions for the RAA isothermal amplification combined with lateral flow chromatography test strip detection method, this invention also further screens and optimizes the primer concentration, reaction time, and reaction temperature of the RAA amplification system.

[0089] I. Optimization of primer concentration

[0090] The methods for extracting genomic DNA from Toxoplasma gondii, Babesia, and Plasmodium were the same as in Example 1. The RAA amplification system and reaction conditions were basically the same as those in Table 6 of Example 2. Specifically, primer mixtures with different concentration gradients (primer concentrations of 400 nM, 300 nM, 200 nM, and 100 nM, corresponding to probe concentrations of 120 nM, 90 nM, 60 nM, and 30 nM) were set for Toxoplasma gondii, Babesia, and Plasmodium, and optimization experiments were conducted separately. The optimal primer concentration for each parasite was selected by observing the intensity and clarity of the T line on the test strips.

[0091] like Figure 5 The results showed that for Toxoplasma gondii, the T-line of the test strip with only the combination of primer 100 nM and probe 30 nM was not clear enough; for Babesia, the intensity and clarity of the T-line of all four primer and probe combinations were good enough; and for Plasmodium malariae, the intensity and clarity of the T-line of all four primer and probe combinations were good enough.

[0092] Subsequently, based on single-parasite optimization, primers for the three parasites were mixed at different concentration ratios for multiple optimization experiments. The results are as follows: Figure 6 As shown, only the three T lines in sequence 2 had the best intensity and clarity. Therefore, the optimal primer and probe concentration combination was finally determined to be: 200 nM primer and 60 nM probe for Toxoplasma gondii; 300 nM primer and 90 nM probe for Babesia; and 400 nM primer and 120 nM probe for Plasmodium malariae.

[0093] II. Optimization of Reaction Temperature

[0094] The genomic DNA extraction methods for Toxoplasma gondii, Babesia, and Plasmodium were the same as in Example 1. The RAA amplification system was the same as in Table 6 of Example 2. Reaction temperatures were set at 25℃, 30℃, 35℃, 37℃, 39℃, 41℃, 43℃, and 45℃, with a fixed reaction time of 15 minutes for each group. The optimal reaction temperature was determined through comparison. Results are as follows: Figure 7 As shown, the stripes are clearest and the signal strength is most obvious at 39℃, therefore the optimal response temperature is 39℃.

[0095] III. Optimization of Reaction Time

[0096] The genomic DNA extraction methods for Toxoplasma gondii, Babesia, and Plasmodium were the same as in Example 1. The RAA amplification system was the same as in Table 6 of Example 2. The reaction times were set at 5 min, 8 min, 10 min, 12 min, 15 min, 18 min, 20 min, and 25 min, respectively, with the reaction temperature fixed at 39℃ for each group. The optimal reaction time was determined by comparison. The results are as follows: Figure 8 As shown, the bands are clearest and the signal intensity is most obvious when the reaction time is 18 minutes, therefore the optimal reaction time is 18 minutes.

[0097] Example 4: Specificity and sensitivity verification of the RAA-LFS method

[0098] I. Specificity Validation

[0099] To verify the specificity of the RAA-LFS multiplex detection method provided in this invention in detecting three bloodborne parasites—Toxoplasma gondii, Babesia, and Plasmodium—without detecting other parasites, this embodiment further uses whole-genome templates of Plasmodium falciparum, Babesia microsporum, Toxoplasma gondii, Trypanosoma cruzi, Clostridium humanis, Echinococcosis, and Taenia solium to perform RAA-LFS to evaluate the method's specificity. Wild-type samples of Plasmodium falciparum, Babesia microsporum, Toxoplasma gondii, and Leishmania donovani were all preserved in our laboratory. Clostridium humanis was kindly provided by the Chinese Center for Disease Control and Prevention, Trypanosoma cruzi by Hangzhou Medical College, and Echinococcosis and Taenia solium by Zhejiang University.

[0100] The genomic DNA extraction methods for various parasites were the same as in Example 1, and the RAA amplification system and reaction conditions were the same as in Example 2. The results are as follows: Figure 9 As shown, only Toxoplasma gondii, Babesia, and Plasmodium were detected positively, while the results for the remaining pathogens were negative, indicating that the method has good specificity.

[0101] II. Sensitivity Verification

[0102] To verify that the detection method provided by this invention has excellent detection effect, 100pg of Toxoplasma gondii, Babesia and Plasmodium genomic DNA were further serially diluted 10-fold and RAA-LFS detection and nested PCR amplification detection were performed respectively. The nested PCR amplification steps were as follows: (1) Reaction system: 1 μl of corresponding parasite genomic DNA, 1 μl of forward and reverse primers (10μM), 12.5 μl of 2× Taq Master Mix, and ddH2O to 25 μl; (2) Reaction conditions: 94℃, pre-denaturation for 3 min; 94℃, denaturation for 30 s; 60℃, annealing for 30 s; 72℃, extension for 30 s; 30 cycles; 72℃, final extension for 5 min; (3) 1 μl of the first round amplification product was added to the same reaction system as the first round (except that the outer primer was replaced with the inner primer) and PCR amplification was performed; the sensitivity of the RAA-LFS method was compared and evaluated.

[0103] The results are as follows Figure 10 and Figure 11 As shown, the RAA-LFS method has a detection limit of 10 fg for both Toxoplasma gondii and Plasmodium, and a detection limit of 100 fg for Babesia. In contrast, nested PCR has a detection limit of 10 fg for Toxoplasma gondii, 100 fg for Babesia, and 1 pg for Plasmodium. The RAA-LFS method achieves a lower detection limit, indicating that it has better sensitivity.

[0104] Example 5: Detection of blood samples using the RAA-LFS method

[0105] To verify the application effect of the RAA-LFS detection method provided by this invention in clinical sample testing, this embodiment simultaneously used the RAA-LFS method and a real-time fluorescence PCR detection kit to extract and detect nucleic acids from more than 3,000 blood samples collected from blood centers in southern Zhejiang. The detection results of the two molecular biology methods in blood samples were compared to evaluate their clinical specificity and sensitivity. The amplification system, reaction conditions, and detection method of RAA-LFS were the same as in Example 2, and the qPCR amplification method was the same as in Example 1. The genomic DNA was extracted from the corresponding blood samples. The detection results are shown in Table 6.

[0106] Table 6 Results of RAA-LFS and qPCR testing of blood samples

[0107]

[0108] The data in the table show that the RAA-LFS detection method established in this invention has completely consistent results with the fluorescence PCR method, indicating that the rapid multiplex RAA-LFS detection method provided by this invention also has superior specificity and sensitivity in clinical applications, and the detection process is simple and efficient, solving the problem of time-consuming and labor-intensive existing technologies.

[0109] Although the present invention has been described in detail above with general descriptions and specific embodiments, the embodiments described are exemplary. The specific features, structures, materials, or characteristics described can be combined and integrated in any suitable manner in one or more embodiments. Based on the present invention, some modifications or improvements can be made, which will be obvious to those skilled in the art. Therefore, all such modifications, improvements, substitutions, or variations made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. The use of a primer-probe combination for preparing a reagent to simultaneously detect Toxoplasma gondii, Babesia, and Plasmodium via RAA-LFS and improve detection sensitivity, characterized in that, The primer-probe combination includes forward primers, reverse primers, and probes for specifically amplifying the 18S rRNA-1 gene of *Toxoplasma gondii*, the 18S rRNA-2 gene of *Babesia*, and the 18S rRNA-3 gene of *Plasmodium*. The forward primer for specifically amplifying the 18S rRNA-1 gene of *Toxoplasma gondii* is the sequence shown in SEQ ID NO. 3, the reverse primer is the sequence shown in SEQ ID NO. 4, and the probe sequence is Dig-CTCAGGTGATTCATAGTAACCGAACGGATCG[dSpacer]GTTGACTTCGGTCTGC-C3 Spacer; the forward primer for specifically amplifying the 18S rRNA-2 gene of *Babesia* is the sequence shown in SEQ ID NO. 8, the reverse primer is the sequence shown in SEQ ID NO. 11, and the probe sequence is 6-FAM-ACATGCTCGAGGCGCGTTTTCGCGTGGCGTT[dSpacer]ATTAGACTTTAACCAA-C3 Spacer; the probe sequence for specifically amplifying the 18S rRNA-3 gene of *Plasmodium* is... The forward primer for the rRNA-3 gene is the sequence shown in SEQ ID NO.15, the reverse primer is the sequence shown in SEQ ID NO.18, and the probe sequence is TAMRA-TTAGAGTTCGATTCCGGAGAGGGAGCCTGAG[dSpacer]AATAGCTACCACATCT-C3 Spacer; the 5' end of each reverse primer is labeled with biotin; the 18S rRNA-1 gene is the sequence shown in SEQ ID NO.43, the 18S rRNA-2 gene is the sequence shown in SEQ ID NO.44, and the 18S rRNA-3 gene is the sequence shown in SEQ ID NO.

45.

2. A primer and probe combination for simultaneous detection of Toxoplasma gondii, Babesia, and Plasmodium based on RAA-LFS, characterized in that, This invention includes forward primers, reverse primers, and probes for specifically amplifying the 18S rRNA-1 gene of *Toxoplasma gondii*, the 18S rRNA-2 gene of *Babesia*, and the 18S rRNA-3 gene of *Plasmodium*. The forward primer for specifically amplifying the 18S rRNA-1 gene of *Toxoplasma gondii* is the sequence shown in SEQ ID NO. 3, the reverse primer is the sequence shown in SEQ ID NO. 4, and the probe sequence is Dig-CTCAGGTGATTCATAGTAACCGAACGGATCG[dSpacer]GTTGACTTCGGTCTGC-C3 Spacer; the forward primer for specifically amplifying the 18S rRNA-2 gene of *Babesia* is the sequence shown in SEQ ID NO. 8, the reverse primer is the sequence shown in SEQ ID NO. 11, and the probe sequence is 6-FAM-ACATGCTCGAGGCGCGTTTTCGCGTGGCGTT[dSpacer]ATTAGACTTTAACCAA-C3 Spacer; the forward primer for specifically amplifying the 18S rRNA-3 gene of *Plasmodium* is SEQ ID NO.

4. The sequence shown in SEQ ID NO.15 is used as the reverse primer, and the sequence shown in SEQ ID NO.18 is used as the probe sequence, which is TAMRA-TTAGAGTTCGATTCCGGAGAGGGAGCCTGAG[dSpacer]AATAGCTACCACATCT-C3 Spacer. The 5' ends of the reverse primers are all labeled with biotin.

3. A kit for simultaneous detection of Toxoplasma gondii, Babesia, and Plasmodium based on RAA-LFS, characterized in that, Includes the primer and probe combination as described in claim 1.

4. A method for simultaneous detection of Toxoplasma gondii, Babesia, and Plasmodium based on RAA-LFS, characterized in that, Amplification is performed using the primer and probe combination as described in claim 2, or the kit as described in claim 3, and the method is for non-disease diagnosis and treatment purposes.

5. The method as described in claim 4, characterized in that, Includes the following steps: (1) Genomic DNA extraction and amplification from samples; (2) Test strip detection.

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