Primer-mediated probe composition, kit and use for quadruple detection of fecal intestinal parasites

Abstract

The application specifically relates to a primer-mediated probe composition, a kit and application for quadruple detection of fecal intestinal parasites. The composition comprises a primer set, a mediated probe and a molecular beacon, and the primer set comprises an upstream primer and a downstream primer. The application provides an efficient solution for multiple detection of intestinal parasitic diseases, and multiple detection of four parasites is completed in a single tube and a single fluorescence channel, effectively filling the gap of high-throughput and low-cost multiple detection technology of intestinal parasites in the clinic, and has significant clinical transformation potential and popularization value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to primer-vector probe compositions, kits, and applications for quadruple detection of fecal intestinal parasites. Background Technology

[0002] Intestinal parasitic diseases are infectious diseases caused by parasites residing in the human intestine. Their transmission is closely related to the natural environment, sanitation conditions, dietary habits, and lifestyle. They are widespread in tropical, subtropical, and temperate regions worldwide, especially in developing countries and areas with scarce sanitation resources. Southern my country (such as Guangxi Zhuang Autonomous Region and Guangdong Province) is a high-prevalence area for key parasites such as Clonorchis sinensis, Clonorchis chinensis, Hookworm, and Strongyloides stercoralis. These diseases not only directly damage the physical health of infected individuals, leading to malnutrition, developmental delays, hepatobiliary complications, and even fatal disseminated infections, but also reduce labor productivity, increase the burden on healthcare, and pose a serious threat to global public health security, population quality improvement, and socio-economic development. Among them, Clonorchis sinensis infection has been classified as a Group 1 human carcinogen by the International Agency for Research on Cancer; Strongyloides stercoralis has a mortality rate as high as 87.1% in immunocompromised populations; and Clonorchis chinensis and Hookworm are prominent public health problems due to their high infection rates and iron deficiency anemia-related harms, respectively. The key to the prevention and control of intestinal parasitic diseases lies in early and accurate diagnosis. Although current clinical treatments can effectively control infection, their effect on reversing chronic damage and complications is limited. Therefore, developing efficient diagnostic technologies has become a core aspect of disease prevention and control.

[0003] Current diagnostic techniques for intestinal parasites have many limitations and fail to meet clinical and public health needs. Etiological examination, as the preferred clinical diagnostic method, relies heavily on microscopic examination (including direct smears and saturated saline flotation). While simple and inexpensive, it has extremely low sensitivity, easily missing low-load infections and intermittently ovulating parasites. Furthermore, it is highly dependent on the expertise of laboratory personnel, making it difficult to differentiate morphologically similar parasites and mixed infections. Immunological methods (such as enzyme-linked immunosorbent assays and immunochromatography) have achieved standardization and speed to some extent, but they cannot distinguish between current and past infections. Antigen detection sensitivity is closely related to the degree of infectivity, and most methods only detect single parasites, making it difficult to meet the clinical needs for differentiating mixed infections.

[0004] The development of molecular biology techniques has provided new pathways for parasite detection. While conventional PCR and real-time quantitative PCR (QPCR) have improved sensitivity and specificity, their throughput per test is limited. Traditional multiplex QPCR is limited by the number of fluorescence channels in the instrument; each additional target requires a new fluorescence channel, leading to an exponential increase in the complexity of fluorophore selection, spectral correction, and system optimization. Furthermore, the synthesis of multicolor fluorescent reporter probes is costly. Digital PCR (ddPCR), while capable of absolute quantification, is expensive, complex, and has low throughput, making it unsuitable as a routine screening method. Next-generation sequencing (NGS / mNGS) technology can detect multiple pathogens unbiasedly, but its procedures are cumbersome, time-consuming, and extremely costly, and data analysis requires a high level of expertise, limiting its application to the auxiliary diagnosis of difficult cases.

[0005] In summary, achieving high-throughput, high-sensitivity, and low-cost simultaneous detection of various intestinal parasites, especially for nucleic acid detection of low-abundance parasites in fecal samples under complex matrix interference, has become a key direction for breaking through the bottleneck of intestinal parasite diagnostic technology. Summary of the Invention

[0006] In view of this, the present invention provides a primer-vector probe composition, kit, and application for quadruple detection of fecal intestinal parasites to solve the above-mentioned technical problems.

[0007] This application provides a primer-vector probe composition for detecting fecal intestinal parasites, comprising a primer set, a vector probe, and a molecular beacon, wherein the primer set includes an upstream primer and a downstream primer, and the upstream primer, the downstream primer, and the vector probe are selected from at least one of the following combinations I to IV: Combination I: The nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the medium probe is shown in SEQ ID NO.9; Combination II: The nucleotide sequence of the upstream primer is shown in SEQ ID NO.3, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4, and the nucleotide sequence of the medium probe is shown in SEQ ID NO.10; Combination III: The nucleotide sequence of the upstream primer is shown in SEQ ID NO.5, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.6, and the nucleotide sequence of the medium probe is shown in SEQ ID NO.11; Combination IV: The nucleotide sequence of the upstream primer is shown in SEQ ID NO.7, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8, and the nucleotide sequence of the medium probe is shown in SEQ ID NO.12; The nucleotide sequence of the molecular beacon is shown in SEQ ID NO.13; The medium probe is chemically modified with NH2C7; The molecular beacon is modified with a fluorescent reporter group at one end and a second fluorescent quencher group at the other end.

[0008] Optionally, combination I is used to detect Clonorchis sinensis (Clonorchis sinensis). Clonorchis sinensis Combination II is used to detect human budding protozoa (…). Blastocystis hominis Combination III is used to detect American hookworm ( Necator Americanus The combined IV is used to detect Strongyloides stercoralis (…). Strongyloides stercoralis ).

[0009] Optionally, the primer set can specifically bind to conserved gene regions of fecal intestinal parasites.

[0010] This application also provides the use of the primer-vector probe composition described above in the preparation of reagents for detecting fecal intestinal parasites.

[0011] This application also provides a kit for detecting fecal intestinal parasites, comprising polymerase, buffer solution, magnesium ions, and a primer-mediator-probe composition as described above.

[0012] Optionally, the molar ratio of the upstream primer to the downstream primer is 450-550:450-550.

[0013] Optionally, the molar ratio of the upstream primer to magnesium ions is 450-550 nM: 1 μM-3 μM.

[0014] Optionally, the molar ratio of the upstream primer to the medium probe is 450-500:350-450.

[0015] Optionally, the molar ratio of the upstream primer to the molecular beacon is 450-500:150-250.

[0016] Optionally, the concentration of the upstream primer is 450-550 nM.

[0017] Optionally, the buffer includes polymerase chain reaction buffer (i.e., PCR buffer).

[0018] The beneficial effects of this invention are: This application successfully achieved simultaneous and accurate detection of Clonorchis sinensis, Clonorchis sinensis, Hookworm, and Strongyloides stercoralis by constructing and optimizing a multiplex detection system for intestinal parasites based on the Single-Tube MeltingCurve Array Method (SMCA). This system, through the innovative combination of target-specific primers, vector probes, reporter probes, and melting curve analysis, overcomes the limitations of traditional detection techniques and exhibits the following advantages: (1) High sensitivity and excellent specificity: Specific primers and vector probes designed based on four parasite-specific conserved genes (clonorchis sinensis COX1 gene, human budding protozoan SSU rRNA gene, hookworm 18S rRNA gene and strongyloides stercoralis 18S rRNA gene) were verified for homology by NCBI Primer-BLAST and confirmed by experiments to have no cross-reactivity. Methodological validation showed that the detection limit of the quadruple system for Clonorchis sinensis was 10 copies / μL, and that of the single system was as low as 5 copies / μL, significantly superior to the detection capability of traditional microscopic examination for low viral load infections. Validation with 139 clinical samples showed that the overall concordance rate between this method and microscopic examination was 95.0%, with a Kappa value of 0.87 (P<0.001), and the positive concordance rate for Strongyloides stercoralis was 100%. The system showed no cross-reactivity with six common enteropathogenic pathogens, including Escherichia coli, Vibrio parahaemolyticus, Salmonella, Giardia lamblia, adenovirus, and Enterovirus 71, demonstrating excellent detection specificity.

[0019] (2) High-throughput detection and ease of operation: It breaks through the technical bottleneck of traditional multiplex qPCR being limited by the number of fluorescence channels in the instrument. It can realize the simultaneous detection of four intestinal parasites in a single tube with a single fluorescence channel. Compared with traditional single-multiplex PCR, which detects one by one, the detection efficiency is greatly improved and the detection cycle of batch samples is significantly shortened. The operation process is simplified. The amplification and melting curve interpretation are completed automatically by the instrument. No professional bioinformatics analysis is required. The requirements for the parasite morphology identification experience of the testing personnel are low. It is suitable for promotion and application in the laboratory departments of hospitals at all levels and primary medical institutions.

[0020] (3) Rapid detection and outstanding clinical applicability: Using the system of this application, the entire process from nucleic acid extraction from fecal samples to result interpretation can be completed within 5 hours, and the detection cycle is significantly shorter than the 24-72 hours of NGS technology; the closed tube operation design can effectively avoid cross-contamination of amplification products and reduce the risk of false positives; at the same time, it successfully detected one case of low viral load infection of Clonorchis sinensis that was missed by microscopic examination, which was verified as true positive by Sanger sequencing, fully highlighting the diagnostic advantages of this method for occult and low viral load infections.

[0021] (4) Significant cost advantages and public health value: The detection cost of the system proposed in this application is significantly lower than that of high-throughput molecular detection technologies such as ddPCR and NGS, which has the cost advantage of large-scale population screening. For areas with high incidence of parasites in southern my country, such as Guangxi Zhuang Autonomous Region, the system proposed in this application can effectively improve the detection efficiency of low-load intestinal parasite infections and reduce chronic complications caused by missed diagnoses (such as Clonorchis sinensis-related cholangiocarcinoma and hookworm disease with iron deficiency anemia). It provides a new technical tool for the clinical precision diagnosis, mixed infection identification and epidemiological monitoring of intestinal parasitic diseases in my country, which has important public health significance.

[0022] In summary, this application provides an efficient solution for the multiplex detection of intestinal parasitic diseases, enabling the detection of four parasites within a single tube and single fluorescence channel. This effectively fills the gap in clinical high-throughput, low-cost multiplex detection technology for intestinal parasites, and has significant clinical translational potential and promotional value. Attached Figure Description

[0023] Figure 1 A schematic diagram illustrating the principle of SMCA technology for detecting intestinal parasites; Figure 2 The figure shows the results of validating the effectiveness and specificity of primers for four parasites. Figure 3 Melting peak results for feasibility verification of the SMCA detection system; Figure 4 Figure showing the results of optimizing the optimal annealing temperature for the SMCA single-component system; Figure 5 The figure shows the results of optimizing the optimal primer concentration for the SMCA single-layer system. Figure 6 The figure shows the results of optimizing the ratio of the mediator probe to the molecular beacon in the SMCA single-layer system. Figure 7 Figure showing the results of optimizing the optimal magnesium ion concentration for the SMCA single-weight system; Figure 8 Figure showing the results of synergistic optimization of molecular beacons and magnesium ion concentration in the SMCA quadruple system; Figure 9 The results of sensitivity verification of the SMCA single-component system for detecting four parasites are shown in the figure. Figure 10 The results of the sensitivity verification of the SMCA quadruple system for detecting four parasites are shown in the figure. Figure 11 The figure shows the results of specific cross-reactivity validation between the SMCA quadruple system and non-target pathogens. Figure 12 The figure shows a comparison of the results of SMCA method and microscopic examination of clinical samples of four parasites. Figure 13This is a diagram showing the Sanger sequencing verification results for samples where the SMCA method and microscopic examination results are inconsistent. Detailed Implementation

[0024] The present invention will be further illustrated by specific examples below. However, it should be noted that the specific material ratios, process conditions and results described in the embodiments of the present invention are only for illustrating the present invention and cannot be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

[0025] This application constructs a multiplex detection system for intestinal parasites based on SMCA technology. Specifically, it includes primers, vector probes (MPs), and universal molecular beacons (MBs) reporter probes designed for four parasite-specific conserved genes. The primers target the coding regions of the conserved parasite genes. The vector probes are designed with a two-segment structure: the 5' end is a fragment complementary to the molecular beacon, and the 3' end is a sequence completely complementary to the target gene, with the end chemically modified with NH2C7. The 5' end of the molecular beacon is labeled with the fluorescent reporter group ROX, and the 3' end is labeled with the quencher group BHQ2. The detection system utilizes the specific melting temperatures (T0) corresponding to different targets. m (Value) to achieve multiple distinctions.

[0026] like Figure 1 As shown, the detection principle of SMCA technology is to design the medium probe corresponding to each target as two segments: a 5'-flap is attached to the 5' end of the medium probe, which is not complementary to the target DNA but is perfectly complementary to the molecular beacon; the 5'-flap is then attached to the 3' end of the medium probe, which is chemically modified with NH2C7 to prevent product elongation. When the PCR reaction begins, the medium probe binds to the target gene, Taq The 5'FEN activity of DNA polymerase catalyzes the cleavage of 5'-flap DNA in a forked double-stranded DNA substrate, producing a 5'-flap DNA fragment. This free 5'-flap fragment becomes the mediator primer for hybridization with the molecular beacon reporter gene. Simultaneously, a fluorescent group is labeled at the 5' end of the molecular beacon reporter gene, and a quenching group is labeled at its 3' end. Different mediator probes are designed for different targets, each with a different 5'-flap. Taq DNA polymerase cleaves each mediator probe into different mediator primers that can bind to the same molecular beacon reporter gene. Multiple mediator primers can hybridize and extend at different positions on a single molecular beacon, producing a series of fluorescent hybrid double strands of different lengths with varying melting temperatures. Specific lengths of fluorescent hybrid double strands correspond to different parasite detection targets. Melting curve analysis, specifically the difference in Tm values, identifies different targets in the sample.

[0027] The present invention will be described in detail below through specific examples and embodiments. It should also be understood that the following embodiments are only for specific illustration of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0028] Example 1: Establishment and optimization of the SMCA technique for detecting intestinal parasites 1. Materials and Experimental Methods 1.1 Sample Source and Collection Preservation 1.1.1 Sample Source Patients hospitalized at the First Affiliated Hospital of Guangxi Medical University from June to December 2025 were selected as the study subjects, and 139 clinical stool samples were collected. All samples were residual samples after direct smear examination using optical microscopy with physiological saline. Inclusion criteria: ① The sample was intact and free from contamination by urine, body fluids, or disinfectant; ② The patient's clinical data was complete, including gender, age, inpatient department, and clinical diagnosis; ③ The microscopic examination result was clear (positive or negative). Exclusion criteria: ① Samples with incomplete clinical data or missing key information; ② Samples that were not refrigerated in time after collection and were at risk of spoilage; ③ Samples with improper microscopic examination procedures or results that could not be confirmed.

[0029] 1.1.2 Sample Collection and Preservation Samples were collected using hospital-specific stool collection containers (clean, dry, non-absorbent, and with a sealed cap). A pea-sized amount of stool (200 μL for liquid samples) was collected and placed into a 1.5 mL sterile, enzyme-free centrifuge tube labeled with a unique identification number. The tube was immediately placed in a -80°C ultra-low temperature freezer for later use. All samples were aliquoted and frozen within 1 hour of collection to prevent nucleic acid degradation.

[0030] 1.2 Experimental Methods 1.2.1 Total DNA extraction from fecal samples The procedure was followed according to the instructions of the Tiangen Biotech Fecal Genomic DNA Extraction Kit, and optimizations were made to address the low efficiency of nucleic acid extraction from fecal samples. The specific steps are as follows: (1) Take the sample out of the -80℃ freezer, let it thaw at room temperature for 5 minutes, weigh 180-220mg of fecal sample (200μL for liquid sample) into a 1.5mL centrifuge tube and place it on ice; (2) Add 500 μL buffer SA, 100 μL buffer SC, 15 μL proteinase K and 0.25 g grinding beads (1 mm in diameter) to a centrifuge tube, place it in a tissue homogenizer, and oscillate at 6 m / s for 30 s. Repeat this cycle for 30 s intervals to ensure that the sample is thoroughly mixed and broken up. (3) Place the centrifuge tube in a 56°C metal water bath and incubate for 15 minutes. During this period, take it out and invert it once every 5 minutes until the solution becomes turbid. (4) Vortex for 15s, centrifuge at 12000rpm for 3min, carefully transfer the supernatant to a new 1.5mL centrifuge tube, add 10μL RNaseA, vortex to mix, and incubate at room temperature for 5min to remove RNA contamination. (5) Add 200 μL of buffer SH, shake vigorously to mix, and then let stand on ice for 5 min; (6) Centrifuge at 12000 rpm for 3 min, aspirate the supernatant into a new 1.5 mL centrifuge tube, add an equal volume of buffer GFA, and gently invert to mix. (7) Slowly add the mixture into the adsorption column CR2 (already placed in the collection tube), centrifuge at 12000 rpm for 30s, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube. (8) Add 500 μL of buffer GD to the adsorption column CR2, centrifuge at 12000 rpm for 30 s, discard the waste liquid, and put the adsorption column back into the collection tube. (9) Add 700 μL of washing solution PW (anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 30 s, discard the waste liquid, and return the adsorption column to the collection tube. (10) Repeat step (9) to wash the adsorption column again; (11) Place the adsorption column CR2 back into the collection tube, centrifuge at 12000 rpm for 2 min to completely remove residual rinsing solution, and place the adsorption column at room temperature for 3 min to dry. (12) Transfer the adsorption column to a new 1.5 mL sterile enzyme-free centrifuge tube, add 30 μL of elution buffer TB to the middle of the adsorption membrane, and let it stand at room temperature for 3 min. (13) Centrifuge at 12000 rpm for 2 min and collect the eluent; add the eluent to the adsorption membrane again, place at room temperature for 2 min, centrifuge at 12000 rpm for 2 min, and elute a second time to improve the nucleic acid recovery rate; (14) The extracted DNA solution should be used immediately for experiments or stored in a -80°C refrigerator for later use.

[0031] 1.2.2 DNA quality assessment DNA purity and concentration were determined using an ultra-micro UV spectrophotometer. A purity of 1.6-2.0 OD260 / 280 ratio was considered acceptable, and a concentration of ≥5 ng / μL was considered acceptable. Unacceptable samples were re-extracted.

[0032] The results showed that after optimized extraction from 139 fecal samples, the DNA purity of 135 samples was within the range of OD260 / 280 of 1.6-2.0, with a purity qualification rate of 97.1%; the DNA concentration ranged from 8.2 to 45.6 ng / μL, with an average concentration of 23.5 ng / μL, all of which met the requirements for subsequent PCR amplification. The 4 unqualified samples were qualified after a second extraction.

[0033] 1.2.3 Design and Synthesis of Primers and Medium Probes This application targets four intestinal parasites (clonorchis sinensis, Clo ... Clonorchis sinensis ), human buddesmus ( Blastocystis hominis American hookworm ( Necator americanus ) and sterile strongyloides ( Strongyloides stercoralis Four highly prevalent intestinal parasites were identified, and a single-tube, single-fluorescent-channel SMCA detection system was constructed to accurately distinguish specific conserved genes. This system included specific primers, vector probes, and a universal molecular beacon reporter system. Their nucleotide sequences are shown in Tables 1 and 2. All primers were synthesized by a biotechnology company. The specific steps are as follows: 1.2.3.1 Target gene selection By searching the NCBI GeneBank database for specific conserved genes of four parasites, the following genes were identified as target genes: Clonorchis sinensis COX1 gene (GeneBank ID: FJ965388.1), Bacillus spp. SSU rRNA gene (GeneBank ID: AY244621.1), Hookworm 18S rRNA gene (GeneBank ID: AF217891.1), and Strongyloides stercoralis 18S rRNA gene (GeneBank ID: AF279916). These genes are highly conserved in their respective parasites and have no homology with the human genome or genes of common gut microorganisms, ensuring the specificity of the detection.

[0034] 1.2.3.2 Primer Design and Synthesis Specific primers were designed using the NCBI Primer-BLAST tool and Primer Premier 5 software. The design principles were: ① Primer length 18-25bp, Tm value 55-65℃; ② Avoid primers forming hairpin structures or dimers; ③ The primers amplify fragments of appropriate length (100-500bp), which facilitates subsequent electrophoresis verification; ④ The primer sequences are completely complementary to the target gene and show no significant homology with non-target genes. Primer sequences are detailed in Table 1. They were synthesized by a biotechnology company, purified by PAGE, and prepared as a 10 μM stock solution, stored at -20°C.

[0035] 1.2.3.3 Design and Synthesis of Mediated Probes (MP) and Molecular Beacons (MB) (1) Vector probe design: A two-segment structure is adopted, with the 5' end being a universal fragment (10-15bp) complementary to the molecular beacon, and the 3' end being a specific fragment (18-22bp) completely complementary to the target gene. The 3' end is chemically modified with NH2C7 to avoid the vector probe itself from extending. (2) Molecular beacon design: The 5' end is labeled with the fluorescent reporter group ROX, the 3' end is labeled with the quencher group BHQ2, the stem length is 6-8 bp, and the loop sequence is complementary to the universal fragment at the 5' end of the medium probe; (3) Sequence verification: The specificity of the vector probe sequence was verified using the NCBI BLAST tool to ensure that it only binds to the corresponding target gene. The melting temperature (T) of the four vector probes was also verified. m The interval between values ​​is >5℃, which facilitates the differentiation of melting curves; (4) Synthesis and purification: The mediator probe and molecular beacon were synthesized by the biotechnology company, purified by HPLC, and prepared as a 10 μM stock solution. They were stored at -20℃ in the dark. The sequences are shown in Table 2.

[0036] Table 1. Nucleotide sequence of primers Note: F indicates the upstream primer, and R indicates the downstream primer.

[0037] Table 2. Nucleotide sequence listing of the medium probes and molecular beacons Note: MB stands for molecular beacon.

[0038] 1.2.4 Validation of primer effectiveness and specificity To verify the effectiveness and specificity of the detection system of this application, effectiveness and specificity verification experiments were conducted, and the specific steps are as follows: 1.2.4.1 Real-time fluorescent PCR amplification Using DNA from four parasite-positive microscopic samples as templates and sterile, enzyme-free water as a negative control, singlet real-time fluorescent PCR amplification was performed using the SYBR Green I dye method to verify primer amplification efficiency and specificity. The reaction system is shown in Table 3, and the reaction procedure is shown in Table 4. A negative control group was also set up. The difference between the negative control group and the experimental group was that the negative control group system did not contain the four parasite-positive template DNA, but instead added an equal amount of sterile, enzyme-free water to make up the volume. The fluorescence intensity test results are shown in Table 4. Figure 2 As shown in Figure A.

[0039] Table 3 Primer Validation System Table 4 Primer Validation Reaction Conditions Depend on Figure 2 As shown in A, all four primer pairs exhibited a single "S"-shaped amplification curve with no obvious delay period, and the negative control showed no amplification curve, indicating that the primer amplification efficiency was good.

[0040] 1.2.4.2 Agarose gel electrophoresis Take 10 μL of the PCR amplification product and perform 4% agarose gel electrophoresis, as follows: (1) Prepare 0.5×TBE working solution: Dilute 10×TBE buffer with sterile enzyme-free water 20 times; (2) Weigh 3g of agarose powder, add 75mL of 0.5×TBE working solution, heat in microwave oven until boiling, shake intermittently until the agarose powder is completely dissolved, and cool to about 60℃. (3) Add 10 μL of 4S GelRed nucleic acid dye, mix gently, pour into a gel mold, insert a 10-well comb, and let it solidify at room temperature for 20 min; (4) Place the gel in the electrophoresis tank and add 0.5×TBE working solution until the liquid level is 1-2 mm above the gel surface; (5) Mix 10 μL of PCR amplification product with 2 μL of 6× Loading buffer and slowly add it to the gel wells. Add 6 μL of 20bp DNA Ladder to the first well and add blank amplification product (sterile enzyme-free water to replace the template) to the last well as a control. (6) Electrophoresis at a constant voltage of 200V for 30 minutes. After electrophoresis, place the gel in a UV gel imaging system to observe whether a single specific band appears. The results are as follows: Figure 2 As shown in B.

[0041] like Figure 2 As shown in B, the amplified product showed a single, clear band at the expected position, without any extraneous bands or primer dimers. The band sizes were 115 bp for Clonorchis sinensis, 119 bp for Bacillus thuringiensis, 477 bp for Hookworm, and 244 bp for Strongyloides stercoralis.

[0042] 1.2.4.3 Sanger sequencing validation PCR products that passed electrophoresis verification were sent to the Biotechnology Department for bidirectional Sanger sequencing. Sequencing results were compared with target gene sequences from NCBI GeneBank using SnapGene 8.0 software. A match greater than 99.5% was considered good primer specificity. Sanger sequencing results are as follows: Figure 2 As shown in C.

[0043] like Figure 2 As shown in Figure C, the sequencing peaks are clear with no overlapping peaks, and the matching degree between the sequencing sequence and the target gene sequence in GeneBank is >99.5%, confirming that the primers have good specificity.

[0044] 1.2.5 Construction of positive plasmids for four parasites (1) Synthesize the corresponding target gene fragments shown in Table 5.

[0045] Table 5. Target gene sequences of four parasites (2) Plasmid transformation and culture: The glycerol strain containing the above 4 parasite target sequences of PUC57 recombinant plasmid was taken out from the -80℃ freezer, thawed at room temperature, and a small amount of glycerol strain was dipped into the inoculation loop in the ultra-clean workbench and evenly spread on LB plates containing 100μg / mL Ampicillin resistance. The plates were incubated in an inverted incubator at 37℃ overnight. (3) Plasmid extraction: Pick a single colony on an LB plate and inoculate it into 5 mL of LB medium containing 100 μg / ml Ampicillin resistance. Incubate at 37℃ and 200 rpm for 15 h. Extract plasmid DNA using a plasmid mini-prep kit (Tiangen Biotech, catalog number DP103-03). Follow the instructions in the kit manual for operation. (4) Plasmid identification: The concentration and purity of the plasmid (OD260 / 280=1.8-2.0) were detected by ultra-micro UV spectrophotometer, and the integrity of the plasmid was verified by 1% agarose gel electrophoresis. Qualified plasmids were stored in a -80℃ refrigerator for later use.

[0046] 1.2.6 Establishment of the SMCA System 1.2.6.1 Basic Formulation of Single SMCA System The total reaction volume of this detection system is 25 μL. The composition of the basic reaction system is detailed in Table 6 (i.e., F+R+P+MB), and the basic amplification and melting reaction procedures are detailed in Table 7. The following groups were set up (if the volume of any group is insufficient, "sterile enzyme-free water" will be added to make up the volume): F+R group: Only the forward primer and the reverse primer are added; F+R+P group: only includes forward primer + reverse primer + medium probe; F+R+P+MB group: only includes forward primer + reverse primer + medium probe + molecular beacon; Clonorchis sinensis ( Clonorchis sinensis Group 1: Only the following groups were added: forward primer + reverse primer + vector probe + molecular beacon + Clonorchis sinensis plasmid template DNA. human budsomnium ( Blastocystis hominis Group 1: Only human cysticercosis single-layer system forward primer + reverse primer + vector probe + molecular beacon + human cysticercosis plasmid template DNA were added; American hookworm ( Necator americanus Group ); only the American hookworm single-layer system forward primer + reverse primer + vector probe + molecular beacon + American hookworm plasmid template DNA were added; Strongyloides stercoralis ( Strongyloides stercoralis Group 1: Only the single-layer system containing *Strongyloides fragilis* forward primer + reverse primer + vector probe + molecular beacon + *Blastocystis humanis* plasmid template DNA was added; The amplification was performed using the basic reaction system and procedure shown in Tables 6 and 7, and melting curve analysis was conducted to obtain the melting curves. The results are as follows: Figure 3 As shown.

[0047] Table 6 Composition of SMCA Single-Basis Reaction System Table 7 Basic Amplification and Melting Reaction Procedure Depend on Figure 3 It can be seen that all four parasites exhibited specific melting peaks without interference from other peaks, and the melting temperature (T) was [missing information]. m The values ​​were 49.85℃ (Clonorchis sinensis), 58.97℃ (Blastocystis humanis), 65.85℃ (Hookworm), and 71.22℃ (Strongyloides stercoralis), with an interpeak interval of >5℃, which can be clearly distinguished, confirming the feasibility of the SMCA system design.

[0048] 1.2.6.2 Single-factor variable optimization Using a single-factor variable method, the annealing temperature, primer concentration, ratio of medium probe to molecular beacon, and magnesium ion concentration were optimized sequentially. + The optimal parameters for each singleton system were determined by evaluating the concentration, the melting peak signal (the rate of change of fluorescence signal relative to temperature (-dF / dT)), and specificity (no impurity peaks). Specifically, under the premise of ensuring that the melting curve is a single sharp main peak (no impurity peaks), the result with the highest peak height was selected as the optimal condition.

[0049] Annealing temperature optimization: Six temperature gradients were set: 55℃, 56℃, 58℃, 60℃, 63℃, and 64℃. Other conditions were set according to the basic formula. Melting curves were plotted using the method described above. The results are as follows. Figure 4 As shown.

[0050] like Figure 4 As shown, 55℃ is the general optimal temperature for the four targets. At this temperature, the melting peak signal intensity is the highest, and there is no non-specific amplification.

[0051] Primer concentration optimization: Six final concentration gradients were set: 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, and 600 nM. Other conditions were the optimized annealing temperature. Melting curves were plotted according to the above method, and the results are as follows. Figure 5 As shown.

[0052] like Figure 5 As shown, the final concentration of primers for Clonorchis sinensis, Clonorchis chinensis, and Strongyloides stercoralis was 500 nM, and the final concentration of primers for Hookworm was 400 nM.

[0053] Optimization of the ratio of medium probe to molecular beacon: With the final concentration of the molecular beacon fixed at 100 nM, four gradients were set for the final concentration of the medium probe: 50 nM, 100 nM, 200 nM, and 400 nM. Other conditions remained optimized. Melting curves were plotted using the method described above, and the results are as follows: Figure 6 As shown.

[0054] like Figure 6 As shown, the optimal final concentration of the medium probe is 400 nM, and the optimal final concentration of the molecular beacon is 100 nM. At this ratio, the melting peak signal is the strongest.

[0055] Mg² + Concentration optimization: Eight concentration gradients were set: 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, and 5 mM. Other conditions were set to the optimized parameters. Melting curves were plotted using the method described above. The results are as follows. Figure 7 As shown, 7A is the melting curve corresponding to Clonorchis sinensis, 7B is the melting curve corresponding to Cynocystis hominis, 7C is the melting curve corresponding to Hookworm, and 7D is the melting curve corresponding to Strongyloides stercoralis.

[0056] like Figure 7 As shown, the optimal Mg² for Clonorchis sinensis, Clonorchis sinensis, and Hookworm is... + The optimal Mg² concentration for *Strongyloides stercoralis* is 2.0-2.5 mM. + The concentration was 5.0 mM.

[0057] The optimized amplification reaction system is shown in Table 8.

[0058] 1.2.6.3 Integration of the Four-Tier SMCA System The optimal parameters of the singlet system were integrated, and the molecular beacon concentrations (100 nM, 200 nM, 300 nM, 400 nM) and Mg² were further optimized. + Concentrations (1.5-5 mM) were used, and melting curves were plotted according to the above method. The optimal reaction conditions for the four parasites were determined based on the clear distinction of melting peaks, balanced signal intensity, and absence of cross-interference. The optimal reaction conditions are shown in Table 9, and the melting curves under these conditions are as follows: Figure 8 As shown, 8A represents MB:100nM,,Mg 2+ 2.0mM, 8B is MB:100nM, Mg 2+ 2.5mM 8C is MB: 200 nM, Mg 2+ :1.5mM, 8D is MB:200nM, Mg 2+ 2.0mM 8E is MB: 300nM, Mg 2+ 2.0mM, 8F is MB: 300nM, Mg 2+ 2.5mM 8G is MB: 400nM, Mg 2+ :2.0mM., 8H is MB:400nM, Mg 2+ 2.5mM.

[0059] Table 8 Optimized Amplification Reaction System Table 9 Optimized amplification and melting reaction procedures like Figure 8 As shown, under these conditions, the melting peaks of the four parasites are clear and independent, without cross-over or superposition, and the signal intensity is balanced, allowing for synchronous differentiation within a single fluorescence channel.

[0060] Example 2 Performance Verification of SMCA Detection System 1. Materials and Experimental Methods 1. Materials and Experimental Methods 1.1 Sample Source and Collection Preservation Same as Example 1 (DNA extracts from 139 clinical fecal samples and four parasite positive plasmid standards).

[0061] 1.2 Experimental Methods 1.2.1 Sensitivity Verification Plasmid serial dilution: Four parasite-positive plasmids were serially diluted to 10⁻⁶. 6copies / μL, 10 5 copies / μL, 10 4 Eight concentrations were set up: copies / μL, 10³ copies / μL, 10² copies / μL, 10 copies / μL, 5 copies / μL, and 1 copy / μL. Three replicate wells were set up for each concentration. (1) Sensitivity detection of single-system: System: Example 1 - Optimized single SMCA system; Procedure: Using serially diluted plasmids as templates and sterile, enzyme-free water as a negative control, amplification was performed according to the optimized reaction program, with the following groups set up: Group A: Plasmid concentration is 10 6 copies / μL; Group b: Plasmid concentration is 10 5 copies / μL; Group C: Plasmid concentration is 10 4 copies / μL; Group d: Plasmid concentration is 10 3 copies / μL; Group e: Plasmid concentration is 10 2 copies / μL; Group f: Plasmid concentration is 10 copies / μL; Group g: The concentration of plasmid is 5 copies / μL; Group h: The concentration of plasmid is 1 copie / μL; The lowest concentration at which a specific melting peak can be stably observed is defined as the limit of detection (LoD). The results are as follows: Figure 9 As shown, 9A is the melting curve corresponding to Clonorchis sinensis, 9B is the melting curve corresponding to Cynocystis hominis, 9C is the melting curve corresponding to Hookworm, and 9D is the melting curve corresponding to Strongyloides stercoralis.

[0062] like Figure 9 As shown, the detection limit for Clonorchis sinensis and Clonorchis chinensis is 5 copies / μL; the detection limit for Hookworm is 10³ copies / μL; and the detection limit for Strongyloides stercoralis is 10² copies / μL.

[0063] (2) Sensitivity detection of the quadruple system: System: Example 1 - Optimized quadruple SMCA system; Procedure: Gradient plasmids of the four parasites were mixed in equal proportions. Using the mixture as a template, the remaining procedures were the same as for the singlet system. The detection limits of each target in the quadruple system were determined. Results are as follows: Figure 10As shown, 10A is the melting curve corresponding to Clonorchis sinensis, 10B is the melting curve corresponding to Cynocystis hominis, 10C is the melting curve corresponding to Hookworm, and 10D is the melting curve corresponding to Strongyloides stercoralis.

[0064] like Figure 10 As shown, the sensitivity decreased slightly due to the competitive inhibition effect of multiplex PCR: 10 copies / μL for Clonorchis sinensis, 10³ copies / μL for Clonorchis sinensis, and 10 copies / μL for Hookworm. 4 copies / μL, Strongyloides stercoralis: 10 3 copies / μL.

[0065] comprehensive Figure 9 and Figure 10 It can be seen that at all detection limit concentrations, the melting peak signal is clear and there are no non-specific impurities, which meets the clinical needs for stool sample testing.

[0066] 1.2.2 Specificity Validation Specificity validation is divided into in-system specificity (no cross-reactivity among the four parasites) and out-of-system specificity (no cross-reactivity with non-target pathogens), as detailed below: (1) Specificity verification within the quadruple system Template: Four parasite positive plasmid standards prepared in Example 1 (all plasmids are commercially available products synthesized by a biotechnology company) (10 of each) 5 (copies / μL), sterile enzyme-free water (negative control); System: The optimized quadruple SMCA system of Example 1 (final molecular beacon concentration 200 nM, Mg²⁺) + Final concentration 2.0 mM); Set up the following groups: Clonorchis sinensis ( Clonorchis sinensis Group 1: The optimized quadruple SMCA system of Example 1, with only Clonorchis sinensis positive template DNA added; human budsomnium ( Blastocystis hominis Group 1: The optimized quadruple SMCA system of Example 1, with only human blastocystis positive template DNA added; American hookworm ( Necator americanus Group 1; Example 1: The optimized quadruple SMCA system, with only hookworm positive template DNA added; Strongyloides stercoralis ( Strongyloides stercoralis Group 1: The optimized quadruple SMCA system of Example 1, with only positive template DNA from Strongyloides stercoralis added; Procedure: After adding the template, amplification was performed according to the optimized reaction program. The melting curves were analyzed to verify whether the melting peaks of the four targets in the mixed template were clearly distinguishable and without overlap. The results are as follows: Figure 11As shown.

[0067] like Figure 11 As shown, after amplification with individual and mixed templates, the melting peaks of the four parasites were clear and independent, with an interpeak interval of >5℃ and no cross-over. The signal intensity of each peak in the mixed template was balanced.

[0068] (2) Quadruple in vitro specificity verification: Template: Genomic DNA extracted from 6 positive clinical samples of non-target pathogens (clinical positive specimens) (concentration 50 ng / μL), sterile enzyme-free water (negative control), and a mixed sample of four parasite plasmids obtained in Example 1 (positive control; specifically, the positive control consists of a concentration of 10...). 5 (This mixture consists of equal volumes of four parasite plasmid standards, measured in copies / μL). System: Specificity validation within the same quadruple system; Set up the following groups: E.Coli Group: The optimized quadruple SMCA system of Example 1 was amplified by adding E. coli DNA as a template; VP group: The optimized quadruple SMCA system of Example 1 was amplified by adding Vibrio parahaemolyticus DNA as a template; G. lambia group: The optimized quadruple SMCA system of Example 1 was amplified by adding Giardia lamblia DNA as a template; ADV group: The optimized quadruple SMCA system of Example 1 was amplified by adding adenovirus cDNA as a template; EV71 group: The optimized quadruple SMCA system of Example 1 was amplified by adding enterovirus 71 DNA as a template; Procedure: After amplification, analyze the melting curve to assess whether non-target pathogens produce specific melting peaks. Results are as follows: Figure 11 As shown.

[0069] like Figure 11 As shown, no specific melting peaks were observed in the six non-target pathogens (Escherichia coli, Vibrio parahaemolyticus, Salmonella, Giardia lamblia, adenovirus, and Enterovirus 71) and the negative control. The melting peak of the positive control (mixed plasmid) was consistent with the expectation, confirming that the system has 100% specificity and no cross-reactivity.

[0070] Example 3: Clinical efficacy validation of the SMCA detection system 1. Materials and Experimental Methods 1. Materials and Experimental Methods 1.1 Sample Source and Collection Preservation Same as Example 1 (DNA extracts and four parasite-positive plasmid standards from 139 clinical fecal samples). Among them, 109 samples were confirmed positive by microscopic examination (ME) (54 cases of Clonorchis sinensis, 21 cases of Clonorchis sinensis, 24 cases of Hookworm, and 10 cases of Strongyloides stercoralis), and 30 samples were negative. The clinical information of the samples was complete, and there was no cross-infection.

[0071] 1.2 Experimental Methods 1.2.1 DNA extraction and quality evaluation of clinical stool samples The procedure was performed in accordance with the instructions of the commercially available fecal genomic DNA extraction kit, and the extraction process was optimized based on the characteristics of the sample. The specific steps were strictly performed in accordance with Example 1.

[0072] The extracted DNA was evaluated using an ultra-micro UV spectrophotometer, and only samples with DNA purity OD260 / 280 in the range of 1.6 to 2.0 and a concentration ≥5 ng / μL were included in subsequent testing.

[0073] 1.2.2 SMCA method for detection Reaction system: The final quadruple SMCA reaction system optimized according to Example 1, with a total reaction volume of 25. μL, the core optimization parameters are a final concentration of 200 nM for the universal molecular beacon reporter probe and Mg². + The final concentration was 2.0 mM, and the concentrations of the remaining components were strictly in accordance with the optimized final system.

[0074] Reaction procedure: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 20 s, 55℃ annealing extension for 60 s, for a total of 50 cycles; cooling at 35℃ for 30 min; followed by melting curve analysis: 95℃ for 60 s, 40℃ for 2 min, and then uniformly increasing the temperature from 40℃ to 95℃, continuously collecting the fluorescence signal of the ROX channel 15 times for every 1℃ increase.

[0075] Result determination: The result is determined based on the melting temperature (Tm) of the characteristic melting peak. The positive determination thresholds are: Clonorchis sinensis 49.85℃, Clonorchis sinensis 58.97℃, Hookworm 65.85℃, and Strongyloides stercoralis 71.22℃. A sample is considered positive if it has a single, clear, and specific melting peak corresponding to the Tm value, and negative if it has no specific melting peak or only non-specific peaks.

[0076] 1.2.3 Gold Standard Comparison Test (Microscopic Examination, ME) The direct smear method using physiological saline was employed for detection: 1-2 drops of physiological saline were placed on a clean glass slide, and direct smears were prepared from different parts of the feces. The smears were observed in a "battlefield" pattern, first using a low-power (10×) optical microscope to examine the entire slide, then using a high-power (40×) microscope to observe at least 10 fields of view. The presence of eggs, cysts, or larvae of the corresponding parasite indicated a positive result. All microscopic examination results were interpreted by qualified senior laboratory personnel. Results are as follows: Figure 12 As shown.

[0077] 1.2.4 Verification of Inconsistent Results For samples where the SMCA and ME detection results were inconsistent, SYBR Green I dye PCR amplification was performed using specific primers for the corresponding parasite. The amplification system and reaction procedure were strictly followed according to Example 1. The amplification products were sent to a biotechnology company for bidirectional Sanger sequencing. The sequencing results were compared with the corresponding target gene reference sequence in GeneBank using the NCBI Primer-BLAST tool. A sequence match of >99.5% was considered a true positive, otherwise it was considered a false positive / false negative.

[0078] 1.2.5 Statistical Analysis A 2×2 contingency table was used to compare the detection results of the SMCA method and ME, and the overall concordance rate, sensitivity (true positive rate), and specificity (true negative rate) for each parasite were calculated. The Kappa test was used to evaluate the consistency between the two methods: Kappa ≥ 0.75 was considered excellent consistency, 0.4–0.75 was moderate consistency, and < 0.4 was poor consistency. Receiver operating characteristic (ROC) curves were plotted, and the area under the curve (AUC) was calculated to evaluate the overall diagnostic efficacy of the system. The clinical detection results for each target parasite are as follows: Figure 12 As shown in AD, the overall consistency results are as follows: Figure 12 As shown in Figures E and F, 12A compares the results of SMCA and ME methods in detecting Clonorchis sinensis; 12B compares the results of SMCA and ME methods in detecting Clonorchis sinensis; 12C compares the results of SMCA and ME methods in detecting Hookworm; 12D compares the results of SMCA and ME methods in detecting Strongyloides stercoralis; 12E shows the overall agreement between SMCA and ME methods; and 12F shows the receiver operating characteristic (ROC) curves for the SMCA method in detecting four parasites. Inconsistent sample validation results are shown below. Figure 13 As shown, 13A is the Sanger sequencing peak diagram of the Clonorchis sinensis-specific primer amplification product, 13B is the melting peak curve of the inconsistent sample, and 13C is the NCBI BLAST comparison screenshot of the sequencing results.

[0079] like Figure 12As shown, regarding overall consistency, among 139 clinical stool samples, microscopic examination detected 109 positive samples and 30 negative samples; the overall concordance rate between the SMCA method and microscopic examination was 95.0% (132 / 139), with a Kappa value of 0.87 (P<0.001), indicating excellent consistency between the two methods. Comprehensive ROC curve analysis showed that the AUC of this quadruple SMCA system was 0.942 (95% CI: 0.89-0.97), demonstrating excellent diagnostic efficacy.

[0080] like Figure 12 As shown, for Clonorchis sinensis, 54 cases were positive by ME method and 49 cases were positive by SMCA method, with a positive concordance rate of 90.7% (49 / 54), sensitivity of 90.7%, specificity of 96.7%, and Kappa value of 0.852 (P<0.001). For Clonorchis sinensis, 21 cases were positive by ME method and 18 cases were positive by SMCA method, with a positive concordance rate of 85.7% (18 / 21), sensitivity of 85.7%, specificity of 100%, and Kappa value of 0.876 (P<0.001). For Hookworm: 24 cases were positive by ME method and 23 cases were positive by SMCA method, with a positive concordance rate of 95.8% (23 / 24), sensitivity of 95.8%, specificity of 100%, and Kappa value of 0.962 (P<0.001). Regarding Strongyloides stercoralis: 10 cases were positive by ME method, and all were detected by SMCA method, with a positive concordance rate of 100% (10 / 10), sensitivity of 100%, specificity of 100%, and Kappa value of 1.000 (P<0.001).

[0081] like Figure 13 As shown, regarding the inconsistent sample validation results, only one Clonorchis sinensis sample was positive by the SMCA method but negative by ME. This sample was negative for ME, and the SMCA method showed a T value corresponding to Clonorchis sinensis. m A distinct and specific melting peak was observed at the value; after qPCR amplification using Clonorchis sinensis-specific primers, the Sanger sequencing results showed a >99.5% match with the Clonorchis sinensis COX1 gene reference sequence, confirming a true positive result for Clonorchis sinensis and representing a low-viral infection case missed by ME testing. No samples of this type of inconsistency were found in *Blastocystis humanis*, *Hookworm*, or *Strongyloides stercoralis*.

[0082] In summary, this application successfully established a novel four-fold simultaneous detection method for four common intestinal parasites based on SMCA technology. This detection strategy is high-throughput, high-specificity, simple to operate, time-saving, and cost-effective. It can also detect cases missed by microscopic examination, providing a new tool for the precise clinical diagnosis of intestinal parasitic diseases, the identification of mixed infections, and epidemiological investigations. It has significant advantages and application value in the field of parasitic disease prevention and control.

[0083] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A primer-vector probe composition for detecting fecal intestinal parasites, characterized in that, The system comprises a primer set, a medium probe, and a molecular beacon, wherein the primer set includes an upstream primer and a downstream primer, and the upstream primer, the downstream primer, and the medium probe are selected from at least one of the following combinations I to IV: Combination I: The nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the medium probe is shown in SEQ ID NO.9; Combination II: The nucleotide sequence of the upstream primer is shown in SEQ ID NO.3, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4, and the nucleotide sequence of the medium probe is shown in SEQ ID NO.10; Combination III: The nucleotide sequence of the upstream primer is shown in SEQ ID NO.5, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.6, and the nucleotide sequence of the medium probe is shown in SEQ ID NO.11; Combination IV: The nucleotide sequence of the upstream primer is shown in SEQ ID NO.7, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8, and the nucleotide sequence of the medium probe is shown in SEQ ID NO.12; The nucleotide sequence of the molecular beacon is shown in SEQ ID NO.13; The medium probe is chemically modified with NH2C7; The molecular beacon is modified with a fluorescent reporter group at one end and a second fluorescent quencher group at the other end.

2. The primer-mediator-probe composition as described in claim 1, characterized in that, Combination I is used to detect Clonorchis sinensis, Combination II is used to detect Bacillus thuringiensis, Combination III is used to detect Hookworm, and Combination IV is used to detect Strongyloides stercoralis.

3. The primer-mediator-probe composition as described in claim 1, characterized in that, The primer set can specifically bind to the conserved gene regions of fecal intestinal parasites.

4. The use of the primer-vector probe composition according to any one of claims 1-3 in the preparation of reagents for detecting fecal intestinal parasites.

5. A kit for detecting fecal intestinal parasites, characterized in that, It includes polymerase, buffer solution, magnesium ions, and primer-mediator-probe composition as described in any one of claims 1-3.

6. The reagent kit as described in claim 5, characterized in that, The molar ratio of the upstream primer to the downstream primer is 450-550:450-550; And / or, the molar ratio of the upstream primer to magnesium ions is 450-550 nM: 1 μM-3 μM.

7. The kit according to claim 6, characterized in that, The molar ratio of the upstream primer to the medium probe is 450-500:350-450.

8. The kit as described in claim 6 or 7, characterized in that, The molar ratio of the upstream primer to the molecular beacon is 450-500:150-250.

9. The reagent kit as described in claim 6, characterized in that, The concentration of the upstream primer is 450-550 nM.

10. The kit according to claim 6, characterized in that, The buffer solution includes a polymerase chain reaction buffer.

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