Primer group, kit and method for LAMP-LFD (loop-mediated isothermal amplification-lateral flow dipstick) detection of cryptosporidium parvum

Abstract

The invention discloses a primer group, a kit and a method for LAMP-LFD (loop-mediated isothermal amplification-lateral flow dipstick) detection of cryptosporidium parvum, and belongs to the technical field of molecular biological detection.The primer group comprises an LAMP inner primer pair and / or an LAMP outer primer pair for detecting a target MEDLE-5 gene; the LAMP inner primer pair comprises a primer MEDLE-5-FIP and a primer MEDLE-5-BIP, and the nucleotide sequences of the primer MEDLE-5-FIP and the primer MEDLE-5-BIP are respectively shown as SEQ ID NO: 11-12 in a sequence table; the LAMP outer primer pair comprises a primer MEDLE-5-F3 and a primer MEDLE-5-B3, and the nucleotide sequences of the primer MEDLE-5-F3 and the primer MEDLE-5-B3 are respectively shown as SEQ ID NO: 9-10 in a sequence table. The primer group provided by the invention is designed by taking the cryptosporidium parvum MEDLE-5 gene as a target gene, can realize rapid detection of cryptosporidium parvum, and plays an important role in immediate prevention and control of cryptosporidium parvum.

Description

Technical Field

[0001] This invention relates to the field of molecular biological detection technology, specifically to a primer set, kit, and method for LAMP-LFD detection of Cryptosporidium microlucum. Background Technology

[0002] Cryptosporidium parvum is an opportunistic pathogenic protozoan belonging to the phylum Apicocyticum. It primarily parasitizes the brush border of intestinal epithelial cells, causing cryptosporidiosis, characterized mainly by watery diarrhea. This disease is an important zoonotic disease, listed by the World Health Organization as one of the six most prevalent diarrheal diseases worldwide. The pathogen is mainly transmitted via the fecal-oral route. The oocysts are highly resistant to the environment and common chlorine disinfectants, easily causing waterborne outbreaks and posing a persistent threat to public health. In immunocompetent individuals, infection often presents as a self-limiting illness. However, in infants, young children, and those with weakened or compromised immune systems (such as HIV / AIDS patients), infection can lead to severe and persistent diarrhea, even life-threatening conditions.

[0003] MEDLE-5 is a secretory protein encoded by the Cryptosporidium microsporidium-specific gene cgd6_5480, belonging to the Cryptosporidium-specific MEDLE protein family. Studies have found that MEDLE family proteins are closely related to the host cell invasion process and host specificity, and are important potential virulence and immunogenic factors. Compared with other family members, MEDLE-5 exhibits significantly different gene expression kinetics; its transcriptional level peaks 2 hours after sporophyte invasion of the host and then rapidly declines, suggesting that its main function may be concentrated in the early host cell contact and adhesion phase. Protein localization studies show that MEDLE-5 is widely distributed across the entire surface of the sporophyte and merozoite, which is significantly different from members such as MEDLE-3, which are mainly concentrated at the anterior end of the sporophyte, suggesting that its mechanism of action may not depend on the typical apical invasion organ. Furthermore, MEDLE-5 exhibits the lowest antibody cross-reactivity with other family members, demonstrating its unique antigenicity. The copy number of the MEDLE gene varies significantly among different Cryptosporidium species. For example, Cryptosporidium minimus, which has broad host adaptability, possesses six MEDLE genes, including MEDLE-5, while Cryptosporidium human, with a narrower host range, contains only one. This characteristic can serve as a reliable molecular marker for developing specific nucleic acid detection methods to identify Cryptosporidium species. Given its secretory characteristics, early expression patterns, and interspecies distribution differences, MEDLE-5 not only serves as an important target for studying the interaction mechanisms between Cryptosporidium and its host but also has the potential to become a molecular target for differentiating species or developing novel diagnostic detection methods.

[0004] Loop-mediated isothermal amplification (LAMP) is a novel isothermal nucleic acid amplification technique. This technique targets six specific regions of the target gene and uses four to six specific primers (including outer primers F3 / B3, inner primers FIP / BIP, and optional loop primers LF / LB). Under the action of Bst DNA polymerase with strand displacement activity, the reaction can be carried out at a constant temperature of 60°C to 65°C, without the need for complex thermal cycling. Its amplification efficiency is extremely high, typically achieving 10-1A19 Up to 10 10 The LAMP technique exhibits exponential amplification, resulting in a 100-fold increase in amplification rate. Therefore, LAMP technology offers significant advantages such as high sensitivity, strong specificity, rapid amplification, and the need for simple temperature control devices like water baths or metal baths, greatly reducing reliance on sophisticated instruments. Lateral Flow Dipstick (LFD) is a mature chromatographic immunoassay platform. It uses capillary action to move the sample across a membrane, where it specifically binds to pre-fixed capture probes, ultimately presenting the result as a visible band. LFD offers advantages such as intuitive and clear result interpretation, simple and rapid operation, no need for additional instruments, and ease of simultaneous detection of multiple targets, making it an ideal terminal display technology for rapid on-site detection. Currently, there are no reports on LAMP-LFD detection of the Cryptosporidium MEDLE-5 gene; therefore, it is necessary to establish a LAMP-LFD detection method for the Cryptosporidium MEDLE-5 gene to achieve rapid determination of whether a sample contains Cryptosporidium, which is of positive significance for the diagnosis, epidemiological investigation, and control of Cryptosporidium. Summary of the Invention

[0005] The purpose of this invention is to provide a primer set, kit, and method for LAMP-LFD detection of Cryptosporidium microsporum, in order to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A primer set for LAMP-LFD detection of Cryptosporidium microsporum includes an inner LAMP primer pair and an outer LAMP primer pair for detecting the target MEDLE-5 gene; the inner LAMP primer pair includes primers MEDLE-5-FIP and MEDLE-5-BIP, whose nucleotide sequences are shown in SEQ ID NO:11-12, respectively; the outer LAMP primer pair includes primers MEDLE-5-F3 and MEDLE-5-B3, whose nucleotide sequences are shown in SEQ ID NO:9-10, respectively.

[0008] Furthermore, the primers MEDLE-5-FIP and MEDLE-5-BIP are labeled with a biotin group and 6-carboxyfluorescein, respectively.

[0009] Another object of the present invention is to provide the application of the above-mentioned primer set for LAMP-LFD detection of Cryptosporidium microsporidium in the preparation of products for detecting Cryptosporidium microsporidium.

[0010] Furthermore, the product is a reagent kit or a test strip.

[0011] Another object of the present invention is to provide a kit for detecting Cryptosporidium microsporidium using LAMP-LFD, which includes the above-mentioned primer set for detecting Cryptosporidium microsporidium using LAMP-LFD.

[0012] Furthermore, the kit also includes DNA polymerase, polymerase buffer, amplification product diluent, test strips, positive control reagent, and negative control reagent.

[0013] Furthermore, the DNA polymerase is Bst II DNA polymerase; the polymerase buffer is LAMP premixed reaction solution; the amplification product dilution solution is ddH2O; and the test strip is a transverse lateral flow single nucleic acid test strip.

[0014] Furthermore, the positive control reagent is the genomic DNA of Cryptosporidium microsporum; the negative control reagent is ddH2O.

[0015] The LAMP-LFD primer set for Cryptosporidium microsporidium detection provided by this invention is designed with the Cryptosporidium MEDLE-5 gene as the target gene. This primer set enables rapid detection of Cryptosporidium microsporidium, playing an important role in the immediate control of Cryptosporidium microsporidium. This technology ensures high specificity by targeting specific targets of the parasite species, enabling rapid amplification under isothermal conditions and requiring only simple equipment. Combined with LFD test strips, the results are visualized and interpreted, making the operation simple. The entire solution is low-cost, easy to use, and suitable for rapid on-site identification and screening. Attached Figure Description

[0016] Figure 1 The image shows the results of the LAMP product fluorescence dye method; in the image, 1 is the negative control, 2 is the amplification result of the MEDLE-2 primer, 3 is the amplification result of the MEDLE-3 primer, and 4 is the amplification result of the MEDLE-5 primer.

[0017] Figure 2 The image shows the agarose gel electrophoresis results of the LAMP products; in the image, 1-4 represent the amplification results of the negative control, MEDLE-2, MEDLE-3, and MEDLE-5 primers, respectively.

[0018] Figure 3Electrophoresis results of the LAMP-LFD primer ratio optimization experiment; in the figure, according to the outer and inner primer concentration ratios (F3:FIP and B3:BIP), 1-6 are 1:1, 1:2, 1:4, 1:6, 1:8 and 1:10 respectively; M: Marker.

[0019] Figure 4 Electrophoresis results of the LAMP-LFD reaction time optimization experiment; in the figure, 1-8 represent LAMP reaction times of 15, 20, 25, 30, 35, 40, 45, and 50 min, respectively; M: Marker.

[0020] Figure 5 The figure shows the results of the LAMP-LFD reaction temperature optimization experiment; in the figure, 1-10 represent the LAMP reaction temperatures of 58, 59, 60, 61, 62, 63, 64 and 65℃ respectively; M: Marker.

[0021] Figure 6 The graph shows the results of LAMP-LFD detection under different instruments, including a PCR instrument, a water bath, and a metal bath, to test the applicability of different instruments. In the graph, A is an example of LAMP-LFD result interpretation; B is the detection result in different instruments.

[0022] Figure 7 The figure shows the results of the sensitivity detection experiment for LAMP-LFD; in the figure, 1 represents a Cryptosporidium microsporidium genome concentration of 25 ng / µL, 2 represents 2.5 ng / µL, and 3 represents 2.5 × 10⁻⁶. -1 ng / µL, 4 represents 2.5 × 10⁴ -2 ng / µL, 5 represents 2.5 × 10⁵ -3 ng / µL, 6 represents 2.5 × 10⁶ ng / µL. -4 ng / µL, 7 indicates the negative control; in the figure, A is the LAMP sensitivity result; B is the PCR sensitivity result; C is the LAMP-LFD sensitivity result.

[0023] Figure 8 The image shows the results of the specific detection experiment for LAMP-LFD. In the image, A represents the electrophoresis results of the specific detection experiment; B represents the test strip results of the specific detection experiment; 1 represents Cryptosporidium microsporum, 2 represents Neosporidium, 3 represents Giardia lamblia, 4 represents Fasciola hepatica, 5 represents Clonorchis sinensis, 6 represents Trichomonas vaginalis, and 7 represents a negative sample.

[0024] Figure 9 This is a graph showing the results of routine PCR testing of clinical samples; in the graph, 1-48: clinical samples; M: Marker; +: positive control; -: negative control.

[0025] Figure 10The image shows the results of LAMP-LFD detection for clinical samples; in the image, A represents the results of double LAMP electrophoresis for clinical samples; B represents the results of double LAMP test strips for clinical samples; 1-48: clinical samples; M: Marker; +: positive control; -: negative control. Detailed Implementation

[0026] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0027] In one embodiment of the present invention, a primer set for LAMP-LFD detection of Cryptosporidium microsporum is provided, comprising an inner LAMP primer pair (FIP / BIP) and an outer LAMP primer pair (F3 / B3) for detecting the target MEDLE-5 gene; the inner LAMP primer pair includes primers MEDLE-5-FIP and MEDLE-5-BIP, the nucleotide sequences of which are shown in SEQ ID NO:11-12, respectively; the outer LAMP primer pair includes primers MEDLE-5-F3 and MEDLE-5-B3, the nucleotide sequences of which are shown in SEQ ID NO:9-10, respectively.

[0028] Preferably, the primers MEDLE-5-FIP and MEDLE-5-BIP are labeled with a biotin group and 6-carboxyfluorescein (6-FAM), respectively; specifically, the 5' end of the FIP of the inner primer pair is modified with biotin, and the 5' end of the BIP is modified with 6-FAM.

[0029] In this embodiment of the invention, amplification is performed using the above-mentioned primer set. By modifying the 5' end of the FIP with biotin, it can also be used to specifically identify the Cryptosporidium microsporidium genome, thereby achieving the purpose of detecting Cryptosporidium microsporidium.

[0030] In another embodiment of the present invention, the application of the above-described primer set for LAMP-LFD detection of Cryptosporidium microsporidium in the preparation of products for detecting Cryptosporidium microsporidium is also provided.

[0031] Preferably, the product is a reagent kit or a test strip.

[0032] In another embodiment of the present invention, a kit for detecting Cryptosporidium microsporidium using LAMP-LFD is also provided, comprising the above-described primer set for detecting Cryptosporidium microsporidium using LAMP-LFD.

[0033] This kit enables rapid detection of Cryptosporidium microsporidium and assists in pathogenicity analysis, playing an important role in the prevention and control of Cryptosporidium microsporidium.

[0034] Preferably, the kit also includes DNA polymerase, polymerase buffer, amplification product diluent, test strips, positive control reagent and negative control reagent.

[0035] Preferably, the DNA polymerase is Bst II DNA polymerase; the polymerase buffer is LAMP premixed reaction solution (5×LAMP Reaction Mix); the amplification product dilution solution is ddH2O; the test strip is a transverse lateral flow single nucleic acid test strip; the positive control reagent is Cryptosporidium microsporidium genomic DNA; and the negative control reagent is ddH2O.

[0036] In this embodiment of the invention, Bst II DNA polymerase and 5×LAMP Reaction Mix were both purchased from Beijing TransGen Biotech Co., Ltd., with catalog number LP301-01-V2.

[0037] In addition, the lateral flow single nucleic acid detection test strip includes a nitrocellulose membrane with a test line and a control line. The test strip can specifically hybridize with biotin-labeled LAMP amplification products via primers labeled with 6-carboxyfluorescein, binding to the test line on the strip coated with biotinylate antibody. It should be noted that the source of the lateral flow single nucleic acid detection test strip is not particularly limited in this embodiment of the invention; commercially available products commonly used in the art are acceptable. For example, in this embodiment, the lateral flow single nucleic acid detection test strip was purchased from Sangon Biotech (Shanghai) Co., Ltd., product number B110202.

[0038] In another embodiment of the present invention, a LAMP-LFD detection method for Cryptosporidium microsporidium for non-disease diagnostic purposes is also provided, which includes the following steps:

[0039] S1. Extract genomic DNA from the sample to be tested;

[0040] S2. Using the genomic DNA of the sample to be tested as a template, LAMP amplification was performed using the primer set for LAMP-LFD detection of Cryptosporidium microlucidum described above, and LAMP amplification products were obtained.

[0041] S3. After diluting the LAMP amplification product, use a test strip to detect it and observe whether the DNA of the sample to be tested contains the DNA sequence of Cryptosporidium microphyllum.

[0042] Preferably, the reaction temperature for LAMP amplification is 58-65℃, more preferably 61℃; the reaction time is 15-50 min, more preferably 25 min; and the molar concentration ratio of the LAMP outer primer pair to the LAMP inner primer pair is 1:(1-10), more preferably 1:6.

[0043] The LAMP-LFD detection method for Cryptosporidium microsporidium provided in this invention has advantages such as fast detection speed, high stability, high specificity, good sensitivity, simple operation, low detection cost, and no need for professional personnel. The detection results are intuitive and easy to interpret, making it suitable for rapid diagnosis in clinical settings and large-scale epidemiological surveys.

[0044] It should be noted that when using the LAMP-LFD-based primer set to detect the sample, the amplification product is detected using a lateral flow single nucleic acid test strip. The test results are then interpreted based on the control line (C) and the test line (T). The result interpretation method is as follows:

[0045] Positive: A red band appears on the control line (C) and the test line (T); the positive result indicates that the sample contains the MEDLE-5 gene and that the sample contains Cryptosporidium microsporidium.

[0046] Negative: A red band appears on the control line (C), and no band appears on the test line (T); the negative result indicates that the sample does not contain the MEDLE-5 gene and does not contain Cryptosporidium microsporidium.

[0047] Invalid: Red bands appear on the control line (C), no bands appear on the test line (T).

[0048] In this embodiment of the invention, the above primer set was used to achieve a temperature as low as 2.5 × 10⁻⁶ within 25 minutes under a constant temperature of 61°C. -2 The test can detect the MEDLE-5 gene in samples with a concentration of ng / μL, and the results can be displayed visually using a side-flow chromatography test strip.

[0049] This invention establishes a loop-mediated isothermal amplification (LAMP) technique based on lateral flow chromatography (LFD) strips. By detecting the MEDLE-5 gene of Cryptosporidium microsporum, it enables rapid detection of the Cryptosporidium microsporum genome in test samples. Specific LAMP primers for the MEDLE-5 gene are designed and labeled with a fluorescent group at the 5' end of the primers. Finally, the LAMP detection is achieved using lateral flow chromatography strip technology. The optimized LAMP-LFD detection system can be used in a water bath, PCR instrument, or metal bath, with a constant temperature reaction at 61°C for 25 min to complete the amplification. The amplified products are visualized within 15 min using the lateral flow chromatography strip. Furthermore, the optimized LAMP-LFD detection system exhibits good specificity, detecting only the MEDLE-5 gene and showing no cross-reactivity with other pathogens. Simultaneously, this detection system demonstrates high sensitivity, with a detection limit for the MEDLE-5 gene reaching 2.5 × 10⁻⁶. -2 ng / μL. When used for actual sample testing, the detection results of the LAMP-LFD detection method established in this embodiment of the invention show a high degree of consistency with the PCR detection results, indicating that the results of this detection method are reliable and can meet the detection requirements of actual samples.

[0050] The following embodiments are implementation examples of the technical solution of the present invention in practical applications, but are not limited thereto. Unless otherwise specified, all materials and reagents involved are purchased from commercial channels; unless otherwise specified, all experimental methods used are conventional methods.

[0051] Example 1: This example describes a method for screening primer sets for the detection of Cryptosporidium microsporidium, as detailed below:

[0052] The MEDLE gene sequences of *Cryptospora microsporum* (MEDLE-2, MEDLE-3, and MEDLE-5) published in GenBank were selected, and three sets of LAMP primer pairs were designed, with the sequence information shown in Table 1. LAMP amplification was performed on the three primer pairs using Bst II DNA polymerase (catalog number: LP301-01-V2) from Beijing TransGen Biotech Co., Ltd., following the operating instructions. *Cryptospora microsporum* genomic DNA was used as the amplification template. The amplification system for each primer pair was as follows: 5×LAMP Reaction Mix 5 μL, Bst II DNA polymerase 2 μL, TS LAMP Green (20×) for quantitative real-time amplification 0.45 μL, FIP / BIP (10 μmol / L) 4 μL, B3 / F3 (10 μmol / L) 1 μL, template 2 μL, and ddH2O to a final volume of 25 μL. The reaction temperature was 60℃, with 60 cycles, and fluorescence signals were collected once per minute. The results are shown in Table 1. Figure 1 As shown.

[0053] The amplification products were detected by 1% agarose gel electrophoresis, and the results are as follows: Figure 2 As shown.

[0054] according to Figure 2 Electrophoresis results showed that the primer set MEDLE-5 exhibited bright and clear bands with the most typical band distribution, resulting in the best amplification results. Therefore, the MEDLE-5 gene was selected as the target gene for primer pair design to detect Cryptosporidium microsporidium. Table 1 shows the LAMP primer sequences to be screened for the MEDLE gene.

[0055] Table 1

[0056]

[0057] Example 2: This example provides a method for detecting Cryptosporidium microsporidium MEDLE-5 gene loop-mediated isothermal amplification (LAMP). The specific steps are as follows:

[0058] 1. Using Cryptosporidium microsporum genomic DNA as a template, a LAMP amplification system (total volume 25 μL) was prepared as follows: 5 μL 5×LAMP Reaction Mix, 2 μL Bst II DNA polymerase, 4 μL inner primer pair FIP / BIP (10 μmol / L), 1 μL outer primer pair B3 / F3 (10 μmol / L), 2 μL template, and ddH2O to a final volume of 25 μL. LAMP amplification was performed using the primer set shown in Table 2. The amplification reaction program was 60℃ for 30 min.

[0059] Table 2

[0060]

[0061] 2. Optimization of LAMP primer concentration ratio, reaction temperature, and reaction time: The LAMP primer concentration ratio, reaction temperature, and reaction time were optimized using a single controlled variable method. The reaction temperatures were set to 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, and 65℃; the reaction times were set to 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, and 45 min; and the outer and inner primer concentration ratios (F3:FIP and B3:BIP) were set to 1:1, 1:2, 1:4, 1:6, 1:8, and 1:10, respectively. The reaction products were detected by 1% agarose gel electrophoresis, and the results are as follows: Figures 3-5 As shown.

[0062] The results showed that the bands were brightest when the reaction time was 25 min, the reaction temperature was 61 ℃, and the ratio of the concentrations of the outer and inner primers (F3:FIP and B3:BIP) was 1:6.

[0063] The final reaction system and reaction procedure are as follows:

[0064] The 25 μL Cryptosporidium microsporidium LAMP reaction system was prepared as follows: 5 μL 5×LAMP Reaction Mix, 2 μL Bst II DNA polymerase, 6 μL inner primer pair FIP / BIP (10 μmol / L), 1 μL outer primer pair B3 / F3 (10 μmol / L), 2 μL template, and 2 μL ddH2O, for a total volume of 25 μL. LAMP amplification was performed using the primer sets listed in Table 2. The LAMP amplification reaction program was: 61℃ for 25 min.

[0065] Take 5 μL of LAMP amplification product, dilute it 20 times with physiological saline, mix well, and drop 80 μL of the diluted reaction product into the sample well of the transverse flow single nucleic acid detection test strip. Record the detection result of the interpretation area within 15 min.

[0066] The diagnostic reagents were subjected to isothermal reactions using a PCR instrument, a metal bath, and a water bath, respectively, to verify the applicability of different instruments. The results are as follows: Figure 6 As shown.

[0067] Figure 6 Figures A and B show that when a red band appears on both the control line (C) and the test line (T), a positive result indicates the presence of the MEDLE-5 gene in the sample, and therefore the presence of Cryptosporidium microsporidium. A negative result indicates the absence of the MEDLE-5 gene in the sample, and therefore the absence of Cryptosporidium microsporidium.

[0068] Example 3: This example provides a method for detecting the sensitivity and specificity of a LAMP-LFD kit, as detailed below:

[0069] The LAMP-LFD kit includes inner primers MEDLE-5-FIP and MEDLE-5-BIP, outer primers MEDLE-5-F3 and MEDLE-5-B3, Bst II DNA polymerase, LAMP premix (5×LAMP Reaction Mix), amplification product dilution buffer (ddH2O), lateral flow singlet nucleic acid detection strips, a positive control reagent (genomic DNA of Cryptosporidium microsporum), and a negative control reagent (ddH2O).

[0070] Dilute the Cryptosporidium microsporidium genome template to 25 ng / µL, 2.5 ng / µL, and 2.5 × 10⁻⁶ ng / µL. -1 ng / µL, 2.5×10 -2 ng / µL, 2.5×10 -3 ng / µL, 2.5×10 -4ng / µL, using the above LAMP-LFD kit, LAMP-LFD detection was performed, and the detection sensitivity was analyzed. The results are as follows: Figure 7 As shown ( Figure 7 In C, bands 1-4 are positive and show red color, while the others do not show red color. Figure 7 Figures A, B, and C show that the LAMP-LFD has a sensitivity of 2.5 × 10⁻⁶ for detecting MEDLE-5. -2 The PCR detection sensitivity for MEDLE-5 was 2.5 ng / μL. The PCR primer pairs used were MEDLE-5-F3 and MEDLE-5-B3 from Table 2.

[0071] In addition, using DNA from Neosporidium, Giardia, Fasciola hepatica, Clonorchis sinensis, Trichomonas vaginalis, and Cryptosporidium microsporum as templates, the primer specificity of the LAMP-LFD system was evaluated according to the above method under optimal system and reaction conditions. The results are as follows: Figure 8 As shown in Figures A and B, when Cryptosporidium microsporidium DNA was used as a template, a bright and clear red band appeared on the detection line T. When Neosporidium, Giardia lamblia, Fasciola hepatica, Clonorchis sinensis, Trichomonas vaginalis, and ddH2O were used as amplification templates, no red band appeared on the detection line T, while a red band appeared on the control line C. The results indicate that the primer set provided in this embodiment of the invention has good specificity for detecting Cryptosporidium microsporidium. This methodology exhibits good specificity, and no cross-reaction occurred between pathogens.

[0072] Example 4: This example is for clinical application, as detailed below:

[0073] Forty-eight cattle feces samples were collected from a cattle farm and tested for Cryptosporidium microsporidium using the industry standard PCR method and the LAMP-LFD method described above. The fecal genome was extracted.

[0074] Table 3

[0075]

[0076] Forty-eight bovine fecal genomic templates were detected by conventional PCR (including primers CF and CR, whose sequences are shown in Table 3, specifically as shown in the sequence listing SEQ ID NO:13-14) and the LAMP-LFD method (using the LAMP-LFD kit provided in Example 3), respectively. The results are as follows: Figure 9 and Figure 10 As shown.

[0077] Figure 9 The results showed that 15 out of 48 samples tested positive by conventional PCR. Figure 10The results from the A study showed that among the 48 samples tested by LAMP, 16 samples (numbered 6, 7, 8, 12, 15, 17, 18, 19, 22, 24, 28, 39, 40, 41, 44 and 46) showed characteristic gradient bands, while the rest did not show characteristic gradient bands. The 16 LAMP-positive amplification products were then analyzed by LFD. Figure 10 The results from the B test showed that all 16 samples exhibited bands at the T-line, indicating a positive result for Cryptosporidium microsporidium, consistent with the standard PCR method. The positive detection rate of the standard PCR method was 31.3%, while the positive detection rate of the LAMP-LFD method was 33.3%, both higher than the industry-recommended standard PCR method.

[0078] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. A primer set for LAMP-LFD detection of Cryptosporidium microsporum, characterized in that, It includes LAMP inner primer pairs and LAMP outer primer pairs for detecting the target MEDLE-5 gene; the LAMP inner primer pairs include primers MEDLE-5-FIP and MEDLE-5-BIP, whose nucleotide sequences are shown in SEQ ID NO:11-12, respectively; the LAMP outer primer pairs include primers MEDLE-5-F3 and MEDLE-5-B3, whose nucleotide sequences are shown in SEQ ID NO:9-10, respectively.

2. The primer set for LAMP-LFD detection of Cryptosporidium microsporum according to claim 1, characterized in that, The primers MEDLE-5-FIP and MEDLE-5-BIP are labeled with a biotin group and a 6-carboxyfluorescein group, respectively.

3. The application of a primer set for LAMP-LFD detection of Cryptosporidium as described in claim 1 or 2 in the preparation of products for detecting Cryptosporidium.

4. The application according to claim 3, characterized in that, The product is a reagent kit or test strip.

5. A LAMP-LFD detection kit for Cryptosporidium microsporidium, characterized in that, The primer set for LAMP-LFD detection of Cryptosporidium microsporidium as described in claim 1 or 2.

6. The LAMP-LFD detection kit for Cryptosporidium microsporidium according to claim 5, characterized in that, The kit also includes DNA polymerase, polymerase buffer, amplification product diluent, test strips, positive control reagent, and negative control reagent.

7. The LAMP-LFD detection kit for Cryptosporidium microsporidium according to claim 6, characterized in that, The DNA polymerase is Bst II DNA polymerase; the polymerase buffer is LAMP premixed reaction solution; the amplification product dilution solution is ddH2O; and the test strip is a transverse lateral flow single nucleic acid test strip.

8. The LAMP-LFD detection kit for Cryptosporidium microsporidium according to claim 6, characterized in that, The positive control reagent is the genomic DNA of Cryptosporidium microphyllum; the negative control reagent is ddH2O.

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