A gene fragment, primer pair, kit and detection method for detection of nosema ceranae

Abstract

The present application relates to the field of biotechnology, in particular to a gene fragment, primer pair, kit and detection method for detection of Nosema ceranae. Firstly, the present application designs a specific primer pair for specific detection of Nosema ceranae based on the polar tube gene NcPTP3 of Nosema ceranae as a target gene, wherein the specific primer pair NcPTP3-13 has good specificity and accuracy for detection of Nosema ceranae. Secondly, the present application further modifies the specific primer pair NcPTP3-13 and provides a modified primer pair NcPTP3-13, which can further improve the sensitivity and visibility of the specific primer pair NcPTP3-13 for detection of Nosema ceranae by combining with a lateral flow test strip, thereby solving the defects in the prior art, detecting the infection of Nosema ceranae in Apis cerana in time, early and accurately, and achieving effective prevention and control of Nosema ceranae.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a gene fragment, primer pair, kit, and detection method for detecting microsporidia in Apiaceae. Background Technology

[0002] Honeybees are resource insects, playing a vital role not only in protecting biodiversity and maintaining ecological balance, but also in the significant economic benefits of their various bee products. Among numerous diseases, microsporidia are parasites with specialized infection mechanisms (polar tubes). After invading the midgut, they can rapidly multiply using the nutrients within the bee's body. Infected adult worker bees exhibit digestive system disorders, shortened lifespans, reduced foraging abilities, and increased susceptibility to pathogens, ultimately leading to a decline in colony numbers and even colony collapse.

[0003] Microsporidia, comprising over 1200 species, are the smallest parasitic protozoa, widely parasitizing arthropods, birds, mammals, and humans. Currently discovered honeybee microsporidia include *Apis simonii*, *Apis bumblebee*, and *Apis orientalis*. (Nosema ceranae, N.ceranae) In addition to the native Chinese honeybee, the main bee species farmed in my country is the Italian honeybee, introduced in the early 20th century. Research has found that *Apis ostreatus* microsporidia can infect both of these bee species with extremely high infectivity. Currently, *Apis ostreatus* microsporidia is troubling the beekeeping industry. This disease has diverse transmission routes and spreads rapidly; an outbreak in a bee colony can cause huge economic losses for beekeepers. There are currently no effective treatments for *Apis ostreatus* infection, making the research into efficient detection technologies for this disease essential.

[0004] There are three main methods for detecting microsporidia in Apis spp.: microscopy, immunology, and molecular biology. First, microscopy involves observing the field of view under an optical microscope for spores. This method is simple, easy to learn, and highly practical, much more efficient than visual observation. However, it requires a high level of experience from the staff, and it is prone to missed detection when the number of spores in the slide is low or in the early stages of infection. The sensitivity and accuracy of the results vary considerably. Furthermore, spore development can be divided into four stages: sporogenous stage, fission stage, sporulation stage, and mature sporulation stage. The first three developmental stages are difficult to observe under a microscope, inevitably leading to missed detections. This limits its effectiveness in effectively and promptly controlling microsporidia in Apis spp. Microscopy also requires a large number of personnel, resulting in high economic costs. Second, regarding immunological detection methods, in 2009, Liu Feng obtained polyclonal rabbit antibodies by immunizing rabbits with total protein from Apis spp. By optimizing the reaction conditions of ELISA, this method was used for the detection of Apis spp. Compared with ordinary optical microscopy, this method significantly improved sensitivity, achieving a spore detection sensitivity of 10. 3The method boasts a high concentration of microsporidia per mL and a short detection cycle, allowing for preliminary identification with the naked eye. These factors effectively shorten the time required for disease diagnosis, facilitating further prevention and control measures. However, the preparation of antiserum and the treatment of *Apis cerana* microsporidia are somewhat cumbersome before detection, and the polyclonal antibodies have low specificity, thus the accuracy of the results needs improvement.

[0005] Molecular biology mainly utilizes PCR amplification technology, which has high sensitivity, good specificity, and can be used for batch operations. However, current research on microsporidia detection mainly focuses on the detection of silkworm microsporidia, while research on Apis cerana microsporidia is very limited. In particular, a detection method with good specificity and accuracy for Apis cerana microsporidia has not yet been reported. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide a gene fragment, primer pair, reagent kit, and detection method for detecting microsporidia in Apiaceae, thereby overcoming the deficiencies in the prior art.

[0007] To achieve the above-mentioned technical effects, the present invention adopts the following technical solution: In a first aspect, the present invention provides a gene fragment for detecting microsporidia in honeybees, the gene fragment having a nucleotide sequence as shown in SEQ ID NO: 1.

[0008] Secondly, the present invention also provides a specific primer pair for detecting microsporidia in honeybees, the specific primer pair being used for specifically detecting gene fragments with nucleotide sequences as described in SEQ ID NO: 1.

[0009] Preferably, the specific primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.2 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.3.

[0010] More preferably, the fluorescent group is 6-FAM.

[0011] Thirdly, the present invention also provides a modified primer pair for detecting microsporidia in Apiaceae, the modified primer pair being used to specifically detect gene fragments with nucleotide sequences as shown in SEQ ID NO.1, and the modified primer pair comprising 5 ’ Upstream primers modified with the first chromogenic group and 5 ' Downstream primers modified with a second chromogenic group at the end.

[0012] Preferably, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3.

[0013] Preferably, the first chromogenic group and the second chromogenic group are any one of fluorescein thiocyanate (FITC), 6-FAM, or biotin, and the first chromogenic group is different from the second chromogenic group.

[0014] Fourthly, the present invention also provides a kit for detecting microsporidia in honeybees, the kit being used to specifically detect gene fragments with nucleotide sequences as described in SEQ ID NO: 1, or the kit comprising specific primer pairs provided in the second aspect above, or the kit comprising modified primer pairs provided in the third aspect above.

[0015] Fifthly, the present invention provides the application of the gene fragment and its encoded protein for detecting *Apis cerana* provided in the first aspect, the specific primer pair for detecting *Apis cerana* provided in the second aspect, the modified primer pair for detecting *Apis cerana* provided in the third aspect, or the kit provided in the fourth aspect, in the prevention and control of *Apis cerana*. Preferably, the application is in the specific detection of *Apis cerana*.

[0016] In a sixth aspect, the present invention also provides a method for detecting microsporidia in honeybees, wherein the method uses the specific primer pair provided in the second aspect above, or the modified primer pair provided in the third aspect above, or the kit provided in the fourth aspect above to detect microsporidia in honeybees.

[0017] Seventhly, the present invention also provides a method for detecting microsporidia in honeybees, the method specifically including the following steps: Obtain the total genome of the bees to be tested; A PCR reaction system was constructed and a PCR reaction was performed. The PCR reaction was used to specifically amplify a gene fragment with a nucleotide sequence as shown in SEQ ID NO: 1. The PCR reaction results are interpreted to determine whether the bees being tested are infected with Apis asiaticus microsporidia.

[0018] Preferably, the total genome of the Eastern honeybee to be tested is obtained by the following method: The midgut portion of the bee's digestive tract to be tested was selected, and the tissue was disrupted using a high-throughput tissue disruptor. The disrupted sample was used as the extraction sample, and the total genome of the bee to be tested was obtained from the extraction sample using a genome extraction kit.

[0019] Preferably, the PCR reaction system includes 12.5 μL of Premix STAR MaxDNA Polymerase, 0.2 μL of upstream primer, 0.2 μL of downstream primer, 0.5 μL of DNA template, 11.6 μL of sterilized ddH2O, and 0.5 μL of DMSO.

[0020] Preferably, the PCR reaction program is as follows: pre-denaturation at 98°C for 5 minutes; annealing at 98°C for 10 seconds, annealing at 15 seconds, annealing at 72°C for 30 seconds, for a total of 22 cycles; extension at 72°C for 10 minutes; more preferably, the annealing temperature is 58.6°C, 60.1°C, 61.9°C, or 63.4°C, with the most preferred temperature being 61.9°C.

[0021] Preferably, the interpretation is achieved by gel electrophoresis or lateral chromatography test strips; More preferably, the interpretation is achieved using lateral chromatography test strips. Specifically, the upstream primer in the PCR reaction system is modified with a Biotin group, the downstream primer is modified with a 6-FAM group, and the lateral chromatography test strip is coated with colloidal gold-labeled anti-FAM antibody and streptavidin, thereby enabling the visual interpretation of PCR results.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: First, this invention designs specific primer pairs for the specific detection of *Apis orientalis* microsporidia based on a novel gene fragment used for detecting this microsporidia. The specific primer pair NcPTP3-13 exhibits good specificity and accuracy in detecting *Apis orientalis* microsporidia. Second, this invention further modifies the aforementioned specific primer pair NcPTP3-13, providing a modified primer pair NcPTP3-13. By combining this modified primer pair NcPTP3-13 with a lateral chromatography test strip, the sensitivity and visibility of the specific primer pair NcPTP3-13 in detecting *Apis orientalis* microsporidia can be further improved. This overcomes the shortcomings of existing technologies, enabling timely, early, and accurate detection of *Apis orientalis* microsporidia infection in bees, achieving effective control of *Apis orientalis* microsporidia. Attached Figure Description

[0023] Figure 1 The PCR detection results of the specific primer pairs NcPTP3-13 and NcPTP3-14 provided in Example 1 of this invention; Figure 2 This invention provides a comparison of the detection results of modified primer pair NcPTP3-13 and modified primer pair NcPTP3-14 using the nucleic acid lateral chromatography test strip provided by this invention and nucleic acid gel electrophoresis. Figure 3The results show the specificity test results of the modified primer pair NcPTP3-13 in Example 3 of this invention; Figure 4 The results of the sensitivity test of NALFS provided in Embodiment 4 of the present invention; Figure 5 The results of the spore quantity sensitivity test of NALFS provided in Example 5 of this invention; Figure 6 This is the experimental result of NALFS for the timeliness of detecting poisoned Eastern honeybees provided in Embodiment 5 of the present invention; Figure 7 The results show the experimental accuracy of the modified primer pair NcPTP3-13 provided in Example 5 of this invention for detecting poisoned Eastern honeybees. Detailed Implementation

[0024] The embodiments of the technical solution of the present invention will now be described in detail with reference to the accompanying drawings. These embodiments are merely illustrative of the technical solution of the present invention and are therefore intended to limit the scope of protection of the present invention.

[0025] The specific embodiments listed in this invention are merely examples, and the invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of this invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of this invention should be covered within its scope. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer should be followed. All reagents or instruments without specified manufacturers are commercially available conventional products.

[0026] To better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art will understand that the present invention can be practiced without certain specific details. In other embodiments, methods, means, apparatus, and steps well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors unavoidable in industrial production.

[0028] Example 1 This embodiment uses the polar tube gene NcPTP3 of the microsporidia fasciculata in Apis orientalis (NCBI accession number XM_024473556.1) as the target gene to design multiple pairs of specific primers. Specific primer pairs (a total of 18 pairs, recorded as NcPTP3-1, NcPTP3-2, NcPTP3-3…NcPTP3-18) capable of amplifying the above target gene were designed using NCBI and Primer 5 software. The primers were synthesized by a bioengineering company, and the detection effectiveness of the above 18 primer pairs was tested, specifically including the following steps: S11: Total genome extraction of microsporidia from Apis orientalis; The midgut of the diseased honeybee was collected using tweezers, ground, and then subjected to gradient centrifugation to initially separate the spores from impurities. Further purification was achieved by Percoll density gradient centrifugation at different concentrations (100%, 75%, 50%, 25%) to obtain pure *Apis orientalis* microsporidia. Finally, 500 μL of ddH2O was added and the mixture was stored at room temperature. This *Apis orientalis* microsporidia suspension was used as the sample for extraction. Take the suspension of *Apis ostreatus* microsporidia, centrifuge at 8000 r / min for 3 min, remove the supernatant and retain the spore precipitate. Add 2 g of 300 μm glass beads to the spore precipitate and use a high-throughput tissue homogenizer to homogenize for 10 min. Extract the total genome of *Apis ostreatus* from the homogenized sample according to the steps described in the fungal genome extraction kit produced by Seville. S12: Primer screening; The total genome of the *Apis oryzae* obtained above was subjected to conventional PCR with the above 18 pairs of primers. The PCR system was as follows: 12.5 μL of Premix STAR MaxDNA Polymerase, 0.2 μL of upstream primer, 0.2 μL of downstream primer, 0.5 μL of DNA template, and 11.6 μL of sterilized ddH2O.

[0029] The PCR program was as follows: 98℃ pre-denaturation for 5 minutes; 98℃ for 10 seconds, annealing for 15 seconds, 72℃ for 30 seconds, for a total of 36 cycles; extension at 72℃ for 10 minutes, with one set of sterile ddH2O templates for each primer pair as a negative control; it should be noted that the above annealing temperatures should be adjusted according to the specific primers.

[0030] The amplified products were detected by electrophoresis on a 1.5% agarose gel. The experimental results are as follows: Figure 1 As shown; Please see Figure 1In the figure, A: modified primer pair NcPTP3-13; M: Maker; 1, 2: no template; 3, 4: template is the total genome of *Apis cerana* microsporidia; B: modified primer pair NcPTP3-14; M: Maker; 1, 2: no template; 3, 4: template is the total genome of *Apis cerana* microsporidia.

[0031] The above experimental results show that two specific primer pairs, NcPTP3-13 and NcPTP3-14, were obtained through screening. Both NcPTP3-13 and NcPTP3-14 can amplify the target band independently without the appearance of primer dimers. No primer dimers were also present when no template was available. Chromogenic groups can be added to the two primer pairs for further experiments. See Example 2 for details.

[0032] The nucleotide sequence of the upstream primer for NcPTP3-13 is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3. NcPTP3-3 was designed using the gene fragment with the nucleotide sequence shown in SEQ ID NO.1 as the target gene. The nucleotide sequence of the upstream primer of NcPTP3-14 is shown in SEQ ID NO.4, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.5.

[0033] Example 2 Based on Example 1 above, this embodiment further modifies and detects the two pairs of primers NcPTP3-13 and NcPTP3-14 initially obtained in Example 1. The specific steps are as follows: S21: Synthesis of modified primer pairs Biotin groups were added to the 5' ends of the upstream primers of NcPTP3-13 and NcPTP3-14, and 6-FAM groups were added to the 5' ends of the downstream primers of NcPTP3-13 and NcPTP3-14. The synthesis was commissioned to a bioengineering company.

[0034] S22: PCR amplification The total genome of the Oriental honeybee microsporidia obtained in Example 1 was subjected to conventional PCR with the two sets of modified primer pairs. The PCR amplification system and amplification procedure were the same as in Example 1, and the PCR amplification products were obtained.

[0035] S23: Detection of modified amplification products The PCR amplification products were detected by electrophoresis on a 1.5% agarose gel, and the results were interpreted.

[0036] In addition, this embodiment also provides another method for result interpretation, namely, using lateral chromatographic strips (NALFS) for result interpretation. The operation method and result interpretation method are as follows: The test strips used are lateral chromatography test strips produced by Hangzhou Youstar Biotechnology Co., Ltd., coated with colloidal gold-labeled anti-FAM antibody and streptavidin. These lateral chromatography test strips include a sample pad, a test line, and a control line arranged sequentially. The sample pad is used to add PCR amplification products. Because the modified primers provided in this invention modify the upstream primers of NcPTP3-13 and NcPTP3-14 with biotin groups and the downstream primers with 6-FAM groups, the PCR amplification products simultaneously carry both biotin and 6-FAM groups, thus allowing a colorimetric reaction to occur in the control and test areas of the lateral chromatography test strip. The result interpretation method for this nucleic acid lateral chromatography test strip is as follows: The result is negative when only the control line appears; the result is positive when both the control line and the test line appear simultaneously. If the quality control line is not present at any time, it indicates that the test results are unreliable.

[0037] The modified amplification products were detected using the aforementioned lateral chromatography test strip. The specific method was as follows: 8 μL of each amplification product was loaded onto the sample pad of the test strip. The test strip was then inserted into a nucleic acid buffer solution containing 80 μL. The results were observed after 10 minutes. The detection results of the agarose gel and lateral chromatography test strip are shown in the attached figure. Figure 2 As shown.

[0038] Please see Figure 2 Where, A: modified primer pair NcPTP3-13; N: no template; P: total genome of *Apis cerana* microsporidia; B: modified primer pair NcPTP3-14; N: no template; P: total genome of *Apis cerana* microsporidia.

[0039] The above experimental results show that the modified primer pair NcPTP3-13 nucleic acid electrophoresis detection results show a single band in the cDNA group, with no primer dimer bands appearing, and the negative control also shows no primer dimer bands. Only the genomic amplification product showed a positive result on the test strip. The modified primer pair NcPTP3-14 nucleic acid electrophoresis detection results show obvious primer dimer bands with and without a template. The test strip results are also affected by the primer dimer; the detection result is positive when there is no template. Therefore, the above experimental results indicate that the modified primer pair NcPTP3-13 has a good detection effect on *Microsporidium orientalis*.

[0040] Example 3 This embodiment, based on Embodiment 2 above, further tests the specificity of the modified primers obtained in Embodiment 2 for NcPTP3-13. The specific steps are as follows: S31: Setup of experimental and control groups One newly emerged Chinese honeybee and one Italian honeybee (not infected with *Apis cerana* microsporidia*) were placed in each 1.5 ml EP tube, chopped with scissors, and the total genome of the Chinese honeybee and Italian honeybee in the experimental group was extracted according to the steps described in the fungal genome extraction kit produced by Seville. Simultaneously, the total genomes of collected yeast cells (GS115), *Bombyx mori* microsporidia, and *Tetratus mori* microsporidia were extracted from each experimental group according to the steps recommended in the fungal genome extraction kit produced by Seville. In addition, the *Cystic honeybee sacbrood virus* (CSBV virus) gene was set up as an experimental group; the CSBV virus genome was obtained from Professor Dang Xiaoqun's group at the Chongqing Key Laboratory of Vector Insects. The control group consisted of the *Apis cerana* microsporidia genome obtained in Example 1.

[0041] S32: Amplification and detection of samples from experimental and control groups The samples from each of the above experimental groups and control groups were subjected to routine PCR with the modified primer pair NcPTP3-13. The PCR system and PCR operation procedure were as described in Example 1.

[0042] The amplified products were detected by electrophoresis on a 1.5% agarose gel. The experimental results are as follows: Figure 3 As shown.

[0043] Please see Figure 3 In the diagram, M represents Maker; 1 represents the genome of the Oriental honeybee microsporidia; 2 represents the genome of the Chinese honeybee; 3 represents the genome of the Italian honeybee; 4 represents the CSBV genome; 5 represents the yeast (GS115) genome; 6 represents the genome of the silkworm microsporidia; and 7 represents the genome of the tussah silkworm microsporidia.

[0044] The above gel electrophoresis results show that the modified primer pair NcPTP3-13 can only amplify samples with genomic DNA of *Apis cerana* as a template. Therefore, the modified primer pair NcPTP3-13 and the detection method provided in this embodiment have high specificity and can effectively identify *Apis cerana*.

[0045] Example 4 This embodiment tests the detection sensitivity of the modified primers for NcPTP3-13, including the following steps: S41: Sample Concentration Measurement and Dilution The total genome concentration of pure Oriental honeybee microsporidia was determined in Case 1, and the total genome was diluted with TEBuffer to 10 ng / uL, 1 ng / uL, 100 pg / uL, 10 pg / uL, 1 pg / uL, and 100 fg / uL, respectively.

[0046] Genome samples of different concentrations were subjected to conventional PCR with the modified primer pair NcPTP3-13. The PCR system was the same as in Example 1, and the amplification program was as follows: 98℃ pre-denaturation for 5 minutes; 98℃ for 10 seconds, 61.9℃ for 15 seconds, 72℃ for 30 seconds, for a total of 22 cycles; extension at 72℃ for 10 minutes; PCR amplification products were obtained.

[0047] S42: Product detection and result interpretation The amplified products were detected by electrophoresis on a 1.5% agarose gel as a control group; the experimental group was detected using lateral chromatography test strips. 8 μL of each PCR amplified product was loaded onto the sample pad of the test strip, and then the test strip was inserted into 80 μL of nucleic acid buffer. The results were observed after 10 minutes. The experimental results are as follows: Figure 4 As shown.

[0048] Please see Figure 4 The NALFS and detection method provided by this invention can successfully detect 5 pg (10 pg / uL, with 0.5 uL as template) of genomic DNA. When detected by nucleic acid gel electrophoresis, a positive signal appears when the amount is greater than 0.5 ng (1 ng / uL, with 0.5 uL as template). The detection sensitivity of NALFS is 100 times higher than that of nucleic acid gel electrophoresis.

[0049] Example 5 This embodiment tests the detection sensitivity of the modified primers for NcPTP3-13, and the steps are as follows: S51: Sample Concentration Measurement and Dilution The amount of spores prepared was 1.2 × 10⁻⁶. 4 1.2×10 3 1.2×10 2 The test samples had different spore counts; the genomes of samples with different spore counts were subjected to conventional PCR with the modified primer pair NcPTP3-13. The PCR system was as follows: Premix STAR MaxDNA Polymerase 12.5uL, upstream primer 0.2uL, downstream primer 0.2uL, DNA template 0.5uL, sterilized ddH2O 11.6uL, DMSO 0.5uL.

[0050] Amplification program: 98℃ pre-denaturation for 5 minutes; 98℃ for 10 seconds, 61.9℃ for 15 seconds, 72℃ for 30 seconds, for a total of 22 cycles; 72℃ extension for 10 minutes.

[0051] S52: Product detection and result interpretation The amplified products were detected by electrophoresis on a 1.5% agarose gel as a control group; the experimental group was detected using the lateral chromatography test strip provided in Example 2. 8 μL of each product was loaded onto the sample pad of the test strip, and then the test strip was inserted into 80 μL of nucleic acid buffer. The results were observed after 10 minutes. The experimental results are as follows: Figure 5 As shown.

[0052] Please see Figure 5 In the figure, M represents Maker; P represents a positive control using the genome of *Apis cerana* as a template; N represents a negative control using the genome of a healthy *Apis cerana*; and C represents a negative control using template-free amplification.

[0053] The above experimental results show that the NALFS provided by this invention can successfully detect 1.2 × 10⁻⁶. 4 The genomic DNA of the spores was analyzed, but no positive signal was found when detected with nucleic acid gel.

[0054] The above examples demonstrate that the NALFS detection sensitivity provided by this invention is significantly higher than that of nucleic acid gel detection.

[0055] Example 6 6.1 Healthy Italian Bees Add Poison Take 200 newly emerged Italian honeybees, treat them with CO2 to stun them, place them in a 48-well plate, and then starve them in an incubator for 2 hours. After starvation, each bee is given 10g of food. 5 The spores were placed in disposable plastic cups and the bees that had been successfully infected were then kept in an incubator.

[0056] 6.2 Sample collection and genome extraction Midgut samples were collected from bees on days 2, 4, 6, 8, and 10 after venom administration. Midgut genome extraction: 2g of 300µm glass beads were added to the sample, and the mixture was homogenized using a high-throughput tissue homogenizer for 10 min. The homogenized samples were then used to extract the total genome from each sample using the fungal genome extraction kit manufactured by Seville. Amplification of the above genome: The above-mentioned genomes were subjected to conventional PCR with the modified primer pair NcPTP3-13, and the PCR system and amplification procedure were as described in Example 5.

[0057] 6.3 Detection and Result Interpretation of Modified Amplification Products The NALFS test strip provided in Example 2 of this invention was used for detection. 8 μL of each of the above-mentioned amplification products was loaded onto the sample pad of the test strip. The test strip was then inserted into a nucleic acid buffer solution containing 80 μL. The results were observed after 10 minutes. The experimental results are as follows: Figure 6 As shown.

[0058] Please see Figure 6 In the figure, N: midgut genome of healthy Italian honeybees as a negative control; C: template-free amplification as a negative control; d: number of days of honeybee infection.

[0059] The above experimental results show that NALFS can successfully detect infection in bees as early as the second day after infection. Currently, when using other infection methods to detect infected bees, obvious microsporidia symptoms usually appear only in the later stages of infection. The detection method provided by this invention facilitates timely detection and control of infection.

[0060] Example 7 The accuracy of the above modified primers for NcPTP3-13 detection was tested using this real-time exchange rate method. The steps included: 7.1 Adding venom to healthy Italian bees Take 50 newly emerged Italian honeybees, stun them with CO2, place them in a disposable plastic cup, starve them for 2 hours, and then feed them a solution containing 10... 6 The spores were added to a sugar solution and then cultured in an incubator for 8 days. The midgut of 10 bees that had been infected with the virus and the midgut of 2 healthy bees were randomly taken and the genome was extracted according to the method in Example 6.2.

[0061] 7.2 Expansion of the total genome: The total genomes of the poisoned bees and healthy bees were subjected to conventional PCR with the modified primer pair NcPTP3-13, respectively. The PCR system and amplification procedure were as described in Example 5.

[0062] 7.3 Interpretation of Test Results The amplified products were detected by electrophoresis on a 1.5% agarose gel as a control; simultaneously, the lateral chromatography test strips provided in Example 2 were used for detection to compare the accuracy of the two methods. The experimental results are as follows: Figure 7 As shown.

[0063] Please see Figure 7 In the figure, P: positive control using the genome of *Apis cerana* as a template; N: negative control using the genome of a healthy honeybee; C: negative control without template amplification. 4, 12: midgut genome of a healthy honeybee; 1-3, 5-11: midgut genome of a diseased honeybee.

[0064] The above experimental results show that the NALFS test strip provided by this invention detected 10 diseased bees, and the nucleic acid test showed that 8 diseased bees. This indicates that the NALFS test strip provided by this invention has very high accuracy, and the accuracy rate of this test reached 100%, which is significantly higher than the nucleic acid electrophoresis test results.

[0065] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A gene fragment for detecting microsporidia in Apiaceae, characterized in that: The gene fragment is the nucleotide sequence shown in SEQ ID NO:

1.

2. A specific primer pair for detecting microsporidia in Apiaceae, characterized in that: The specific primer pair is used for the specific detection of gene fragments with nucleotide sequences as described in SEQ ID NO:

1.

3. The specific primer pair for detecting microsporidia in Apiaceae as described in claim 2, characterized in that: The specific primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.2 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.

3.

4. A modified primer pair for detecting microsporidia in Apiaceae, characterized in that: The modified primer pair is used to specifically detect gene fragments with nucleotide sequences as shown in SEQ ID NO.1, and the modified primer pair includes an upstream primer with a first labeling group modified at the 5' end and a downstream primer with a second labeling group modified at the 5' end.

5. The modified primer pair for detecting microsporidia in Apiaceae as described in claim 4, characterized in that: The nucleotide sequence of the upstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

3.

6. The modified primer pair for detecting microsporidia in Apiaceae as described in claim 4, characterized in that: The first labeling group and the second labeling group are any one of fluorescein thiocyanate (FITC), 6-FAM, or biotin, and the first labeling group is different from the second labeling group.

7. A kit for detecting microsporidia in Apiaceae, characterized in that: The kit is used for the specific detection of gene fragments with nucleotide sequences as described in SEQ ID NO: 1, or the kit has specific primer pairs as described in any one of claims 2 to 3, or the kit has modified primer pairs as described in any one of claims 4 to 6.

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