A composition, kit, and application for detecting microsporidia in ocular keratoconjunctivitis.

Abstract

This invention provides a composition, kit, and application for detecting microsporidia in ocular keratoconjunctivitis, relating to the field of biodetection technology. The composition includes an upstream primer set and a downstream primer set. The upstream primer set includes a first upstream primer and a second upstream primer. The downstream primer set includes a first downstream primer, a second downstream primer, a third downstream primer, and a fourth downstream primer. Six species of microsporidia that can infect the eye are divided into two groups. One group includes target sequences from *Microsporidia keratosa*, *Microsporidia truncatula*, *Microsporidia helenosa*, *Microsporidia enterica*, and *Microsporidia tracheobronchia*, sharing the first upstream primer. The other group includes the target sequence from *Anopheles mosquitoes*, each corresponding to a separate second upstream primer. The downstream primers are divided into four sets, enabling non-genotypic detection of the six microsporidia species. The method possesses high specificity, high sensitivity, and good anti-interference ability, making it suitable for detecting ocular microsporidia infections.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a composition, kit, and application for detecting microsporidia in ocular keratoconjunctivitis. Background Technology

[0002] Microsporidian keratitis is usually associated with trauma or environmental exposure and poses a potential zoonotic risk, particularly through contact with animals such as birds. The onset of microsporidian keratitis is insidious, with initial symptoms lacking specificity, mainly manifesting as redness of the eye, photophobia, and a foreign body sensation. Clinical signs are difficult to distinguish from viral keratitis or superficial punctate keratitis, easily leading to misdiagnosis, missed diagnosis, and prolonged disease course.

[0003] Among the reported cases, *Vittaforma corneae* accounts for the highest proportion of microsporidian keratitis, followed by *Encephalitozoon cunicululi*, *Encephalitozoon hellem*, *Anncaliia algerae*, *Encephalitozoon intestinalis*, and *Trachipleistophora hominis*.

[0004] In current nucleic acid testing, the 18S rRNA gene of microsporidia is a commonly used target. However, the conserved region of this gene shows differences in sequence homology among primers for six known pathogenic microsporidia. If a single upstream primer is used, the primer binding efficiency is low, resulting in false negatives. Ocular samples (such as conjunctival scrapings and corneal scrapings) are rich in hemoglobin, mucin, and various eye drop components, which have a concentration-dependent amplification effect on PCR inhibition. As the number of primers in the system increases, the probability of non-specific adsorption increases. Therefore, it is difficult to simultaneously cover common ocular pathogenic microsporidia.

[0005] Given the infection status of microsporidia in keratoconjunctivitis, the development of microsporidia nucleic acid detection kits is of great clinical significance for the early diagnosis of keratoconjunctivitis, the setting of highly sensitive detection indicators, and targeted treatment methods.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] One of the objectives of this invention is to provide a composition for detecting microsporidia in ocular keratoconjunctivitis, thereby solving the technical problem of the lack of substances in the prior art that cover the detection range of Microsporidia keratosa, Microsporidia truncatula, Microsporidia helenii, Microsporidia enterica, Microsporidia trachei, and Microsporidia anopheles.

[0008] A second objective of this invention is to provide the application of the above-described composition in the preparation of products for detecting or assisting in the detection of whether a sample contains microsporidia.

[0009] The third objective of this invention is to provide a reagent.

[0010] The fourth objective of this invention is to provide a reagent kit.

[0011] The fifth objective of this invention is to provide a method for detecting or / and assisting in the detection of microsporidia for purposes other than disease diagnosis.

[0012] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a composition for detecting microsporidia in ocular keratoconjunctivitis, the composition comprising an upstream primer set and a downstream primer set; The upstream primer set includes a first upstream primer that specifically binds to the target sequences of Microsporidium corneum, Microsporidium rabbit encephalitis, Microsporidium helensonii, Microsporidium enteroencephalitis, and Microsporidium tracheosporidioides, and a second upstream primer that specifically binds to the target sequence of Microsporidium anopheles. The downstream primer set includes a first downstream primer for specifically amplifying the target sequence of Microsporidium cornealis, a second downstream primer for specifically amplifying the target sequence of Microsporidium enterocytitis, Microsporidium rabbit encephalitis, or Microsporidium helenii, a third downstream primer for specifically amplifying Microsporidium human tracheobronchiolitis, and a fourth downstream primer for specifically amplifying the target sequence of Microsporidium anopheles. The target sequences of *Microsporidium cornealis* are shown in SEQ ID NO: 12; the target sequences of *Microsporidium rabbitis*, *Microsporidium helenii*, and *Microsporidium enteritidis* are shown in SEQ ID NO: 13; the target sequence of *Microsporidium trachei* is shown in SEQ ID NO: 14; and the target sequence of *Anopheles mosquitois* is shown in SEQ ID NO: 15.

[0013] Furthermore, the nucleotide sequence of the first upstream primer is shown in SEQ ID NO: 1, and the nucleotide sequence of the second upstream primer is shown in SEQ ID NO: 2; The nucleotide sequence of the first downstream primer is shown in SEQ ID NO: 3, the nucleotide sequence of the second downstream primer is shown in SEQ ID NO: 4, the nucleotide sequence of the third downstream primer is shown in SEQ ID NO: 5, and the nucleotide sequence of the fourth downstream primer is shown in SEQ ID NO: 6.

[0014] Preferably, it further includes a probe group, which includes a first probe and a second probe; The first probe is used to detect the amplification products of the target sequences of Microsporidium cornea, Microsporidium rabbit encephalitis, Microsporidium helenii, Microsporidium enteroencephalitis, or Microsporidium anopheles; the second probe is used to detect the amplification products of the target sequence of Microsporidium human tracheobronchiolitis. Preferably, the nucleotide sequence of the first probe is shown in SEQ ID NO: 7, and the nucleotide sequence of the second probe is shown in SEQ ID NO: 8.

[0015] Furthermore, it also includes an internal standard primer set, which includes an upstream primer with a sequence as shown in SEQ ID NO.9 and a downstream primer with a sequence as shown in SEQ ID NO.10; Preferably, it also includes an internal standard probe for detecting the amplification products of the internal standard primer set; Preferably, the sequence of the internal standard probe is shown in SEQ ID NO.11.

[0016] Furthermore, the first probe, the second probe, and the internal standard probe are each independently connected to a fluorescent reporter group; Preferably, the fluorescent reporter group includes any one of FAM, VIC, HEX, ROX, or CY5; Preferably, the first probe, the second probe, and the internal standard probe are each independently connected to a quenching group; Preferably, the quenching group includes any one of BHQ1, BHQ2, TAMRA, or DABCYL; Preferably, the 5′ end of the first probe, the second probe, and the internal standard probe is connected to a fluorescent reporter group, and the 3′ end is connected to a quencher group.

[0017] Secondly, the present invention provides the application of the above-described composition in the preparation of products for detecting or assisting in the detection of whether a sample contains microsporidia. Preferably, the microsporidia include at least one of Microsporidia keratosa, Microsporidia rabbitis, Microsporidia helenii, Microsporidia enterica, Microsporidia trachei, or Microsporidia anopheles. Preferably, the sample is from the eye.

[0018] Preferably, the sample includes vitreous scrapings and / or conjunctival scrapings.

[0019] Thirdly, the present invention provides a reagent comprising the above-described composition.

[0020] Fourthly, the present invention provides a kit comprising the above-described composition or the above-described reagent.

[0021] Furthermore, it also includes reagents used for PCR amplification; Preferably, the reagents used for PCR amplification include UDG enzyme, Taq enzyme, and Mg. 2+ At least one of the following: ions, PCR buffer, dNTPs, or dUTPs; Preferably, the kit also includes negative and / or positive controls; Preferably, the positive control is a plasmid containing a target, the target including at least one of the following: Microsporidium corneal striatum target sequence, Microsporidium rabbit encephalitis target sequence, Microsporidium helenii encephalitis target sequence, Microsporidium enteroencephalitis target sequence, Microsporidium human tracheobronchiolitis target sequence, or Microsporidium anopheles target sequence. Preferably, the anion control includes purified water; Preferably, the kit also includes nucleic acid extraction reagents and / or nucleic acid purification reagents.

[0022] Fifthly, the present invention provides a method for detecting or / and assisting in the detection of microsporidia for non-disease diagnosis purposes, comprising using DNA of the sample to be tested as a template, applying the above-mentioned composition or the above-mentioned reagent or the above-mentioned kit to perform PCR amplification, and determining whether the sample to be tested contains microsporidia based on the Ct value of the amplification. If the primers for detecting microsporidia show amplification and the Ct value is <37, and the internal standard primers show amplification and the Ct value is <35, then the sample is positive for microsporidia; if the primers for detecting microsporidia do not show amplification, but the internal standard primers show amplification and the Ct value is <35, then the sample is negative for microsporidia.

[0023] Furthermore, the sample to be tested is from the eye; Preferably, the microsporidia include at least one of Microsporidia keratosa, Microsporidia rabbitis, Microsporidia helenii, Microsporidia enterica, Microsporidia trachei, or Microsporidia anopheles.

[0024] This invention provides a composition for detecting microsporidia in ocular keratoconjunctivitis. Six species of microsporidia that can infect the eye are divided into two groups. One group includes target sequences from *Microsporidia keratosa*, *Microsporidia truncatula*, *Microsporidia helenii*, *Microsporidia enterica*, and *Microsporidia tracheobronchia*, all sharing a first upstream primer. The other group includes a target sequence from *Anopheles mosquitoes*, each corresponding to a separate second upstream primer. The downstream primers are divided into four groups according to amplification specificity requirements to ensure amplification efficiency, successfully achieving non-genotypic detection of the six microsporidia species. Through specific matching of the upstream and downstream primer groups, the composition exhibits high specificity, high sensitivity, and good anti-interference ability, making it suitable for detecting ocular microsporidia infections. Attached Figure Description

[0025] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0026] Figure 1 The image shows the amplification gradient curve of rabbit encephalitis microsporidia (NR_183208.1) provided in Example 3 of this invention, where 1 represents 500 copies / mL, 2 represents 1000 copies / mL, 3 represents 10000 copies / mL, and 4 represents 100000 copies / mL. Figure 2 The amplification gradient curve of Microsporidium keratosum (PV789692.1) provided in Example 3 of the present invention is shown, where 1 represents 500 copies / mL, 2 represents 1000 copies / mL, 3 represents 10000 copies / mL, and 4 represents 100000 copies / mL. Figure 3 The amplification gradient curve of *Pyrrosia lingua* (LC274956.1) provided in Example 3 of this invention is shown, where 1 represents 500 copies / mL, 2 represents 1000 copies / mL, 3 represents 10000 copies / mL, and 4 represents 100000 copies / mL. Figure 4 The amplification gradient curve of Anopheles microsporidia (AF069063.1) provided in Example 3 of the present invention is shown, where 1 represents 500 Copies / mL, 2 represents 1000 Copies / mL, 3 represents 10000 Copies / mL, and 4 represents 100000 Copies / mL. Figure 5 This is a cross-pathogen curve of microsporidia provided in Embodiment 4 of the present invention; Figure 6 The amplification curves of microsporidia samples detected by different primer and probe sets provided in Comparative Example 1 of this invention are shown. Detailed Implementation

[0027] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0028] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0029] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] In one aspect, the present invention provides a composition for detecting microsporidia in ocular keratoconjunctivitis, the composition comprising an upstream primer set and a downstream primer set; The upstream primer set includes a first upstream primer that specifically binds to the target sequences of Microsporidium corneum, Microsporidium rabbit encephalitis, Microsporidium helensonii, Microsporidium enteroencephalitis, and Microsporidium tracheosporidioides, and a second upstream primer that specifically binds to the target sequence of Microsporidium anopheles. The downstream primer set includes a first downstream primer for specifically amplifying the target sequence of Microsporidium cornealis, a second downstream primer for specifically amplifying the target sequence of Microsporidium enterocytitis, Microsporidium rabbit encephalitis, or Microsporidium helenii, a third downstream primer for specifically amplifying Microsporidium human tracheobronchiolitis, and a fourth downstream primer for specifically amplifying the target sequence of Microsporidium anopheles. The target sequences of *Microsporidium cornealis* are shown in SEQ ID NO: 12; the target sequences of *Microsporidium rabbitis*, *Microsporidium helenii*, and *Microsporidium enteritidis* are shown in SEQ ID NO: 13; the target sequence of *Microsporidium trachei* is shown in SEQ ID NO: 14; and the target sequence of *Anopheles mosquitois* is shown in SEQ ID NO: 15.

[0031] The composition for detecting microsporidia in ocular keratoconjunctivitis provided by this invention divides six species of microsporidia that can infect the eye into two groups. One group includes target sequences of *Microsporidia keratosa*, *Microsporidia truncatula*, *Microsporidia helenii*, *Microsporidia enterica*, and *Microsporidia tracheobronchia*, all sharing a first upstream primer. The other group includes target sequences of *Anopheles mosquitois*, each corresponding to a separate second upstream primer. The downstream primers are divided into four groups according to amplification specificity requirements to ensure amplification efficiency, successfully achieving non-genotypic detection of the six microsporidia species. Through the specific matching of upstream and downstream primer groups, it possesses high specificity, high sensitivity, and good anti-interference ability, making it suitable for the detection of microsporidia ocular infections.

[0032] In some specific embodiments, the nucleotide sequence of the first upstream primer is shown in SEQ ID NO: 1, and the nucleotide sequence of the second upstream primer is shown in SEQ ID NO: 2; The nucleotide sequence of the first downstream primer is shown in SEQ ID NO: 3, the nucleotide sequence of the second downstream primer is shown in SEQ ID NO: 4, the nucleotide sequence of the third downstream primer is shown in SEQ ID NO: 5, and the nucleotide sequence of the fourth downstream primer is shown in SEQ ID NO: 6.

[0033] In some specific embodiments, a probe set is also included, which includes a first probe and a second probe; the first probe is used to detect the amplification products of the target sequence of *Microsporidium cornealis*, the target sequence of *Microsporidium rabbitis*, the target sequence of *Microsporidium helenii*, the target sequence of *Microsporidium enterica*, or the target sequence of *Anopheles mosquitois*; the second probe is used to detect the amplification product of the target sequence of *Pyrrosia lingua*.

[0034] In SEQ ID NO: 3 and SEQ ID NO: 7, Y represents pyrimidine C or T to match base variations at the target sequence site. This, combined with other primers and / or probe sets, enables detection with high sensitivity, high specificity, high accuracy, and good anti-interference ability.

[0035] In some specific embodiments, the nucleotide sequence of the first probe is shown in SEQ ID NO: 7, and the nucleotide sequence of the second probe is shown in SEQ ID NO: 8.

[0036] In some specific embodiments, the system further includes an internal standard primer set, which includes an upstream primer with the sequence shown in SEQ ID NO. 9 and a downstream primer with the sequence shown in SEQ ID NO. 10; in some specific embodiments, the system further includes an internal standard probe for detecting the amplification product of the internal standard primer set; in some specific embodiments, the sequence of the internal standard probe is shown in SEQ ID NO. 11.

[0037] In some specific embodiments, the 5′ ends of the first probe, the second probe, and the internal standard probe are each independently connected to a fluorescent reporter group; Preferably, the fluorescent reporter group includes any one of FAM, VIC, HEX, ROX, or CY5; Preferably, the 3' ends of the first probe, the second probe, and the internal standard probe are each independently connected to a quenching group; Preferably, the quenching group includes any one of BHQ1, BHQ2, TAMRA, or DABCYL.

[0038] According to another aspect of the present invention, the use of the above-described composition in the preparation of products for detecting or assisting in the detection of whether a sample contains microsporidia is also provided; In some specific embodiments, the microsporidia include at least one of Microsporidia keratosa, Microsporidia rabbitis, Microsporidia helenii, Microsporidia enterica, Microsporidia trachei, or Microsporidia anopheles. In some specific embodiments, the sample is derived from the eye. In some specific embodiments, the sample includes vitreous scrapings and / or conjunctival scrapings.

[0039] According to another aspect of the present invention, a reagent comprising the above-described composition is also provided.

[0040] The components in the composition exist individually or in a mixture.

[0041] According to another aspect of the present invention, a kit is also provided, comprising the composition described above, or the reagent described above.

[0042] In some specific embodiments, reagents for PCR amplification are also included; in some specific embodiments, the reagents for PCR amplification include UDG enzyme, Taq enzyme, and Mg... 2+ At least one of ions, PCR buffer, dNTPs, or dUTPs.

[0043] Specifically, in reagents used for PCR amplification, UDG enzyme and Taq enzyme can be mixed as a PCR reaction solution; Mg 2+Ions, PCR Buffer, dNTPs, and dUTPs can be mixed as another set of PCR reaction solutions.

[0044] In some specific implementations, the kit also includes negative and / or positive controls; In some specific embodiments, the positive control is a plasmid containing a target, the target including at least one of the following: *Microsporidium cornealis* target sequence, *Microsporidium rabbitis* target sequence, *Microsporidium helenii* target sequence, *Microsporidium enteritidis* target sequence, *Microsporidium tracheobronchiolus* target sequence, or *Anopheles mosquitois* target sequence; in some specific embodiments, the negative control includes purified water.

[0045] In some specific implementations, the kit also includes nucleic acid extraction reagents and / or nucleic acid purification reagents.

[0046] Specifically, nucleic acid extraction reagents are used to extract nucleic acid substances from the sample to be tested; nucleic acid purification reagents are used to purify the nucleic acid substances extracted from the sample to be tested.

[0047] According to another aspect of the present invention, a method for detecting or / and assisting in the detection of microsporidia for non-disease diagnosis purposes is also provided, comprising using DNA of the sample to be tested as a template, applying the above-described composition or the above-described reagent or the above-described kit to perform PCR amplification, and determining whether the sample to be tested contains microsporidia based on the Ct value of the amplification. If the primers for detecting microsporidia show amplification and the Ct value is <37, and the internal standard primers show amplification and the Ct value is <35, then the sample is positive for microsporidia; if the primers for detecting microsporidia do not show amplification, but the internal standard primers show amplification and the Ct value is <35, then the sample is negative for microsporidia.

[0048] In some specific implementations, the sample to be tested is taken from the eye.

[0049] In some specific embodiments, the microsporidia include at least one of Microsporidia keratosa, Microsporidia rabbitis, Microsporidia helenii, Microsporidia enterica, Microsporidia trachei, or Microsporidia anopheles.

[0050] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0051] Example 1 Reagent Kit The kit consists of PCR reaction solution 1, PCR reaction solution 2, negative control, and positive control.

[0052] PCR reaction solution 1 consists of primers and probes, PCR buffer, dNTPs, and Mg. 2+The ionic composition of the PCR Buffer is as follows: the PCR Buffer consists of a buffer system including Tris-HCl, KCl, glycerol, and BSA, and was purchased from Zhengzhou Matris Biotechnology Co., Ltd. (PCR Buffer, catalog number: EP10011-03).

[0053] PCR reaction solution 2 is an enzyme mixture consisting of thermosensitive UDG enzyme (catalog number: EN10004) and inhibition-resistant hot-start Taq DNA polymerase (catalog number: EP10023-01), with a volume of 2.4 μL / test, purchased from Zhengzhou Mattress Biotechnology Co., Ltd.

[0054] The negative control is physiological saline, and the positive control is a plasmid containing both the target and internal standard.

[0055] The preparation of PCR reaction solution 1 and PCR reaction solution 2 are shown in Table 1, the primer and probe nucleic acid sequence information is shown in Table 2, and the target sequence for target amplification is shown in Table 3.

[0056] In this embodiment, both the target microsporidia and the internal standard probe are labeled with fluorescent groups. The probe corresponding to the microsporidia is labeled with the FAM group, the internal standard probe is labeled with the HEX / VIC group, and the quenching group is BHQ2.

[0057] Table 1. Preparation of the reaction system

[0058] Prepare PCR reaction solution 1 and PCR reaction solution 2 according to the table above, and add the negative control with physiological saline as the matrix and the positive control containing the target and internal standard plasmids to form the whole kit.

[0059] Table 2 Primer and probe sequences

[0060] In the sequences shown in SEQ ID NO:3 and SEQ ID NO:7, Y represents pyrimidine C or T.

[0061] Table 3 Amplification Target Sequences

[0062] Example 2 Detection Method Perform the detection using the kit provided in Example 1 according to the following steps.

[0063] 1. Sample processing and loading Take 200 μL of the test sample (test sample containing microsporidia pathogens), negative control (containing normal saline), and positive control (containing plasmid) into a 2.0 mL centrifuge tube, and use the nucleic acid extraction or purification reagent of Zhengzhou Antu Bioengineering Co., Ltd. (No. Yu Zheng Xie Bei 20180037) to extract and purify nucleic acid according to the instructions. The internal standard needs to be added to the nucleic acid extraction or purification reagent for extraction.

[0064] Use the extracted nucleic acid above as the amplification template for PCR amplification respectively. Specifically, take 5 μL and add it to the reaction tube, configure the PCR reaction solution according to the dosage shown in Table 1, and extract 20 μL of the PCR reaction solution and add it to the corresponding reaction tube to obtain the amplification solution.

[0065] 2. PCR Amplification Reaction On the Hongshi SLAN-96P fully automatic medical PCR detection system, set the PCR reaction program, and the amplification program is shown in Table 4.

[0066] Table 4 Amplification Program

[0067] 4. Result Judgment The result judgment criteria are shown in Table 5. If the corresponding target fluorescence curve shows a typical S-shaped curve and the Ct value < 37, the corresponding target can be judged as positive. If there is no fluorescence curve and the Ct value is NoCt or Ct > 40, it is judged as negative. If 37 ≤ Ct ≤ 40, the judgment result is suspicious and the sample needs to be retested. If the retest result is still in this interval and the amplification curve shows a typical S-shaped curve, it is judged as positive.

[0068] Table 5 Result Judgment Rules

[0069] Example 3 Verification of the Detection Sensitivity Effect among Microsporidia Subspecies In this example, the 18S gene sequences of 6 microsporidia subspecies were analyzed. Among them, the gene sequences of Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis have high homology and small variation. The plasmid was synthesized based on the NCBI sequence number of Encephalitozoon cuniculi (NR_183208.1); the plasmids corresponding to Nosema corneal NCBI sequence number (PV789692.1), Trachipleistophora anthropophthera NCBI sequence number (LC274956.1), and Nosema anopheles NCBI sequence number (AF069063.1) were synthesized respectively.

[0070] Following the kit in Example 1 and the detection method in Example 2, the synthesized plasmid was serially diluted and used as the test sample for verification. The concentrations were set at four gradients: 100,000 copies / mL, 10,000 copies / mL, 1,000 copies / mL, and 500 copies / mL, with 20 replicates. The amplification curves are shown below. Figures 1-4 As shown in Table 6, the Ct values ​​were determined according to the rules in Table 5. The detection rate reached 100%, confirming that this kit has excellent detection capability for samples with a concentration of 500 Copies / mL.

[0071] Table 6 Sensitivity Tests

[0072] Example 4 Specificity Test This embodiment selected other pathogens in keratitis, including EB virus, cytomegalovirus, herpes zoster virus, herpes simplex virus type I, herpes simplex virus type II, adenovirus, enterovirus 71, and Coxsackievirus CA16 (all from the standard material of the China National Institutes for Food and Drug Control), as samples for specificity testing. Specific sample concentrations are shown in Table 7. The detection was performed according to the kit provided in Example 1 and the method in Example 2, and the amplification curves are shown below. Figure 5 As shown in Table 7, the Ct values ​​showed no cross-reactivity, confirming that the present invention has good specificity for Microsporidium cornealis, Microsporidium rabbitis, Microsporidium helenii, Microsporidium enteroencephalitis, Microsporidium tracheosporium, and Microsporidium anopheles.

[0073] Table 7 Specificity Tests

[0074] Example 5 Anti-interference test In this embodiment, exogenous drugs and endogenous substances were used as interfering agents to test the anti-interference effect. The exogenous drugs included levofloxacin eye drops, tobramycin eye drops, chloramphenicol eye drops, erythromycin eye ointment, acyclovir eye drops, ganciclovir ophthalmic gel, olopatadine eye drops, emestin fumarate eye drops, pranoprofen eye drops, and rifampicin eye drops, as shown in Table 7. The endogenous substances included hemoglobin, whole blood, and mucin.

[0075] Microsporidia were detected using the kit provided in Example 1 and the method in Example 2 (the sample consisted of a mixture of plasmids provided in Example 3, with a concentration of 10,000 copies / mL for each plasmid). Interference agents were added to the interference group (one drop for eye drops and one swab for gel), while no interference agents were added to the control group. The results are shown in Tables 8 to 11. The target Ct < 37 and the internal standard Ct < 35, both of which were positive, indicating that this method has good anti-interference ability against both exogenous drugs and endogenous substances.

[0076] Table 8 Drug Interference Tests

[0077] Table 9 Hemoglobin Interference Test

[0078] Table 10 Whole Blood Interference Test

[0079] Table 11 Mucin Interference Test

[0080] Example 6 Clinical Validation Five conjunctival scraping samples from patients with keratoconjunctivitis clinically diagnosed by microsporidiosis were tested according to the kit provided in Example 1 and the method in Example 2. The results are shown in Table 12.

[0081] Table 12 Clinical Testing

[0082] The results showed that microsporidia targets were detected in all clinical microscopic keratitis samples, consistent with the microscopic findings, with an accuracy rate of 100%. This demonstrates the clinical application value of the proposed method in keratoconjunctivitis and its reference significance for the detection of microsporidia infection in keratitis patients.

[0083] Comparative Example 1 This comparative example provides primer and probe sequences for detecting microsporidia as shown in Table 13. The probe is labeled with the FAM group, the internal standard probe is labeled with the HEX / VIC group, and the quencher group is BHQ2. The primer and probe sequences shown in Table 13 are used instead of the primer and probe sequences for microsporidia shown in Table 2. The amounts are the same as in Example 1. The volume of ultrapure water is adjusted to maintain the total system at 20 μl. Other reagents are the same as in Example 1.

[0084] Table 13 Primer and probe sequences

[0085] The kits of Comparative Example 1 and Example 1 were used respectively, and the detection was performed according to the method of Example 2. The sample was composed of a mixture of plasmids provided in Example 3, and the concentration of each plasmid was 10,000 Copies / mL.

[0086] Amplification curves as follows Figure 6 As shown, compared with Example 1, the fluorescence value of the amplification curve in Comparative Example 1 was reduced to varying degrees; the low-value region curve of Comparative Example 1 did not exhibit a typical S-shaped curve, and the amplification height in the low-value region was reduced by more than half compared with Example 1, resulting in a flatter curve; the Ct value in the median region of Comparative Example 1 was lower than that of Example 1. This indicates that Comparative Example 1 has technical problems such as low amplification efficiency, elevated detection limit, increased false negative risk, and impaired sensitivity.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition for detecting microsporidia in ocular keratoconjunctivitis, characterized in that, The composition includes an upstream primer set and a downstream primer set; The upstream primer set includes a first upstream primer that specifically binds to the target sequences of Microsporidium corneum, Microsporidium rabbit encephalitis, Microsporidium helensonii, Microsporidium enteroencephalitis, and Microsporidium tracheosporidioides, and a second upstream primer that specifically binds to the target sequence of Microsporidium anopheles. The downstream primer set includes a first downstream primer for specifically amplifying the target sequence of Microsporidium cornealis, a second downstream primer for specifically amplifying the target sequence of Microsporidium enterocytitis, Microsporidium rabbit encephalitis, or Microsporidium helenii, a third downstream primer for specifically amplifying Microsporidium human tracheobronchiolitis, and a fourth downstream primer for specifically amplifying the target sequence of Microsporidium anopheles. The target sequences of *Microsporidium cornealis* are shown in SEQ ID NO: 12; the target sequences of *Microsporidium rabbitis*, *Microsporidium helenii*, and *Microsporidium enteritidis* are shown in SEQ ID NO: 13; the target sequence of *Microsporidium trachei* is shown in SEQ ID NO: 14; and the target sequence of *Anopheles mosquitois* is shown in SEQ ID NO:

15.

2. The composition according to claim 1, characterized in that, The nucleotide sequence of the first upstream primer is shown in SEQ ID NO: 1, and the nucleotide sequence of the second upstream primer is shown in SEQ ID NO: 2; The nucleotide sequence of the first downstream primer is shown in SEQ ID NO: 3, the nucleotide sequence of the second downstream primer is shown in SEQ ID NO: 4, the nucleotide sequence of the third downstream primer is shown in SEQ ID NO: 5, and the nucleotide sequence of the fourth downstream primer is shown in SEQ ID NO:

6. Preferably, it further includes a probe group, which includes a first probe and a second probe; The first probe is used to detect the amplification products of the target sequences of Microsporidium corneal striatum, Microsporidium rabbit encephalitis, Microsporidium helensonii, Microsporidium enteroencephalitis, or Microsporidium anopheles. The second probe is used to detect the amplification product of the target sequence of *Pyrrosia lingua*. Preferably, the nucleotide sequence of the first probe is shown in SEQ ID NO: 7, and the nucleotide sequence of the second probe is shown in SEQ ID NO:

8.

3. The composition according to claim 1 or 2, characterized in that, It also includes an internal standard primer set, which includes an upstream primer with the sequence shown in SEQ ID NO.9 and a downstream primer with the sequence shown in SEQ ID NO.10; Preferably, it also includes an internal standard probe for detecting the amplification products of the internal standard primer set; Preferably, the sequence of the internal standard probe is shown in SEQ ID NO.

11.

4. The composition according to claim 3, characterized in that, The first probe, the second probe, and the internal standard probe are each independently connected to a fluorescent reporter group; Preferably, the fluorescent reporter group includes any one of FAM, VIC, HEX, ROX, or CY5; Preferably, the first probe, the second probe, and the internal standard probe are each independently connected to a quenching group; Preferably, the quenching group includes any one of BHQ1, BHQ2, TAMRA, or DABCYL.

5. The use of the composition according to any one of claims 1 to 4 in the preparation of products for detecting or assisting in the detection of whether a sample contains microsporidia; Preferably, the microsporidia include at least one of Microsporidia keratosa, Microsporidia rabbitis, Microsporidia helenii, Microsporidia enterica, Microsporidia trachei, or Microsporidia anopheles. Preferably, the sample is from the eye.

6. A reagent, characterized in that, Includes the composition according to any one of claims 1 to 4.

7. A reagent kit, characterized in that, Includes the composition according to any one of claims 1 to 4, or the reagent according to claim 6.

8. The reagent kit according to claim 7, characterized in that, It also includes reagents used for PCR amplification; Preferably, the reagents used for PCR amplification include UDG enzyme, Taq enzyme, and Mg. 2+ At least one of the following: ions, PCR buffer, dNTPs, or dUTPs; Preferably, the kit also includes negative and / or positive controls; Preferably, the positive control is a plasmid containing a target, the target including at least one of the following: Microsporidium corneal striatum target sequence, Microsporidium rabbit encephalitis target sequence, Microsporidium helenii encephalitis target sequence, Microsporidium enteroencephalitis target sequence, Microsporidium human tracheobronchiolitis target sequence, or Microsporidium anopheles target sequence. Preferably, the anion control includes purified water; Preferably, the kit also includes nucleic acid extraction reagents and / or nucleic acid purification reagents.

9. A method for detecting or / aiding the detection of microsporidia for non-disease diagnosis purposes, characterized in that, The method includes using the DNA of the sample to be tested as a template, applying the composition of any one of claims 1 to 4, the reagent of claim 6, or the kit of claim 7 or 8 to perform PCR amplification, and determining whether the sample to be tested contains microsporidia based on the Ct value of the amplification. If the primers for detecting microsporidia show amplification and the Ct value is <37, and the internal standard primers show amplification and the Ct value is <35, then the sample is positive for microsporidia; if the primers for detecting microsporidia do not show amplification, but the internal standard primers show amplification and the Ct value is <35, then the sample is negative for microsporidia.

10. The method according to claim 9, characterized in that, The sample to be tested was taken from the eye; Preferably, the microsporidia include at least one of Microsporidia keratosa, Microsporidia rabbitis, Microsporidia helenii, Microsporidia enterica, Microsporidia trachei, or Microsporidia anopheles.

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