Primer group, kit and method for simultaneously detecting 11 microorganisms and diagnosing chronic endometritis intrauterine infection
By designing primer sets and real-time fluorescence PCR technology, detection kits for chronic endometriitis pathogens have been developed, which solves the problem of detection difficulties in the prior art, achieves rapid and accurate pathogen detection, and improves diagnosis and treatment efficiency and treatment effect.
Patent Information
- Application Number
- CN202510704010.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-29
- Publication Date
- 2025-08-22
AI Technical Summary
The prior art is difficult to detect pathogens of chronic endometriitis efficiently, quickly and accurately, resulting in difficulty in diagnosis and lag in treatment.
The primer group was designed to target common pathogens of chronic endometriitis, and using real-time fluorescence PCR technology, and developed kits for the detection of endometrial tissue samples. It contains multiple pairs of primers to cover 11 pathogens. It combines with UNG enzyme/dUTP anti-pollution system to achieve fast and accurate pathogen detection.
Efficient, rapid and accurate detection of 11 chronic endometriitis pathogens has been achieved, reducing the time and cost of diagnosis and treatment, improving diagnosis and treatment efficiency, and avoiding the unreasonable use of antibiotics.
Smart Images
Figure FT_1 
Figure SMS_1 
Figure SMS_2
Abstract
Description
Technical Field
[0001] The present application relates to the field of molecular biology, specifically, to a primer set, a kit, and a method for simultaneously detecting 11 types of chronic endometritis. More specifically, it relates to a primer set and its use, a kit and its use, and a method for detecting chronic endometritis. Background Art
[0002] Chronic endometritis (CE) is a persistent inflammation caused by bacterial pathogens in the uterine cavity, but it is often overlooked because of its mild clinical symptoms. CE is often clinically silent and rarely suspected and diagnosed, although it may be accompanied by symptoms such as pelvic pain, dysfunctional uterine bleeding, dyspareunia, and bleeding.
[0003] CE is a "silent killer." Although clinically mild, it has a significant impact on women. An increasing number of studies have found a significant increase in the incidence of CE in conditions such as abnormal uterine bleeding, endometrial hyperplasia, and intrauterine adhesions, suggesting that CE may be involved in the development and progression of these conditions. The incidence of CE ranges from 0.2% to 46% in different populations, and in infertile individuals, the incidence can be as high as 57%. CE is highly prevalent in patients with unexplained infertility, recurrent implantation failure, and unexplained recurrent miscarriage. Antibiotic treatment targeting CE can improve reproductive outcomes in these patients. Therefore, the diagnosis and treatment of CE are of great clinical significance in gynecology and reproductive medicine.
[0004] Epidemiological studies have shown that the most common causes of CE are Chlamydia trachomatis, Enterococcus faecalis, Escherichia coli, Trichomonas vaginalis, Klebsiella pneumoniae, Mycoplasma hominis, Neisseria gonorrhoeae, Staphylococcus aureus, Streptococcus, Mycoplasma genitalium, and Ureaplasma urealyticum.
[0005] Diagnosis of CE is difficult due to the absence of typical clinical or ultrasound manifestations. Classical diagnostic techniques for CE rely on histology, primarily the identification of plasma cells in histopathological sections. However, this approach is limited, requiring detection only when patients undergo histological examination for other reasons and is subject to numerous factors, such as the menstrual cycle and physician experience. Hysteroscopy and microbiological culture are also commonly used to diagnose CE. Hysteroscopic diagnosis of chronic endometritis relies on subjective features determined by the reproductive endoscopist, such as stromal edema, focal or diffuse epithelial congestion, and / or the presence of micropolyps. Identification of endometrial pathogens through microbiological culture is currently the only clinical method that can provide objective information on pathogens. Endometrial bacterial culture is not routinely performed in clinical practice because it requires a long incubation time and not all microorganisms that cause chronic endometritis can be cultured.
[0006] The diagnosis of CE still depends on the method used. To improve and personalize the state of the art in the diagnosis and treatment of chronic endometritis, we sought to develop a molecular diagnostic tool for chronic endometritis based on real-time polymerase chain reaction (PCR). This tool overcomes the limitations of existing detection methods and accurately detects pathogens, which is of great significance for improving the diagnosis and management of asymptomatic chronic endometritis and guiding precise medication. Summary of the Invention
[0007] The present application aims to solve the technical problems in existing clinical practice. To this end, the inventors designed primers for the gene-specific conserved sequences of the most common pathogens of chronic endometritis, including Enterococcus faecalis, Escherichia coli, Streptococcus, Staphylococcus, Gardnerella vaginalis and Mycoplasma, as well as genital pathogens associated with sexually transmitted infections, such as Ureaplasma urealyticum, Chlamydia trachomatis and Neisseria gonorrhoeae, and provided a real-time fluorescence PCR detection method for rapid and low-cost detection of genes of 11 pathogens, which can detect nucleic acids extracted from endometrial tissue samples. This product solves the problem of limitations of current detection methods for pathogens of chronic endometritis and is urgently needed in clinical practice. There is currently a gap in detection products in this field on the market.
[0008] The symptoms of chronic endometritis caused by different pathogens are similar, but the treatments for different pathogens vary greatly. Clinicians need a combination test that covers most of the pathogens of chronic endometritis so that they can determine the infection pathogen and prescribe the right medicine. This application designs multiple pairs of primers for each pathogen. After repeated screening of multiple pairs of primers for each pathogen, this application ultimately provides a primer set for detecting 11 chronic endometritis pathogens. The detection efficiency is high and consistent, the test results are accurate, and the pathogen diagnosis can be completed quickly and at low cost.
[0009] Specifically, this application provides the following technical solutions:
[0010] In the first aspect of the present application, the present application proposes a primer set, which is used to detect the pathogens of chronic endometritis: Chlamydia trachomatis, Enterococcus faecalis, Escherichia coli, Trichomonas vaginalis, Klebsiella pneumoniae, Mycoplasma hominis, Neisseria gonorrhoeae, Staphylococcus, Streptococcus, Mycoplasma genitalium, and Ureaplasma urealyticum.
[0011] In some examples of the present application, the aforementioned primer set may further include at least one of the following additional technical features:
[0012] In some examples of the present application, the aforementioned primer set sequences are shown in SEQ ID NO: NO:1-2, NO:3-4, NO:5-6, NO:7-8, NO:9-10, NO:11-12, NO:13-14, NO:15-16, NO:17-18, NO:19-20, NO:21-22, NO:23-24 (Table 1).
[0013] In some preferred examples of the present application, the primer sequences for detecting Chlamydia trachomatis are shown in SEQ ID NO: 1-2;
[0014] In some preferred examples of the present application, the primer sequences for Enterococcus faecalis are shown in SEQ ID NO: 3-4;
[0015] In some preferred examples of the present application, the primer sequences for detecting Escherichia coli are shown in SEQ ID NO: 5-6;
[0016] In some preferred examples of the present application, the primer sequences for detecting Trichomonas vaginalis are shown in SEQ ID NOs: 7-8;
[0017] In some preferred examples of the present application, the primer sequences for detecting Klebsiella pneumoniae are shown in SEQ ID NOs: 9-10;
[0018] In some preferred examples of the present application, the primer sequences for detecting Mycoplasma hominis are shown in SEQ ID NOs: 11-12;
[0019] In some preferred examples of the present application, the primer sequences for detecting Neisseria gonorrhoeae are shown in SEQ ID NO: 13-14;
[0020] In some preferred examples of the present application, the primer sequences for detecting Staphylococcus are shown in SEQ ID NO: 15-16;
[0021] In some preferred examples of the present application, the primer sequences for detecting Streptococcus are shown in SEQ ID NOs: 17-18;
[0022] In some preferred examples of the present application, the primer sequences for detecting Mycoplasma genitalium are shown in SEQ ID 19-20;
[0023] In some preferred examples of the present application, the primer sequences for detecting Ureaplasma urealyticum are shown as SEQ ID NOs: 21-22.
[0024] In some preferred examples of the present application, the primer sequences for detecting human internal reference GAPDH are shown in SEQ ID NOs: 23-24.
[0025] Table 2: Primer set sequences
[0026]
[0027] Based on the aforementioned primer set, this application pioneered the use of endometrial tissue samples for CE-related pathogen detection, and the detection results were highly consistent with those of the positive control strain.
[0028] In some examples of the present application, the aforementioned kit further includes an internal reference primer.
[0029] In some preferred examples of the present application, the aforementioned internal reference primer sequences are shown as SEQ ID NOs: 31-32.
[0030] In some examples of the present application, the aforementioned kit further includes: reaction reagents, positive controls, and negative controls.
[0031] In some examples of the present application, the aforementioned reaction reagents include: HotStart Taq enzyme, UNG enzyme, dATP, dUTP, dCTP, dGTP, and MgCl2.
[0032] This application uses a UNG enzyme / dUTP anti-contamination system to reduce contamination interference caused by the previous PCR reaction product.
[0033] In addition to the above reagents, the kit may also include some commonly used reagents, such as enzyme-free water, instructions, etc.
[0034] This kit is the first to use endometrial tissue samples for CE-related pathogen detection. It can efficiently and evenly detect nearly all pathogens that can cause chronic endometritis, potentially improving the diagnosis and treatment rate of chronic endometritis and enabling women of childbearing age to receive accurate and effective treatment as soon as possible.
[0035] The kit in this application utilizes fluorescent PCR technology to detect 11 pathogens associated with CE in a single sampling and single test, providing results within three hours. It demonstrates high specificity and a sensitivity of up to 20 copies / μL. This supports accurate and timely treatment of CE, reducing the need for patients to make multiple trips to the hospital for diagnosis and treatment, while also alleviating physician workload, improving diagnostic and treatment efficiency, and reducing costs.
[0036] The kit of the present application can detect known CE infection pathogens at one time, thus avoiding the irrational use of antibiotics.
[0037] In the third aspect of the present application, the present application proposes the use of any primer set of the first aspect or any kit of the second aspect in detecting CE-related pathogens.
[0038] The aforementioned primer sets or kits have the advantages of high sensitivity and strong specificity in detecting CE-related pathogens and are suitable for endometrial tissue samples.
[0039] In the fourth aspect of the present application, the present application proposes a method for detecting CE-related pathogens.
[0040] According to an embodiment of the present application, the aforementioned method includes: using the primer set of any example in the first aspect or the kit of any example in the second aspect to amplify the sample to be tested; detecting the amplification product to determine the type of pathogen in the aforementioned sample to be tested; wherein, the sample to be tested is the DNA of an endometrial tissue sample.
[0041] In some examples of the present application, the aforementioned method may further include at least one of the following additional technical features.
[0042] In some preferred examples of the present application, the sample to be tested is DNA from an endometrial tissue sample.
[0043] In some examples of the present application, the aforementioned amplification is performed in an amplification reaction system, in which the final concentration of magnesium chloride is 2 mM-3 mM.
[0044] In some examples of the present application, in the aforementioned amplification reaction system, the final concentration of the primer is 100 nM-1000 nM.
[0045] Optionally, the reaction procedure of the amplification reaction is as shown in Table 3:
[0046] Table 3 Reaction procedure
[0047]
[0048] In some examples of the present application, the positive control of the aforementioned kit is a plasmid containing amplified gene sequences corresponding to the above-mentioned 11 CE pathogens and the internal reference gene GAPDH, or a DNA template mixture obtained by extracting DNA from all bacteria by lysis, and the added concentration is 5000-10000 genomes / well.
[0049] Those skilled in the art will appreciate that the reaction temperature, reaction time or number of cycles may be fine-tuned and optimized based on actual experimental requirements.
[0050] In some examples of the present application, the test results include a fluorescence curve and a Ct value. If the Ct value is not greater than a first predetermined threshold, it indicates that the sample is positive for chronic endometritis microorganisms; if the Ct value is not less than a second predetermined threshold, it indicates that the sample is negative for chronic endometritis microorganisms; if the Ct value is greater than the first predetermined threshold but less than the second predetermined threshold, the sample is retested.
[0051] In some examples of the present application, the aforementioned predetermined threshold may be a cut-off value obtained after testing a large number of samples.
[0052] Those skilled in the art will appreciate that differences in sample size may result in variations in the cut-off value. In some examples of the present application, the first predetermined threshold is selected from 37, and the second predetermined threshold is selected from 40.
[0053] This method uses primers designed to target conserved regions of 11 known CE pathogens, resulting in strong specificity. It can detect target gene sequences at concentrations up to 20 copies / μL, demonstrating high sensitivity. It can simultaneously detect all 11 known CE pathogens, providing high throughput. It is compatible with endometrial tissue samples, utilizing a disposable, sterile endometrial sampler for non-invasive sampling. The testing process is simple and rapid, with results completed in under three hours from specimen submission. Results are clear and objective, and qualitative analysis is also possible. A single test can identify the CE pathogen, helping physicians precisely target the pathogen and improving diagnostic and treatment efficiency. The entire testing process is free of toxic or hazardous substances, and requires no post-processing of the PCR product, making it harmless to both the operator and the environment.
[0054] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become obvious from the description below, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS
[0055] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the accompanying drawings show the melting curve profiles and melting curve temperatures of independent amplicons of 11 pathogens + 1 internal reference, and the specific melting temperature Tm values of the pathogens and internal reference are determined to distinguish between specificity (positive) and nonspecificity (negative).
[0056] Figure 1 Melting curve profiles and melting curve temperatures of independent amplicons for 11 pathogens and 1 internal control.
[0057] Obviously, the drawings described above are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work. Specific embodiments
[0059] Example 1: CE pathogen primer testing and melting curve temperature
[0060] This example uses a primer list, and the primers are synthesized by GenScript.
[0061] 1. The technical method of this application is used to detect 11 CE-related pathogens. The reagents include the following parts:
[0062] 1) Primers: Add primers for 11 pathogens and one pair of internal reference primers to the corresponding detection wells.
[0063] 2) PCR MIX: PCR buffer containing 2.5 mM MgCl2, dATP, dUTP, dCTP, dGTP, HotStartTaq enzyme, and UNG enzyme.
[0064] 3) Samples: Plasmids containing amplified gene sequences corresponding to the 11 CE-related pathogens listed above plus DNA extracted from normal human endometrial tissue samples were used as samples. A 96-well plate was used, with one well per sample and 11 wells for microbial primers plus one well for internal reference primers.
[0065] 4) In the PCR reaction system, the final concentration of primers in the amplification system is 100 nM-1000 nM. The sequences of primers and probes are shown in Table 2, and the reaction system is shown in Table 4.
[0066] Table 4 Reaction system
[0067]
[0068] 2. Detection operation process
[0069] 1) Sample Preparation and Loading: Prepare the reaction system according to Table 3. Add 10 μl of PCR MIX to each tube. Add different primer pairs to numbered PCR reaction tubes. Then add the corresponding pathogen amplification plasmid and enzyme-free water to make a 20 μL system. Cap the tubes and place them in a fluorescent quantitative PCR instrument for fluorescent PCR detection.
[0070] 2) Amplification: Set the PCR amplification reaction conditions in the instrument to: 37°C, 2 min; 95°C, 5 min; 95°C, 15 s; 60°C, 45 s; 45 cycles.
[0071] 3) Analysis: After the reaction is completed, the baseline is set to automatic adjustment, and the test results are analyzed based on the amplification curve and Ct value.
[0072] 4) Results: All tubes tested had Ct values ≤ 30. Melting curve profiles and temperatures of independent amplicons for 11 pathogens and one internal control were determined, and specific melting temperatures (Tm) for the pathogens and internal control were determined to distinguish between specific (positive) and nonspecific (negative) pathogens (see Supplementary Figure 1).
[0073] Example 2: Multiplex qPCR specificity testing
[0074] Since multiplex fluorescence quantitative PCR requires the detection of three or four pathogens and one internal reference gene in one system, 2.5 μL of plasmid standards containing pathogen-specific sequences at a concentration of 2×103 copies / μL were mixed as templates according to Table 4 to perform specificity tests within the multiplex fluorescence quantitative PCR detection wells. The results showed no cross-reactions between pathogens in each group, indicating that the detection primers had good specificity in multiplex fluorescence quantitative PCR. 5 μL of plasmid standards containing pathogen-specific sequences at a concentration of 2×103 copies / μL were mixed as templates according to Table 4 to perform specificity tests within the multiplex fluorescence quantitative PCR detection kit. The results showed no interference reactions between pathogens in each group, indicating that the detection primers had good specificity in multiplex fluorescence quantitative PCR.
[0075] Table 5
[0076] Intra-well specificity In-kit specificity 2.5uL of each of the four pathogen standard mixtures in the wells was added to 15uLTEBufer, with a total volume of 25μL and a final concentration of 200 copies / uL Mixture of 10 pathogen standards other than the pathogen corresponding to the target gene: 5uL each, final concentration is 200 copies / uL
[0077] Example 3: CE pathogen detection process
[0078] 1. The tested primers were matched into primer sets and the technical method of the present application was used to detect 11 CE-related pathogens. The kit reagents included the following parts:
[0079] 1) PCR MIX 1: PCR buffer, dATP, dUTP, dCTP, dGTP, and MgCl2; HotStart Taq and UNG enzymes; and primers for Chlamydia trachomatis, Enterococcus faecalis, Escherichia coli, and Trichomonas vaginalis labeled with FAM, HEX, Cy5, and ROX, respectively.
[0080] 2) PCR MIX 2: PCR buffer, dATP, dUTP, dCTP, dGTP, and MgCl2; HotStart Taq and UNG enzymes; and primer sets for four pathogens: Klebsiella pneumoniae, Mycoplasma hominis, Neisseria gonorrhoeae, and Staphylococcus aureus, each labeled with FAM, HEX, Cy5, or ROX fluorescence.
[0081] 3) PCR MIX 3: PCR buffer, dATP, dUTP, dCTP, dGTP, and MgCl2; HotStart Taq and UNG enzymes; primers for Streptococcus, Mycoplasma genitalium, and Ureaplasma urealyticum; and an internal reference primer labeled with FAM, HEX, Cy5, and ROX fluorescence, respectively.
[0082] 2. Test samples:
[0083] 1) Sample + PCR MIX1
[0084] 2) Sample + PCR MIX2
[0085] 3) Sample + PCR MIX3
[0086] 3. Positive control:
[0087] 1) Plasmid + PCR MIX1 containing the amplified gene sequences of four CE-related pathogens: Chlamydia trachomatis, Enterococcus faecalis, Escherichia coli, and Trichomonas vaginalis;
[0088] 2) Plasmid + PCR MIX2 containing amplified gene sequences of four CE-related pathogens: Klebsiella pneumoniae, Mycoplasma hominis, Neisseria gonorrhoeae, and Staphylococcus aureus;
[0089] 3) Plasmids containing amplified gene sequences of three CE-related pathogens, Streptococcus, Mycoplasma genitalium, and Ureaplasma urealyticum, and normal endometrial tissue sample DNA + PCR MIX3.
[0090] 4. Negative control: RNase-free water + PCR MIX 1.
[0091] In the PCR reaction system, the final concentration of the primer set in the amplification system is 100nM-1000nM. The sequences of the primers are shown in Table 2.
[0092] 5. Kit operation process
[0093] 1) Sample Preparation: Sample DNA was extracted from endometrial tissue samples. 5 μL of negative and positive controls were added to separate PCR reaction tubes. PCR MIX was added to each tube to form a system. The tubes were capped and placed in a fluorescence quantitative PCR instrument for fluorescence PCR detection.
[0094] 2) Amplification: Set the PCR amplification reaction conditions in the instrument to: 37°C, 2 min; 95°C, 5 min; 95°C, 15 s; 60°C, 45 s; 45 cycles.
[0095] 3) Analysis: After the reaction is completed, the baseline is set to automatic adjustment, and the test results are analyzed based on the amplification curve and Ct value.
[0096] 6. Validity determination:
[0097] 1) Control quality control: The Ct value of the RNase-free water test is Undet or ≥40, and the Ct value of the positive control test is ≤32. Otherwise, the experiment is considered invalid.
[0098] 2) Internal standard gene: When using PCR MIX3, the Ct value of the internal standard gene ROX fluorescence channel in the sample detection well should be ≤30, and the amplification curve should have a clear exponential growth period.
[0099] 7. Interpretation of results:
[0100] Target gene: If the Ct value of the sample detection well is Undet or ≥40, the sample result is judged to be negative, and the sample to be tested does not contain target gene DNA or the content is below the detection limit; if the Ct value of the sample detection well is ≤37, the sample result is judged to be positive for the corresponding pathogen, and the sample test is successful; if the Ct value of the sample detection well is 37-40, a retest is required. If the retest Ct value is <40 and the amplification curve has a clear exponential growth period, it is judged to be positive; the rest are negative.
[0101] Example 4: Clinical sample detection
[0102] 1. Endometrial Tissue Sample Collection
[0103] 1) For use by medical personnel or trained professionals. Before use, make sure the packaging is intact.
[0104] 2) Place the patient in the lithotomy position, retract the sampling brush completely into the withdrawal tube, and gently insert the sampling brush through the cervix into the uterus until the tip of the instrument is flush with the fundus of the uterus.
[0105] 3) Pull the extraction tube back until it reaches the handle, and rotate the sampling brush clockwise for 6-8 turns.
[0106] 4) Push the sampling tube along the sampling brush to the brush head, then remove the sampling brush. Immerse the entire sampling brush in the cell preservation solution. Withdraw the sampling tube until it reaches the handle, exposing the sampling brush to the preservation solution. Swish the sampling brush in the preservation solution at least 10 times. Hold the sampling tube and move the sampling brush up and down in the tube at least 10 times to fully remove adhered cells and tissue. Tighten the cell preservation solution tube cap. Place the capped cell preservation solution tube in the sample bag and seal it.
[0107] 2. Sample DNA Extraction
[0108] 1) Processing Materials: Centrifuge the endometrial exfoliated cell sample collection tube at 900g for 5 minutes, remove part of the supernatant, transfer the remaining 1.5ml sample to an EP tube, and centrifuge at 12000r for 1 minute and 30 seconds. Remove the supernatant and retain the cell pellet. Add 300 μl of buffer GA and vortex until thoroughly mixed.
[0109] 2) Add 30 μl of Proteinase K solution and mix thoroughly. If tissue cells are not completely dissolved after adding Proteinase K and mixing, incubate at 56°C for 30 minutes until the tissue is dissolved. Briefly centrifuge to remove any water droplets on the inner wall of the tube cap before proceeding to the next step.
[0110] 3) Add 300 μl of Buffer GB, mix thoroughly by inversion, and incubate at 70°C for 10 min. The solution should become clear. Centrifuge briefly to remove water droplets from the inner wall of the tube cap.
[0111] 4) Add 200 μl of anhydrous ethanol and vortex thoroughly for 15 seconds. Flocculent precipitation may occur at this time. Briefly centrifuge to remove water droplets on the inner wall of the tube cap.
[0112] 5) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (place the adsorption column in a collection tube), centrifuge at 12,000 rpm for 1 minute and 30 seconds, discard the waste liquid, and place the adsorption column CB3 back into the collection tube for collection.
[0113] 6) Add 500 μl of buffer GD to the adsorption column CB3 (please check whether anhydrous ethanol has been added before use). Centrifuge at 12,000 rpm for 1 minute and 30 seconds. Discard the waste liquid and collect the adsorption column CB3.
[0114] 7) Add 600 μl of rinse solution PW to the adsorption column CB3 (please check whether anhydrous ethanol has been added before use). Centrifuge at 12,000 rpm for 1 minute and 30 seconds. Discard the waste liquid and collect the adsorption column CB3.
[0115] 8) Repeat step 7.
[0116] 9) Place the adsorption column CB3 back into the collection tube and centrifuge at 12,000 rpm for 2 minutes and 30 seconds. Discard the waste liquid and leave the adsorption column CB3 uncapped at room temperature for 5 minutes to completely dry any remaining rinse solution in the adsorption material.
[0117] Note: The purpose of this step is to remove the residual rinse solution in the adsorption column. The residual ethanol in the rinse solution will affect the subsequent enzyme reaction (enzyme digestion, PCR, etc.) experiments.
[0118] 10) Transfer the adsorption column CB3 to a clean centrifuge tube. Add 75 μl of elution buffer TE dropwise to the middle of the adsorption membrane. Incubate at room temperature for 15 minutes. Centrifuge at 12,000 rpm for 2 minutes and 30 seconds. Collect the solution into a new 1.5 ml centrifuge tube with a label.
[0119] 11) Repeat step 13. (Add 75ul of elution buffer)
[0120] 12) Take 2 μl of the extract and use Nanodrop to measure the DNA concentration and record it.
[0121] 13) The desired DNA solution is now available. Store at -20±5°C until use. For long-term storage, store at -70°C.
[0122] 3. Sample testing and result interpretation: Sample loading and result interpretation were performed according to the testing process in Example 3.
[0123] 4. Test Results: Twelve clinical samples were collected and tested using qPCR. The test results are recorded and shown in Table 6.
[0124] Table 6 CE clinical sample test results
[0125] Sample No. Clinical diagnosis (pathology) Test results Detection of pathogens 1 Chronic endometritis Positive Streptococcus 2 Chronic endometritis Positive Escherichia coli 3 Chronic endometritis Positive Staphylococci, Streptococci 4 Chronic endometritis Positive Escherichia coli 5 Chronic endometritis Positive Mycoplasma hominis 6 Chronic endometritis Positive Escherichia coli 7 Chronic endometritis Positive Ureaplasma urealyticum 8 Chronic endometritis Negative NA 9 Chronic endometritis Positive staphylococcus 10 Chronic endometritis Positive staphylococcus 11 Chronic endometritis Positive Enterococcus faecalis 12 Chronic endometritis Negative NA
[0126] 5. Results Analysis
[0127] Among the 12 pathologically diagnosed CE cases, 10 were positive when tested with this kit, and 2 were not detected with any of the 11 pathogens included in this kit, suggesting that they were probably infected with other pathogens.
[0128] In this invention, a single sampling and testing process can complete the inspection process. Multiplex testing covers the vast majority of pathogens associated with CE. Application to clinical diagnosis allows for timely diagnosis of the condition and early, rapid, and precise treatment of the disease. Endometrial tissue samples are collected using a non-invasive endometrial sampler. Multiplex testing confirms the presence of the infectious pathogen, allowing for precise medication and shortening recovery time.
[0129] It should be noted that the method of collecting pathogen DNA from endometrial tissue samples using a non-invasive endometrial sampler is the core of the present invention. Ordinary technicians in this field are inspired by this patent to change, modify, replace, add and reduce the above-mentioned pathogen combination without departing from the principles and purpose of this application.
[0130] Throughout this specification, reference to terms such as "one embodiment," "some embodiments," "examples," "specific examples," or "some examples" means that a specific feature, structure, material, or characteristic described in conjunction with that embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
[0131] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application without departing from the principles and purpose of the present application.
Claims
1. A primer set for simultaneous detection of 11 microorganisms for diagnosis of intrauterine infection of chronic endometritis, characterized in that: Used to detect pathogens related to intrauterine infection in chronic endometritis; Among them, the pathogens of chronic endometritis include: Chlamydia trachomatis, Enterococcus faecalis, Escherichia coli, Trichomonas vaginalis, Klebsiella pneumoniae, Mycoplasma hominis, Neisseria gonorrhoeae, Staphylococcus, Streptococcus, Mycoplasma genitalium, and Ureaplasma urealyticum.
2. The primer set according to claim 1, characterized in that The primer set sequences are as follows: SEQ ID NO: 1-2, NO: 3-4, NO: 5-6, NO: 7-8, NO: 9-10, NO: 11-12, NO: 13-14, NO: 15-16, NO: 17-18, NO: 19-20, NO: 21-22, NO: 23-24; Preferably, the primer sequences for detecting Chlamydia trachomatis are shown in SEQ ID NO: 1-2; Preferably, the primer sequences for Enterococcus faecalis are shown in SEQ ID NO: 3-4; Preferably, the primer sequences for detecting Escherichia coli are shown in SEQ ID NO: 5-6; Preferably, the primer sequences for detecting Trichomonas vaginalis are shown in SEQ ID NO: 7-8; Preferably, the primer sequences for detecting Klebsiella pneumoniae are shown in SEQ ID NOs: 9-10; Preferably, the primer sequences for detecting Mycoplasma hominis are shown in SEQ ID NO: 11-12; Preferably, the primer sequences for detecting Neisseria gonorrhoeae are shown in SEQ ID NOs: 13-14; Preferably, the primer sequences for detecting Staphylococcus are shown in SEQ ID NOs: 15-16; Preferably, the primer sequences for detecting Streptococcus are shown in SEQ ID NOs: 17-18; Preferably, the primer sequences for detecting Mycoplasma genitalium are shown in SEQ ID NOs: 19-20; Preferably, the primer sequences for detecting Ureaplasma urealyticum are shown in SEQ ID NO: 21-22; Preferably, the primer sequences for detecting human internal reference GAPDH are shown in SEQ ID NOs: 23-24.
3. A kit, characterized in that include: The primer set according to claim 1 or 2.
4. The kit according to claim 3, wherein Further comprising: reaction reagents, positive controls and negative controls; Optionally, the reaction reagents include: HotStart Taq enzyme, UNG enzyme, dATP, dUTP, dCTP, dGTP, and MgCl2.
5. Use of the primer set according to any one of claims 1 to 2 or the kit according to any one of claims 3 to 4 in detecting microorganisms of chronic endometritis.
6. A method for detecting microorganisms in chronic endometritis, characterized in that: include: Amplifying the sample to be tested using the primer set according to any one of claims 1 to 2 or the kit according to claim 5; The amplified product is detected to determine the type of urogenital tract pathogen in the sample to be tested; wherein the sample to be tested is DNA in an endometrial tissue sample.
7. The method according to claim 6, characterized in that The sample to be tested is DNA in an endometrial tissue sample.
8. The method according to claim 7, characterized in that The amplification is performed in an amplification reaction system, wherein the final concentration of magnesium chloride is 2 mM-3 mM; In the amplification reaction system, the final concentration of the primer is 100nM-1000nM; Optionally, the reaction procedure of the amplification reaction is as shown in Table 1: Table 1 Reaction procedure.
9. The method according to any one of claims 6 to 8, characterized in that: The test results include a fluorescence curve and a Ct value; if the Ct value is not greater than a first predetermined threshold, it indicates that the sample to be tested is positive for chronic endometritis microorganisms; if the Ct value is not less than a second predetermined threshold, it indicates that the sample to be tested is negative for chronic endometritis microorganisms; if the Ct value is greater than the first predetermined threshold and less than the second predetermined threshold, the sample to be tested is retested.