A DNA probe for detecting nucleic acid of pathogens of urogenital tract infection and its application

Abstract

The present invention discloses a DNA probe for detecting nucleic acids of pathogens of urogenital tract infections and its application. The pathogens of urogenital tract infections include Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium. The DNA probe comprises probe H1 and probe H2. The DNA probe is mixed with a target sequence to undergo a catalytic hairpin self-assembly reaction. The 5' end of probe H1 is modified with digoxigenin, and the 5' end of probe H2 is modified with biotin. The present invention utilizes the DNA probe and target sequence to catalyze the hairpin self-assembly reaction, combined with an immunochromatographic test strip, to rapidly, highly sensitively, and specifically detect Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium in an isothermal, enzyme-free amplification environment.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, in particular to a DNA probe for detecting nucleic acid of pathogens of urogenital tract infection and application thereof. Background Art

[0002] Chlamydia trachomatis (CT) infection is currently the most frequently diagnosed sexually transmitted infection (STD), with approximately 130 million new cases worldwide each year, particularly among adolescents. Infection in women can cause symptoms such as pelvic inflammatory disease, urethritis, ectopic pregnancy, fallopian tube obstruction, and ectopic pregnancy, and can also increase the risk of cervical cancer. In men, symptoms include urethritis, epididymitis, and conjunctivitis. Furthermore, C. trachomatis infection can cause reactive arthritis, neonatal ophthalmia, and pneumonia.

[0003] Currently, the main clinical methods for detecting Chlamydia trachomatis include culture, immunoassays, and polymerase chain reaction (PCR). Culture is a traditional etiological method, but it is time-consuming and susceptible to false negatives due to factors such as sample size and delivery time. Immunoassays are simple to perform and require less time, but their specificity and sensitivity are suboptimal. PCR offers high sensitivity and specificity, but requires specialized personnel and specific instrumentation, making it difficult to widely use in grassroots units and remote areas.

[0004] Neisseria gonorrhoeae (NG) is the causative agent of gonorrhea. Failure to promptly diagnose and treat NG can lead to serious complications, making early diagnosis and treatment crucial for controlling its spread. Laboratory diagnostic techniques play a crucial role in the diagnosis and treatment of gonorrhea. Currently, commonly used laboratory diagnostic methods include smear microscopy, isolation and culture, immunological methods, molecular biological testing, and rapid tests. Direct smear microscopy of secretions using Gram stain is often performed, but this has its limitations. Bacterial culture is the gold standard and currently the most reliable method for diagnosing gonorrhea. However, culturing N. gonorrhoeae is difficult, and bacteriological culture alone is of limited diagnostic value.

[0005] Mycoplasma genitalium (MG) is a sexually transmitted infection pathogen that can cause symptoms or complications such as cervicitis, pelvic inflammatory disease, prostatitis, appendicitis, proctitis, urethritis, arthritis, and tubal infertility. Literature reports indicate that the positive rate for MG in male patients with symptomatic non-gonococcal urethritis is 15% to 25%. Among male patients with urethritis symptoms at STD clinics in Nanjing, my country, the positive rate was 19.7%, and the positive rate for MG in non-chlamydial, non-gonococcal urethritis reached 41.4%.

[0006] The clinical manifestations of Mycoplasma genitalium lack specificity, and its diagnosis relies on laboratory etiological diagnosis. Mycoplasma genitalium lacks a cell wall and is invisible under Gram stain microscopy. Furthermore, in vitro culture is extremely difficult and takes months, making isolation and culture impractical for routine clinical practice. The genome of Mycoplasma genitalium is highly similar to that of Mycoplasma pneumoniae. Due to cross-reactivity, serological tests have poor specificity. Furthermore, in the absence of standard diagnostic reagents, serological tests are limited to studies evaluating the association between Mycoplasma genitalium and pelvic inflammatory disease and infertility. Summary of the Invention

[0007] In order to address the deficiencies mentioned in the above background technology, the purpose of the present invention is to provide a DNA probe for detecting nucleic acids of pathogens of urogenital tract infections and its application, which can quickly, cheaply and highly sensitively detect Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] A DNA probe for detecting nucleic acids of pathogens of urogenital tract infection, wherein the pathogens of urogenital tract infection include Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium. The DNA probe includes probe H1 and probe H2. The DNA probe is mixed with a target sequence to perform a catalytic hairpin self-assembly reaction. The 5' end of probe H1 is modified with digoxigenin, and the 5' end of probe H2 is modified with biotin.

[0010] The pathogen of urogenital tract infection is Chlamydia trachomatis. The DNA probe uses the gene shown in SEQ ID NO.1 as the detection target. Probe H1 is CT-H1 with a sequence shown in SEQ ID NO.2, and probe H2 is CT-H2 with a sequence shown in SEQ ID NO.3.

[0011] The pathogen of urogenital tract infection is Neisseria gonorrhoeae. The DNA probe uses the gene shown in SEQ ID NO.4 as the detection target. Probe H1 is NG-H1 with a sequence as shown in SEQ ID NO.5, and probe H2 is NG-H2 with a sequence as shown in SEQ ID NO.6.

[0012] The pathogen of urogenital tract infection is Mycoplasma genitalium. The DNA probe uses the gene shown in SEQ ID NO.7 as the detection target. Probe H1 is MG-H1 with a sequence shown in SEQ ID NO.8, and probe H2 is MG-H2 with a sequence shown in SEQ ID NO.9.

[0013] The invention discloses an application of a DNA probe for detecting nucleic acid of pathogens of urogenital tract infection in the preparation of a product for detecting Chlamydia trachomatis.

[0014] The invention discloses an application of a DNA probe for detecting nucleic acid of pathogens of urogenital tract infection in the preparation of a product for detecting Neisseria gonorrhoeae.

[0015] The invention discloses an application of a DNA probe for detecting nucleic acid of pathogens of urogenital tract infection in the preparation of a product for detecting Mycoplasma genitalium.

[0016] A method for detecting nucleic acid of pathogens of urogenital tract infection, comprising the following detection steps:

[0017] (1) Collect vaginal secretions and immerse the swab head in a sampling tube containing cell preservation solution;

[0018] (2) Take 250 μL of the above-mentioned sample to be tested and add it to the test tube containing the lyophilized powder of H1 and H2. After thorough mixing, place it in a 35°C water bath and react for 15 minutes.

[0019] (3) After the water bath, take 80 μL of the above reaction solution and add it dropwise to the sample wells of the immunoassay test strip. After 5-10 minutes, insert the analysis strip into the fluorescence detector and read the fluorescence values ​​of the test line and the quality control line.

[0020] Beneficial effects of the present invention:

[0021] The present invention utilizes an isothermal, enzyme-free signal amplification system in conjunction with immunoassay test strips to detect nucleic acids from Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium. This method allows for rapid, inexpensive, and highly sensitive detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium. This method does not require complex pre-test preparations or PCR instrumentation, thus enabling immediate testing of Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium. Unlike PCR, the present invention utilizes an enzyme-free signal amplification system, eliminating the need for costly DNA polymerases. This significantly reduces testing costs, making it suitable for large-scale screening and immediate testing, and can meet the needs of primary care hospitals that lack PCR equipment for rapid testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention will be further described below with reference to the accompanying drawings.

[0023] Figure 1 This is a specific step of catalyzing the hairpin DNA self-assembly reaction of the present invention;

[0024] Figure 2 This is the principle of the immunoassay test strip of the present invention for detecting H1-H2 double-stranded hybrids that are double-modified with Dig and Bio;

[0025] Figure 3 The feasibility analysis results of the present invention's non-reducing polyacrylamide gel electrophoresis verification of the catalytic hairpin DNA self-assembly reaction for detecting Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium;

[0026] Figure 4 It is the sensitivity analysis result of the nucleic acid detection method for Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium using the isothermal enzyme-free signal amplification system combined with the immunoassay test strip of the present invention;

[0027] Figure 5 The invention discloses a specific analysis result of a method for detecting nucleic acids of Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium using an isothermal enzyme-free signal amplification system combined with an immunoassay test strip. DETAILED DESCRIPTION

[0028] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0029] Example 1

[0030] Preparation of lyophilized detection probe powder

[0031] The present invention performs multiple sequence alignment based on the published sequences of Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium, and screens out a conserved sequence of 22 bases in the gene as a detection target. Two probes are designed based on the conserved sequence of the Chlamydia trachomatis TRPR gene, respectively denoted as CT-H1 and CT-H2. Two probes are designed based on the conserved sequence of the Neisseria gonorrhoeae porA pseudogene, respectively denoted as NG-H1 and NG-H2. Two probes are designed based on the conserved sequence of the Mycoplasma genitalium MgpB gene, respectively denoted as MG-H1 and MG-H2. The 5' end of each of the above-mentioned probes H1 is labeled with digoxin (Dig), while the 5' end of the H2 probe is modified with biotin (Biotin, Bio). The relevant sequences are shown in Table 1 below:

[0032] Table 1 Probe sequences

[0033]

[0034] The prepared probes were2+ After the buffer solution was dissolved to 100 nM / L, annealing treatment was performed to keep each probe in hairpin structure. The annealing temperature was: slowly cooled from 95°C to 25°C at a rate of 1°C / min. After annealing treatment, H1 probe and H2 probe were taken and eluted with TAE / MgCl2. 2+ Dilute to 10 nM / L in buffer. Mix 150 μL of diluted H1 and 50 μL of released H2 in an EP tube. Place the mixture in a vacuum freeze dryer for freeze drying.

[0035] Example 2

[0036] Sensitivity analysis of the nucleic acid detection method for Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium using an isothermal enzyme-free signal amplification system combined with immunoassay test strips

[0037] Single-stranded DNA was synthesized according to the selected detection sequence to analyze the detection sensitivity of the system. 2+ The buffer solution was diluted to 100 μM / L, and then gradient diluted (1nM~1fM) with vaginal secretion specimens from normal healthy people. Take 250 μL of gradient target DNA solution respectively, add it to the gene probe freeze-dried powder, mix thoroughly, and place it in a 37°C water bath for reaction for 15 minutes. After the reaction is completed, take 80 μL of the reaction solution and add it dropwise to the sample well of the immunoassay test strip (prepared by Nanjing Donna Biotechnology Co., Ltd.). After 5-10 minutes, the fluorescence analyzer reads the fluorescence values ​​of the test line (T-line) and the quality control line (C-line). The results are as follows. Figure 4 As shown, the cutoff value was set as the mean of the negative control fluorescence value + 3 standard deviations. The negative control fluorescence values ​​for CT, NG, and MG were 100.5 ± 17.2, 121.3 ± 23.3, and 175.3 ± 52.8, respectively. The resulting cutoff values ​​were 152.1, 191.2, and 333.7, respectively. Based on the cutoff values, the sensitivity (i.e., the minimum detectable concentration) of the detection method of the present invention is 1 fM.

[0038] Example 3

[0039] Specificity analysis of the nucleic acid detection method for Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium using an isothermal non-enzymatic signal amplification system combined with immunoassay test strips

[0040] According to the selected detection sequence ( Figure 3 ) to synthesize single-stranded DNA, randomly generate a single-base mutation sequence (denoted as SMTD) and a double-base mutation sequence (denoted as DMTD), and synthesize single-stranded DNA respectively. The sequences are shown in Table 2. Each sequence was separated by TAE / Mg 2+The buffer solution was diluted to 100 pM of the test solution, 250 μL of the test solution was added to the test tube, mixed thoroughly, and then reacted in a 37°C water bath for 15 minutes. After the reaction, 80 μL of the reaction solution was added dropwise to the sample well of the immunoassay test strip (manufactured by Nanjing Donna Biotechnology Co., Ltd.). After 5-10 minutes, the fluorescence value of the test line (T-line) and the quality control line (C-line) was read on a fluorescence analyzer. The results are as follows. Figure 5 As shown, T, DMTD, SMTD and buffer were added to four tubes respectively. The detection method of the present invention has a high degree of sequence specificity. A single base mutation can cause a significant decrease in the detected fluorescence value. The fluorescence value is equivalent to that of the double-mutated control sequence and is close to that of the blank control group.

[0041] Table 2 Sequences used in specificity analysis

[0042]

[0043] Table Notes: Mutation sites are marked with underlines.

[0044] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the invention as claimed. Sequence Listing <110> Southeast University <120> A DNA probe for detecting nucleic acid of pathogens of urogenital tract infection and its application <140> 2022103337064 <141> 2022-03-30 <160> 9 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty two <212> DNA <213> Artificial Sequence <400> 1 agttaatcaa gctctacaag at 22 <210> 2 <211> 47 <212> DNA <213> Artificial Sequence <400> 2 atcttgtaga gcttgattaa ctaagactca gacagttaat caagctc 47 <210> 3 <211> 44 <212> DNA <213> Artificial Sequence <400> 3 gattaactgt ctgagtctta gttaatcaag ctcaagactc agac 44 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <400> 4 cgtattgtcc gcactgccgt tt 22 <210> 5 <211> 47 <212> DNA <213> Artificial Sequence <400> 5 aaacggcagt gcggacaata cgtgaaccta agccgtattg tccgcac 47 <210> 6 <211> 44 <212> DNA <213> Artificial Sequence <400> 6 acaatacggc ttaggttcac gtattgtccg cactgaacct aagc 44 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <400> 7 cttgaaacaa taacaacttc tc 22 <210> 8 <211> 47 <212> DNA <213> Artificial Sequence <400> 8 gagaagttgt tattgtttca agcagccacg aaccttgaaa caataac 47 <210> 9 <211> 44 <212> DNA <213> Artificial Sequence <400> 9 gtttcaaggt tcgtggctgc ttgaaacaat aaccagccac gaac 44

Claims

1. A DNA probe for detecting nucleic acid of pathogens of urogenital tract infection, characterized in that: The urogenital tract infection pathogens include Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium. The DNA probes include probe H1 and probe H2. The DNA probes are mixed with the target sequence to perform a catalytic hairpin self-assembly reaction. The 5' end of probe H1 is modified with digoxigenin, and the 5' end of probe H2 is modified with biotin. The pathogen of urogenital tract infection is Chlamydia trachomatis, the DNA probe uses the gene shown in SEQ ID NO.1 as the detection target, the probe H1 is CT-H1 with the sequence shown in SEQ ID NO.2, and the probe H2 is CT-H2 with the sequence shown in SEQ ID NO.3; The pathogen of urogenital tract infection is Neisseria gonorrhoeae, the DNA probe uses the gene shown in SEQ ID NO.4 as a detection target, the probe H1 is NG-H1 with a sequence shown in SEQ ID NO.5, and the probe H2 is NG-H2 with a sequence shown in SEQ ID NO.6; The pathogen of urogenital tract infection is Mycoplasma genitalium, the DNA probe uses the gene shown in SEQ ID NO.7 as the detection target, the probe H1 is MG-H1 with a sequence shown in SEQ ID NO.8, and the probe H2 is MG-H2 with a sequence shown in SEQ ID NO.

9.

2. Use of the DNA probe for detecting nucleic acid of urogenital tract infection pathogens according to claim 1 in the preparation of a product for detecting Chlamydia trachomatis.

3. Use of the DNA probe for detecting nucleic acid of urogenital tract infection pathogens according to claim 1 in preparing a product for detecting Neisseria gonorrhoeae.

4. Use of the DNA probe for detecting nucleic acid of urogenital tract infection pathogens according to claim 1 in the preparation of a product for detecting Mycoplasma genitalium.

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