Chlamydia trachomatis detection system based on fluorescence immunochromatography test strip

Abstract

The invention discloses a chlamydia trachomatis detection system based on a fluorescence immunochromatography test strip, and belongs to the technical field of nucleic acid detection. The chlamydia trachomatis detection system comprises a nucleic acid probe and a fluorescence immunochromatography test strip (FIC), the nucleic acid probe comprises a probe H1 and a probe H2; the sequence of the probe H1 is as shown in SEQ ID NO. 2; the sequence of the probe H2 is as shown in SEQ ID NO. 3; the fluorescence immunochromatography test paper comprises a sample pad, a combination pad, a nitrocellulose membrane and an absorption pad, and the combination pad is coated with nano microspheres which are double-labeled by AlexaFluor647 fluorescein and avidin. According to the detection system, a nucleic acid probe and target RNA mixed catalysis hairpin self-assembly reaction is combined with a fluorescence immunochromatography test strip, so that a professional does not need to operate equipment for detection, the operation is simple, the consumed time is short, and the detection cost is obviously reduced; the fluorescence immunochromatography test strip has low requirements on the concentration of chlamydia trachomatis and has high detection sensitivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection, and in particular relates to a Chlamydia trachomatis detection system based on a fluorescent immunochromatographic test strip. Background Art

[0002] Chlamydia trachomatis (CT) is one of the most common bacterial sexually transmitted pathogens worldwide. The incidence of CT infection has continued to rise in recent years, becoming a major global public health concern. CT infection is highly insidious, particularly in women, with over 75% of infected individuals experiencing no apparent clinical symptoms. This significantly increases the risk of cryptic transmission. If left undiagnosed and untreated, CT infection can lead to a range of serious complications, including pelvic inflammatory disease, infertility, ectopic pregnancy, and chronic pelvic pain in women, while also significantly increasing the risk of HIV transmission. CT can also cause trachoma, a chronic conjunctivitis that can lead to blindness, through ocular infection. Although screening programs have been established in some countries, limited testing methods in resource-poor regions result in low rates of early diagnosis. Traditional testing methods are often complex, equipment-dependent, and slow to produce results, making them inadequate for rapid screening at the grassroots level. Therefore, developing a highly sensitive, specific, portable, and user-friendly diagnostic tool is crucial for improving the early detection of CT infection and controlling community transmission.

[0003] Currently, CT detection methods primarily include cell culture, direct fluorescent antibody assay, enzyme-linked immunosorbent assay (ELISA), serological testing, and nucleic acid amplification technology. Cell culture, the traditional "gold standard," takes 5-7 days and has a sensitivity of only 50%-70%. Its high cost and complex operation limit its clinical application. The direct fluorescent antibody assay is low-cost and does not require a cold chain, but it is susceptible to artifacts and relies on operator experience, resulting in low sensitivity and accuracy. The ELISA rapidly detects CT antigens using a kit with a sensitivity of 65%-75%, but is costly and prone to false positives due to antibody cross-reactivity. Serological testing, due to a delayed immune response, lacks sensitivity during the acute phase of infection and is cumbersome to perform, making it unsuitable for routine screening. Nucleic acid amplification technology has become mainstream due to its high sensitivity and specificity, but it relies on sophisticated instruments, specialized personnel, and standardized laboratories. It has multiple steps and a long processing time, is prone to false negatives and false positives, and is difficult to scale up in resource-constrained areas. Existing methods suffer from deficiencies in sensitivity, cost, ease of use, and equipment dependency, making them difficult to meet the needs of rapid screening at the grassroots level.

[0004] Therefore, a Chlamydia trachomatis detection system based on fluorescent immunochromatographic test strips was proposed. Summary of the Invention

[0005] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a Chlamydia trachomatis detection system based on a fluorescent immunochromatographic test strip, which solves the problems in the prior art.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A Chlamydia trachomatis detection system comprises: a nucleic acid probe and a fluorescent immunochromatographic test strip;

[0008] The nucleic acid probes include: probe H1 and probe H2; the sequence of probe H1 is shown in SEQ ID NO.2; the sequence of probe H2 is shown in SEQ ID NO.3;

[0009] The fluorescent immunochromatographic test paper comprises: a sample pad, a conjugation pad, a nitrocellulose membrane and an absorption pad; the conjugation pad is coated with nanoparticles double-labeled with AlexaFluor647 fluorescent pigment and avidin.

[0010] Furthermore, the 3' end of the sequence of the probe H1 is modified with digoxigenin; and the 3' end of the sequence of the probe H2 is modified with biotin.

[0011] Furthermore, the nitrocellulose membrane is marked with a detection line and a quality control line; the detection line is sprayed with anti-digoxigenin monoclonal antibody, and the quality control line is sprayed with biotin.

[0012] Furthermore, the molar ratio of the probe H1 to the probe H2 is 3:1.

[0013] Furthermore, the detection target sequence of Chlamydia trachomatis is shown as SEQ ID NO.1.

[0014] Application of probes H1 and H2 in the preparation of a Chlamydia trachomatis detection kit.

[0015] Furthermore, the sequence of the probe H1 is shown as SEQ ID NO.2; the sequence of the probe H2 is shown as SEQ ID NO.3.

[0016] Furthermore, the 3' end of the sequence of the probe H1 is modified with digoxigenin; and the 3' end of the sequence of the probe H2 is modified with biotin.

[0017] Furthermore, the Chlamydia trachomatis detection kit also includes a fluorescent immunochromatographic test strip; the fluorescent immunochromatographic test strip includes: a sample pad, a conjugation pad, a nitrocellulose membrane and an absorption pad; the conjugation pad is coated with nanoparticles double-labeled with AlexaFluor647 fluorescent pigment and avidin.

[0018] Furthermore, the nitrocellulose membrane is marked with a detection line and a quality control line, the detection line is sprayed with anti-digoxigenin monoclonal antibody, and the quality control line is sprayed with biotin.

[0019] Beneficial effects of the present invention:

[0020] 1. The detection system proposed in the present invention utilizes a nucleic acid probe and target RNA mixed to catalyze a hairpin self-assembly (CHA) reaction combined with a fluorescent immunochromatographic test strip. It does not require professional personnel to operate the equipment for detection, is simple to operate, and takes less time, significantly reducing detection costs.

[0021] 2. The fluorescent immunochromatographic test strip of the present invention is designed with a sample pad, a conjugation pad, a nitrocellulose membrane, and an absorption pad. The conjugation pad is coated with nanospheres dual-labeled with AlexaFluor647 fluorescein and avidin, and the nitrocellulose membrane is sprayed with anti-digoxigenin monoclonal antibody and biotin to form a detection line and a quality control line, respectively. It can efficiently capture and detect the hybrid double strands formed by probes H1 and H2 and the target RNA, thereby requiring a low concentration of Chlamydia trachomatis 16S rRNA, with a minimum detection limit of 10 fmol / L, showing a highly sensitive detection effect.

[0022] 3. The detection system of the present invention ensures high specificity for the detection of Chlamydia trachomatis 16S rRNA by specific hybridization of probe H1 (modified with digoxigenin at the 3' end, the sequence of which is shown in SEQ ID NO. 2) and probe H2 (modified with biotin at the 3' end, the sequence of which is shown in SEQ ID NO. 3) with the target RNA (SEQ ID NO. 1), combined with the high selectivity of the catalytic hairpin assembly (CHA) reaction and the precise recognition of the H1+H2 hybrid double strand by the anti-digoxigenin monoclonal antibody of the fluorescent immunochromatographic test strip detection line, and can effectively distinguish single-base or double-base mutation sequences, significantly reducing false positive interference. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0024] Figure 1 Schematic diagram of the catalytic hairpin self-assembly reaction principle of the present invention;

[0025] Figure 2 Schematic diagram of the principle of the fluorescent immunochromatographic test strip of the present invention for detecting H1+H2 double-stranded hybrids double-modified with digoxigenin and biotin;

[0026] Figure 3 This is the feasibility analysis result of using the catalytic hairpin self-assembly reaction to detect Chlamydia trachomatis 16S rRNA verified by gel electrophoresis of the present invention;

[0027] Figure 4 This is the feasibility analysis result of the fluorescent system detection and verification of the catalytic hairpin self-assembly reaction for detecting Chlamydia trachomatis 16SrRNA;

[0028] Figure 5 This is a schematic diagram of the sensitivity analysis results of the catalytic hairpin DNA self-assembly combined with fluorescence immunochromatographic test strip detection system of the present invention;

[0029] Figure 6 Schematic diagram of specific analysis results of the catalytic hairpin DNA self-assembly combined with fluorescence immunochromatographic test strip detection system of the present invention;

[0030] Figure 7 The present invention is a method for detecting Chlamydia trachomatis 16S rRNA in clinical specimens using the method of the present invention combining catalytic hairpin DNA self-assembly with a fluorescent immunochromatographic test strip detection system. DETAILED DESCRIPTION

[0031] 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.

[0032] Example 1

[0033] In this example, the preparation process of the CHA detection probe is described;

[0034] Catalyzed Hairpin Assembly (CHA) is a typical isothermal enzyme-free nucleic acid amplification technology that relies on the specific recognition ability of target molecules to trigger cyclic self-assembly between hairpin structures, thereby achieving signal amplification. The reaction principle of CHA is as follows Figure 1 As shown, the CHA platform consists of two partially complementary hairpin probes, H1 and H2, each composed of a toe region, a stem region, and a loop region. The complementary sequences of the two are stably enclosed by the stem region, keeping the probes in a thermodynamically stable state and preventing nonspecific spontaneous hybridization. When the target T is present, it first binds specifically to the toe region of H1, opening the hairpin structure of H1, exposing the complementary region and pairing with H2 to form a stable H1-H2 double-stranded complex. Subsequently, the T molecule is released through a strand displacement mechanism, triggering a new reaction cycle until either H1 or H2 is depleted.

[0035] Based on the published 16S rRNA sequence of Chlamydia trachomatis, a multiple sequence alignment was performed to screen out a conserved sequence of 24 bases as the detection target. The sequence is shown in Table 1 below:

[0036] Two probes, designated H1 and H2, were designed based on conserved sequences of Chlamydia trachomatis 16S rRNA. The 3' end of probe H1 was labeled with digoxin (Dig), while the 3' end of probe H2 was modified with biotin (Biotin). The relevant sequences are shown in Table 1 below:

[0037] Table 1 Sequences of target RNA and DNA probes

[0038]

[0039]

[0040] The two prepared probes (H1 and H2) were dissolved in TNaK buffer (140 mM NaCl, 5 mM KCl, and 20 mM Tris, pH 7.5) to a concentration of 1 nM / L. Annealing was then performed to maintain the hairpin structure of each probe. The annealing temperature was 95°C, and the probes were cooled naturally to room temperature. After annealing, the hairpins were stored at -20°C.

[0041] Example 2

[0042] In this example, the feasibility of the catalytic hairpin self-assembly system was verified by electrophoresis.

[0043] The system was verified by 12% non-reducing polyacrylamide gel electrophoresis. Probes H1 and H2 were annealed and diluted to 1 μM with the target detection sequence. 30 μL of probe H1, 10 μL of probe H2 and 10 μL of target sequence were mixed thoroughly and reacted in a 30°C water bath for 30 minutes.

[0044] The mixture was mixed with 10× loading buffer (containing 50 mM Tris-HCl, pH 6.8, 10% glycerol, and 0.02% bromophenol blue) in a 9:1 ratio (volume ratio), diluted to a 1× working concentration, and 20 μL was loaded for electrophoresis at 100 V for 90 minutes. The electrophoresis gel was stained with EB (ethidium bromide) for 10 minutes and imaged using a gel imager. The gel image showed bands in lanes 1-7, which were: probe H1, probe H2, target detection sequence, probe H1 + target sequence mixture, probe H2 + target sequence mixture, probe H1 + probe H2 mixture, and probe H1 + probe H2 + target sequence mixture.

[0045] like Figure 3As shown, lane 6 shows that when there is no target RNA, the two probes H1 and H2 almost do not spontaneously bind, lane 4 shows that the target RNA can open the hairpin binding of probe H1 to form probe H1+target RNA, and lane 7 shows that when probe H1, probe H2 and target RNA are added, H1+H2 hybrid double strands can be formed; this shows that: the catalytic hairpin self-assembly reaction can be specifically triggered by the target RNA, probes H1 and H2 efficiently form hybrid double strands in the presence of target RNA, and maintain a low background signal in the absence of target RNA, verifying the feasibility and specificity of the system for detecting Chlamydia trachomatis 16S rRNA.

[0046] Example 3

[0047] In this example, the feasibility of the catalytic hairpin self-assembly system was verified by a fluorescence system.

[0048] Probe H2, labeled with a fluorescent group (FAM) and a quencher (BHQ1), was synthesized and purified by Shanghai Sangon Biotechnology Co., Ltd. 1 nmol / L H1 (30 μL), 1 nmol / L H2 (10 μL), and 1 nmol / L target RNA (10 μL each) were mixed. The reaction temperature was set at 30°C, and 60 cycles were performed with a 30-second interval. Detection was performed using a 7500 Fast Real-Time PCR System (Applied Biosystems, USA).

[0049] like Figure 4 As shown, the real-time fluorescence quantitative curve shows that when there is no target RNA, the background fluorescence value formed by H1+H2 is low. When the target sequence and probe are present at the same time, the probe H1 and H2 hairpins are opened in sequence, and finally a large number of H1+H2 hybrid double strands are formed, thereby generating an obvious fluorescence curve.

[0050] The above results show that the catalytic hairpin self-assembly reaction can be specifically triggered by the target RNA. Probes H1 and H2 efficiently form hybrid double strands in the presence of target RNA, generating a significant fluorescence signal, while maintaining low background fluorescence in the absence of target RNA, verifying the high sensitivity and specificity of the system in the detection of Chlamydia trachomatis 16S rRNA.

[0051] Example 4

[0052] This embodiment introduces a fluorescent immunochromatographic test strip.

[0053] The fluorescent immunochromatographic test strip includes: a sample pad, a conjugation pad, a nitrocellulose membrane and an absorption pad, and each part is connected in sequence to form a chromatographic flow path. The sample pad is located at the starting end of the test strip and is used to receive the reaction liquid sample; the conjugation pad is adjacent to the sample pad and is used to fix the fluorescent marker; the nitrocellulose membrane is connected to the conjugation pad for signal detection; the absorption pad is located at the end of the test strip and is used to drive the unidirectional flow of the liquid. Sample pad: located at the starting end of the test strip, made of hydrophilic fiber material, absorbs the reaction liquid and transfers it to the conjugation pad. Among them:

[0054] Conjugate pad: adjacent to the sample pad, coated with nanoparticles dual-labeled with AlexaFluor647 fluorescent dye and avidin. Avidin specifically binds to D-biotin on probe H2 and carries a fluorescent signal.

[0055] Nitrocellulose membrane: Connect the binding pad and the absorption pad, set up the detection line (sprayed with anti-digoxigenin monoclonal antibody, capture probe H1) and the quality control line (sprayed with D-biotin, verifying the chromatography function).

[0056] Absorbent pad: Located at the end, made of absorbent cellulose material, it drives the reaction liquid to flow in one direction through capillary action.

[0057] The preparation process of the fluorescent immunochromatographic test strip includes: mixing 300nm nanospheres with 50mM MES buffer, washing by centrifugation and resuspending by ultrasound. Add freshly prepared NHS (6μL, 20mg / mL) and EDC (3μL, 20mg / mL), mix and incubate for 20 minutes, wash twice with MES buffer and resuspend. Mix avidin and AlexaFluor647 fluorescent pigment with the activated microspheres, incubate at room temperature for 2 hours, add blocking solution and mix for 1 hour, wash twice and resuspend in Tris buffer, and store at 4°C. Another set of microspheres was coupled with anti-digoxigenin monoclonal antibodies in a similar manner. The avidin-AlexaFluor647-microsphere complex and the anti-digoxigenin antibody-microsphere complex were mixed in a ratio of 15:1 and incubated at 5μL / cm 2 Prepare a conjugate pad by spraying onto a glass fiber membrane and drying at 34 ± 3°C, humidity ≤ 40% for 16 hours. Mount a nitrocellulose membrane (NC membrane) onto a PVC substrate. After equilibration for 30 minutes, spray anti-digoxigenin monoclonal antibody (test line) and D-biotin (control line) and dry under the same conditions for 16-20 hours. Assemble the absorbent pad, nitrocellulose membrane, conjugate pad, and sample pad in this order. Cut the strips and seal with desiccant in an aluminum foil bag for storage.

[0058] Example 5

[0059] In this example, the sensitivity analysis of Chlamydia trachomatis 16S rRNA detection using a catalytic hairpin self-assembly system combined with a fluorescent immunoassay test strip is described;

[0060] According to the target detection sequence, the DNA target chain is synthesized (the DNA target chain is a single strand), and the synthesized DNA target chain is washed with TNaK+Mg 2+ Dissolve in buffer to 100 nM and dilute with vaginal swab storage solution as diluent, with the concentration diluted from 10 nM to 10 fM;

[0061] 100 μL of the DNA target strand solution was mixed thoroughly with 100 μL of probe H1 (30 nmol / L) and 100 μL of probe H2 (10 nmol / L) of appropriate concentrations, and reacted in a 35°C water bath for 30 minutes to obtain a reaction solution;

[0062] 100 μL of the reaction solution was added dropwise to the fluorescent immunochromatographic test strip, and the fluorescence values ​​at the test line and the quality control line were read on a fluorescence detector after 15 minutes.

[0063] Fluorescence results such as Figure 5 As shown, as the target concentration decreases, the fluorescence value also decreases. The lowest detectable target concentration in this example is 10 fmol / L. It is found that there is a good linear relationship between the fluorescence value (Y) and the target RNA concentration (X). The linear equations are: Y = 0.2683*X + 2.316 (R 2 =0.9496), and the cutoff value (mean value of negative samples plus 3×standard deviation) was 521.8.

[0064] Example 6

[0065] In this example, the specificity of the Chlamydia trachomatis detection method using an entropy-driven enzyme-free amplification system combined with a fluorescent immunochromatographic test strip was analyzed.

[0066] According to the target RNA, single-base mismatch target (T-SM), single-base addition target (T-SI), single-base deletion target (T-SD), double-base mismatch target (T-DM), double-base addition target (T-DI), and double-base deletion target (T-DD) were designed. The sequences are shown in Table 2:

[0067] Table 2 Probe sequences used for specific detection

[0068]

[0069]

[0070] The six base mutation sequences were diluted to 1 nmol / L with vaginal swab preservation solution, and 100 μL of mutant RNA was mixed thoroughly with 100 μL of probe H1 (30 nmol / L) and 100 μL of probe H2 (10 nmol / L). The mixture was reacted in a 35°C water bath for 15 minutes to obtain a reaction solution.

[0071] Take 100 μL of the reaction solution and add it dropwise to the fluorescent immunochromatographic test strip. After 15 minutes, read the fluorescence values ​​at the test line and the quality control line on the fluorescence detector.

[0072] Test results such as Figure 6 As shown in the figure, the fluorescence values ​​of several mutant RNAs were significantly reduced, and there was a 4* gap between them and the target RNA, indicating that the detection method has excellent selectivity and high identification ability for Chlamydia trachomatis 16S rRNA.

[0073] Example 7

[0074] In this example, a catalytic hairpin self-assembly system combined with a fluorescent immunoassay test strip is described for the detection of clinical samples;

[0075] The process of catalytic hairpin self-assembly system combined with fluorescent immunoassay strips to detect clinical samples is as follows Figure 2 As shown, when target T is present in the sample, it generates an H1-H2 complex through the CHA reaction principle. The generated H1-H2 complex carries digoxigenin and biotin labels, binds to the fluorescent nanospheres when flowing through the binding pad, and is then captured by anti-digoxigenin antibodies on the detection line to generate a fluorescent signal; the unbound nanospheres are fixed by biotin on the quality control line to form an internal reference. Sensitive quantitative detection of target T can be achieved by measuring the difference in fluorescence intensity between the detection line and the quality control line using a fluorescent immunoassay quantitative analyzer. 38 clinical positive samples and 62 negative samples were used to verify whether the invention can be used for clinical Chlamydia trachomatis infection screening. Figure 7 As shown, the results of three independent tests showed that the obtained results had a specificity of 100% and a sensitivity of 89.47% compared with the PCR results, with an overall accuracy of 96%. This proves that the entropy-driven enzyme-free amplification system combined with the fluorescent immunochromatographic test strip can be used for clinical detection of Chlamydia trachomatis.

[0076] Throughout this specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these 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.

[0077] 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.

Claims

1. A Chlamydia trachomatis detection system, characterized in that: include: Nucleic acid probes and fluorescent immunochromatographic test strips; The nucleic acid probes include: probe H1 and probe H2; the sequence of probe H1 is shown in SEQ ID NO.2; the sequence of probe H2 is shown in SEQ ID NO.3; The fluorescent immunochromatographic test paper comprises: a sample pad, a conjugation pad, a nitrocellulose membrane and an absorption pad; the conjugation pad is coated with nanoparticles double-labeled with AlexaFluor647 fluorescent pigment and avidin.

2. A Chlamydia trachomatis detection system according to claim 1, characterized in that: The 3' end of the sequence of the probe H1 is modified with digoxigenin; the 3' end of the sequence of the probe H2 is modified with biotin.

3. A Chlamydia trachomatis detection system according to claim 1, characterized in that: The nitrocellulose membrane is marked with a detection line and a quality control line; the detection line is sprayed with anti-digoxigenin monoclonal antibody, and the quality control line is sprayed with biotin.

4. A Chlamydia trachomatis detection system according to claim 1, characterized in that: The molar ratio of the probe H1 to the probe H2 is 3:

1.

5. A Chlamydia trachomatis detection system according to claim 1, characterized in that: The detection target sequence of Chlamydia trachomatis is shown in SEQ ID NO.

1.

6. Application of probe H1 and probe H2 in the preparation of a Chlamydia trachomatis detection kit.

7. The use according to claim 6, characterized in that The sequence of the probe H1 is shown in SEQ ID NO.2; the sequence of the probe H2 is shown in SEQ ID NO.

3.

8. The use according to claim 7, characterized in that The 3' end of the sequence of the probe H1 is modified with digoxigenin; the 3' end of the sequence of the probe H2 is modified with biotin.

9. The use according to claim 6, characterized in that The Chlamydia trachomatis detection kit further comprises a fluorescent immunochromatographic test strip; the fluorescent immunochromatographic test strip comprises: a sample pad, a conjugation pad, a nitrocellulose membrane and an absorption pad; the conjugation pad is coated with nanoparticles double-labeled with AlexaFluor647 fluorescein and avidin.

10. The use according to claim 9, characterized in that The nitrocellulose membrane is marked with a detection line and a quality control line. The detection line is sprayed with anti-digoxigenin monoclonal antibody, and the quality control line is sprayed with biotin.

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