Primer composition for detecting chlamydia trachomatis, gonococcus and ureaplasma urealyticum and use thereof

Abstract

The application provides a primer composition for detecting chlamydia trachomatis, gonococcus and ureaplasma urealyticum and application thereof, and relates to the technical field of nucleic acid determination or detection methods.The primer composition provided by the application comprises a first primer and a second primer for detecting chlamydia trachomatis, a third primer and a fourth primer for detecting gonococcus and a fifth primer and a sixth primer for detecting ureaplasma urealyticum.The primer composition provided by the application can realize the detection of the above-mentioned three pathogens, and has the characteristics of good specificity, high sensitivity and good repeatability.Based on the primer composition, the application further combines an immunochromatography test strip detection technology, so that rapid and efficient detection of chlamydia trachomatis, gonococcus and ureaplasma urealyticum can be realized, the operation is simple and relatively portable, and the primer composition can be used for home self-detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nucleic acid detection methods, and particularly relates to primer compositions for detecting Chlamydia trachomatis, Neisseria gonorrhoeae and Ureaplasma urealyticum and application thereof. BACKGROUND

[0002] Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Ureaplasma urealyticum (UU) are three common pathogens causing sexually transmitted diseases, and widely exist in the genitourinary system infections. The three pathogens can cause urethritis, cervicitis, pelvic inflammatory disease, infertility, prostatitis and a series of serious diseases, which seriously affect the reproductive health of the population, especially in the childbearing age population. Due to the certain overlapping of the three in clinical symptoms, single detection is easy to cause missed diagnosis or misdiagnosis, so there is an urgent need in the clinic for a means of simultaneous joint detection of CT, NG and UU.

[0003] At present, common detection methods include bacterial culture method, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) and the like. Although the traditional culture method is reliable, it is tedious and time-consuming, and has strict requirements for the survival conditions of the pathogens, especially for the isolation of CT and UU; the immunological method such as ELISA is affected by the specificity of antigen-antibody reaction, and has certain false positive or false negative problems; and the PCR technology has high sensitivity and good specificity, but requires professional instruments and equipment and operators, and is difficult to meet the instant detection needs of primary medical institutions and resource-limited areas.

[0004] Nowadays, the infection rate of sexually transmitted diseases caused by CT, NG and UU pathogens continues to rise. The three pathogens have high overlap in clinical symptoms, often leading to misdiagnosis and missed diagnosis, posing a serious challenge to patient health and public health management. At the same time, due to the privacy sensitivity of sexually transmitted diseases, many suspected infected people are reluctant to actively go to medical institutions for detection, further increasing the risk of transmission. Therefore, there is an urgent need for a simple operation, fast detection, low cost, and on-site use of multiple pathogen joint detection method. In recent years, as a new type of nucleic acid detection method, the isothermal amplification technology (such as LAMP, RPA, etc.) has gradually become an important technical path for field detection and basic application due to its simple reaction conditions, no need for complex thermal cycling equipment, high amplification efficiency and other advantages. Under constant temperature, the technology realizes the efficient amplification of target nucleic acid through specific primers and polymerase, and combines with endonuclease and modified probe to recognize and cut specific sequences, and generates amplification products that can be recognized with labeled primers, and finally realizes visual result interpretation through test paper strip color development or small fluorescence detector, with high sensitivity, strong specificity, wide application scene and other characteristics. The combination of isothermal amplification and visual detection technology not only improves the simplicity of operation and the intuitiveness of results, but is more suitable for home self-testing, on-site rapid screening and other application environments. However, the current visual detection based on isothermal amplification mainly focuses on the detection of single pathogen, and there is still a lack of mature products or technical systems that can simultaneously detect CT, NG and UU three pathogens. Therefore, it has important clinical application value and social significance to develop an isothermal amplification-visual rapid detection method that can simultaneously detect the three sexually transmitted disease pathogens in clinical samples. SUMMARY

[0005] The present application provides a composition for simultaneously detecting Chlamydia trachomatis, Neisseria gonorrhoeae and Ureaplasma urealyticum and its application.

[0006] In a first aspect, the present application provides a composition for detecting Chlamydia trachomatis, Neisseria gonorrhoeae and Ureaplasma urealyticum, comprising a first primer and a second primer for detecting Chlamydia trachomatis, a third primer and a fourth primer for detecting Neisseria gonorrhoeae, and a fifth primer and a sixth primer for detecting Ureaplasma urealyticum, wherein:

[0007] The structure general formula of the first primer, the second primer, the third primer, the fourth primer, the fifth primer and the sixth primer is shown as formula 1:

[0008] 5'-Y-n(a)-n(b)-n(c)-3' formula 1;

[0009] In formula 1, Y represents a labeling group, n(a) and n(c) represent polynucleotide fragments with different nucleotide sequences, and n(b) represents a connecting sequence;

[0010] In the first primer, n(a) represents a polynucleotide fragment having a nucleotide sequence of SEQ ID NO: 4, and n(c) represents a polynucleotide fragment having a nucleotide sequence of SEQ ID NO: 5.

[0011] In the second primer, n(a) represents a polynucleotide fragment having a nucleotide sequence of SEQ ID NO: 6, and n(c) represents a polynucleotide fragment having a nucleotide sequence of SEQ ID NO: 7.

[0012] In the third primer, n(a) represents a polynucleotide fragment having a nucleotide sequence of SEQ ID NO: 8, and n(c) represents a polynucleotide fragment having a nucleotide sequence of SEQ ID NO: 9.

[0013] In the fourth primer, n(a) represents a polynucleotide fragment having a nucleotide sequence of SEQ ID NO: 10, and n(c) represents a polynucleotide fragment having a nucleotide sequence of SEQ ID NO: 11.

[0014] In the fifth primer, n(a) represents a polynucleotide fragment having a nucleotide sequence of SEQ ID NO: 12, and n(c) represents a polynucleotide fragment having a nucleotide sequence of SEQ ID NO: 13.

[0015] In the sixth primer, n(a) represents a polynucleotide fragment having a nucleotide sequence of SEQ ID NO: 14, and n(c) represents a polynucleotide fragment having a nucleotide sequence of SEQ ID NO: 15.

[0016] The primer composition as described above, the labeling group is independently selected from at least one of a fluorescent group, biotin, digoxin. Further, the fluorescent group is selected from at least one of FITC, TAMRA, FAM, CY3, CY5, Texas Red.

[0017] The primer composition as described above, the linker sequence is independently selected from one of TT, TTT, TTTT, TTTTT, TTTTTT.

[0018] The primer composition as described above, wherein the first primer is a single-stranded DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 16 modified with biotin at the 5' end; the second primer is a single-stranded DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 17 modified with a fluorescent group FAM at the 5' end; the third primer is a single-stranded DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 18 modified with biotin at the 5' end; the fourth primer is a single-stranded DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 19 modified with digoxin at the 5' end; the fifth primer is a single-stranded DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 20 modified with biotin at the 5' end; and the sixth primer is a single-stranded DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 21 modified with a fluorescent group FITC at the 5' end.

[0019] In a second aspect, the present application provides a product comprising the primer composition as described above.

[0020] The product as described above can be a kit for detecting Chlamydia trachomatis, Neisseria gonorrhoeae and Ureaplasma urealyticum.

[0021] In a third aspect, the present application provides use of the primer composition as described above or the product as described above in the preparation of a product for detecting Chlamydia trachomatis, Neisseria gonorrhoeae and Ureaplasma urealyticum.

[0022] In a fourth aspect, the present application provides a method for simultaneously detecting multiple pathogens including Chlamydia trachomatis, Neisseria gonorrhoeae and Ureaplasma urealyticum for non-diagnostic purposes, the method comprising:

[0023] extracting DNA from a sample to be detected to obtain a DNA sample to be detected;

[0024] performing an amplification reaction on the DNA sample to be detected using the primer composition as described above to obtain an amplification product;

[0025] detecting the amplification product, and determining whether the sample to be detected contains the pathogens according to the detection result.

[0026] The method as described above, wherein the sample to be detected is a urogenital tract swab from a subject, for example, a human cervical secretion swab or a human urethral secretion swab.

[0027] The method as described above, wherein the DNA polymerase used in the amplification reaction is a Bst polymerase, for example, Bst 2.0, Bst 3.0, etc. Further, the DNA polymerase is Bst 2.0.

[0028] As described above, the amplification reaction is carried out under a constant temperature environment of 55-65°C.

[0029] As described above, the amplification product is added to the sample area of ​​the colloidal gold immunochromatographic test strip, and the presence of at least one of Chlamydia trachomatis, Neisseria gonorrhoeae, and Ureaplasma urealyticum in the sample is determined by whether the corresponding bands develop color.

[0030] In this article, the term "non-diagnostic purpose" refers to something that is not intended to obtain information about whether an individual is infected with the aforementioned pathogens and suffers from the corresponding disease, i.e., it is not for the direct purpose of obtaining a disease diagnosis or health status.

[0031] Fifthly, the present invention also provides a portable detection device based on isothermal PCR reaction, comprising:

[0032] The box contains colloidal gold test strips and has an observation window located above the colloidal gold test strips. The top of the box has a liquid outlet, and the upper surface of the box has a tube seat with the liquid outlet located inside the tube seat.

[0033] A constant temperature heating element is disposed on the top surface inside the box body and located below the tube seat;

[0034] The first stepped tube is connected to the tube seat at its lower part, and a reaction chamber that runs vertically through the bottom surface of the first stepped tube is provided.

[0035] The second stepped tube is connected to the first stepped tube at its lower part, and the bottom surface of the second stepped tube has a liquid inlet.

[0036] An end cap is fastened to the top of the second stepped tube, and the end cap is provided with a drip tube, the bottom end of which is located inside the inlet.

[0037] A sealing plug is used to seal the drip outlet of the drip tube.

[0038] As described above, the detection device has a TYPE-C interface on one side of the housing, which is connected to the constant temperature heating element.

[0039] The detection device described above includes an upper box and a lower box that are connected to each other.

[0040] As described above, in the detection device, a first slide rail is provided on the side wall of the tube seat, and a cylindrical first slider is provided on the first stepped tube, which can slide along the first slide rail.

[0041] As described above, the detection device has first limiting bosses at both ends of the first slide rail to limit the position of the first slider within the first slide rail.

[0042] As described above, in the detection device, a second slide rail is provided on the side wall of the first stepped tube, and a cylindrical second slider is provided on the second stepped tube, which can slide along the second slide rail.

[0043] As described above, the detection device has second limiting bosses at both ends of the second slide rail, which are used to limit the position of the second slider within the second slide rail.

[0044] In the detection device described above, a transparent plastic film is provided on the observation window.

[0045] In the detection device described above, the inner wall of the reaction chamber is configured as a rack shape.

[0046] As described above, the bottom surface of the second stepped tube is provided with a sealing boss, which is used to cover the top of the reaction chamber after the second stepped tube is rotated.

[0047] This invention utilizes isothermal amplification technology based on melting temperature differences, combining PCR primer design strategies with the isothermal amplification advantages of LAMP. It selects nucleic acid regions within the pathogen genome target sequence where the melting temperature (Tm) varies significantly, and designs a primer pair (D and P) with a melting temperature higher than the reaction temperature, while localized regions of the target sequence have melting temperatures lower than the reaction temperature. At a constant temperature (e.g., 55-65℃), the primers efficiently anneal to the partially melted target sequence regions and initiate a chain reaction. Amplification does not rely on heat denaturation or coenzymes, requiring only Bst DNA polymerase. Because the template melting temperature varies, only specific target sequences open and are recognized at that temperature, improving specificity and reducing false positives. This invention provides a rapid, isothermal, visualized, and triple isothermal detection method for sexually transmitted infections (STDs) involving Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Ureaplasma urealyticum (UU) in clinical samples. It offers advantages such as high specificity, accurate results, low cost, and short detection time, making it suitable for home testing and other applications. Attached Figure Description

[0048] Figure 1 This is the isothermal reaction carrier used in this invention; wherein, the left figure is a left view and the right figure is a top view;

[0049] Figure 2 This is a schematic diagram of the structure of a portable detection device based on isothermal PCR reaction provided in an embodiment of the present invention;

[0050] Figure 3 This is an exploded schematic diagram of the portable detection device based on isothermal PCR reaction provided in this embodiment of the present invention;

[0051] Figure 4This is a longitudinal cross-sectional schematic diagram of the portable detection device based on isothermal PCR reaction provided in this embodiment of the present invention;

[0052] Figure 5 This is a schematic diagram of the upper box body provided in this embodiment of the present invention;

[0053] Figure 6 This is a schematic diagram of the lower box body provided in this embodiment of the present invention;

[0054] Figure 7 This is a top view schematic diagram of the first stepped tube provided in this embodiment of the present invention;

[0055] Figure 8 This is a bottom view of the first stepped tube provided in this embodiment of the present invention;

[0056] Figure 9 This is a top view schematic diagram of the second stepped tube provided in this embodiment of the present invention;

[0057] Figure 10 This is a bottom view of the second stepped tube provided in this embodiment of the present invention;

[0058] Figure 11 The lyophilized microspheres used in the reagents of this invention;

[0059] Figure 12 This is a specific test result for herpes simplex virus type I;

[0060] Figure 13 This is a specific test result for Chlamydia pneumoniae;

[0061] Figure 14 This is a specific test result for Mycoplasma pneumoniae;

[0062] Figure 15 Results of specific detection of human genomic DNA;

[0063] Figure 16 The results show the sensitivity of quality control samples containing different concentrations of CT-positive plasmids.

[0064] Figure 17 The results show the sensitivity of quality control samples containing different concentrations of CT and NG positive plasmids.

[0065] Figure 18 The results show the sensitivity of quality control samples containing different concentrations of CT and UU positive plasmids.

[0066] Figure 19 The results show the sensitivity of quality control samples containing different concentrations of CT, NG, and UU positive plasmids.

[0067] Figure 20The results of fluorescent PCR for quality control samples containing different concentrations of CT-positive plasmids are shown.

[0068] Figure 21 The results of fluorescent PCR for quality control samples containing different concentrations of NG-positive plasmids are shown.

[0069] Figure 22 The results of fluorescent PCR for quality control samples containing different concentrations of UU-positive plasmids;

[0070] Figure 23 This is a repeatability test result for a quality control sample containing a high concentration of CT-positive plasmids;

[0071] Figure 24 This is a repeatability test result for a quality control sample containing a medium concentration of CT-positive plasmids;

[0072] Figure 25 This is a repeatability test result for a quality control sample containing a low concentration of CT-positive plasmids;

[0073] Figure 26 For repeatability test results containing high concentrations of CT and NG positive plasmids;

[0074] Figure 27 The results are for repeatability testing of quality control samples containing medium concentrations of CT and NG positive plasmids.

[0075] Figure 28 For repeatability test results containing low concentrations of CT and NG positive plasmids;

[0076] Figure 29 For repeatability test results of quality control samples containing high concentrations of CT and UU positive plasmids;

[0077] Figure 30 The results are for repeatability testing of quality control samples containing medium concentrations of CT and UU positive plasmids;

[0078] Figure 31 The results are for repeatability testing of quality control samples containing low concentrations of CT and UU positive plasmids;

[0079] Figure 32 For repeatability test results of quality control samples containing high concentrations of CT, NG, and UU positive plasmids;

[0080] Figure 33 The results are for repeatability testing of quality control samples containing medium concentrations of CT, NG, and UU positive plasmids.

[0081] Figure 34 This is a repeatability test result for a quality control sample containing low concentrations of CT, NG, and UU positive plasmids.

[0082] Explanation of reference numerals in the attached figures:

[0083] 1 is the box body, 101 is the upper box body, 102 is the lower box body, 2 is the constant temperature heating element, 3 is the first stepped tube, 301 is the first slider, 302 is the second slide rail, 303 is the second limiting boss, 4 is the second stepped tube, 401 is the second slider, 402 is the sealing boss, 5 is the end cap, 501 is the drip tube, 6 is the sealing plug, 7 is the colloidal gold test paper, 8 is the observation window, 9 is the liquid outlet, 10 is the tube seat, 1001 is the first slide rail, 1002 is the first limiting boss, 11 is the reaction chamber, 12 is the TYPE-C interface, and 13 is the colloidal gold test paper placement platform. Detailed Implementation

[0084] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, embodiments of this invention, and should not be construed as limiting the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. In the description of this invention, it should be understood that the terminology used is for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0085] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0086] Example 1: Preparation of a portable detection device based on isothermal PCR reaction

[0087] Please refer to Figures 1 to 4 This embodiment provides a portable detection device based on isothermal PCR reaction, including a box body 1, an isothermal heating element 2, a first-step tube 3, a second-step tube 4, an end cap 5, and a sealing plug 6.

[0088] The box 1 contains colloidal gold test strips 7 and has an observation window 8 located above the colloidal gold test strips 7. The top of the box 1 has an outlet 9 and a tube seat 10 is provided on the upper surface of the box 1, with the outlet 9 located inside the tube seat 10.

[0089] The constant temperature heating element 2 is disposed on the top surface inside the box 1 and is located below the tube seat 10;

[0090] The lower part of the first-step tube 3 is connected to the tube seat 10, and the bottom surface of the first-step tube 3 is provided with a reaction chamber 11 that runs vertically through the tube.

[0091] The lower part of the second-step tube 4 is connected to the first-step tube 3, and the bottom surface of the second-step tube 4 is provided with a liquid inlet.

[0092] The end cap 5 is fastened to the top of the second-step tube 4, and the end cap 5 is provided with a drip tube 501, the bottom end of which is located inside the inlet.

[0093] The sealing plug 6 is placed at the drip outlet of the drip tube 501.

[0094] Specifically, the entire housing of the detection device, including the box 1, the first-step tube 3, the second-step tube 4, and the tube base 10, is made of thermoplastic ABS. ABS has high strength and good mechanical and thermal properties, ensuring that the housing does not deform at 70°C during the isothermal PCR reaction. ABS's good thermal conductivity allows the enzyme to react fully within the reaction chamber 11.

[0095] In this embodiment, the box body 1 is a square box body, and a TYPE-C interface 12 is provided on one side of the box body 1. The TYPE-C interface 12 is connected to the constant temperature heating element 2.

[0096] Specifically, the constant temperature heating element 2 uses a ceramic resistance heating element found in existing technologies, such as an electric heating wire core constant temperature heating element, which can achieve a constant output temperature of 70°C under a 5V working voltage. Utilizing the thermal conductivity of ABS material, the temperature of the reaction chamber 11 is maintained above 60°C but will not exceed 70°C, ensuring the normal occurrence of the enzyme reaction. The constant temperature heating element 2 is powered via a TYPE-C interface 12, which can be powered by a power bank or a mobile phone charger. The heating time is 15-20 minutes.

[0097] Please refer to Figures 5 to 6 In this embodiment, the box body 1 includes an upper box body 101 and a lower box body 102 that are connected to each other.

[0098] Specifically, the upper box 101 and the lower box 102 are connected by a snap-fit ​​or screws. An observation window 8 is located on the upper box 101, and a transparent plastic film is placed on the window 8 to facilitate observation of the results displayed by the colloidal gold test strip 7, while also providing a seal. The colloidal gold test strip 7 is pre-placed inside the box 1. A colloidal gold test strip placement platform 13 is located at the bottom of the lower box 102. The platform 13 has a slot, and the colloidal gold test strip 7 is placed within this slot. The platform 13 supports the colloidal gold test strip 7 and prevents it from shifting within the box 1, thus ensuring accurate observation of the measurement results.

[0099] In this embodiment, a first slide rail 1001 is provided on the side wall of the tube base 10, and a cylindrical first slider 301 is provided on the first stepped tube 3. The first slider 301 can slide along the first slide rail.

[0100] Specifically, the tube seat 10 and the upper box body 101 are integrally formed. The tube seat 10 is a circular tube, and its bottom surface is the upper surface of the upper box body 101. The liquid outlet 9 is located at a non-center position on the bottom surface of the tube seat 10. The liquid outlet 9 can be a square opening with a chamfered top edge to facilitate the flow of liquid into the liquid outlet 9. The bottom of the liquid outlet 9 extends into the box body 1 to facilitate the flow of liquid through the liquid outlet 9 onto the colloidal gold test paper 7. The colloidal gold test paper 7 is located below the liquid outlet 9.

[0101] Two symmetrically arranged first slide rails 1001 are provided on the side wall of the tube seat 10. First limiting bosses 1002 are provided at both ends of the first slide rails 1001. The first limiting bosses 1002 and the end faces of the first slide rails 1001 form a limiting groove. When the first slider 301 is engaged in the limiting groove, its position within the first slide rail 1001 is restricted. When the first slide rail 1001 is located in the limiting groove at one end, the reaction chamber 11 is aligned with the liquid outlet 9. When the first slide rail 1001 is located in the limiting groove at the other end, the bottom surface of the reaction chamber 11 is in sealed contact with the bottom surface of the tube seat 10, ensuring that the contents of the reaction chamber 11 do not flow from its bottom end. Furthermore, the reaction chamber 11 is located directly above the constant temperature heating element 2, allowing for better heating of the reaction chamber 11.

[0102] The first stepped tube 3 is a stepped cylinder, and two first sliders 301 are symmetrically arranged, each located within its respective first slide rail 1001. Rotating the first stepped tube 3 allows the two limiting grooves of the first slider 301 within the first slide rail 1001 to switch.

[0103] Please refer to Figures 7 to 8 In this embodiment, the inner wall of the reaction chamber 11 is configured as a toothed rack.

[0104] Specifically, the reaction chamber 11 is located inside the first-step tube 3, with openings at both its top and bottom. The inner wall of the reaction chamber 11 is uniformly arranged in a rack-like shape, similar to an internal gear structure. The reaction chamber 11 is pre-filled with lyophilized amplification enzyme microspheres. The rack-like structure prevents the lyophilized amplification enzyme microspheres from moving, ensuring that the dropped sample does not contact the microspheres. Both the top and bottom of the reaction chamber 11 are provided with annular grooves, each containing a sealing ring. These sealing rings maintain a seal between the reaction chamber 11 and the bottom surface of the tube base 10 and the second-step tube 4.

[0105] Please refer to Figures 9 to 10In this embodiment, a second slide rail 302 is provided on the side wall of the first stepped tube 3, and a cylindrical second slider 401 is provided on the second stepped tube 4. The second slider 401 can slide along the second slide rail 302. Second limiting bosses 303 are provided at both ends of the second slide rail 302 to limit the position of the second slider 401 within the second slide rail 302.

[0106] Specifically, two symmetrically arranged second slide rails 302 are provided on the side wall of the first stepped tube 3. Second limiting bosses 303 are provided at both ends of the second slide rails 302. The second limiting bosses 303 and the end faces of the second slide rails 302 form a limiting groove. When the second slider 401 is engaged in the limiting groove, its position within the second slide rail 302 is restricted. When the second slider 401 is located in one of the limiting grooves, the reaction chamber 11 is aligned with the dripping pipe 501. A sealing boss 402 is provided on the bottom surface of the second stepped tube 4. Rotating the second stepped tube 4 covers the top of the reaction chamber. When the second slider 401 is located in the limiting groove at the other end, the top surface of the reaction chamber 11 contacts the sealing boss 402 of the second stepped tube 4, ensuring a tight seal between the reaction chamber 11 and the bottom surface of the second stepped tube 4.

[0107] The second-step tube 4 is also a stepped cylinder. The end cap 5 is fastened to the top of the second-step tube 4. The space formed by the end cap 5 and the second-step tube 4 is a buffer zone, which can prevent pathogen aerosols from spreading to the external environment. The drip tube 501 of the end cap 5 is tubular, and its bottom end is directly inserted into the liquid inlet.

[0108] In this embodiment, the sealing plug 6 is a rubber plug with a connector. The end cap 5 has a connection hole, and the connector is snapped into the connection hole, which can prevent the sealing plug 6 from being lost after being removed from the drip tube 501.

[0109] When using the portable detection device based on isothermal PCR reaction of this embodiment for HPV virus detection, the device is a single-use device. The reaction chamber 11 is pre-filled with lyophilized microspheres of amplifying enzyme. The bottom of the reaction chamber 11 is pre-sealed to the tube seat 10, and the top of the reaction chamber 11 is aligned with the dropper 501. The sealing plug 6 is removed, and two to three drops (approximately 40 μL) of sample are added through the dropper 501. After 10 seconds, the sealing plug 6 is resealed. At this point, the sample has entered the reaction chamber 11, and the lyophilized microspheres of amplifying enzyme dissolve within it. The reaction chamber 11 has a space of approximately 0.5 ml. After the sample and enzyme are mixed, the second-step tube 4 is rotated to seal the top of the reaction chamber 11, making it a closed chamber. The isothermal heating element 2 is energized to heat the reaction chamber 11 for 15 to 20 minutes to ensure the normal occurrence of the enzyme reaction. Rotate the second-step tube 4 to align the dropper 501 with the reaction chamber 11, remove the sealing plug 6, and inject the diluent into the reaction chamber 11. The diluent is approximately 50 times the volume of the sample, about 2 ml. After injecting the diluent, replace the sealing plug 6 to prevent aerosols containing pathogens from spreading out of the shell. Simultaneously, rotate the first-step tube 3 to align the bottom of the reaction chamber 11 with the outlet 9. The diluted sample is absorbed by the colloidal gold test paper 7 through the outlet 9. The test is completed through the absorbent layer of the colloidal gold test paper 7, and the test results are displayed. The tester observes the test results through the observation window 8.

[0110] The preparation method of the colloidal gold test strip 7 for triple nucleic acid detection of sexually transmitted diseases includes the following steps:

[0111] (1) Preparation of colloidal gold (AuNPs) and gold-labeled trapping probes:

[0112] AuNPs were prepared by the reduction method using trisodium citrate, and their particle size was 40 nm. 2.0 mL of a 194 mmol / L sodium citrate solution was rapidly added to 100 mL of boiling 0.1% HAuCl4 solution. The formation of AuNPs was indicated when the solution color changed from purple to red. The solution was cooled to room temperature and stored at 4°C.

[0113] Preparation of the gold-labeled capture probe: 20 μL of 100 μmol / L antibiotin monoclonal antibody was slowly added dropwise to 500 μL of 40 nm AuNPs. The mixture was incubated in the dark for 16 h. Then, 56 μL of 10 mmol / L phosphate buffer (PB Buffer) (NaH2PO4 / Na2HPO4, pH=7.4) and 92 μL of 2 mol NaCl solution were added dropwise. After incubation for another 8 h, the mixture was centrifuged for 30 min (4 ℃, 16100 g) to remove the supernatant. The precipitate was washed with 0.3 mol / L NaCl solution and 10 mmol / L PB Buffer. Finally, the precipitate was dissolved in 10 mmol / L PB Buffer containing 0.3 mol / L NaCl and stored at 4 ℃ protected from light.

[0114] (2) Preparation of nitrocellulose membrane (NC membrane): 1.0 mg / mL goat anti-mouse IgG polyclonal antibody was sprayed onto the NC membrane to form control line C; 0.8 mg / mL anti-FAM monoclonal antibody was sprayed onto the NC membrane to form control line T1; 0.8 mg / mL anti-digoxin monoclonal antibody was sprayed onto the NC membrane to form control line T2; and 0.8 mg / mL anti-FITC monoclonal antibody was sprayed onto the NC membrane to form control line T3.

[0115] (3) Place the absorbent paper, the treated sample pad, the obtained gold label pad, and the NC membrane on the PVC base plate in a certain order. Place the assembled PVC base plate in an oven at 37 ℃ and dry it for 12 h. Then cut and assemble the strips to form the triple nucleic acid detection colloidal gold test strip for sexually transmitted diseases 7.

[0116] Example 2: Primers designed based on isothermal amplification reaction for the detection of three pathogens

[0117] 1. *Chlamydia trachomatis* Sweden5 (GenBank accession number FM865442), *Neisseria gonorrhoeae* FA1090 (GenBank accession number AJ223447), and *Ureaplasma urealyticum* ATCC 33698 (GenBank: AF085729) were selected as reference strains. The selected target sequences are shown in Table 1.

[0118] Table 1. Target sequences of three pathogens

[0119]

[0120] In Table 1, the nucleotide sequences marked with underline and superscript are the same as or reverse complementary to the primers.

[0121] 2. Nested primers were designed for the target sequences of specific Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Ureaplasma urealyticum (UU). The outer and inner primers of the nested primers were ligated into a pair of primers for isothermal amplification. Based on the nucleotide sequence of the target, outer and inner primers were designed to obtain the P1+D1 outer primer and the P2+D2 inner primer. Primers P2 and P1 were ligated in the 5'-3' direction and sequence to obtain primer P, and primers D2 and D1 were ligated in the 5'-3' direction and sequence to obtain primer D. The ligation uses 4-6 identical bases (T) as linkers. Primers P and D can be used for isothermal amplification of nucleic acids.

[0122] Biotin was used to label the 5' end of primer P for detecting CT, NG, and UU pathogens. FAM was used to label the 5' end of primer D for detecting CT pathogens, digoxigenin was used to label the 5' end of primer D for detecting NG pathogens, and FITC was used to label the 5' end of primer D for detecting UU pathogens. This facilitates the capture or detection of pathogen-specific probes during the detection process. The primer sequences for detecting the three pathogens are shown in Table 2.

[0123] Table 2. Primers for detecting three pathogens

[0124]

[0125] Example 3: Detection of pathogens using the above primer composition.

[0126] Total DNA was extracted from human cervical secretion swabs. The total DNA was thoroughly mixed with lyophilized pellets and reacted in a 60℃ constant temperature device for 8-10 minutes to obtain the reaction product. 1 mL of sample diluent was added to the device, and the device was rotated to bring the reaction product into contact with the sample end of the colloidal gold test strip. The results of the control line and the test line were observed after 15 minutes.

[0127] Each 25µL reaction mixture includes 12.5µL of 2×LAMP Premix Buffer (purchased from Baorui Biotechnology, catalog number HW205-P01), 2.5µL of 10×Primer mix (containing 10 µM CT-P, 10 µM CT-D, 10 µM NG-P, 22 µM NG-D, 10 µM UU-P, and 10 µM UU-D in TE Buffer), 1µL of Bst2.0 HS (8U / µL), 0.25µL of RNase H II (50mU / µL), and 3.75µL of ddH2O. After mixing the reaction solutions, the reaction mixture is... Figure 11 The freeze-dried microspheres shown are used for small-batch production and storage.

[0128] The method for determining the results of isothermal nucleic acid detection provided by this invention is as follows: (1) When the sample contains CT, both the T1 line and the C line are red, indicating a positive result for Chlamydia trachomatis infection; (2) When the sample contains NG, both the T2 line and the C line are red, indicating a positive result for Neisseria gonorrhoeae infection; (3) When the sample contains UU, both the T3 line and the C line are red, indicating a positive result for Ureaplasma urealyticum infection; (4) When the sample contains both CT and NG, both the T1 line and the C line are red, indicating a positive result for Ureaplasma urealyticum infection; (5) When the sample contains both CT and UU, the T1, T3, and C lines are all red. (6) When the sample contains both CT and NG / UU, the T1, T2, T3, and C lines are all red, indicating a positive result for Chlamydia trachomatis, Neisseria gonorrhoeae, and Ureaplasma urealyticum infection. (5) When the sample does not contain CT / NG / UU, only the C line is red, indicating a negative result. (6) When the C line does not show color, the test strip is considered invalid.

[0129] Example 4: Specificity Detection

[0130] Common pathogens sharing the same infection sites as Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Ureaplasma urealyticum (UU) infections—herpes simplex virus type I, Chlamydia pneumoniae, and Mycoplasma pneumoniae—were selected using human genomic DNA quantitative standard material (NIM-RM4035) as specificity control. These materials were purchased from Guangzhou Bondsheng Biotechnology Co., Ltd. and the National Standard Material Resource Sharing Platform, respectively. Specificity was assessed using the detection method provided in Example 2, with enzyme-free sterile water as a blank control. Three replicates were performed for each sample. The specificity of the established detection method was determined based on the test strip results.

[0131] The results are as follows Figures 12-15As shown, the detection results for herpes simplex virus type I, Chlamydia pneumoniae, Mycoplasma pneumoniae, and human genomic DNA were all negative, indicating that the primer composition provided by this invention has specificity.

[0132] Example 5: Sensitivity Detection

[0133] Specific conserved region sequences covering the primer regions of CT, NG, and UU pathogens were inserted into the PMD18-T vector plasmid to obtain positive plasmids. The CT positive plasmid quality control sample (4 × 10⁻⁶) was then used. 7 (copies / mL), NG positive plasmid quality control (content 2.9×10) 7 (copies / mL), UU-positive plasmid quality control (content 3.5 × 10⁻⁶) 7 To perform sensitivity testing, reconstitute 1 mL of RNase-free water and then perform a 10-fold serial dilution to obtain concentrations of 10. 7 -10 2 DNA solutions of different concentrations (copies / mL) were used as templates, and the detection was performed using the method provided in Example 2. The detection results are as follows: Figures 16-19 As shown.

[0134] Simultaneously, commercially available gonococcal (NG), chlamydia trachomatis (CT), and ureaplasma urealyticum (UU) nucleic acid detection kits (fluorescent PCR method) (purchased from Hangzhou Borui Technology Co., Ltd.) were used to detect the three plasmid quality control samples. The detection results are as follows: Figures 20-22 As shown, the sensitivity of the two methods is compared.

[0135] like Figures 16-22 As shown, the method provided by this invention is applicable when the target nucleic acid concentration is 10. 2 It can still stably generate amplification signals even at copies / mL, and its detection sensitivity is at the same level as existing fluorescent PCR methods (such as real-time fluorescent PCR), and it has equivalent detection capabilities.

[0136] Example 6: Repeatability Detection

[0137] Use CT-positive plasmid quality control material (content 4×10⁻⁶). 7 (copies / mL), NG positive plasmid quality control (content 2.9×10) 7 (copies / mL), UU-positive plasmid quality control (content 3.5 × 10⁻⁶) 7 For repeatability testing, reconstitute the sample with 1 mL of RNase-free water and then perform 10-fold serial dilutions to obtain high (1×10⁻⁶ copies / mL) values. 6 copies / mL), medium (1×10) 4copies / mL), low (1×10) 2 Three concentrations of standards (copies / mL) were tested three times in each round of testing, and the results were observed to be consistent.

[0138] like Figures 23-34 As shown, the detection results of standards with different concentrations are consistent, indicating that the method provided by this invention has good consistency.

[0139] In the description of this invention, it should be noted that the terms "upper", "lower", "front", "rear", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the system or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0140] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "assembly," "setup," and "connection" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0141] In the description of this invention, it should be noted that relational terms such as "first" or "second" are used merely to distinguish one entity or operation from another, and do not necessarily require such an actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also elements not expressly listed, and elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0142] 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 of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer composition for detecting Chlamydia trachomatis, Neisseria gonorrhoeae, and Ureaplasma urealyticum, characterized in that, This includes primers 1 and 2 for detecting Chlamydia trachomatis, primers 3 and 4 for detecting Neisseria gonorrhoeae, and primers 5 and 6 for detecting Ureaplasma urealyticum, wherein: The general structural formulas of the first primer, second primer, third primer, fourth primer, fifth primer, and sixth primer are shown in Equation 1: 5'-Yn(a)-n(b)-n(c)-3' Equation 1; In Formula 1, Y represents a labeling group, n(a) and n(c) represent polynucleotide fragments with different nucleotide sequences, and n(b) represents a linker sequence; In the first primer, n(a) represents a polynucleotide fragment with the nucleotide sequence SEQ ID NO:4, n(b) represents a polynucleotide fragment with the nucleotide sequence TTTTT, and n(c) represents a polynucleotide fragment with the nucleotide sequence SEQ ID NO:5; In the second primer, n(a) represents a polynucleotide fragment with the nucleotide sequence SEQ ID NO:6, n(b) represents a polynucleotide fragment with the nucleotide sequence TTTTT, and n(c) represents a polynucleotide fragment with the nucleotide sequence SEQ ID NO:7; In the third primer, n(a) represents a polynucleotide fragment with the nucleotide sequence SEQ ID NO:8, n(b) represents a polynucleotide fragment with the nucleotide sequence TTTTT, and n(c) represents a polynucleotide fragment with the nucleotide sequence SEQ ID NO:9; In the fourth primer, n(a) represents a polynucleotide fragment with the nucleotide sequence SEQ ID NO:10, n(b) represents a polynucleotide fragment with the nucleotide sequence TTTTT, and n(c) represents a polynucleotide fragment with the nucleotide sequence SEQ ID NO:

11. In the fifth primer, n(a) represents a polynucleotide fragment with the nucleotide sequence SEQ ID NO:12, n(b) represents a polynucleotide fragment with the nucleotide sequence TTTT, and n(c) represents a polynucleotide fragment with the nucleotide sequence SEQ ID NO:13; In the sixth primer, n(a) represents a polynucleotide fragment with the nucleotide sequence SEQ ID NO:14, n(b) represents a polynucleotide fragment with the nucleotide sequence TTT, and n(c) represents a polynucleotide fragment with the nucleotide sequence SEQ ID NO:

15.

2. The primer composition according to claim 1, characterized in that, The labeling group is independently selected from at least one of fluorescent groups, biotin, and digoxigenin.

3. The primer composition according to any one of claims 1-2, characterized in that, The first primer is a single-stranded DNA molecule with a nucleotide sequence modified with biotin at the 5' end, as shown in SEQ ID NO:

16. The second primer is a single-stranded DNA molecule with a nucleotide sequence modified with the fluorescent group FAM at the 5' end, as shown in SEQ ID NO:

17. The third primer is a single-stranded DNA molecule with a 5' end modified with biotin as shown in SEQ ID NO:18; The fourth primer is a single-stranded DNA molecule with a 5' end modified with digoxigenin, as shown in SEQ ID NO:

19. The fifth primer is a single-stranded DNA molecule with a 5' end modified with biotin as shown in SEQ ID NO:20; The sixth primer is a single-stranded DNA molecule with a 5' end modified with the fluorescent group FITC, as shown in SEQ ID NO:

21.

4. The product, characterized in that, Includes the primer composition according to any one of claims 1-3.

5. The use of the primer composition according to any one of claims 1-3 or the product according to claim 4 in the preparation of products for detecting Chlamydia trachomatis, Neisseria gonorrhoeae and Ureaplasma urealyticum.

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