LAMP (loop-mediated isothermal amplification) primer group and kit for three-item detection of venereal disease
By designing a LAMP primer set suitable for high-temperature amplification at 70℃, the problems of insufficient primer thermostability and low detection limit were solved, achieving high sensitivity and specificity for three sexually transmitted disease tests, reducing the false positive rate, and providing an accurate disease diagnosis solution.
Patent Information
- Application Number
- CN202511562312.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-29
- Publication Date
- 2025-12-23
AI Technical Summary
Existing LAMP detection technology suffers from insufficient primer thermostability under high-temperature amplification conditions, resulting in low template binding rate and detection limit. This leads to non-specific binding and false positive results, especially when procedures are not standardized in primary laboratories, which increases the risk of cross-reaction and false positives.
A LAMP primer set for the detection of three sexually transmitted diseases (Uu, CT, NG) was designed, including a specific primer combination targeting the UreB gene of Ureaplasma urealyticum, the gyrA gene of Chlamydia trachomatis, and the glnA gene of Neisseria gonorrhoeae. It is suitable for high-temperature amplification at 70℃, which improves the thermal stability and specificity of the primers and reduces non-specific binding.
It achieves high sensitivity and specificity for the three sexually transmitted disease tests at 70℃, reduces the false positive rate, and provides an accurate disease diagnosis solution.
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Figure CN121182997A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular biology technology, specifically to a LAMP primer set and kit for the detection of three sexually transmitted diseases. Background Technology
[0002] Ureaplasma urealyticum (Ureaplasma urealyticum) Ureaplasma urealyticum Ureaplasma gondii (UU) is a common pathogen causing urogenital tract infections. Its infection is closely associated with non-gonococcal cervicitis and urethritis in women, as well as neonatal infections, and it has the highest incidence rate in coastal areas of my country. Furthermore, UU easily develops resistance to antibiotics, and positive results can still be observed after traditional treatment. Therefore, highly sensitive detection techniques (such as PCR) are needed to clarify the pathogen load and activity, avoid the overuse of antibiotics, and reduce the burden on patients. Reliable detection methods can also provide data support for epidemiological studies and help develop regional prevention and control strategies.
[0003] Chlamydia trachomatis ( chlamydia trachomatis Chlamydia trachomatis (CT) infection is one of the most common sexually transmitted diseases worldwide, with asymptomatic infections accounting for 80% to 90% of cases. This can easily lead to missed diagnoses and serious complications such as pelvic inflammatory disease, infertility, and ectopic pregnancy. CT can be transmitted vertically from mother to child, causing neonatal conjunctivitis or pneumonia; early detection can effectively block transmission and improve prognosis. Furthermore, while CT is sensitive to antibiotics, drug resistance is becoming increasingly apparent. High-sensitivity detection technologies (such as quantitative PCR and immunofluorescence assays) can accurately assess pathogen load, guide individualized treatment, and reduce the risk of drug resistance. At the public health level, promoting screening (especially among sexually active individuals) helps reduce community transmission rates and optimize the allocation of medical resources.
[0004] Neisseria gonorrhoeae ( Neisseria gonorrhoeae Gonorrhea (gonococcus) infection is one of the most common sexually transmitted diseases worldwide. Approximately 60% of female patients are asymptomatic but can still transmit the pathogen, making it a major source of neonatal gonorrhea and community transmission. Early detection can effectively block mother-to-child vertical transmission and reduce the spread of asymptomatic infections. Secondly, gonorrhea easily leads to serious complications such as urethritis, pelvic inflammatory disease, and infertility. Accurate detection (such as culture methods and PCR technology) can achieve early diagnosis and treatment, improving prognosis. Furthermore, gonococci are increasingly resistant to antibiotics, and traditional smear methods are prone to missed detection, while molecular biological detection (such as PCR) can highly sensitively identify pathogen DNA, guiding personalized medication and reducing the risk of drug resistance. The World Health Organization (WHO) recommends culture methods as the "gold standard" for the diagnosis of gonorrhea in women, while immunochromatography is suitable for rapid screening at the grassroots level.
[0005] Loop-mediated isothermal amplification (LAMP) is a highly efficient, rapid, and specific nucleic acid amplification technique first proposed by Notomi et al. in 2000 (see "Notomi, T., et al. (2000). Loop-mediated isothermal amplification of DNA. NucleicAcids Research, 28(12), e63."). LAMP utilizes a DNA polymerase with strand displacement activity (such as Bst DNA polymerase) and a set of specifically designed primers to achieve exponential amplification of the target DNA sequence under isothermal conditions (typically 60-65°C). Unlike traditional polymerase chain reaction (PCR), LAMP does not require thermal cycling equipment and only requires a constant temperature heating device to complete the reaction. Therefore, it is particularly suitable for on-site testing or point-of-care diagnosis with limited resources (see the literature "Mori, Y., & Notomi, T. (2009). Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. Journal of Infection and Chemotherapy, 15(2), 62-69").
[0006] LAMP primers typically include two outer primers (F3 and B3), two inner primers (FIP and BIP), and two optional loop primers (LF and LB). These primers can recognize multiple specific regions on the target DNA sequence, thus ensuring high specificity and sensitivity of the reaction (see “Nagamine, K., Hase, T., & Notomi, T. (2002). Accelerated reaction by loop-mediated isothermal amplification using loopprimers. Molecular and Cellular Probes, 16(3), 223-229”). LAMP reaction products are a series of DNA fragments with different lengths and structures, and the results are usually interpreted by turbidity detection, fluorescent dyes, or colorimetry (see “Tomita, N., Mori, Y., Kanda, H., & Notomi, T. (2008). Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nature Protocols, 3(5), 877-882”). Due to its ease of operation, speed (usually completed within 30-60 minutes), and low equipment requirements, LAMP technology has been widely used in pathogen detection, food safety monitoring, environmental microbiology analysis, and genetic disease diagnosis (see the literature "Parida, M., Sannarangaiah, S., Dash, PK, Rao, PVL, & Morita, K. (2008). Loop-mediated isothermalamplification (LAMP): principle, features, and future prospects. Journal of Clinical Microbiology, 46(2), 610-614").
[0007] Currently, loop-mediated isothermal amplification (LAMP) for Ureaplasma urealyticum (Uu), Chlamydia trachomatis (Ct), and Neisseria gonorrhoeae (NG) offers advantages such as speed and low equipment dependence. However, due to the high primer specificity requirements and high sensitivity to contamination, LAMP detection still carries the risk of false positives and cross-reactions. At conventional reaction temperatures (60-65°C), the aerosols from LAMP amplification products easily contaminate the environment, especially in primary laboratories where improper operation may lead to false positive results. Increasing the amplification incubation temperature can reduce aerosol contamination, and high-temperature amplification can effectively reduce primer dimer formation, improving detection specificity, especially suitable for complex samples (such as blood or sputum containing inhibitors). However, at high temperatures (e.g., 70°C), primer thermostability is insufficient, leading to non-specific binding; simultaneously, high temperatures reduce primer-template binding efficiency, significantly degrading the detection limit. Summary of the Invention
[0008] This invention provides a LAMP primer set and kit for the detection of three sexually transmitted diseases, in order to solve the problems of insufficient primer thermostability, low template binding rate and low detection limit under high temperature amplification conditions.
[0009] In a first aspect, the present invention provides a LAMP primer set for the detection of three sexually transmitted diseases (Uu, CT, NG), wherein the LAMP primer set includes a first primer set targeting the UreB gene of Ureaplasma urealyticum, a second primer set targeting the gyrA gene of Chlamydia trachomatis, and a third primer set targeting the glnA gene of Neisseria gonorrhoeae. The first primer set includes a first outer primer pair, a first inner primer pair, and a loop primer; the first outer primer pair includes a forward outer primer and a reverse outer primer, the nucleotide sequences of which are shown in SEQ ID NO. 1-2, respectively; the first inner primer pair includes a forward inner primer and a reverse inner primer, the nucleotide sequences of which are shown in SEQ ID NO. 3-4, respectively; the nucleotide sequence of the loop primer is shown in SEQ ID NO. 5; The second primer set includes a second outer primer pair, a second inner primer pair, and a loop primer; the second outer primer pair includes a forward outer primer and a reverse outer primer, the nucleotide sequences of which are shown in SEQ ID NO. 6-7, respectively; the second inner primer pair includes a forward inner primer and a reverse inner primer, the nucleotide sequences of which are shown in SEQ ID NO. 8-9, respectively; the nucleotide sequence of the loop primer is shown in SEQ ID NO. 10; The third primer set includes a third outer primer pair, a third inner primer pair, and a loop primer pair; the third outer primer pair includes a forward outer primer and a reverse outer primer, with nucleotide sequences as shown in SEQ ID NO. 11-12, respectively; the third inner primer pair includes a forward inner primer and a reverse inner primer, with nucleotide sequences as shown in SEQ ID NO. 13-14, respectively; the loop primer pair includes a forward loop primer and a reverse loop primer, with nucleotide sequences as shown in SEQ ID NO. 15-16, respectively.
[0010] Secondly, the present invention also provides a kit for detecting three sexually transmitted diseases (Uu, CT, NG), the kit comprising the above-mentioned LAMP primer set.
[0011] In an alternative embodiment, the kit further includes a high-temperature Bst enzyme and an amplification buffer.
[0012] In one alternative embodiment, the amplification buffer comprises Tris-HCl buffer (pH 8.8), MgSO4, KCl, (NH4)2SO4, dNTPs, and / or fluorescent dye.
[0013] In one alternative embodiment, the kit further includes a negative control; the negative control is enzyme-free water.
[0014] In one optional embodiment, the kit further includes a positive control; the positive control is a quality control of Uu, CT, and NG.
[0015] In one alternative embodiment, the kit further includes quality control materials; the quality control materials include the UreB gene, gyrA gene, and glnA gene.
[0016] Thirdly, the present invention also provides a method for detecting three sexually transmitted diseases (Uu, CT, NG), wherein the method is not for the purpose of disease diagnosis and treatment, and the method includes: using the above-mentioned kit to test the sample.
[0017] In one optional implementation, the amplification conditions in the LAMP detection method are: constant temperature incubation at 68~72℃ for 30~60 min.
[0018] In one optional implementation, the amplification conditions in the LAMP detection method are: incubation at a constant temperature of 70°C for 30-60 minutes.
[0019] In one optional embodiment, the method includes: incubation at 37°C for 2-5 min, incubation at a constant temperature of 70°C for 30-60 min, inactivation at 85°C for 5 min, and melting at the melting end.
[0020] Fourthly, the present invention also provides the application of the above-mentioned LAMP primer set, the above-mentioned kit, or the above-mentioned detection method in the detection of three sexually transmitted diseases (Uu, CT, NG), wherein the application is not for the purpose of disease diagnosis and treatment.
[0021] The technical solution of this invention has the following advantages: 1. This invention provides a LAMP primer set for the detection of three sexually transmitted diseases (Uu, CT, NG), wherein the LAMP primer set includes a first primer set targeting the UreB gene of Ureaplasma urealyticum, a second primer set targeting the gyrA gene of Chlamydia trachomatis, and a third primer set targeting the glnA gene of Neisseria gonorrhoeae; the first primer set includes a first outer primer pair, a first inner primer pair, and a circular primer UreB-LB; the first outer primer pair includes a forward outer primer UreB-F3 and a reverse outer primer UreB-B3, the nucleotide sequences of which are shown in SEQ ID NO. 1~2, respectively; the first inner primer pair includes a forward inner primer UreB-FIP and a reverse inner primer UreB-BIP, the nucleotide sequences of which are shown in SEQ ID NO. 3~4, respectively; the nucleotide sequence of the circular primer UreB-LB is shown in SEQ ID NO. As shown in SEQ ID NO. 5; the second primer set includes a second outer primer pair, a second inner primer pair, and a loop primer gyrA-LB; the second outer primer pair includes a forward outer primer gyrA-F3 and a reverse outer primer gyrA-B3, with nucleotide sequences as shown in SEQ ID NO. 6~7 respectively; the second inner primer pair includes a forward inner primer gyrA-FIP and a reverse inner primer gyrA-BIP, with nucleotide sequences as shown in SEQ ID NO. 8~9 respectively; the nucleotide sequence of the loop primer gyrA-LB is shown in SEQ ID NO. 10; the third primer set includes a third outer primer pair, a third inner primer pair, and a loop primer pair; the third outer primer pair includes a forward outer primer glnA-F3 and a reverse outer primer glnA-B3, with nucleotide sequences as shown in SEQ ID NO. 11~12 respectively; the third inner primer pair includes a forward inner primer glnA-FIP and a reverse inner primer glnA-BIP, with nucleotide sequences as shown in SEQ ID NO. As shown in SEQ ID NOs 13-14, the loop primer pair includes a forward loop primer glnA-LF and a reverse loop primer glnA-LB, with nucleotide sequences shown in SEQ ID NOs 15-16, respectively. The LAMP primer set provided by this invention exhibits good thermostability, is suitable for 70℃ high-temperature LAMP amplification detection systems, reduces non-specific binding, and possesses good detection sensitivity and specificity. The limit of detection (LOD) for the Uu primer set is 50 C / R; the LOD for the CT primer set is 60 C / R; and the LOD for the NG primer set is 50 C / R. Furthermore, the primer sets show no cross-reactivity with other pathogens. This invention's LAMP primer set provides an innovative solution for the accurate diagnosis of sexually transmitted diseases.
[0022] 2. This invention also provides a kit for detecting three sexually transmitted diseases (Uu, CT, NG), the kit comprising the aforementioned primer set. The LAMP primer set used in the kit exhibits good thermostability, is suitable for LAMP amplification detection systems at 70°C, reduces non-specific binding, and possesses good detection sensitivity and specificity. Therefore, the kit of this invention provides an innovative solution for the accurate diagnosis of sexually transmitted diseases. Attached Figure Description
[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0024] Figure 1 This is a validation of the amplification performance of LAMP primer sets targeting the Uu-UreB gene. Figure 1 In the middle, (A) blank group; (B) positive group.
[0025] Figure 2 This is a validation of the amplification performance of LAMP primer sets targeting the CT-gyrA gene detection. Figure 2 In the middle, (A) blank group; (B) positive group.
[0026] Figure 3 This is a validation of the amplification performance of LAMP primer sets targeting the NG-glnA gene. Figure 3 In the middle, (A) blank group; (B) positive group.
[0027] Figure 4 This is a validation of the thermal stability of the LAMP primer set targeting the Uu-UreB gene. Figure 4 In the examples, the primer sets are as follows: orange represents the positive amplification curve, and blue represents the blank amplification curve; the primer sets for Comparative Example 1-1 are as follows: orange represents the positive amplification curve, and gray represents the blank amplification curve; the primer sets for Comparative Example 1-2 are as follows: green represents the positive amplification curve, and orange represents the blank amplification curve; the primer sets for Comparative Example 1-3 are as follows: purple represents the positive amplification curve, and gray represents the blank amplification curve.
[0028] Figure 5 This is a validation of the thermal stability of the LAMP primer set targeting the CT-gyrA gene detection. Figure 5In the examples, the primer sets are as follows: purple represents the positive amplification curve, and gray represents the blank amplification curve; the primer sets of comparative examples 1-4 are as follows: green represents the positive amplification curve, and gray represents the blank amplification curve; the primer sets of comparative examples 1-5 are as follows: orange represents the positive amplification curve, and fluorescent green represents the blank amplification curve.
[0029] Figure 6 This is a validation of the thermal stability of LAMP primer sets targeting the NG-glnA gene detection. Figure 6 In the examples, the primer sets are as follows: yellow represents positive amplification curves and purple represents blank amplification curves; the primer sets for comparative examples 1-6 are as follows: red represents positive amplification curves and gray represents blank amplification curves; the primer sets for comparative examples 1-7 are as follows: red represents positive amplification curves and yellow represents blank amplification curves.
[0030] Figure 7 It is a sensitivity test of LAMP primer sets for detecting the Uu-UreB gene.
[0031] Figure 8 This is a sensitivity test for LAMP primer sets targeting the CT-gyrA gene.
[0032] Figure 9 It is a sensitivity test for LAMP primer sets targeting the NG-glnA gene.
[0033] Figure 10 It is a detection rate test of LAMP primer sets targeting the Uu-UreB gene.
[0034] Figure 11 It is a detection rate test of LAMP primer set for targeted CT-gyrA gene detection.
[0035] Figure 12 It is a detection rate test of LAMP primer sets targeting the NG-glnA gene.
[0036] Figure 13 It is a specific detection method using LAMP primer sets targeting the Uu-UreB gene. Figure 13 In the middle, (A) NG and CT; (B) syphilis, herpes simplex virus type 2 and influenza A virus.
[0037] Figure 14 It is a specific detection method using LAMP primer sets targeting the CT-gyrA gene. Figure 14 In the following, (A) UU and NG; (B) Mycoplasma pneumoniae and Chlamydia psittaci.
[0038] Figure 15 It is a specific detection method using LAMP primer sets targeting the NG-glnA gene. Figure 15In the middle, (A) UU and CT; (B) meningococcus and syphilis. Detailed Implementation
[0039] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.
[0040] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.
[0041] Information on reagent materials used in the embodiments of this invention: Enzyme-free water (purchased from Shanghai Sangon Biotech); RT-LAMP Dye Assay Kit (UDG plus) was purchased from Yisheng Biotechnology Co., Ltd. (item number: 13762ES60); for details on high-temperature Bst enzyme and high-temperature enzyme dilution solution, please refer to the literature "FADS-Based Directed Evolution of a Robust Bst DNA Polymerase Adapting High-Temperature Loop-Mediated Isothermal Amplification (HT-LAMP)"; plasmid DNA and primers were synthesized by Sangon Biotech.
[0042] Example 1: A LAMP primer set for the detection of three sexually transmitted diseases (Uu, CT, NG) This embodiment provides a LAMP primer set for the detection of three sexually transmitted diseases (Uu, CT, NG). The LAMP primer set consists of a first primer set targeting the UreB gene of Ureaplasma urealyticum, a second primer set targeting the gyrA gene of Chlamydia trachomatis, and a third primer set targeting the glnA gene of Neisseria gonorrhoeae. The first primer set consists of a first outer primer pair, a first inner primer pair, and a loop primer UreB-LB; the first outer primer pair consists of a forward outer primer UreB-F3 and a reverse outer primer UreB-B3, with nucleotide sequences as shown in SEQ ID NO. 1~2, respectively; the first inner primer pair consists of a forward inner primer UreB-FIP and a reverse inner primer UreB-BIP, with nucleotide sequences as shown in SEQ ID NO. 3~4, respectively; the nucleotide sequence of the loop primer UreB-LB is shown in SEQ ID NO. 5; The second primer set consists of a second outer primer pair, a second inner primer pair, and a loop primer gyrA-LB; the second outer primer pair consists of a forward outer primer gyrA-F3 and a reverse outer primer gyrA-B3, with nucleotide sequences as shown in SEQ ID NO. 6-7, respectively; the second inner primer pair consists of a forward inner primer gyrA-FIP and a reverse inner primer gyrA-BIP, with nucleotide sequences as shown in SEQ ID NO. 8-9, respectively; the nucleotide sequence of the loop primer gyrA-LB is shown in SEQ ID NO. 10; The third primer set consists of a third outer primer pair, a third inner primer pair, and a loop primer pair; the third outer primer pair consists of a forward outer primer glnA-F3 and a reverse outer primer glnA-B3, with nucleotide sequences as shown in SEQ ID NO. 11-12, respectively; the third inner primer pair consists of a forward inner primer glnA-FIP and a reverse inner primer glnA-BIP, with nucleotide sequences as shown in SEQ ID NO. 13-14, respectively; the loop primer pair consists of a forward loop primer glnA-LF and a reverse loop primer glnA-LB, with nucleotide sequences as shown in SEQ ID NO. 15-16, respectively (primer set sequences are shown in Table 1).
[0043] Table 1 Primer sequences
[0044] Example 2: A kit for detecting three sexually transmitted diseases (Uu, CT, NG). This embodiment provides a kit for detecting three sexually transmitted diseases (Uu, CT, NG), which consists of high-temperature Bst enzyme, amplification buffer, 10× mixed primers (Example 1) and a negative control group.
[0045] The final concentration of the high-temperature Bst enzyme in the high-temperature enzyme dilution solution is 50 U / μL; The amplification buffer consists of 20 mM Tris-HCl buffer (pH 8.8), 4.4 mM MgSO4, 48 mM KCl, 44 mM (NH4)2SO4, 0.48 mM dNTPs and 2 μL of fluorescent dye SYBR-16. The 10× mixed primers are shown below: UU-UreB: Composed of UreB-F3-1 (10μM, 2μL), UreB-B3-1 (10μM, 2μL), UreB-FIP-1 (10μM, 16μL), UreB-BIP-1 (10μM, 16μL), UreB-LB-1 (10μM, 4μL) and enzyme-free water (60μL); CT-gyrA: consists of gyrA-F3-1 (10 μM, 2 μL), gyrA-B3-1 (10 μM, 2 μL), gyrA-FIP-1 (10 μM, 16 μL), gyrA-BIP-1 (10 μM, 16 μL), gyrA-LB-1 (10 μM, 4 μL) and enzyme-free water (60 μL); NG-glnA: Composed of glnA-F3-1 (10μM, 2μL), glnA-B3-1 (10μM, 2μL), glnA-FIP-1 (10μM, 16μL), glnA-BIP-1 (10μM, 16μL), glnA-LF-1 (10μM, 4μL), glnA-LB-1 (10μM, 4μL) and enzyme-free water (56μL).
[0046] The negative control was enzyme-free water.
[0047] Example 3: A method for detecting three sexually transmitted diseases (Uu, CT, NG). This embodiment provides a method for detecting three sexually transmitted diseases (Uu, CT, NG). The method uses the kit from Example 2, and the detection process includes the following steps: 1. Synthesize plasmid DNA carrying the target gene separately, and dilute the synthesized plasmid DNA with enzyme-free water to 4 ng / mL. Convert 4 ng / mL to copies / mL using the conversion formula. The result: The concentration of Ureaplasma urealyticum plasmid DNA is 2.2 × 10⁻⁶. 13 The concentration of Chlamydia trachomatis plasmid DNA was 2.1 × 10⁻⁶ copies / mL. 13 The concentration of Neisseria gonorrhoeae plasmid DNA was 2.2 × 10⁻⁶ copies / mL. 13 copies / mL; dilute the plasmid DNA template to a final concentration of 2.0 × 10⁻⁶. 4 copies / mL; 2. The plasmid DNA was detected using the kit described in Example 2. Amplification was performed using a 25 μL system, with the following reagent volumes: 6.25 μL amplification buffer, 2.5 μL 10× mixed primers, 1 μL high-temperature Bst enzyme, 5 μL template, and 10.25 μL enzyme-free water. 3. After the system is configured, use a real-time fluorescence PCR instrument (ABI Q1 real-time fluorescence PCR instrument) to perform real-time fluorescence PCR. Based on the real-time fluorescence signal obtained, determine whether Uu, CT, or NG can be detected. The amplification conditions for real-time PCR were as follows: incubation at 37°C for 2-5 min, incubation at 70°C for 30-60 min, inactivation at 85°C for 5 min, and melting at the melting end.
[0048] Comparative Example 1-1: A LAMP primer set for Uu detection This comparative example provides a LAMP primer set for Uu detection, the primer set sequences of which are shown in SEQ ID NO.17~22 (primer set sequences are shown in Table 2).
[0049] Table 2 Primer sequences
[0050] Comparative Examples 1-2: A LAMP primer set for Uu detection This comparative example provides a LAMP primer set for the detection of Uu. The primer set can be found in the literature (Shen, JN., Ye, JY., Lao, MX. et al. Evaluation of the real-time fluorescence loop-mediated isothermal amplification assay for the detection of Ureaplasmaurealyticum. AMB Expr 12, 16 (2022)), and its nucleotide sequence is shown in SEQ ID NO.23~28 (primer set sequence is shown in Table 3).
[0051] Table 3 Primer sequences
[0052] Comparative Examples 1-3: A LAMP primer set for Uu detection This comparative example provides a LAMP primer set for the detection of Uu. The primer set can be found in the literature (Shen, JN., Ye, JY., Lao, MX. et al. Evaluation of the real-time fluorescence loop-mediated isothermal amplification assay for the detection of Ureaplasmaurealyticum. AMB Expr 12, 16 (2022)), and its nucleotide sequence is shown in SEQ ID NO.29~34 (primer set sequence is shown in Table 4).
[0053] Table 4 Primer sequences
[0054] Comparative Examples 1-4: A LAMP primer set for CT detection This comparative example provides a LAMP primer set for CT detection, the primer set sequences of which are shown in SEQ ID NO.35~39 (primer set sequences are shown in Table 5).
[0055] Table 5 Primer sequences
[0056] Comparative Examples 1-5: A LAMP primer set for CT detection This comparative example provides a LAMP primer set for CT detection. The primer set is described in the literature (Xu, X., Jia, Y., Li, R. et al. Rapid and simultaneous detection of multiple pathogens in the lower reproductive tract during pregnancy based on loop-mediated isothermal amplification-microfluidic chip. BMC Microbiol 22, 260 (2022)), and its nucleotide sequence is shown in SEQ ID NO.40~44 (primer set sequence is shown in Table 6).
[0057] Table 6 Primer sequences
[0058] Comparative Examples 1-6: A LAMP primer set for NG detection This comparative example provides a LAMP primer set for NG detection, the primer set sequences of which are shown in SEQ ID NO.45~50 (primer set sequences are shown in Table 7).
[0059] Table 7 Primer sequences
[0060] Comparative Examples 1-7: A LAMP primer set for NG detection This comparative example provides a LAMP primer set for NG detection. The primer set can be found in the literature (Edwards T, Burke PA, Smalley HB, Gillies L, Hobbs G. 2014. Loop-Mediated Isothermal Amplification Test for Detection of Neisseria gonorrhoeae in Urine Samples and Tolerance of the Assay to the Presence of Urea. J Clin Microbiol. 2014 Jun;52(6):2163-5), and its nucleotide sequence is shown in SEQ ID NO.51~54 (primer set sequence is shown in Table 8).
[0061] Table 8 Primer sequences
[0062] Comparative Examples 2-1 to 2-7: A kit for detecting three sexually transmitted diseases (Uu, CT, NG). This comparative example provides a kit for detecting three sexually transmitted diseases (Uu, CT, NG). The kit is based on Example 2, but the primer sets are replaced with the primer sets of Comparative Examples 1-1 to 1-7.
[0063] Comparative Examples 3-1 to 3-7: A method for detecting three sexually transmitted diseases (Uu, CT, NG). This comparative example provides a method for detecting three sexually transmitted diseases (Uu, CT, NG). The method is based on Example 3, in which the kit of Example 2 is replaced with the kits of Comparative Examples 2-1 to 2-7 respectively.
[0064] Experiment Example 1: Primer set performance test In this experiment, plasmid DNA from UU, CT, and NG was used as the test samples to perform performance tests on the primer sets in Example 1 and Comparative Examples 1-1 to 1-7. The specific process is as follows: 1. Dilute the plasmid DNA template to a final concentration of 2.0 × 10⁻⁶. 4 copies / mL; 2. The plasmid DNA was detected using the one-step RT-LAMP Dye Assay Kit (UDG plus) manufactured by Yisheng Biotechnology Co., Ltd. Amplification was performed using a 25 μL system, with the following reagent volumes: Bst Buffer 6 μL, 10× mixed primers 2.5 μL, high-temperature Bst enzyme 1 μL, template 5 μL, and enzyme-free water 10.5 μL. 3. After the system is configured, use a real-time fluorescence PCR instrument (ABI Q1 real-time fluorescence PCR instrument) to perform real-time fluorescence PCR. Based on the real-time fluorescence signal obtained, refer to the positive control and negative control (see Table 3) to determine whether Uu, CT or NG can be detected (see Table 4 for the judgment criteria). The amplification conditions for real-time PCR were as follows: incubation at 37°C for 2-5 min, incubation at 65°C for 30-60 min, inactivation at 85°C for 5 min, and melting at the ends. 4. Performance verification and evaluation criteria: Primer sets with a positive time of less than 15 minutes for positive samples and no obvious peak in the blank group are considered to have good amplification effect.
[0065] 5. Experimental Results: Using Yisheng's commercial reagents, at 65℃ and a template concentration of 100 C / R (2.0 × 10⁻⁶), 4 The test results (using 5 μL of a template per 1000 mL) showed that the primer sets of the example and comparative examples could be amplified normally. Figures 1-3 As shown.
[0066] Figure 1 In A, among the four sets of UU primers, only the blank group of primer set 1 of Example 1 did not show a peak; the primer set of Comparative Example 1-1 only showed a slight fluorescence change at the end of the amplification; the primer sets of Comparative Example 1-2 and Comparative Example 1-3 both showed very obvious fluorescence fluctuations, that is, the blank group showed non-specific amplification. Figure 1 In sample B, all four primer sets for UU amplified normally at 100°C / R. Furthermore, the average positive time for all test samples was ≤10 min. The average positive time for the primer set in Example 1 was 5 min; the average positive time for the primer set in Comparative Example 1-1 was 9 min; the average positive time for the primer set in Comparative Example 1-2 was 6 min; and the average positive time for the primer set in Comparative Example 1-3 was 6 min. In conclusion, all four primer sets for UU amplified normally. Except for the primer set in Example 1, the other primer sets exhibited non-specific amplification of the blank group.
[0067] Figure 2 In A, among the three primer sets for CT, only the blank set of primer set 1 in Example 1 did not show non-specific amplification; although the amplification curves of primer sets 1-4 in Comparative Examples did not change significantly, the melting curves showed very obvious characteristic peaks; primer sets 1-5 in Comparative Examples only showed slight fluorescence fluctuations at the end of the amplification stage. Figure 2In sample B, all three primer sets for CT amplified normally, and the average positive time for the test group was ≤10 min. However, there was a significant difference between comparative examples 1-4 and comparative examples 1-5 (i.e., poor intra-batch reproducibility). In conclusion, all three primer sets for CT amplified normally and can proceed to the next step of testing.
[0068] Figure 3 In A, none of the blank groups of the three primer sets of NG showed a clear peak trend; Figure 3 In Example B, the average positive time for the test groups using primer sets 1 (Example 1) and primer sets 1-6 (Comparative Examples 1-6) was ≤10 min; while for primer sets 1-7 (Comparative Examples 1-7), which did not contain loop primers, the average positive time was 11 min. In conclusion, all three primer sets for NG can amplify normally and can proceed to the next step of testing.
[0069] Experiment Example 2: High-Temperature Stability Test of Primer Sets This experiment tested the high-temperature stability of the primer sets in Example 1 and Comparative Examples 1-1 to 1-7. The specific process is as follows: Using plasmid DNA as the test sample, the sample was diluted to a concentration of 100 C / R with enzyme-free water. Real-time PCR was performed on the test sample using the methods described in Example 3 and Comparative Examples 3-1 to 3-7. The performance verification and evaluation criteria were the same as in Experiment 1. The detection results are as follows: Figures 4-6 As shown.
[0070] Figure 4 In A, after increasing the incubation temperature to 70°C, only the primer set of Example 1 showed no nonspecific amplification; the peak trends of the blank sets of Comparative Example 1-1 and Comparative Example 1-2 were slightly weaker than those at 65°C, but still existed; the peak trend of the blank set of Comparative Example 1-3 did not change compared to 65°C. Figure 4 In Example B, the average positive time of the test groups using primer sets 1 of Example 1, primer sets 1-1 of Comparative Example 1-2, and primer sets 1-2 was ≤10 min; while the positive time of the test group using primer sets 1-3 of Comparative Example 1-3 increased to 14 min.
[0071] Figure 5 In group A, after increasing the incubation temperature to 70℃, the three sets of CT primers did not show significant changes in fluorescence values in the amplification curves. Only comparative examples 1-4 (average positive time 24 min) and comparative example 1-5 (average positive time 28 min) showed positive time values. Furthermore, comparative examples 1-4 and 1-5 primers also exhibited distinct small characteristic peaks in the melting curves. Figure 5 In B, the average positive time of the three primer groups for CT was ≤10 min, and the consistency between the test samples was improved compared with 65℃.
[0072] Figure 6 In A, after increasing the incubation temperature to 70℃, no peaks were observed in the blank group of the three primer sets NG; Figure 6 In B, all three primer sets of NG amplified normally at 70℃. The average positive time for primer set 1 in Example 1 was 6 min; the average positive time for primer sets 1-6 in Comparative Examples was 17 min; and the average positive time for primer sets 1-7 in Comparative Examples was 23 min.
[0073] In summary, the primer set shown in Example 1 exhibits good high-temperature stability.
[0074] Experiment Example 3: Primer set sensitivity test This experimental example demonstrates the sensitivity testing of the primer set from Example 1. The specific procedure is as follows: Using plasmid DNA as the test sample, plasmid DNA templates for Uu, CT, and NG were serially diluted to 5-50000 C / R, 6-60000 C / R, and 5-50000 C / R, respectively, using Copies / R as the unit of measurement. The test samples were then subjected to quantitative real-time PCR using the method described in Example 3. The detection results are shown in [Figure 3]. Figures 7-9 .
[0075] Figure 7 The results showed that the limit of detection for UU was 50 C / R; Figure 8 The results showed that the lowest detection limit for CT was 60 C / R; Figure 9 The results showed that the limit of detection for NG was 50 C / R.
[0076] Experiment Example 4: Detection Rate Test of Lowest Detection Limit This experimental example tests the detection rate of the primer set of Example 1 with the lowest detection limit. The specific process is as follows: Using plasmid DNA as the test sample, the test sample was diluted to the lowest detection limit with enzyme-free water (Example 3). Twenty plasmid DNA samples were tested for each pathogen. Real-time quantitative PCR was performed on the test samples using the method described in Example 3. The detection results are shown in [Figure 3]. Figures 10-12 .
[0077] Figure 10 The results showed that the UU primer set was detected in 20 out of 20 plasmid DNA samples, with a detection rate of 100%, and no peak was observed in the blank group, indicating that the primer set had good amplification efficiency and no non-specific amplification. Figure 11 The results showed that the CT primer set was detected in 19 out of 20 plasmid DNA samples, with a detection rate of 95%, and no peak was observed in the blank group, indicating that the primer set had good amplification efficiency and no non-specific amplification. Figure 12The results showed that the NG primer set was detected in 20 out of 20 plasmid DNA samples, with a detection rate of 100%, and no peak was observed in the blank group, indicating that the primer set had good amplification efficiency and no non-specific amplification.
[0078] Experiment Example 5: Primer set specificity detection This experimental example demonstrates the specificity detection of the primer set from Example 1. The specific procedure is as follows: 1. Specificity detection using UU primer set: Plasmid DNA was used as the test sample. The test sample was diluted with enzyme-free water to a concentration of 100 C / R. The plasmid DNA templates for CT, NG, syphilis, herpes simplex virus type 2, and influenza A virus (synthesized by Shanghai Sangon Biotech) were amplified using the UU primer set described in Example 1 and the method described in Example 3. The detection results are shown in […]. Figure 13 ; Figure 13 As shown in Figures A-B, the UU primer set in Example 1 can only amplify the UU template and cannot amplify CT, NG, syphilis, herpes simplex virus type 2, and influenza A virus, indicating that it has good specificity.
[0079] 2. CT primer set specificity detection: Using plasmid DNA as the test sample, the sample was diluted to a concentration of 100 C / R with enzyme-free water. The plasmid DNA templates of CT, NG, Mycoplasma pneumoniae, and Chlamydia psittaci were amplified using the UU primer set described in Example 1 and the method described in Example 3. The detection results are shown in […]. Figure 14 ; Figure 14 As shown in Figures A-B, the UU primer set in Example 1 can only amplify the UU template and cannot amplify CT, NG, Mycoplasma pneumoniae, and Chlamydia psittaci, indicating that it has good specificity.
[0080] 3. NG primer set specificity detection: Using plasmid DNA as the test sample, the sample was diluted to a concentration of 100 C / R with enzyme-free water. The CT, NG, meningococcal, and syphilis plasmid DNA templates were amplified using the UU primer set described in Example 1 and the method described in Example 3. The detection results are shown in [Figure 1]. Figure 15 ; Figure 15 As shown in Figures A-B, the UU primer set in Example 1 can only amplify the UU template and cannot amplify CT, NG, meningococcus, and syphilis, indicating that it has good specificity.
[0081] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.
Claims
1. A LAMP primer set for the detection of three sexually transmitted diseases, characterized in that, The LAMP primer set includes a first primer set targeting the UreB gene of Ureaplasma urealyticum, a second primer set targeting the gyrA gene of Chlamydia trachomatis, and a third primer set targeting the glnA gene of Neisseria gonorrhoeae. The first primer set includes a first outer primer pair, a first inner primer pair, and a loop primer; the first outer primer pair includes a forward outer primer and a reverse outer primer, the nucleotide sequences of which are shown in SEQ ID NO. 1-2, respectively; the first inner primer pair includes a forward inner primer and a reverse inner primer, the nucleotide sequences of which are shown in SEQ ID NO. 3-4, respectively; the nucleotide sequence of the loop primer is shown in SEQ ID NO. 5; The second primer set includes a second outer primer pair, a second inner primer pair, and a loop primer; the second outer primer pair includes a forward outer primer and a reverse outer primer, the nucleotide sequences of which are shown in SEQ ID NO. 6-7, respectively; the second inner primer pair includes a forward inner primer and a reverse inner primer, the nucleotide sequences of which are shown in SEQ ID NO. 8-9, respectively; the nucleotide sequence of the loop primer is shown in SEQ ID NO. 10; The third primer set includes a third outer primer pair, a third inner primer pair, and a loop primer pair; the third outer primer pair includes a forward outer primer and a reverse outer primer, with nucleotide sequences as shown in SEQ ID NO. 11-12, respectively; the third inner primer pair includes a forward inner primer and a reverse inner primer, with nucleotide sequences as shown in SEQ ID NO. 13-14, respectively; the loop primer pair includes a forward loop primer and a reverse loop primer, with nucleotide sequences as shown in SEQ ID NO. 15-16, respectively.
2. A kit for detecting three sexually transmitted diseases, characterized in that, The kit includes the LAMP primer set as described in claim 1.
3. The reagent kit according to claim 2, characterized in that, The kit also includes a high-temperature Bst enzyme and an amplification buffer.
4. The reagent kit according to claim 3, characterized in that, The amplification buffer contains Tris-HCl buffer, MgSO4, KCl, (NH4)2SO4, dNTPs and / or fluorescent dyes.
5. The reagent kit according to any one of claims 2 to 4, characterized in that, The kit also includes quality control materials; the quality control materials include the UreB gene, gyrA gene, and glnA gene.
6. A method for detecting three sexually transmitted diseases, wherein the method is not for the purpose of diagnosing or treating the disease, characterized in that, The method includes: using the kit according to any one of claims 2 to 5 to test the sample to be tested.
7. The LAMP detection method according to claim 6, characterized in that, The amplification conditions in the LAMP detection method are: constant temperature incubation at 68~72℃ for 30~60 min.
8. The application of the LAMP primer set of claim 1, the kit of claims 2-5, or the detection method of claim 6 or 7 in the three tests for sexually transmitted diseases, wherein the application is not for the purpose of disease diagnosis and treatment.