Multi-copy nucleic acid target fragment for detecting treponema pallidum and application

Abstract

The invention discloses a multi-copy nucleic acid target fragment for detecting treponema pallidum, the nucleic acid target fragment comprises a nucleic acid fragment as shown in SEQ ID NO: 1, and application of the multi-copy nucleic acid target fragment in nucleic acid detection of various nucleic acid amplification platforms. The invention further relates to a primer designed based on the target fragment, a detection kit and application of the primer and the detection kit in syphilis diagnosis and curative effect evaluation in all stages. The multi-copy nucleic acid target fragment TMF7 provided by the invention can be detected in a sample with treponema pallidum greater than or equal to 6 copies / ml, has very high sensitivity, and is remarkably superior to Tpp47 and polA (greater than or equal to 1000 copies / ml); through clinical verification, the detection rate of TMF7 in blood plasma, saliva and cerebrospinal fluid of syphilis patients in all stages is remarkably higher than that of existing Tpp47 and polA targets; in addition, the method is suitable for detecting treponema pallidum in all samples, and industrialization and large-scale popularization are easy.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of molecular detection and diagnosis, and particularly relates to a high-sensitivity nucleic acid target fragment of Treponema pallidum and application thereof in detection of Treponema pallidum. BACKGROUND

[0002] Syphilis is a chronic systemic sexually transmitted disease caused by Treponema pallidum (TP). Treponema pallidum Currently, syphilis is still a major public health challenge. In 2016, about 6 million new cases of syphilis were reported worldwide, and the incidence rate continued to rise in both high-income countries and low- and middle-income countries. In China, syphilis has been ranked among the top three in the list of notifiable infectious diseases for decades, with more than 500,000 cases reported annually. If not diagnosed and treated in time, syphilis can cause irreversible damage to tissues and organs. Currently, the diagnosis of syphilis mainly relies on serological detection, but this method has obvious limitations: once specific antibodies to Treponema pallidum are produced, they will be positive for life; non-Treponema pallidum antibody detection is prone to false positives or false negatives; both antibody tests cannot reliably determine the current infection status or response after treatment. Therefore, there is an urgent need for more accurate diagnostic tools to improve patient outcomes and block disease transmission.

[0003] The current diagnosis of syphilis mainly relies on serological detection, but has obvious limitations: 1) False positive problem: physiological conditions (such as pregnancy) and various diseases (such as autoimmune diseases, tumors, etc.) can cause biological false positives; 2) False negative problem: false negatives may occur in early (5-7 weeks after infection) and late syphilis; 3) Serum fixation phenomenon: some patients remain serologically positive for a long time even after regular treatment, making it difficult to determine whether they are infectious; 4) Insufficient evaluation of therapeutic effect: antibody titers cannot objectively reflect the treatment response and cannot directly evaluate the clearance of pathogens in the patient's body.

[0004] Direct pathogen detection methods (such as dark field microscopy DFM, direct fluorescent antibody staining DFA, and immunohistochemistry IHC) are limited by specific sampling sites and syphilis stages (such as primary syphilis chancre and secondary syphilis flat warts). Although nucleic acid amplification testing (NAATs) can provide direct evidence of pathogens, most existing PCR methods use single-copy genes such as Tpp47 and polA as detection targets, which have insufficient sensitivity and low detection rates in samples with low pathogen load (such as plasma and cerebrospinal fluid), limiting their application in clinical diagnosis, therapeutic evaluation, and follow-up management.

[0005] Therefore, there is an urgent need for a new target fragment with high sensitivity, high specificity and high detection rate in different sample types to realize the routine application and POCT landing of NAATs in the etiological detection, clinical diagnosis and treatment of syphilis patients. SUMMARY

[0006] One object of the present application is to provide a high-sensitivity nucleic acid target fragment of Treponema pallidum and its application in the detection of Treponema pallidum, aiming to solve the technical problems of insufficient sensitivity and low detection rate in low pathogen load samples (such as patient saliva, plasma and cerebrospinal fluid).

[0007] The present application provides a nucleic acid target fragment for detecting Treponema pallidum, which is derived from the shared sequence of the gene cluster (tprC / D / E / F / G / I / J) of Treponema pallidum tpr 72 bp, repeated 7 times, and the nucleic acid comprises the nucleic acid fragment shown in SEQ ID NO: 1.

[0008] A primer pair capable of amplifying the nucleic acid fragment for nucleic acid amplification detection of Treponema pallidum. The primer pair comprises an outer primer and an inner primer: The outer primer comprises TMF7-Outer-F of SEQ ID NO: 2 and TMF7-Outer-R of SEQ ID NO: 3; and the inner primer comprises TMF7-Inner-F of SEQ ID NO: 4 and TMF7-Inner-R of SEQ ID NO: 5.

[0009] A probe for the multi-copy nucleic acid target fragment, characterized in that the probe is complementary to the sequence SEQ ID NO: 1 of the multi-copy nucleic acid target fragment.

[0010] A vector containing the above-mentioned multi-copy nucleic acid target fragment.

[0011] An isolated host cell containing the above-mentioned vector or the exogenous nucleic acid target fragment integrated in the genome.

[0012] A kit for detecting Treponema pallidum, the kit containing the above-mentioned multi-copy nucleic acid target fragment primer pair, or the above-mentioned probe, or the above-mentioned vector.

[0013] The above-mentioned various vectors are used for non-diagnostic detection of Treponema pallidum intermediates. An application system of multiple copy nucleic acid target fragments, characterized in that the system comprises an extraction module, an amplification module and a determination module: the system comprises an extraction module, an amplification module and a determination module: the extraction module refers to extracting nucleic acid from a test sample; the amplification module uses an amplification system platform designed for SEQ ID NO: 1 for amplification or signal amplification; the determination module is to determine whether specific signals exist to indicate whether Treponema pallidum nucleic acid exists in the sample.

[0014] The module-based method comprises the following steps: S1 extracting nucleic acid from a test sample; The test sample comprises plasma, cerebrospinal fluid, saliva, and skin lesion exudate; The DNA extraction step is as follows: (1) Clean or disinfect the skin of the sampling site before extraction, and the patient collecting saliva needs to fast for half an hour.

[0015] (2) Add the collected and preserved sample into an EP tube; (3) Add protease K into the EP tube, mix well with a vortex mixer, and centrifuge until no liquid adheres to the tube wall; (4) Add lysis buffer to the above EP tube, mix well with a vortex mixer, and centrifuge briefly until no liquid adheres to the tube wall; (5) Place the EP tube in a water bath; (6) Add anhydrous ethanol into the EP tube, mix well with a vortex mixer, and centrifuge briefly until no liquid adheres to the tube wall; (7) Move the solution in the above EP tube to a separation column, centrifuge, and pour out after completion; (8) Repeat step (7) until all samples pass through the separation column, and use a new collection tube for the last time; (9) Add a specific washing buffer to the separation column, centrifuge, and pour out the waste liquid in the collection tube; (10) Add a specific washing buffer to the separation column, centrifuge, and pour out the waste liquid in the collection tube, and then centrifuge again under certain conditions; (11) Place the filter column in a new EP tube, open the lid, and let the anhydrous ethanol evaporate fully; (12) Add elution buffer to the filter column, stand for a while, centrifuge, discard the filter column after centrifugation is completed, and the liquid obtained in the EP tube is the extracted DNA solution, which is stored in a frozen state for use. S2 using nested PCR designed for SEQ ID NO: 1 for amplification or signal amplification: The first round of nested PCR amplification reaction conditions are as follows: 95℃ pre-denaturation for 4 minutes; then 25 cycles of 95℃ denaturation for 1 minute, 65℃ annealing for 30 seconds, and 72℃ extension for 45 seconds; and finally 72℃ terminal extension for 7 minutes.

[0016] S3 determines whether specific signals exist to indicate whether Treponema pallidum nucleic acid exists in the sample.

[0017] (1) Place the prepared agarose gel in the electrophoresis tank, pay attention to the direction of the gel, and place the DNA loading hole in the negative direction; (2) Add an appropriate amount of electrophoresis buffer to the electrophoresis tank; (3) Take the DNA marker and the sample to be tested with a pipette gun and add it to the sample tank of the gel; (4) Cover the electrophoresis tank cover, connect the power supply and perform electrophoresis; (5) After electrophoresis is completed, place the gel plate in the nucleic acid gel imager to take a photo, and record the results.

[0018] Preferably, the sample to be tested is selected from one or more of plasma, serum, whole blood, saliva, cerebrospinal fluid, urine, semen, skin exudate or a cotton swab (from skin mucosa, oral cavity, urethra, vagina, anus, anal canal, etc.).

[0019] In addition, the detection kit: contains primers / probes / crRNA, polymerase and buffer system, quality control and instructions, which can be selected with DNA extraction reagent and POCT related hardware equipment; the samples include but are not limited to human and animal body fluids, tissues, etc., such as blood (plasma / serum / whole blood), saliva, cerebrospinal fluid, urine, semen, skin tissue and exudate, etc., and environmental samples, etc., and has a wide range of applications.

[0020] The beneficial effects of the present application are: The nucleic acid target fragment TMF7 and the primers thereof provided by the present application can be detected in a sample of Treponema pallidum ≥ 6 copies / ml, have very high sensitivity, and are significantly better than Tpp47 and polA (≥ 1000 copies / ml); and through clinical verification, the detection rate of TMF7 and the primers in the plasma, saliva and cerebrospinal fluid of patients with syphilis of various stages is significantly higher than that of the existing Tpp47 and polA target points; in addition, the present application is suitable for detection of Treponema pallidum in all samples; and is easy to industrialize and widely popularize. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 : Schematic diagram of target identification and its application in clinical sample detection.

[0022] Figure 2 : Schematic diagram of K-mer based repeat sequence screening.

[0023] Figure 3 : Specificity and sensitivity analysis of TMF in TP-DNA detection.

[0024] Figure 4 : Patient enrollment and sample collection.

[0025] Figure 5 : Sensitivity analysis of TMF in detection of plasma samples from syphilis patients.

[0026] Figure 6 : Sensitivity analysis of TMF in detection of saliva samples from syphilis patients.

[0027] Figure 7 : Sensitivity analysis of TMF in detection of cerebrospinal fluid from different types of neurosyphilis patients. DETAILED DESCRIPTION

[0028] The present application will now be described in further detail with reference to the accompanying drawings. It is to be emphasized that the examples are for illustrative purposes only and are not intended to limit the scope of the present application. The main features of the present application can be used in various embodiments without deviating from the scope of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to one of ordinary skill in the art. Furthermore, any method and material similar or equivalent to those described herein can be used in the present application, and such equivalents are considered to be within the scope of the present application and covered by the claims. The preferred methods and materials described herein are only for demonstration.

[0029] I. Target sequence screening and specificity and sensitivity verification: Since the sensitivity of nucleic acid detection depends on the DNA load of the target fragment, we hypothesized that the multi-copy fragment (TMF) on the Treponema pallidum genome has a higher copy number in the sample, and thus has higher detection sensitivity than single-copy target points (such as Tpp47 and polA). As shown in Figure 1 Treponema pallidum initially invades the human body through minor skin and mucous membrane damage, enters the blood through the lymph nodes, and then spreads to multiple tissues and organs throughout the body. Body fluid samples are collected, and Treponema pallidum DNA (TP-DNA) is detected by PCR.

[0030] As shown in Figure 2As shown, by K-mer analysis of the genome of Nichols strain of Treponema pallidum, three multi-copy fragments (TMFs) were identified. Among them: 1) TMF7: 72 bp, repeated 7 times in the genome, corresponding to the shared sequence of tprCDEFGIJ (SEQ ID NO: 1, GenBank: CP004010.2); 2) TMF4: 368 bp, repeated 4 times, corresponding to the shared region of tprCDFI and its upstream sequence; 3) TMF3: 924 bp, repeated 3 times, corresponding to the shared region of tprEGJ and its upstream sequence.

[0031] The specific information is shown in the following Table 1: Table 1 Related information of multi-copy fragments (TMFs) in the genome of Treponema pallidum

[0032] By comparing and analyzing 88 T. pallidum whole genome sequences in the NCBI public database, it was found that the above-mentioned three TMFs were highly conserved. Further BLAST analysis showed that these TMFs had no homology with the human genome and other spirochetes (including Borrelia burgdorferi, T. denticola and Leptospira spp.), and only existed in the same sequence in T. paraluiscuniculi. It is worth noting that T. paraluiscuniculi only infects rabbits and does not cause human disease, so it does not affect the specificity of the present application in clinical diagnosis.

[0033] To verify the specificity of the above TMFs, primers were designed according to the TMF sequences and real-time PCR was performed. The results showed that amplification signals could be detected in T. pallidum DNA templates, while no amplification signals were detected in T. denticola (10 4 copies), Leptospira spp. (10 4 copies) and human DNA (10 4 copies) templates. The results showed that TMF7, TMF4 and TMF3 had high specificity for T. pallidum.

[0034] To further analyze the sensitivity of TMF, a series of genomic dilutions (10 7To 0.75 copies / ml), the nested PCR primers were designed according to the TMF sequence, and the specific sequence was shown in Table 1, and the nested PCR was used for detection.

[0035] The specific steps of the nested PCR (nPCR) were as follows: 1. DNA extraction In this embodiment, the DNA extraction in the collected samples (including plasma, cerebrospinal fluid, saliva, and skin lesion exudate) was performed by using QIAamp DNA Blood Mini Kit of QIAGEN Company, and the detailed steps were shown as follows: (1) Before the experiment, the biological safety cabinet was irradiated with ultraviolet light for more than 30 minutes for disinfection, and the patients for saliva collection needed to fast for half an hour.

[0036] (2) 1 ml of the collected and preserved sample was taken, and 500 ul was taken by using a pipette gun and added into two 2 ml EP tubes; (3) 50 ul of protease K was added into the 2 ml EP tube, and a vortex mixer was used for fully mixing, and a short centrifugation was performed until no liquid adhered to the wall of the tube; (4) 500 ul of Buffer AL was added into the above EP tube (if there were crystals in the buffer AL, the crystals could be dissolved by water bath at 56°C for several minutes), and a vortex mixer was used for fully mixing, and a short centrifugation was performed until no liquid adhered to the wall of the tube; (5) The EP tube was placed in a water bath, and water bath was performed at 56°C for 30 minutes; (6) 500 ul of anhydrous ethanol was added into the EP tube, and a vortex mixer was used for fully mixing, and a short centrifugation was performed until no liquid adhered to the wall of the tube; (7) The solution in the above EP tube was moved to a separation column, 700 ul was taken by using a pipette gun each time, and then centrifugation was performed at 6000 g for 1 min, and the liquid was poured out after completion; (8) Step (7) was repeated until all the samples passed through the separation column, and a new collection tube was used for the last time; (9) 500 ul of AW1 was added into the separation column, and centrifugation was performed at 6000 g for 1 min, and the waste liquid in the collection tube was poured out; (10) 500 ul of AW2 was added into the separation column, and centrifugation was performed at 20000 g for 3 min, and the waste liquid in the collection tube was poured out, and then centrifugation was performed at 20000 g for 1 min; (11) The filter column was placed in a new 1.5 ml EP tube, the cap was opened, and volatilization was performed for 5 min to fully volatilize the anhydrous ethanol; (12) Add 100ul Buffer AE into the filter column, stand for 1min, then centrifuge at 12000g for 3min, after centrifugation, discard the filter column, the liquid in the 1.5ml EP tube is the extracted DNA solution, which is stored in the freezer at -20 degrees for later use.

[0037] 2. Primer sequences of each gene in nPCR The PolA gene and TPP47 gene primers used in nPCR were synthesized by Shanghai Shengong Bioengineering Co., Ltd. According to the data and methods provided on the report provided by the company, the primers were dissolved into a storage solution with a concentration of 100 umol / L using ddH2O, and stored in a freezer at -20 degrees for later use. The concentration of the outer primer used in the nPCR of this experiment was 5 umol / L, and the concentration of the inner primer was 20 umol / L. The above storage solution was diluted according to the corresponding multiple using ddH2O, and the required concentration was obtained. The primer sequences of PolA gene and TPP47 gene are shown in Table 2.

[0038] Table 2 Primer sequences of each gene in nPCR

[0039] 3. nPCR The reaction system and amplification program of nPCR are as follows. Each time the experiment is performed, a positive control (Treponema pallidum Nichols strain genome) and a negative control (ddH2O) are performed. After adding the reaction system according to the table, the instrument is placed for amplification.

[0040] 3.1 PCR system The specific reaction system is shown in Table 3 and Table 4: Table 3 nPCR first round reaction system

[0041] Table 4 nPCR second round reaction system

[0042] 3.2 PCR amplification program The specific amplification conditions are shown in Table 5 and Table 6: Table 5 nPCR first round amplification program

[0043] Table 6 nPCR second round amplification program

[0044] 4. Agarose gel electrophoresis 4.1 Preparation of 1.5% agarose gel (1) Weigh 1.5g of agarose using an electronic analytical balance, place it in an Erlenmeyer flask, and add 100ml of 1×TAE solution (prepared by diluting 50×TAE solution with ultrapure water according to the ratio). (2) After covering with sealing film, put the conical flask into the microwave oven to heat. Then you can take it out and shake the conical flask to ensure that the agarose is completely melted. If it is not completely melted, you can heat it again. (3) Cool the agarose solution to 60°C at room temperature, add 10 μL of nucleic acid dye, and shake well. (4) Place the acrylic inner tank in the horizontal mold, install the baffle, insert the comb, and slowly pour the prepared agarose solution into the mold. The thickness of the gel is about 3-5 mm. Be careful to prevent air bubbles from forming in the gel. (5) Let stand at room temperature for 30 minutes, and after the gel cools and solidifies, slowly pull out the comb and set aside for use.

[0045] 4.2 Electrophoresis and Gel Imaging (1) Place the prepared agarose gel in the electrophoresis tank, paying attention to the direction of the gel, with the DNA loading wells placed in the negative direction; (2) Add an appropriate amount of electrophoresis buffer to the electrophoresis tank, about 1 mm above the gel surface; (3) Take 6 μL of DNA marker and the sample to be tested into the sample well of the gel using a pipette. Be gentle when adding the sample and change the pipette tip for different samples. (4) Cover the electrophoresis tank and turn on the power. The electrophoresis conditions are 120V and 30 minutes. (5) After electrophoresis, take the gel plate and place it in a nucleic acid gel imaging instrument to take pictures and record the results.

[0046] like Figure 3 As shown, the results indicate that TMF7 can be detected in samples with ≥6 copies / ml; the detection limit for TMF4 is ≥100 copies / ml; while the detection limits for TMF3, Tpp47, and polA are all ≥1000 copies / ml. These results demonstrate that the TMF7 target fragment of this embodiment is significantly superior to existing targets in the detection of Treponema pallidum, providing higher sensitivity. Under the same experimental conditions, the detection sensitivity of TMF7 is at least two orders of magnitude higher than that of the tpp47 and polA genes.

[0047] II. Sample Processing and DNA Extraction 1) Collection and storage: 2) Extraction: 1 ml per sample, QIAamp DNA Mini Blood Kit extraction (or equivalent); final elution volume 100 µl; carrier RNA added as necessary to boost recovery.

[0048] 3) Negative and positive controls: Collect blood, saliva or water from normal people, extract the genome as a negative control, and extract the genome of Nichols strain as a positive control.

[0049] III. Analysis of the clinical detection performance of TMF (using nested PCR detection as an example) 1. Patient inclusion and sample collection From January 1, 2021 to December 31, 2024, 210 patients with untreated syphilis diagnosed by laboratory were prospectively included in this study, and all patients had positive treponema pallidum particle agglutination test (TPPA) (This study was approved by the Ethics Committee of Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University and Shanghai Dermatology Hospital. All participants signed a written informed consent form). Among the 210 syphilis patients, different types of clinical samples were collected, including plasma, saliva, cerebrospinal fluid and part of the patient's skin lesion exudate. Among them, 210 plasma samples, 210 saliva samples, 208 cerebrospinal fluid samples and 10 skin lesion exudate samples were collected. The sample design covers different stages of syphilis and different types of body fluids, which can comprehensively evaluate the applicability of TMF7 target fragments in clinical detection. The detection results are shown in the subsequent examples. Figure 4 Different sample types (plasma, saliva, cerebrospinal fluid, exudate) and different syphilis types (primary syphilis, secondary syphilis, latent syphilis, neurosyphilis, all cases) case number column chart: Plasma: 7 cases of primary syphilis, 42 cases of secondary syphilis, 69 cases of latent syphilis, 92 cases of neurosyphilis, and 210 cases of all cases.

[0050] Saliva: 7 cases of primary syphilis, 42 cases of secondary syphilis, 69 cases of latent syphilis, 92 cases of neurosyphilis, and 210 cases of all cases.

[0051] Cerebrospinal fluid (CSF): 6 cases of primary syphilis, 41 cases of secondary syphilis, 69 cases of latent syphilis, 92 cases of neurosyphilis, and 208 cases of all cases.

[0052] Exudate: 6 cases of primary syphilis, 3 cases of secondary syphilis, 0 cases of latent syphilis, 1 case of neurosyphilis, and 10 cases of all cases.

[0053] ​2. Sensitivity analysis of TMF in detecting plasma samples of syphilis patients The plasma samples of syphilis patients at different stages were detected by nested PCR, and the detection rates of TMF7 target fragment and existing Tpp47 and polA target points were compared. As shown in the table, the results are as follows: Figure 5 • Primary syphilis: the detection rate of TMF7 is 100.0%, which is significantly higher than that of Tpp47 (71.4%) and polA (71.4%); • Secondary syphilis: the detection rate of TMF7 is 76.2%, which is also significantly higher than that of Tpp47 (35.7%) and polA (35.7%); • Latent syphilis: the detection rate of TMF7 is 23.2%, while the detection rates of Tpp47 and polA are only 4.3% and 2.9%; • Neurosyphilis: the detection rate of TMF7 is 20.7%, which is significantly higher than that of Tpp47 (4.3%) and polA (2.2%); • All syphilis patients: the plasma detection rate of TMF7 is 35.2%, which is much higher than that of Tpp47 (12.9%) and polA (11.4%).

[0054] The above results show that the TMF7 target fragment provided by the present application has a higher detection rate in the plasma samples of syphilis patients at different stages, and is significantly better than the existing single-copy target points Tpp47 and polA. Therefore, the TMF7 target fragment has obvious advantages in the detection of clinical plasma samples.

[0055] 3. Sensitivity analysis of TMF in detecting saliva samples of syphilis patients The saliva samples of syphilis patients at different stages were detected by nested PCR, and the detection rates of TMF7 target fragment and existing Tpp47 and polA target points were compared. As shown in the table, the results are as follows: Figure 6 • Primary syphilis: the detection rate of TMF7 is 42.9%, which is equivalent to that of Tpp47 (42.9%) and polA (42.9%); • Secondary syphilis: the detection rate of TMF7 is 100.0%, which is also higher than that of Tpp47 (81.0%) and polA (83.3%); • Latent syphilis: the detection rate of TMF7 is 42.0%, while the detection rates of Tpp47 and polA are only 20.3% and 23.2%; ​​• Neurosyphilis: The detection rate of TMF7 was 41.3%, which was significantly higher than that of Tpp47 (13.0%) and polA (14.1%); • All syphilis patients: The saliva detection rate of TMF7 was 53.3%, which was significantly higher than that of Tpp47 (30.0%) and polA (31.9%).

[0056] In summary, the detection rate of the TMF7 target fragment in saliva samples is overall better than that of the existing Tpp47 and polA, especially in the stages of secondary syphilis, latent syphilis and neurosyphilis, which can provide additional etiological evidence in addition to serological detection.

[0057] 4. Detection sensitivity analysis of TMF in cerebrospinal fluid of patients with different types of neurosyphilis The cerebrospinal fluid samples of non-neurosyphilis, suspected neurosyphilis, asymptomatic neurosyphilis and symptomatic neurosyphilis patients were detected, and the detection rates of TMF7 target fragment and Tpp47, polA were compared. As shown in the table, the results are as follows: Figure 7 • Non-neurosyphilis patients: The detection rate of TMF7 was 16.1%, which was significantly higher than that of Tpp47 (4.6%) and polA (1.1%); • Suspected neurosyphilis patients: The detection rate of TMF7 was 34.5%, which was significantly higher than that of Tpp47 (3.4%) and polA (6.9%); • Asymptomatic neurosyphilis patients: The detection rate of TMF7 was 62.2%, while Tpp47 and polA were 2.2% and 4.4%, respectively; • Symptomatic neurosyphilis patients: The detection rate of TMF7 was 78.7%, which was significantly better than that of Tpp47 (17.0%) and polA (12.8%); • Overall: In the CSF of all diagnosed neurosyphilis cases (asymptomatic neurosyphilis and symptomatic neurosyphilis), the detection rate of TMF7 was 70.7%, while that of Tpp47 and polA was only 9.8% and 8.7%, respectively.

[0058] The results show that the TMF7 target fragment described in the present application has significant advantages in cerebrospinal fluid samples, especially in symptomatic and asymptomatic neurosyphilis patients, with a detection rate much higher than that of existing single copy target points, which can provide more reliable molecular basis for the auxiliary diagnosis and follow-up of neurosyphilis.

[0059] The above results support that TMF7 significantly improves the detection rate in various samples and stages, especially in CSF-neurosyphilis, and can be used for efficacy evaluation. ​

[0060] The embodiment can be extended as follows: a probe, which is complementary to the sequence of SEQ ID NO: 1 of the multiple copy nucleic acid target fragment.

[0061] a carrier, which contains the multiple copy nucleic acid target fragment, or the primer pair, or the probe described above, as a positive control for preparing a quantitative standard.

[0062] an isolated host cell, which contains the carrier or the exogenous nucleic acid target fragment integrated into the genome, as a stable quality control source for producing the target fragment.

[0063] a kit, which contains the multiple copy nucleic acid target fragment primer pair, or the probe, or the carrier described above.

[0064] The TMF7 can be used in a kit for detecting Treponema pallidum nucleic acid in a test sample, and is commercially applicable.

[0065] The above only describes the preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can make equivalent replacements or changes within the technical range disclosed by the present application, according to the technical solutions and inventive concepts of the present application, which should be covered within the protection scope of the present application.

Claims

1. A multi-copy nucleic acid target fragment for detecting Treponema pallidum, characterized in that, The multicopy nucleic acid target fragment comprises a nucleic acid molecule with the base sequence shown in SEQ ID NO:

1.

2. Based on the multi-copy nucleic acid target fragment of claim 1, characterized in that: The multi-copy nucleic acid target fragment is a 72 bp sequence, with a total of 7 copies on the chromosome, located at the tprC, tprD, tprE, tprF, tprG, tprI, and tprJ loci.

3. A primer pair based on the multi-copy nucleic acid target fragment of claim 1, characterized in that, The primer pair can amplify the nucleic acid fragment for nucleic acid amplification detection of Treponema pallidum.

4. The primer pair according to claim 3, characterized in that: The primer pair includes: External primers and internal primers: The outer primers include the TMF7-Outer-F sequence as SEQ ID NO:2 and the TMF7-Outer-R sequence as SEQ ID NO:3; the inner primers include the TMF7-Inner-F sequence as SEQ ID NO:4 and the TMF7-Inner-R sequence as SEQ ID NO:

5.

5. A probe based on the multi-copy nucleic acid target fragment of claim 1, characterized in that, The probe targets the complementary sequence in SEQ ID NO:1 of the multi-copy nucleic acid target fragment of claim 1.

6. A carrier, characterized in that: The vector contains a multi-copy nucleic acid target fragment as described in any one of claims 1 to 2, or a primer pair as described in claim 3 or 4, or a probe as described in claim 5.

7. An isolated host cell, characterized in that: The isolated host cells contain the vector of claim 6 or the genome in which the exogenous nucleic acid target fragment of claim 1 or 2 is integrated.

8. A kit for detecting Treponema pallidum, the kit comprising the multi-copy nucleic acid target fragment primer pair as described in claim 3 or 4, or the probe as described in claim 5, or the vector as described in claim 6.

9. The use of the multi-copy nucleic acid target fragment according to any one of claims 1-2, comprising the primer pair of the multi-copy nucleic acid target fragment according to claim 3 or 4, or the probe according to claim 5, or the vector according to claim 6, or the kit according to claim 8, characterized in that: The purpose described is as a reagent for detecting Treponema pallidum.

10. An application system based on the multi-copy nucleic acid target fragment according to any one of claims 1 to 9, characterized in that, The system includes an extraction module, an amplification module, and a determination module: the extraction module extracts nucleic acid from the sample being tested; the amplification module uses an amplification system platform designed for SEQ ID NO:1 to amplify or amplify the signal; and the determination module determines whether a specific signal is present to indicate the presence of Treponema pallidum nucleic acid in the sample.

11. The method for applying the multi-copy nucleic acid target fragment according to claim 10, characterized in that: In the extraction module, the samples to be tested include plasma, cerebrospinal fluid, saliva, and exudate from skin lesions; The DNA extraction steps are as follows: (1) Clean or disinfect the skin at the sampling site before extraction. Patients collecting saliva should fast for half an hour. (2) Add the collected and preserved samples into EP tubes respectively; (3) Add proteinase K to the EP tube, mix thoroughly with a vortex mixer, and centrifuge until no liquid adheres to the tube wall; (4) Add lysis buffer to the above EP tube, mix thoroughly with a vortex mixer, and centrifuge briefly until no liquid adheres to the tube wall; (5) Place the EP tube in a water bath and incubate it in water; (6) Add anhydrous ethanol to the EP tube, mix thoroughly with a vortex mixer, and centrifuge briefly until no liquid adheres to the tube wall; (7) Transfer the solution in the EP tube to the separation column, centrifuge, and then pour out the liquid. (8) Repeat step (7) until all samples pass through the separation column. After the last entry into the separation column, replace with a new collection tube. (9) Add washing buffer to the separation column, centrifuge, and pour out the waste liquid in the collection tube; (10) Add washing buffer to the separation column, centrifuge, pour out the waste liquid in the collection tube, and then perform empty separation; (11) Place the filter column into the new EP tube, open the cap, and allow the anhydrous ethanol to evaporate fully; (12) Add elution buffer to the filter column, let it stand, and then centrifuge. After centrifugation, discard the filter column. The liquid obtained in the lEP tube is the extracted DNA solution. Freeze and store for later use.

12. The method for applying the multi-copy nucleic acid target fragment according to claim 11, characterized in that: The amplification system platform is nested PCR. The first round of nested PCR amplification reaction conditions are 95℃ pre-denaturation for 4 minutes; followed by 25 cycles, each cycle including 95℃ denaturation for 1 minute, 65℃ annealing for 30 seconds, 72℃ extension for 45 seconds; and finally 72℃ final extension for 7 minutes.

13. The method for applying the multi-copy nucleic acid target fragment according to claim 12, characterized in that: The conditions for the second round of nested PCR amplification were as follows: pre-denaturation at 95°C for 4 minutes; followed by 30 cycles, each cycle consisting of denaturation at 95°C for 1 minute, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, and a final extension at 72°C for 7 minutes.

14. The method for applying the multi-copy nucleic acid target fragment according to claim 11, characterized in that: The specific procedures in the determination module include: (1) Place the prepared agarose gel in the electrophoresis tank, paying attention to the direction of the gel, with the DNA loading wells placed in the negative direction; (2) Add an appropriate amount of electrophoresis buffer to the electrophoresis tank; (3) Use a pipette to pick up the DNA marker and the sample to be tested and add them into the sample well of the gel; (4) Cover the electrophoresis tank and turn on the power to perform electrophoresis; (5) After electrophoresis, take the gel plate and place it in a nucleic acid gel imaging instrument to take pictures and record the results.

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